FIELD: medicine, molecular biology, polypeptides.
SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.
EFFECT: valuable medicinal properties of polypeptide.
21 cl, 92 dwg, 14 tbl, 24 ex
The SCOPE of the INVENTION
The present invention relates to the isolation, purification and recombinant or chemical synthesis of proteins that affect the survival, proliferation, differentiation or maturation of hematopoietic cells, in particular progenitor cells platelets. The present invention particularly relates to the cloning and expression of nucleic acids encoding a protein ligands capable of binding to and activation of mpl, a member of the superfamily of cytokine receptors. The present invention further relates to the use of these proteins alone or in combination with other cytokines for the treatment of diseases of the blood, including thrombocytopenia.
BACKGROUND of the INVENTION
I. the blood System
Hematopoietic system produces Mature highly specific blood cells, known to be essential for the life of all mammals. These Mature cells include red blood cells, specialized for the transport of oxygen and carbon dioxide; T - and b-lymphocytes are responsible for cell and mediated by antibodies of the immune response; blood platelets or thrombocytes, specialized for the formation of blood clots; and granulocytes and macrophages, specialized as traps or as accessory cells to fight infection. Granulocytes further subdivide the by: neutrophils, eosinophils, basophils and mast cells, specialized cells types, with discrete functions. It should be noted that all these specialized cells derived from the same common primitive cell type, referred to as pluripotent (or totipotent) stem cells are primarily found in the bone marrow (Dexter et al., Ann. Rev. Cell Biol., 3: 423-441 ).
Mature highly specialized blood cells must be produced continuously in large quantities throughout the life of the mammal. The vast majority of these specialized cells is intended to remain functionally active only in a period of from several hours to several weeks (Cronkite et al., Blood Cells, 2: 263-284 ). Thus, a continuous update of the Mature blood cells, themselves primitive stem cells, as well as any intermediate or destined for the differentiation of progenitor cell lines lying between primitive and Mature cells are necessary to maintain normal blood cells, which are required by the mammal.
In the centre empathically system is a pluripotent stem cell(cell). These cells are relatively few in number and they are subject to renewal by the proliferation of obtaining the child's stem cells or transform is described over a series of stages of differentiation in the increasingly Mature, partially differentiated cells, clearly forming a highly Mature cell(cells) of blood.
For example, certain multipotential progenitor cells, referred to as CFC-Mix, derived from stem cells undergo proliferation (self-renewal) and development with obtaining colonies, containing all the different myeloid cells: erythrocytes, neutrophils, megakaryocytes (precursors of platelets, macrophages, basophils, eosinophils and mast cells. Other progenitor cells of the lymphoid lineage differentiation undergo proliferation and development in T-cells or b-cells.
In addition, between CFC-Mix progenitor cells and myeloid cells are progenitor cells of another level, intermediate kometiani in relation to its offspring. These partially differentiated progenitor cells are classified on the basis of their offspring that they produce. That is, the known direct precursors of the myeloid cells are erythroid colony-forming units (CFU-e) for erythrocyte, granulocyte/macrophage colony-forming cells (GM-COCS) for neutrophils and macrophages, megakaryocyte colony-forming cells (Meg-COCS) for megakaryocytes, eosinophil colonies-forming cells (EOS-COCS) for eosinophils and basophil Colonie-forming cell is (Baz-COCS) for the fat cells. Other cells precursors, intermediate between pluripotent stem cells and Mature blood cells, are known cells (see above), or cells, which will probably be open, with varying degrees of limited differentiation and the ability to self-update.
A fundamental principle of normal empathically cellular system, apparently, is the reduced ability to self-renewal, such as loss of multipotentiality and acquisition of limited differentiation and maturation. Thus, one end of the spectrum empathically cells lies pluripotent stem cell, showing the ability of self-renewal and differentiation into all the various subject-specific differentiation of progenitor cells. This ability is the basis of therapy of bone-marrow-transplant, where a primitive stem cells restore the full population empathically system cells. The other end of the spectrum lie vysokoosnaschennoe differentiation of progenitor cells and their progeny, which have lost the ability of self-renewal, but acquired the functional capacity of Mature cells.
The proliferation and development of stem cells and partially differentiated progenitor cells is under the close control of the m different gemopoeticheskoi growth factors or cytokines. The role of these factors in vivo are complex and poorly understood. Some growth factors such as interleukin-3 (IL-3), is able to stimulate as multipotential stem cells and kometiani progenitor cells of multiple differentiated lines, including, for example, the megakaryocytes. Initially it was assumed that other factors, such as granulocyte/macrophage, Colonie-stimulating factor (GM-CSF), are limited in their effect on GM-KOK. However, later it was found that GM-CSF also affects the proliferation and development interalia megakaryocytes. Thus, it was found that IL-3 and GM-CSF have overlapping activities, although with differing potency. It was recently revealed that interleukin-6 (IL-6) and interleukin-11, although not have a noticeable influence on Meg-kolonialapologie themselves, act synergistically with IL-3 stimulation of maturation of megakaryocytes (Yonemura et al., Exp. Hematol., 20:1011-1016 ).
Therefore, gemopoeticescoe growth factors can influence the growth and differentiation of one or more differentiated lines may overlap with other growth factors in influencing individual progenitor cell line, or may act synergistically with other factors.
It is also likely that gemopoeticescoe growth factors can show their effects is as different stages of development, cells from a totipotent stem cell, various komitirovannyh progenitor cells with limited differentiation to Mature blood cells. For example, erythropoietin (EPO), apparently causes the proliferation of Mature erythroid progenitor cells. IL-3, apparently, does its effect earlier, affecting primitive stem cells and intermediate partially differentiated progenitor cells. Other growth factors such as stem cell factor (FGC), can influence the development of even more primitive cells.
Later it will be clear that the new hematopoietic growth factors that affect the survival, proliferation, differentiation or maturation of any blood cells or their precursors, would be useful, especially to help restore impaired hematopoietic system, which is a consequence of the disease or radiation or chemotherapy.
II. Megakaryocytes - production of platelets
Regulation of megakaryocytopoiesis and production of platelets considered Mazur, Exp. Hematol., 15: 248  and Hoffman, Blood, 74: 1196-1212 . In short, the pluripotent stem cells in the bone marrow differentiate into megakaryocyte, erythrocyte and miliitary cell lines. It is assumed that there is a hierarchy komitirovannyh megakaryocytic progenitor cells among stem cells and megakaryocyte identified, at least three classes megakaryocytic progenitor cells, namely megakaryocytes hearth-forming units (OPE-MK), megakaryocytes, Colonie-forming units (KFE-MK) and megakaryocyte progenitor cells light density (MK-KFE-PL). Maturation of megakaryocytes in itself represents the continuity of development, which has been divided into stages based on conventional morphological criteria. The earliest recognizable members megakaryocytes (MK or Meg) family are megakaryoblast. These cells, originally from 20 to 30 microns in diameter, have a basophilic cytoplasm and slightly irregular nucleus with lost, somehow, reticular chromatin and multiple nucleoli. Late megakaryoblast can contain up to 32 cores (polyploidy), but the cytoplasm remains sparse and immature. The onset of maturation, the nucleus becomes more segmented and pinnatisectum, the cytoplasm increases in quantity and becomes more acidophilus and granular. Most Mature cells of this family can give the appearance of wybodaeth platelets on its periphery. Usually less than 10% of megakaryocytes are under blast and more than 50% are Mature. On conventional morphological classification commonly used for megakaryocyte series, megacar Octobrist is the earliest form; promagisterial or basophilic megakaryocyte is an intermediate form; and Mature (acidophilus, granular or producing platelet megakaryocyte) is the latest form. Mature megakaryocytes spreading the filaments in the cytoplasm in the sinusoidal space, where they are detached and fragmented into individual platelets (Williams et al., Hematology, 1972).
In megakaryocytopoiesis seem to involve multiple regulatory factors (Williams et al., Br. J. Haematol., 52:173  and Williams et al., J. Cell Phisiol., 110: 101 ). Early level megakaryocytopoiesis postulated as mitotic related to cell proliferation and the initiation of the colony of KFE-MK, but not to the number of platelets (Burstein et al., J. Cell Phisiol., 109:333  and Kimura et al., Exp. Hematol., 13: 1048 ). The late stage of maturation is not mitotic associated with nuclear polyploidization and cytoplasmic maturation, and appears to be regulated by a feedback mechanism, depending on the number of peripheral platelets (Odell et al.. Blood, 48: 765  and Ebbe et al., Blood, 32: 787 ).
Discussed the existence of a separate and specific Colonie-stimulating factor megakaryocytes (MK-CSF) (Mazur, Exp. Hematol., 15: 340-350 ). However, most authors believe that the processes that are so vital to survival, as the production of platelets, would be regulated by the cytokine(the mi), responsible solely for this process. The hypothesis that there is megakaryocyte/platelet-specific cytokine(s), represented the basis for the search for more than 30 years, but to this day no one cytokine, such a unique MK-CSF (SRW)has not been purified, sequenced and installed in the study.
Although, it was reported that MK-CSF was partially purified with experimentally obtained thrombocytopenia (Hill et al., Exp. Hematol., 14: 752 ), the air-conditioned environment of the kidney of a human embryo [KC] (McDonald et al., J. Lab. Clin. Med., 85: 59 ), from extracts of human urine with a plastic anemia and idiopathic thrombocytopenic purpura (Kawakita et al., Blood, 6: 556 ) and plasma (Hoffman et al., J. Clin. Invest., 75: 1174 ), in most cases, their physiological function is still unknown.
As potentiators megakaryocytes was used, air-conditioned environment of the spleen cells activated by mitogen from Phytolacca American (PWM SpCM), and cell lines of myelomonocytic mouse WEHI-3 (WEHI-3CM). PWM SpCM contains the factors that enhance the growth KFE-MK (Metcalf et al., Pro. Natl. Acad. Sci., USA, 72:1744-1748 ; Quesenberry et al., Blood, 65:214 ; Iscove, N.N., Hemapoetic Cell Differentiation, ICN-UCLA Simposia on Molecular and Cell Biology, Vol. 10, Golde et al., eds. [New York, Academy Press] pp 37-52 , one of which is interleukin-3 (IL-3), multidifferential Colonie-stimulating factor (multi-CSF) (Burstein, Blood Cells 11:469 ). Other factors in the environment have not yet been defined and selected. WEHI-3 is a myelomonocytic cell line mouse, secreting a relatively large number of IL-3 and a small amount of GM-CSF. It was found that IL-3 promotes the growth of a wide range gemopoeticheskoi cells (Ihle et al., J. Immunol., 13:282 ). It was also found that IL-3 has synergy with many well-known hematopoietic hormones or growth factors (Barteliaez et al., J. Cell Physiol., 122:362-369  and Warren et al., Cell, 46:667-674 ), including erythropoietin (EP), and interleukin-1 (IL-1), in the induction of early multipotent precursors in the formation of very large mixed hematopoietic colonies.
The air-conditioned environment of the lung, bone, cell lines, macrophages, peritoneal exsudate mouse cells and in embryonic cells of human kidney were found other sources of potentiation megakaryocytes. Despite some conflicting evidence (Mazur, Exp. Hematol., 15:340-350 ), there is evidence (Geissler et al., Br.J.Haematol., 60: 233-238 ), which is likely to activated T-lymphocytes than monocytes play a reinforcing role in megakaryocytopoiesis. These data indicate that the secrets of activated T-lymphocytes, such as interleukins, can be regulatory factors in the development of MK (Geissler et al., Exp. Hematol., 15: 845-853 ). The kind of research Meg is curiosities when using purified erythropoietin EPO (Vainchenker et al., Blood, 54:940 ; McLeod et al., Nature, 261: 492-4 ; Williams et al., Exp. Hematol., 12: 734 ) indicates that this hormone has a reinforcing effect on the formation of colonies MK. It was also shown and is free from serum and containing serum cultures, and in the absence of accessory cells (Williams et al., Exp. Hematol., 12: 734 ). It was postulated that the EPO is involved to a greater extent on one - or two-cell stage of megakaryocytopoiesis in contrast to the action of the PWM S that participates in the four-cell stage of development of megakaryocyte. The interaction of these factors on both the early and late phases of development of megakaryocyte remains to be claried.
From the data obtained in several laboratories, it is assumed that only multidifferential factors, which are GM-CSF and IL-3, and, to a lesser extent, IL-6 stimulating factor b-cells, have individually MK-Colonie-stimulating activity (Ikebuchi et al., Proc. Natl. Acad. Sci. USA, 84:9035 ). Recently, several authors have reported that IL-11 and the inhibitory factor leukemia (PHIL) act synergistically with IL-3 on the growth of megakaryocytes and ploidy (Yonemura et al., British Journal of Hematology, 84:16-23 ; Burstein et al., J. Cell. Physiol., 153:305-312 ; Metcalf et al., Blood, 76:50-56 ; Metcalf et al., Blood, 77:2150-2153 ; Bruno et al., Exp. Hematol., 19:378-381 ; Yonemura et al., Exp. Hematol., 20:1011-1016 ).
Other materials represent the work of interest, include: Eppstein et al., US Patent No. 4962091; Chong, US Patent No. 4879111; Fernandes et al., U.S. Patent No. 4604377; Wissler et al., US Patent No. 4512971; Gottlieb, US Patent No. 4468379; Bennet et al., US Patent No. 5215895; Kogan et al., US Patent No. 5250732; Kimura et al., Eur. J. Immunol., 20(9): 1927-1931 ; Secor et al., J. of Immunol., 144 (4): 1484-1489 ; Warren et al., J. of Immunol., 140(1): 94-99 ; Warren et al., Exp. Hematol., 17(11): 1095-1099 ; Bruno et al., Exp. Hematol., 17(10): 1038-1043 ; Tanikawa et al., Exp. Hematol., 17 (8): 883-888 ; Koike engineering Germany et al., Blood, 75 (12): 2286-2291 ; Lotem, Blood, 75(5): 1545-1551 ; Rennik et al., Blood, 73(7): 1828-1835 ; and (Clutterbuck et al., Blood, 73(6): 1504-1512 .
Platelets are a key element in the mechanism of blood clotting. Depletion of circulating levels of platelets is called thrombocytopenia has been observed in various clinical conditions and diseases. Thrombocytopenia is usually defined when the platelets below 150×109per liter. Most of the causes of the disease thrombocytopenia can be subdivided into three categories based on the life span of platelets, namely: (1) impaired production of platelets by the bone marrow, (2) sequestration of platelets in the spleen (splenomegaly), or (3) increased destruction of platelets in the peripheral circulation (i.e. autoimmune thrombocytopenia or chemo - and radiation therapy). In addition, in patients receiving large volumes quickly entered depleted platelets blood products, thrombocytopeni whom I can develop due to cultivation.
Clinical manifestation of bleeding with thrombocytopenia depends on the severity of thrombocytopenia, its causes and possible correlations with impaired coagulation. In General, patients with a score of platelet counts between 20 and 100×109per liter have an enhanced risk of posttraumatic bleeding when the platelet count below 20×109per liter, may be spontaneous bleeding. These latter patients are candidates for transfusion of platelets, accompanied immune or viral risk. For any of these degrees of thrombocytopenia bleeding tends to be more severe when the cause is reduced products than at the increased breakdown of platelets. In the latter situation, the accelerated turnover of platelets leads to the appearance in the circulation younger, big and hemostatically more efficient platelets. Thrombocytopenia may be due to various violations, are briefly described below. A more detailed description can be found in Schafner, A.I., Thrombocytopenia and Disorders of Platelet Function, Internal Medicine, 3rdEd., John J. Hutton et al., Eds., Little Brown and Co., Boston/Toronto/London .
(a) Thrombocytopenia due to decreased production of platelets
Causes of congenital thrombocytopenia include constitutional aplastic anemia (Fanconi syndrome) and congenital amegakaryocytic trombi is topenia, which may be associated with malformation of the skeleton. Acquired disorders product platelets are caused or hypoplasia of megakaryocytes, or ineffective thrombopoiesis. Megakaryocytes hypoplasia may be due to various conditions, including bone aplasia (including idiopathic form or myelosuppression chemotherapeutic agents or radiation therapy), myelofibrosis, leukemia and invasion in bone marrow tumors or granulomas. In some situations, toxins, infectious agents or drugs can affect thrombocytopenia relatively selectively; examples include alternating thrombocytopenia caused by alcohol or certain viral infections, and moderate thrombocytopenia associated with the introduction of casinovip diuretics. Finally, inefficient trombozitopoez, secondary to megaloblastic processes (deficiency of folic acid or B12), can also cause thrombocytopenia, usually accompanied by anemia and leukopenia.
The current treatment of thrombocytopenia caused by reduced production of platelets, depends on the definition and treatment, determining the causes of bone marrow failure. Infusion of platelets is usually reserved for patients with serious complications with bleeding or during the performance of surgical operations, because isoimmunization can lead to refractoriness to the infusion of platelets. Bleeding of the mucous membrane, manifested in severe thrombocytopenia, can be reduced by oral or intravenous administration of antifibrinolytic agents. However, thrombotic complications can develop if used antifibrinolytic agents in patients with disseminirovannam intravenous koagulyatsiami (DCK).
(b) Thrombocytopenia due to trapping in the spleen
Enlargement of the spleen, resulting from any cause, may be accompanied by mild to moderate thrombocytopenia. This is largely a passive process (hypersplenism) capture of platelets, in contrast to the active destruction of platelets by the spleen in immunopositive thrombocytopenia, discussed below. Although the most common cause of hypersplenism is congestive enlargement of the spleen due to hypertension of the portal vein due to alcoholic cirrhosis, other forms of congestion, infiltration or lymphoproliferative splenomegaly are also associated with thrombocytopenia. The expense of platelets in the result only of hypersplenism in General does not fall below 50×109per litre.
(c) Thrombocytopenia due nimmanoradee destruction of platelets
Thrombocytopenia which may be due to accelerated destruction of platelets in various non-immunological processes. Violations of this type include disseminirovanne intravenous coagulation, intravascular prosthetic devices, extracorporeal circulation, thrombotic microangiopathy, such as thrombotic purpura platelet. In all these situations circulating platelets, which are delayed or on artificial surfaces, or violated an intim vessel, or disposed of at these sites, or dissolved and then prematurely cleared the reticulo-endothelial system. The disease or disorder, which may occur dissimilatory intravascular coagulation (DCK), described in detail Braunwald et al. (eds), Harrison''s Priciples of Internal Medicine, 11thEd., p.1478, McGrow Hill . Intravenous prosthetic devices, including heart valves, intra-aortic balloons, can cause mild, to moderate, destructive thrombocytopenia and intermittent thrombocytopenia; in patients subject to cardiopulmonary bypass or dialysis, may be due to the utilization of or damage to the platelets in the extracorporeal circulation.
(d) Immune thrombocytopenia induced by drugs
More than 100 drugs involved in immunologically mediated thrombocytopenia. Characterized, however, only quinidine, hin is h, gold, sulfonamides, cefalotin and heparin. Caused by drugs thrombocytopenia is often very severe and usually occurs rapidly within a few days, until the patient takes the predisposing medicatio.
(e) Immune (autoimmune) thrombocytopenic purpura (ITP)
ITP in adults is a chronic disease characterized by autoimmune destruction of platelets. Antibodies are usually IgG, although it was reported about other immunoglobulins. Although detected at ITP autoantibodies bound to the platelet membrane with GPIIbIIIain most cases, the specificity of the antigen platelets are not identified. In the reticuloendothelial system of the spleen and liver was observed extravascular destruction of sensitized platelets. Although more than half of all cases of ITP is idiopathic, many patients sick rheumatic or autoimmune diseases (for example, erythromatosus systemic lupus erythematosus), or lymphoproliferative diseases (e.g. chronic lymphocytic leukemia).
(f) HIV-induced ITP
ITP is increasing in the usual complication of HIV infection (Morris et al., Ann. Intern. Med., 96: 714-717 ) and can be observed at different stages of development of the disease and in patients who diagnosed with acquired immunodeficiency syndrome (AIDS), have AIDS-dependent complex, and in people living with HIV, but without the syndrome AIDS. HIV infection is transmitted disease, is uniquely characterized by severe deficiency of the cellular immune functions, as well as accompanied by concomitant infections and malignant neoplasms. The primary immunologic disorders resulting from HIV infection is a progressive reduction and functional debility of T-lymphocytes expressing cell surface glycoprotein CD4 (Lane et al., Ann. Rev. Immunol., 3: 477 ). The loss of CD4 helper/induction of T-cell function, obviously, profound defects in cellular and humoral immunity, leading to concomitant infections and malignancy, characterizing AIDS (N. Lane, above). Although the mechanism of ITP associated with AIDS, is unknown, it seems that it is different from the mechanism of ITP, not associated with HIV infection. (Walsh et al., N. Eng. J. Med., 311: 635-639 ; and Ratner, Am. J. Med., 86: 194-198 ).
IV. Modern therapy of thrombocytopenia
Therapeutic approaches to the treatment of patients with thrombocytopenia are determined by the severity and rapidity of the clinical situation. Treatment is the same for HIV-dependent and not zavisimoi from HIV thrombocytopenia, and although used a variety of therapeutic approaches, therapy remains controversial.p> Account thrombocyto patients diagnosed with thrombocytopenia successfully increases under the influence of corticosteroids (e.g. prednisolone) therapy, but most patients curing is incomplete or there are relapses, when dosage glucocorticoid is reduced or treatment is interrupted. Relying on patients with HIV-associated ITP, some researchers have suggested that therapy with glucocorticoids can lead to susceptibility to AIDS. Glucocorticoids are commonly used if the account platelet count falls below 20×109/liter or when there are spontaneous bleeding.
For patients refractory to glucocorticoids, have successfully applied the compound 4-(2-chlorophenyl)-9-methyl-2-[3-(morpholinyl)-3-propanone-1-yl]6N-thieno[3,2,f][1,2,4]triazolo-[4,3,a][1,4]diazepin (WEB 2086) for the treatment of severe cases of ITP, not related to HIV. Patients who have an account with platelets 37000 - 58000/ µl, were treated WEB 2086 and after two weeks of treatment the expense of platelets increased to 14000-190000/ µl. (EP 361077 and Lohman et al., Lancet, 1147 ).
Although the optimal treatment of acquired amegakaryocytic thrombocytopenic purpura (MTE) is not defined, it has been shown that, antithymocyte globulin (ATG) equine antisera to the tissue of the thymus person gives long-lasting complete remission (Trimble et al., Am. J. Hematol., 37: 126-127 ). But not avnei publication noted, what hematopoietic effects of APG can be attributed to thimerosal, which operates mainly on proteins as carrying mercury (Panella et al., Cancer Research, 50: 4429-4435 ).
It was reported on good results in splenectomy. When splenectomy removed the main plot destruction of platelets and the main source of production of autoantibodies in many patients. This operation leads to long-term, not requiring treatment, remissi from a large number of patients. However, because surgical intervention, in General, and in immunocompromised patients is avoided, splenectomy is recommended only in severe cases of thrombocytopenia (eg, severe HIV-associated ITP)in patients who are unable to respond within 2-3 weeks of treatment with glucocorticoids, or not achieving the long-term response after termination of treatment with glucocorticoids. Based on current scientific evidence, it is not clear predisposes whether splenectomy patients to AIDS.
In addition prednizolonovuyu therapy and splenectomy certain cytotoxic agents, namely vincristine, azidothymidine (AST, zid.ovud.ine), also show promise in the treatment of ITP induced HIV; however, these results are preliminary.
Later it will be clear that one of the ways to treat thrombocytopenia could be the receiving agent, the FPIC of the service to accelerate the differentiation and maturation of megakaryocytes or their predecessors in forms producing platelets. Were spent considerable efforts to search for such an agent, usually referred to as "thrombopoietin" (TA). Other names of the TA usually encountered in the literature include: stimulating factor trombozitopoez (FTS); Colonie-stimulating factor megakaryocytes (MK-CSF); megakaryocyte-stimulating factor and potentiator megakaryocytes. The TA activity was detected early enough, already in 1959 (Rak et al., Med. Exp., 1: 125), but also by ongoing attempts to characterize and purify the agent. Despite the existing data on the partial purification TA-active polypeptide (see, for example, Tayrien et al., J. Biol. Chem., 262: 3262  and Hoffman et al., J. Clin. Invest. 75: 1174 ), some authors have postulated that the TA is inherently not a separately existing reality, but rather it's just a polyfunctional manifestation of the known hormones (IL-3, Sparrow et al. Prog. Clin. Biol. Res., 215: 123 ). Regardless of its form or nature of the molecule, showing thrombopoetin activity, would be of significant therapeutic value. Although neither protein has not been clearly identified as TA, considerable interest surrounded recent discovery that the mpl, the natural receptor cytokines may mediate thrombopoiesis signal.
V. Mpl is megakaryocytopoiesis receptros cytokines
It is assumed that h is about the proliferation and maturation of hematopoietic cells is tightly regulated by factors that positively or negatively modulate the proliferation and differentiation predifferentiated pluripotent stem cells. These effects are mediated by way of the high-affinity binding of extracellular protein factor with specific cell surface receptors. These surface receptors show some homology and are usually classified as members of the superfamily of cytokine receptors. Members of this family include receptors for IL-2 (β and γ-chain) (Hatakeayma et al., Science 244: 551-556 ; Takeshita et al., Science 257: 379-382 ), IL-3 (Itoh et al.. Science, 247: 324-328 ; Gorman et al., Proc. Natl. Accad. Sci. USA, 87: 5459-5463 ; Kitamura et al., Cell 66: 1165-1174 [1991a]; Kitamura et al., Proc. Natl. Acad. Sci. USA, 88: 5082-5086 [1991b]), IL-4 (Mosley et al., Cell 59: 335-348 ), IL-5 (Takaki et al., EMBO J. 9: 4367-4374 ; Tavernier et al., Cell, 66: 1175-1184 ), IL-6 (Yamasaki et al., Science 241: 825-828 ; Hibi et al., Cell 63:1149-1157 ), IL-7 (Goodwin et al., Cell, 60: 941-951 ), IL-9 (Renault et al., Proc. Natl. Acad. Sci. USA, 89: 5690-5694 ), granulocyte-macrophage, Colonie-stimulating factor (GM-CSF) (Gearing et al., EMBO J. 8: 3667-3676 ; Hayashida et al., Proc. Natl. Acad. Sci. USA, 244: 9655-9659 ), granulocyte, Colonie-stimulating factor (G-CSF) (Fukunaga et al., Cell, 61: 341-350 [a]; Fukunaga et al., Proc. Natl. Acad. Sci. USA, 87: 8702-8706 [1990b]; Larsen et al., J. Exp. Med., 172: 1559-1570 ), EP (D'andrea et al., Cell, 57: 277-285 ; Jones et al., Blood, 76: 31-35 ), the factor inhibiting leukemia (PHIL) (Gearing et al., EMBO J. 10: 2839-2848 ), oncostatin M (OSM) (Rose et al., Proc. Natl. Acad. Sci. USA, 88: 8641-8645 ) and the prolactin receptor (Boutin et al., Proc. Natl. Acad. Sci. USA, 88: 7744-7748 ; Edery et al., Proc. Natl. Acad. Sci. USA, 86: 2112-2116 ), growth hormone (GH) (Leung et al., Nature, 330: 537-543 ), and ciliary neurotropic factor (CNTF) (Davis et al., Science 253: 59-63 ).
Members of the superfamily of cytokine receptors can be grouped into three functional categories (for a review, see Nicola et al., Cell, 67:1-4 ). The first class includes the receptors with a single chain, such as the receptor for erythropoietin (EP-R) or the receptor Colonie-stimulating factor, granulocyte (G-CSF-R), which bind ligand with high affinity due to the extracellular domain, and generate intracellular signals. The second class of receptors, the so-called α-subunit that includes the receptor for interleukin-6 (IL-6-R), the receptor for granulocyte-macrophage, Colonie-stimulating factor (GM-CSF-R), the receptor for interleukin-3 (IL-3-Rαand other members of the superfamily of cytokine receptors. These α-subunit bind ligands with low affinity, but not able to transmit an intracellular signal. High-affinity receptors capable of transmitting the generated signal heterodimers, between α-subunit and a member of the third class of cytokine receptors, labeled β-subunit, namely βwithtotal β-subunit for the three α-subunits of the IL-3-Rα and GM-CSF-R.
Evidence that mpl is a member of supersam istwa cytokine receptors follows from the homology sequence (Gearing, EMBO J. 8: 3667-3676 ; Bazan, Proc. Natl. Acad. Sci. USA, 87: 6834-6938 ; Davis et al., Science 253: 59-63  and Vigon et al., Proc. Natl. Acad. Sci. USA, 89: 5640-5644 ) and its ability to transmit proliferative signals.
Deduced sequence of the protein with the molecular cloning of c-mpl mouse discovers that this protein is homologous to other cytokine receptors. The intracellular domain contains a 465 amino acid residues and comprises two subdomains, each with four highly conserved with cysteine, and a specific structure in the N-terminal subdomain and the C-terminal subdomain. The ligand-binding extracellular subdomain is supposed to be folded structural geometry that is similar to the double β-barrel-shaped. This duplicated the extracellular domain is vysokogermetichnym signal transmitting circuit that is common to IL-3, IL-5 and GM-CSF receptors, as well as low-affinity binding domain PHIL (Vigon et al., Oncogene, 8:2607-2615 ). Thus, the mpl may belong to the low-affinity ligand-link class of cytokine receptors.
Comparison of mpl mouse and Mature mpl P of the man found that these two proteins are 81% identity in the sequence. More specific N-terminal and C-terminal extracellular subdomains are 75 and 80% identity in the sequence, respectively. The most conservative region t is aetsa cytoplasmic domain, showing 91% identity to the amino acid sequence of 37 amino acids, which are completely identical in the region near the transmembrane domain of both species. Thus, it is reported that the mpl is one of the most conservative members of the superfamily of cytokine receptors (Vigon, ibid.).
Evidence that mpl is a functional receptor, is able to transmit proliferative signals, it follows from the construction of chimeric receptor containing the extracellular domain of the cytokine receptor with high affinity to known cytokines, together with the cytoplasmic domain of mpl. Because not informed of any known ligand for the mpl, it was necessary to construct a high-affinity chimeric ligand-binding extracellular domain of the class of cytokine receptors such as IL-4-R or G-CSF-R. Vigon et al., ibid, joined the extracellular domain of G-CSF-R with both transmembrane and cytoplasmic domains of c-mpl. IL-3 dependent cell line BAF/B03 (Ba/F3), was transfected with a chimeric G-CSF-P/mpl together with control G-CSF-R full length. Cells transfected with a chimeric, grew equally well in the presence of the cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSF-R also grew well in the presence of IL-3 and G-CSF. All cells were killed in the absence of growth factors. Similar experimental the options were held Skoda et al., EMBO J., 12(7): 2645-2653 , in which both the extracellular and transmembrane domains of the receptor for IL-4 (RF-IL-4), were fused to the cytoplasmic domain of mpl mouse, and transfected into the IL-3-dependent Ba/F3 cell line mouse. Cells Ba/F3 transfected with wild type RH-IL-4, normally shared in the presence of species-specific either IL-4 or IL-3. Ba/F3 cells transfected with RH-IL-4/mpl, normally shared in the presence of RH-IL-4 (in the presence or absence of IL-3), showing that Ba/F3 cells mpl cytoplasmic domain contains all the elements necessary for the transmission of proliferative signals.
These experiments with chimeras demonstrate the ability to conduct a proliferative signal in the cytoplasmic domain of mpl, but silent, do not pay attention, can bind the ligand extracellular domain of mpl. These data include at least two possibilities, namely, mpl is the receptor with one chain (first class), similar to the EP-P or G-CSF-R, or is the signal-transmitting β-subunit (third class), requiring α-subunit, like IL-3 Skoda et al., ibid.).
VI. The mpl ligand is thrombopoetin (TA)
As described above, it was hypothesized that the serum contains a unique factor, sometimes referred to as thrombopoetin (TA), which acts synergistically with other cytokines to stimulate growth isorevenue megakaryocytes. Despite significant efforts by numerous groups, no such natural factors has not been isolated from serum or any other source. Although it is unknown whether mpl directly link megakaryocyte-stimulating factor, recent experiments show that mpl is involved in the transfer of proliferative signal factor or factors in the serum of patients with aplastic bone marrow (Mathia et al., Blood, 82(5):1395-1401 ).
Proof that a unique colony forming factor distinct from IL-1α, IL-3, IL-4, IL-6, IL-11, SCF, FL, G-CSF and GM-CSF, reports proliferative signal through the mpl, it follows from the research distribution expresii C-mpl in primitive and komitirovannyh hematopoietic cell lines, and antisense (using nucleotide reverse sequence) studies on one of these cell lines.
Using reverse transcriptase (RT)-PCR in immunodeciency hematopoietic human cells, Mathia et al., ibid, showed that a strong signal mpl mRNA was only detected in CD34+treated cells, megakaryocytes and platelets.
CD34+, purified from bone marrow (KM) cells represent approximately 1% of all cells KM and enriched primitive and kometiani progenitor cells of all differencewas the lines (namely, erythroid, granulometrically and megakaryocytes).
It was shown that oligodeoxynucleotide mpl reverse sequence inhibit the formation of colonies of megakaryocytes from pluripotent CD34+cultivated in serum of patients with aplastic bone marrow (a rich source of Colonie-stimulating activity of megakaryocytes [MK-KSA]). The same oligodeoxynucleotide reverse sequence had no effect on the formation of erythroid or granulometrically colonies.
Directly whether links mpl ligand and does factor in serum that causes megakaryocytes through mpl, is still unknown. It was suggested, however, that if the mpl acts directly by binding the ligand, its amino acid sequence would be similar to the highly conserved and there are species with cross-reactivity with certain identical sequence between the extracellular domains mpl human and mouse (Vigon et al., ibid ).
VII. The subject inventions
From the point of view of the subsequent will be clear that there is a real and continuing need in the isolation and identification of molecules capable of stimulating proliferation, differentiation and maturation hematopoetic cells, especially megakaryocytes or their predecessors lateritius use in the treatment of thrombocytopenia. Apparently, such a molecule is the ligand mpl and, therefore, there is a need for allocation of such ligand (ligands)to assess its role (roles) for the growth and differentiation of cells.
Accordingly the object of this invention to provide a pharmaceutically pure molecule that can stimulate division, differentiation and/or maturation of megakaryocytes in the Mature form, producing platelets.
Another object is to obtain a molecule in a form suitable for therapeutic use in the treatment of hematopoietic disorders, particularly thrombocytopenia.
Further, the object of the present invention is the isolation, purification and specific determination of ligands capable of binding in vivo receptor superfamily of cytokines, known as mpl, to transmit proliferative signals.
Still another object is to obtain nucleic acid molecules encoding such protein ligands, and the use of these nucleic acid molecules to produce mpl binding ligands in recombinant cell culture, for diagnostic and therapeutic use.
Another object is to obtain derivatives and modified forms of the protein ligands, including amino acid sequence variants, variations which you types of glycoproteins and their covalent derivatives.
An additional object is to obtain fused types of polypeptides, combining the mpl ligand and a heterologous protein, and their covalent derivatives.
Another additional object is to obtain a variant form of the polypeptide, combining the mpl ligand with amino acid additions, and substitutions of the sequence VC to obtain protein, is able to regulate the division and growth of both progenitor cells platelets and red blood cells.
Another object is the preparation of immunogens to increase antibodies against ligands mpl or combined types, as well as antibodies able to bind similar ligands.
These and other objects of the invention will be apparent to the ordinary worker when considering the parts as a whole.
The objects of the invention are obtained by obtaining the selected mammalian protein that stimulates megakaryocytopoiesis proliferation and maturation, denoted by the "mpl ligand" (ML) or thrombopoietin" (TA), capable of stimulating the proliferation, maturation and/or differentiation of megakaryocytes in the Mature form, producing platelets.
This substantially homogeneous protein can be purified from a natural source, the method comprising: (1) the contacting of the plasma source containing the olukolu mpl ligand for purification on immobilized receptor polypeptide, specific mpl or mpl fused protein, immobilized on a substrate, under conditions when the purified molecule of mpl ligand selectively adsorbed on the immobilized polypeptide receptor, (2) washing the immobilized receptor polypeptide and its substrate to remove readsorbing material, and (3) flushing molecules mpl ligand to the immobilized receptor polypeptide, to which he was adsorbed, the elution buffer. Natural source, preferably, is plasma mammals or urine containing the mpl ligand. The mammal is not necessarily aplastic, and immobilized receptor is a fusion of mpl and IgG.
The preferred protein, stimulating megakaryocytopoiesis proliferation and maturation, optional, is selected substantially homogeneous mpl ligand polypeptide obtained by synthetic or recombinant means.
Polypeptide "mpl ligand" or "TA" of the present invention preferably has at least 70% identical to the sequence relative to the amino acid sequence of purified, substantially homogeneous polypeptide ligand mpl pigs and at least 80% identical to the sequence in relation to EP-domain polypeptide ligand mpl pigs. Selectively mpl ligand of the present and the gain is Mature mpl ligand person (MLC), with the Mature amino acid sequence represented in figure 1 (Th. No.1), or a variant, or as posttranscriptional modified form, or a protein having about 80% identical to the sequence relative to the Mature ligand mpl person. Optional choice of the mpl ligand is a fragment, especially amino end (N-end), or a fragment EP-domain" Mature mpl ligand person (MLC). Preferably, a fragment of the amino-end remains substantially the entire sequence ML of a person between the first and fourth cysteine residues, but may contain significant insertion, deletion or replacement outside of this region. In accordance with this implementation of a fragment of the polypeptide can be represented by the formula:
where MLC(7-151) represents the amino acid sequence of TA person (TFC) from CIS7to CIS151inclusive; X represents the amino group of Cys7or one or more amino end(s) of amino acid(s) residue(s) Mature MLC, or amino acid residue, propagating, in addition, such as Met, Tyr, or a leader sequence containing, for example, designated for proteolytic cleavage (namely, Factor XA or thrombin); and Y represents a carboxy end group CIS151or one or more end(s) of amino acid(s) residue(the) Mature MLC, or, in addition, their extension.
Polypeptide ligand mpl or the optional fragment can be fused to heterologous polypeptide (Chimera). Preferred the heterologous polypeptide is a cytokine, Colonie-stimulating factor or interleukin or its fragment, in particular the kit-ligand (KL), IL-1, IL-3, IL-6, IL-11, FL, GM-CSF or PHIL. The preferred sampling the heterologous polypeptide is a chain of immunoglobulin, in particular human IgG1, IgG2, IgG3, IgG4, IgE, IgD, IgM or fragments thereof, particularly including the domains of the heavy chain of IgG.
Another aspect of the present invention is a composition comprising the selected mpl agonist possessing biological activity and, preferably, capable of stimulating the incorporation of labeled nucleotides (a, namely,3H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected mpl person. Agonist mpl is not necessarily biologically active mpl ligand, but preferred is its ability to stimulate the inclusion of35S in circulating platelets in the study of the recovery of platelets mouse.
Suitable mpl agonists include MLC153, MLC(RA, RA), MLC, MLC, MLC, MLM, MLM, MLM, MLP and MLP or fragments thereof.
In another embodiment the present invention describes the selection of antibodies that are able to communicate with all possible is consistent with the mpl ligand. The selected antibody can contact the mpl ligand, can be optionally connected to the second polypeptide, and the antibody or the connection can be used for isolation and purification of mpl ligand from sources as described above for immobilized mpl. In a further aspect of this embodiment of the invention describes a method of determining the mpl ligand in vivo, comprising contacting the antibody with the sample, in particular a serum sample suspected on the content of the ligand and binding, if it occurs.
In a further embodiment of the invention describes the selection of nucleic acid molecules, encoding the mpl ligand or its fragments, nucleic acid molecule which, optionally, may be mecena detektivami radical and a molecule of nucleic acid has a sequence that is complementary to, or hybridized in moderate to highly stringent conditions with a nucleic acid molecule having a sequence encoding the mpl ligand. Preferred nucleic acid molecules are those that encode the mpl ligand of human, pig and mouse, and include DNA and RNA, and genomic and cDNA. In a further aspect of this embodiment, the nucleic acid molecule is DNA encoding the mpl ligand, and further comprising a replicable vector, is where the DNA is operatively linked with the control sequence, recognized by the host, which transformed vector. DNA does not necessarily represent cDNA having the sequence represented in figure 1 5’-3’ (Th. No.2), 3’-5’, or fragments thereof. This aspect further includes host cells, preferably Cho cells transformed by the vector, and a method of using DNA to stimulate the production of mpl ligand, preferably including the expression of cDNA encoding the mpl ligand, in the culture of the transformed host cells, and return the mpl ligand from the host cells or the culture of the host cells. The mpl ligand obtained in this way is preferably mpl ligand person.
The invention further includes a method of treating animals with hematopoietic violation, particularly thrombocytopenia, comprising the administration to a mammal a therapeutically active amount of mpl ligand. The mpl ligand, optionally, is introduced in combination with a cytokine, in particular with Colonie-stimulating factor or interleukin. The preferred Colonie-stimulating factors or interleukins include: kit-ligand (KL), PHIL, G-CSF, GM-CSF, M-CSF, FL, IL-1, IL-3, IL-6 and IL-11.
The invention further includes a method of isolation and purification TA (ML) of TA-producing microorganisms, including:
(1) the destruction or lysis of cells containing TA,
(2) long is inoe separating soluble material from insoluble material, containing TA,
(3) the solubilization of TP from insoluble material by using solubilizing buffer
(4) the Department solubilizing TA from the other soluble and insoluble material,
(5) putting TP in oxidative/reductive buffer, and
(6) the Department correctly laid TP from neurodiverse TA.
The method describes the solubilization of insoluble material containing TA, chaotropes agent when chaotropic agent is selected among the salts of guanidine, sodium thiocyanate or urea. The method further describes what the solubilized TA is separated from other soluble and insoluble material by means of one or more of the steps selected from among centrifugation, gel filtration and chromatography with reversed phase. The stage of laying the method of obtaining describes oxidative/rehabilitation buffer containing oxidizing and reducing agent. In General, the oxidizing agent is oxygen or a compound containing at least one disulfide bond, and the reducing agent is a compound containing at least one free sulfhydryl group. Preferably, the oxidant is selected from oxidized glutathione (GSSG) and cysteine, and the reducing agent is selected from the restored glutathione (GSH) and cysteine. More preferably, the oxidizing agent which is oxidized glutathione (GSSG), and the reducing agent is restored glutathione (GSH). Also is preferred that the molar ratio of oxidizing agent to be equal to or greater than that of the reducing agent. Oxidative/reductive buffer further comprises a detergent, preferably selected from CHAPS and CHAPSO present, at least at the 1%level. Oxidative/reductive buffer further comprises NaCl, preferably in the range of concentration from about 0.1-0.5 M, and glycerol, preferably in a concentration higher than 15%. the pH of the oxidative/reductive buffer lies preferably in the range of from about pH 7.5 to pH of 9.0, and the installation phase is carried out at 4 degrees within 12-48 hours. Stage styling gives biologically active TA, in which the disulfide bond is formed between nearest to the amino end of Cys and Cys closest to the carboxy end of the EP domain.
The invention further includes a method of purification of biologically active TA from a microorganism, including:
(1) lysis, at least the extracellular membrane of the microorganism,
(2) treatment of the lysate containing TA chaotropes agent,
(3) laying the TA and
(4) the Department of impurity and wrong current TA from the right way the current TA.
Brief description of drawings
Figure 1 shows the deduced amino acid sequence (Th. No.1) mpl ligand (MLC), cDNA and to drowsey nucleotide sequence (Th. No.2). Nucleotides are numbered from the beginning of each line. 5’ and 3’ untranslated region are indicated by letters at the bottom. Amino acid residues are numbered above the sequence, starting from Series 1 of mpl ligand (ML) of the Mature protein sequence. Communications intended AXONE 3 as indicated by the arrows, and the potential site of N-glycosylation is separated by a partition. Residues of cysteine marked by the dotted line above the sequence. The underlined sequence corresponds to the N-terminal sequence mpl ligand purified from the plasma of pigs.
Figure 2 shows how the definitions used to enable3H-thymidine in the mpl ligand. To determine whether the mpl ligand in various sources cell mplPBa/F3 left to starve without IL-3 for 24 hours in a humidified incubator at 37°in the presence of 5% CO2and air. After fasting without IL-3 cells were placed in culture plates with 96 cells with or without dilution of the samples and were cultured for 24 hours in a cell culture incubator. In each cell was added 20 µl of RPMI medium free from serum, containing 1 MX3H-thymidine and leave for 6-8 hours. The cells are then collected on a filter plate with 96 cells and washed with water. Then I calculated the score on the filters.
Figure 3 shows the effect of pronase, DTT and nahrawan is the ability of the APS to stimulate cell proliferation of Ba/F3-mpl. For hydrolysis of APS by pronasol pronase (Boehringer Mannheim) or bovine serum albumin was tied to Affi-gel (Biorad) and separately incubated with the TSA for 18 hours. After that, the gel was removed by centrifugation, and supernatant investigated. APS also warmed up to 80°C for 4 minutes, or treated with 100 μm DTT, and then were dialyzed against PBS.
Figure 4 shows the elution of the active mpl ligand with columns Phenyl-Toyoperl, Blue-Sepharose and Ultralink-mpl. Fractions 4-8 with mpl affinity column were the fractions with peak activity, loirevalley with the column.
Figure 5 shows SDS-gelelectrophoresis on polyacrylamide gel (SDS-PAGE) fractions, buyrevia with Ultralink-mpl column. To 200 μl of each fraction 2-8 was added 1 ml of acetone containing 1 mM HCl at -20°C. After 3 hours at -20°the samples were centrifuged and the precipitate washed 2 times with acetone at -20°C. thereafter, the acetone precipitate was dissolved in 30 μl of SDS-solubilizing buffer, treated with 100 μm DTT and warmed up at 90°C for 5 minutes. The samples were then dispersed on a 4-20% polyacrylamide gel and proteins were visualized by silver colouring.
6 shows the elution of the active mpl ligand after SDS-PAGE. Fraction 6 after mpl-affinity column was dispersed on 4-20% SDS-polyacrylamide gel under non conditions. After electrophoresis the gel was cut into 12 equal to the share and electrolytically, as described in the examples. Electroelution samples were dialyzed in PBS and examined at 1/20 dilution. Mr standards used to calibrate the gel consisted of 12 standards Novex Mark.
Fig.7 shows the effect of the APS, devoid of mpl ligand, megakaryocytes in humans. APS, devoid of mpl ligand was obtained by passing 1 ml in 1 ml mpl-affinity column (700 μg mpl-IgG/ml NHS-Superose, Pharmacia). Culture of peripheral stem cells were treated with 10% APS 10% APS, devoid of mpl ligand, and were cultured for 12 days. Megakaryocytes was calculated as described in the examples.
Fig shows the effect of mpl-IgG stimulation APS of megakaryocytopoiesis in humans. Culture of peripheral stem cells were treated with 10% APS and were cultured for 12 days. At 0, 2 and 4 days added mpl-IgG (0.5 μg) or ANP-R-IgG (0.5 μg). After 12 days was calculated megakaryocytes, as described in the examples. Graphics built upon average values of duplicate samples in brackets valid data takes.
Fig.9 shows both strands of the fragment 390 base pairs of human genomic DNA encoding the mpl ligand. Shows the deduced amino acid sequence "AXONE 3" (Pet. No:3), coding sequence (Th. No: 4) and its complement (PEFC. No: 5).
Figure 10 shows the deduced amino acid sequence is the Mature ligand mpl person (MLC) (Th. No: 6) and Mature human erythropoietin (APC) (Th. No: 7). The predicted amino acid sequence for the ligand mpl person is in series with the sequence of human erythropoietin. Identical amino acids are separated by a partition, and gaps introduced for optimal location marked by hatching. Designated for potential N-glycosylation sites are underlined by a straight line for MLC and the broken line for APC. Two cysteine important for activity of erythropoietin, marked in bold dotted line.
11 shows the deduced amino acid sequence of the Mature isoforms of mpl ligand person MLC (PEFC. No: 6), MLC (PEFC. No: 8), MLC (PEFC. No: 9) and MLC (PEFC. No: 10). Identical amino acids are separated by a partition, and gaps introduced for optimal location marked by hatching.
Figa, 12B and 12C show the effect of ligand nipl person on the proliferation of Ba/F3 cells (A), the number of megakaryocytopoiesis in humans in vitro, calculated using labeled IgG mouse monoclonal antibodies specific for glycoproteins GPIIbIIIamegakaryocyte (In), and the definition of thrombocytopoiesis in mice, analyzed by reductive synthesis of platelets (C).
Cells two hundred and ninety-three were transfected according to CaPO4method (Gorman, in DNA Cloning: A New Approach 2:143-190 ), only the vector pRK5, pRK5-MLC or pRK5-the LCH 153during the night (pRK5-MLC153was obtained by introduction of a stop codon after residue 153 MLC method ROC). Then Wednesday was balanced for 36 hours and examined it for stimulation of proliferation of Ba/F3-mpl cells as described in Example 1 (A) or megakaryocytopoiesis in humans in vitro (In). Megakaryocytopoiesis was calculated using labeled125IIgG monoclonal antibody mouse (HP1-ID) to a specific glycoproteins GPIIbIIIaas described (Grant et al., Blood, 69: 1334-1339 ). The effect of partially purified recombinant ML (RML) for the production of platelets in vivo (C) was determined using the survey of rehabilitation synthesis of platelet described McDonald, TR OEWG. Sc. Exp. Biol. Med. 144: 1006-1012 (1973). Partially purified RML received 200 μl of conditioned medium containing recombinant ML. Wednesday conceded through the column with 2 ml of Blue-Sepharose, equilibrated PBS, the column was washed in PBS and were suirable PBS containing 2 M urea and NaCl, everyone. Fractions with activity were dialyzed in PBS and created a concentration of 1 mg/ml BSA, free from endotoxin. The samples contained less than one unit of endotoxin/ml Mice were injected with on of 64000 32000 16000 units RML or just filler. Each group consisted of six mice. Presents the mean and standard deviation in each group. P value was determined by paired T-test comparing the medians.
F. g compares the action isoforms and variants of mpl ligand person in the study of proliferation of Ba/F3 cells. Were investigated MLC, mock, MLC, MLC(RA, RA) and MLC153at various dilutions, as described in Example 1.
Figa, 14B and 14C show the deduced amino acid sequence (Th. No: 1) ligand mpl person (MLC) or TA man (CCC) human genomic DNA, the coding sequence (Th. No: 11). The nucleotides and amino acid residues are numbered from the beginning of each line.
Fig shows SDS-PAGE (electropores in polyacrylamide gel in the presence of sodium dodecyl sulphate), peeled 293-rmlc332and purified 293-rmlc153.
Fig shows the nucleotide sequence: coding cDNA (PEFC. No: 12) and deduced amino acid sequence (Th. No: 13) open reading frame isoforms ML mouse. This isoform Mature mpl ligand mouse contains a 331 amino acid residue, four fewer than the natural full length MLM, and therefore designated as MLM. Nucleotides are numbered from the beginning of each line. Amino acid residues are numbered above the sequence, starting with Series 1. Sites of potential N-glycosylation sites are underlined. Residues of cysteine marked by the dotted line above the sequence.
Fig shows the cDNA sequence (Th. No: 14) and the predicted protein sequence (Th. No: 15) of this isoform ML mouse (MLM). Nucleotides are numbered began what each line. Amino acid residues are numbered above the sequence, starting with Series 1. This isoform Mature mpl ligand mouse contains a 335 amino acid residues, and it seems that is a ligand mpl full length, denoted by MLM. The signal sequence is indicated emphatic hatch and possible splitting of the arrow. 5’ and 3’ untranslated region marked located below the letters. Two detected deletions as a result of alternative splicing (MLM and MLM) are underlined. Four cysteine residue is marked by hatching. Seven potential sites of N-glycosylation separated by a partition.
Fig compares the deduced amino acid sequence MLC isoforms ML of a person's school. No: 9) and isoforms ML mouse, marked MLM (PEFC. No: 16). The predicted amino acid sequence of mpl ligand person is located along the sequence of mpl ligand mouse. Identical amino acids are separated by a partition, and gaps introduced for optimal location in a row and marked by hatching. Amino acids are numbered from the beginning of each line.
Fig compares the predicted amino acid sequence of Mature ML isoforms ML mouse (PEFC. No: 17), ML pigs (Pet. No: 18) and ML human (PEFC. No: 6). Amino acid sequences are located along with the breaks, tokenname hatching, inserted for the optimal location. Amino acids are numbered from the beginning of each line, separated by a partition identical amino acids. Places of possible N-glycosylation sites are marked darkened walls, and cysteine residues are indicated by dashes. Structure with conservative dibasic amino acids, which is the place for potential cleavage by proteases, underlined. Found the fourth amino acid deletion observed in all three species (ML) separated fat partition.
Fig shows the cDNA sequence (Th. No: 19) and the predicted sequence (Th. No:18) isoforms Mature protein ML pigs (LFM). This isoform of mpl ligand pigs contains 332 amino acid residue and, presumably, is the mpl ligand pigs full length, denoted by PD. Nucleotides are numbered from the beginning of each line. Amino acid residues are numbered above the sequence, starting with Series 1.
Fig shows the cDNA sequence (Th. No: 20) and the predicted sequence (Th. No: 21) isoforms Mature protein ML pigs (MLS). This isoform of mpl ligand pigs contains 228 amino acid residues and is a shape with four divisions residues compared to mpl ligand full length, denoted MLS. The nucleotides of pronumerals the beginning of each line. Amino acid residues are numbered above the sequence, starting with Series 1.
Fig compares the deduced amino acid sequence ML pig with a full-length isoforms LFM (PEFC. No: 18) and isoforms ML pigs indicated MLS (PEFC. No: 21). The predicted amino acid sequence for ML is a number sequence MLS. Identical amino acids are separated by partition walls, and gaps introduced for optimal location marked by hatching. Amino acids are numbered from the beginning of each line.
Fig shows essential properties of plasmids p3V15. ID.LL.MLORF ("full length" or TA332used for transfection of the host CHO-DP12 cells for the production of SNO-rh332.
Fig shows essential properties of plasmids p3V15.ID.LL.MLEPO-D ("truncated" or TA153used for transfection of the host CHO-DP12 cells for the production of SNO-rh153.
Figa, 25V and 25C shows the effect .colirhin (Meth-1, 153) on platelets (A), red blood cells (b) and white blood cells (C) normal mice. Two groups of six females S B6 mice were daily injected with either PBS buffer or 0.3 ág E. coli-rhTn (Meth-1, 153) (100 μl/P.K.). On day 0 and 3-7 days were taken in 40 ml of blood from the eye of the sinus. This blood was immediately dissolved in 10 ml of commercial diluent and received a full account of the AOC and to Serrono Baker Heiaatology Analyzer 9018. Data are presented as mean ± standard error of the definition.
Figa, 26C and 26C show the effect of E. coli-rhTu (Meth-1, 153) on platelets (A), red blood cells (b) and white blood cells (C) exposed to sublethal dose mice. Two groups of 10 females S B6 mice were irradiated in a sublethal dose of 750 cGy of gamma radiation from a source with137Cs, which received daily injections of either PBS buffer or 3,0 µg E. coli-rh-1, 153) (100 μl/P.K.). On day 0 and in the subsequent interim periods were selected in 40 µl of blood from the ocular sinus. This blood was immediately dissolved in 10 ml of commercial diluent and received a full account of blood on Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard error of the definition.
Figa, 27B and 27C shows the effect of SNO-rh332on platelets (A), red blood cells (erythrocytes) (b) and white blood cells (leukocytes) (C) normal mice. Two groups of six females S B6 mice were daily injected with either PBS buffer or 0.3 ág SNO-rh332,) (100 μl/P.K.). On day 0 and 3-7 days were taken in 40 ml of blood from the eye of the sinus. This blood was immediately dissolved in 10 ml of commercial diluent and received a full account of blood on Serrono Baker Hematology Analyzer 9018. Data are presented as mean ± standard error of the definition.
Fig curve shows dozozawisim the th response to various forms rtpc, obtained from different cell lines. Curves dose-dependent responses to rtpc of the following cell lines: TFC332from SNO (full length from ovary cells Chinese hamster); Tpmet-1153 (derived E. coli, a truncated form with N-terminal methionine); Tpch332(TA full length of 293 cells); without Meth 155 E. coli (a truncated form [rtpc155] from E. coli without end-methionine). Groups of 6 female SW mice daily were injected with in 7 days rtpc depending on the group. Every day was selected by 40 µl of blood from the ocular sinus for a full account of the blood. The data presented above represent the maximum effects observed at various introductions, except (meth 153 E. coli)that occur on the 7th day of administration. In the above-mentioned "metal 153 E. coli" the maximum effect was observed on day 5. Data are presented as mean ± standard error of the definition.
Fig shows a comparison of the response curves dose-response relationships of activities rtpc of full length and truncated forms rtpc produced by Cho cells with a truncated form of E. coli. Groups of 6 female SW mice were daily injected at 0.3 µg rtpc different types. For 2-7 days took the blood from the eye of the sine for a full blood count. The treated group are the following: TA153a truncated form of TP from E. coli; TA332(mixed fraction)TS full length, containing approximately 80-90% of TP full length and 10-20% cut shape; TP(30K fraction) = purified truncated fraction of the original "mixed drug; TP(K fraction) = cleaned fraction TP full length of the original "mixed drug. Data are presented as mean ± standard error of the definition.
Fig is a picture showing the study KIRA ELISA for determination of TP. The drawing shows the MPL Chimera/Rse.gD and the corresponding portion of the parent receptor, as well as the final design (right side of drawing) and a smooth chart (left side of drawing)showing the relevant stages of the research.
Fig is a smooth chart studies KIRA ELISA, showing each step in the definition.
Figa-32L describes the nucleotide sequence (Th. No:22) vector p3VI17.ID.LL expression used for the expression of Rse.gD in Example 17.
Fig is a schematic representation of the preparation of plasmid pMP1.
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is schematic is a mere representation of the preparation of plasmid RMR.
Fig is a table of the five best in the expression of TS clones of the plasmid RMR Bank plasmids (Pet. No: 23, 24, 25, 26, 27 and 28).
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is a schematic representation of the preparation of plasmid RMR.
Fig is a schematic representation of the preparation of plasmid RMR.
DETAILED description of the INVENTION
Basically, the following words or phrases have the above definition, when used in the description, examples and claims.
"Chaotropic agent" refers to a compound which in aqueous solution and suitable concentrations may cause changes in the spatial configuration or conformation of the protein, at least partially destroying the forces responsible for the support of normal secondary or tertiary structure of the protein. Such compounds include, for example, urea, guanidine-Hcl and sodium thiocyanate. To provide conformational effects on protein typically requires a high concentration, 4-9 M, of these compounds.
"Cytokine" is a term that is characteristic of proteins secreted by one cell population which act on another cell as intercellular mediators. Examples of such cytokines are lymphokines, Monokini and normal polypeptide hormones. The cytokines include: th the Mont growth insulin-like growth factors, human growth hormone, N-methionyl the human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, prolactin, the glycoprotein hormone stimulants, such as follicle-stimulating hormone (FSH), thyrostimulin hormone (TSH), and luteinizing hormone (LH), hematopoietic growth factor, growth factor liver, fibroblast growth factor, prolactin, placental lactogenic, tumor necrosis factor α (TNF-α or TNF-β), Mullerian-inhibiting substance associated with gonadotropin protein mouse inhibin, activin, endothelial growth factor vascular integrin, growth factor neurons, such as NGF-β, platelet growth factor, transforming growth factors (TGF)such as TGF-α and TGF-β, insulin-like growth factor-1 and -2, erythropoietin (EP), osteoinductive factors, interferons such as interferon-α, -β and -γ, Colonie-stimulating factors (CSF)such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL)such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12 and other polypeptide factors including CSF, PHIL and kit-ligand. As used herein, the following terms are intended to encompass proteins from natural sources or recombinant Faure the cell lines. Similarly, the terms are intended to encompass biologically active equivalent derivatives, namely, differing in amino acid sequence one or more amino acids, or type, or degree of glycosylation.
"Mpl ligand", "polypeptide ligand mpl", "ML", thrombopoetin" or "TP" are used herein interchangeably and include any polypeptide which is capable of binding to mpl, a member of the supergroup cytokine receptors, and has biological properties of ML, as shown below. An example of biological features is the ability to stimulate the incorporation of labeled nucleotides (namely,3H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected mpl P person. Another example of biological features is the ability to stimulate the inclusion of35S in circulating platelets in the study of reductive synthesis of platelets. This definition encompasses a polypeptide selected from the source mpl ligand, such as aplastic plasma pigs, described here, or other animals, including humans, or obtained by recombinant ways or methods of synthesis, and contains a variety of forms including functional derivatives, fragments, alleles, isoforms and its analogs.
"A fragment of the ligand ML" or "fragment TA" - this is the hour of the ü naturally find Mature mpl ligand full length, or the sequence TA, having a deletion in one or more amino acid residues, or deprived of carbohydrate parts. Deletion of amino acid residue (residue) can occur anywhere in the protein, including the N-terminal and C-terminal end, or inside. The fragment has at least one biological property in common with the mpl ligand. Fragments of the ligand mpl will typically have a serial sequence at least 10, 15, 20, 25, 30, or 40 amino acid residues that are identical sequence of mpl ligand isolated from a mammal, including ligand isolated from aplastic plasma pig, or a ligand of a person, or a ligand of a mouse, especially their EP-domain. Typical examples of N-terminal fragments are MLC or TA (Met-11-153).
"Versions of mpl ligand" or "variants of the sequence of mpl ligand", as defined here, means a biologically active mpl ligand, as defined below, having less than 100% identity in the sequence with the mpl ligand isolated from recombinant cell culture or aplastic plasma pig, or a ligand of a person having deduced the sequence described in figure 1 (Th. No:l). Typically, biologically active mpl ligand will have an amino acid sequence having at least 70% identity with the amino acid sequence is lnasty mpl ligand, isolated from aplastic plasma swine or Mature ligand mouse, or human, or their fragments (see figure 1 [Th. No:l]), preferably at least about 75%, more preferably at least 80%, even more preferably at least 85%, and even more preferably at least 90%, and most preferably at least 95%.
"Chimeric mpl ligand" is a polypeptide containing the mpl ligand full length or one or more fragments, fused or associated with the second a heterologous polypeptide, or one or more fragments. Chimera has at least one biological property in common with the mpl ligand. The second polypeptide is usually a cytokine, an immunoglobulin or its fragment.
"Isolated mpl ligand", "highly purified mpl ligand" and "substantially homogeny the mpl ligand" are used interchangeably and refer to the mpl ligand, which was purified from a source to mpl ligand or has been obtained by recombinant means or methods of synthesis and has been substantially purified from other peptides or proteins (1), with at least 15 and preferably 20 amino acid residues C-terminal or internal amino acid sequence, using the sequencing machine with a rotating reactor, or the best commercially available amino acid sequencing machine, or using published with osobov according to the modifications on the date of filling this application, or (2) until a homogeneous state by using SDS-PAGE in non or reducing conditions using Coomassie blue or, preferably, silver staining. Homogeneous condition here means less than 5% contamination with other proteins.
"Biological property"when used in connection with the "mpl ligand" or "isolated mpl ligand", indicates the presence of thrombopoetin activity, or the presence of in vivo effector or antigenic function or activity that is directly or indirectly related to or caused by mpl ligand (one way or another natural or denatured conformation), or its fragment. Effector properties include: linking mpl and any binding activity vector, agonism or antagonism mpl, particularly in the transmission of proliferative signals, including replication, function, regulation of DNA, modulation of the biological activity of other cytokines, activation of receptors (especially cytokine), deactivation, direct and inverse regulation, regulation of cell growth and differentiation, and the like. Antigenic function means the possession epitope or antigenic site that is capable of cross-reacting with antibodies generated against the natural ligand mpl. The main antigenic property of the polypeptide ligand mpl is that it binds to affinity, at the very the least, about 106l/mol, with the antibody produced against the mpl ligand isolated from aplastic plasma pigs. Typically, the polypeptide binds to affinity of at least about 107l/mol. Most preferably, antigenically active polypeptide ligand mpl was a polypeptide that binds with antibodies produced against mpl ligand having one of the above effector functions. Antibodies used to define "biological activity"are polyclonal rabbit antibodies produced in the preparation of the mpl ligand isolated from recombinant cell culture or from aplastic plasma pigs with complete adjuvant Freund'a subcutaneous injection of the drug and increasing the immune response by intraperitoneal injection of the drug, until a plateau in the titer of antibodies to the ligand mpl.
"Biologically active"when used in connection with either the term "mpl ligand"or "isolated mpl ligand"means a ligand mpl or polypeptide that exhibits thrombopoetin activity, or has the effector function of the mpl ligand isolated from aplastic plasma pigs or expressed in recombinant cell culture, are described here. The main known effector function of mpl ligand or polypeptide is binding to mpl and stimulat what I include labeled nucleotides ( 3H-thymidine) into the DNA of IL-3 dependent cells Ba/F3 transfected mpl P person. Another well-known effector function of mpl ligand or polypeptide is the ability to stimulate the inclusion of35S in circulating platelets in the study of inverse binding of platelets in mice. Another well-known effector function of mpl ligand is the ability to stimulate in vitro megakaryocytopoiesis in humans, which can be calculated by using radioactively labeled antibodies specific for glycoprotein GPllblllamegakaryocyte.
"Percent identity of amino acid sequence, whereas the sequence of mpl ligand, is defined here as the percentage of amino acid residues selected identical sequence to residues of the sequence of mpl ligand isolated from aplastic plasma pig, or a ligand for a mouse or a person having the amino acid sequence set origin, described in figure 1 (Th. No:1), after straightening the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the identical sequence. None of N-terminal or C-terminal, or internal expanded the th, deletions or insertions in the sequence of mpl ligand should not be construed as affecting sequence identity or homology. Thus, examples of biologically active polypeptides of mpl ligand must have identical sequences, including: shall-mpl ligand, Pro-mpl ligand and Mature mpl ligand.
"Microeconomie mpl ligand" can be performed by any suitable conventional method, if the method is sufficiently sensitive. In one of these ways purified polypeptides derived from the SDS gel, or end-stage GHUR sequeiros directly by automated splitting (phenylisothiocyanate) Edman, using the model of the sequencing machine A Applied Biosystems gas phase, equipped with a 120A phenylthiohydantoin (FGF) amino acid analyzer. In addition, fragments of mpl ligand, obtained by chemical means (namely, with CNBr, hydroxylamine, 2-nitro-5-thiocyanato-atom) or enzymatic (namely trypsin, Clostridium, staphylococcal protease) cleavage and subsequent purification of the fragments (namely, by GHWR), can be sequenced in the same way.
FGF amino acids was investigated using data systems ChromPerfect (Justice Innovations, Palo Alto, CA). The interpretation of the sequences was done on the computer VAX 11/785 Digital Equipment Co. as described Henzel et al., J. Chromatography, 404: 41 to 52 . Equal shares fractions GHUR, optional, can be subjected to electrophoresis in a 5-20% SDS-PAGE, transferred under the influence of electric current on the PDVF membrane (ProBlott, AIB, Foster City, CA) and painted Kumasi diamond blue (Matsurdiara J. Biol. Chem., 262: 10035-10038 . Identified by the color of a specific protein is cut from the blot and N-terminal sequence was processed using a sequencing machine with a gas phase, described above. For internal sequence of the protein fraction GHUR dried under vacuum (SpeedVac), resuspended in a suitable buffer and subjected to interaction with CYANOGEN bromide, Liz-specific enzyme Lys-C (Wako Chemicals, Richmond, VA)or ASP-N (Boehringer Mannheim, Indianapolis, IN). After hydrolysis of the obtained proteins sequeiros in a mixture or after separation GHUR if vacuumed on a C4 column in a gradient of propanol and 0.1% TFA, before sequencing in the gas phase.
"Thrombocytopenia" is defined as the number of platelets in the blood is below 150×109per litre.
"Tromboticheskoe activity" is defined as the biological activity, which includes the acceleration of proliferation, differentiation and/or maturation of megakaryocytes or predecessors of megakaryocytes in thrombocytopathies shape of these cells. This activity can be measured in various studies, including research in stanowiacego synthesis of mouse platelets in vivo, study the induction of cell surface antigen platelets, measured by antiplatelet immunoassay (anti-GPllblllafor megakaryoblastic cell human leukemia (QMS), and a study of the induction of polyploidization in line megakaryoblastic cells (DAMI).
"Thrombopoietin" (TP) is defined as a compound that thrombopoetin activity or is able to increase the number of platelets in the serum of mammals. TA mostly able to increase the amount of endogenous platelet count at least 10%, more preferably 50%, and most preferably, is able to increase the number of platelets in humans, more than up to 150×109per liter of blood.
"Nucleic acid selected mpl ligand" is RNA or DNA containing more than 16 and preferably 20 or more, sequential nucleotide bases that encode biologically active mpl ligand or a fragment, which is complementary to a RNA or DNA, or hybridization with RNA or DNA and keeping a stable connection under moderate to stringent conditions. Data RNA or DNA free, at least from a single source impurity nucleic acid with which it is normally associated in the natural environment and, preferably, substantially free from any other RNA or the NC mammals. The phrase "free, at least from a single source impurity nucleinate acid with which it is usually connected" includes the place in which usually are nucleic acid source, or natural cell, but in different chromosomal position, or otherwise connected with a sequence of nucleic acids, usually not found in the source cells. An example of a nucleic acid selected mpl ligand is an RNA or DNA that encode biologically active mpl ligand, having at least 75% identity in the sequence, more preferably at least 80%, even more preferably 85%, and even more preferably 90%, and most preferably 95% identity in the sequence with the mpl ligand of human, mouse, or pig.
"Control sequence", when related to the expression of the mean DNA sequences necessary for the expression of operationally linked to the coding sequence in a particular host organism. Control sequence, applicable, for example, prokaryotes, include a promoter, an optional operator sequence, a binding site of the ribosome, and possibly other, as yet poorly understood sequence. The cells of eukaryotes are known to use promoters, polyadenylation signals usilitel.
"Operationally linked", when it refers to nucleic acids, means that the nucleic acids are in a functional relationship with other sequences of nucleic acids. For example, DNA for predpolagavshegosja or secretory leader is operationally linked to DNA for a polypeptide if it is expressed as preprotein that participates in the secretion of the polypeptide; a promoter or amplifier is operationally linked with a coding sequence if it affects the transcription of the sequence; or the binding site of the ribosome, operationally linked with a coding sequence if it is to enhance the broadcast. In General, "operationally linked" means that the concatenated DNA sequences in touch, and in the case of a secretory leader in contact and in the phase of reading. However, the amplifiers should not touch. Scalebane accompanied by legacies in sites suitable for restriction analysis. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with generally accepted practices.
"Exogenous"when it refers to an item, means the sequence of nucleic acid that is foreign to the cell, or homologous to the cell but in this position in the nucleic acid the notches of the owner, where element is usually not detected.
"Cell", "cell line" and "cell culture" are used interchangeably here, and this designation include all descendants of cells or cell lines. Thus, for example, terms such as "transformants" and "transformed cells"include the primary subject cell and derive from it the culture, regardless of the number of subcultures. It is also clear that all descendants may not be completely identical in composition to DNA due to deliberate or inadvertent mutations. Descendants of mutants having the same function or biological activity as screened for usually transformed cells, are also included. Where are assumed to be different designations, they will be removed from the context.
"Plasmids" - Autonomous molecules can replicate circular DNA, showing independent replication of nature, and here marked with a small letter "p", which is placed before and/or after the uppercase letters and/or numbers. The original plasmids here either commercially available, openly available without restrictions, or can be obtained from such available plasmids in accordance with the described methods. In addition, in the technique known other equivalent plasmids that will be available for the ordinary worker.
"Limited enzymatic rassal the separation", when refers to DNA, refers to catalytic cleavage fosfolipidnyh relations enzyme that reacts only on certain positions or sites in DNA sequences. Such enzymes are called restriction endonucleases". Each restriction endonucleases recognize specific DNA sequence called "restriction site", which exhibits bilateral symmetry. The various restriction enzymes used herein are commercially available, and the conditions applied to reactions, cofactors and other requirements as established by the suppliers of enzymes. Restrictive enzymes are usually indicated by abbreviations composed of capital letters and follow her other letters representing the microorganism from which each restriction enzyme was originally obtained, and then a number indicating a specific enzyme. Usually use about 1 mg of plasmid or DNA fragment with about 1-2 units of enzyme in about 20 μl of buffer solution. Suitable buffers and the number of substrate-specific restriction enzymes are determined by the manufacturer. Usually use incubation for about 1 hour at 37°With, but you can modify them in accordance with the instructions of the supplier. After incubation, the proteins or polypeptides are removed by extraction with phenol and chloroform, and R is salena nucleic acid is extracted from the aqueous fraction by precipitation with ethanol. Cleavage with restriction enzymes can be continued by the hydrolysis of bacterial alkaline phosphatase limit 5 fosfato to prevent two restriction the split ends of the DNA fragment from "circularities" (forming a ring) or forming a closed loop, which could interfere with the insertion (insert) another DNA fragment at the restriction site. Unless otherwise stated, the cleavage of the plasmid does not follow 5 concavum dephosphorylation. Conventional methods and reagents for dephosphorylate described in detail in sections 1.56-1.61 Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring Harbor Laboratory Press, 1989].
"Exit" or "selection" of a given fragment of DNA from a split restriction residue means the selection of the split balance by electrophoresis on polyacrylamide or agarose gel, identification of the fragment of interest by comparison of its mobility relative to the marker DNA fragments of known molecular weight, distinguishing it from the gel section containing the desired fragment, and separating gel and DNA. These methods are well known. For example, see Lawn et al., Nucleic Acids Res., 9: 6103-6114  and Goeddel et al., Nucleic Acids Res., 8: 4057 .
Southern analysis or Southern blotting is the method by which confirms the presence of DNA sequences in the remainder of the DNA or in the composition, the soda is containing DNA after cleavage with restriction endonuclease by hybridization with known labeled oligonucleotide or DNA fragment. Southern analysis typically involves electrophoretic separation of DNA fragments and agarose gel, denaturation of the DNA after electrophoretic separation and transfer of DNA to nitrocellulose, nylon or other suitable membrane substrate for analysis using radioactively labeled, biotinylated or enzyme-labeled probe as described in sections 9.37-9.52 Sambrook et al., ibid.
"Nothern analysis" or "Nothern blot is a method used to identify RNA sequences that hybridize with known probe such as an oligonucleotide, a DNA fragment, cDNA or fragment or RNA fragment. Probe Machen radioactive isotope, such as32P, or by biotinidase, or enzymatic. The analyzed RNA usually electrophoretic separated on agarose or polyacrylamide gel, transferred to nitrocellulose, nylon or other suitable membrane, and hybridizing probe using conventional methods, well known methods, such as those described in sections 7.39-7.52 Sambrook et al., ibid.
"Ligation" - the process of education fosfolipidnyh relations between the two nucleic acid fragments. For ligation of the two fragments of the ends of these fragments must be the compatible with each other. In some cases, the ends will be compatible directly after endonuclease cleavage. However, you may need to first convert the floating ends, usually resulting in the process endonuclease cleavage, to align the ends to make them compatible for ligation. To align the ends of the DNA is treated in a suitable buffer for at least 15 minutes at 15°With about 10 parts of the Klenow fragment of DNA polymerase I or T4 DNA polymerase in the presence of four deoxyribonucleotide of triphosphates. Then the DNA purified by ekstraktsiei phenol-chloroform and precipitation with ethanol. DNA fragments that must be legirovanyh, are placed in solution in approximately equimolar amounts. The solution also contains ATP, legasey buffer and ligase, such as T4 DNA ligase at about 10 units per 0.5 μg DNA. If this DNA must be Legerova in the vector, the vector is first leveled by splitting a suitable restriction endonuclease (endonuclease). Then straightened into a line fragment associated with bacterial alkaline phosphatase or phosphatase from calf intestine to prevent Smolevichi during the ligation.
"Obtaining DNA from cells means isolation of plasmid DNA from a culture of host cells. Usually, to obtain DNA uses the method I large and small - scale obtain plasmids described in sections 1.25-1.33 Sambrook et al., ibid. After receiving the DNA, it can be purified using methods well known in the art, such as described in section 1.40 Sambrok et al., ibid.
"Oligonucleotides" are short-length, single-or double-strand polyethoxylated that are chemically synthesized according to known chemical methods, such as phosphocreatine, fosfatnyi or phosphorimetry using solid-phase techniques such as described in EP 266032, published 4 may 1988, or via deoxynucleoside H-phosphonate intermediates as described Froeler et al., Nucl. Acids Res., 14: 5399-5407 ). Further methods include the polymerase chain reaction, described below, and other autoprimer ways and synthesis of oligonucleotides on a solid substrate. All of these methods described in Engels et al., Agnew. Chem. Int. Ed. Engi., 28: 716-734 (1989). These methods are applied, if known, the full sequence of nucleic acids in a gene or the available sequence of nucleic acids, complementary to the coding strands. Alternatively, if the target amino acid sequence is known, you can display the possible nucleic acid sequences, using the known and the preferred encoding residues for each amino acid residue. The oligonucleotides were then purified on polyacrylamide gel.
"Polymerase chain reaction, or "ROC" refers to a method or process, in which a small amount of a specific fragment of nucleic acid, RNA and/or DNA propagated as described in U.S. Patent No.4683195, published on July 28, 1987, In General, if you want to access sequence information from the ends and the region of interest or beyond required can be created such oligonucleotide primers; these primers for the amplification will be identical or similar sequences threads of the opposite direction of the template. 5’ nucleotides of the two primers can be the same with all amplifierarava material. The ROC can be used for reproduction of specific RNA sequences, specific DNA sequences from whole genomic DNA and cDNA, read from the entire sequence of the RNA of the cell, bacteriophage or plasmid, etc. In General, see Mullis et al., Cold Spring Harbor Symp. Quant Biol., 51: 263 ; Eriich, ed., PCR Technology (Stockton Press, NY, 1989). As used here, the ROC is supposed to be one, but not the only, example of a method of polymerase reactions of nucleic acids for breeding of the sample nucleic acid, comprising the use of a known nucleic acid as a primer and a nucleic acid polymerase to multiply or production of specific nucleic acid fragments.
"Stringent conditions" are those when (1) prom the Cai employ low ionic strength and high temperature, for example, of 0.015 M NaCl/0,0015 M sodium citrate/0.1% of NaDodSO4(SDS) at 50°or (2) employ during hybridization, denaturing agent, such as formatid, for example, 50% (vol./about.) formated with 0.1% bovine serum albumin/01%, Ficoll/0.1% polyvinylpyrrolidone/50 mM phosphate buffer at pH 6.5 with 750 mM NaCl, 75 TM sodium citrate at 42°C. Another example is use of 50% of formamide, 5×SSC (0.75 M NaCl, Of 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×solution Denhardt'a processed ultrasound DNA salmon sperm (50 µg/ml), 0,1% SDS, and 10% dextran sulfate at 42°C, with wash in 0.2×SSC and 0.1% SDS.
"Moderately stringent conditions" are described by Sambrook et.al., ibid, and include the use of washing solution and hybridization conditions (namely, temperature, ionic strength and %SDS)less stringent than described above. Examples of moderately stringent conditions are conditions such as incubation over night at 37°C in a solution containing 20% of formamide, 5×SSC (150 mm NaCl, 15 mm trisodium citrate), 50 mm sodium phosphate (pH 7,6), 5×solution Denhardt'a, 10% dextran sulfate, and 20 μl/ml denatured cut DNA salmon sperm, followed by washing the filters in 1×SSC at about 37-50°C. the Skilled worker disassemble, how to bring the temperature, ionic strength, etc. as necessary for coordination of such factors as the Lina probe and the like.
"Antibodies" (AT) and "Immunoglobulin" (IG) is a glycoprotein having the same structural characteristics. While antibodies show specificity in binding with specific antigens, immunoglobulin and include antibodies and other antibody molecules that lack antigen specificity. The polypeptides of the latter species, for example, are produced by the lymphatic system at low levels and at high levels for myeloma.
"Natural antibodies and immunoglobulins" are usually heterotetrameric glycoproteins weight of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds between heavy chains differ in different isotypes of immunoglobulins. Each heavy and light chain also is usually located inside the chain disulfide bridges. Each heavy chain has at one end a variable domain (VT), following after numerous constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end; the constant domain of the light chain is located along the first constant domain of the heavy chain and light variabel the first domain is located along together with the variable domain of the heavy chain. It is assumed that the specific amino acid residues form the boundary between the light and heavy chain variable domains (Clothia et al., J. Mol. Biol., 186: 651-663 ; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82: 4592-4596 ).
The term "variable" refers to the fact that certain parts of the variable domain of the differ extensively in sequence among antibodies and are used for binding and (give) specificity of each particular antibody for its particular antigen. However, variability is not equally distributed across the variable domains of antibodies. It is concentrated in three segments, called domains of definition of complementarity (KLO) or hypervariable regions in both: and in the light chain and heavy chain variable domains. More highly conserved part of the variable domains is called the working frame (RR). The variable domains of the natural heavy and light chains each contains four areas of PP, largely adapted to the βfolded configuration, connected by three KLO, which form hinge joints and in some cases form part of the β-folded structure. KLO in each chain are supported together in close proximity to the RR field and KLO from the other chain, contribute to the formation of the binding site of the antigen to the antibody (see Kaba et al., Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD, ). The constant domains are not involved directly in binding the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody-dependent antibodies cellular toxicity.
Cleavage of antibodies with papain produces two identical fragments that bind antigen, called "Fab" fragments, each with one binding site of the antigen, and the remaining "Fc" fragment, whose name reflects its ability to easily crystallize. Treatment with trypsin gives F(ab’)2the fragment that has two connected region of binding antigen, and is still capable of cross-linking antigen.
"Fv" is the minimum antibody fragment which contains a complete site recognition and binding of the antigen. This area consists of a variable domain of the dimer of one heavy and one light chain in a tight non-covalent linkages. In this configuration, in which three interact KLO each of the variable domains, set the binding site of the antigen on the surface of the VT-VLdimer. All together six KLO give the antibody binding specificity of the antigen. However, even a single variable domain (or half of an Fv, containing only three KLO, specific for an antigen) has the ability to resposne the project and bind antigen, although at a lower affinity than the full binding site.
Fab fragment also contains the constant domain of the light chain and the first constant domain (SN) the heavy chain. Fab’ fragments differ from Fab fragments by the addition of a few residues at the carboxy-end of the heavy chain CN domain, including one or more cysteines from the area of the loop antibodies. Fab’-SH is the designation for Fab’in which the residue of cysteine constant domain are three free tylnej group. Fragments F(ab’)2antibodies are usually produced as pairs of Fab’ fragments which have cysteine in the loop between them. It is also known another chemical compound fragments of antibodies.
The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be defined to one or two clearly distinct types, called Kappa and lambda (λ), on the basis of amino acid sequences of their constant domains.
Immunoglobulins can be divided into different classes depending on their amino acid sequence of the constant domain of the heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further subdivided into subclasses (isotypes: IgG-1, IgG-2, IgG-3 and IgG-4; IgA-1 and IgA-2. The constant domains of the heavy chain, which correspond to different the classes of immunoglobulins are called α , Delta, Epsilon, γ and μ respectively. Structures of subunits and three-dimensional configurations of different classes of immunoglobulins are well known.
The term "antibody" is used in its broadest sense and specifically covers some monoclonal antibodies (including agonist and antagonist antibodies), antibody composition with polyepitopic specificity, as well as antibody fragments (namely, Fab, F(ab’)2and Fv) and so on, until they exhibit the desired biological activity.
The term "monoclonal antibody", as used herein, refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally observed mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, as it is directed against a single antigenic site. Moreover, in contrast to conventional (polyclonal) antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized g is britaney culture, not contaminated by other immunoglobulins. Modified "monoclone" notes the character of the antibody as obtained from a substantially homogeneous population of antibodies, and should not be construed as desired antibodies in any other specific way. For example, the monoclonal antibodies to be used in accordance with the present invention, can be obtained hybridoma method first described by Kohler &Milstein, Nature, 256: 495 (1975), or obtained by recombination of DNA (see, for example, U.S. Patent No.4816567 [Cabilly et.al.]).
The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins)in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequence in antibodies derived from certain species or belonging to a particular class or subclass of antibody, while the remainder of the chain (chain) is identical with or homologous to corresponding sequence in antibodies derived from another species or belonging to another class or subclass antibodies as well as fragments of such antibodies, as they exhibit the desired biological activity (U.S. Patent No.4816567 (Cabilly et al.); and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-6855 ).
"Humanized" forms of inhuman (e.g., murine) antibodies are chimeric immunoglobulins, immune is globulinemia chains or fragments thereof (such as Fv, Fab, Fab’, F(ab’)2or other antigen binding subsequences of antibodies)which contain minimal sequence derived from nonhuman immunoglobulin. In the main part, humanized antibodies are human immunoglobulins (antibodies recipient), in which areas of the region that defines complementarity (KLO) of the recipient are replaced by parts from (KLO) nonhuman species (donor antibodies), such as a mouse, rat or rabbit having the desired specificity, affinity and capacity. In some cases the remains of the operating framework for the Fv of the human immunoglobulin are replaced by corresponding inhuman remnants. Furthermore, humanized antibodies may comprise residues that are not found either in the antibodies of the recipient, nor made in the KLO, or sequences of the working frame. These modifications are made to further improve and streamline the presentation of the antibodies. Basically, the humanized antibody will comprise substantially all of at least one and usually two mutable domains in which all or substantially all of the KLO regions correspond to the non-human immunoglobulin and all or substantially all of the FR regions are those consistent sequences of human immunoglobulin. Humanized antibody optimally Boo the ut also include, at least part of a constant region of immunoglobulin (Fc), which is typical for human immunoglobulin. For further details, see: Jones et al., Nature, 321: 522-525 ; Reichmann et al., Nature, 332: 323-329 ; and Presta, Curr. Op. Struct. Biol., 2: 593-596 ).
"He immunogenic in humans" means that under the conditions of contacting the polypeptide on a pharmaceutically acceptable carrier and therapeutically effective amounts with a suitable cloth a person's state of sensitivity or resistance polypeptide up to the second injection of the polypeptide after appropriate latent period (namely, from 8 to 14 days) is not shown.
II. The preferred embodiment of the invention
Preferred peptides of the present invention are substantially homogeneous polypeptide(s), referring to a ligand(s) mpl or thrombopoetin (TA), which are the property of binding to mpl, a member of the superfamily of cytokine receptors, possessing the biological property of stimulating the incorporation of labeled nucleotides (3H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected mpl P person. The preferred ligand(s) mpl is selected protein(s)with hematopoietic, especially megakaryocytopoiesis or thrombocytopoiesis activity - namely, capable of stimulating the proliferation, maturation and/or trim is intialy immature megakaryocytes or their precursors in Mature producing platelets form. The most preferred polypeptides of the present invention are ligand(s) mpl person, including their fragments with hematopoietic megakaryocytopoiesis or thrombocytopoiesis activity. This ligand(s) mpl person is not necessarily devoid of glycosylation. Other preferred ligands mpl are "domain EP" MLC, touching as MLC153or TFC153, a truncated form MLC and touching MLC or TPC, and Mature full length polypeptide having the amino acid sequence shown in figure 1 (Th. No:1), referring to how MLC, MLC332or TFC332and variants are biologically active substitutes MLC(R153, R154A).
Optional preferred polypeptides of the present invention are biologically or immunologically active variants of mpl ligands, selected from among MLC, MLC, MLC, MLM, MLM, MLM, LFM IMLS.
Optional preferred polypeptides of the present invention is a biologically active variant(s) of mpl ligand, which has an amino acid sequence having at least 70% identical to the amino acid sequence with the mpl ligand (see figure 1 [Th. No: 1]), the mpl ligand mouse (see Fig [Th. No: 12 and 13]), recombinant mpl ligand pigs (see Fig [Th. No: 18]), or the mpl ligand pig isolated from aplastic the coy plasma pigs, preferably, at least 75%, more preferably at least 80%, even more preferably at least 85%, and even more preferably at least 90%, and most preferably at least 95%.
The mpl ligand isolated from aplastic plasma pigs, has the following characteristics:
(1) Partially purified ligand eluted from the column with gel filtration as PBS; PBS, containing 1% SDS or PBS containing 4 M MgCl2when Mr 60000-70000;
(2) the Activity of the ligand is disturbed by pronasol;
(3) the Ligand is stable at low pH (2,5), SDS 0.1% and 2M urea;
(4) the Ligand is a glycoprotein that is based on its ability to bind to various latinovibe speakers;
(5) Highly purified ligand eluted with SDS-PAGE in non conditions with Mr 25000-35000. Fewer activity also eluted at Mr 18000-22000 and 60000;
(6) Highly purified ligand is divided in reducing conditions in SDS-PAGE in the form of a doublet with Mr 28000 31000 and;
(7) the Amino-terminal sequence of strips in 18000-22000, 28000 31000 and is the same SPAPPACDPRLLNKLLRDDHVLHGR (PEFC. No: 29); and
(8) the Ligand is bound and eluted with the following columns
Ether 650m Toyoperl,
Butyl 650 m Toyoperl,
Phenyl 650 m Toyoperl, and
Phenyl-Sefa is oz.
Preferred polypeptides of the mpl ligand are those that encode genomic or cDNA having the amino acid sequence described in figure 1 (Th. No: 1).
Other preferred naturally occurring biologically active polypeptides of the mpl ligand of the present invention include ligand shall-mpl, the ligand Pro-mpl, Mature mpl ligand fragments of mpl ligand and their glycosylated variants.
Still other preferred polypeptides of the present invention include sequence variants and chimeras of mpl ligand. Usually the preferred sequence variants and chimeras of mpl ligand are biologically active variants of mpl ligand, which have an amino acid sequence having at least 70% sequence identity with the ligand mpl or mpl ligand isolated from aplastic plasma pigs, preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, and even more preferably at least 90%, and most preferably at least 95%. An example of a preferred version of the mpl ligand is N-terminal domain variant MLC (referred to as "EP-domain" because of its homology with erythropoietin). Preferred VC-domain MLC includes approximately 153 first aminokislot the x residue of the Mature MLC and marked as MLC 153. The preferred sequence variants MLC optionally includes one in which one or more primary or two basic amino acid residue(s) in the C-terminal domain are replaced by non-core amino acid residue(s) (e.g., hydrophobic, neutral, acidic, aromatic, Gli, About and such). The preferred sequence of the C-terminal domain variant MLC includes one in which Arg residues 153 and 154 replaced by residues Ala. This option is designated as MLC332(R153A, R154A). Alternative preferred option MLC includes either MLC332or MLC153in which amino acid residues 111-114 (QLPP or LPPQ) removed or replaced with different tetrapeptide sequences (e.g., AGAG, or the like). Subsequent mutants with divisions marked as ΔMLC332or ΔMLC153.
The preferred Chimera is a merger between the mpl ligand or its fragment (defined below) with a heterologous polypeptide or its fragment. For example, MLC153may be fused with a fragment of IgG to improve the time half-life in plasma or with IL-3, G-CSF or VC to obtain molecules with increased thrombopoetin or chimeric hematopoietic activity.
Alternative preferred chimeric ligand mpl person is "Chimera ML-EP house is n", including N-terminal 153 to 157 residues replaced by one or more, but not all, of the remains of the EP man, set in rows, as shown in Fig. 10 (Th. No: 7). In the present embodiment, the Chimera MLC would be about 153-166 residues in length, in which individual or grouped in a block of residues from the sequence of electronic signature of a person added or substituted for the sequence MLC in terms of the corresponding location in the range shown in Fig. 10 (Th. No: 6). The approximate block-sequence insertion in the N-Kontsevoy part MLC would include one or more sites of N-glycosylation in the regulations (EP) 24-26, 38-40 and 83-85; one or more predicted aliphatic α-helical folds in provisions (EP) 9-22, 59-76, 90-107 and 132-152; and other highly conserved region, including the N-terminal and C-terminal region, and the position of the residues (EP) 44-52 (see, for example. Wen et al., Blood, 82: 1507-1516  and Boissel et al., J. Biol. Chem., 268(21): 15983-15993 ). Supposedly, this "Chimera ML-EP domain will have mixed thrombopoetin-eritropoeticescoe (TEP) biological activity.
Other preferred polypeptides of the present invention include fragments of the mpl ligand having a sequence sequence at least, 10, 15, 20, 25, 30 or 40 amino acid residues that are identical sequence of mpl ligand isolated from aplastic plasma pigs or leagues the NDA mpl person, described here (see, for example, Table 14, Example 24). A preferred fragment of mpl ligand is a ligand of a person, where ML[1-X], where X is 153, 164, 191, 205, 207, 217, 229 or 245 (see Fig. 1 [Th. No: 1] sequence of residues 1-X). Other preferred fragments of the mpl ligand include those obtained by chemical or enzymatic hydrolysis or cleavage of purified ligand.
Another preferred aspect of the invention is a method of purification of molecules of mpl ligand, comprising contacting the source of the mpl ligand containing molecules of mpl ligand, immobilized receptor polypeptide, especially mpl, or fused polypeptide mpl, in terms whereby the purified molecule ligand mpl are selectively adsorbed onto the immobilized receptor polypeptide, washing the immobilized substrate to remove readsorbing material, and elution of the purified molecules to the immobilized receptor polypeptide elution buffer. When the immobilized receptor is preferably fused mpl-IgG, a source containing the mpl ligand, can be plasma.
Alternatively, the source containing the mpl ligand is a recombinant cell culture, where the concentration of mpl ligand in the culture medium and cell lysate, in General, higher than in plasma or other prirodnomatematicki. In this case, the above mpl-IgG immunoaffinity way, although still useful, usually not necessary, can be applied as known in the art to more traditional methods of protein purification. In short, the preferred method of cleaning, providing substantially homogeneous mpl ligand, includes: removal of residue particles as the host cells, and fragments of the lysate, for example, by centrifugation or ultrafiltration; protein, optionally, can be concentrated using a commercially available filter for protein concentration; further separation of the ligand from other impurities by one or more of the steps selected from: immunoaffinity, ion exchange (for example, DEAE or matrix containing carboxymethyl or sulfhydryl groups), Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, Lentil lectin-Sepharose, WGA-Sepharose, Con a-Sepharose, Ether-Toyoperl, Butyl Toyoperl, Phenyl-Toyoperl, protein And Sepharose, SDS-PAGE, IHVR with reversed phase (namely, the silica gel with the added aliphatic groups), chromatography or chromatography on a molecular sieve of Sephadex, or chromatography in size; and precipitation with ethanol or ammonium sulfate. In any of the previous stages for the inhibition of proteases can be activated inhibitor of proteases, such as methylphenylsulfonyl (MPSF).
In another predpochtitel the nom embodiment of the present invention describes the selection of antibodies the ability to communicate with the mpl ligand. The preferred selected antibody to the mpl ligand is a monoclonal (Kohler and Milstein, Nature, 256: 495-497 ; Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Burdon et al., Eds, Volume 13, Elsevier Science Publishers, Amsterdam ; and Huse et al., Science, 246: 1275-1281 ). The preferred selected antibody to the mpl ligand, which binds to the mpl ligand with an affinity of at least about 106l/mol. More preferably, the antibody binds with an affinity of at least about 107l/mol. More preferably, the antibody is generated against mpl ligand having one of the above effector functions. The selected antibody capable of binding to mpl ligand, may be optionally fused with a second polypeptide, antibody, or their alloy may be used for isolation and purification of mpl ligand from a source described above for the immobilized polypeptide mpl. In a further preferred aspect of this embodiment of the invention describes a method for the detection of mpl ligand in vitro or in vivo, comprising contacting the antibody with the sample, in particular a serum sample suspected on the content of the ligand, and detecting if there is binding.
Still further, in the preferred embodiment of the invention describes the allocation of a nucleic acid molecule that encodes l is GAND mpl or its fragment, a nucleic acid molecule which can be mechina or not mecena find on the radical, and a nucleic acid molecule having a sequence that is complementary to, or hybridizes in stringent or moderately stringent conditions with a nucleic acid molecule having a sequence encoding the mpl ligand. Preferred nucleic acid ligand mpl is RNA or DNA that encodes a biologically active mpl ligand having at least 75% identical to the sequence, more preferably at least 80%, even more preferably at least 85%, and even more preferably at least 90%, and most preferably at least 95% identical to the sequence with the mpl ligand. The preferred molecule selected nucleic acid is a DNA sequence encoding a biologically active mpl ligand selected from: (a) DNA-based coding region of the gene of mpl ligand mammals (namely, DNA comprising the nucleotide sequence presented in figure 1 (Th. No: 2) (or the complement or a fragment of it) in low to moderate hardness. Thus, a further aspect of the present invention includes DNA that hybridizes in conditions with low to moderate hardness with DNA encoding polypeptides is of Uganda mpl.
In a further preferred embodiment of the present invention the nucleic acid molecule is a cDNA encoding the mpl ligand, and further including can replicate the vector in which cDNA operatively linked with the control sequence, identified by a host transformed by the vector. This aspect further includes a host cell transformed by the vector, and the usage of cDNA for the impact on production of mpl ligand, comprising the expression of a cDNA encoding the mpl ligand in the culture of the transformed host cells, and the allocation of mpl ligand from the culture of host cells. The mpl ligand, thus obtained, is preferably substantially homogeneous mpl ligand person. Preferred host cells for the production of mpl ligand are cells of the ovary hamster (Cho).
The invention further includes a preferred method for treating a mammal having an immunological or hematopoietic disease, particularly thrombocytopenia, comprising the administration to a mammal a therapeutically effective amount of mpl ligand. The mpl ligand, optionally, is introduced in combination with a cytokine, in particular, with Colonie-stimulating factor or interleukin. The preferred Colonie-stimulating factors or interleukins include: kit-ligand, PHIL, G-CSF, GM-CSF, the-CSF, EP, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL-11.
III. Ways to get
Production of platelets, have long been studied by some authors, is under the control of numerous specific differentiating humoral factors. It was postulated that the two different activity of the cytokine, denoted as Colonie-stimulating factor (Meg-CSF) and thrombopoetin, regulate megakaryocytes and trombozitopoez (Williams et al., J. Cell Physiol., 110: 101-104 ; Williams et al., Blood Cells, 15: 123-133 ; and Gordon et al. Blood, 80: 302-307 ). In accordance with this hypothesis, Meg-CSF stimulates the proliferation of precursor megakaryocytes, whereas thrombopoetin first affects the maturation of more differentiated cells and, ultimately, on the release of platelets. Since the 1960-ies data on induction and detection of both activities - Meg-CSF and thrombopoietin in plasma, serum and urine of animals and humans, after thrombocytopenic attacks are well documented (Odell et al., Proc. Soc. Exp. Biol. Med., 108: 428-431 ; Nakeff et al., Acta Haematol., 54: 340-344 ; Specter, Proc. Soc. Exp. Biol., 108: 146-149 ; Schreiner et al., J. Clin. Invest., 49: 1709-1713 ; Ebbe, Blood, 44: 605-608 ; Hoffman et al., N. Engi. J. Med., 305: 533 ; Straneva ey al., Exp. Hematol., 17: 1122-1127 ; Mazur et al., Exp. Hematol., 13: 1164 ; Mazur et al., J. Clin. Invest., 68: 733-741 ; Sheiner et al., Blood, 56: 183-188 ; Hill et al., Exp. Hematol., 20: 354-360 ; and (Hegyi et al., Int. J. Cell. Cloning, 8: 236-244 ). It was noted that these and the performance show specificity towards differentiating lines and distinct from the activities of known cytokines (R.J. Hill et al., Blood, 80: 346 ; Erickson-Miller C.L. et al., Brit. J. Haematol., 84: 197-203 ; Staneva J.E. et al., Exp. Hematol., 20: 4750 ; and J. Tsukada et al., Blood, 81: 866-867 ). After that attempts to clear the Meg-CSF or thrombopoetin from thrombocytopenic plasma or urine were unsuccessful.
In accordance with the above observations, describing thrombocytopenic plasma, it was found that aplastic plasma pigs (APS), obtained from irradiated pigs, stimulates megakaryocytopoiesis in humans in vitro. It was found that this stimulatory activity prevents the soluble extracellular domain of C-mpl, confirming that the TSA is a potential source of natural mpl ligand (ML). Currently, the mpl ligand was successfully purified from plasma APS, and information about the amino acid sequence was used to identify cDNA ML mouse, pig and human. These sequences ML homologous erythropoietin and have Meg-CSF, and thrombopoietin-like activity.
1. Purification and identification of mpl ligand from plasma
As stated above, aplastic plasma of different species, as reported, contains activities that stimulate hematomas in vitro, however, as previously reported, no hematopoietic stimulating factor from plasma was not allocated. One source aplastic plasma is one that is obtained from oblucheny the x pigs. This aplastic plasma pigs (APS) stimulates hematomas in humans in vitro. To determine whether the AFA ligand mpl, its effect was investigated by measuring include3H-thymidine in Ba/F3 cells transfected mpIP (Ba/F3-mpl) of a person, the method shown in figure 2. APS encourages the inclusion of3H-thymidine in Ba/F3-mpl cells, but not in the control Ba/F3 cells (i.e., atransferrinemia mpl P person). In addition, no other activity in normal plasma pigs were observed. These results underline that the APS contains a factor or factors that transmit proliferative signals via the mpl receptor and, therefore, can be a natural ligand for this receptor. This was further confirmed by the discovery that treatment of APS soluble mpl-IgG suppresses stimulatone effect of APS on Ba/F3-mpl cells.
The activity of APS, apparently, is a protein, because pronase, DTT or heating destroys the activity of APS (figure 3). The activity is also medialiteracy. However, the activity was stable at low pH values (pH 2.5 V for 2.5 hours)and was shown to be the binding and elution with different latinatranny speakers, noting that she was a glycoprotein. Further, in order to shed light on the structure and identification of this activity, she was cleared of APS by affinity using mpl-IgG Chimera./p>
APS has processed according to the Protocol outlined in Examples 1 and 2. In short, the mpl ligand was purified using chromatography on hydrophobic interaction (GC), chromatography with immobilized dye and chromatography mpl on affinity. Release of activity at each stage is shown in figure 4, and the degree of purification is presented in Table 1. The total activity release after mpl-affinity column was approximately 10%. Fractions with peak activity (F6) after mpl-affinity columns was according to the definition of specific activity of 9.8×106units/mg Total clearance of 5 liters APS was about 4×106time (0.8 units/mg to 3.3×106units/mg), with 86×10° the extent of the loss in protein (from 250 g to 3 g). Was determined the specific activity of the ligand, lirovannomu with mpl-affinity column, component 3×106units/mg.
Cleaning mpl ligand
|Sample||Volume ml||Protein mg/ml||Units/ml||Units||The specific soil. active. units/mg||Output %||The degree of purification|
|Phenyl||4700||0,8td align="center"> 40||200000||50||94||62|
|mpl(ál) (Fractions 5-7)||12||5×10-4||1666||20000||3300000||10||4100000|
Protein was determined by Bradford method. The protein concentration in fractions 5-7 with elyuirovaniya mpl was measured based on the intensity of the color silver SDS-gel. One unit of activity was defined as the ability to induce 50% maximal stimulation of proliferation of Ba/F3-mpl cells.
Analysis of the fractions after elution with mpl-affinity column by SDS-PAGE (4-20%, Novex gel), dispersed in reducing conditions revealed the presence of several proteins (figure 5). Proteins that were painted silver more intensively, given the resolution of the seeming Mr to 60000 55000, 30000, 28000 and 18000-22000. To determine which of these proteins stimulates the proliferation of Ba/F3-mpl cells, proteins were suirable from the gel as described in Example 2.
The results of these experiments showed that most of the activity eluted with sliced gel, which included proteins with Mr 28000-32000, lower activity was swerves from the field gel 18000-22000 (6). The only proteins that are visible in this area had Mr 30000, 28000 is 18000-22000. To identify and retrieve the protein sequences of the proteins expressed in this region of the gel (namely, the bands at 30, 28 and 18 to 22 kDa), three of these protein was subjected to electroblotting on PVDF and sequenced as described in Example 3. The obtained amino acid sequence presented in Table 2.
Amino-terminal sequence of mpl ligand
|(S)P A P P A(C)D PRLLNKLLRD D(H/S)V L H(G)R L||.No: 30|
|(S)P APPAXDPRLLNKLLRD D(H)V L(H)G R||.No: 31|
|X P A P P A X D P R L X (N) (K)||.No: 32|
Analysis with the use of computer found the novelty of these amino acid sequences. Since all three sequences were close, it is estimated that 30, 28 and 18 to 22 kDa proteins are related and are possibly p is slishnimi forms of the same new protein. Moreover, this protein(and) was likely candidate natural mpl ligand, as activity, dispersed in SDS-PAGE, were in the same areas (28000-32000) 4-20% gel. In addition, partially purified ligand migrates with Mr 17000-30000, when it is subjected to gel chromatography on a column of Superose 12 (Pharmacia). It was hypothesized that different Mr forms of the ligand are the result of proteolysis, or differences in glycosylation or other post - or pre-translational modifications.
As described previously, antisense RNA mpl person cancels megakaryocytes in cell cultures of human bone marrow-enriched CD 34+progenitor cells without affecting the differentiation of other differentiated cell lines (Methia et al., ibid.). These results confirm that the mpl receptor may play a role in the differentiation and proliferation of megakaryocytes in vitro. To further elucidate the role of mpl ligand in megakaryocytopoiesis were compared the effects of APS and APS, devoid of mpl ligand, megakaryocytes human in vitro. The effect of APS on megakaryocytopoiesis man was determined using a modification of the research megakaryocytopoiesis as a liquid suspension as described in Example 4. In this study, peripheral stem cells (PSC) was treated with APS before and after mpl-IgG affinity chromatography. Stimulation of megakaryocytopoiesis p the activity GPII bIIIawas calculated by using125I-anti-IIbIIIandantibodies (7). As shown in Fig.7, 10% APS causes approximately 3-fold stimulation, whereas APS, devoid of mpl ligand, had no effect. It is noteworthy that the TSA, devoid of mpl ligand, is not induced proliferation of Ba/FS-mpI cells.
In another experiment, soluble mpl-human IgG added at 0, 2 and 4 days of culture containing 10% APS, neutralized stimulatory effect of APS on megakaryocytopoiesis person (Fig). These results point out that the mpl ligand plays a role in the regulation of megakaryocytopoiesis in humans and, therefore, can be used to treat thrombocytopenia.
2. Molecular cloning mpl ligand
Based on the amino acid sequence of amino end, derived from proteins of 30, 28 and 18 to 22 kDa (see Table 2 above), were constructed two oligonucleotide primernih pool, used for amplification of genomic DNA pigs by the method of the ROC. Was residene that if the amino acid sequence of amino end is encoded by a single exon, then it would be expected that the correct PCR product length 69 base pairs. The DNA fragment of this size was identified and subcloned into pGEMT. The oligonucleotide sequence of the ROC primer and obtained three clones shown in Example 5. Amino acid p is the sequence (PRLLNKLLR [Th. No: 33]) of a peptide intermediate coded primers was identical to that obtained when the protein sequence amino-end of the ligand pigs (see residues 9-17 for sequences of proteins 28 and 30 kDa pigs above).
A synthetic oligonucleotide derived from the sequence of the ROC fragment used for screening the genomic library of human DNA. Was designed and synthesized oligonucleotide from 45 bases, designated as pR45, based on the sequence of a fragment of the ROC. This had the following nucleotide sequence:
5'GCC-GTG-AAG-GAC-GTG-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG-3’ (Th. No: 34).
This nucleotide was used for screening the genomic library of human DNA in λgem12 under low stringency hybridization and wash conditions in accordance with Example 6. Positive clones were selected, dies purified and analyzed by the methods of restriction mapping and southern blotting. Fragment of 390 base pairs EcoRI-Xbal, which hybridized with 45 member, was subcloned into pBluescript SK-. DNA sequencing of this clone confirmed that this DNA encodes the selected mpl ligand human homologous pork. The sequence of human DNA and deduced amino acid sequence presented in Fig.9 (PEFC. No: 3 and 4). The predicted position of introns in the genomic sequence, also marked with what recami, define natural Exxon (Exxon 3").
Based on sequence "AXONE 3" person (Example 6), was synthesized oligonucleotide corresponding to the 3’ and 5’ ends of the sequence axona. These two primers were used in the selected mpl ligand person ROC reaction using as a template cDNA derived from various human tissues. The expected sizes of the right product, the ROC was 140 base pairs. After analysis of the product of the ROC in 12% polyacrylamide gel was discovered a fragment of the cDNA of the expected size in the library, cDNA derived from the kidney of an adult human cells, 293 human embryonic kidney human, and cDNA derived from fetal human liver.
The following was sifted cDNA library embryonic liver (7×106clones in the lambda DR2 with the same 45-membered oligonucleotide used for screening genomic libraries or cDNA libraries of embryonic liver at low stringency hybridization conditions. One clone with the inclusion of 1.8 thousand bases was selected for further analysis. Using the procedure described in Example 7, were obtained nucleotide and deduced amino acid sequence of mpl ligand person (MLC). These sequences are presented in figure 1 (Th. No: 1 and 2).
3. The structure of the mpl ligand person (MLC)
P is the sequence of the cDNA of mpl ligand person (MLC) (figure 1 [Th. No: 2]) contains 1774 nucleotide following the end of the poly(A). It contains 215 nucleotides of 5’ untranslated sequences and 3’ untranslated region of 498 nucleotides. Estimated initiating codon at (216-218) the position of the nucleotides located within a coherent sequence, favorable for initiation of translation in eukaryotes. The open reading frame is 1059 nucleotides in length and encodes a polypeptide of 353 amino acid residues starting at 220 nucleotide position. N-end of the predicted amino acid sequence is vysokoriskovannym and probably corresponds to the signal peptide. Computer analysis of the predicted amino acid sequence (von Heijne et al., Eur. J. Biochem., 133: 17-21 ) notes the potential for splitting the signal peptidases between residues 21 and 22. Cleavage at this position would give the Mature polypeptide of 332 amino acid residues beginning with amino-terminal sequence obtained from mpl ligand purified from the plasma of pigs. Predicted molecular weight replicationmanager ligand of 332 amino acid residues is about 38 kDa. There are 6 potential sites for N-glycosylation and 4 cysteine residue.
Comparison of the sequence of mpl ligand sequence databank Genbank revealed the 23% identity between 153 aminobenzamide remnants of Mature mpl ligand person and erythropoietin (Fig. 10 [Th. No: 6 and 7]). If to take into account conservative substitutions, this area MLC shows 50% similarity with the human erythropoietin (APC). Both APC and MLC contain four cysteine. Three of the 4 cysteines conservative in MLC, including first and last cysteine. Experiments on point-directed mutagenesis showed that the first and last cysteine erythropoietin form a disulfide bond, which is required for operation (Wang, F.F. et al., Endocrinology, 116: 2286-2292 ). By analogy, the first and last cysteine MLC can also form a significant disulfide bonds. None of glucosylrutin sites is not conservative at MLC. All potential sites of glycosylation MLC are located in the carboxy-terminal half of the polypeptide MLC.
Like APC, mRNA MLC not contain any coherent sequence AAUAAA polyadenylation or regulatory element AUUUA, which is present in the 3’ untranslated region of many cytokines, and thereby affect mRNA stability (Shaw et al., Cell 46: 659-667 ). Northern blot analysis revealed a low level of a single 1.8 thousand reasons RNA transcript MLC and in fetal and adult liver. After a long incubation can be determined and in adult kidney weak band of the same size. For comparison, human erythropoietin expressively in fetal liver and adult kidney and liver (Jacobs et al., Nature, 313: 804-809  and Boudurant et al., Molec. Cell. Biol., 6: 2731-2733 ) in response to hypoxia.
The importance of the C-terminal region MLC remains necessary for clarification. Based on the presence of six potential sites for N-linked glycosylation and the ability of the ligand to contact lectin-affinity column, this area MLC, apparently, is glycosylated. In some experiments by elution from the gel were observed activity at resolution Mr of about 60,000, which can be glycosylated molecule full length. C-terminal region, therefore, can stabilize and increase the half-life of circulating MLC. In the case of erythropoietin deglycosylation form has full biological activity in vitro, but has significantly reduced the half-life in plasma compared to glycosylated erythropoeitin (Takeuchi et al., J. Biol. Chem., 265: 12127-12130 ; Narhi et al., J. Biol. Chem., 266: 23022-23026  and Spivack et al., Blood, 7: 90-99 ). The C-terminal domain MLC contains two sequences of two basic amino acids [Arg-Arg structure in terms 153-154 and 245-246], which could serve as possible sites for processing. Cleavage at these sites could be responsible for the formation of 30, 28 and 18 to 22 kDa forms of ML, isolated from the APS. It is important that the sequence Apr153-Apr154observed immediately after erythropoietin-like the th domain ML. These observations note that ML full length can be a precursor which is subjected to limited proteolysis with the formation of the Mature ligand.
4. Isoforms and variants mpl ligand person
Isoforms, or alternative splicing forms, mpl ligand people were determined by the method of the ROC in adult human liver. In brief, primers were synthesized corresponding to each end, as well as selected regions coding for the sequence MLC. These primers were used in the ROC for multiplying the RNA of adult human liver, as described in Example 10. In addition to the full form, marked MLC were detected or displayed three other shape MLC, MLC and MLC. Bred Mature amino acid sequence of all four isoforms presented at the 11 (Th. No: 6, 8, 9, and 10). MLC 116 has a nucleotide deletion at position 700, which leads to amino acid deletions, and to shift the frame. Now cDNA encodes a Mature polypeptide, which is the length of 265 amino acids and is derived from the sequence MLC with 139 amino acid residue. Finally, MLC has a deletion in 12 (pairs) nucleotides after 618 provisions nucleotide (also found in sequences from mouse and pig [see below]), and a deletion at 116 base pairs found in MLC. While (indicated by the Oh MLC) the person was not selected clones with deletions in only 12 base pairs (after nucleotide 619), this form, presumably, exists because this isoform was identifitsirovana and mouse, and pig (see below), and besides, she was identified in conjugation with 116 nucleotide deletion in MLC.
To determine whether ML full length necessary for biological activity, were designed and option MLC with the substitution, in which a sequence of two basic amino acids Apr153-Apr154replaced by two alanine residue, and a truncated form MLC, "EP-domain. Using the ROC, as described in Example 10, was constructed replaced by a variant with two key amino acids in the sequence Apr153-Apr154marked as MLC(VA, R154A). Using the ROC was also created a truncated form EP-domain MLC153by introducing a stop codon after Arg.
5. Expression of recombinant mpl ligand person (rmlc) in transtorno transfected cells embryonic human kidney (293)
To confirm that the cloned human cDNA encodes a ligand for the mpl, the ligand was expression in 293 cells mammals, using expression vectors R5-MLC or pRK5-MLC153in the direct suppression of the early promoter of cytomegalovirus. It was found that supernatant of transtorno transfected cells 293 embryonic kidney of man will stimuliruyut the inclusion of 3H-thymidine in Ba/F3-mpl cells but not in parental Ba/F3 cells (figa). Medium from 293 cells, transfected with only the pRK vector does not contain this activity. Add mpl-IgG to the environment prevents stimulation (data not shown). These results indicate that the cloned cDNA encodes a functional ML human (MLC).
To determine whether EP-domain" itself to contact and activate the mpl was expressed a truncated form MLC, rmlc153in 293 cells. It was found that supernatant transfected cells have activity similar to that found in supernatant cells expressing MLC full length (figa), showing that the C-terminal end of the ML is not required for binding and activation of C-mpl.
7. Megakaryocytopoiesis and mpl ligand
It was hypothesized that megakaryocytes regulated at the many cellular levels (Williams et al., J. Cell Physiol., 110: 101-104  and Williams et al., Blood Cells, 15: 123-133 ). This is based mainly on the observation that certain hematopoietic growth factors stimulate the proliferation of progenitor megakaryocytes, while others, mainly showing the effect on maturation. The data presented here confirm that ML acts as a proliferative factor and as a factor of maturation. What ML stimulates the proliferation of progenitors mega is arilton, confirmed in several ways evidence. First, APS and stimulates the proliferation and maturation of megakaryocytes human in vitro, and this stimulation is completely suppressed mpl-IgG (Fig.7 and 8). Moreover, inhibition of the formation of colonies of C-mpl antisense nucleotides (Methia et al., Blood, 82: 1395-1401 ), and the discovery that C-mpl can transmit proliferative signals in cells that are transfected (Skoda et al., EMBO, 12: 2645-2653  and Vigon et al., Oncogene, 8: 2607-2615 ), also notes that ML stimulates proliferation. The visible expression of C-mpl throughout all stages of the differentiation of megakaryocytes (Methia et al., Blood, 82: 1395-1401 ) and the ability of recombinant ML quickly to stimulate production of platelets in vivo notes that ML also affects maturation. The ability to activate the recombinant ML creates opportunities for a more thorough evaluation of its role in the regulation of megakaryocytopoiesis and thrombocytopoiesis, as well as its ability to influence other differentiating hematopoietic lines.
8. The selection of the gene of mpl ligand (TA) of a person
Clones of human genomic DNA TA gene was isolated by the method of screening the genomic library of the human X-Geml2 with pR45 in conditions of low stringency or high stringency with a fragment corresponding to the 3’ half of the human cDNA, encoding the mpl ligand. Was allocated two overlapping l is MBDA clone length of 35 thousand grounds. Were cloned and sequenced two overlapping fragments (BamHl and EcoRI), containing the complete gene TA (see figa, 14B and 14C).
The structure of the human gene consists of 6 Aksenov 7 thousand bases of genomic DNA. Connection all connections to the exon/intron consist of coherent structures characteristic of mammalian genes (Shapiro, M.B., et al., Nucl. Acids Res. 15: 7155 ). The exon 1 and exon 2 contain 5’ untranslated sequence and the initial four amino acids of the signal peptide. The remaining secretory signal and the first 26 amino acids of the Mature protein is encoded by 3 Exxon. Full carboxyl domain and 3’ untranslated, as well as -50 amino acids erythropoietin-like domain encoded by exon 6. Four amino acids included in the deletion observed between MLC-2 (TP-2), are encoded at the 5’ end of axona 6.
Analysis of human genomic DNA using the Sauthern blot noted that TA gene present in a single copy. Chromosomal location of the gene was determined using fluorescence in situ hybridization (FISH), which mapped the chromosome 3q27-28.
9. Expression and purification of TA cells 293
Production and purification ML or TA from 293 cells described in Example 19. In brief, cDNA corresponding to the full system with open frame, was obtained using the ROC using pRK5-hmpl I. the Product of the ROC was purified and cloned between what customi Clal restriction and Xbal plasmids pRK5tkneo (derived vector pRK5, modified for expression of the gene of resistance to neomycin, in terms of suppression of the promoter of the thymidine kinase) to obtain a vector pRK5tkneo.ORF (vector encoding a complete open reading frame).
A second vector encoding the homologous domain of electronic signature was obtained in the same way, but using different ROC primers to produce the final design called pRK5-tkneoSn-A.
These two constructs were transfected into cells embryonic human kidney using Saro4method and were selected clones resistant to neomycin, who grew up confluentes state. The expression ML153or ML332the air-conditioned environment of these clones was evaluated by a study of proliferation of Ba/F3-mpl.
Cleaning rmlc332conducted as described in Example 19. In brief, 293-rmlc332conditioned medium was applied on the column with a Blue-Sepharose (Pharmacia), which was immediately washed with buffer containing 2 M urea. The column was suirable buffer containing 2 M urea and 1 M NaCl. The amount of after elution of the column with a Blue-Sepharose is then directly applied on the column with WGA-Sepharose, washed with 10 column volumes of a buffer containing 2 M urea and 1 M NaCl, and was suirable the same buffer containing 0.5 M N-acetyl-D-glucosamine. The eluate from the column with WGA-Sepharose was applied to the C4-GHUR column (Synchron, Inc.) and suirable continuous gr is dantom propanol. During electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE) of purified 293-rmlc332migrates in the gel in the form of a broad band in the range of 68-70 kDa (see Fig).
Cleaning rclc153also conducted as described in Example 19. In brief, 293-rmlc153air-conditioned environment conceded through a column of Blue-Sepharose, as described for rmlc332. The eluate from the column with a Blue-Sepharose directly caused by mpl-affinity column as described above. Suirvey with mpl-column rmlc153, was purified to a homogeneous state, using C4-GHUR column down under the same conditions as for rmlc332. On SDS-PAGE of purified rmlc153accelerated 2 large and 2 small bands with Mr of about 18000-22000 (see Fig).
10. The mpl ligand mouse
The DNA fragment corresponding to the region encoding the mpl ligand of the person obtained by means of the ROC, cleared and marked in the presence of32R-d and32R-d. These probes were used for screening 106clones of the cDNA library of mouse liver in λGT10. Was isolated and sequenced clone mouse (Fig [Th. No: 12 and 13]), containing 1443 base pairs inclusion. The presumed initiation codon in position 138-141 nucleotides were in a coordinated sequence, favorable for initiation of translation in eukaryotes (Kozak, M.J. Cell Biol., 108:229-241 ). The sequence is determined by the quality control Department shall itoi frame read in 1056 nucleotides, predicting primary product of translation of 352 amino acids. Fringe this open-reading frames 137 5’ nucleotide and 247 3’ nucleotides naschityvalos sequence. The end of the poly(A) after the 3’ untranslated region absent, which notes that the clone apparently not completed. N-end of the predicted amino acid sequence is highly hydrophobic and, apparently, represents the signal peptide. Computer analysis (von Heijne, G. Eur. J. Biochem., 133: 17-21 ) noted a possible place to break for signal peptidases between 21 and 22 residues. The gap in this place could give the Mature polypeptide of 331 amino acids (35 kDa), identical MLM331(or MLM for the reason described above). The sequence contains 4 cysteine, all conservative in the sequence of a person, and 7 possible sites of N-glycosylation, 5 of which are conservative in the sequence of human rights. Again, as MLC, all seven possible sites of N-glycosylation sites are located in the C-terminal half of the protein.
When compared with ML of a man, was found significant identity and the nucleotide and derived amino acid sequence from EP-domain data ML. However, when the deduced amino acid sequence of ML human and mouse placed in a row, it was found that the sequence we is and has tetrapeptide a deletion between residues 111 and 114, the corresponding 12 nucleotide deletion after 618 nucleotide position, visible from both cDNA, human (see above) and pigs (see below). So have been investigated for more clones to detect possible isoforms ML mouse. One clone encodes derived 335 amino acid sequence of the polypeptide containing "lost" tetrapeptide LPLQ. This form is submitted ML mouse full length and marked as MLM or MLM335. Nucleotide and deduced amino acid sequence MLM presented on Fig (PEFC. No: 14 and 15). This cDNA clone contains 1443 base pairs are terminated poly(a) end. It shows an open reading frame of 1068 base pairs, bordered 134 bases 5’ and 241 the basis of 3’ untranslated sequence. The presumed initiation codon lies in the position of the nucleotides 138-140. The open reading frame encodes a predicted protein of 356 amino acids, 21 the first of which are vysokoshirotnymi and apparently function as secretory signal.
Finally, the third selected and sequenced clone mice, it was found that it contains 116 nucleotide deletions, corresponding MLC. This isoform of mouse, therefore, was marked MLM. Comparison of the deduced amino acid sequences of these two isoforms is shown in Fig (PEFC. No: 9 and 16).
General the identity of amino acid sequence between ML human and mouse (Fig [Th. No: 6 and 17]) is 72%, but this homology is distributed unevenly. The area designated as "EP-domain (amino acid residues 1-153 for the sequence of human and 1-149 mouse) is more conservative (86% homology)than the carboxy-terminal region of the protein (62% homology). It may further specify that only "EP-domain is important for the biological activity of the protein. Interestingly, the two structures of the two basic amino acids, found in MLC, only the structure of the two basic amino acids immediately following the EP-domain" (residues 153-154) in the sequence of a person, is present in the sequence of the mouse. This is consistent with the possibility that ML full length may represent a protein precursor which is subjected to limited proteolysis with getting Mature ligand. Alternatively, proteolysis between Arg153-Apr154can accelerate the excretion MLC.
The expression vector containing the full coding sequence of MLM, they have transferrable in 293 cells as described in Example 1. Conditioned medium from these cells stimulated the inclusion of3H-thymidine in Ba/F3 cells expressing mpl and human and mouse, but had no action on the parent (without mpl) cell line. This indicates that the cloned cDNA ML mouse to direet functional ligand, which is able to activate both ML receptor (mpl), mouse and human.
11. The mpl ligand pigs
cDNA ML pigs (LFM) identified by the ROC RACE as described in Example 13. cDNA from 1342 base pairs, the product of the ROC, was detected in the kidney and subcloned. Was sequenced several clones and found 332 amino acid residue for encoding mpl ligand pigs, denoted as PD (or PD332)having the nucleotide and deduced amino acid sequence shown in Fig. 20 (Th. No: 18 and 19).
Was again identified the second form, marked MLS encoding a protein with 4 deletions of amino acid residues (228 amino acid residues) (see Fig [Th. No: 21]. Comparison of amino acid sequences of the LFM and MLS showed that the latter form is identical except that tetrapeptide QLPP corresponding to residues 111-114, inclusive, has been removed (see Fig [Th. No: 18 and 19]). The loss of four amino acids found in cDNA ML and mouse, and pig, is observed exactly in the same position in the predicted proteins.
Comparison of the predicted amino acid sequence of the Mature ML human, mouse and pig (pig [Th. No: 6, 17 and 18] notes that in General the sequence is identical and comprise 72 percent between mouse and man, 68 percent between mouse and pig and 73 percent between pig is her man. Homology significantly higher in the amino-terminal half ML (homologous domain EP). This domain from 80 to 84 percent identical between any two species, whereas the carboxy-terminal half (carbohydrate domain) are identical only 57-67 percent. The structure of the two basic amino acids, which could provide a site for cleavage by proteases present in the carboxyl end of the homologous domain of erythropoietin. This structure is conserved in the three species in this position (Fig [Th. 6, 17 and 18]). Second segment with two bases is present at position 245 and 246 in humans and is not present in the sequence of a mouse or a pig. The sequence ML mouse and pig contains 4 cysteine, all conservative in the sequence of human rights. There are seven areas of possible N-glycosylation sites in the ligand mouse and six ml of pigs, 5 of them conservative sequence of human rights. All possible sites of N-glycosylation are again in C-terminal half of the protein.
12. Expression and purification of TA cells of the Chinese hamster ovary (Cho)
Used the expression vectors for transfection of Cho cells indicated: pSVIS.ID.LL.MLORF (full length or TA332), and p3V15.ID.LL.MLEPO-D (truncated or TA153). Having the attitude of these plasmids is presented on Fig and 24.
Procedures Tr is speccie described in Example 20. In brief, cDNA corresponding to the full open reading frame of TP obtained by the ROC. The product of the ROC was purified and cloned between the two restriction sites (Clal and Sall) plasmids p3V15.ID.LL. to obtain the vector pSV15.ID.LL.MLORF. The second design appropriate to the homologous domain of the electronic signature received in the same way, but using a different reverse primer (EPOD.Sal). The final design for vector encoding homologous domain EP at TA called pSV15.ID.LL.MLEPO-D.
These two designs were linearizable with Notl and transferrable in cells of Chinese hamster ovary cells CHO-DP12, European patent 307247, published March 15, 1989) by electroporation. Were elektroporcelany 107cells using BRL apparatus for electroporation (350 volts, 350 MT, low capacity) in the presence of 10, 25 or 50 mg DNA, as described (Andreason, G.L. J. Tissue Cult. Meth. 15, 56, ). The day after transfection cells were stratified in DHFR selective medium (DMEM-F12 with high glucose 50:50 without glycine, 2 mm glutamine, 2-5% cialisbuynow calf serum). After 10-15 days, individual colonies were transferred to a Board with 96 cells and left to grow until confluently. Expressio MA153or ML332was determined in the conditioned medium from these clones using the study of proliferation of Ba/F3-mpl cells (described in Example 1).
Method of cleaning and allocation of TA from the culture the liquid grown cells SNO described in Example 20. In short, the culture fluid after growing cells (CGC) was applied on a column of Blue Sepharose (Pharmacia) in a ratio of about 100 l CZK per liter of granules media. Then the column was washed 3-5 volumes of column buffer containing 2.0 M urea. Then suirable TA 3-5 volumes of column buffer containing 2.0 M urea and 1 M NaCl.
The volume of the eluate from the column of Blue Sepharose containing TA, put then on a column Wheat Germ Lectin Spharose (Separate with the lectin wheat germ) (Pharmacia), equilibrated eluting buffer Blue Sepharose, in a ratio of from 8 to 16 volumes of the eluate per ml volume of the granules Blue Sepharose. Then the column was washed with 2-3 volumes of equilibration buffer. Then suirable TA 2-5 volumes of buffer containing 2.0 M urea and 0.5 M N-acetyl-D-glucosamine.
Then the eluate from the column with Weat Germ Lectin containing TA, was acidified and added With12E8to a final concentration of 0.04%. The final volume was applied on the column C4 reversed-phase equilibrated with 0.1% TFA, 0,04%12E8when loading of about 0.2 to 0.5 mg of protein per ml of media.
Protein was suirable two-phase linear gradient of acetonitrile containing 0.1% TFA and 0.04% With12E8and created a volume-based data SDS-PAGE.
Volume with C4 lit and diafiltration against approximately 6 volumes of buffer on Amicon YM or similar ultrafiltration membrane having useche is their molecular weight from 10,000 to 30,000 daltons. The final diafiltrate can then be directly processed or further concentrated by ultrafiltration. Diafiltrate/concentrate is usually adjusted to the final concentration with 0.01% tween-80.
All or part of diafiltrate/concentrate, equivalent to 2-5% of the calculated volume of the column, then applied on a column of Sephacryl S-300 HR (Pharmacia), equilibrated with buffer containing 0.01% tween-80, and chromatographic. The fractions containing TA, which is free from aggregates and degradation products, then combined on the basis of data from SDS-PAGE. The final volume was filtered and kept at 2-8°C.
13. Methods of transformation and induction of the synthesis of TP in the microorganism and selection, cleaning and stacking the resulting TA
Construction of expression vector of the TA in E. coli is described in detail in Example 21. In brief, plasmids RMR, RMR, RMR, RMR and RMR all created for the expression of the first 155 amino acids TA downstream young leader, which varies for different designs. Leaders get mainly for the initiation of translation of high level and quick cleaning. Plasmids RMR-1, -T8, -21, -22, -24, -25 created for the expression of the first 153 amino acids TA downstream of the initiating methionine and only differ in codon used for the first 6 amino acids of TP, whereas plasmid RMR is derived from RMR-1,in which the carboxyl end of TP increased by two amino acids. All of the above plasmid will produce intracellular expression of high levels of TP in E. coli under conditions of induction of the tryptophan promoter (Yansura, D.G. et al.. Methods in Enzymology (Goeddel, D.V., Ed.) 185:54-60, Academic Press, San Diego ). Plasmid pMP1 and RMR are intermediate in the construction of the above plasmids intracellular expression of TP.
The above expression plasmids TA were used to transform E. coli, using the method of thermal shock with CaCl2(Mandel, M. et al., J. Mol. Biol., 53:159-162 ) and other methods described in Example 21. In summary form, the transformed cells were first grown at 37°to achieve an optical density of cultures at 600 nm) of approximately 2-3. Then the culture was diluted and after cultivation under aeration was added acid. The culture was then left to continue growth with aeration for the next 15 hours, after which cells were collected by centrifugation.
Methods of isolation, purification and laying below for obtaining biologically active, the current TA of the person or its fragments, as described in Examples 22 and 23, can be applied to any variant of the TA, including N - and C-terminal extended forms. Other methods suitable for providing recombinant or synthetic TP, can be found in the following patents: Builder et al., U.S. patent 4511502; Jone et al. U.S. patent 4512922; Olson, U.S. Patent 4518526 and Builder et al., U.S. patent 4620948; for a General description of the exits and placement for various recombinant proteins expressed in insoluble form in E. coli.
A. Obtaining insoluble TA
Organism like E. coli expressing TA encoded by any appropriate plasmids were fermentatively in the conditions in which TA is deposited in insoluble "refractile or refractive corpuscles". Optionally, the cells are first washed in buffer for cell disruption. Usually, about 100 g of cells resuspendable in 10 volumes of buffer cell destruction (namely, 10 mm Tris, 5 mm EDTA, pH 8) using, for example, a homogenizer of the type Polytron, and the cells were centrifuged at 5000×g for 30 minutes. Then the cells were literally using any conventional methods, such as tonic shock, scoring, cyclic punching, chemical or enzymatic methods. For example, the washed above the sediment cells can be resuspendable other 10 volumes of buffer for cell disruption using a homogenizer, and then the cell suspension was passed through LH Cell Disrupter (LH Iceltech, Inc.) or through a Microfluidizer (Microfluidics International) in accordance with the instructions. Particles of matter containing TA, then separated from the liquid phase and optionally washed with any suitable liquid is. For example, the suspension of cell lysate may be centrifuged at 5000×g for 30 minutes, resuspension and, optionally, centrifuged a second time to obtain a precipitate refractile Taurus. The washed precipitate may be used immediately or, optionally, stored at freezing (when, for example, -70°).
C. Solubilization and purification of Monomeric TA
Insoluble TP in the sediment refractile Taurus then solubilizers in the solubilization buffer. The solubilization buffer contains chaotropic agent, and usually create a buffer when the basic pH values, and contains a reducing agent to increase the output of Monomeric TA. Approximate chaotrope agents may include urea, guanidine-Hcl and sodium thiocyanate. Preferred chaotropes agent is guanidine-Hcl. Concentration chaotropes agent usually 4-9 M, preferably 6-8 M pH solubilizing buffer are supported by any suitable buffer in the pH range from about 7.5 to 9.5, preferably to 8.0, and 9.0, and most preferably, 8,0. Preferably, solubilisers the buffer also contains a reducing agent to promote the formation of Monomeric forms of TA. Suitable reducing agent includes organic compounds containing a free thiol (RSH). Exemplary reducing agents include dithiothreitol (DTT), diti is aritra (DTA), mercaptoethanol, glutathione (GSH), the group probably facilitates and cysteine. Preferred regenerating agent is dithiothreitol (DTT). The solubilization buffer can, optionally, contain a mild oxidizing agent (e.g., molecular oxygen) and a suitable salt for the formation of Monomeric TA through sulfatases. In this embodiment the end-TA-3-sulfonate further formed in the presence of a redox buffer (e.g., GSH/GSSH) for education properly stowed TA.
Protein TA usually further purified using, for example, centrifugation, chromatography in gel filtration and chromatography on a column with reversed phase.
According to the illustrations the following procedures give satisfactory output Monomeric TA. Sediment refractile Taurus resuspendable in about 5 volumes by weight of solubilization buffer (20 mm Tris, pH 8, with 6-8 M guanidine and 25 mm DTT) and stirred for 1-3 hours or overnight at 4°to solubilize protein TA. High concentrations of guanidine are also applicable, but in General lead to a slightly reduced output compared with guanidine. After solubilization, the solution was centrifuged at 30000×g for 30 minutes to obtain a clear supernatant containing the denatured Monomeric protein TA. Then the supernatant was chromatographically by the gel is istratii on the column with Superdex 200 (Pharmacia, 2,6×60 cm) at a speed of current of 2 ml/min, and was suirable protein 20 mm Na phosphate, pH 6.0, in the presence of 10 mm DTT. Fractions containing Monomeric denatured protein TA loirevalley between 160 and 200 ml, were combined. Further protein TA was purified on prepreparation column C4 reversed-phase (2×20 cm VYDAC). Samples were applied at a speed of 5 ml/min to a column equilibrated with 0.1% TFA (triperoxonane acid) with 30% acetonitrile. Protein was suirable linear gradient of acetonitrile (30-60% for 60 minutes). Cleaned restored protein eluted at approximately 50% acetonitrile. This material is used for laying the purpose of obtaining biologically active variant of TA.
C. Stacking system for obtaining biologically active form.
After solubilization and further purification of the TA received a biologically active form by placing denatured Monomeric TA in redox buffer. Due to the high activity of the TA (premaxilla stimulation in the study on Ba/F3 cells is achieved at approximately 3 PG/ml), it was possible to get biologiske active material, using many different buffers, detergents, and redox conditions. However, in most conditions received only a small amount of properly laid material (<10%). To construct the definition of the production method, it is desirable to have the output of the laid material, at least 10%, more preferably 30-50%, and most preferably >50%. It was found that many different detergents, including Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, SDS, sarkosyl, tween 20 and tween 80, Twitterget 3-14 and others to obtain at least some appropriately laid material. However, among the most preferred detergents were those from the family of CHAPS (CHAPS and CHAPSO), which, as it was found that best worked by the reaction stacking, and limited protein aggregation and improper formation of disulfide bonds. The most preferred was the level of CHAPS more than about 1%. Sodium chloride is required for best yield, with an optimum level of between 0.1 M and 0.5 M Were preferred the presence of EDTA (1-5 mm) in a redox buffer to limit catalyzed by metal oxidation (and aggregation), which was observed in some preparations. The concentration of glycerol is more than 15% has created optimal conditions for laying. For maximum yield was mandatory to have a redox couple in a redox buffer containing both oxidized, and the recovered organic thiol (RSH). Suitable redox couples include mercaptoethanol, glutathione (GSH), C is steamine, cysteine and their corresponding oxidized form. Preferred redox couple were glutathione (GSH) : oxidized glutathione (GSSH) or cysteine : cystine. The most preferred redox couple was glutathione (GSH) : oxidized glutathione (GSSH). In General, high yield was observed when the molar ratio of the oxidized member of the redox pair was equal to or exceeded the molar ratio of the recovered member of the redox pair. Storing these options TA has the optimum pH value between 7.5 and about 9. Organic solvents (e.g. ethanol, acetonitrile, methanol) in concentrations of 10-15% or below were indifferent. High levels of organic solvents increased the number of misfolded forms. In General, applicable were Tris and phosphate buffers. Incubation at 4°also gave an increased level stacked properly TA.
Exit when placing 40-60% (based on the amount recovered and denatured TA used in the reaction styling is typical for the preparation of the TA, which was purified by use of the first C4 stage. The active material may be obtained from a less purified preparations (for example, immediately after the column Superdex 200, or after the primary extrac the AI refractile Taurus), although the yield is lower due to the increased deposition and the influence of non-TA proteins during the process of laying the TA.
Since the TA contains four cysteine residue, it is possible to obtain three different disulfide version of this protein:
version 1: disulfide bonds between cysteine residues 1-4 and 2-3;
version 2: disulfide bonds between cysteine residues 1-2 and 3-4;
version 3: disulfide bonds between cysteine residues 1-3 and 2-4.
During the initial study determination of conditions for laying by chromatography with reversed phase C4 was divided several different peaks containing protein TA. Only one of these peaks had a significant biological activity, as defined when using Ba/F3 cells. In accordance with these terms of styling were optimized for preferred this version. In these conditions incorrectly put version was less than 10-20% of the total Monomeric TA obtained in step solubilization.
By mass spectrometry and sequencing of the protein was determined pattern of disulfide bonds for biologically active TA representing 1-4 and 2-3, where cysteine numbered in order from the amino end. Picture of cross-connections is in accordance with known disulfide bonds related molecules erythropoietin
D. Biological activity of recombinant laid TA
Laid and cleaned the TA has activity in research and in vitro and in vivo. For example, in a study on the cells Ba/F3 premaxillary stimulation of thymidine incorporation in Ba/F3 cells for TA (Meth-11-153) was reached at a 3.3 PG/ml (0.3 PM). Based on the mpl receptor study ELISA premaxillary activity was observed at 1.9 ng/mg (120 PM). The ordinary and myelosupression animals, obtained close to lethal irradiation by x-rays, laid TP (Meth-11-153) was highly active (activity was observed at doses of less than 30 ng/mouse) by stimulating the production of new platelets. Similar biological activity was detected for other forms of TA, arranged in accordance with the above methods (see Fig, 26 and 28).
14. Methods of measurement thrombopoetin activity
Tromboticheskoe activity can be measured in various studies, including the study of mpl ligand on cells Ba/F3 described in Example 1, the study of restorative synthesis of platelets in mouse in vivo study of the induction of cell surface antigen platelet measurement method antiplatelet immunoprotein (anti-GIIbIIIand) megakaryoblastic cell lines (QMS) human leukaemia (see Sato et al., Brt. J. Haematol., 72: 184-190 ) (see also the study of megakaryocytopoiesis as a liquid suspension as described in Example 4), and the induction of polyploidization on megakaryoblastic cell lines (DAMI) (see Ogura et al., Blood, 72(1): 49-60 ). Maturation of megakaryocytes from immature forms, mainly, not synthesizing DNA cells, morphologically recognizable megakaryocytes involves a process that includes the appearance of cytoplasmic organelles, the acquisition of membrane antigens (GPIIbIIIa), internal replication and release of platelets, as described in the prerequisites. Specifically differentiating stimulator line (for example, the mpl ligand) maturation of megakaryocytes, could be expected to induce at least some of these changes have immature megakaryocytes, resulting in the release of platelets and facilitate thrombocytopenia. Thus, the study was designed to measure these parameters in immature megakaryocyte cell lines, for example, QMS and DAMI cells. The study of the QMS defines the appearance of specific markers of platelet GPIIbIIIaand the output of platelets. In determining DAMI (Example 15) measured endoreplication as higher ploidy is the hallmark of Mature megakaryocytes. Recognizable megakaryocytes have a value of ploidy 2N, 4N, 8N, 16N, 32N, etc. Finally, the study of restorative synthesis of platelets in mice in vivo (Example 16) it is useful to demonstrate that the introduction of the investigated compounds (here mpl ligand) leads to an increase in platelet count.
There were developed two additional in vitro studies for measuring the activity of TA. The first is the study of the activation of receptor kinase (KIRA) using ELISA method, in which cells transfected Cho-mpl Chimera-Rse, and measured the phosphorylation of tyrosine Rse using ELISA method after keeping part mpl Chimera with the mpl ligand (see Example 17). The second method is an ELISA based on the detection of the receptor, in which ELISA plates coated with rabbit serum against human IgG capture the chimeric receptors of the human mpl-IgG that bind analyzed mpl ligand. Biotinylated monoclonal antibody rabbit to the mpl ligand (TA155used to determine the bound ligand mpl, which was measured using the streptavidin-peroxidase as described in Example 18.
18. Biological response of normal and sublethal irradiated mice on the introduction of TP in vivo
Both normal and irradiated in a sublethal dose mice were injected truncated and full length TP, was isolated from the cells of the Chinese hamster ovary (Cho), E. coli and embryonic cells of human kidney (293). Both Faure is s TA obtained from these three farmers, stimulated the production of platelets in mice, however, TA full length, selected from SNO, apparently, causes the strongest response in vivo. These results emphasize that proper glycosylation carboxy-terminal domain may be required for optimal activity in vivo.
Form "Met" domain EP (Meth at -1 position +153 of the first residue TA man)produced in E. coli (see Example 23)daily were injected with normal females S B6 mice, as described in the explanatory notes to figa, 25V and 25S. These drawings show that produced in E. coli, deglycosylation truncated form TA, arranged as described above, is capable of stimulating an approximately twofold increase the production of platelets from normal mice without affecting the population of red and white blood cells.
These molecules injected daily sublethal irradiated (137Cs) females S B6 mice, as described in the legend to Fig. 26A, 26C and 26C, stimulated the release of platelets and reduced the period of extreme depression, but had no effect on erythrocytes or leukocytes.
Form TA full length, produced in cell SNO entered daily normal females S B6 mice as described above in the legend Figo, 27B and 27C, caused approximately a five-fold increase in the product of the AI platelets from normal mice without affecting the population of erythrocytes or leukocytes.
(c) SNO-rtpc332; E. coli-rtpc (Meth-1, 153); 293-rtpc332and
Was built curve dose response to the introduction of normal mice rtpc from different cell lines (Cho-rtpc332; E. coli-rtpc(Meth-1, 153); 293-rtpc332and .li-rtpc155as described in the caption to Fig. 28. This drawing shows that all studied forms of molecules stimulate the production of platelets, but the form of full length, produced in the cells SNO has the greatest activity in vivo.
(d) CHO-rtpc153, SNO-rtpc "circumcised" and SNO-rtpc332
Was also constructed curve dose response to the introduction of normal mice of different forms rtpc produced in the cells of AIDS to navigation (Aton-rtpc153, SNO-rtpc "circumcised" and SNO-rtpc332as described in the caption to Fig. 29. This drawing shows that all tested forms of molecules from cells SNO stimulate the production of platelets, but that the form of the full length of 70 Kda is most active in vivo.
16. General recombinant drug and ligand rnpl
The mpl ligand, preferably, obtained according to conventional recombinant methods, which include the production of the polypeptide ligand mpl, by culturing cells transfected for expression of the nucleic acid ligand mpl (usually by transferowania cell expression vector) and allocation polypeptides cells. But not necessarily predictable that the mpl ligand can be obtained by homologous recombination or methods of producing recombinant, utilizing control elements introduced into cells already containing DNA encoding the mpl ligand. For example, a strong promoter/amplifying element, an inhibitor or an exogenous element, modulating transcription, can be introduced into the genome of the alleged host cells in sufficient proximity and orientation to influence the transcription of DNA encoding the desired polypeptide ligand mpl. The control element does not encode the mpl ligand, rather, DNA is similar in nature to the genome of the host cell. The next element eliminates cells that creates the receptor polypeptide of the present invention, or to increase, or to decrease the level of expression of the desire.
Thus, the invention regards a method of producing mpl ligand, including the introduction of modulatory element transcription in the genome of cells containing in the molecule a nucleic acid ligand mpl, in sufficient proximity and oriented molecule of nucleic acid, to influence their transcription, with a further optional step, comprising culturing cells containing a modulatory element transcription and nucleic acid molecule. The invention also considers the host cell, terzidou natural molecule nucleic acid ligand mpl, operable linked to exogenous control sequences recognized by a host cell.
A. isolation of DNA that encodes a polypeptide ligand mpl
DNA encoding a polypeptide ligand mpl, can be obtained from any cDNA library derived from tissue suspected on the contents of the mpl ligand and expressing it on detektiruya level. Gene mpl ligand can also be obtained from a genomic DNA library or by oligonucleotide synthesis in vitro from full nucleotide or amino acid sequences.
The library was screened with probes designed to identify the gene of interest or the protein encoded by them. For libraries expressed cDNA suitable probes include monoclonal or polyclonal antibodies that recognize and specifically associated with the mpl ligand. For cDNA libraries, suitable probes include oligonucleotides of about 20-80 bases in length that encode known or suspected areas of cDNA mpl ligand from the same or different species; and/or complementary or homologous cDNA or fragments thereof that encode the same or a similar gene. Suitable probes for screening cDNA libraries include, but are not limited to, oligonucleotides, cDNA, or fragments thereof that encode the same or a similar gene, and/or homologous genomic DNA is whether their fragments. The screening of cDNA or genomic library with the selected probe may be conducted using conventional procedures, as described in paragraphs 10-12, Sambrook et al., ibid.
An alternative method for isolation of the gene encoding the mpl ligand, is the technique of the ROC, as described in section 14, Sambrook et al., ibid. This method requires the use of oligonucleotide probe, which hybridizes with DNA encoding the mpl ligand. Strategies for selecting the nucleotides described below.
The preferred practice of the method of the present invention is the use of a careful selection of the nucleotide sequences to screen cDNA libraries from various tissues, preferably human or kidney pig (adult or embryonic), or lines of liver cells. For example, a cDNA library of the cell line of embryonic human liver was screened with oligonucleotide probes. Alternatively, it may be screened with oligonucleotide probes genomic library of a man.
Oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous to minimize false positive responses. Effective nucleotide sequence(s) are usually developed based on the areas of mpl ligand having the least redundant codon. Oligonuclear the IDA can be obtained from one or many locations. The use of the nucleotides is especially important when screened a library of form, which is preferably used codon is unknown.
Oligonucleotide must be marked so that it can be detected by hybridization with DNA screening libraries. The preferred method of labeling is to use ATP (namely, γ32P) and polynucleotide kinase to include radioactive labels to the 5’ end of the oligonucleotide. However, there may be used other ways of labeling oligonucleotides, including, but not limited to, biotinylation or enzymatic labels.
Of special interest is a nucleic acid ligand mpl, which encodes the complete polypeptide ligand mpl. In some preferred embodiments the nucleic acid sequence includes the natural signal sequence of mpl ligand. Nucleic acid having all the sequences of the codons of the protein, obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence.
C. Amino acid sequence variants of the natural ligand mpl
Amino acid sequence variants of the mpl ligand obtained by introducing appropriate nucleotide changes into the DNA of mpl ligand or by synthesis in itro desired polypeptide ligand mpl. Such variants include, for example, the form of deletion, or insertion, or substitution of residues within the amino acid sequence of mpl ligand pigs. For example, part of the carboxy-end of the Mature ligand mpl full length can be removed by proteolytic cleavage in vivo and in vitro, or by cloning and expression of a fragment or DNA encoding the mpl ligand full length for obtaining biologically active variant. Provides for the use of any combination of deletions, insertions and substitutions to achieve the final design to the final design showed the desired biological activity. The amino acid changes also may alter post-translational processes of the mpl ligand, such as change the order or position glycosylases sites. To develop options for the amino acid sequence of mpl ligand position of the site of mutation and the nature of the mutation will depend on the modification features (characteristics) of the mpl ligand. The sites of mutation can be modified individually or in series, for example, by: (1) first, change the selected conservative amino acids and then more radical selected depending on the achieved result, (2) deleting the target residue, or (3) inserting residues of the same class or another, adapted to the plot, or comb the nation elections 1-3.
The method is useful for identification of certain residues or regions of the polypeptide ligand mpl, which is preferred for localization of mutagenesis is called "scanning mutagenesis of alanine, as described by Cunningham and Wells, Science, 244: 1081-1085 . Here, a residue or group of target residues were identified (namely, charged residues such as arg, ASP, GIS, Lys, and Glu) and replaced by any, but preferably neutral or negatively charged amino acid (most preferably alanine or polyalanine) to influence the interaction of amino acids with the surrounding aqueous environment inside or outside cells. Then those domains, demonstrating functional sensitivity to the substitutions, improved by further introduction or other variants of domains or sites for a replacement. Thus, despite the fact that the plot for the introduction of variations in the amino acid sequence is predetermined, the nature of the mutation per se (as such) does not require to be defined. For example, to improve the performance of mutations in a given area carried out the scan Ala(Nina) or performed random mutagenesis at the target codon or region and the expressed variants of mpl ligand was screened for the optimal combination of desired activity.
There are two basic possibilities of variation in the design of aminotic is now sequence variants: the location of the site of mutation and the nature of the mutation. For example, variants of the polypeptide ligand mpl options include forms the sequence of mpl ligand, and may represent naturally occurring alleles (which will not require manipulation of DNA ligand mpl)or predetermined mutant forms obtained by DNA mutation engine, or to achieve alleles, or not found in nature option. In General, the location and nature of the selected mutation will depend on the modified characteristics of the mpl ligand.
Amino acid sequence deletions generally range from about 1 to 30 residues, more preferably from about 1 to 10 residues, and they are usually related. Alternatively, the amino acid sequence deletions may also include part or all of the carboxy-terminal glycosylated domain. Amino acid sequence deletions may also include one or more of the first 6 amino-terminal residues of the Mature protein. Amino acid sequence deletions, optionally, comprises one or more residues in the loop, which is "nodes spiral". Related deletions are usually in even numbers of residues, but singular or odd numbers deletions are within this framework. For modification of the activity of mpl ligand can be introduced deletions in the region of low homology between the ligand and the mpl, which accounts for the greater part of the identical sequence. Or deletions can be introduced into the region of low homology between the polypeptide ligand mpl person and mpl ligand other mammals that have the greatest sequence identity to the mpl ligand person. Deletions of the polypeptide ligand mpl mammal in the region of significant homology with the mpl ligand of another mammal will be more likely to be more significantly modify the biological activity of mpl ligand. The number of sequence deletions will be selected so as to maintain the Quaternary structure of mpl ligand in an domain, for example, beta-folded or alpha-helical.
Amino acid sequence insertions include amino - and/or carboxy-terminal fusions, varying in length from one residue to a polypeptide containing a hundred or more residues, as well as insert inside a sequence of one or more amino acid residues. Insert into the sequence (i.e. insertions within the Mature sequence of mpl ligand) may vary in size, generally from about 1 to 10 residues, more preferably from 1 to 5, most preferably from 1 to 3. Exemplary preferred merger consists of a mpl ligand or its fragment and another cytokine or its fragment. On the specimens of the end inserts include Mature mpl ligand with N-terminal methionine residue, the artifact of the direct expression of the Mature ligand mpl in recombinant cell culture, and fusion of a heterologous N-terminal signal sequence to the N-end of the Mature molecule ligand mpl to enhance secretion of the Mature mpl ligand from the recombinant host. Such signal sequences are usually obtained from and due to homology with the assumed form of the host cell. Suitable sequences include STII or Ipp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells.
Other options inserts molecule ligand mpl include the fusion to the N - and C-ends of ligada mpl immunogenic polypeptides (namely, not endogenous to the host, which this fusing tasks is introduced, for example, a bacterial polypeptide, such as beta-lactamase, or an enzyme encoded by the trp locus of E. coli, or yeast protein, and C-terminal fusing tasks with proteins having a long duration half-life, such as the constant region of the immunoglobulin (or another region of the immunoglobulin, albumin, or ferritin, as described in WO 89/02922 published on 6 April 1989
The third group of variants is a variant of amino acid substitutions. These variants have at least one remote amino acid residue in the molecule ligand mpl, and a different residue inserted in its place. Stations naibolshim is of interest for substitutional mutagenesis include sites, identified as active areas(OK) ligand ml, and sites where the amino acids found in other analogues are significantly different in terms of the amount of the side chain, charge, or hydrophobicity, but where there is a high degree of identity in a selected area, among the mpl ligands in different species, and/or inside one of the mpl ligand various members of the loved ones animals.
Another area of interest is one in which particular residues of the ligand mpl received from various family members and/or animal species, identical with one member. These sites, especially those drawn from the sequence of at least three other identical conservative areas, replaced by a relatively conservative manner. Such conservative substitutions are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then introduced a more substantial replacement designated as exemplary substitutions in Table 3, or as described below in reference to classes of amino acids, and the resulting products had been sifted.
Substantial modifications in function or immunological identity of the mpl ligand was accompanied by selecting substitutions that differ significantly in their effect on maintaining (a) structure the market fundamentals of the polypeptide in the area of the substitution, for example, in the form of folds or helical conformation, (b) the charge or hydrophobicity of the molecule at the target area, or (b) the volume of the side chain. Naturally detectable residues are divided into groups based on common properties of the side chain:
(1) hydrophobic: norleucine, Met, Ala, Val, Dei, Il;
(2) a neutral hydrophobic: Cys, Ser, Tra;
(3) acidic: ASP, Glu;
(4) basic: ASN, GLn, GIS, Lys, Arg;
(5) residues that influence chain orientation: Gli Pro;
(6) aromatic: TRP, Tyr, and hair dryers.
Non-conservative substitutions will entail replacement of the members of one of these classes on the other. Such substituted residues may also be introduced in the areas of conservative substitutions or, more preferably, in the remaining (non-conservative) areas.
In one embodiment of the invention is desirable to inactivate one or more sites of cleavage by a protease that is present in the molecule. These areas were identified by checking the coding amino acid sequences in the case of trypsin, for example, residues of arginine or lysine. When the sites of cleavage by the protease identified and turned into inactive for proteolytic cleavage by replacing the target residue to another residue, preferably a basic residue such as glutamine, or hydrophobic residue, such as serine; by UD the population balance or by insertion of a Proline residue immediately after the rest.
In another embodiment of any methionine residues, other than the initial methionine residue signal sequence, or any residue that is located inside the three N-terminal or C-terminal residues with respect to each such residue methionine is replaced by another residue (preferably in accordance with Table 3) or deleted. Alternatively, about 1-3 residues introduced in such close proximity to the site.
Any cysteine residues not involved in maintaining the proper conformation of the ligand mpl, can also be replaced, usually to serine, to increase the stability of the molecule oxidation and prevent aberrant cross-linking. It was found that the first and fourth cysteine in the VC domain, numbered from the amino end, necessary to maintain the proper conformation, but that the second and third do not. Accordingly, the second and third cysteine in VC domain can be replaced.
The nucleic acid molecules encoding amino acid sequence variants of the mpl ligand, has received a variety of ways known in the methodology. These methods include, but are not limited to selection from a natural source (in the case of naturally occurring amino acid sequence variants) or by obtaining mediated oligonucleotide (or site-directed) mutagenesis, Mut the Genesis of the ROC, and cassette mutagenesis previously obtained variant or invariant version of the polypeptide ligand mpl.
Mediated by oligonucleotide mutagenesis is a preferred method for obtaining DNA variants of mpl ligand with the substitution, deletion and insertion. These methods are well known in the art, as described by Adelman et al., DNA, 2: 183 . In brief, DNA ligand mpl changed by hybridization of the oligonucleotide encoding the desired mutation to a DNA matrix, where the matrix is single-stranded form of a plasmid or bacteriophage containing neizmennoy or native DNA sequence of mpl ligand. After hybridization used DNA polymerase to synthesize a full second complementary thread to the matrix, which will, thus, enable the oligonucleotide primer, and will code a selected change in the DNA ligand mpl.
In General, used the oligonucleotides of at least 25 nucleotides in length. The optimal nucleotide will be from 12 to 15 nucleotides that is fully complementary to the matrix on either side of the nucleotide(s)coding for the mutation. This gives confidence that the nucleotides hybridize properly to single-stranded DNA molecule matrix. Nucleotides are easily synthesized using methods known in the art, such as described by Crea et al., Proc. Natl. Acad. Sci. USA, 75: 5765 .
The template DNA may be the ü obtained using those vectors, which are either derived from bacteriophage vector M13 (applicable commercially available vectors MTR and MTR), or those vectors that contain a single-stranded phage replication as described by Viera et al., Meth. Enzymol., 153: 3 . Thus, the DNA will mutate, can be inserted into one of these vectors to create a single-strand of the matrix. Obtaining single-stranded matrix described in Sections 4.21-4.41 Saiabrook et al., Molecular Cloning: A Laboratory Mannual (Cold Spring Harbor Laboratory Press, NY 1989).
Alternatively, a matrix of single-stranded DNA can be obtained by denaturirovannyj double-strand DNA plasmids (or other)using conventional techniques.
To change the natural DNA sequence (for amino acid sequence variants, for example) nucleotide hybridized with single-stranded matrix under suitable hybridization conditions. Then add the enzyme polimerizuet DNA, usually Klenow fragment of DNA polymerase I for the synthesis of complementary strands of a matrix, using nucleotides as primers for synthesis. Thus, formed heteroduplex molecule, such that one strand of DNA encodes the mutated form of mpl ligand, and the other strand (the original matrix) encodes a natural, unaltered sequence of mpl ligand. Then heteroduplex molecule transformed into a suitable host cell, usually procure is the such as E. coli JM101. After growing the cells, they were transferred onto agarose plates and screened using the nucleotide primer, radioactively labeled with 32-phosphate to identify the bacterial colonies that contain the mutated DNA. The mutated region was then removed and placed into a suitable vector to obtain protein, mainly the expression vector of the type commonly used to transform a suitable host.
Way, directly above, can be modified so that heteroduplex molecule created in both strands of the plasmid containing the mutation(s). Modification of the following: single-stranded oligonucleotide was senatoriable to single-stranded matrix, as described above. Consisted of a mixture of three deoxyribonucleotides, desoxyephedrine (datf), deoxyribofuranosyl (DSTF) and desoxyepothilone (TTF) with a modified thio-dezoksiribozimov, the so-called DStv-(as) (which may be supplied by Amersham Corporation). This mixture was added to a matrix-oligonucleotide complex. Adding DNA polymerase to this mixture was given strand of DNA that is identical to the matrix, except for the mutated bases. In addition, this new strand of DNA will contain DSTF-(as) instead of dstp, which serve to protect them from cleavage with restriction endonucleases.
After tearing one filament matrix is voiniagovo of heteroduplex using an appropriate restriction enzyme thread matrix can be split by ExoIII nuclease or other appropriate nuclease after area, which contains the site(s)targeted for mutagenesis. Then the reaction was stopped, leaving the molecule, which is only partially single-stranded. Then form a complete double-strand heteroduplex DNA using DNA polymerase in the presence of all four deoxyribonucleotides, ATP and Dnknikaza. This homoduplex molecule can then be transformed into a suitable host cell, such as E. coli JM101, as described above.
DNA encoding the mutant mpl ligand with more than one substituted amino acid, can be produced in one of several ways. If amino acids are located close together in the polypeptide chain, they can be motivovany simultaneously using one oligonucleotide that encodes all of the desired replacement of amino acids. If, however, amino acids are located at some distance from each other (separated by more than about ten amino acids), are much more difficult to obtain a single oligonucleotide that encodes all of the desired changes. Instead, it can be used one or two alternative ways.
In the first method, for each of the replaced amino acids have received a separate oligonucleotide. Then the oligonucleotides at the same time senatoriable to the single-stranded DNA matrix, and the second strand of DNA, which is synthesized on the matrix, will be Kadirova the desired replacement of amino acids.
An alternative method involves two or more cycles of mutagenesis to obtain the desired mutant. The first round is as described for the single mutants: for the matrix used wild type DNA oligonucleotide encoding the first desired replacement(s) amino acids were senatoriable to the matrix and then received heteroduplex the DNA molecule. In the second round of mutagenesis was used mutant DNA obtained in the first cycle of mutagenesis on the matrix. Thus, the matrix contains one or more mutations. Then this matrix was senatoriable oligonucleotide encoding the desired amino acid substitution(s), and now received a strand of DNA encodes the mutation from both the first and second cycles of mutagenesis. This final DNA can be used as a matrix in the third cycle of mutagenesis, etc.
Mutagenesis of the ROC is also suitable for making amino acid variants of the polypeptide ligand mpl. As the following discussion applies to DNA, it is clear that this technique is also applicable to RNA. The technique of the ROC as a whole refers to the following procedures (see Eriich, ibid, section R. Higuchi, p.61-70). When used a small number of the matrix as the starting material in the ROC, it is possible to use primers from the corresponding regions of the template DNA, which slightly differed in sequence, on which I receive relatively large quantities of specific DNA fragments which differed from the sequence matrix only in the positions where the primers differed with the matrix. For the introduction of mutations in DNA plasmids one of the primers were designed to overlap the position of the mutation and the content of the mutation; the sequence of the other primer must be identical to the fragment sequencing opposite strands of the plasmid; but this sequence can be located somewhere far in the DNA plasmids. It is preferable, however, that the sequence of the second primer was located within 200 nucleotides from the first, so that at the end of the full amplificare DNA bound to the primer could be easily sequenced. Amplification of the ROC, using pairs of primers, only describes the results in a population of DNA fragments which differ by the position of the mutation specific primer, and possibly at other positions, because the copy of the matrix to some extent prone to error.
If the ratio of the matrix material of the product is extremely low, there is a huge majority of the fragments of DNA products for incorporation of the desired mutation(s). The material of the product used to replace the corresponding region in the plasmid, which serves as the matrix of the ROC, using the normal DNA technologies. Mutations in separate provisions of the can b is to be introduced at the same time, by using the second mutant primer or the second ROC with different mutant primers and legirovaniem the two end fragments of the ROC at the same time to a portion of a vector in three- (or more) parts of the notes.
In a specific example, mutagenesis of the ROC matrix plasmid DNA (1 µg) was linearizable by cleavage with a restriction endonuclease that has a unique plot of recognition DNA plasmids outside amplificare area. This material, 100 ng added to the ROC mixtures containing buffer ROC, which contains four deoxynucleotides and which is included in the sets GeneAmp® (obtained from Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmol of each oligonucleotide primer, to a final volume of 50 µl. The reaction mixture was covered with 35 ál of mineral oil. The reaction mixture was denaturiruet for 5 minutes at 100°With, quickly placed on ice and then added under a film of mineral oil 1 μl DNA polymerase, Thermus aquaticus (Taq) (5 units/μl, obtained from Perkin-Elmer Cetus). Then the reaction mixture was placed in a DNA Thermal Cycler (obtained from Perkin-Elmer Cetus)programmed as follows:
2 min 55°C
30 sec 72°With, then 19 cycles:
30 sec 94°C
30 sec 55°C
30 sec 72°C.
At the end of the reaction, the vial was removed from temperature cilitator, and the aqueous phase pereneslis new bottle, was extracted with phenol/chloroform (50:50 by volume) and besieged ethanol, DNA was isolated according to conventional methods. This material was immediately subjected to a suitable treatment for inclusion in the vector.
Another way of getting a cassette mutagenesis, based on the technique described by Wells et al., Gene, 34: 313 . The starting material is the plasmid (or other vector)comprising motorway DNA ligand mpl. Identify intended for mutation codon DNA ligand mpl. There must be a special place for restriction endonuclease, on each side of the site(s), specific for mutations. If such sites for restriction does not exist, they must be obtained using the above-mediated oligonucleotide way mutagenesis, to introduce them at the appropriate location in the DNA of the mpl ligand. After that, the restriction sites were introduced into the plasmid, the plasmid was cut off on these areas for their linearization. Using conventional methods, synthesized double-strand oligonucleotide encoding the sequence of the DNA between the restriction sites, but containing the desired mutation(s). Two strands synthesized separately and then hybridized together using conventional methods. This double-strand oligonucleotide is called a cartridge. This cassette is designed to have 3’ and 5’ ends, to the that are compatible with the ends of the linearized plasmid thus, to directly ligitamate plasmid. This plasmid now contains the mutated DNA sequence of mpl ligand.
C. Introduction of nucleic acid in the vector can replicate
Nucleic acid (e.g., cDNA or genomic DNA)encoding a natural polypeptide or variant polypeptide ligand mpl, introduced in can replicate the vector for further cloning (amplification of the DNA) or for expression. Many vectors are available, so the selection of the appropriate vector will depend on: (1) will it be used for DNA amplification or for the expression of DNA, (2) the amount of nucleic acid for introduction into the vector, (3) transforming the vector to the host cell. Each vector contains various components, depending on its function (amplification of the DNA or the expression of DNA) and cells of the host with which it must be compatible. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, sample replication, one or more marker genes, an amplifying element, the promoter and the final transcription of the sequence.
(i) Component signal sequence, the mpl Ligand of the present invention can be expressed not only directly, but also fused with a heterologous polypeptide, predpochtitel is but the signal sequence, or other polypeptide having a specific site for cleavage at the N-end of the Mature protein or polypeptide. In General, the signal sequence may be a component of the vector, or it may be part of a DNA ligand mpl, which is put into the vector. Selected heterologous signal sequence should be such that is recognized and subjected to processing (i.e. cleaved signal peptidase) by the host cell. For the host cells of prokaryotes, which do not recognize, do not expose the signal processing sequence of the natural ligand mpl, the signal sequence was replaced with the selected signal sequence prokaryotes, for example, from the group of alkaline phosphatase, penitsillinazy, Ipp, or leaders resistant to heat enterotoxin II. For yeast secretion the natural signal sequence may be replaced by, for example, the yeast invertase, alpha factor, or acid phosphatase leaders, the leader of glucoamylase .albicans (EP 362179, published 4 April 1990), or the signal described in WO 90/13646, published November 15, 1990, In mammalian cell expression the natural signal sequence (i.e. predpolagavshegosja mpl ligand, which usually leads to the secretion of mpl ligand from its natural mammalian cells in vivo)is to satisfy the recreational, although it may be applicable to other signal sequences mammals, such as signal sequences from other polypeptides of the mpl ligand, or from the same form of mpl ligand other species, signal sequences from mpl ligand, signal sequences from secreted polypeptides of the same or related species, as well as secretory leaders of the virus, for example, gD signal herpes simplex.
(ii) the starting Point of the component replication
Both expressed and cloned, the vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. In General, the cloned vectors this sequence is the only one that enables the vector to replicate independently of the chromosomal DNA of the host and includes the replication origin or offline can replicate the sequence. Such sequences are well known for various bacteria, yeast and viruses. The replication origin of the plasmid pBR322 is applicable for most Gram-negative bacteria, the point of starting plasmid 2 μ applicable for yeast, and various viral start point (SV40, polyoma, adenovirus, VSV or BPV) are applicable for cloning vectors in mammalian cells. In General, the starting point of the components is that replication is not required for expression vectors in mammals (usually can be used a starting point SV40 only because it contains the early promoter).
Most expression vectors are Shuttle vectors, i.e. they are capable of replication in at least one class of organisms, but can be transfected for expression in another organism. For example, the vector is cloned in E. coli and then the same vector was transferrable in yeast or mammalian cells for expression, not even paying attention to the fact whether he is able to independent replication in chromosomes of the host cell.
DNA can also be amplified by introducing into the host genome. This is easily done using the species Bacillus as the host, for example, by including in the vector a DNA sequence that is complementary to a sequence found in the genomic DNA of Bacillus. Transferowania Bacillus vector leads to homologous recombination with the genome and integration into the DNA of the mpl ligand. However, the allocation of genomic DNA encoding the mpl ligand, is more complex than exogenously replicated vector because it requires cleavage by the restriction enzyme to cut DNA ligand mpl.
(iii) the Selection of components of the gene
Expressed and cloned vectors should contain a selection gene, also known breeding marker. This gene encodes a protein required for the air traffic management or growth of transformed host cells in a selective culture medium. The host cells, normal vector containing a selection gene will not survive in the culture medium. Usually selection genes encode proteins that (a) give resistance to antibiotics or other toxins, e.g. ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (C) provide critical nutrients not available from complex environments, such as the gene encoding racemase D-alanine for Bacilli.
One example of a selection scheme that uses drugs to delay the growth of host cells. Those cells that are successfully transformed heterologous gene, Express a protein which imparts resistance to drug drug and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin (Southern et al., J Molec. Appi. Genet., 1: 327 ), mycophenolate acid (Mulligan et al., Science 209: 1422 ) or hygromycin (Sugden et al., Mol. Cell. Biol., 5: 410-413 ). In the three examples above, participate bacterial genes under controlled conditions eukaryotes transfer resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycofenolate acid) or hygromycin respectively.
Other examples of suitable breeding markers for mammalian cells are those that autosmooth identify cellular components to extract nucleic acid ligand mpl, such as dihydrofolate reductase (DHFR) or thymidine kinase. Transformed mammalian cells were placed in a pressure of selection, in which only survive especially adapted transformed cells by capturing the token. The selection pressure on transformed cells, applied by culturing under conditions in which the concentration of selection agent in the medium is successively changed, leads through this amplification of both genes, breeding gene and the DNA that encodes the polypeptide ligand mpl. Amplification is the process by which genes with greater demand for the production of proteins that are critical for growth, are repeated many times in tandem in the chromosomes consistently breeding of recombinant cells. Thus, the synthesized increased the number of mpl ligand from the amplified DNA.
For example, cells transformed by selective DHFR gene, first identified in the cultivation of all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. When using wild-type DHFR received and multiplied the appropriate number of host cells from Chinese hamster ovary (Cho), deficient in DHFR activity, as described Uriaub and Chasin, Proc. Natl. Acad. Sci. USA, 77: 4216 . Then transformi is consistent cells maintained with increasing levels of Mtx. This led to the synthesis of multiple copies of the DHFR gene and, consequently, multiple copies of other DNA containing expressed by vectors, such as DNA encoding the mpl ligand. The technique of amplification may be used with any more or less suitable host, for example, ATS No. CCL61 Cho-K1, despite the presence of endogenous DHFR, if, for example, used a mutant DHFR gene, which is highly resistant to Mtx (EP 117060). Alternatively, the host cells (in particular, the master of the wild type, which contain endogenous DHFR) transformed or transformed together with the DNA sequence, encoding the mpl ligand, wild-type DHFR protein, and another breeding marker such as aminoglycoside 3’ phosphotransferase (aft), can be selected by growing cells in medium containing a selection agent for breeding marker such as aminoglycoside antibiotic, such as kanamycin, neomycin, or G418. Cm. U.S. patent No.4965199.
A suitable selection gene for use in yeast is trp1 gene present in the plasmid Yrp7 (Stinchomb et al., Nature, 282: 39 ; Kingsman et al., Gene, 7: 141 ; or Tschemper efc al., Gene, 10: 157 ). Gene trp1 produces a breeding marker for a mutant strain of yeast lacking the ability to grow in the absence of tryptophan, for example, ATS No. 44076 or RER-1 (Jones, Genetics, 85: 12 ). Damage trp1 in the genome drag is of evich of host cells creates an effective environment for detecting transformation when grown in the absence of tryptophan. Similarly, La-deficient strain of yeast (FTSS No.20622 or 38626) are complemented by known plasmids bearing the gene La.
(iv) Promoter component
Expression and cloning vectors usually contain promoters that are recognizable by the host organism and operable linked with the nucleic acid ligand mpl. The promoters are untranslated sequence located in the reverse direction (5’) to the start codon of a structural gene (generally from 100 to 1000 base pairs), which controls the transcription and translation of a particular nucleic acid sequence, such as the sequence of the nucleic acid ligand mpl to which they are operable linked. Such promoters are usually divided into two classes, induced and constitutive. Induced promoters are promoters that initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of nutrients, or a change in temperature. Currently, there are a large number of promoters recognized by a variety of potential cells masters. These promoters operative concatenate to the DNA encoding the mpl ligand, by removing the promoter from the source DNA by splitting restriction fer entom and enable the selected sequence of the promoter in the vector. And the natural promoter sequences of mpl ligand, and many of the heterologous promoters may be used to direct amplification and/or expression of the DNA of the mpl ligand. However, preferred heterologous promoters, as they generally permit greater transcription and higher expression of mpl ligand compared with the natural promoter of the mpl ligand.
The promoters suitable for use with the owners prokaryotes include β-lactamase or lactose system promoters (Chang et al., Nature, 275: 615 ; and Goeddel et al., Nature, 281: 544 ), alkaline phosphatase promoters or tryptophan (trp) (Goeddel, Nucleic Acida Res., 8: 4057  and EP 36 776) and hybrid promoters such as the tac promoter (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 ). However, applicable to other known bacterial promoters. Their nucleotide sequences are published, thereby enabling the skilled worker operable to sew them to DNA encoding the mpl ligand (Siebenlist et al. Cell, 20: 269 )using linkers or adapters to obtain any desired restriction site. The promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operable sewn to DNA encoding the polypeptide ligand mpl.
Known promoter sequences for eukaryotes. Virtually all eukaryotic genes and EUT AT-rich region, located approximately 25 to 30 bases in the opposite direction from the site where transcription is initiated. Another sequence found 70 to 80 bases in the opposite direction from the start of transcription of many genes is SHSAT region, where X may be any nucleotide. On the 3’ end of many eukaryotic genes contain a sequence of AATAAA, which may be a signal, in addition to the poly And the end of the 3’ end of the coding sequence. All these sequences applicable for insertion into expression vectors eukaryotes.
Examples of suitable sequences of vectors for use with cells of the owners of the yeast include the promoters for 3-phosphoglycerate (Hitzeman et al., J. Biol. Chem., 255: 2073 ), or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Res., 7: 149 ; and Holland, Biochemistry, 17: 4900 ), such as enolase, glyceraldehyde-3-phosphatedehydrogenase, glucokinase, piruvatcarboksilazy, phosphofructokinase, glucose-6-fortismere, 3-phosphoglyceromutase, piruwatkinaza, triazolopyrimidine, phosphoglucomutase and glucokinase.
Other yeast promoters, which are induced promoters having the additional advantage of transcription controlled by growth conditions, are the promoter region for alcoholdehydrogenase is 2, sociogram C, acid phosphatase, digestive enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphatedehydrogenase and the enzymes responsible for the utilization of maltose and galactose. Suitable vectors and promoters for use in gene expression in yeast are further described in Hitzeman et al., EP A. Amplifiers yeast also have an advantage when used with yeast promoters.
Transcription of mpl ligand from the vectors in host cells of the mammal is controlled, for example, by promoters obtained from the genomes of viruses such as virus polyoma, the smallpox virus in poultry (Patent UK 2211504, published 5 July 1989), adenovirus (such as adenovirus 2), the virus bovine papilloma virus sarcoma birds, cytomegalovirus, a retrovirus, hepatitis b virus and most preferably simian vacuolating virus 40 (SV 40), from heterologous mammalian promoters, e.g. the actin promoter or the promoter of the immunoglobulin promoters thermal stability, and from the promoter normally associated with the sequence of mpl ligand, provided such promoters are compatible with the systems of the host cell.
Early and late promoters of SV40 virus received an accepted way in the form of restriction fragments SV 40, which also contain a viral point n the early replication of SV 40. Fiers et al. Nature, 273: 113 ; Mulligan and Berg, Science, 209: 1422-1427 ; Pevlakis et al., Proc. Natl. Acad. Sci. USA, 78: 7398-7402 . The immediate early promoter of human cytomegalovirus received an accepted way in the form of a HindIII E restriction fragment. Greenaway et al. Gene, 18: 355 to 360 above . The system of the expression of the DNA of the mammal host, using the virus bovine papilloma as a vector described in U.S. Patent No.4419446. Modification of this system is described in U.S. Patent No.4601978, see also Gray et al., Nature, 295: 503-508  about the expression of cDNA encoding immune interferon in cells of monkeys; Reyes et al., Nature, 297: 598-601  about the expression of cDNA (3-human interferon in mouse cells under the control of timeintensive promoter from herpes simplex; Canaani and Berg, Proc. Natl. Acad. USA, 79: 5166-5170  about gene expression β1-human interferon in cell culture mouse and rabbit; and German et al., Proc. Natl. Acad. Sci. USA, 79: 6777-6871  on the expression of the bacterial CAT sequences in CV-1 cells, monkey kidney, fibroblasts of chicken embryo, cells of the Chinese hamster ovary, cells HeLa cells and NIH-3T3 mouse, using the repetition of the long end of the rous sarcoma virus in the form of the promoter.
(v) Component amplifier
Transcription of DNA encoding the mpl ligand of the present invention, higher eukaryotes often increase through the introduction of amplification of a sequence in the vector. Enhancers are CIS-dei is adequate DNA elements, usually about from 10 to 300 base pairs that act on a promoter to increase its transcription. Amplifiers are relatively oriented and independent position detecting 5’ (Laimins et al., Proc. Natl. Acad. Sci. USA, 78: 993 ) and 3’ (Lusky et al., Mol. Cell Bio., 3: 1108 ) localization of the transcription unit, within an intron (Banerji et al., Cell, 33: 729 ), as well as in the coding sequence (Osborne et al., Mol. Cell Bio., 4: 1293 ). Many of amplifying sequences from mammalian genes are now known (globin, elastase, albumin, α-fetoprotein and insulin). Usually, however, use the amplifiers from viruses of eukaryotic cells. Examples include the amplifier SV 40 late field position to start replication (base pairs 100-270), the amplifier of the early promoter of the cytomegalovirus, the amplifier polimi of the late field position to start replication and adenovirus enhancers. Cm. also Yaniv, Nature, 297: 17-18  the amplifying elements for activation of eukaryotic promoters. The amplifier can be embedded in the vector 5’ or 3’ position to the coding sequence of mpl, but preferably is located in the 5’ segment from the promoter.
(iv) Transcription component termination
Expressing the vectors used in the cells of the owners eukaryotes (yeast, fungi, insect, plant, animal, human or nucleated cells from other mnogokletochnogo) will also contain sequences necessary to stop transcription and for stabilizing the mRNA. Such sequences of 5’, sometimes 3’ untranslated regions of eukaryotic or viral DNA or RNA is widely available. These areas contain segments of nucleotides transcribed in the form of fragments of polyadenylation in the untranslated portion of the mRNA that encodes the ligand mpl.
(vii) Design and analysis of vectors
Construction of suitable vectors containing one or more of the above components, includes the usual technique of ligation. Selected plasmids or DNA fragments splintered, got tails and connected in the form desired to obtain the desired plasmids.
For analysis and validation sequences in the constructed plasmids used ligation mixture to transform E. coli K12 strain 294 (ATSS No.31446)and successful transformants, where necessary, were selected for resistance to ampicillin or tetracycline. Plasmids obtained from transformants was analyzed by cleavage with restriction endonucleases, and/or sequenced by the method of Messing et al., Nucleic Acids Res., 9: 309 , or by the method of Maxam et al., Methods in Enzymology, 65: 499 .
(viii) Transient expression vectors
Particularly useful in the practice of the present invention are vectors, which are provided for transient expression of DNA in mammalian cells encodes a polypeptide ligand mpl. In General, transient expression involves the use of expression vectors that are able to effectively replicate in host cells, so that the host cells have accumulated the downregulation of multiple copies of the vector and, in turn, synthesized high levels of a desired polypeptide encoded by expressed by the vector. Sambrook et al., ibid, str-16.22. Transient expression system containing a suitable expression vector and the host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNA, as well as the rapid screening of such polypeptides with the desired biological or physiological properties. Thus, the transition expression is particularly useful in the invention for purposes of identifying analogs and variants of the polypeptide ligand mpl, which have the biological activity of the polypeptide ligand mpl.
(ix) Examples of convenient vector cells of vertebrates
Other methods, vectors, and host cells suitable for synthesis of ligand mpl in recombinant cell culture, vertebrates are described in Gething et al., Nature, 293: 620-625 ; Mantel et al., Nature, 281: 40-46 ; Levinson et al., EP 117060; EP 117058. A particularly useful plasmid for the expression of mpl ligand in the culture of mammalian cells is pRK5 (EP 307247; U.S. Patent No.5258287) or pSVI6B (PCT Publication No. WO 91/08291).
D. From the PRS and transformation of host cells
Suitable host cells for cloning or expression vectors in this paper are prokaryotes, yeast or higher eukaryotes, as described above. Suitable prokaryotes include eubacteria, such as Gram-negative or Gram-positive, for example, E. coli, Bacilli such as .subtilis, Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serrata marcescans. One preferred cloning host E. coli is E. coli 294 (ATSS No.31446), although applicable also to other strains, such as E. coli B, E. coli X1776 (ATSS No.31537) and E. coli W3110 (ATSS No.27325). These examples are illustrative rather than limiting. The preferred host cell must secrete minimal amounts of proteolytic enzymes. Alternative, applicable methods of cloning in vitro, for example the ROC or the reactions of other polymerases nucleic acids.
In addition to prokaryotes suitable hosts for encoding the mpl ligand vectors are prokaryotic microbes such as filamentous fungi or yeast. The most commonly used among lower eukaryotic hosts microorganisms are Saccharomyces cerevisiae, or common Baker's yeast. However, a large number of other genera, species, and strains are commonly available and applicable here, such as Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 ; EP 139383, published 2 may 1985), in the form of Kluyveromyces hosts (U.S. Patent No.4943529), yet is how, e.g., K. lactis (Louvencourt et al., J. Bacteriol., 737 ), K. fragilis, K. bulgaricus, K. termotolerans, and K. marxianus, yarrowia [EP 402226], Pichia pastoris (EP 183070; Sreekrishna et al., J. Basic Micribiol., 28: 265-278 , Candida, Nrichoderma reesia (EP 244234), and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 ; Tiburn et al., Gene, 26: 205-221 ; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 ) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479 ).
Appropriate cell hosts for the expression of glycosylated ligand mpl come from multicellular organisms. These cells are the masters capable of sophisticated processing and glycosylase activity. In principle, any culture of the cells of higher eukaryotes is healthy, whether it's culture vertebrates or invertebrates. Examples of invertebrate cells include plant and insect cells. Identified numerous strains and variants of baculoviruses and the appropriate cell hosts permissive (allowing) insects from such owners, as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombix mori. See, for example, Lukow et al., Bio/Technology, 6: 47-55 ; Miller et al., Genetic Engineering, Setlow et al., eds., Vol.8 (Plenum Publishing, 1986) pp.277-279; Maeda et al., Nature, 315: 592-594 . Freely available a variety of viral strains for transfection, for example, the L-1 variant of Autographa californica NPV and the strain Bm-5 Bombix mori NP, such viruses can be used here as viruses in accordance with the present invention, particularly for transfection of cells Spodoptera frugiperda.
Can be used as hosts for the culture of plant cells of cotton, maize, potato, soybean, Petunia, tomato and tobacco. Typically, plant cells transferout by incubation with certain strains of the bacterium Agrobacterium tumefaciens, which has previously processed for DNA content of mpl ligand. During incubation of the plant cell culture with A. tumefaciens, the DNA encoding the mpl ligand, is transferred into the plant host cell so that it becomes transfected, and will, under certain conditions, to Express the DNA of the mpl ligand. In addition, the available regulatory and signal sequences compatible with plant cells, such as the promoter of nopaline synthetase and a signal sequence for polyadenylation. Depicker et al., J. Mol. Appl. Gen., 1: 561 . In addition, segments of DNA isolated from the field of the opposite direction of the T-DNA 780 gene capable of activating or increasing the levels of transcription expressed in plants genes in recombinant DNA-containing plant tissue, EP 321196, published on June 21, 1989
However, the greatest interest in vertebrate cells, in recent years, the proliferation of cells pozvonochnyh the x in culture (tissue culture) has become a routine method (Tissue Culture, Academic Press. Kruse and Patterson, editors ). Examples of useful cell lines owner of a mammal are line CV-1 monkey kidney transformed by SV40 (COS-7, ATCC CRL 1651); the line of embryonic human kidney (293 or 293 cells, subclavian for growth in suspensions culture, Gracham et al., J. Gen Virol., 36:59 ); kidney cells baby hamster (KSS, ATCC CCL 10); the cells of the Chinese hamster ovary/-DHFR (Cho, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 ); Sertoli cells of the mouse (TM, Mather, Biol. Reprod., 23:243-251 ); cells monkey kidney (CV1 ATCC CCL 70); kidney cells African green monkeys (VERO-76, ATCC CRL-1587); cell cervical carcinoma human (HELA, ATCC CCL 2); cells of the kidney of the dog (MDCR, ATCC CCL 34); cells of buffalo rat liver (BRL, ATCC CRL 1442); cells of the human lung (W138, ATCC CCL 75); the cells of the human liver (ner G2, HB 8065); tumors of the mammary glands of a mouse (MMT 060562, ATCC CCL51); TRI cells (Mather et al.. Annals N.Y. Acad. Sci., 383: 44-68 ); cells, MRC 5; FS4 cells; and a line human hepatoma (ner G2).
The host cells were transferrable and mainly transformed the above-described expression vectors or cloning of the present invention and cultured in conventional nutrient medium, modified accordingly, to induce the promoters, selecting transformants, or amplificatoare genes encoding the desired sequence.
Transfection refers to the capture of the expression vector cell hosaina is regardless encodes or not he actually expressed any sequence. The ordinary skilled worker known for the numerous methods of transfection, such as how to Saro4or electroporation. Successful transfection is recognized when there is any indicator of the operation that this vector is inside the cell.
Transformation means introducing DNA into an organism so that the DNA is capable of replication either as an extrachromosomal element or integrated into the chromosome. Depending on the host cells were transformation uses conventional methods suitable for such cells. Treatment includes calcium using calcium chloride as described in section 1.82 Sambrook et al., ibid, mainly using prokaryotes or other cells which contain substantial barrier of the cell wall. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al. Gene, 23: 315  and WO 89/05859, published June 29, 1989 in Addition, plants can be transferout using ultrasonic treatment, as described in WO 91/00358 published 10 January 1991 For mammalian cells without such cell walls, the preferred method of deposition of calcium phosphate on Graham anad van der Eb, Virology, 52: 456-457 . General aspects of the systems is transformatsii cells of mammalian hosts described Axel in U.S. Patent No.4399216, published August 16, 1983, Transformation of yeast normally held in accordance with the method of Van Solingen et al., J. Bact., 130: 946  and Hsiao et al., Proc. Natl. Acad. Sci. USA, 76: 3829 . However, there may be used other methods for introducing DNA into cells, such as injection engines, electroporation or fusion of protoplasts.
That is, the Cultivation of cells of hosts
Prokaryotic cells used to produce the polypeptide of the mpl ligand of the present invention, cultured in a suitable medium, as described in General in Sambrook et al., ibid.
Cell owners mammals, used to obtain the mpl ligand of the present invention, can be grown in different environments. Commercially available medium such as Ham''s F10 (Sigma), minimal sufficient medium ([MEM], Sigma), RPMI-1640 (Sigma) and modified Dulbecco Wednesday Needle ([DMEM], Sigma) are suitable for culturing the host cells. In addition, can be used as culture media for the host cells of any of the media described in Ham and Walles, Meth. Enz., 58: 44 , Barnes and Sato, Anal. Biochem., 102: 255 , U.S. patent No.4767704; 4657866; 4927762; or 4560655; WO 90/03430; WO 87/00195; U.S. patent Re. 30985; or jointly expected U.S.S.N. 07/592 107 or 07/592 141, both completed on 3 October 1990, the descriptions of which are incorporated herein by reference. Any of these media may be supplemented if necessary with hormones and/or other factors R is one hundred (such as insulin, transferrin or growth factor epidermis), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine or thymidine), antibiotics (such as drug Gentamycin™), trace elements (defined as inorganic compounds usually present at final concentrations in microgramos area), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that must be known to skilled workers. Culture conditions such as temperature, pH and the like, and those previously applied for selection of host cells for the expression will be obvious to the ordinary skilled worker.
The host cells referred to the present invention include cells in culture in vitro as well as animal cells of the host.
F. Determination of the amplified/expressed gene
Amplification and/or expression may be measured by calculating the transcription of mRNA directly in the sample, for example, conventional the Southern blot, northern blot testing (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205 ), the dot blotting (DNA analysis), or in situ hybridization, using appropriate labeled probes based on the sequences described here. Can be used according to CNAE label, the most common radioisotopes, namely32R. However, can be applied to other equipment, such as using a modified Biotin nucleotides for introduction into polynucleotide. The Biotin then serves as the binding site avidin or antibodies, which can be observed by a wide variety of labels, such as radionuclides, fluorescent labels, enzymes or the like. Alternatively, it may be applied antibodies that recognize specific duplexes, including DNA duplexes, RNA duplexes, and hybrid duplexes, DNA-RNA, or DNA duplexes protein. These antibodies, in turn, can be too efficient, and the determination may be carried out where the duplex is contacted with the surface so that upon the formation of duplex on the surface, it is possible to determine the presence of antibodies bound to the duplex.
An alternative for the direct calculation of the expression product of the gene expression may be measured by immunological methods, such as immunohistochemical staining of pieces of fabric and study of cell culture or body fluids. The technique immunohistochemical staining samples of cells are produced, usually with dehydration and fixation, followed by reaction with a labeled antibody specific for the bound gene product, where the labels Oba is but visually detectable, such as enzymatic labels, fluorescent labels, luminescent labels, and the like. Particularly sensitive staining technique suitable for use in the present invention, described by Hsu et al. Am. J. Clin. Path., 75: 734-738 .
Antibodies applicable for immunohistochemical staining and/or research sample fluids may be either monoclonal and polyclonal, and can be obtained from any mammal. Conveniently, when antibodies can be obtained against a natural polypeptide ligand mpl or against a synthetic peptide based on the DNA sequences obtained here, as described further below.
G. Purification of the polypeptide ligand mpl
The mpl ligand is preferably isolated from the culture medium as a secretory polypeptide, although it also can be isolated from the lysate host cells, if the immediate expression is not accompanied by a secretory signal.
When the mpl ligand expressed in other recombinant cell, different in origin from human cells, the mpl ligand is completely free of proteins or polypeptides of human nature. However, you still need to clear the mpl ligand from other recombinant proteins or polypeptides cells to obtain the drug, which essentially homogeneous, as by itself, the mpl ligand. In the first stage cultural the current environment or the lysate was centrifuged to remove particles of residual cells. Then divided the membrane fraction and soluble proteins. Alternatively, it may be used commercially available filter for concentrating proteins (for example, the filter cell Amicon or Millipore Pellicone). The mpl ligand can then be purified from the soluble fraction of proteins from the membrane fraction of the culture lysate according to whether the mpl ligand membrane-associated. According to the mpl ligand was purified from other soluble proteins and polypeptides by vysalivaniya and exchange or chromatographic methods, using different gel matrices. These matrices include: acrylamide, agarose, dextran, cellulose and other adopted for the purification of proteins. Exemplary chromatographic procedures, applicable for protein purification include: immunoaffinity (for example, anti-hmpl ligand Mab), recuperative (e.g., mpl-IgG or protein And Sepharose), chromatography on hydrophobic interaction (GC), for example, ether, butyl, or phenyl Toyopearl), chromatography on a lectin (e.g., Con a-Sepharose, lentil-lectin-Sepharose), split by size (e.g., Sephadex G-75), cation - and anion-exchange column (for example, DEAE or carboxymethyl - and sulfopropyl-cellulose), and liquid chromatography with high-resolution reversed-phase (RP-GHUR) (see, for example, Urdal et al., J. Chromatog., 296:171 , which used two stages OF W is BP for purification of recombinant IL-2 people). Other cleaning steps, optionally, include: precipitation by ethanol precipitation with ammonium sulfate, chromatofocusing, preparative SDS-PAGE and the like.
Options mpl ligand, in which the residues are deleted, inserted, or replaced, received the same manner as the natural ligand mpl, taking into account any significant changes in properties that occur at the options. For example, the preparation of mpl ligand, fused with another protein or polypeptide, such as a bacterial or viral antigen, facilitates purification; can be used immunoaffinity column containing antibody to the antigen adsorption fused polypeptide. Immunoaffinity column, such as column with polyclonal anti-mpl ligand rabbit, can be applied to adsorption version of the mpl ligand by binding it to at least one immune epitope. Alternatively, the mpl ligand can be purified by affinity chromatography using purified mpl-IgG bound to (preferably) immobilized resin such as Affi-gel 10 (Bio-Rad, Richmond, CA) or similar, in a way well known in the art. For inhibition of proteolytic cleavage in the cleaning process can be used inhibitor of proteases, such as phenylmethylsulfonyl (PMSF), and antibiotics may be included to prevent the growth with uchenykh contamination. Experienced in the technique, the employee will realize that the cleaning methods applicable to natural mpl ligand or its variants may require modification for assessing changes-mpl ligand or its variants upon expression in recombinant cell culture.
N. Covalent modifications of the polypeptide ligand mpl
Covalent modifications of the polypeptide ligand mpl included in the scope of the present invention. And natural mpl ligand and amino acid sequence variants of the mpl ligand can be covalently modified. One type of covalent modification included within the scope of the present invention is a fragment of mpl ligand. Fragments of a version of the mpl ligand having up to about 40 amino acid residues can be covalently obtained by chemical synthesis or by enzymatic or chemical cleavage of the polypeptide ligand mpl full length. Another type of covalent modification of the mpl ligand or its fragments is the introduction into the molecule by reaction with the target amino acid residues of mpl ligand or its fragments, organic agent, forming derivative, which is capable of reacting with selected side chain or the N - or C-terminal residues.
Residues of cysteine often react with α-galvanically (and corresponding amines), such as Chloroacetic to the slot or chloracetamide, obtaining carboxymethyl or carboxylatomethyl derivatives. Residues of cysteine also modify in reaction with BROMOTRIFLUOROMETHANE, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetylation, N-alkylamide, 3-nitro-2-pyridyldithio, methyl-2-pyridyldithio, p-chloromercuribenzoate, 2-chloromercuri-4-NITROPHENOL, or chloro-7-nitrobenzo-2-oxa-1,3-diazoles.
Residues of histidine modify in reaction with diethylpyrocarbonate at pH of 5.5 to 7.0 because this agent is relatively specific for the side chain of histidine. Applicable is paraprofessional; the reaction is preferably carried out in 0.1 M cacodylate sodium at pH 6.0.
Lisini and aminobenzene residues react with succinic or other carboxylic acid anhydrides. Deriving from these agents has an effect on the change of the charge of lysine residues. Other suitable agents for obtaining derivatives of aminecontaining residues include imidiately, such as methylphenidate; pyridoxal phosphate; pyridoxal; chlorobromide; trinitrobenzenesulfonic acid; O-methylisoleucine; 2,4-pentandiol and catalyzed transaminase reaction with glyoxylate, being.
Residues of arginine change in the reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butandiol, 1,2-C is clohexane and ninhydrin. Preparation of derivatives of arginine residues requires that the reaction was carried out in alkaline conditions because of the high values of the pKaguanidino functional groups. Furthermore, these reagents may react with the groups of lysine as well as with Upsilon-amino groups of arginine.
You can get especially interesting specific modification of tyrosine residues with the introduced spectral labels into tyrosine residues in the reaction with aromatic diazonium compounds or tetranitromethane. Most widely for the formation of species O-acetyltyrosine and 3-nitro derivatives are N-acetylimidazole, tetranitromethane respectively. To obtain the labeled proteins for use in radioimmunodetection idiot tyrosine residues using125I or131I, which fits the above described method with chloramine-T.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified in the reaction with carbodiimide (R-N=C=N-R’, where R and R’ are different alkyl groups, such as 1-cyclohexyl-3-(morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Moreover, the remains of aspartyl and glutamyl turn in asparaginyl or glutaminyl remains in the reaction with the ammonium ion.
Derivatives with bifunctional agents is applicable to cross-stitching is of Uganda mpl with a water-insoluble matrix substrate or surface for use in the methods of purification of antibodies anti-mpl ligand, and Vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetate)-2-Penilaian, glutaric aldehyde, esters of N-hydroxysuccinimide, for example, esters with 4-azidoaniline acid, homobifunctional imidiately, including esters of disuccinimidyl, such as 3,3’-dithiobis(succinimidylester) and bifunctional maleimide, such as bis-N-maleimido-1,8-octane. Agents for obtaining derivatives, such as methyl-3-[(p-azidophenyl)dithio]propionamide give photoactivated intermediates, which are capable of forming cross-links in the presence of light. Alternatively, the activated water-insoluble matrices such as activated cyanogenmod carbohydrates and activated substrates described in U.S. Patent No.3969287; 3691016; 4195128; 4247642; 4229537 and 4330440, used for immobilization of proteins.
Glutaminyl and asparaginyl remains often desamuduru to the appropriate glutamine and bartiloro residues, respectively. These residues desaminase in neutral or alkaline conditions. Dezaminirovanie form of these residues fall within the scope of the present invention.
Other modifications, including hydroxylation of Proline and lysine, phosphorylation of hydroxyl groups of residues serine or threonine, methylation α-amino group of the side chains of lysine, arginine and histidine is (..Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 ), acetylation of the N-terminal amine, and amidation of any terminal carboxyl groups.
Another type of covalent modification of the polypeptide ligand mpl included in the scope of the present invention is to change the natural pattern of glycosylation of the polypeptide. By altering is meant deleting one or more carbohydrate radicals found in the natural ligand mpl, and/or adding one or more glycosylation sites not found in the natural ligand mpl.
Glycosylation of peptides is usually the N - or C-bound. N-linked refers to the attachment of a carbohydrate moiety to the side chain of the arginine residue. Sequence tripeptides asparagine-X-serine and asparagine-X-threonine, where X represents any amino acid except Proline, are the recognition sites for the enzyme attaching a carbohydrate moiety to the side chain of asparagine. Thus, the presence of both of these Tripeptide sequences in the polypeptide creates a potential glycosylation sites. O-linked glycosylation refers to the attachment of one of the sugars N-atsetilgalaktozamin, galactose, or xylose to hydroxynicotinate, most usually to serine or threonine, although it can use the change 5-hydroxyproline or 5-hydroxylysine.
Addition of glycosylation sites to the polypeptide ligand mpl is conveniently achieved by modifying the amino acid sequence such that it contains one or more serine residues in the above Tripeptide sequences (for N-linked glycosylation sites). Changes can also be made by adding or substitution by, one or more residues of serine or threonine to the natural sequence of mpl ligand (for O-linked glycosylation sites). To facilitate the amino acid sequence of mpl ligand preferably change through changes at the DNA level, particularly by mutations to DNA that encodes a polypeptide ligand mpl at a pre-selected basis, so that those obtained codons will be to translate the desired amino acids. Mutation and DNA can be obtained using the methods described above under the heading "Amino acid sequence variants of the mpl ligand."
Other ways of increasing the number of carbohydrate radicals in the mpl ligand are chemical or enzymatic sewing glycosides to the polypeptide. These methods have the advantage that they do not require the acquisition of the polypeptide in the host cell, which has the capacity for N - and O-linked glycosylation. Depending on the used method of presian the sugar I(a) can be attached to (a) arginine and histidine, (b) free carboxyl group, (C) free sulfhydryl groups such as cysteine, (d) free hydroxyl groups such as serine, threonine, or hydroxyproline, (e) aromatic residue such as phenylalanine, tyrosine or tryptophan, or (f) the amino group of glutamine. These methods are described in WO 87/05330, published on September 11, 1987, and Alpin and Wriston, CRC Crit. Rev. Biochem., pp.259-306 .
Removal of the carbohydrate moiety present in the polypeptide ligand mpl, can be accomplished chemically or enzymatically. Chemical deglycosylation requires keeping polypeptide with the compound triftormetilfullerenov acid or equivalent connection. This treatment results in the cleavage of most or all sugars except the articulated sugar (N-acetylglucosamine or O-atsetilgalaktozamin), leaving polypeptide neizmennym. Chemical deglycosylation described by Hakimuddin et al., Arch. Biochem. Biophys., 259: 52  and Edge et al., Anal. Biochem., 118: 131 . Enzymatic cleavage of carbohydrate residues of the polypeptide can be achieved with the use of various endo - and ectoparasites as described Nhotakura et al., Meth. Enzymol., 138: 350 .
Glycosylation at potential sites of glycosylation can be prevented when using connection tunicamycin as described Duskin et al., J. Biol. Chem., 257: 3105 . Tunicamycin to lock the t formation of protein-N-glycosidic bonds.
Another type of covalent modification of mpl ligand involves sewing polypeptide ligand mpl to one of a variety of non-protein polymers, such as polyethylene glycol, polypropyleneglycol or polyoxyalkylene, according to the methods described in U.S. Patent No.4640835; 4496689; 4301144; 4670417; 4791192 or 4179337. The polypeptides of the mpl ligand, covalently grafted to the above-mentioned polymers, designated here as pegylated interferon (PEG=PEG - polyethylene glycol attached to PEG, approx. translator) polypeptides of the mpl ligand.
It will be clear that will require a certain screening the obtained variants of mpl ligand for the selection of the optimal variant for binding to mpl with immunological and/or biological activity as defined above. You can sift stability in recombinant cell culture or in plasma (e.g., relative to proteolytic cleavage), high affinity to members of the mpl, oxidation stability, ability to secretariats in increased amounts, and the like. For example, changes in the immunological character of the polypeptide of the mpl ligand, such as affinity for a given antibody, is determined by immunoprotein competitive type. Other potential modifications of the properties of the protein or polypeptide, such as oxidative/reductive or thermal stability, hydrophobicity, or play inchiostri to proteolytic degradation, determined by methods well known from the technique.
17. General methods of preparation of antibodies to the ligand mpl
Preparation of antibodies
(i) polyclonal antibodies
Polyclonal antibodies to the polypeptides of the mpl ligand or fragments generally are produced in animals by multiple subcutaneous (s/C) or intraperitoneal (/b) injection of mpl ligand and adjuvant. May be acceptable accession of mpl ligand or a fragment containing the target amino acid sequence to a protein that immunogene the views, which are subjected to immunization, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or trypsin inhibitor from soybeans, using a bifunctional or modifying agent, for example maleimidophenylmethacrylates ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residue), glutaraldehyde, succinic acid anhydride, SOCl12or R1N=C=NR, where R and R1are alkyl groups.
Animals subjected to immunization against the polypeptide of the mpl ligand or fragments, immunogenic conjugates, or derivatives by combining 1 mg or 1 μg of peptide or conjugate (for rabbits or mice, respectively) with 3 volumes complete adjuvant Freund'and intradermal injections of a solution in many places. After a month the animal reimmunized 1/5 Il is 1/10 the original amount of peptide or conjugate with complete adjuvant Freund'a by subcutaneous injection in many places. After 7 or 14 days at an animal take blood, and study on serum antibody titer to the mpl ligand. Animals reimmunized, while the title will not be released on the plateau. Preferably, the animal reimmunized conjugate of the same ligand mpl, but conjugated to a different protein and/or by using different cross-linking reagents. The conjugates can also be produced in recombinant cell culture in the form of a fused protein. Aggregating agents such as alum, is also used to enhance the immune response.
(ii) Monoclonal antibodies
Monoclonal antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical except for possible natural observed mutations that may be present in minor amounts. Thus, modification of the "monoclonal" notes the character of the antibody as not present in the discrete mixture of antibodies.
For example, monoclonal antibodies to the mpl ligand according to the invention can be obtained using the hybridoma method first described by Kohler and Milstein, Nature, 256: 495 , or can be obtained by the method with recombinant DNA (U.S. Patent No.4816576 [Cabilyet al.]).
In the hybridoma method, a mouse or other suitable animal host, such as a hamster, subjected to immunization, according to the above described here, for which yavleniya lymphocytes, producing or capable of producing antibodies that will specifically bind the protein used for immunization. Alternatively, the lymphocytes can immunize in vitro. Cells then fuse with myeloma cells using a suitable fused agent, such as polyethylene glycol, to form cells hybridoma (Coding, Monoclonal Antibodies: Principles and Practice, pp.59-103 [Academic Press, 1986]).
Thus obtained hybridoma cells are sown and grown in a suitable culture medium that preferably contains one or more compounds that inhibit the growth or survival rasplavlennykh, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme gipoksantin-guanidine-phosphoribosyl-transferase (HGPRT [HGPRT] or HPRT [GMT)], the culture medium for the hybridomas typically will include gipoksantin, aminopterin and thymidine (HAT medium), substances which inhibit growth of HGPRT-deficient cells.
Preferred myeloma cells are those that are effectively fused, support stable high level expression of antibody by the selection producing antibodies cells, and are sensitive to a medium such as HAT medium. Among these preferred cell lines myeloma is line myeloma mice, such as derived from the MORSE-21 or the RS-11 tumor mouse available at the Salk Institute Cell Distribution Center, San Diego, California USA, and SP-2 cells available from the American Type Culture Collection, Rockville, Maryland, USA. Cell line the human myeloma and heteromyinae mouse and man is also described as producing monoclonal human antibodies (Kozbor, J. Immunol., 133: 3001 ; Broder et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New York, 1987).
Cultural environment in which to grow hybridoma cells is examined for the production of monoclonal antibodies directed against the ligand mpl. Preferably, the binding specificity of monoclonal antibodies produced by cells of hybridoma is thus or research associate in vitro, such as radioimmunoassay (RIA), or in research on fermentation-sewn immunoabsorbent (ELISA).
The affinity of binding of the monoclonal antibody can, for example, be determined from analysis of Scatchard (Scatchard) no Munson and Pollard, Anal. Biochem., 107: 220 .
After identification of hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity, the clones can be subclinical by procedures limited breeding and cultivation by the usual methods (Goding, ibid.). A suitable culture medium for this purpose includes, for example, the modified Dulbecco Wednesday Needle or medium RPMI-1640. In addition, cells hybridoma can is to grow in vivo in the form of ascitic tumors in animals.
Monoclonal antibodies secreted by the subclones, convenient stand out from the culture medium, ascitic fluid, or serum by conventional methods for purification of immunoglobulins, such as, for example, protein a-Sepharose, chromatography on hydroxylapatite, gelelectrophoresis, dialysis, or chromatography on affinity.
DNA encoding the monoclonal antibodies of the present invention, is easily detected, and sequeiros using conventional methods (e.g., by using oligonucleotide probes that are capable of specific binding to genes encoding the heavy and light chains of an antibody of a mouse). Hybridoma cells according to the invention serve as a preferred source of such DNA. To establish the synthesis of monoclonal antibodies in the recombinant host cell once the selected DNA may be included in the vector expresii, which is then transferout in the cell host, such as COS cells, monkey cells Chinese hamster ovary (Cho or myeloma cells that do not produce otherwise protein immunoglobulin. DNA also can be modified, for example, by substituting the coding sequence for the heavy and light chain constant domain of human rights in the homologous location in the sequence of the mouse (Cabilly et al., ibid.; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81: 6851 ), or by kovalan the aqueous join the immunoglobulin, codereuse the sequence of all or part of the coding sequence nimmanahaeminda polypeptide.
Typically, such nimmanahaeminda polypeptides replace the constant domains of the antibodies according to the invention or their substituted for the variable domains of the combined area of the antibodies according to the invention to create a chimeric bivalent antibody containing one combined plot of antigen with specificity to the mpl ligand, and the other United parcel antibodies having specificity for a different antigen.
Chimeric or hybrid antibodies can also be obtained in vitro using known methods of chemical synthesis of proteins, including those that use cross-linking agents. For example, immunotoxins can be constructed using the exchange reaction with disulfides, or by formation of a thioester linkages. Examples of suitable reagents for these purposes include aminothiols and methyl-4-mercaptopyrimidine.
For use in the diagnosis of antibodies according to the invention will usually be in the state detected by the radical. Detectable moiety can be any one of which is capable of producing, either directly or indirectly, the detected signal. For example, the detectable moiety may be a radioisotope, such as3H,13C,32P,35S or125I fluores youdim or chemiluminescense connection such as fluorescein, isothiocyanate, rhodamine, or luciferin; radioactive isotopic label such as, for example,125I32P,14With or3H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
Any known technique may be applied for separately attaching antibodies to find the radical, including the methods described by Hunter, et al., Nature, 144: 945 ; David, et al., Biochemistry, 13: 1014 ; Pain, et al., J. Immunol. Meth., 40: 219 ; and Nygren, J. Histochem. and Cytochem., 30: 407 .
Antibodies of the present invention can be applied in any known techniques, such as the study of competitive binding, direct, and indirect research type of sandwich, and immunoprecipitation study. Zola, Monoclonal Antibodies: A Manual of Techniques, pp.147-158 (CRC Press, Inc., 1987).
Study competitive binding based on the ability of the standard (which can be a mpl ligand or its immunologically reactive part) to compete with testing a test specimen (mpl ligand) binding with a limited amount of antibody. The number of mpl ligand in the test sample is inversely proportional to the amount of standard that is associated with antibodies. To facilitate determining the amount of standard that will be associated antibodies is usually translated into an insoluble state before or after to the competitive binding so that the standard and the test substance, which binds to the antibody, can be easily separated from the standard and analyte that remains unbound.
Study type sandwich involves the use of two antibodies, each capable of binding to a different immunogenic portions or epitopes defined protein (mpl ligand). In the study type of sandwich test, the sample binds to the first antibody, which is immobilized on a solid substrate, and thereafter a second antibody binds to a test specimen, thus forming an insoluble complex of three parts. David and Greene, U.S. Patent No.4376110. The second antibody may be in itself observed detektivami radical (direct research type of sandwich), or can be measured using an anti-immunoglobulin antibody that observed detektivami radical (indirect research type of sandwich). For example, the first type of study type of sandwich is the study of ELISA, in this case detektivami radical is an enzyme (e.g. horseradish peroxidase).
(iii) Humanized antibodies and human antibodies
Ways with humanized and human antibodies are well known in the art. In General, the humanized antibody has entered into him amino acid residues from a source that is is inhuman. These inhuman amino acid residues are commonly referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially to hold, following the method of Winter and co-authors (Jones et al., Nature, 321: 522-525 ; Reichmann et al., Nature, 332: 323-327 ; Verhoeyen et ai.. Science, 239: 1534-1536 ), by replacing the CDRs (remains constant domain, approx. translator) rodents or sequence of the corresponding CDR sequence of the antibody is human. Thus, such "humanized" antibodies are chimeric antibodies (Cabilly et al., there, where changed substantially less than the corresponding sequence of nonhuman species than an intact human variable domain. In fact, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues (operating framework) are replaced by residues from analogous sites of antibodies rodents.
The choice of the variable domains of a person, both light and heavy, for use with obtaining humanized antibodies is very important to reduce antigenicity. In accordance with the so-called "best-fit" (best match) chain variable domain of antibodies rodent screened against the full library of known sequences of the variable home is a new person. The sequence of the person that is closest to that of the rodent is then accepted as a working framework (FR) for the humanized antibody (Sims et al., J. Immunol., 151: 2296 ; Chotia and Lesk, J. Mol. Biol., 196: 901 ). Other methods use a specific operating limits derive from a coherent sequence of all human antibodies of a particular subgroup of light and heavy chains. The same working frame can be used for several humanized antibodies (Carter et al., Proc. Nati. Acad. Sci. USA, 89: 4285 ; Presta et al., J. Immunol., 151: 2623 ).
Further important that antibodies were humanized with retention of high affinity and other favorable biological properties. To achieve this goal in accordance with the preferred methods humanized antibody obtained by the analysis of the related sequences and different designed humanized products using three-dimensional models of kin and humanized sequences. Three-dimensional models of immunoglobulin widely available in the art and familiar to the skilled worker. Available computer programs that illustrate and represent the possible three-dimensional conformational structures of selected candidate sequences of immunoglobulins. Consideration of these views allows to consider the likely role of the residues in the function is the operation of the proposed sequence of the immunoglobulin, that is, the analysis of residues that influence the ability of the proposed immunoglobulin contact with the antigen. In this direction remains of FR (operating framework) can be selected and combined with consistent and import sequences so as to obtain the desired characteristics of the antibodies, such as increased affinity for the antigen(s)target. In General, the CDR residues that are directly and most substantially involved in influencing antigen binding. For further details, see Application U.S. Serial No.07/934373, filed August 21, 1992, which is part of application serial No.07/715272 filed June 14, 1991
Alternatively, at the present time it is possible to obtain transgenic animals (e.g. mice)that are capable of immunization, of producing a full repertoire of human antibodies in the absence of endogenous production of immunoglobulins. For example, described that the homozygous deletion in the gene region of the compound (JH) the heavy chain of the antigen in chimeric and germ line mutant mice results in complete inhibition of endogenous production of antibodies. Transplantation of the total number of gene germline immunoglobulin in such germ-line mutant mice will result in the production of antibodies person when replacing the antigen. See, for example, Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551-255? ; Jakobovita et al., Nature, 62: 255-258 ; Bruggermann et al. Year in Measurement., 7: 3 . Human antibodies can also be produced in the solidity of the phage libraries (Hoogenboom and Winter, J. Mol. Biol., 227: 381 ; Marks et al., J. Mol. Biol., 222: 581 ).
(iv) Bespecifically antibodies
Bespecifically antibodies are monoclonal, preferably humanized or human antibodies that have the ability to bind at least two different antigens. Ways to get bespecifically antibodies known in the art.
Traditionally, the products of the recombinants bespecifically antibodies based on simultaneous expression of two pairs of immunoglobulin heavy chain - light chain, where the two heavy chains have different specificity (Millstein and Cuello, Nature, 305: 53-539 ). Due to the randomness of the diversity of the heavy and light chains of immunoglobulins these hybridoma (quadroma) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bespecifically structure. Purification of the correct molecule, which is usually performed through stages affinity chromatography, is more than difficult and gives a low yield of product. Similar methods are described in PCT publications No. WO 93/08829 (opublikowany may 13, 1993) and Trauneker et al., EMBO J., 10: 3655-3659 .
In accordance with various and preferred approaches of the variable domains of the antibodies with the desired what pecifically binding (combined sites of antibody-antigen) alloys with sequences of the constant domains of immunoglobulin. Alloy preferably within the constant domain of the heavy chain immunoglobulin comprising at least part of the hinge regions CH2 and CH3. It is preferable to have the first constant region of the heavy chain (SN)containing the site necessary for binding to the light chain representing at least one of splavlenii. DNA encoding fused heavy chain immunoglobulin and, if desirable, the light chain immunoglobulin is injected into a single expression vector and co transferout in a suitable host organism. This allows for more flexibility in OKRUGA reform of mutual relations of the three fragments of the polypeptides in the embodiment, when there are unequal parts chains of three polypeptides in the design, giving optimum output. However, it is possible to introduce the coding sequence for two or three chains of polypeptides in one expression vector when the expression of at least two polypeptide chains in equal relations leads to high yields or when the ratios are of no particular value. In the preferred embodiment of this approach bespecifically antibodies comprise a heavy chain hybrid immunoglobulin single binding specificity in the first handle, and a hybrid pair of heavy chain - light chain immunoglobulin (providing a second binding specificity) in the hand grip. It was found that this asymmetric structure facilitates the separation of the desired bespecifically connections from unwanted combinations of chains of immunoglobulins, as the presence of light chain immunoglobulin in only one half of bespecifically molecules provides an easy way to split. These approaches are described in outstanding application serial No.07/931811, filed August 17, 1992
For further details get bespecifically antibodies see, for example, Suresh et al., Methods in Enzymology, 121: 210 .
(v) Heteroconjugate antibodies
Heteroconjugate antibodies are also included in the scope of the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. Such antibodies, for example, was supposed to target the immune system to unwanted cells (U.S. Patent No.4676980) and for the treatment of HIV infection (PCT publication No.WO 91/00360 and WO 92/00373; EP 03089). Heteroconjugate antibodies can be obtained using the methods of covalent cross-linking. Suitable cross-linking agents are well known in the art and described in U.S. Patent No.4676980 in numerical order methods cross sewing.
IV. Therapeutic use megakaryocytopoiesis protein ligand mpl
Biologically active mpl ligand having hematopoietic effector function, is described here as megakaryocytopoiesis or thrombopoiesis protein (TP), can be used in a sterile pharmaceutical preparation for stimulation megakaryocytopoiesis or thrombopoetin activity in patients suffering from thrombocytopenia, in breach of the products, capture or increased breakdown of platelets. Associated with thrombocytopenia hypoplasia of the bone marrow (e.g., aplastic anemia after hemoterapia or bone marrow transplantation) can be effectively treated by the compounds of the present invention, as well as violations such as dissimilatory intravascular coagulation (DIC=DIC), immune thrombocytopenia (including HIV-induced ITP and non-HIV-induced ITP), chronic idiopathic thrombocytopenia, hereditary anaemia, myelodysplasia, and thrombotic thrombocytopenia. In addition, data megakaryocytopoiesis proteins can be useful in the treatment of myeloproliferative platelet diseases, as well as thrombocytosis after inflammatory conditions and iron deficiency.
The preferred use of megakaryocytopoiesis or thrombocytopathies protein (TP) of the present invention is: mielotoksicskie chemotherapy in the treatment of leukemia or solid tumors, and cynomolgus monkeys) (suppressive myeloid cell line - approx. translator) chemotherapy is ri autologous or allogeneic bone marrow transplantation, idiopathic aplastic anemia, hereditary thrombocytopenia and immune thrombocytopenia.
Still other disorders suitable for treatment megakaryocytopoiesis proteins of the present invention, include defects or damages platelets as a result of taking drugs, poison or activation on artificial surfaces. In these cases, you can use these compounds to stimulate "shedding - end thrombocytopoiesis" new "intact" platelets. For a more complete enumeration of suitable applications, see above, in particular the section (a)-(f) and the references cited here.
Megakaryocytopoiesis proteins of the present invention can be applied by themselves or in combination with other cytokines, hematopoietins, interleukins, growth factors or antibodies for the treatment of the above disorders and diseases. Thus, these compounds can be used in combination with other proteins or peptides having thrombopoetin activity, including G-CSF, GM-CSF, PHIL, M-CSF, IL-1, IL-3, erythropoietin (EP), kit-ligand, IL-6 and IL-11.
Megakaryocytopoiesis proteins of the present invention is prepared in a mixture with a pharmaceutically acceptable carrier. Data therapeutic compositions can be administered intravenously or through the nose or lungs. Compositions that can the same be entered parenterally or subcutaneously at will. The systematic introduction of therapeutic composition should be pyrogen-free and present a parenterally applicable solution, which is considered to pH, isotonicity and stability. These conditions are known to the skilled in the technique of the employee. In short, the dose formulations of the compounds of the present invention are prepared for storage or use by mixing the compounds with the desired degree of purity, with physiologically applicable carriers, excipients or stabilizers. Such materials are nontoxic to recipients at the dosages and concentrations, and include buffers such as phosphate, citrate, acetate and other organic acid salts; antioxidants such as ascorbic acid; peptides or low molecular weight (less than about 10 residues), such as polyalanine, proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, gisagara and other carbohydrates including cellulose or its derivatives, glucose, mannose or dextrins; chelating agents such as EDTA; polyalcohols, xylytol such as mannitol or sorbitol; counterions such as sodium and/or nonionic surfactants (surface is about-active substances), such as tween, Pluronics or polyethylene glycol.
About 0.5 to 500 mg of the compound or mixture megakaryocytopoiesis protein as a free acid or a base, or pharmaceutically acceptable salt is mixed with a physiologically acceptable diluent, carrier, filler, adhesive, binder, preservative, stabilizer, flavor, etc. as provided for conventional pharmaceutical practices. The amount of the active ingredient in this composition is to obtain a suitable dosage in the range of the norm.
A sterile composition for injection can be formulated according to conventional pharmaceutical practice. For example, it may be desirable breeding or suspension of the active compound in a diluent, such as water or naturally occurring vegetable oil like sesame, peanut or cotton, or synthetic fatty diluent, such etiloleat or similar. Can be enabled buffers, preservatives, antioxidants and the like in accordance with accepted pharmaceutical practice.
Suitable examples of long release preparations include semi-permeable the matrices or solid hydrophobic polymers containing the polypeptide, the matrix which represents the views of prisoners in the form of particles, for example captivity is, or microcapsules. Examples of long-term release of matrices include polyesters, hydrogels (for example, poly(2-gidroximetilbenzoat)as described by Langer et al., J. Biomed. Mater. Res., 15: 167-277  and Langer, Chem. Tech., 12: 98-105 , or poly(vinylalcohol)), polylactide (U.S. Patent No. 3773919, EP 58481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 ), neraspadayuschikhsya the ethylene vinyl acetate (Langer et al., ibid.), disintegrating the copolymer of lactic acid and of glycolic acid, such as Lupron Depot™ (injectable microspheres composed of copolymer of lactic acid and of glycolic acid and acetate leuprolide), and poly-D-(-)-3-hydroxipropionic acid (EP 133988).
While polymers such as ethylene-vinyl acetate and a polymer of lactic acid-glycolic acid enable release molecules for over 100 days, certain hydrogels release proteins for shorter time period. When enclosed in capsules proteins remain in the body for a longer time, they can denaturing or aggregate in the aging in humid conditions at 37°leading to loss of biological activity and possible changes in immunogenicity. You can plan a rational strategy for stabilizing protein-dependent mechanisms involved. For example, if the aggregation mechanism, not only is no intramolecular formation of S-S linkages through disulfide interchange, the stabilization can be achieved by modifying sulfhydryl residues, lyophilisation from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer composition of matrices.
Long-releasing composition megakaryocytopoiesis protein also include enclosed in liposomal trap megakaryocytopoiesis protein. Liposomes containing megakaryocytopoiesis proteins, receive such methods: DE 3218121; Epstein et al., Proc. Natl. Acad. Sci. USA, 82: 3688-3692 ; Hwang et al., Proc. Natl. Acad. Sci. USA., 77: 4030-4034 ; EP 36676; EP 143949; EP 142641; Japanese patent application 83-118008; U.S. Patent No.4485045 and 4544641; and EP 102324. Typically, liposomes are unilamellar type, small (about 200-800 angstroms), in which the lipid content is greater than 30 mol.% cholesterol, the selected proportion selected for optimal therapy megakaryocytopoiesis protein.
Dosage will be determined by the supervising physician when considering various known factors that modify the action of drugs, including the severity and type of disease, body weight, sex, diet, time and route of admission, other mechanisms and other relevant clinical factors. Usually day mode will vary from 0.1 to 100 μg/kg of body weight. Preferably, the dosage will vary from 0.1 to 50 µg/kg body weight. More preferred the equipment, the initial dose will vary from 1 to 5 mcg/kg/day. Optionally, the dose level will be the same as for other cytokines, in particular, G-CSF, GM-CSF and FL. Therapeutically effective dosages can be determined by methods of in vitro or in vivo.
It is obvious that any expert on the basis of the previous description and illustrating it with examples, can make and use the present invention in its entirety without any additional descriptions. Therefore, the following working examples describe only the preferred variants of the present invention and should not be construed as a limitation of the invention.
Partial purification of porcine mpl ligand
Depleted platelet plasma collected from normal pigs or anemic pigs with aplasia. Aplasia in pigs induced by irradiation whole body irradiation at 900 centigray (cGy) using a linear accelerator at a flow of energy of 4 MeV. Irradiated pigs within 6-8 days were injected intramuscular injection of Cefazolin. Then the animals under General anesthesia were removed blood in full and this blood was Gaprindashvili and centrifuged at 1800×g for 30 minutes, resulting in a received depleted platelet plasma. Peak megakaryocytopoiesis activity was observed after 6 DN is th after irradiation.
Plasma obtained from irradiated pigs with aplasia, was treated with 4 M NaCl and stirred at room temperature for 30 minutes. The resulting precipitate was removed by centrifugation at 3800 rpm in a Sorvall RC3B, and the supernatant was loaded onto a column of Phenyl-Toyopearl (220 ml), equilibrated in 10 mm NaPO4containing 4M NaCl. The column was washed with this buffer until until And280he reached values less than 0.05, then the column was suirable dH2O. Suirvey peak of protein was diluted dH2O so that its conductivity was 15 ICES, then it was loaded into a column of Blue-Sepharose, equilibrated (240 ml) in PBS. Then the column was washed with 5 column volumes of PBS and 10 mm NaPO4(pH 7,4)containing 2M urea. Proteins were suirable column with 10 mm NaPO4(pH 7,4)containing 2M urea and 1 M NaCl. Suirvey peak Elka were treated with 0.01%by octylglucoside (n-octyl β - D glucopyranoside) and 1 mm each of EDTA and Pefabloc (Boehinger Mannheim) and directly loaded in tandem-connected speakers with CD4 - IgG (Capon REVIEW/ et al. Nature 337: 525-531 (1989)) and mpl-IgG Ultralink (Pierce) (see below). After sample loading, the column with CD4-IgG (2 ml) was removed, and the column with mpl - IgG (4 ml) was washed with 10 volumes each of PBS and PBS containing 2M NaCl, and suirable in 0.1m glycine-HCl (pH of 2.25). Fractions were collected into 1/10 volume of 1 M Tris-HCl (pH 8.0).
Analysis buervenich fractions with mpl-affinity column, carried out by means of electrophoresis in polyacrylamide gel with sodium dodecyl sulfate (LTOs-SDS page) (4-20%, Novex gel) in terms of recovery, revealed the presence of several proteins (figure 5). Proteins that were painted silver, with the greatest intensity, had an apparent molecular mass Mg66000, 55000, 30000, 28000 and 140,000. In order to determine which of these proteins stimulate the proliferation of Ba/F3-mp1 - cell cultures, these proteins were suirable of the gel, as described below in Example 2.
Ultralink affinity column
10-20 mg mpl-IgG or CD4-IgG in PBS was combined with 0.5 g Ultralink resin (Pierce), as described in the manufacturers ' instructions.
Konstruirovanie and expression of mpl-IgG
A chimeric molecule containing the complete extracellular domain of mpl person (amino acids 1-491) and Fc-fragment molecules IgGI person, expressed in 293 cells. cDNA fragment encoding amino acids 1-491 mpl man, were obtained using polymerase chain reaction (PCR) from cDNA of megakaryocytes QMS and sequenced. Clal site was inserted in the 5’-end, and BstEll site was inserted in the 3’-end. This fragment was cloned above IgGl-Fc-coding region in the vector Bluescript between Clal sites and BstEll, after partial hydrolysis of PCR product by the enzyme BstEll, because the DNA that encodes the extracellular domain of mpl, there are two other BstEll site. BstEll site introduced at the 3’-end PCR product mpl was designed so that the Fc-region had the same reading frame, and that the extracellular domain of mpl. This construct was subcloned into the vector PK5-tkneo between Clal sites and Xbal, and transfusional in embryonic cells, human kidney 293 method using calcium phosphate. These cells were collected at 0.4 mg/ml G418 and individual clones were isolated. The expression of mpl-IgG from the selected clones was evaluated by means of people. Fc-specific ELISA. Most vysokodispersnyi clone had the expression level of 1-2 mg/ml mpl-IgG.
Cells, expressible mpl Ba/F3
cDNA corresponding to the complete coding region mpl F, cloned in pPKS-tkneo, which was then linearizable enzyme Notl, and transferrable in IL-3-dependent cell line Ba/Fe by electroporation (1×107cells 9605F, 250V). Three days were selected in the presence of 2 mg/ml G-418. Cells were collected in the form of pools or received individual clones by serial dilution in 96-well plates. Selected cells maintained in RPM1 containing 15% FBS, 1 mg/ml G-418, 20 mm glutamine, 10 mm HEPES and 100 μg/ml Pen-Strep. The expression of mpl P in the selected clones was assessed by fluorescent method of dividing cells (FACS analysis) using rabbit polyclonal antibodies against mpl R.
Analysis for the presence of mpl ligand Ba/F3
Analysis for the presence of mpl ligand was performed, as shown in figure 2. To determine the presence of mpl-ligand from various sources of cells mpl P-Ba/F3 were cultured in medium containing IL-3 for 24 hours at a cell density of 5×105cells/ml in humid is th camera at 37° C in an atmosphere of 5% CO2and air. After growing cells in medium containing IL-3, these cells were planted in 96-well tablets at a density of 50,000 cells in 200 μl medium in the presence or in the absence of diluted samples and were cultured for 24 hours in an incubator for cell cultures. Then in the last 6-8 hours to each well was added to serum-free RPMl-medium (20 μl)containing 1 µci3H-thymidine. After that, cells were harvested in 96-well tablets with filter GF/C, and washed 5 times with water. The filters were analyzed in the counter Packard Top Count in the presence of 40 μl of scintillation fluid (Microscint 20).
Highly purified porcine mpl ligand
The Protocol gel-elution
An equal number affiliatesensor mpl-ligand (fraction 6, allerona with mpl-IgG-column) and 2× buffer laemmli's method was mixed at room temperature in the absence of reducing agent and as quickly as possible was loaded on 4-20% Novex polyacrylamide gel. The sample was not heated. As a control on the adjacent track was performed electrophoresis buffer without ligand. The gel was subjected to electrophoresis at 135 volts for about 2 1/4 hours at 4-6°C. First missed buffer at room temperature. Then the gel was removed from the tray and the plate on one side of the gel was removed.
Replica of the gel to the nitrocellulose was used to track the way. A piece of nitrocellulose, wetted with distilled water, carefully placed upside down on the surface of the gel so that it does not form bubbles. On the nitrocellulose and the plate of the gel did tentative labels for replica can be accurately repositioned after application. After about 2 minutes the nitrocellulose was carefully removed and the gel was wrapped in plastic film and placed in the refrigerator. The nitrocellulose was stained with a dye for full Biorad protein gold (gold) first by mixing 3×10 ml of 0.1% tween 20+0.5 M NaCl+0.1 M Tris-HCl (pH 7.5) for about 45 minutes, and then 3×10 ml of purified water for 5 minutes. Then added a gold dye and left for the development of the painting until the strip in the standards did not become visible. After this replica was rinsed with water, placed on top of the polyethylene film on the gel, gently combined with the estimated labels. On the plate of the gel were marked position standards Novex and held the line that indicates the designated cut. Then the nitrocellulose and a polyethylene film was removed and the gel was cut along these lines with a sharp razor blade. The cuts continued outside lanes of the gel samples so that they could be used to determine the positions of the slices when staining of the gel. After UD is of slices remaining gel was stained with silver and regulations standards and labels were measured. The molecular weight corresponding to the provisions of the cut, was determined on the basis of standards Novex.
12 Slices of the gel were placed in a cell of two devices for electrosurgery Biorad model 422. In these cells used membrane, cut-off molecular weight of 12-14 kDa. As an eluting buffer was used with 50 mm ammonium bicarbonate +0.05% of LTOs (approximate pH of 7.8). One liter of buffer was cooled for approximately 1 hour before using it in a cold room at 4-5°C. the gel Slices were suirable at 10 mA/cell (initial voltage 40 V) in a cold room at 4-6°C. the Elution was performed approximately 4 hours. Then cells were carefully removed and the liquid on top of the Frith was removed by pipette. Then the camera for elution was taken out and all the liquid on top of the membrane were removed with a pipette. The liquid in the membrane were removed by pipette Pipetman and maintained. Then the membrane was added 50 μl of purified water, stirred and removed until until dissolve all the crystals-ordinator. After this wash was combined with the above the stored liquid. The full volume of the sample elution was 300-500 ál of the gel slice. The samples were placed in 10 mm 12-14 To-cut-off dialysis tubing Spectrapor 4, which previously within a few hours were filled with purified water. Dialysis was performed overnight at 4-6°against 600 ml of buffered F. what statom saline (PBS, approximately 4 mm in potassium) for 6 samples. Then the next morning, the buffer was replaced and the dialysis continued for another 2.5 hours. Then, the samples were removed from dualization bags and placed in tubes for microcentrifuge. These test tubes for 1 hour were placed on ice, centrifuged for 3 minutes at speed (rpm) at around 14K and supernatant was carefully removed from the besieged ordinator. These supernatant was placed approximately 1 hour on ice and again centrifuged for 4 minutes. Then supernatant were diluted in saline solution, phosphate buffered, and were analyzed by the activity. The remaining samples were frozen at -70°C.
Microeconomie ligand mpl pigs
Fraction 6 (2.6 ml) of mpl-IgG-affinity column was concentrated on Microcon-10 (Amicon). In order to avoid absorption of mpl ligand on Microcon the membrane was washed for 1% of the LTO and fraction 6 was added 10% - ordinator. After concentration of the fractions 6 on Microcon (20 ml) was added a buffer to the sample (20 μl) (2) and received volume (40 ál) were loaded on one track 4-20% gradient acrylamide gel (Novex). Then the gel was subjected to electrophoresis in accordance with the Protocol Novex. After this pre-electromotively gel was balanced for 5 minutes in 10 mm S-buffer (CAPS=3-(cyclohexylamino)-1-propanesulfonic acid), pH 11/0, containing 10%methanol. Electroblotting on the membrane Immobilon-PSQ (Millipore) Provo is or for 45 minutes at a constant current of 250 mA in the cell for electrophoretic migration (32) BioRad Trans-Blot. PVDF-membrane was stained with 0.1% of Kumasi blue R-250 in 40%methanol with 0.1%acetic acid for 1 minute, after which the dye was washed for 2-3 minutes using 10%acetic acid in 50%methanol. Proteins that were visible in 18000-35000-region blot, had Mr=30000, and 22000 28000.
Bands of proteins 30, 28 and 22 kDa were subjected to sequencing. This was carried out by automated sequencing on the sequencing machine model A (Applied Biosystem)with PTH analyzer, operating in real time. The specified sequencing machine was modified for the introduction of 80-90% of the sample (Rodriguez, J. Chromatogr., 350: 217-225 ). For regulating the optical density in the UV range to the solvent And acetone was added (≈12 µl/l). Electromotively proteins sequenced in the Blott cartridge. Peaks were integrated using the software Justic Innovations using interfaces Nelson Analitycal 970. The interpretation of sequences were performed on the vAX device 5900 (Henzel et al., J. Chromatogr., 404: 41 to 52 ). N-terminal sequence (using the one-letter code; the remains uncertain given in parentheses) and the number of the obtained material (in square brackets) are presented in Table 4.
Analysis megakaryocytes in liquid suspension culture
Peripheral stem cells man (PSC) (obtained with the consent of patients) 5-fold diluted 1M M-medium (Gibco), and centrifuged for 15 minutes at 800×g at room temperature. Cellular precipitate resuspendable 1 MDM and covered with a layer on top of the 60% Percoll (density of 1.077 g/ml) (Pharmacia), and then centrifuged for 30 minutes at 800×g. Mononuclear cell low density was aspirated at the border and washed 2×1 MDM, after which they were sown at a density of (1-2)·106cells/ml in medium 1 MDM containing 30% FBS (final volume 1 ml)in 24-hole tablets for culturing tissue cultures (Costar). Then added (10%) ARR or ARR, not containing mpl ligand, and the cultures were grown for 12-14 days in a humid chamber at 37°C in an atmosphere of 5% CO2and air. Then the culture was incubated in the presence of 10% of the RDA with added 0.5 μg mpl-IgG, was added on days 0, 2 and 4. Remove mpl ligand from the RDA was carried out by passing the RDA through mpl-IgG-affinity column.
To quantify megakaryocytopoiesis in these liquid suspension cultures used the modified method Jong Clemente, etc. and radioactively labeled mouse monoclonal IgG antibody (HP1-1D) to Gpllbllla(supplied Dr.Nichols, Mayo Clinic). 100 mcg HP1-D (see Grant, C. Et al., Blood 69: 1334-1339 ) were subjected to radioactive tagging of 1 MCI of Na125using Enzzmobeads (Biorad, Richmond, CA) according to manufacturer's instructions. Radioactively labeled HP1-1D kept the ri 70° C in PBS containing 0.01% octylglucoside. Typical specific activity was 1-2-10° n.a./min/µg (>95%, precipitated with 12.5%trichloroacetic acid).
Liquid suspension cultures were prepared in triple duplicate for each experimental stage. After 12-14 days of incubation, 1 ml of the cultures was transferred into a 1.5-ml Eppendorf tubes, then centrifuged at 800×g for 10 minutes at room temperature, and the resulting cellular precipitation resuspendable in 100 µl of PBS containing 0.02% of EDTA and 20% calf serum. To resuspending cultures were added to 10 ng125l-HPI-ID 50 ál analytical buffer and incubated for 60 minutes at room temperature (RT), shaking occasionally. After that, the cells were collected by centrifugation at 800×g for 10 minutes at RT and washed 2x buffer for analysis. The cell sediment was placed for 1 minute in a gamma counter for counting (Packard). Nonspecific binding was assessed by adding 1 μg unlabeled HP1-1D, followed by keeping the mixture for 60 minutes before adding labeled HP1-1D. Specific binding was defined as total125l-l-lD-binding minus binding in the presence of excess unlabeled HP1-1D.
Oligonucleotide PCR primers
Based on the amino-terminal amino acids of the second sequence, obtained for proteins of 30, 28 and 18 to 22 kDa, designed degenerate oligonucleotides as PCR primers (PCR-polymerase chain reaction) (see Table 5). Was synthesized two pools of primers: one notional 20-dimensional pool, encoding amino acid residues 2-8 (mpl 1) and antisense 21-dimensional pool, complementary to sequences encoding amino acids 18-24 (mpl 2).
Porcine genomic DNA isolated from lymphocytes of swine peripheral blood was used as template for PCR. 50 µl reaction mixture contained: 0,8 µl of porcine genomic DNA in 10 mm Tris-Hcl (pH 8.3), 50 MM KCl, 3 mm MgCl2, 100 µg/ml BSA, 400 μm dNTP, 1 μm of each primerno pool and 2.5 units of Taq polymerase Tao. Initial denaturation of the matrix was carried out at 94°C for 8 minutes, followed by 35 cycles of 45 sec at 94°C, 1 min at 55°C and 1 min at 72°C. the Final cycle was performed for 10 minutes at 72°C. PCR products were separated by electrophoresis on 12%polyacrylamide gel and visualized by staining with ethidiumbromid. It is reasonable to assume that if the amino-terminal amino acid sequence is encoded by a single exon, the correct PCR product, obviously, is 69 BP DNA fragment of this size was suirable from the gel and was zablokirovali in pGEMT (Promega). Sequences of three clones shown the following:
Porcine genomic DNA fragments with a length of 69 BP
5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC STATEAS TGCCTCGTGA
3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACT
TGACCACGTT CAGCACGGC [69 BP] Th. NO: 37
ACTGGTGCAA GTCGTGCCG .NO: 38
5'CCAGCACCTC CGGCATGTGA CCCCCGACTC STATEAS TGCTTCGTGA
3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT
CGACCACGTC CATCACGGC [69 BP] .NO: 39
GCTGGTGCAG GTAGTGCCG .NO: 40
Gene ligand mpl person
Based on the results obtained as described in Example 5, were designed and synthesized a 45-dimensional deoxyoligonucleotide marked pR45, for screening of the genomic library. The specified 45-Mer had the following sequence:
5’ GCC-GTG-AGG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG-3’ (.NO: 28)
This oligonucleotide was subjected to32R-tagging using (γ32P)-ATP and T4 kinase and used to screen genomic DNA libraries in λdem 12 in stringent conditions of hybridization and washing (see Example 7). Positive clones were collected, plaques were purified and analyzed by restriction mapping and southern blotting. Clone No. 4 was selected for further analysis.
BamHl-Xbal fragment (2,8 kV), which was hybridisable with 45-Merom, was subcloned into the vector pBluescript SK-. Partial DNA sequencing of this clone was performed using oligonucleotide primers specific to the DNA sequence of the porcine mpl ligand. Obtained the selected confirmed that was isolated DNA encoding a human hormone ligand mpl pigs. In this sequence was discovered EcoRl restriction site, which allowed the authors of this proposal to allocate 390 BP-EcoRl-Xbal fragment from 2,8kV discharge-VAT-Xbal fragment to sublimirovanny in the vector pBluescript SK-.
Both strands of the fragment sequenced. Human DNA sequence and deduced amino acid sequence shown in Fig.9 (PEFC. NOS 3 and 4). The predicted position of introns in the genomic sequence is also shown by the arrows and determine the expected exon (exon 3").
The study predicted amino acid sequence confirmed that serine residue is the first amino acid of the Mature mpl ligand, as determined from direct analysis of amino acid sequence. It is assumed that directly above from this codon is the signal sequence that is involved in secretion of Mature mpl ligand. Region encoding a signal sequence, obviously, is interrupted by an intron at nucleotide position 68.
This exon probably ends at nucleotide position 196 in the direction 3`. Therefore, this exon encodes the sequence of 42 amino acids, 16 of which, obviously, are part of a signal sequence, and 26 are part of the Mature ligand inpl h the rights.
Full-size cDNA of mpl ligand person
Based on the sequence of exon 3 (Example 6) was synthesized two nondegenerate oligonucleotide corresponding to the 3`- and 5`-ends of the sequence of exon 3 (table 6).
Non-degenerate people. cDNA oligonucleotide PCR primers
|Prampram: 5'GCT AGC TCT AGA AAT TGC TCC TCG TGG TCA TTC TGC T 3’||.N: 43|
|Obratil: 5’ CAG TCT GCC GTG AAG GAC ATG G-3’||PEFC. NO: 44|
These two primers were designed for polymerase chain reaction, which was performed in the conditions described in Example 5, using as template DNA from different Chelkans libraries or 1 ng cDNA clone (Quick DNA, Clonetech) from various tissues. The expected size of the correct PCR products on a 12%polyacrylamide gel, the DNA fragment of the expected size was detected in cDNA libraries derived from a kidney of an adult individual cells from fetal kidney 293 and DNA derived from fetal human liver (Clonetech cat.№7171-1).
cDNA library from fetal liver DR2(Clonetech cat. No. n 1151 x) skanirovali using the same 45-dimensional oligonucleotide that was used for screening of the genomic library. The oligonucleotide was labeled (γ32R)-Asia-Pacific the BL is reattaching polynucleotide-T4 kinase. The library was skanirovali in hybridization conditions of low stringency. The pre-filters were hybridisable within 2 hours, and then was hybridisable with probe overnight at 42°With 20%formamide, 5×SSC, 10x denhardt's solution, 0.05 M phosphate (pH 6.5), 0.1% sodium pyrophosphate, 50 μg/ml treated with ultrasound sperm DNA, salmon for 16 hours. Then the filters were washed in 2×SSC, and then once in 0.5×SSC, 0,1% - ordinator at 42°C. the Filters were exposed to x-ray film (Kodak) overnight. Positive clones were collected, plaques were purified and the size of the insert was determined by polymerase chain reaction using oligonucleotides flanking BamHl-Xbal-fragment cloned in λ DR2 (Clonetech cat.№6475-1). As the source matrix used 5 ál ragovoy culture. Initial denaturation was performed for 7 minutes at 94°C, followed by 30 cycles of amplification (1 min at 94°C, 1 min at 52°C and 1.5 min at 72°). The final elongation was performed for 15 minutes at 72°C. Clone No. FL2b had a 1.8-to-box and was selected for further analysis.
"Salvation" plasmid pDR2 (Clonetech, DR2 & pDR2 cloning and Expression System Library Protocol Handbook, p.42)contained λ FR-phage shoulders, was carried out as described in the manufacturer's instructions (Clonetech, DR2 & pDR2 cloning and Expression System Library Protocol Handbook, p.29-30). Restriction the initial analysis of the plasmid pDR2-FL2b, carried out using enzymes BamHl and Xbal, revealed the presence of a BamHl restriction site within the insert about the position 650. Hydrolysis of plasmid enzymes BarnHl-Xbal cut the box into two fragments, one of 0.65 kV and one of 1.15 square DNA sequence was determined using matrix three different classes, derived from the plasmid pDR2-FL2b. DNA sequencing the double-stranded plasmid DNA was performed using the automated fluorescent DNA sequencing machine AV (Applied Biosystems, Foster City, California) using standard sequencing using labeled with dye dideoxynucleosides terminator (dye-terminator) and synthesized in the traditional way of primers for "walking on the genome" (Sanger et al., Proc. Nati.Acad.Sci. USA, 74: 5463-5467 ; Smith et ai., Nature, 321: 674-679 ). Direct sequencing of PCR-amplified fragments derived from plasmids was performed using the sequencing machine AS using conventional primers and labeled terminator. Single-stranded matrix was generated using the vector M13 Janus (DNASTAR, Inc., Madison, Wisconsin) (Burland et al., Nucl. Acids res., 21: 3385-3390 ). BamHI-Xbal (1,15 kV) and BamHl (0,65 kW)fragments were isolated from a plasmid pDR2, the ends were built using DNA polymerase T4 in the presence of deoxynucleotides, and then was subcloned into the Smal site M13 Janus. Sequencing was carried out by ACC is accordance with the standard method, involving the use of dye-labeled universal and reverse M13 primers or primers for "walking on the genome and dye-terminators. Conventional non-automatic sequencing was carried out according to standard methods on single-stranded M13 DNA using primers for "walking on the genome and the traditional technology of dideoxy-termination (Sanger et al., Proc. Natl.Acad.Sci. USA, 74: 5463-5467  using32P-labeled α-dATP and Sequenase (United States Biochemical Corp. Cleveland, Ohio). The Assembly of DNA sequences was performed using sequencing machine v2.1bl2 (Gene Codes Corporation. Ann Arbor, Michigan). Nucleotide and deduced sequence hML presented in figure 1 (.NO: 1).
The selection of the gene of mpl ligand person (SRW)
Genomic people. DNA clones of the gene SRW was isolated by screening genomic libraries in X-Geml2 using pR45 previously described oligonucleotide probe, and low stiffness (see Example 7), or in conditions of high stringency using a fragment corresponding to the 3’-half of Chelkans, encoding the mpl ligand (from the BamHl site to the 3’-end). There were two overlapping clone lambda length of 35 to. Two overlapping fragments (BamHl and EcoRl)containing the complete gene SRW, were subcloned and sequenced. The structure of this human gene consists of 6 exons 7 to. Romney DNA (figa, B and C). The borders of all the exon-intron joints corresponded to the consensus element set for mammalian genes (Shapiro M.B., Nucl.Acids Res. 15:7155 ). Exon 1 and exon 2 containing 5-noncoding sequence and the initial four amino acids of the signal peptide. The remaining secretory signal and the first 26 amino acids of the Mature protein is encoded in exon 3. Full carboxy-domain and 3’-noncoding region, and -50 amino acids erythropoietin-like domain coded in exon 6.. Four amino acids included in the deletion observed in hML-2 (hTPO-2), encoded in the 5'-end of exon 6.
Transient expression of mpl ligand person (hML)
In order to sublimirovanny full-sized inserts contained in pDR2-FL2b, plasmid completely hydrolyzed by the enzyme Xbal, and then partially enzyme Bamhl. The DNA fragment corresponding to 1.8 to-insert was subjected to gel purification and was subcloned into pRK5 (pRK5-hmpl 1) (see U.S. Patent No. 5258287 to construct pRK5) under control pretannage promoter of cytomegalovirus. DNA from design pRK5-hmpl 1) received a REG-method and transfusional in human embryonic kidney cells 293, supported in a modified method of Dulbecco environment Needle (DMEM), to which were added: nutrient mixture F-12, 20 mm Hepes (pH 7,4) and 10% fetal bovine serum. Cells were transfiere the Ana calcium phosphate method, as described (Corman, S. , in DNA Cloning: A Practical Approach (Glover, D.M., ed.) Vol.11, pp.143-190, IRL Press, Washington, D.C.). 36 hours after transfection, the supernatant of transfected cells were analyzed for proliferative activity (see Example 1). The supernatant of 293 cells, transfected with only one vector pRK, did not stimulate any of the cells Ba/F3 or cells Ba/F3-mpl (figa). Supernatant from cells transfected with pRK5-hmpl 1, did not give any effect in the cells Ba/F3, but found a significant stimulation of cell proliferation in Ba/F3-mpl (figa), suggesting that this cDNA encodes a functionally active mpl ligand person.
Isoforms hML2, hML3 and hML4 people ligand
To identify forms hML produced as the result of alternative splicing, synthesized primers corresponding to each end of the coding sequence hML.
These primers were used to aplicatii RNA of adult human liver using polymerase chain reaction at room temperature (RT-PCR). In addition, were constructed and similarly used internal primers flanking the desired area (see below). Direct sequencing of the ends of the PCR product revealed a single sequence that exactly matches the sequence of cDNA isolated from a library of fetal human liver (see IG (.NO: 1)). However, the area is located near the end of the EPO-domain (in the middle of the PCR product)was detected complex sequences, suggesting the existence of possible splicing in this region. To highlight these options splicing primers shown in Table 7 and flanking the desired area, were used in PCR as matrices for the cDNA of the adult human liver.
PCR primers for separation of isoforms ML human
|phmpllcdna.31: 5’ TGTGGACTTTAGCTTGGGAGAATG3’||(.NO 45)|
|pbx4.f2: 5’ GGTCCAGGGACCTGGAGGTTTG3’||(.NO 46)|
PCR products were subclinically on blunt ends in M13. Sequencing of individual subclones revealed the existence of at least three isoforms ML. One of them hML (also denoted hML332) is the longest and corresponds exactly to the sequence, selected from a library of fetal liver. Sequence isoforms people mpl ligand from the longest to the shortest (hML-4) shown in figure 11 (.NO 6, 8, 9, and 10).
Construction and transient expression of isoforms of people Mpl ligand and its variants substitution hML2, hML3, and hML (R153A, R154A)
Isoforms hML2 and hML3 and substitution options (options replacement) hML (R153A, R154A) designed from hML using is the use of recombinant PCR technique, described Russell Higuchi, in PCR Protocols, A guide to Methods and Applications Acad. Press, M.A.Innis, D.H.Gelfand, J.J.Sninsky & T.J.White Editors.
All structures flanking the primers were as shown in Table 8, and "overlapping" of the primers were as shown in Table 9.
All PCR-aplicatii was performed using ready-to-use DNA polymerase (Stratagene) under the following conditions: initial denaturation of the matrix was carried out at 94°C for 7 minutes, and then spent 30 cycles: 1 min at 94°C; 1 min at 55°S; 1.5 min at 72°C. the Final cycle was continued for 10 minutes at 72°C. the Obtained PCR product is hydrolyzed in Clal-Xbal-enzymes, subjected to purification on a gel and cloned in pRK5tkneo. The 293 cells were transfusional various constructions described above, and the supernatant from these cells was analyzed on the proliferation of Ba/F3-mpl. In this analysis of hML-2 and hML-3 did not show appreciable activity, but the activity of hML (R153A, R154A) was similar to the activity of hML, which suggests that the activity does not require processing in this dibasic site (smfg).
cDNA of murine mpl ligand, mML, mML-2, inML-3
The selection of cDNA mML
The DNA fragment corresponding to the complete coding region colligenda mpl, were obtained using PCR and then subjected to gel purification and m is the treatment method of random priming in the presence of 32P-dATP and32P-dCTP. This probe was used for screening cDNA libraries from mouse liver in GT10 (Clontech cat. No. m a). Duplicate filters were hybridisable in 35% formamide, 5×SSC, 10× denhardt's solution, 0.1% of LTOs of 0.05 M sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 100 μg/ml treated with ultrasound DNA salmon sperm, in the presence of the probe during the night. Filters were washed in 2×SSC, and then once washed in 0.5×SSC, 0,1% - ordinator at 42°C. Hybridizers phage were purified by the method of plaques, and cDNA inserts were zablokirovali in the EcoRl site of plasmid Bluescript SK-. Clone "LD", containing a 1.5 KB insert, was selected for further analysis and both strands sequenced as described above for people. ML cDNA. Nucleotide and deduced amino acid sequence of clone LD presented on Fig (SEQ ID NOS 1 and 11). Deduced sequence of the Mature ML from this clone had a length of 331 amino acid residues was identified as mL331(or mML-2 reasons below). A significant degree of identity of both the nucleotide and derived amino acid sequence was observed in the EPO-like domain of these ML. However, when comparing the deduced primary amino acid sequences of human and murine ML was found that the mouse sequence has a deletion in the four peptide located between people. mod is AMI 111-114, and the corresponding deletion of 12 nucleotides, located behind the nucleotide at position 618 and observed in both as a human. (see above), and pork (see below) cDNA. In this regard, to detect possible mouse m-l-isoforms were investigated additional clones. One clone "14" had a 1.4 KB insert corresponding to the derived amino acid sequence of 335 amino acids, which was absent tetrapeptide LPLQ. This form, which, obviously, is a full-sized mouse ML, was marked mML or mL335. Nucleotide and deduced amino acid sequence for ML presented at Fig (SEQ ID NOS 12 and 13). And finally, was isolated and sequenced clone "12". This clone had a deletion in 116 nucleotides corresponding .L3, and therefore it was designated ML-3. Comparison of the deduced amino acid sequences of these two isoforms is shown in Fig.
Expression of recombinant mML.
Expressing vectors for murine ML was obtained essentially as described in Example 8. Clones encoding mMl and mML-2, was subclinically a pRK5-tkneo expressing vector mammal, which provides the expression under control of the CMV promoter and SV40 polyadenylation signal. Received expressing vectors. MMlpRKtkneo and mML2pRKtkneo were short transliterowany in 293 cells by the method using calcium phosphate. After kratkofil the Noah transfection medium was kondicionirovanie within five days. Cells were maintained in DMEM with high glucose content and added 10%fetal calf serum.
Expression of the murine mpl (mmpl) in the cells Ba/F3.
Stable cell lines expressing C-mpl, bshi obtained by transfection mmpl-pRKtkneo in much the same way as described in Example 1 for the human mpl. For this expressing vector (20 μg, linearized)containing the full sequence, encoding the mpl (R.C. Skoda et al., EMBO J. 12: 2645-2653 (1993)), was transfusional cells Ba/F3 by electroporation (5×106cells, 250 Volts, 960 μf) with subsequent selection of resistance to neomycin using 2 mg/ml G418. The mpl expression was assessed by flow cytometry using antisera against mouse mpl-IgG. Cells Ba/F3 maintained in the medium RPM1 1640 from cells WEH1-3B used as a source 1L-3. Supernatant from 293 cells, short-transfected with both mML and mMl-2, were analyzed F3-cells, transfected with both mmpl and hrnpl as described in Example 1.
cDNA of porcine mpl ligand, pML and RM-2
cDNA pork ML (pML) were isolated using PACE-PCR. For this purpose were designed olioo dT-primer and 2 specific primers based on the sequence of exon gene pork ML, encoding the amino-end of the ML and isolated from the serum of pigs with aplasia. Then from various tissues aplas the practical pigs received and amplified cDNA. PCR product of cDNA consisting of 1342 BP and detected in kidney tissue was subcloned. Several clones were sequenced, resulting in it was found that they encode the Mature porcine mpl ligand (not including the full signal secretion). It was found that the cDNA encodes 332 amino acids of the Mature protein (pML332)having the sequence shown in Fig (SEQ ID NOS: 9 and 6).
The allocation of the pML gene and cDNA.
Genomic clones of the gene pork ML were isolated by screening genomic libraries pigs in EMBL3 (Clontech Inc.) with pR45. The library was skanirovali method basically described in Example 7. Several clones were isolated and the exon encoding amino acid sequence identical to the sequence obtained from purified ML, sequenced. CDNA pork ML was obtained in accordance with the modified scheme for PAGE-PCR. Starting from the sequence of a porcine gene ML were designed two ML-specific primer. Polyadenylated MRNA was isolated from renal tissue aplastic pigs, as described above. CDNA was obtained by reverse transcription using BamdT-primer:
(BamdT: 5’ GACTCGAGGATCCATCGATTTTTTTTTTTTTTTTT 3’
(Th. NO: 55),
against polyadenine-tail of mRNA. The initial series of PCR amplification (28 cycles for 60 sec at 95°C for 60 sec at 58°and within 90 seconds pri° C) was performed using Mg-specific direct primer
(h-forward-1: 5’ GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3’)
(Th. NO: 43)
and primer BAMAD
(BAMAD: 5’ GACTCGAGGATCCATCG 3’)
(Th. NO: 56)
in 100 ml of reaction mixture (50 mm KCl, 1,5 VgCl, 10 mm Tris, pH 8.0, 0.2 mm dNTPs and 0.05 units/ml of polymerase, Perkin (Elmer Amplitaqst)). Then the PCR product is hydrolyzed by the enzyme Clal, were extracted with phenol-chloroform (1:1), precipitated with ethanol and ligated into the Bluescript vector (0.1 mg) (Stratagene Inc.), which was cut by enzymes Clal and Kpnl. After 2-hour incubation at room temperature for one-fourth of the mixture for ligation was added directly to the second series of PCR (22 cycle, as described above)by using the second ML-specific forward primer-1.
(forward-1: 5’ GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3’
(Th. NO: 57)
and T3-21 (oligonucleotide that binds to a sequence adjacent to the area multiple klonirovania in the vector Bluescript SK-):
(5’ CGAAATTAACCCTCACTAAAG 3’
(Th. NO: 58)
The obtained PCR product is hydrolyzed by enzymes Xbal and Clal and was subcloned into Bluescript SK-. Several clones from independent PCR reaction sequence.
And again identified the second form (indicated by the pML-2)encoding a protein with a deletion of 4 amino acid residue (328 amino acid residues) (see Fig (SEQ ID NO: 21)). Comparison of amino acid sequences of pML and pML-2 has shown, these pan-sequences are identical except in the form of pML-2 no tetrapeptide QLPP corresponding to residues 111-114, which were deletethread (see Fig (SEQ ID NOS: 18 and 21)). Thus, deletion of 4 amino acids was observed in rat, human and porcine cDNA, with the same provisions of the predicted proteins.
The analysis of cells QMS on TRO (thrombopoietin) inducing the expression of platelet antigen GPIIbIIIa
Cells QMS is maintained in the environment RMPI 1640 (Sigma), which were added 10%fetal bovine serum and 10 mm glutamine. To obtain drugs for analysis cells were collected, washed and resuspendable at a density of 5×105cells/ml in serum-free medium GIF, which was added 5 mg/l bovine insulin, 10 mg/l APO-transferrin, 1x trace elements. Then to each well of flat-bottomed 96-well tablet was added to the standard SRW or experimental samples at the appropriate dilution in 100 μl volumes. Then, to each well was added 100 μl of QMS-cell suspension and the plates were incubated for 48 hours in the incubator at 37°C in an atmosphere of 5% CO2. After incubation, the tablets were subjected to centrifugation at 1000 rpm at 4°C for 5 minutes. Supernatant was discarded and each well was added FITC-conjugated (FITC-fluoresceinisothiocyanate) monoclonal anti who ate 2D2 to GPII bIIIa. After 1-hour incubation at 4°With tablets centrifuged at 100 rpm for 5 minutes. Supernatant containing unbound antibody was discarded and each well was added 200 μl of 0.1% BSA-PBS-washing. Stage leaching with 0.1% BSA-PBS was repeated three times. The cells are then analyzed FASCAN using the standard one-parameter analysis with a measure of the relative intensity of fluorescence.
Analysis of DAMI cells on thrombopoetin (TRO) by measuring antimitotics activity of these cells on 96-well tablets for micrometrology
The DAMI cells were maintained in medium IMDM + 10% horse serum (Gibco) with the addition of this Wednesday 10 mm glutamine, 100 ng/ml of penicillin G and 50 μg/ml streptomycin. To obtain drugs for the analysis of cells collected. Washed and resuspendable (with density 1×106cells/ml) in IMDM + 1% horse serum. To DAMI-cell suspension in 96-well round-bottom tablet was added 100 μl of TRO-standard or experimental samples. Then cells were incubated for 48 hours at 37°C in an incubator in the presence of 5% CO2. After incubation, the tablets were subjected to centrifugation in a centrifuge Sorvall 6000B at 1000 rpm for 5 minutes at 4°s Supernatant was discarded and repeated stage leaching 200 Microlitre PBS and 0.1% BSA. Then the entrances were fixed by adding 200 ál of ice 70% ethanol-PB and resuspendable by aspiration. After 15 minutes of incubation at 4°the plates were centrifuged at 2000 rpm for 5 minutes and to each well was added 150 μl of 1 mg/ml RNase containing 0.1 mg/ml propitiated and 0.05% Tween-20. After 1-hour incubation at 37°C was assessed by changes in the composition of DNA using flow cytometry. Quantitative measurement of polyploidy was performed according to the following equation
The normalized coefficient of polyploidy (NPR)=
Thrombopoetin (TRO) in vivo analysis (analysis on re-binding of murine platelets)
In vivo analysis to determine (35S) producing platelets
The C57BL6 mice (obtained from Charies River) were injected with intraperitoneal (IP), 1 ml goat antisera against mouse platelets (6 amp.) (on day 1) for producing thrombocytopenia. On days 5 and 6 mice were administered two intraperitoneal injections of factor or PBS for control. On day 7 the mice were injected intraperitoneally injected 30 µci Na
KIRA-ELIS-analysis on thrombopoetin carried out by measuring the phosphorylation mpl-Rse.gD-chimeric receptor
Receptor people. ml was described Vigon and others (PNAS, USA, 89: 5640-5644 91992)). For use in the KIRA-ELISA-analysis described in this application, designed chimeric receptor containing the extracellular domain (ECD) of the mpl receptor and the transmembrane (TM) and intracellular (ICD) domains Rse (Mark et al., J. of Biol. Chem. 269(14): 10720-10728 (1994)). With carboxy-terminal flagellaris the polypeptide (i.e. Rse.gD). Cm. Fig and 31, are presented for a schematic description of the analysis.
(a) Obtaining exciting agent
Monoclonal antibody to gD (clone V) was produced against the peptide, glycoprotein D of herpes simplex virus (Paborsky et al., Protein Engineering 3(6): 547-553 ). Cleaned the original drug was brought to a concentration of 3.0 mg/ml in phosphate buffered saline (PBS), pH 7.4 and 1/0 ml-aliquots were stored at -20°C.
(b) Obtaining antibodies against phosphotyrosine
Monoclonal antibody against phosphotyrosine (clone 4G10) was purchased from UBI (Lake Placid NY) and was biotinilated using highly branched Biotin-N-hydroxysuccinimide (Biotin-X-NHS, Research Organics, Cleveland, OH)
The mpl ligand was obtained using recombinant techniques is NC, described in this application. The purified ligand mpl kept as stock solution at 4°C.
(d) Obtaining a nucleic acid Rse.gD
Synthetic double-stranded oligonucleotides were used to rekonstruirovaniya sequence that encodes a C-terminal 10 amino acids (880-890) pers-Rse with the addition of 21 amino acids, containing the epitope for antibodies V and a stop codon. Table 10 presents the final sequence of the synthesized part of a hybrid gene
Synthetic DNA ligated with cDNA encoding amino acids 1-880 people-Rse, Pstl site, beginning with nucleotide 2644 published cDNA sequences person-Rse (Mark et al. Journal of Biological Chem. 269(14): 10720-10728, and Hindl I-sites in polylinker expressing vector pSV17.ID.LL (see Fig A-L; SEQ ID NO: 22), to create expressing plasmids pSV17.ID.Rse.gD. In short, expressing plasmid includes dicistronic primary transcript, which contains the sequence encoding DHFR and a limited 5'-site splitinto (donor site) and 3'-site of the splicing acceptor site), and this sequence is the sequence that encodes Rse.gD. Full-size (displayserver) the transcript contains the DHFR as the first open reading frame, and therefore produces a protein DHFR, which allows the t to select stable transformants.
(e) Obtaining a nucleic acid mpl-Rse.gD
Expressing plasmid pSV.ID.Rse.gD., produced as described above was modified in order to obtain the plasmid pSV. ID.M.tmRd.6, which contains the coding sequence of the ECD .ml (amino acids 1-491), connected with the transmembrane domain and intracellular domain Rse.gD (amino acids 429-911). To attach the coding sequence portion of the extracellular domain .ml to the coding sequence part Rse conducted dvukhstadiinoi reaction cloning by PCR using synthetic oligonucleotides, as described by Mark and others (J. Biol. Chem. 267: 26166-26171 91992)). Primers used in the first PCR reaction were: M1
(5’ TCTCGCTACCGTTTACAG 3’)
(Th. NO: 61)
(5’ CAGGTACCCACCAGGCGGTCTCGGT 3’)
(Th. NO: 62)
with mpl-cDNA-matrix; and R1
(5’ GGGCCATGACACTGTCAA 3’)
(Th. NO: 63)
(5’ GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT 3’)
(Th. NO: 64)
with Rse-cDNA-matrix. Pvull-Smal fragment of this part of the connection used to construct a full-sized chimeric receptor.
(f) Transformation of cells
Plasmid pSV. ID.M. tmRd.6, which was linearized at a unique Notl site in the plasmid backbone, was introduced into cells DP12.CHO (EP 307247, published March 15, 1989) by electroporation. After extraction with phenol/chloroform DNA precipitated with ethanol and resuspendable in 20 μl of 1/10 Tris-ED is K. Then, 10 μg of DNA were incubated with 107cells SNO R in 1 ml of PBS on ice for 10 minutes, and then subjected to electroporation at 400 Volts and 330 μf. The cells were returned for 10 minutes on ice, and then were sown on non-selective medium. After 24 hours, cells were transferred to medium without containing nucleoside, for selection of stable DHFR+-clones.
(g) Selection of transformed cells for use in the KIRA-ELISA
Clones expressing MPL/Rse.gD identified by Western blotting of whole cell lysates after fractionation using LTO-SDS page using antibodies V, which detects an epitope tag gD.
Cells were cultured in F12/DMEM (Gibco/BPL, Life Technologies, Grand Island, NY). In these environments was added 10% diafiltration FBS (HyClone, Logan, Utah), 25 mm HEPES and 2 mm L-glutamine.
Mpl-Rse.gD-transformed cells DP12.CHO were sown in holes (3×104cells per well) in flat-bottomed 96-well plate in a 100 μl of medium and were cultured overnight at 37°C in 5% CO2. The next morning, supernatant decantation, and tablets lightly blotted with a paper towel. Then to each well was added 50 μl of medium containing either experimental samples, or 200, 50, 12,5, 3,12, 0,78, 0,19, 0,048 or 0 ng/ml mpl ligand. Cells were stimulated for 30 minutes at 37°, supernatant decantation, and the tablets again after the ka blotted with a paper towel. For cell lysis and solubilization of chimeric receptors in each well was added 100 μl of buffer for lysis. Buffer for lysis consisted of 150 mm NaCl containing 50 mm HEPES (Gibco), 0/5% Triton-X (Gibco), 0.01% thimerosal, 30 KIE/ml Aprotinin (ICN Biochemicals, Aurora, OH), 1 mm 4-(2-amino-ethyl)-benzosulfimide hydrochloride (AEBSF; ICN Biochemicals), 50 μm leupeptin (ICN Biochemicals) and 2 mm orthovanadate sodium (Na3VO4; Sigma Chemicals Co, St. Lous, MO), pH 7.5. Then the tablet gently stirred on a shaker for tablets (Belico, Instruments, Vineland, NJ) for 60 minutes at room temperature.
When cells were solubilization, ELISA-microplates (Nunc Maxis, Inter Med, Denmark), sensitized overnight at 4°With a monoclonal antibody V against gD (5,0 mg/ml in 50 mm carbonate buffer, pH of 9.6, 100 μl/well), decantation, blotted with a paper towel and blocked with 150 μl/well blocking buffer (PBS containing 0.5% BSA (Intergen Company, Purchase, NJ) and 0.01% thimerosal) for 60 minutes at room temperature with gentle stirring. After 60 minutes anti-gD56-sensitized tablets were washed 6 times with wash buffer (PBS containing 0.05% tween-20 and 0.01% thimerosal) using automatic devices tablets (ScanWasher 300, Skatron Insrtuments, Inc., Sterling, VA).
The lysate containing the solubilized MPL/Rse.gD, removed from the culture plate to micrometrology in anti-gD V-senzibilizirana is nye and blocked ELISA-wells (85 μl/well) and incubated for 2 hours at room temperature, stirring gently. Unbound mpl-Rse.gD was removed by washing with wash buffer in each well was added 100 μl of biotinylated 4G10 (anti-phosphotyrosine), diluted 1:18000 buffer for dilution (PBS containing 0.5% BSA, 0.05% tween-20, 5 MM EDTA and 0.01% thimerosal), i.e. 56 ng/ml After 2-hour incubation at room temperature the tablets were washed and to each well was added 100 μl conjugated to horseradish peroxidase (NRRO) streptavidin (Zymed Laboratores, S. San Francisco, CA)diluted at a ratio of 1:60000 in the buffer for cultivation. The plates were incubated for 30 minutes at room temperature, stirring gently. Free aydinbey conjugate is washed and to each well was added 100 μl of freshly prepared substrate solution (tetramethylbenzidine) (TMB); 2-component substrate kit; Kirkegaard &Perry, Gaithersburg, MD). The mixture was left for 10 minutes for reaction, after which the color development was stopped by adding 100 μl/well of 1.0 M H3RHO4. The optical density at 450 nm was determined by comparison with the optical density at 650 nm (AB450/650) using the tablet reader (vmax) (Molecular Devices, palo Alto, CA)controlled by a Macintosh Centris 650 (Apple Computers, Cupertino, CA) and Deltasoft software (Biometallics, Inc., Princeton, NJ).
A standard curve was built by stimulating cells dp12.trkA,B or C.gD 200, 50, 12,5, 0,78, 0,19, 0,048 �if 0 ng/ml mpl ligand and represented using Deltasoft) in the form of a graph according to TRO (ng/ml) from (AB 450/650)±average deviation. The concentration of the sample was obtained by interpolation of optical density on the standard curve and were expressed as activity SRW (ng/ml).
It was found that the mpl-ligand capable of activating chimeric receptor mpl-Rse.gD ligand-specific manner depending on the concentration. In addition, analysis of the KIRA-ELISA on mpl-Rse.gD, as has been established for almost 100% calcaverage (shown) or 100% plasma (not shown)that allows easy screening of the patient and RK-samples.
The total value of ES for TRO
Summary of TPO EC50's
|the shape of the cells TRO Form (cells)||AS mass./about. (wt/vol.)||IS by both molarity (molarltv)|
|No TRO 332 (293)||2.56 nq/ml||67.4 PM PM|
|Mi TRO 332 (293)||3.69 nq/ml||97.1 PM PM|
|No TRO 153 (293)||~41 nq/ml||~1.08 PM PM|
|No TRO 155 (.li)||0.44 nq/ml||11.6 PM PM|
|No TRO 153met (E.li)||0.829 nq/ml||21.8 PM|
ELISA analysis, carried out using a receptor for thrombopoietin (SRW)
ELISA-plates were senzibilizirani F(ab’)2-fragments of rabbit antibodies against human IgG (Fc) carbonate is Ohm buffer (pH 9,6) at a temperature of 4° C during the night. Then the plates were blocked with 0.5% bovine serum albumin in PBS at room temperature for one hour. The yield obtained from the fermenter containing the chimeric receptor mpl-IgG, was added to the tablets, after which the resulting mixture was incubated for 2 hours. Twofold serial dilutions (0,39-25 ng/ml) standard (BO332produced in 293 cells using a concentration determined by quantitative amino acid analysis) and serially diluted samples in 0.5%bovine serum albumin and 0.05% Tween-20 was added to the tablets and the mixture was incubated for 2 hours. Associated TPO were detected using purified protein And biotinylated rabbit antibodies to TPO155that were produced in E. coli (incubation time = 1 hour), and then with streptavidin-peroxidase (incubation time = 30 minutes) and 3,3`,5,5`-tetramethylbenzidine used as substrate. The optical density was read at 450 nm. Between stages tablets washed. For data analysis, a standard curve was built using the program Kaleidagraph curve with four parameters. Concentration of samples was calculated using a standard curve.
Expression and purification of solid radioactive waste from the cells 293
1. Obtaining expressing vectors 293 cells
cDNA corresponding to the full open reading frame TRO was obtained using polymerase chain reaction, using primers corresponding oligonucleotides (table).
PCR primers 293
|Cla.FL.F: 5'ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG-3’||(Th. NO: 65)|
|hmpll-R: 5’ GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3’||(Th. NO: 48)|
PRKS-hrnpl I (described in Example 9) was used as matrix for the reaction in the presence of ready-to-eat (Stratagene) DNA polymerase. Initial denaturation was carried out for 7 minutes at a temperature of 94°and then using 25 cycles of amplification (1 min at 94°C, 1 minute at 55°and 1 minute at 72°). The final elongation was performed for 15 minutes at a temperature of 72°C. the PCR product was purified and cloned between the restriction sites Clal and Xbal plasmids pRK5tkneo, and the vector originating from pRK5, modified so that it expressed the gene of resistance to neomycin under control timeintensive promoter with getting open reading frame vector pRK5tkneo.ORF. The second design appropriate to the homologous domain of the EPO, was obtained in the same manner, but using Cla.FL.F as a forward primer and the corresponding reverse primer:
Arg.STOP.Xa: 5’ TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3’
The final design meant pRKS-tkneoEPO-D. the Sequence of both structures was examined by the method described in Example 7.
2. Transfection of embryonic human kidney cells
These 2 designs were transfusional in embryonic stem cells human kidney by the method using Saro4as described in Example 9. 24 hours after transfection, the selection of clones resistant to neomycin, initiated in the presence of 0.4 mg/ml G418,10 and 15 days single colony was transferred into 96-well plates, and then left for cultivation to the condition of continuity. The expression of m l153or ML332the air-conditioned environment from these clones was analyzed using analysis on the proliferation of Ba/F3-mpl (as described in Example 1).
3. Cleaning rhML332.
293-rhL332-air-conditioned environment was applied on a column of Blue Sepharose (Pharmacia), which was balanced in 10 mm sodium phosphate at pH 7.4 (buffer A). After that, the column washed with 10 column volumes of 2M buffer and 2M buffer C. Then the column was suirable buffer containing 2M NaCl and 1M NaCl. Collection after elution of Blue-Sepharose was immediately applied on the column with WGA-Sepharose, equilibrated in buffer A. the Column with WGA-Sepharose was washed washed with 10 column volumes of 2M buffer containing 2M NaCl and 1 M NaCl, and then suirable the same buffer containing 0.5 m N-acetyl-D-glitch the Zamin. The eluate WGA-Sepharose was applied to VRGH-column C4 (Synchrom, Inc.), equilibrated in 0.1% triperoxonane acid. URGH-column C4 was suirable speed propranololum gradient (0-25, 25-35 and 35-70%). Selected rhML332was subjected to elution in 28-30% propanolol gradient. Purified by electrophoresis in SDS page with LTOs rhL332moved in a broad band in 68-80 kDa region of the gel (smfg).
4. Cleaning rhML153
293-rhL153-air-conditioned environment was subjected to separation on a column of Biue-Sepharose, as described in the method for rhL332. The eluate Blue-Sepharose was immediately put on the mpl-affinity column as described above. RhML153, polyanovsky on mpl-affinity column purified to obtain a homogeneous mass using URGH-column C4 in the same conditions described for rhL332. RhML153purified by electrophoresis in SDS page with LTOs were divided into 2 major and 2 minor bands with mass (Mr)equal to about 18000-21000 (see Fig).
Expression and purification of solid radwaste from SNO
1. Clean expressroute vectors SNO
Constructed expressing the vectors used in electroporation, as described below:
pSVI5. ID.LL.MLORF (full or h332), and pSVl5.ID.LL.MLEPO-D (truncated or h153). Relevant distinctive features of these plasmids is presented on Fig and 24.
2. Getting expressi USA vectors SNO
cDNA corresponding to the full open reading frame hTPO, were obtained using the polymerase chain reaction using oligonucleotide primers listed in Table 12.
PCR primers for repressiruyuschy vector in Cho
|Cla.FL.F2 5’ ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3’||(Th. NO: 47)|
|ORF.Sal 5’ AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3’||(Th. NO: 67)|
PRKS-hmpI I (described in Example 7, and 9) was used as matrix for the reaction in the presence of ready-to-eat (Stratagene) DNA polymerase. Initial denaturation was carried out for 7 minutes at 94°and then using 25 cycles of amplification (1 min at 94°C, 1 minute at 55°and 1 minute at 72°). The final elongation was performed for 15 minutes at a temperature of 72°C. the PCR product was purified and cloned between the restriction sites Clal and Sall plasmids pSVI5.ID.LL obtaining vector pSVI5.ID.LL.MLORF. The second design appropriate to the domain of EPO was obtained in the same manner, but using Cla.FL.F2 as a forward primer and a reverse primer specified below:
EPOD.Sal 5’ AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3’ (Th. NO: 68)
The final design meant pSVI5.ID.LL.MLEPO-D. the Sequence of both structures were examined, as the description is but in Example 7, and 9.
Mainly coding sequences for full-length and truncated ligand was inserted in the multiple cloning site expressing vector pSVI5.ID.LL CHO cells. This vector contained the region early promoter/enhancer of SV40 virus, the modified website splicing, contains the mouse DHFR cDNA; a multiple cloning site for introduction of a desired gene (described in this case, the sequence SRW) the SV40 polyadenylation signal; the site of initiation of replication and gene beta-lactamase for selection of the plasmid and its amplification in bacteria.
3. The methods for generating stable cell lines CHO, expressroute recombinant TPO332and TPO153person
A. Description related cell lines CHO
The host cell line CHO (Chinese hamster ovary), used for the expression of molecules of the solid described in the present invention as CHO-DP12 (see EP 307247, published March 15, 1989). Cell line mammal clonal selected in the transfection kinship lines (CHO-KI DUX-BII (DHFR)is obtained from Dr.Frank Lee Stanford University with the permission of Dr. L.Chaisi) vector expressing preproinsulin, obtaining clones with reduced insulin requirements. These cells were also negative for DHFR, and the clones can be selected for the presence of sequences DHFR-cDNA-vector PU is eat cultivation on the environment, not containing nucleotide additives (glycine, gipoksantina and thymidine). Was mainly used selection system stable expression cell lines CHO.
b. The method of transfection (electroparty)
Cell lines expressing TRO332and TPO153, was obtained by transfection of DP12 cells by electroporation (see, for example, Adreason, G.L. J.Tiss. Cult.Meth., 1556 (1993)) using linearized plasmids pSVI5.ID.LL.MLORF or pSVI5.ID.LL.MLEPO-D, respectively. Three reaction mixture restrictively enzymes were taken for each cutting plasmids; 10, 25 and 50 μg of vector hydrolyzed by the enzyme NOTI standard methods used in molecular biology. Only one restriction site was located in the vector in the area of the vessel 3 and in the area outside the field of transcription ligand TRO (see Fig). Incubation 100 µl of the reaction mixture was carried out overnight at 37 ° °C. the next day the mixture once were extracted with phenol/chloroform/isoamyl alcohol (50:49:1)and then precipitated with ethanol, placing on dry ice. The procedure was performed for approximately one hour. The precipitate was collected by 150-minute microcentrifuge, and then drained. Lineitemno DNA resuspendable in 50 ál medium DMEM-F12 (1:1) Hema containing standard antibiotics and 2 mm glutamine.
Cells DP112, is grown in a suspension medium, collected once washed in the environment that was used to resuspendable DNA, and finally, resuspendable in this medium at a concentration of 107cells with 750 ál. Aliquots of cells (750 μl) and each lineitemno DNA mixture was incubated together at room temperature for one hour, and then transferred into the chamber BRL to electroporation. Each reaction mixture was subjected to electroporation standard device BRL to electroporate at 350, 330 UF and low capacity. After electroporation cells were left in this unit for 5 minutes and then placed on ice and carried out additional incubation for 10 minutes. The cells are then processed by electroporation, was transferred to a 60 mm Cup for cell culture containing 5 ml of complete growth medium for cells Cho (high glucose; DMEM-F12 (50:50), not containing glycine, with the addition of 1x average, 2 mm glutamine and 5%fetal bovine serum), and then cultured overnight in an incubator for culturing cells in the presence of 5% CO2.
C. Method of selection and screening
The next day cells were separated from tablets using trypsin standard methods, and then was transferred into a 150-mm tablets with cell cultures containing DHFR-selective medium (DMEM-F12 (1:1) Hema described above which contains 2 percent or 5-percent cialisbuynow fetal bovine serum, but not containing glycine, gipoksantina and thymidine; was used standard DHFR-selective medium). Then the cells from each 60-mm tablet was transferred into a 5/150 mm tablets. The obtained cells were incubated for 10-15 days (with one replacement medium) at 37 ° °in the presence of 5% CO2until then, have not yet been obtained clones having dimensions sufficient to transfer them to the 96-well tablets using sterile yellow tips on Pipettmann installed in 50 ml of These cells were left for cultivation to the condition of continuity (usually 3-5 days), after which the plates were trypsinization and 2 copies of the original tablets were again produced. Two of these copies in a short period of time kept in the refrigerator, and cells in each well was diluted 50 Microlitre 10% fetal bovine serum in dimethyl sulfoxide. Sample 5-day air-conditioned serum-free medium was analyzed on the basis of confluently holes of the third tablet for the expression of TPO by analyzing the activity of the cells Ba/F. Most visokoaktivnie clones confirmed through this analysis, brought again to the condition of continuity in 150-mm T-flasks and then transferred to a group of cell cultures to adapt the suspension, re-analysis and the establishment of the Bank.
d. AmplificationSeveral cell lines with the highest titer selected by the method described above, were subjected to amplification using standard methotrexate with obtaining clones with a higher titer. Clones of cells SNO multiplied and were sown in 10-cm tablets at 4 concentrations of methotrexate (i.e. 50, 100, 200 and 400 nm) by two or three numbers of cells (105,5×105 and 106 cells per well). Then the culture was incubated at 37 ° °in the presence of 5% CO2and educated clones was transferred into a 96-well tablets for subsequent analysis. Several clones with high titers selected in the selection, again, was treated with high concentrations of methotrexate (i.e. 600, 800, 1000 and 1200 nm) and in accordance with the method described previously, the clones with resistance, left, and then transferred into 96-well plates and analyzed.
4. The cultivation of stable cell lines Cho, expressionsa recombinant TPO332and TRO153person
The collected cells were thawed and cell populations were propagated using standard methods of culturing cells or in serum-free medium or in medium containing serum. After breeding to obtain sufficient cell density, the cells were washed to remove culture medium. Cells were cultured using any mill is artego way includes: periodic cultivation, cultivation injection or continuous cultivation at 25-40°at neutral pH in the presence of dissolved O2at least at 5% until until the accumulated constitutive secretory TRO. Then, the liquid culture was separated from the cells by mechanical mixing, for example, centrifugation.
5. Purification of recombinant TPO man of cell suspension SNO
The collected cell suspension culture (HCCF) was directly applied on the column, Fast Flow (Pharmacia) with a Blue-Sepharose 6, equilibrated in 0.01 M phosphate (pH 7/4) and 0.15 M sodium chloride in the ratio of approximately 100 liters HCCF 1 liter resin and linear flow rate of approximately 300 ml/h/cm2. Then the column washed with 3-5 column volumes of equilibrated buffer, then 3-5 column volumes of 0.01 M sodium phosphate (pH 7.4) and 2.0 M urea. Then TRO was suirable 3-5 column volumes of 0.01 M sodium phosphate (pH 7.4), 2.0 M urea and 1.0 M sodium chloride.
Pool Blue-Sepharose containing the solid was applied on the column with Separate PS and lectin from wheat (Wheat Germ Lectin Sepharose 6MB, (Pharmacia), equilibrated in 0.01 M phosphate (pH 7.4), 2.0 M urea and 1.0 M sodium chloride in respect of 8-16 ml of pool Wheat Germ Lec. 1 ml of resin at a flow rate of approximately 50 ml/h/cm2. The village is E. this column was washed with 2-3 column volumes of equilibrated buffer. Then TRO was suirable 2-5 column volumes of 0.01 M sodium phosphate at pH 7.4, 2.0 M urea and 0.5 M N-acetyl-D-glucosamine.
Pool lectin wheat brought up to a final concentration of 0.04% With12E8and 0.1% triperoxonane acid (TFA). The obtained pool was applied to a reversed-phase column C4 (Vydac 214TP1012), equilibrated in 0.1% triperoxonane acid (TFA) and 0.04% With12E8when loading of approximately 0.2 to 0.5 mg of protein per one millimeter resin, and at a flow rate equal to 157 ml/h/cm2.
Protein was suirable in two-phase linear gradient of acetonitrile containing 0.1%triperoxonane acid and 0.04% C12E8. The first phase was treated with a linear gradient of 0-30% acetonitrile for 15 minutes, and the second phase was treated with a linear gradient of 30-60% acetonitrile in 60 minutes. TRO was suirable approximately 50%acetonitrile. The obtained pool was subjected to electrophoresis in SDS page with LTOs.
Pool C4 diluted in two volumes of 0.01 M sodium phosphate at pH 7.4 and 0.15 M sodium chloride, and then was diafiltration against approximately 6 volumes of 0.01 M sodium phosphate (pH 7.4) and 0.15 M sodium chloride on the membrane for ultrafiltration Amicor MIND or equivalent membrane having a molecular weight in 10000-30000 Yes. The resulting diafiltrate can be immediately processed by ultrafiltration, followed by concentration. mesh of diafiltrate and concentrate brought up to a final concentration of 0.01% tween-80.
All this mix or her part with 2-5% computed column volume was applied on a column of HP with Sephacryl S-300 (Pharmacia), equilibrated in 0.01 M sodium phosphate (pH 7.4), 0.15 M sodium chloride and 0.01% Tween-80, and then chromatographically at a flow rate of approximately 17 ml/h/cm2. The fractions containing the solid and does not contain product aggregation and products of proteolytic cleavage, was joined in the pool by electrophoresis in SDS page with LTOs. A new pool has been filtered through to 0.22-mm filter Millex-GY or similar filter, and then kept at a temperature of 2-8°C.
Transformation and induction of protein synthesis TRO in E. coli 1. Construction of vectors for the expression of TPO in E.coli
All plasmids RMR, RMR, RMR, RMR and RMR designed so that they expressed the first 155 amino acids TRO below from a small leader sequence, which is situated among the different designs. Mainly, leader sequences provide a high level of translation initiation and quick cleaning. Plasmids RMR-1, -T8, -21, -22, -24, -25 designed so that they are expressed first 153 amino acids TRO below from the initiating methionine and differ from each other only by using the first 6 codons of amino acids TRO, while plasmid RMR is proizvodi the e RMR-1, in which the carboxy-end of the SRW extended by two amino acids. All of the above plasmids produce high levels of intracellular expression TRO E. coli after induction of tryptophan promoter (Yansura, D.G. et al., Methods in Enzymology (D.V. Goeddel Ed) 185: 54-60, Academic Press, San Diego ). Plasmids pMPI and pMP172 are intermediate in the construction of the above plasmids intracellular expression TRO.
(a) Plasmid pMPI
Plasmid pMPI was a vector for secreting the first 155 amino acids TRO and was constructed by ligating the 5 DNA fragments, as shown in Fig. The first of them was the vector pPho21, in which a small Mlul-BamHl fragment was removed. pPho21 is a derivative phGHl (Chano, C.N. et al., Gene 55: 189-196 ), in which gene of human growth hormone is replaced with a gene phoA E. coli, and Mlul restriction site was designed in to a sequence encoding a signal sequence STll have amino acids 20-21.
The following two fragments, Hinfl-Pstl fragment (258 BP) DNA from pRK5-hmpl 1 (Example 9), encoding amino acids TRO 19-103 and the following synthetic DNA encoding amino acids 1-18:
(Th. NO: 69)
(Th. NO: 70)
pre-ligated using DNA ligase T4 and the second segment is hydrolyzed by the enzyme Pstl. The fourth fragment p is estables a Pstl-Haelll fragment (152 BP) from plasmid pRKShmpll, the coding amino acids 104-155 TRO. The last was Stul-BamHl fragment (412 BP) from plasmid RRO containing lambda terminator of transcription, as described above (Scholtissek, S. Et al., NAP 15: 3185 ).
(b) Plasmid RMR
Plasmid RMR designed so that it is expressed first 155 amino acids SRW with a leader sequence of 13 amino acids, containing part of the signal sequence STll. This plasmid was constructed by ligating all three DNA fragments, as shown in Fig, and the first of them was the vector pVEG31, in which a small Xbal-Sphl fragment was removed. Vector pVEG31 was a derivative pHGH207-l (de Boer, H.A. et al., in Promoter Structure and Function (Rodrigues, P.L. and Chamberlain, M.J., Eo), 462, Praege, New York ), in which gene of human growth hormone replaced gene vascular endothelial growth factor (identical to the fragment of the vector can be obtained from plasmid pHGH207-1).
The second part of the ligation was a synthetic DNA duplex with the sequence specified below:
(Th. NO: 71)
(Th. NO: 72)
The last part was a Mlul-Sptl-fragment (1072 BP) from RMR encoding 155 amino acids TRO.
(c) Plasmid RMR
Plasmid RMR designed for the expression of the first 155 amino acids TRO below leader after whom outermost, containing 7 amino acids of the signal sequence STll, 8 his-tag residues and the site of cleavage factor XA. As shown in Fig, RMR designed by ligating all three DNA fragments, the first of which was an above-described vector pVEG31 from which has been removed a small Xbal-Sptl-fragment. The second plasmid was a synthetic DNA duplex with the sequence specified below:
(Th. NO: 73)
(Th. NO: 74)
The latter plasmid was a Boll-Sptl-fragment (1064 BP) from pMP11 that encodes a 154 amino acids of TPO. Plasmid pMP11 identical plasmid RMR except that in the signal sequence STll have been replaced by several codons (this fragment can be obtained from RMR).
(d) Plasmid RMR
Plasmid RMR was similar expressing vector RMR except that the site of cleavage by factor XA in the leader sequence is replaced by the site of cleavage by thrombin. As shown in Fig, RMR designed by ligating all three DNA fragments. The first one was an above-described vector pVEG31, which was removed a small Xbal-Sptl-fragment. The second was a synthetic DNA duplex with the sequence, the decree is nnow below:
(Th. NO: 75)
(Th. NO: 76)
The latter plasmid was a Bgll-Sptl-fragment (1064 BP) plasmid RMR described above.
(e) Plasmid RMR
Plasmid RMR is secreting vector for the first 153 amino acids TRO and an intermediate for design RMR. As shown in Fig RMR received through co-ligation of three DNA fragments, the first of which was the vector pLS321amB, which was removed a small EcoRl-Hindlll fragment. The second was an EcoRl-Hbal-fragment (946 BP) above plasmids RMR. The last part was a synthetic DNA duplex with the following sequence:
5`-TCCACCCTCTGCGTCAGGT (PEFC. NO: 77)
GGAGACGCAGTCCATCGA-5’ (Th. NO: 78)
(f) Plasmid RMR
Plasmid RMR designed for the expression of the first 153 amino acids TRO after methionine initiating the broadcast. This plasmid was used in the form of Bank plasmids, in which the first 6 codons TRO randomized in the third position of each codon, and then designed, as shown in Fig, by ligating all three DNA fragments. The first fragment was a previously described vector pLEG31, which was removed a small Xbal-Sptl-fragment. The second was a synthetic DNA duplex, to whom were first treated with DNA polymerase 1 (fragment maple), and then hydrolyzed by enzymes Xbal and Hinfl, and finally, this duplex encoding the methionine initiation, randomized in the first six codons of SRW.
ACACTGGAGGCTGTTCTCAGTAAA (PEFC. NO: 79) CAAGAGTCATTTGACGAAGCACTGAGGGTACAGGAAG-5’ (Th. NO: 80)
The third piece was a Hinfl-Sptl-fragment (890 BP) plasmid RMR that encodes amino acids 19-153 TRO.
RMR-plasmid Bank, approximately 3700 clones, re-transformed in LB-tablets with high molecular tetracycline (50 μg/ml) for selection of clones that initiates the broadcast (Yansura, D.G. et al., Methods: A Companion to Methods in Enzymology 4:151-158 ). From 8 colonies plated in tablets with high molecular tetracycline, 5 most-producing colonies in the interval between the expression of TPO were subjected to DNA sequencing; the results are shown in Fig (SEQ ID NOS: 23, 24, 25, 26, 27 and 28)
(g) Plasmid RMR
Plasmid RMR designed for the expression of the first 155 amino acids TRO, merged with a leader sequence consisting of 7 amino acids of the signal sequence STll, which is the site of cleavage by factor XA. This plasmid was designed as shown in Fig by legirovanija all three parts of the DNA, the first of which was a previously described vector pVEG31, which was removed a small Xbal-Sptl-fragment. The second consisted of the following synthetic grass is th DNA duplex:
5` CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (PEFC. NO: 81)
TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-5’ (Th. NO: 82)
The last piece in legirovanii was a Bgll-Sphl fragment (1064 BP) previously described plasmids RM.
(h) Plasmid RMR
Plasmid RMR expresses the first 155 amino acids TRO below the leader sequences consisting of 9 amino acids of the signal sequence and STll dibasic site Lys-Arg. This dibasic site provides the removal of the leader sequence through protease ArgC, This plasmid was designed, as shown in Fig through co-ligation of three DNA fragments. The first of these fragments was a previously described vector pLEG31, which was removed a small Xbal-Sptl-fragment. The second segment consisted of the following synthetic DNA duplex:
5’ CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (PEFC. NO: 83)
TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5’ (Th. NO: 84)
The last piece after ligating was a Bgll-Sphl fragment (1064 BP) previously described plasmids RM.
(i) Plasmid RMR
Plasmid RMR was a derivative RMR-1, where the carboxy-ends were located two additional amino acids TRO. As shown in Fig, this plasmid was constructed by ligating two DNA, and the first fragment represented a previously described vector RMR, W which has been removed a small Xbal-Apal fragment. The second fragment after ligating was a Xbal-Apal fragment (316 BP) plasmid RMR-1.
2. Transformation and induction vectors in the expression of TPO in E.coli
The above TRO-expressing plasmids used for transformation of strain IS E. coli (w3110 tnΔ rpoHtslnΔlΔ galE) using the method of heat shock using CaCl2(Handel, et al., J.Mol.Biol., 53:159-162, ). Transformirovaniya cells were first cultured at 37 ° °C in LB-medium containing 50 μg/ml of carbenicillin, until the optical density (600 nm) of the culture reached approximately 2-3. Then LB culture was diluted 20×M9 medium containing 0,49% Casinocity (wt./about.) and 50 μg/ml of carbenicillin. After a 10-hour cultivation at a temperature of 30°With aeration to the culture was added indole-3-acrylic acid to a final concentration of 50 µg/ml After the culture was grown for a further 15 hours at a temperature of 30°C With aeration and in this period of time the cells were collected by centrifugation.
Production of the biologically active TPO (Met-11-153) in E. coli
The following procedure for producing biologically active restored TPO (Met-11-153) can be carried out by analogy with other options TRO, including N - and C-terminal extended form (see PR the measures 23).
A. Recovering the insoluble solid radwaste (Met-11-153)
TRO expressing cells E.coli (Met-11-153) and encoded by the plasmid RMR-1, were subjected to fermentation, as described above. Approximately 100 g of cells resuspendable in 1 liter (10 volumes) of buffer for disintegration of cells (10 mm, 10 mm Tris, 5 mm add (pH 8) using a homogenizer transmitter station, and then the cells were centrifuged at 5000 g for 30 minutes. The washed cell precipitate again resuspendable in 1 l of buffer for disintegration of the cells using a homogenizer transmitter station, and then the cell suspension was passed through a disintegrator cells LH (LH Inceltech, Inc.) or through pseudocritical Microfluidizer (Microfluidics International) according to the manufacturer's instructions. The resulting suspension was centrifuged at 5000 g for 30 minutes, and then resuspendable and centrifuged again to obtain the washed precipitate refractive Taurus. The washed residue was used immediately or pre-frozen at -70°C.
C. Solubilization and purification of Monomeric TPO (Met-11-153)
The above precipitate resuspendable in 5 volumes of 20 mm Tris (pH 8)containing 6-8 M guanidine and 25 mm DTT (dithiothreitol), then was stirred for 1-3 hours or overnight at a temperature of 4°obtaining solubilizing protein TRO. There is also a high concentration of the (6-8 M) urea, however, as a result received a lower output compared with guanidine. After solubilization, the solution was centrifuged at 30000 g for 30 minutes and got the clear supernatant containing the denatured Monomeric protein TRO. After that, the supernatant was chromatographically by gel filtration on a column of Superdex 200 (Pharmacia, 2,6×60 cm) at a flow rate of 2 ml/minute, and then have suirable 20 mm sodium phosphate (pH 6) and 10 mm dithiothreitol (DTT). Fractions containing Monomeric denatured protein TRO was suirable (160-200 ml) and combined. In addition, protein TRO was purified using prepreparation reversed-phase chromatography on columns C4 (2×20 cm; Vydac). The sample was applied at 5 ml/min to a column equilibrated in 0.1% triperoxonane acid (TFA), elwira 30%acetonitrile. After this protein was suirable gradient of acetonitrile (30-60% for 60 minutes). Then cleaned and restored protein was suirable approximately 50%acetonitrile. The obtained product was used for the re-laying of obtaining biologically active variant TRO.
C. production of the biologically active TPO (Met-11-153)
Approximately 20 mg of Monomeric, reduced and denatured protein TRO in 40 ml of a mixture of 0.1%triperoxonane acid and 50%acetonitrile was dissolved in 360 ml of buffer for stacking, it is certainly containing reagents:
50 mm Tris;
0.3 M sodium chloride;
5 mm add;
Detergent 2% CHAPS;
5 Mm oxidized glutathione;
1 mm restored glutathione;
the pH increased to 8.3.
After mixing the buffer for laying gently stirring his at a temperature of 4°12-48 hours for maximum output the correct disulfide-linked forms TRO (see below). The resulting solution was acidified triperoxonane acid to a final concentration of 0.2%, and then filtered through a filter of 0.45 μm or 0.22 micron, and finally, was added 1,10 volume of acetonitrile. This solution directly using pump was inserted in the reversed-phase column C4, and then cleaned and laid TRO was suirable using the same gradient program described above. Laid biologically active TRO was suirable in the presence of approximately 45% acetonitrile in the same conditions. Incorrect disulfide-linked forms TRO was loirevalley before. The final purified TPO (Met-11-153), having a purity of more than 95%, as evidenced by the analysis carried out by electrophoresis in SDS page with LTOs were analyzed by reverse-phase chromatography on columns C4. For in vivo studies the material purified by column C4, were dialyzed in physiologically compatible buffers. Were also used isotonic buffer (10 mm ACET is tons of sodium (pH 5.5)), 10 mm sodium succinate (pH 5.5) or sodium phosphate (pH 7.4)) containing 150 mm sodium chloride and 0.01% tween 80.
Due to the high activity of TPO in the analysis of Ba/F3 (half maximal stimulation was achieved at approximately 3 PG/ml)was obtained biologically active material using many other buffers, detergents, and redox conditions. However, even in better conditions was obtained only a small amount of properly laid material (<10%). For the industrial manufacture of this product, it is desirable that its output was at least 10%, more preferably 30-50%, and most preferably >50%. Many other detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS, CHAPSO, LTOs, sarkosyl, tween 20 and tween 80, Twitterget 3-14 and others) were analyzed on the effectiveness of maintaining a high level of spontaneous archivaria protein. Of these detergents were found, only the family of CHAPS (CHAPS and CHAPSO) can be used in the reaction collapse in order to limit the aggregation of the protein and formation of incorrect disulfide bonds. Most preferably, the levels CHAPS was more than 1%. For best yield optimal levels of sodium chloride should be 0.1 M and 0.5 M. the Presence of EDTA (1-5 mm) limits the number catalyzed by metal oxidation (and aggregation) which is observed when using certain drugs. The concentration of glycerol, comprising more than 15%, give optimal conditions for the folding of the molecule. For maximum yield it is important to present both oxidized and reduced glutathion, oxidized and reduced cysteine as a redox reagent vapors. Higher output is observed mainly in the case when the molar quantity of the oxidized reagent is equal to or exceeds the number of recovered reactant in a redox pair. The optimum pH value for minimizing these TRO-options is in the range from 7.5 to about 9. Organic solvents (e.g. ethanol, acetonitrile, methanol) permitted at concentrations of 10-15% or below. Higher levels of organic solvents increase the possibility of formation of misfolded forms. Typically used Tris and phosphate buffers. Incubation at 4°also provides higher levels correctly laid TRO.
Usually for drugs TRO, which were cleared during the first C4-stage, levels of recovery of protein folding is 40-60% (based on the number of recovered and denatured TPO used in the reaction collapse. The active material can be obtained with less pure preparations (for example, the received neposredno is but after column with Sephadex 200 or after the initial extraction of the refractive Taurus), although the outputs in this case is less due to extensive precipitation and the influence of other proteins (no TRO) during the recovery process of protein folding.
Because TPO (Met-11-153) contains 4 cysteine residue, can be produced in three different disulfide variant of this protein:
Option 1: disulfides between cysteine residues 1-4 and 2-3
Option 2: disulfides between cysteine residues 1-2 and 3-4
Option 3: disulfides between the cysteine Acadamy 1-3 and 2-4
In the initial studies to determine the conditions for the restoration of laying several different protein peaks containing protein TRO, were separated by C4 reversed-phase chromatography. As determined by analysis on the cells Ba/F3, only one of these peaks was found significant biological activity. Then re-laying were optimized to obtain preferred this option. In these circumstances, options are incorrect phasing of the received total Monomeric TRO was less than 10-20%.
Using mass spectroscopy, protein sequencing, it was found that for a biological active SRW type of disulfide bonds is a 1-4 and 2-3 (i.e. option 1). Aliquots of the various C4-resolved peaks (5-10) hydrolyzed by trypsin (molar ratio of three is Cena to protein was (1:25). Mixture for hydrolysis were analyzed by laser mass spectrometry by desorption ionization using matrix both before and after DTT-recovery. After recovery were detected mass corresponding to the largest trypticase peptides TRO. In unrecovered samples of some of these masses were missing and observed the new mass. Lots of new peaks corresponded mainly to the sum of the individual trypticase peptides present in disulfide pair. Thus, it can certainly be argued that disulfide type recombinant again collapsed biologically active SRW corresponds to variant 1-4 and 2-3. This assumption is consistent with the known disulfide pattern related molecules erythropoietin.
D. Biological activity of recombinant again folded TPO (Met-11-153)
Again folded and purified TPO (Met-11-153) possessed activity both in vivo and in vitro assays. In Ba/F3 premaxilla stimulation of thymidine incorporation into the cells Ba/F3 was achieved at the 3/3 PG/ml (0.3 PM). In ELISA-based receptor mpl premaxilla activity was observed at 1.9 ng/ml (120 PM). The normal and myelosuppression animals, obtained by x-ray irradiation doses close to lethal, TPO (Met-11-153) had high active is thew (this activity was observed at doses of 30 ng/mouse) to stimulate the production of new platelets.
The production of other biologically active variants TRO in E.coli
In E. coli was produced three different ways TRO, which was a purified and re-stacked biologically active forms, described below.
(1) MLF - 13 residues from the bacterial signal sequence ST11 is connected with the N-terminal domain TRO (residues 1-155). The obtained sequence was a
where the leader sequence is underlined, and C·······C means Cys7-Cys151. This option was designed to obtain tyrosine for radioactive iodination TRO for studies of receptors and biological research.
(2) H8MLF - 7 residues from the sequence ST11, 8 histidine residues and a sequence of factor XA-enzymatic degradation IEGR were attached to the N-terminal domain (residues 1-155) TRO. This sequence represents:
where the leader sequence is underlined, and C·······C means Cys7-Cys151. This option when cleaning and packing can be processed by factor XA, which will split the sequence IEGR after arginine residue, which will result in a variant TRO 155 residues with turalei serine N-terminal amino acid.
(3) T-H8MLF - received, as described above for option (2), except that the thrombin-sensitive sequence IEPR is connected with the N-terminal domain TRO. The result was obtained sequence:
where the leader sequence is underlined, and C·······C means CIS7-CIS151. This option after cleaning and styling can be treated with thrombin to obtain N-terminal natural option TRO of 155 residues.
A. Recovery, solubilization and purification of biologically active Monomeric variants TRO: (1), (2) and (3)
All variants were expressed in E. coli. Most of this variation was obnarujeno in refractive corpuscles, as described in Example 22 for TPO (Met-11-153). Identical procedures for recovery, solubilization and purification of Monomeric TRO variants were performed as described in Example 22. The conditions for the recovery of protein folding were identical to the conditions used for TPO (Met-11-153), where the total yield amounted to 30-50%. After restoring styling TRO variants were purified using a C4 reverse-phase chromatography in 0.1% THF using a gradient of acetonitrile as described previously. All TRO-options (deproteinizirovanny form) had biological activity in BA/F3-analyses of promaxyl the th activity 2-5 PM.
C. Proteolytic processing options (2) and (3) with the formation of the active N-terminal TRO (1-155)
Were constructed above TRO-options (2) and (3)that contains the enzymatic ottsepleny leader peptide, located in front of the normal N-terminal amino acid residue of solid radwaste. After packing and cleaning options (2) and (3)as described above, each variant was subjected to hydrolysis by the corresponding enzyme. At the stage of C4 reversed-phase chromatography acetonitrile was removed by blowing the solution a slight stream of nitrogen. After this, two options were treated with either factor XA or thrombin, as described below.
In the case of option (2) not containing acetonitrile solution was added 1M Tris buffer (pH 8) to a final concentration of 50 mm and, if necessary, the pH was brought to 8. Then added NaCl and CaCl2to 0.1 M and 2 M, respectively. Factor XA (New England Biolabs) was added in such a quantity that the molar ratio of enzyme to the variant was 1:25-1:100. The sample was incubated for 1-2 hours at room temperature to achieve maximum cleavage, which was estimated by the change of migration on LTO-gel, reflecting the loss of the leader sequence. Then the reaction mixture was purified by C4 reversed-phase chromatography using the same gradient and the same conditions, which is described above for the purification of correctly installed options. Under these conditions, unsplit option was separated from the split option (2). It has been shown that the N-terminal amino acids are SPAPP, which suggests that the removal of N-terminal leader sequence was successful. Factor XA was also generate a variable number of internal cleavage in TRO-dominan; and the cleavage was observed after agrinova residue at position 118, which formed an additional N-terminal sequence TTAHKDP (SEQ ID NO 88). On nereguliruemyi LTO-gels was observed a single band of approximately 17000 Yes for option cleaved by factor XA; and on reducing gels revealed two bands with molecular weight of about 12000 and 5000 Da, which corresponds to cleavage at arginine 118. These observations also confirm the fact that the two parts of the molecule are linked together by a disulfide bond between 1 and 4 cysteine residues, as revealed in experiments on cryptococcoma splitting described above. In BA/F3-analysis of purified TRO-option (1-155) after removal of N-terminal leader sequence and internal cleavage had premaxillary activity of 0.2-0.3 PM. The intact variant with a leader sequence had premaxillary activity 2-4 PM.
For option (3) used buffer for hydrolysis, consisting of 0 mm Tris, pH 8; 2% CHAPS; 0.3 M NaCl; 5 mm EDTA and human or bovine thrombin (Calbiochem) at a ratio (by weight) of the enzyme and TRO-option 1:25-1:50, respectively. The hydrolysis was carried out at room temperature for 2-6 hours. The digestion was assessed by LTOs-gels, as described above for the reaction of cleavage by factor XA. During this time, mainly achieved more than 90%removal of the leader sequence. The obtained solid was purified on a C4 reversed-phase columns as described above and amino acid sequencing revealed that this TRO is the desired N-aminocore. In this case, as in the case of factor XA, as described above, there was very small (<5%) of the number of internal cleavage in the same arginine-ser / thr connection. Obtained TRO-protein has a high biological activity in BA/F3-analyses premaxillae responses were observed at 0.2 to 0.4 gr of protein. In ELISA-based analysis of the mpl receptor, this protein had premaxillary reply at 2-4 ng/ml of purified protein (120-240 gr), whereas the intact variant containing a leader sequence, was 5-10 times lower activity in both assays. For in vivo studies of HPLC-purified cleaved protein were dialyzed in a physiologically acceptable buffer (150 mm NaCl, 0/01% tween 80 and 10 mm sodium succinate, pH 5.5 or 10 mm sodium acetate, pH 5.5 or 10 mm sodium phosphate, the N 7,4). In the case of HPLC-and LTO-gels of purified protein remained stable for several weeks at 4°C. At normal and myelosuppression mice, this cleared SRW with authentic N-terminal sequence had high activity and stimulate the production of platelets at doses already 30 ng per mouse.
Synthetic ligand mpl
Although the human mpl ligand (hML) were usually obtained using recombinant methods, but it can also be synthesized by enzymatic ligation of a synthetic peptide fragments in accordance with the methodology described below. Synthetic production of hML allows to introduce unnatural amino acids or synthetic functional groups, such as polyethylene glycol. Previously for efficient ligation of esters of peptides in aqueous solution was designed mutant subtilisin (serine protease) BPN, subtilase (S221C/P225A) (Abrahmsen et al., Biochem., 30: 4151-4159 ). In the present work it was shown that synthetic peptides can be enzymatic legirovanyh sequential method for producing enzymatically active long peptides, such as ribonuclease A (Jackson et al., Science ). This technique, which is described in more detail below, allows the chemical synthesis of proteins, which could previously be done is look at using recombinant DNA technology.
The overall strategy of Intesa hL153using subtilase presented in Scheme 1. Starting with a fully released peptide corresponding protected at the N-terminal, C-terminal fragment of the protein, was added under the action of subtilase activated at the end of a complex ester of the peptide. After completion of the reaction the product was isolated using reverse-phase HPLC, and the protective group at the N-Terminus was deleted. Then ligated following peptide fragment, after which it was unblocked and the process is repeated again until, until there was obtained a full-size protein. This process is similar to solid-phase peptide synthesis, which is protected at the N-Terminus and activated at the C-end of the peptide attached to the N end of the previous peptide, resulting in a synthesized peptide in the C→N. However, due to the fact that each connection adds up to 50 residues, and products emit after each ligation, this method allows to synthesize proteins with a much higher degree of purity and with an acceptable output.
Scheme I: Strategy synthesis hML using subtilase
On the basis of our knowledge regarding the specificity of sequence subtilase and amino acid sequence of a biologically active is th "EPO-domain hML we divided hML 153seven fragments of length in residues 18-25. To determine the most appropriate areas of connection for ligating 18-25 meters were synthesized tetrapeptide for a test ligation. The results of such tests ligation presented in Table 13.
The test ligation hML. Donor and nucleophilic peptides at 10 mm in 100 mm Tricine (7,8) with 22°C. Then add the ligase to a final concentration of 10 μm of 1.6 mg/ml stock solution (~70 µm) and the mixture for ligation was left overnight for reaction. Outputs are expressed as % of ligation in relation to the donor hydrolysis of peptides.
|Site||Donor(DK-K-NH2)||Nucleophile-NH2,||% hydrolysis||% ligating|
|Site||Donor (glc-K-NH2)||Nucleophile-NH2||% Hydrolysis||% Ligation|
|1(23/24)||HVLH (SEQ ID NO: 89)||SRLS (SEQ ID NO: 90)||92||08|
|(22/23)||SHVL (SEQ ID NO: 91)||HSRL (SEQ ID NO: 92)||48||52|
|2(46/47)||AVDF (SEQ ID NO: 93)||SLGE (SEQ ID NO: 94)||22||78|
|3(69/70)||AVTL (SEQ ID NO: 95)||LLEG (SEQ ID NO: 96)||53||47|
|(89/90)||LSSL (SEQ ID NO: 97)||LGQL (SEQ ID NO: 98)||95||05|
|(88/89)||C(acm)LSS (SEQ ID NO: 99)||LLGQ (SEQ ID NO: 100)||00||00|
|(90/91)||SSLL (SEQ ID NO: 101)||GQLS (SEQ ID NO: 102)||45||55|
|(88/89)||CLSS (SEQ ID NO: 103)||LLGQ (SEQ ID NO: 100)||90||10|
|5(107/108)||LQSL (SEQ ID NO: 104)||LGTQ (SEQ ID NO: 105)||99||01|
|(106/107)||ALQS (SEQ ID NO: 106)||LLGT (SEQ ID NO: 107)||70||30|
|6(128/129)||NAIF (SEQ ID NO: 108)||LSFQ (SEQ ID NO: 109)||60||40|
On the basis of these experiments using subtilase can be successful ligation of peptides is illustrated in Table 14. To prevent self-ligation, you must use the right protection for N-end of each donor ester of the peptide. As a protective group, we chose isonicotinic (iNOC) (Veber et al., J.Org.Chem., 42: 3286-3289 ), since it is water soluble, can be entered into the last stage solid-phase synthesis and is stable in respect of anhydrous HF, which is used to release and cleavage of the peptides from the solid-phase poly the EPA. In addition, this group can be removed from the peptide after each ligation in mild reducing conditions (Zn/CH3CO2H) to obtain the free N-Terminus for subsequent ligation. Ester, glycolic-lysyl-amide (glc-K-NH2) was used for activation-end on the basis of previous experiments, which showed that it can be effectively allerban using subtilase (Abrahmsen et al., Biochem., 30: 4151-4159 ). INOC-protected and activated glc-K-amidon peptides can be synthesized using standard solid-phase methods, as shown in Scheme 2. These peptides are ligated sequentially until will not be produced complete protein, after which the final product is subjected to reaction laying in vitro. Based on homology with EPO disulfide pairs, obviously, are formed between cysteine residues 7 and 157 and between 28 and 85. The oxidation of disulfides can be carried out by simply mixing the recovered material for several hours in an atmosphere of oxygen. The folded material is then purified by HPLC and the fractions containing the active protein, collect and lyophilized. Alternatively, the disulfides may be variously secured to control subsequent oxidation between specific disulfide pairs. Protection cysteines 7 and 151 acetamidomethyl (cm) groups should ensure oxidation of the 28 and 85. acm Group can then be removed and the remaining 7 and 151 oxidized. Conversely, residues 28 and 85 may be of the ACM-protected and oxidized, if for the correct packing is necessary for subsequent oxidation. Cysteine 28 and 85 can be replaced, but not necessarily, other natural or unnatural residue, non-CIS, to ensure proper oxidation cysteines 7 and 151.
Peptide fragments used for the total synthesis using subtilase
1 (.NO: 110)
2 (.NO: 111)
3 (.NO: 112)
4 (Th. NO: 113)
5 (Th. NO: 114)
6 (Th. NO: 115)
7 (Th. NO: 116)
Ligation of peptides was carried out at 25°C in 100 mm Tricine, pH 8 (svezheprigotovlenvy and degassed by vacuum filtration through a 5 μm filter). Usually C-terminal fragment was dissolved in buffer (2-5 mm peptide) was added 10x mother solution of subtilase (1 mg/ml in 100 mm Tricine, pH 8) to bring the final concentration of the enzyme to ~5 μm. Then add 3-5 molar excess glc-K-activated donor peptide (firmly in the form), was dissolved and the resulting mixture was left at 25°C. Over the course of ligation was monitored by analytical reverse-phase C18-HPLC (CH3SP/N2O-gradient 0/1% TFA). The ligation products were purified using preparative HPLC and was liofilizovane. Removing the protective isonicotinamide (iNOC) groups was carried out by mixing HCl-activated zinc dust-protected peptide in acetic acid. Zinc dust was removed by filtration and the acetic acid evaporated under vacuum. The resulting peptide can then be used directly for the next stage of ligating and the process is repeated. Synthetic hML153can be Legerova in accordance with procedures similar to the procedures described above for synthetic or recombinant hML154-332in the result that can be produced by synthetic or semi-synthetic full hML.
Synthetic hML has many advantages compared to the recombinant. Natural side chains can be introduced to improve the activity or specificity. Polymer functional groups, such as polyethylene glycol, can be introduced to increase duration. For example, the polyethylene glycol may be attached to lysine residues of individual fragments (table 14) before or after the implementation of one the or more stages of ligation. Sensitive to the action of protease peptide bond can be removed or modified to improve stability in vivo. In addition, to facilitate structure determination can be synthesized derivatives with heavy atoms.
Scheme 2: Solid-phase synthesis of peptide fragments for segment ligation
(a) Lysyl-parametermessagefile (MVNA) resin 1 (0,063 IEC/g, Advanced Chem.Tech.) stirred with bromoxynil acid (5 EQ.) and diisopropylcarbodiimide (5 EQ.) for 1 hour at 25°in dimethylacetamide (DMA), resulting in a received bromoacetate derivative 2. (b) Resin intensively washed with DMA, and a separate Sun-protected amino acid (3 EQ., Bachem) was atrificial by mixing with sodium bicarbonate (6 EQ.) in dimethylformamide (DMF) for 24 hours at 50°s, s which receives glycolate-phenylalaninamide resin 3. Amino-acetylated resin 3 was washed with dimethylformamide (3) and dichloromethane (CH2Cl2) (3) and after washing the resin can be stored at room temperature for several months. Then the resin (3) was loaded in an automatic peptide synthesizer (Applied Biosystems 430 A) and peptides increased in accordance with standard procedures of solid-phase synthesis (5). (C) N-α-Boc-group was removed with the Astor 45%triperoxonane acid in CH 2CL2. (d) Subsequent BOC-protected amino acid (5 equiv.) pre-activated using hexafluorophosphate benzotriazol-1-yl-oxy-Tris-(dimethylamino)-phosphonium (BOP, 4 EQ.) and N-methylmorpholine (NMM, 10 EQ.) in DMA and spent a response within 1-2 hours. (e) the Final N-α-Boc-group was removed (TFA/CH2Cl2), resulting in received 4 and introduced isonicotinamide (iNOC) protective group as described above (4) by mixing 4-isonicotinoyl-2-4-dinitrophenyl-carbonate (3 EQ.) and NMM (6 EQ.) in DMA for 24 hours at 25°C. (f) After cleavage and release of the peptide by treatment of anhydrous HF (5% anisole/5% ethylmercury) for one hour at 0°received iNOC-protected glycolic-CIS-amide-activated peptide 5, which was purified using reverse-phase C18-HPLC (CH3SP/N2About the gradient of 0.1% TFA). The identity of all substrates was confirmed by mass spectrometry.
Addition to legal status
The present invention has a legal status in accordance with the above description and readily available references and source materials. However, the applicants were deposited in the American type culture collection (ATSS), Rockville, MD., USA, the following cell line:
Escherichia coli DH10B-pBSK-hmpl 1 to 1.8, ADS, reg. No. CRL 69575, deposited on February 24, 1994.
Plasmid pSV15.ID.LL.MLORRF, ATSS the er. No. CRL 75958, deposited on December 2, 1994.
Cells CHO DP-12, ML 1/50 MRV (mechan. No. 1594), ATSC reg. No. CRL 11770, deposited on 6 December 1994.
The deposition was carried out in accordance with the Budapest Treaty on the International agreement concerning the Deposit of microorganisms for purposes of patent procedure (Budapest Treaty). The maintenance of viable cultures is guaranteed for 30 years from the date of Deposit. These microorganisms, in accordance with the Budapest Treaty will be available to the public without any restrictions after issuance of the patent on the application. Availability of the deposited strain should not be construed as a license for carrying out the invention, because it is contrary to the rights granted by any government in accordance with the patent laws of this state.
It must be borne in mind that illustrated and described above, embodiments of the invention should be considered as preferred, and it can be made various changes, substitutions and modifications, does not extend, however, beyond being and scope of the following claims. Therefore, it should be noted that the protection provided by the patent deed, limited only by the attached claims and their equivalents.
All cited works are introduced in the present description is the exploits of links.
1. Substantially homogeneous polypeptide mpl ligand selected from the group consisting of
(a) a polypeptide fragment represented by the formula
X represents aminobenzene the amino group or the amino acid(s) residue(s)selected from the group
MPPA(SEQ ID NO: 117),
MARR (SEQ ID NO: 118),
MPAPPA (SEQ ID NO: 119),
MSPAPPA (SEQ ID NO: 120),
APPA (SEQ ID NO: 121),
PAPPA (SEQ ID NO: 122),
SPAPPA (SEQ ID NO: 123),
Y represents carboxykinase carboxypropyl or amino acid(s) residue(s)selected from the group
VRRA (SEQ ID NO: 124),
VRRAP (SEQ ID NO: 125),
VRRAPP (SEQ ID NO: 126),
VRRAPPT (SEQ I D NO: 127),
VRRAPPTT (SEQ ID NO: 128),
VRRAPPTTA (SEQ ID NO: 129),
VRRAPPTTAV (SEQ ID NO: 130),
VRRAPPTTAVP (SEQ ID NO: 131),
VRRAPPTTAVPS (SEQ ID NO: 132),
VRRAPPTTAVPSR (SEQ ID NO: 133),
VRRAPPTTAVPSRT (SEQ ID NO: 134),
VRRAPPTTAVRSRTS (SEQ ID NO: 135),
VRRAPPTTAVRSRTSL (SEQ ID NO: 136),
VRRAPPTTAVRSRTSLV (SEQ ID NO: 137),
VRRAPPTTAVRSRTSLVL (SEQ ID NO: 138),
VRRAPPTTAVRSRTSLVLT (SEQ ID NO: 139),
VRRAPPTTAVRSRTSLVLTL (SEQ ID NO: 140),
VRRAPPTTAVRSRTSLVLTLN (SEQ ID NO: 141),
VRRAPPTTA VRSRTSLVLTLNE (SEQ ID NO: 142),
VRRAPPTTAVRSRTSLVLTLNEL (SEQ ID NO: 143),
VRRAPPTTAVRSRTSLVLTLNELP (SEQ ID NO: 144),
aminobenzene and inoculate sequence, containing one or more of the remaining residues of the human ML, presented in figure 1 (SEQ ID NO: 1)and its conjugated with PEG forms;
(b) the chimeric polypeptide containing the mpl ligand, amino acid sequence which includes amino acid residues 1 to x of figure 1 (SEQ ID NO: 1), where X represents an amino acid residue in position 153, 155, 164, 174, 191, 205, 207, 217, 229, 245 or 332, fused with a heterologous polypeptide; and
(b) a polypeptide containing the N-terminal residues from 1 to about 153-157 hML, replaced remaining human EPO, added or replacement of N-terminal residues of hML in positions selected from the group comprising 1, 3-6, 9, 13-15, 17-28, 30, 32-34, 36 to 41, 43-50, 53-59, 61-68, 71, 73, 75-80, 82-88, 90-96, 98-99, 102-103, 105-107, 110, 112-113, 115-119, 121-130, 132-133, 135, 137-140, 143-150 and 152-156 in accordance with the alignment shown in figure 10;
moreover, the specified polypeptide stimulates the incorporation of labeled nucleotides (3H-thymidine) into the DNA of IL-3-dependent cells Ba/F3 transfected with human mpl P; or stimulates incorporation into circulating platelets35S.
2. The polypeptide according to claim 1, characterized in that the amino acid sequence of the specified polypeptide fragment contains
SPAPPACDLRVLSKLLRDSHVL (SEQ ID NO: 110),
HSRLSQCPEVHPLPTPVLLPAVDF (SEQ ID NO: 111),
SLGEWKTOMEETKAODILGAVTL (SEQ ID NO: 112),
LLEGVMAARGQLGPTCLSSLL (SEQ ID NO: 113),
GQLSQVRLLLGALQS (SEQ ID NO: 114),
LLGTQLPPQGRTTAHKDPNAIF (SEQ ID NO: 115) or
LSFQHLLRGKVRFLMLVGGSTLCVR (SEQ ID NO: 116)
or combinations thereof.
3. The polypeptide according to claim 1, characterized in that it does not contain oligosaccharide groups.
4. The polypeptide according to claim 1, characterized in that hTPO selected from the group consisting of hML (SEQ ID NO: 6), hML153 (SEQ ID NO: 1, group 1-153), hML (R153A, R154A) (SEQ ID NO: 1, R153A, R154A), hML2 (SEQ ID NO: 8), hML3 (SEQ ID NO: 9), hML4 (SEQ ID NO: 10), mML (SEQ ID NO: 15), mML3 (SEQ ID NO: 16), pML (SEQ ID NO: 18) and pML2 (SEQ ID NO: 21).
5. The polypeptide according to claim 1, characterized in that it has at least 80%sequence identity with any of the polypeptide, amino acid sequence which includes amino acid residues 1 to x of figure 1 (SEQ ID NO: 1), where X is chosen from the group 153, 155, 164, 174, 191, 205, 207, 217, 229, 245 and 332.
6. The polypeptide according to claim 5, characterized in that X represents a 153.
7. The polypeptide according to claim 1, wherein the heterologous polypeptide in the composition of the chimeric polypeptide is a polypeptide immunoglobulin.
8. The polypeptide according to claim 1, wherein the heterologous polypeptide in the composition of the chimeric polypeptide is a polypeptide interleukin.
9. The polypeptide according to claim 1 encoded by a nucleic acid containing a sequence having at least 70%sequence identity with any of the nucleic acid represented in figure 1 (SEQ ID NO: 2), or complement the nuclear biological chemical (NBC her sequence.
10. The nucleic acid molecule encoding a polypeptide ligand mpl, characterized in claim 1.
11. The nucleic acid molecule of claim 10, characterized in that it encodes a polypeptide described in item 5.
12. The nucleic acid molecule of claim 10, characterized in that it contains the nucleotide sequence of an open reading frame presented in figure 1 (SEQ ID NO: 2).
13. Expressing a vector containing a nucleic acid molecule encoding a polypeptide ligand mpl, characterized in claim 4, optional functionally associated with the control sequences recognized by the host-cell, transformed by the specified vector.
14. A method of obtaining antibodies against the polypeptide ligand mpl involving the immunization of a mammal and isolation of antibodies, characterized in that for immunization using the polypeptide described in item 5.
15. A method of obtaining a polypeptide ligand mpl, characterized in claim 5, providing for the cultivation of host cells and selection of a specified polypeptide, characterized in that cultivation use cell host transformed by the vector, described in item 13, and where the selected polypeptide optionally contains oligosaccharide groups.
16. The method according to clause 15, where the polypeptide of the mpl ligand isolated from the host cell
17. The method according to clause 15, where the polypeptide of the mpl ligand isolated from the culture medium of the host cell.
18. The method of determining the presence of the gene encoding the polypeptide ligand mpl described in claim 1, in a test sample containing nucleic acid, involving the hybridization of the DNA probe with a nucleic acid test sample, characterized in that as a DNA probe using a DNA molecule having at least 80%homology with sequence SEQ ID NO: 2 or its fragment.
19. Composition for stimulating the proliferation, maturation and/or differentiation of megakaryocytes, containing the active agent and pharmaceutically acceptable carrier, wherein the active agent is a polypeptide ligand mpl, characterized in claim 1.
20. The composition according to claim 19, characterized in that it further comprises a therapeutically effective amount of an agent selected from the group consisting of a cytokine, colony stimulating factor and interleukin.
21. The composition according to claim 20, characterized in that the said agent is selected from KL, LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 and IL-11.
FIELD: genetic engineering, molecular biology, pharmacy.
SUBSTANCE: invention relates to methods of genome screening and can be used for identification of pharmacological agent in vegetable extract. Method is realized by treatment of cells with a vegetable extract, isolation of protein or RNA from these cells, identification of isolated protein or RNA wherein their concentration differs from that in untreated cells and detection of compound(s) in indicated vegetable extracts. Then cells are treated with the found compound(s), protein or RNA are isolated from cells treated with this compound(s) and compound(s) are identified that cause the stimulation or inhibition of expression of protein or RNA wherein their concentration differs from that in untreated cells. Invention provides carrying out the characterization of biological properties of vegetable extract and to detect the individual compound(s) that elicit unknown or disclosed biological property of this extract.
EFFECT: improved identifying method.
FIELD: biotechnology, genetics.
SUBSTANCE: invention relates to methods used for detecting low frequency mutations occurrence in gene encoding cytochrome b. Method involves isolation of DNA from known fungi for constructing oligonucleotide probe or primer. Then polymerase chain reaction (PCR) is carried out for assay of binding the nucleotide probe with amplicon generated by this reaction, or the presence of amplicon is detected that is generated as result of PCR using indicated primers. Invention provides rapid and precise detection of mutations conferring resistance of fungus against fungicide.
EFFECT: improved diagnostic methods for detecting mutations.
24 cl, 18 dwg, 14 tbl, 18 ex
FIELD: biotechnology, medicine, proteins.
SUBSTANCE: invention describes new polypeptide in isolated form relating to subfamily of superfamily human immunoglobulins (Ig-Sf). This polypeptide shows at least 70% of homology level with amino acid sequence of murine molecules CRAM-1 or CRAM-2 regulated by the confluence of adhesive (figures 3, 6 are represented in the claim). Also, invention relates to antibodies showing specificity with respect to the polypeptide. Antibodies and soluble polypeptide can be used for treatment of inflammation and tumors. Invention describes polynucleotide or oligonucleotide encoding the full-size polypeptide or its moiety and represents primer, probe, anti-sense RNA and shows the nucleotide sequence that is identical conceptually with human CRAM-1. Invention provides preparing new adhesive proteins from superfamily Ig-Sf that are regulated at the transcription level in endothelium by effect of tumors. Invention can be used for treatment of different diseases, in particular, inflammatory responses.
EFFECT: valuable medicinal properties of polypeptide.
19 cl, 33 dwg, 1 ex
FIELD: medicine, psychiatry.
SUBSTANCE: one should isolate DNA out of lymphocytes of peripheral venous blood, then due to the method of polymerase chain reaction of DNA synthesis one should amplify the fragments of hSERT locus of serotonin carrier gene and at detecting genotype 12/10 one should predict the risk for the development of hallucino-delirious forms of psychoses of cerebro-atherosclerotic genesis.
EFFECT: more objective prediction of disease development.