Streptokinase mutants and their covalently modified forms
SUBSTANCE: invention relates to biochemistry, in particular, to novel mutants of streptokinase. Claimed are both mutant streptokinase polypeptides and fusion proteins, possessing streptokinase activity. Claimed streptokinases are included into formulations of pharmaceutical compositions, suitable for treatment of blood circulation diseases, in particular thromboses.
EFFECT: invention makes it possible to obtain more effective mutant polypeptides with streptokinase activity in treatment of blood circulation diseases.
20 cl, 37 tbl, 10 ex
The technical field to which the invention relates.
The present invention relates to mutant streptokinase and covalently modified forms. The present invention makes use of streptokinase and its related variants obtained by replacing, attaching, removing or creating structures by merging domains, modified PEG homogeneous, site-specific and particular way that allows their use as improved protein therapeutics.
The present invention relates to the covalent attachment of PEG to cysteine variants of streptokinase, it muteena, species or products merge with fibrin, when using reagents based on the derivatives of PEG, reacting with thiol groups. Can be also used different values pKaalpha-amino groups to conduct specific conjugation of the PEG at acidic pH with the aim of obtaining monopegylated variants of streptokinase or its Malinov.
The present invention relates also to identify various cysteine variants of streptokinase or its mutants, including covalent related options on the basis of structural and functional information (Wang et al., 1998). Structural comparison with other single-plasminogen activator, staphylokinase (SAK), the decree which provides for substantial similarity to the alpha-domain SC, despite the fact that both proteins do not possess significant homology sequences at the amino acid level (Rabijns et al., 1997). This indicates that the plasminogen activators from various sources maintain the same type of structural styling, even if they differ considerably in their polypeptide sequence. It can be expected that the evolutionary constraint retains structural integrity, because the bacterial activators of plasminogen are protein cofactors, therefore, they use a variety of contact points required for conformational activation of zymogen. This structural similarity further increases the scope of the present invention, since the methods used in this case, can be applied to bacterial activators of plasminogen other types or species, which are involved in the activation of plasminogen. Therefore, the "rules" here for the biologically active cysteine variants of streptokinase, will be used for the production of biologically active cysteine variants of various other forms of streptokinase. Conjugation of these cysteine variants PEG derivative that can react with cysteine leads to the same advantages of using derivatives in the present research the research Institute. Principles of site definition, which is cysteine, mainly based on the idea of selecting residues that fall either in a loop or spiral, or in the border area between structured and flexible areas. For determining the surface distance used by the program DSSP. The program DSSP (Kabsch et al., 1983) defines the secondary structure, geometric properties and the availability of proteins in relation to the solvent defined by the atomic coordinates in Protein Data Bank format. DSSP sets the accessibility of each residue in square angstroms. Surface accessibility of amino acid residues streptokinase was deciphered from the data on the crystal structure, high-resolution streptokinase in complex with microplasmin (Wang et al., 1998, PDB ID 1BML). For areas that were missed in this structure (175-181 and 252-262) in determining the surface distance using the crystal structure of the isolated beta-domains (Wang et al., 1999, PDB ID 1c4p).
Cysteine variants of streptokinase, it mutiny derived species and its covalently modified forms further chemically modified by the addition of reagents for sulfhydryl groups with subsequent empirical testing for significant biological activity, along with the acquisition of new properties, such as lower the Naya immunogenicity or reaction with anti-ck antibodies, reduced sensitivity to proteolysis, increased survival in vivo, etc. More specifically, the present invention relates to the production of engineered variants of streptokinase for the application for use in pharmaceutical compositions for the treatment of vascular diseases.
The level of technology
The formation of a thrombus (blood clot) in the blood vessels can cause blockage of blood vessels, leading to fatal consequences. The formation of the clot and its dissolution is a strictly controlled process of homeostasis. Any deviation from normal blood clotting leads to various clinical pathological condition, such as stroke, embolism of the lungs, deep vein thrombosis and acute myocardial infarction. Pathophysiological condition developing in breach of the blood clotting process, require nizamedinova clinical intervention. The most commonly practiced medical intervention consists in the introduction of thrombolytic agents (Collen et al., 1988; Collen, 1990; Francis and Mafder, 1991). The most commonly used thrombolytic agents include streptokinase SK (SK), urokinase (UK) and tissue plasminogen activator (TPA). Previously there have been numerous pharmacoeconomic evaluation of using different thrombolytics for a sharp correction is th myocardial infarction (Mucklow, 1995; Gillis and Goa, 1996). Banerjee et al., 2004, reviewed the clinical use of streptokinase and its application as the best choice of drug for treatment. With regard to clinical effectiveness, as streptokinase and TPA equally are good drugs, but due to the fact that streptokinase several orders of magnitude cheaper, and better in respect of the half-life in vivo, it is worldwide is the preferred thrombolytic (Sherry and Marder, 1991, Wu et al., 1998). In addition, the use of TRA somewhat more likely to cause a stroke, the most common side effects for both drugs. However, streptokinase, being a bacterial protein, is the nature of the antigenic and may cause clinical complications such as allergic reactions or bleeding. In addition, the half-life of circulating streptokinase (15-30 min) is not sufficient for effective thrombolite (Wu et al., 1998).
Despite all this recent thrombolytic therapy with fibrinolytic agents such as streptokinase (SK), tissue plasminogen activator (TPA) or urokinase (UK) completely changed the clinical application of various diseases of the circulatory system, for example, deep vein thrombosis, embolism blood vessels of the lungs and acute myocardial infarction. These agents exert their fibrinoliticeski the properties through the activation of plasminogen PG (PG) in circulation by cleavage of the labile peptide bonds between residues 561 and 562 PG. In the inactive zymogen becomes active, the serine protease, plasmin PN (PN), which then circulates in the system and acts on fibrin, destroying the latter with the formation of soluble degradation products. Here it should be mentioned that himself PN is not able to activate PG to MO; this reaction is catalyzed by highly specific proteases, such TRA, complex SK-plasminogen and UK, they all have extremely high specificity with respect to protein substrates, namely the ability to cleave the labile peptide bond in GHG highly specific way. However, unlike the UK and TPA, SK lacks proteolytic activity and activates GHG MO "indirectly", i.e. forming a first vysokoaffinnye equimolar complex with PG, known as an activator complex (reviewed Castellino, F. J., 1981). Then activator complex, acts as a protease, which cleaves other molecules of substrate PG to MO.
Despite the enormous benefits of therapeutic use of streptokinase and other bacterial thrombolytics has several disadvantages that limit the applicability of these polypeptide drugs. These disadvantages include their susceptibility to degradation by proteolytic enzymes, low half-life in the circulation, small storage period, fast in the maintenance of the kidneys and their ability to generate neutralizing antibodies. These shortcomings also sometimes inherent in many other polypeptide drugs are inherently different from the polypeptides of the person. This aspect is in General considered Roberts et al.; 2002. Have been made various attempts to address these shortcomings polypeptide drugs, for example, a change in amino acid sequence to reduce proteolysis or antigenicity, attaching polypeptides to domains globulin or albumin for longer half-life (Osborn et al., 2002).
These methods are of little use to solve problems and carry additional charges. The main breakthrough in this area is pegylation of proteins, providing the only solution to many problems. PEG (polyethylene glycol) is formed by polymerization of several repeating subunits of ethylene glycol, leading to linear or branched polymers PEG preplanned molecular masses. Being covalently conjugated with PEG, proteins or polypeptides exhibit improved pharmacokinetic or pharmacodynamic properties, such as increased water solubility, decrease renal clearance and often significant decrease of immunoreactivity (Moreadith et al., 2003, Doherty et al., 2005, Basu et al., 2006). Conjugation with PEG also makes the molecule less susceptible to proteolysis. Humanitarianassistance with the receptor or interaction with surface proteins of cells, due to the accession of the PEG, also contributes to the suppression of adverse immunological effects. Paglierani drugs are also more stable in a wide range of pH and temperature changes (Monfardini et al. 1995). The use of PEG approved by the FDA for therapeutic agents and shows that he practically has no toxicity and is excreted in unchanged form or through the kidneys, either together with the faeces. Useful properties of conjugation with PEG can be potentially reported IC in order to make it more effective and safe thrombolytic. Attempt paglierani IC using relatively nonspecific reaction of the chemical modifications described in the literature (Ms. Rajagopalan et al., 1985). Therapeutic applications of such modifications is strictly limited to the high anomalous ability of plasminogen activation. In addition, poorly controlled nature of the modification, which may be heterogeneous. The reason for this heterogeneity are chemical transformations used to modify the PEG, which is not address specific modification site. Therefore, this strategy modifications are not used to produce improved thrombolytic drugs on the basis of the UK.
The term streptokinase used wherever collectively in the text, refers to variants of streptokinase, any of its f the purpose ground receiving stations fragments, functional muteena, isolates from different samples and the condensation products obtained by joining oligo - or polypeptides of natural or synthetic origin.
It is known that various functional groups in the protein molecule, can be used for the introduction of the PEG. The most frequently used methods is the derivatization lysine residues or cysteine residues in proteins. Alpha-amino group at the N-terminal fragment can also be used for a single homogeneous conjugation of PEG to proteins (Baker et al., 2006). However, the use of cysteine residues for attachment of PEG groups is particularly preferred, because potentially SH-groups can be used as a target site-specific manner, especially if the protein contains, or may be arranged in such a way that it contained a very limited number of cysteine residues. It is no exaggeration to assume that conjugation with PEG becomes an art form when the protein is devoid of any cysteines, because it is a virtual blank canvas for accession, the introduction of cysteine or replacement of a cysteine for site-specific "painting" PEG, or scenery proteins. Because potentially joining cysteines it does not contain cysteine substrate may have nebago etoe effect on the function of a protein. Therefore, the choice of sites for receiving cysteine variants requires careful planning and execution. As opposed to, say, pegylation, based on a modification of the lysine, while the chemistry of this reaction is well known that heterogeneity is a significant drawback. In the case of SC large number of lysine residues are evenly distributed along the polypeptide and therefore limit the ability of a homogeneous site-specific PEG conjugation. Interesting that in the streptokinase molecule no natural cysteine (Malke et al., 1985), so that it becomes possible to obtain different variants of streptokinase. In addition, natively folded covalent derivatives of SK generated at the confluence with fibrillazione domains (reference US Patent 7163817). This gives the possibility of obtaining various options specific to blood clots streptokinase containing a free cysteine in the absence of the actual factors that interfere with the normal folding of the protein, rich in cysteine (all cysteine residues involved in disulfide bond formation). Enter the free cysteine(s) may interact with various reagents on tirinya groups, including PEG, with the formation of cysteine adducts of these proteins.
Streptokinase (SK) is a species designation secretory protein, formed the centre of the PTO hemolytic streptococci, able to induce lysis of plasma clots (Tillet and Gamer, 1933). Therefore, it can easily and economically be obtained from its main source, or when using rDNA technology of suitable heterologous hosts, SC very cost-effective and therefore is the main thrombolytic drug connection, especially for global markets responsive to price. It is shown that SC is very effective in the clinical treatment of acute myocardial infarction accompanied by thrombosis of the coronary arteries (1SIS-3, 1992), and acts as a thrombolytic agent for more than three decades. However, it has several disadvantages. It is known that the plasmin generated during activation of plasminogen, streptokinase-mediated splits the streptokinase soon after its introduction (Ms. Rajagopalan et al., 1985, Wu et al., 1998). This limits the half-life of streptokinase in vivo approximately 15 min (Wu et al., 1998). Although streptokinase remains in the bloodstream much longer than the selected other thrombolytic drug, TPA (with a half-life equal to less than 5 min; Ross, 1999; Ouriel, 2002), it is not enough for effective therapy (Wu et al., 1998). Because of the deficiencies associated with the rapid excretion in vivo available plasminogen activators, attempts to obtain improved recombinant PR is spodnie these compounds (Nicolini et al., 1992, Adams et al., 1991, Lijnen et al., 1991; Marder, 1993, and Wu et al., 1998). Despite its internal problems streptokinase remains the preferred drug, especially in developing countries, because of its relatively low cost (for example, approximately US $ 50 or less on the course of treatment compared to nearly US $ 1500 for TRA).
First it is shown that streptokinase causes the lysis of blood clots (Tillet and Gamer (1933)). However, it was later shown that the fibrinolytic activity of the SC due to its ability to activate plasminogen person PPP (HPG), described Castellino, 1979. Streptokinase is mainly secreted β-hemolytic group a, C and G streptococci. SK represents the activator GHG man, although she is not a protease, rather it is associated with PG/MO person and attracts other molecules HPG as a substrate and turns them into a product, MO. Last circulates in the bloodstream. The plasmin, being non-specific protease, generalized and intermediate generation MO after injection of IC leads to the widespread destruction of various factors of the blood, which leads to the risk of bleeding, as well as to the dissolution of the ECM and basement membrane (BM) and an increase in bacterial invasiveness in the secondary centers of infection in the host's body (Esmon and Mather, 1998; Uihteenmaki et al., 2001). Thus, there is an acute necessity is the cost to minimize side effects design improved analogues of the UK.
Currently, SC intensively used as a thrombolytic medicines all over the world, because it is an effective solvent of fibrin clots, but it has its own limitations. SC is a protein produced from hemolytic streptococci, its use in humans induces immunogenicity (McGrath and Patterson, 1984; McGrath et al., 1985; Schweitzer et al., 1991). It is known that high titers of anti-SA immunoglobulin (Ig) generated after the first SC injection, remain with the patient during the period from several months to several years (Lee et al., 1993). Thus, anti-SK antibodies significantly limit its application as a prolonged therapy or due to neutralization of the SC in the introduction (Spottal and Kaiser, 1974; Jalihal and Morris, 1990), or due to the fact that it raises allergic reactions (McGrath and Patterson, 1984; McGrath et al., 1985).
As mentioned above, the use of streptokinase thrombolytic therapy prevents the relatively short half-life (a few minutes) of this protein in vivo (which is actually observed for all currently used thrombolytic drugs), in addition to its immunogenicity. It is shown that the foreign proteins when introduced into the bloodstream of the vertebrate often rapidly excreted through the kidneys. This situation becomes even bol is e acute in the case of streptokinase, when gradually increasing doses of protein (in order to exceed the rapid neutralization caused by the antibody) can significantly increase the likelihood of an allergic response/s, which makes the re-introduction largely ineffective and dangerous. Usually attempts to solve these problems in the literature are well documented, where it is shown that to obtain the improved medicinal substances suitable various physical and chemical modification, for example, see: Mateo, C. et al., 2000, Lyczak, J. C. & Morrison, S. L. 1994, Syed, S. et. al.; 1997, Allen, T. M. 1997. The most promising of them is currently the modification of therapeutic proteins covalent joining polyalkylbenzene polymers, e.g. polyethylene glycol (PEG). PEG is neoantigenic, inert polymer and it is known that it increases the half-life of circulating proteins in the body (Abuchowski et al., 1984; Hershfield, 1987; Meyers et al., 1991). It provides long-lasting action of medicinal substances in their use. Believe that conjugation of PEG to proteins increases their overall size and therefore reduces the rapid renal elimination. In addition, attaching a PEG attached to a protein or polypeptide greater solubility in water and increases their stability in vivo, along with greatly reduced and is monogenist and increased stability in vivo (Katre et al., 1987; Kcitre, 1990). U. S. Pat. No.. 4,179,337 discloses the use of PEG or polipropilenglikol attached to proteins, to obtain physiologically active non-immunogenic water soluble polypeptide composition.
Although the chemistry of conjugation of PEG is mostly universal, the key location of the polymers PEG in therapeutic protein is paramount to achieving successful results. The availability of information about the three-dimensional structure with functional active areas identified through various studies in solution, helps in the development of the plan of introduction of mutations to keep the functionality the same.
Complete amino acid sequence SK is defined sequential degradation analysis on Adminu fragments SC received bromocyanogen and enzymatic methods (Jackson and Tang, 1982). The results showed that a molecule with a molecular mass Mrequal 47408 Yes, contains 415 amino acids in a single polypeptide chain of amino acid sequence.
The nucleotide sequence from S. equisimilis H46A (strain prototype for receiving the IC, which are most often used therapeutically for the treatment of humans) was sequenced Malke with employees in 1985. It was also studied the transcriptional control of this gene and described functional Ana is from its complex promoter (Grafe et al., 1996). Consequently, there is significant information for the effective use of this gene in order with confidence to receive streptokinase in a relatively non-pathogenic microbes.
The purpose of the invention
The main purpose of the present invention is the provision of mutant streptokinase, with increased efficiency due to a more prolonged action and low immunoreactivity.
Another objective of the present invention is the selective modification of streptokinase to improve its pharmacokinetic properties and give it the properties of a therapeutically effective thrombolytic and increased proteolytic stability and longer half-life in vivo.
Another objective of the present invention is the selective modification of streptokinase in order to retain the desired biological properties inherent to unmodified molecule.
Another purpose of this invention is the provision of a derivative of the IC, which provides a longer lasting and more effective fibrinolysis in a particular site with reduced immunoreactivity.
Another purpose of this invention is the provision of a method of obtaining paglierani cysteine variants of streptokinase or its active Malinov or molecules of hybrid molecules of activator PLA is minogin in pure and biologically active form.
Disclosure of inventions
Therefore, the present invention provides mutant streptokinase, its functional fragments or covalently modified forms. Derivatives include polypeptides belonging to SC, where one or more cysteine residues substituted by one or more nonessential amino acids preferably, derivatives contain a cysteine residue is replaced with an amino acid selected from amino acids included in the area of the loop, the ends of alpha helices and even in the field, forming a secondary structure, or area where cysteine residues attached to the N-end-or-end of the protein molecule, in the presence or absence of amino acid extensions.
The present invention includes common methods of selection, production and application of streptokinase exhibiting increased proteolytic stability, increased half-life from plasma and reduced immunogenicity. Derivatives have modified amino acid sequences, but actually retain their biological activity. The present invention also describes cysteine variants of streptokinase, covalently associated with one or more molecules of polyethylene glycol (PEG) of various molecular weights, for example, 5, 10, 20 and 40 kDa. One of the embodiments of the present invention Casa is tsya pharmaceutical compositions options paglierani streptokinase together with suitable inert fillers, stabilizers and carriers known in the art for effective distribution in the body in the treatment of various diseases of the circulatory system.
The implementation of the invention
The present invention is based on the experimental results, indicating that the covalent addition of one or more molecules of PEG to strategically substituted or added cysteine residues in the molecule streptokinase leads to biologically active paglierani the streptokinase with increased proteolytic stability and longer half-life and less immunoreactivity compared with native streptokinase. The localization and the number of molecules with PEG-conjugation can be selected using cysteine variants, designed in such a way that they could not inhibit the catalytic function (activation of plasminogen) in the streptokinase and its active variants, including streptokinase specific to blood clots (in this context can refer to U. S. Patent 7163817, which describes the structure and design options streptokinase specific to blood clots, increased fibrinolytic activity due to the introduction fibrinspecific domains on either or both ends of the protein molecule SC). Replacement, connection or introduction of one or not is how many molecules of cysteine in the streptokinase and streptokinase polypeptide, specific to blood clots, creates conditions for the desired localization when attaching a PEG of different molecular masses of the polypeptides provided that replacement and/or accession conduct "strategically" completely, avoiding loss of functional properties, and leads to production of new useful properties that are not in unmodified native protein. The choice of the location of the PEG design, based on the surface distance of the selected site and its structural and functional significance.
Paglierani streptokinase of the present invention have greater applicability as therapeutic agents, as well as large advantages compared with native molecule(s), as preserving native or nearly native biological activity, they have a longer latency period compared with the first, which quickly disintegrate in vitro in the presence of plasmin (plasminogen), and after injection, with the result that they are very rapidly eliminated from the bloodstream. On the contrary, paglierani streptokinase show significantly higher proteolytic stability, are immunoreactive and removed from the bloodstream in vivo only after significantly increased period of time compared with native (naegeliana) proteins.
Therefore, paglierani with whom replikins of the present invention is effective for the treatment of subjects with impaired blood circulation, for example, with venous or arterial thrombosis, myocardial infarction and so on, having the advantage that paglierani streptokinase of the present invention have the potential to increase efficiency by increasing the duration and reduction of immunoreactivity, which gives the possibility of re-introduction due to the minimal antigenicity. The number, size and localization of the groups(s) PEG can be used in this way to reconstruct streptokinase for implementation of different half-life times, so you can use them according to the requirements of a specific disease/clinical syndrome, or a specific patient.
The present invention includes selective modification of streptokinase for pharmaceutical applications as to strengthen their pharmacokinetic properties, and to provide a therapeutically effective thrombolytic drugs. This invention also includes a mutant streptokinase, natural or artificial variants that retain the desired biological properties of the native unmodified molecules. All derivatives of the present invention can be obtained by expressionism sequence of recombinant DNA that encodes a desired derived host cells, for example, prokaryotic cells x is zaina, for example, E. coli or in eukaryotic host cells, for example, yeast cells, or mammalian cells, using the commonly used materials known in the art. Information on DNA sequences for encoded streptokinase of different drugs can be obtained from published data. We studied the polymorphism of streptokinase and explained his role in the pathogenesis (Malke H, 1993). Also conducted a molecular epidemiological study to determine the distribution of the gene streptokinase group a streptococcal strains of different M types and other drugs Streptococcus. The majority of the studied strains in this study showed positive streptokinase activity according to the overlay test casein-plasminogen. The cumulative results of these studies show that there is considerable heterogeneity among streptokinase obtained from different streptococcal drugs (Huang et al.; 1989). You can use any of the available variants of streptokinase, which are able to activate plasminogen to mutagenesis of cysteine and subsequent modifications reactive sulfhydryl reagents.
New DNA sequences encoding the mutants and species derivatives can be similarly cloned and expressed, the AK and in the case of natural forms. Then streptokinase, obtained by expression in genetically engineered host cells can be purified and, if desired, converted into pharmaceutical compositions in the usual way.
As a preferred aspect of the present invention streptokinase, expressed by recombinant react with the target reactive agents on Tilney group under conditions that allow the accession of balance, reactive Tilney group, sulfhydryl group introduced cysteine residues in streptokinase.
The term "reactive Tilney group" is defined here as any compound having a reactive group or able to be activated by formation of a reactive group able to join the sulfhydryl group (-SH) of cysteine residue. Among such compounds include polymers, for example, polypropylenglycol and PEG, polymers based on carbohydrates and polymeric amino acids and derivatives of Biotin. The connection is subject to conjugation, can be activated sulfhydryl residues, for example, a sulfhydryl group, thiol, triflate, tresilian, aziridine or oxirane or preferably by iodoacetamide or maleimide. Conjugating group may have a different molecular weight, preferably, from 500 to 40000 for the PEG. One of the most important features of the present invention is to make the positional selectivity of the pegylation or other ttings while maintaining the functional properties of the protein.
Thus, the present invention provides mutant streptokinase polypeptide with the amino acid sequence selected from the group consisting of SEQ ID NO: 1-24, where at least one cysteine residue substituted or embedded. Table 34 presents the residues corresponding to residues of SEQ ID NO: 1, which, perhaps, are intolerant to mutation or replacement.
In one embodiment of the present invention obtained mutant streptokinase is a functional fragment having SEQ ID NO: 2-6.
In one embodiment of the present invention obtained mutant streptokinase are mottainai streptokinase having SEQ ID nos: 7-19.
In one embodiment of the present invention obtained mutant streptokinase is a species variants of streptokinase having SEQ ID NO: 20-21.
In one embodiment of the present invention for species variants of streptokinase homology of amino acid sequence is 75% -100% relative to the native streptokinase having SEQ ID NO: 1.
In another embodiment of the present invention, at least one cysteine residue substituted for at least one of aminos what slotow, located in at least one region of streptokinase selected from the group consisting of: loop 48-64, loops 88-97, region 103-106 or 119-124 or spiraleobraznymi region 196-207, or petrobrazi region 170-181, or petrobrazi region 254-264, or super helical conformation was the area 318-347, or area 360-372 in SEQ ID NO: 1 or its Malinov or their functional fragments, where the specified derivative possesses a biological activity, measured by the standard test.
* In another embodiment of the present invention the polypeptide mutant streptokinase contains from one to three cysteine substitutions, where cysteine replacement is an acid, localized, at least in one area of the corresponding native sequence of amino acid residues streptokinase (SEQ ID NO: 1), in the field, selected from the group consisting of loop amino acid residues 48-64, loop amino acid residues 88-97, region amino acid residues 102 to 106, the field of amino acid residues 119-124, spiraleobraznymi region amino acid residues 196-207, petrobrazi region amino acid residues 170-181, petrobrazi region amino acid residues 254-264, super helical conformation was the field of amino acid residues 318-347 and the region of amino acid residues 360-372, where the mutant can activate plasminogen.
As used here, the term "appropriate to ejstvujuschij" used to refer to the numbered positions in the reference protein, for example, streptokinase (SK) wild-type or SEQ ID NO: 1, and these provisions in the desired protein (for example, mutant SC) that align with the provisions in the reference protein. Thus, when the amino acid sequence of the tested IC, i.e., SEQ ID NO: 2, 3, 4, 5, and so on, aligned with the amino acid sequence of the reference protein, i.e., SEQ ID NO: 1, amino acids in the sequence of the tested IC, which "correspond to" certain enumerated provisions of the reference sequence SK is such that is consistent with these provisions in the sequence of the reference IC, but not necessarily in these exact numbers the provisions of the sequence of the reference IC. For example, the mutant Gly34Cys in SEQ ID NO: 4 has to "match" the mutant Gly49Cys in SEQ ID NO: 1.
** In another embodiment, the present invention uses a mutant, referred to in paragraph * optionally containing fibrinolitikami domain attached to the C-end, N-end or to both ends.
In another embodiment, the present invention uses the mutant, which was mentioned in the previous embodiment, where fibrinolitikami domain contains at least one substitution of a cysteine.
*** In another embodiment, the present invention uses the mutant, which was mentioned in the previous embodiment, where fibre the binding domain attached to the mutant streptokinase with flexible binding oligopeptides.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant streptokinase further comprises at least one additional amino acid substitution and amino acid substitution corresponds to the amino acid substitution selected from the group consisting of Asn90Ala, HisI07Ala, Serl08Ala, Asp227Tyr, Asp238Ala, Glu240Ala, Arg244Ala, Lys246Ala, Leu260Ala, Lys278Ala, Lys279Ala and Asp359Arg in SEQ ID NO: 1.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant streptokinase contains a deletion, and the deletion corresponds to amino acid deletions selected from the group consisting of Asn90, Asp227 and Asp359.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant further comprises amino acid extension at the N and/or C-end.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant streptokinase contains at least one cysteine mutation in the position corresponding to G49, S57, A, 188, S93, D95, D96, DI02, S105, D120, K, D122, E, K, D173, D174, L179, DI81, S205, A, 1254, N255, K, K, S258, L260, E, K, F287, D303, L32I, L326, A, D347, D360 or R372 in SEQ ID NO: 1.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where mutant strept the kinase contains at least two cysteine mutation at position the corresponding G49; S57, A, 188, S93, D95, D96, 0102, S105, D120, K, D122, E, K, D173, D174, L179, D181, S205, A, 1254, N255, K, K, S258, 1260, E, K, F287, D303, L321, L326, A, 0347, D360 or R372 SEQ ID NO: 1.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant streptokinase contains three cysteine mutation in the position corresponding to G49, S57, A, 188, S93, D95, D96, D102, S105, D120, K, D122, E, K, L173, D174, L179, D181, S205, A, 1254, N255, K, K, S258, L260, E, K, F287, D303, L321, L326, A, D347, D360 or R372 in SEQ ID NO: 1.
In another embodiment, the present invention uses a mutant, referred to in paragraph *, where the mutant is able to treat a disease or disorder selected from the group consisting of myocardial infarction, vascular thrombosis, pulmonary embolism, stroke, vascular complications, angina, transient ischemic attack, deep vein thrombosis, thrombolytic reocclusion after the procedure, coronary angioplasty, peripheral vascular thrombosis, heart surgery, vascular surgery, heart failure, syndrome X and diseases in which a narrowing of at least one coronary artery.
In another embodiment, the present invention uses a mutant, referred to in paragraph * optionally containing a reactive group that interacts with cysteine substituted for men is our least one cysteine mutants.
** In another embodiment, the present invention uses the mutant, which was mentioned in the previous embodiment, where the reactive group that interacts with the cysteine is polyethylene glycol (PEG).
In another embodiment, the present invention uses the mutant, which was mentioned in the previous embodiment, where PEG is a linear or branched polymer with a molecular weight in the range from 5,000 daltons to 40,000 daltons.
In another embodiment, the present invention uses the mutant, which was mentioned in the previous embodiment, where the mutant has an increased proteolytic stability as compared naegeliana mutant streptokinase.
In another embodiment, the present invention uses a mutant, referred to in paragraph **, where the mutant has reduced antigenicity and low immunogenicity in vivo compared naegeliana mutant streptokinase.
In another embodiment, the present invention uses a mutant, referred to in paragraph **, where the mutant has a slow renal clearance and increased half-life in vivo compared naegeliana mutant streptokinase.
*** Another variant of implementation relates to fused to the polypeptide, and fused polypeptide content the t domain of streptokinase, containing from one to three cysteine substitutions, where cysteine replacement is fibrinolysinum domain or at least one area corresponding native amino acid sequence of streptokinase (SEQ ID NO: 1), and this area is selected from the group consisting of loop amino acid residues 48-64, loop amino acid residues 88-97, region amino acid residues 102 to 106, the field of amino acid residues 119-124, spiraleobraznymi region amino acid residues 196-207, petrobrazi region amino acid residues, 170-181, petrobrazi region amino acid residues 254-264, super helical conformation was the field of amino acid residues 318-347 and the region of amino acid residues 360-372 in SEQ ID NO: 1, where the mutant can activate plasminogen to bind fibrin.
In another embodiment, the present invention uses a mutant, referred to in the preceding embodiment, where fibrinolitikami domain attached to the N-end domain of streptokinase, With the end of the domain of streptokinase or to both ends of the domain of streptokinase.
In another embodiment, the present invention uses a mutant, referred to in the preceding embodiment, where fibrinolitikami domain attached to the N-end domain of streptokinase and fused polypeptide contains at least one cysteine mutation selected from the group consisting of the C W16, A17, D62, G80, GI66, SI57, AI8I, 1205, S210, D212, D213, D219, D222, D237, K, D239, E, K. 273, D290, D29I, L296, D298, S322, 1371, N372, K, K, S375, L377, E, K, F404, D420, L438, L443, A450, D464, D477 and R489 in SEQ ID NO: 22.
In another embodiment, the present invention uses a mutant, referred to in the preceding embodiment, where fibrinolitikami domain attached to the N-end domain of streptokinase and fused polypeptide contains at least one cysteine mutation selected from the group consisting of G49, S57, A, I88, S93, D95, D96, D102, S105, D120, K, D122, E, K, D173, D174, L179, D181, S205, A, I254, N255, K, K, S258, L260, E, K, F287, D303, L321, L326, A, D347, D360, R372, N, A, D447 and G465 in SEQ ID NO: 23.
In another embodiment, the present invention uses a mutant, referred to in paragraph ***, where fibrinolitikami domain is connected both to the N-end and C-end domain of streptokinase and fused polypeptide contains at least one cysteine mutation selected from the group consisting of W16, A17, D62, G80, G166, S157, AI8I, 1205, S21O, D212, D213, D219, D222, D237, K. 238, D239, E, K. 273, D290, D291, L296, D298, S322, 1371, N372, K, K, S375, L377, E, K, F404, D420, L438, L443, A450, D464, D477, R489, N, A, D564 and G582 in SEQ ID NO: 24.
In another embodiment, the present invention uses a mutant, referred to in paragraph ***, where the mutant is able to treat a disease or disorder selected from the group consisting of myocardial infarction, vascular thrombosis, pulmonary embolism, stroke, vascular female oslojnenny is, angina, transient ischemic attack, deep vein thrombosis, thrombolytic reocclusion after the procedure, coronary angioplasty, peripheral vascular thrombosis, heart surgery, vascular surgery, heart failure, syndrome X and diseases in which a narrowing of at least one coronary artery.
**** In another embodiment, the present invention uses a mutant, referred to in paragraph * * * additionally contain reactive towards cysteine group, substituted by at least one cysteine mutants.
In another embodiment, the present invention uses a mutant, referred to in the preceding embodiment, where reactive towards cysteine group is polyethylene glycol (PEG).
In another embodiment, the present invention uses a mutant, referred to in the preceding embodiment, where PEG is a linear or branched polymer with a molecular weight in the range from 5,000 daltons to 40,000 daltons.
In another embodiment, the present invention uses a mutant, referred to in paragraph****where the mutant has an increased proteolytic stability as compared naegeliana mutant streptokinase.
In another embodiment of the present invention and the use of mutants, referred to in paragraph****where the mutant has reduced antigenicity and low immunogenicity in vivo compared naegeliana mutant streptokinase.
In another embodiment, the present invention uses a mutant, referred to in paragraph****where the mutant has a slow renal clearance and increased half-life in vivo compared naegeliana mutant streptokinase.
In yet another embodiment, the present invention SEQ ID NO: 22-24 are covalently modified hybrid polypeptide containing at least one functional fragment of streptokinase (SK) and fibrillazione domains 4 and 5, fibrillazione domains (FBDs) 1 and 2 fibronectin person.
In yet another embodiment of the present invention, a functional fragment of the IC and these fibrillazione domains associated flexible connecting Oligopeptide.
In yet another embodiment of the present invention described above, the mutant contains amino acid extension at the N and/or C-end.
In yet another embodiment of the present invention at least an amino acid selected from the group consisting of: G49, S57, A, I88, S93, D95, D96, D102, S105, D120, K, D122, E, K, D173, D174, L179, D181, S205, A, 1254, N255, K, K, S258, L260, E, K, F287, D303, L321, L326, A, D347, D360, R372 replaced by balance C is steina, where the specified derivative possesses a biological activity, measured by standard tests.
In yet another embodiment of the present invention at least an amino acid selected from the group consisting of: W16, A17, D62, G80, G166, S157, A, I205, S210, D212, D213, D219, D222, D237, K, D239, E, K, D290, D291, L296, D298, S322, 1371, N372, K, K, S375, L377, E, K, F404, 0420, L438, L443, A450, D464, D477, R489 in SEQ ID NO: 22, is replaced by a cysteine residue, where the specified derivative possesses a biological activity, measured by standard tests.
In yet another embodiment of the present invention at least an amino acid selected from the group consisting of: G49, S57, A, 188, S93, 095, D96, D102, S105, D120, K, D122, E, K, D173, D174, L179, D181, S205, A, 1254, N255, K, K, S258, L260, E, K, F287, D303, L321, L326, A, D347, D360, R372, N, A, D447, G465 in SEQ ID NO: 23, is replaced by a cysteine residue, where the specified derivative possesses a biological activity, measured by standard tests.
In yet another embodiment of the present invention at least an amino acid selected from the group consisting of: W16, A17, D62, G80, G166, S157, A, 1205, S210, D212, D213, D219, D222, D237, K, D239, E, K, D290, D291, L296, D298, S322, 1371, N372, K, K, S375, L377, E, K, F404, D420, L438, L443, A450, D464, D477, R489, N, A, D564, G582 in SEQ ID NO: 24, replaced by a cysteine residue, where the specified derivative possesses a biological activity, measured by standard tests.
Even the bottom of the embodiment of the present invention substituted cysteine residue is modified reactive towards cysteine group.
In yet another embodiment, the present invention cysteine residue is modified with polyethylene glycol.
In yet another embodiment of the present invention mentioned above, the PEG molecule is a linear or branched polymer with a molecular weight in the range from 5,000 daltons to 40,000 daltons.
In yet another embodiment of the present invention described above derivative has increased proteolytic stability as compared with the original non-modified counterparts.
In yet another embodiment of the present invention described above derivative has low antigenicity and low immunogenicity in vivo compared to the original non-modified counterparts.
In yet another embodiment of the present invention described above has derived a slow renal clearance, which increases the half-life in vivo than the original non-modified counterparts.
In yet another embodiment of the present invention the pharmaceutical composition contains at least one cysteine variants, optionally together with pharmaceutically acceptable(and) inert(and) filler(s).
In yet another embodiment of the present invention the pharmaceutical composition is the use of the ima for the treatment of diseases or disorders, selected from the group consisting of myocardial infarction, vascular thrombosis, pulmonary embolism, stroke, vascular complications, angina, transient ischemic attack, deep vein thrombosis, thrombolytic reocclusion after the procedure, coronary angioplasty, peripheral vascular thrombosis, heart surgery, vascular surgery, heart failure, syndrome X and diseases in which a narrowing of at least one coronary artery.
Especially the present invention is characterized paglierani cysteine variants of streptokinase or its Malinov or hybrid molecules of plasminogen activator containing polypeptide communication between streptokinase (SK), or modified forms of IC, or their respective parts, is able to activate plasminogen (PG), with fibrillazione areas fibronectin person selected from fibrinspecific domains fibronectin person (for example, a pair of domains 4 and 5, or domains 1 and 2, or their modified forms), due to the fact, that activators of hybrid plasminogen have the ability to independently contact the fibrin and therefore be specific to blood clots due to their increased affinity for the substance a blood clot, i.e. fibrin (U. S. Patent no. 7163817).
It provides a mono-, or bi-, or multipayline the s cysteine variants of streptokinase or its protestirovannyx forms, not only active in terms of their ability to activate GHG, but also reveal new and unexpected functional properties. For example, biparentally cysteine variant of IC, in which additional cysteine molecules are located in the two end parts of the polypeptide, i.e., the N - and C-ends, shows an unexpected property with respect to its ability to activate plasminogen person, consisting in the fact that it has a significantly lower rate of activation of plasminogen (PG) compared to unmodified SK, but is fully able to activate plasminogen as well as unmodified SK, a few minutes after the end of the lag-period, when the tests of PG activation in vitro. Failure to semiaccurate nezamecheno (as in the case of native unmodified IC, which activates GHG almost in contact) due to platensimycin action type. In contrast, activation of native SK need no plasmin, and it activates almost as soon as it forms a complex with PG. Thus, after injection into the body, is derived SK travels through the bloodstream, while still in an inactive or partially active state. However, it is preferably activated in the immediate vicinity of the thrombus at the time of his contact with the blood clot, which, ka is known, formulated with plasmin, while the overall flow is not saturated (free plasmin is rapidly inactivated when "open" circulation due to the presence plastisizing of Sechenov [serine proteinase inhibitors], for example, alpha-2-antiplasmin and alpha-2-macroglobulin), which thus eliminates or significantly reduces systemic activation of GHGs occurring synchronously with the introduction of natural SC, which now activates the PG in the introduction, accompanied by side effects, such as bleeding and large-scale destruction of various protein components of the cardiovascular system. This property, i.e., placentalia activation with increased half-life and low immunogenicity and antigenicity, should lead not only to an overall reduced formation of free plasmin in the overall flow, but also to the ability to thrombolytic to be re-introduced in various diseases of the circulatory system at relatively low doses, which avoids unwanted immune reactions. The total result is longer lasting and more effective fibrinolysis in the target assumes a much lower therapeutically effective dose of thrombolytic agent by minimizing side effects, such as reducing immunoreactive and reducing the negative consequences of hemorrhagic complications, often observed for normal SC.
The present invention provides paglierani cysteine variants of streptokinase or its Malinov or hybrid molecules of plasminogen activator containing polypeptide communication between streptokinase (SK), or modified forms of SK, or its relevant parts, which possess biological activity in vitro comparable to the biological activity of native streptokinase, a specific test for the activation of plasminogen, the activity decreases, if in some cases it is well compensated by the increase in time-life is derived and/or speed reduction clearance.
The present invention provides paglierani cysteine variants of streptokinase with the capacity to activate plasminogen only after passing the lag-period of time for more than 5 minutes after the appropriate plasminogen animal or person will be subjected to the action of plasminogen activator.
The present invention provides a prokaryotic or eukaryotic cells transformed or transfetsirovannyh expressing vectors that ekspressirovali streptokinase, Malinov or covalently modified forms clone genes that can Express the cysteine variants of streptokinase or the e Malinov or hybrid molecules of plasminogen activator. For efficient expression of the DNA sequence encoding streptokinase, it mutiny and covalently modified forms optimise taking into account codon sets of preferences used for expression of the hosts of bacterial or yeast nature.
The present invention details a method of obtaining paglierani cysteine variants of streptokinase or its active Malinov or hybrid molecules of plasminogen activators in pure and biologically active form for clinical and scientific applications.
The present invention recognizes that when the pegylation of these cysteine variants use matrix polynucleotide, which is used for the expression of SK polynucleotide encoding IC, modified by mutagenesis, using a known used for DNA biochemical or chemical synthetic methods or combinations thereof, to an activator activity of plasminogen persisted.
The present invention takes into account the fact that cysteine variants of SK or her versions of the forms which are subjected to pegylation, but also saves additional fibrillazione domains connected by a polypeptide bonds, so that the resulting chimeric/fused polypeptides along with the manifestation of the ability to activate plasminogen also have fibrillazione its the problem. Linking between fibrillazione domains can occur directly or via a short linker peptide regions consisting of patches of amino acid sequence, which is not conformationally rigid, and is flexible, for example, mainly consisting of Gly, Ser, Asn, GIn, and amino acids.
Cysteine variants of SK or its mutiny or covalently modified forms Express in E. coli, using standard plasmid under the control of a strong promoter, for example, tac, trc, T7 RNA polymerase, etc. that also have other well-known properties needed to induce high level expression of introduced reading frame, which encodes the IC or its mutiny or covalently modified analogues of the UK.
Cysteine variants of SK or its mutiny or species variants or covalently modified forms Express in yeast expressing systems using standard plasmid, where the N-terminal signal peptide optimize for efficient extracellular secretion of the Mature polypeptide. The information sequence for these signal peptides can be obtained by secretory proteins in yeast expressing systems. Additionally, such information can also be obtained by other recombinant protein is, which Hyper-secreted and contain optimized signal sequence.
The present invention provides a method in which crude cell lysates, obtained using either chemical, mechanical or enzymatic methods based on cells containing single, double or triple cysteine variants of SK, or chimeric polypeptides SC, is subjected to oxidation in air or catalytic oxidation of the thiol-disulfide reagent or thiol-disulfide oxidative refolding, catalyzed by the enzyme to collapse in their biologically active conformation containing the native pair cysteine (covalently modified forms SK), freeing up extra cysteine(s) from participation in the chemical modification of sulfhydryl groups.
The present invention provides a method in which crude cell lysates, obtained using either chemical, mechanical or enzymatic methods based on cells containing single, double or triple or multiple cysteine variants covalently modified forms of IC, is subjected to oxidation or refolding using the mixture of reduced and oxidized glutathione or other similar reagents, which are used for such reactions oxidative folding through the om thiol-disulfide exchange for example, cysteine and cystine, with appropriate redox potential, which allows covalently modified forms of IC to fold into their biologically active conformation, releasing additional cysteine(s) from participation in the chemical modification of sulfhydryl groups.
Cysteine variants of SK, it mutiny or covalently modified forms Express in eukaryotic organisms, for example, animal or plant cells, using standard genetic methods or as included genetic units in the core genome or as an Autonomous genetic elements, well known in this field to obtain a high level of expression of the integrated open reading frame, which encodes the IC or its mutiny or covalently modified forms.
The present invention provides a method in which the targeted cysteine variant of IC or chimeric plasminogen activator protein IC can be used for thrombolytic therapy or prevention of various vascular thrombosis. The activator may be formulated in compositions in accordance with routine procedures as a pharmaceutical composition(s), adapted for the introduction of man, and may include, but are not limited to stabilizers, for example, serum albumin human is and, mannitol and so on, solubilizers agents or anesthetic agents such as lignocaine, etc. and other agents or their combinations in order to stabilize and/or to facilitate the introduction of derivatives in vivo.
The present invention provides a pharmaceutical composition comprising a targeted cysteine variant of IC or hybrid plasminogen activators and stabilizers, which includes, but is not limited to serum human albumin, mannitol, etc., and solubilizers agents, anesthetic agents, and so on
The present invention will be described in more detail in the following examples, which, however, should not limit the scope of the invention. For specialists in the art of the obvious possibility of use within the scope of the present invention other derivatives, combinations and improvements. Thus, it is likely that such work or careful repetition should lead to similar or superior properties even other variants of streptokinase, which are not disclosed in the present invention, and to treat other isolates of human and non-human origin.
The most common methods used in examples
Typically use molecular methods and techniques well known in the Asti molecular biology and the science of proteins. They are readily available from various standard sources, such as textbooks and collection protocols related to this technical field, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (II.sup.nd edition), Cold Sparing Harbor Press, New York, 1989; McPherson, M. J., Quirke, P., and Taylor, G. R., [Ed.] PCR: A Practical Approach, IRL Press, Oxford, 1991, Current Protocols in Protein Science, published by John Wiley & Sons, Inc. For immunological experiments refer to a textbook and a collection of protocols from Iinmu no chemical Protocols, Hudson L, Hay FC (1989) 3rd ed. Blackwell Scientific. However, this does not preclude a detailed explanation in the context of specific experiments describing the present invention, especially when carried out modifications of known procedures specified in the examples, if necessary.
Cloning of the gene of the IC is performed in expressing the vector-based promoter T7 RNA polymerase, pET-23d and transformed into Escherichia coli strain BL21 (DE3 obtained from Novagen Inc. (Madison, WI). Thermostable DNA polymerase (Pfu), restrictase, T4 DNA ligase and other enzymes that modify DNA, obtained from New England Biolabs (Beverly, MA). Oligonucleotide primers provided by one component: Biobasic Inc., Canada, Integrated DNA technologies, US or Sigma-Aldrich, US. Purification and DNA extraction PCR-amplified products from agarose gel carried out using kits available from Qiagen GmbH (Germany). Automated DNA sequencing using similar fluorescent screens the dyes are conducted on a 16-capillary DNA sequencing machine Applied·Biosystems 3130×1 Genetic Analyzer. Glu-plasminogen or receive from Roche Diagnostics GmbH (Penzberg, Germany), or extracted from human plasma by affinity chromatography (Deutsch and Mertz, 1970). N-terminal amino acid sequence determined using an Applied Biosystems Sequencer Model 476A b 491 clc. Urokinases, EACA. Cyanoborohydride sodium, L-lysine provided by Sigma Chemical Co., St. Louis, USA. Penyagolosa 6XL, DEAE-Sepharose (fast flow) buy Pharmacia Biotech, Uppsala, Sweden, at that time, as a while, granules Ni-NTA at Qiagen. All other reagents have the highest degree of chemical purity.
Layering casein-plasminogen for determination of ck activity: the Activity of various derivatives of IC is determined by the layering of casein and plasminogen person in soft agar. The original way Malke and Ferretti, 1984; was modified, when the stain of purified SK (0.5 microgram) was applied directly to the observed deepening on plates with LB-Amp agar. Then Cup incubated at 37°C for 10 minutes; after that, the casein-HPG-agarose is applied, carefully pouring the mixture of solutions a and b at the top of the cups containing spots. A solution obtained by heating 1 g of skim milk in 15 ml of 50 mm Tris-HCl buffer (pH 7.5), and then kept at 37°C in a water bath until further use. The solution obtained by heating of 0.38 g of agarose in 15 ml of 50 mm Tris-HCl buffer (pH 7.5) at 50°C. After cooling the solution to 37°C add 3 ál of Triton X-100 (,04%.about.) and 200 μg HPG. Then the plate is incubated at 37°C and observed for the formation of zone of clearance (education halos), due to the hydrolysis of casein with subsequent activation of the HPG.
To determine the proteolytic stability of each paglierani derived and native SK incubated with proteolytic enzymes, such as trypsin or plasmin. The concentration of the protease used in the reaction, vary in the range 500-10,000 times the concentration of proteins. Reactions carried out with shaking at 25°C for two to four hours. The same number trypsinization protein for both test and control placed on plates with LB-Amp agar and measuring the residual activity after stratifying on Cup casein-plasmalogen.
Analysis of proteins by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS-PAGE): SDS-PAGE carried out essentially according to Laemmli, U. K., 1970, with minor modifications, if needed. Briefly, the protein samples obtained by mixing equal volumes of sample and buffer with a twofold increased concentration (0.1 M Tris-HCI, pH 6,8; 6% SDS; 30% glycerol; 15% beta-mercaptoethanol and 0.01% dye bromophenol blue). For carrying non electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate in the sample buffer does not include beta mercaptoethanol. Before applying the gel on the specimens heated in boiling water bath for 5 minutes. Heterogeneous system gel typically contains 5% (concentration of acrylamide) at separations of proteins and 10% during the decomposition of the gel. Electrophoresis is performed using the Laemmli buffer at a constant current equal to the first 15 mA, up until samples of the concentrate, and then at 30 mA until the process is complete. After completion of electrophoresis, the gel is dipped in a 0.1% solution Kumasi Blue R250 in the system methanol : acetic acid : water (4:1:5) with gentle shaking and then discolor in decolorizing solution (20% methanol and 10% glacial acetic acid) until such time as the background will not be transparent.
Paglierani proteins can be stained with iodine according to the standard method, developed Kurfiirst (Kurfiirst M. M., 1992), according to which specifically stained molecules of PEG. For staining of purified derivatives of PEG-iodine immediately after completion of the electrophoresis, the gel is soaked in a 5% solution of glutaraldehyde (Merck) for 15 minutes for fixation. After this, the gel is stained to determine the PEG as follows. Firstly, the gel is placed in a solution of 20 ml of perchloric acid (0,I M) for 15 minutes and then 5 ml of a 5% solution of barium chloride and add 2 ml of 0.1 M solution of iodine (Merck, Titrisol 9910). After a few minutes the streak painted paglinawan protein. For double staining for e is foresa in polyacrylamide gel in the presence of sodium dodecyl sulfate gels, painted with iodine, then paint Kumasi Blue R250, using the Laemmli Protocol described above.
Kinetic analyses (Shi et al., 1994, Wu et al., 1987, Wohl et al., 1980) are used to determine the activation of HPG modified PEG and unmodified SK or its covalent derivatives, especially when it is necessary to determine the kinetic constants. PEG-modified or unmodified IC or covalently modified forms in different concentrations (10 nm - 200 nm) is added to a final volume of 100 microliters multilauncher tablet containing 1-2 μm HPG in the buffer for the quantitative determination (50 mm Tris-HCI buffer, pH 7.5, containing 0.5-1 mm chromogenic substrate and 0.1 M NaCl). Used chromogenic substrate (S-2251, Roche Diagnostics GmbH, Germany) is platensimycin and gives the splitting of the yellow colored product, the content of which can be determined at 405 nm.
Aliquots of protein add after adding in the hole all the other components and the first spectrophotometric absorption is taken for zero. Then determine the change in absorption at 405 nm as a function of time in the tunable microplate reader for Versa-Max from Molecular Devices, USA. The appropriate dilution of S. equisimilis streptokinase obtained using WHO, Hertfordshire, U. K., used as a standard sample for calibration in international units on the I unknown drugs.
The test to determine the stationary kinetic constants HPG activator activity of PEG-modified and unmodified IC and its covalently modified forms.
To determine kinetic parameters for HPG activation in the buffer for the quantitative determination containing different concentrations of HPG (varying in the range of from 0.035 to 2.0 micromol) add a fixed amount of PEG-modified and unmodified IC and its covalently modified forms of 0.05-0.1 nanomoles) in advance of the tablet, as described above. Then spectrophotometrically measured change in absorbance at 405 nm for 10-40 min at 25°C. Then the standard methods to calculate the kinetic parameters of activation of HPG from the plot of inverse values of the Michaelis-Menton (Wohl et al., 1980).
Various paglierani SC or her mutiny and native SK, labeled with radioactive iodine, Iodine125(I125) get in a Perkin Elmer Singapore Pte Ltd., when using the method with the participation of Imogene (1,3,4,6-tetrachloro-3α-6α-diphenylpyrrole) (Fraker & Speck, 1978). According to this method, used Fraker and Speck, ionogen dissolved in chloroform and applied on the walls of the tube of borosilicate glass by evaporation of the solvent by means of a jet of nitrogen gas. For iodination of the protein solution in phosphate buffer (PBS) is added to trunk is, covered iodophenol, and mixed with I125. Approximately 10-30 min, protein, labeled with radioactive iodine, is separated from free radioactive iodine by desalting on a column of Sephadex G25 (fine) (Amersham Biosciences).
Design and construction of vector pet, containing the gene SC (PET-23d-CK), described Nihalani et al.; (1998). They include cloning the gene SC Streptococcus equisimilis H46A in pBR 322 (Pratap et al., 1996) with subsequent sublimemovies into pet-23d, expressing a vector containing a high-performance binding site of ribosomes from T7 phage main capsid protein (Studier and Moffatt, 1986), and additional modification of the 5' end of the gene for the ability to minimize the formation of secondary structure. A vector is contiguous position inside the frame initiation codon for Met at the beginning of the open reading frame that encodes the UK, in order to Express the protein as Met-CK. For details you can refer to multi-stories et al.; 2007 (US Patent no. 7163817). SC mutiny can be constructed using the updated matrix, as in the case of the UK, in order to obtain a high intracellular expressing ability. This scheme receive a shortened derivative of the IC other way explained in detail Nihalani et al., 1998. In addition to sequencing the gene authenticity of the IC and its ukorochenniy the derivatives also installed gas-phase sequencing of N-terminal amino acids of proteins on protein sequencing machine Applied Biosystems-Perkin Elmer model A or 491 clc.
Design covalently modified forms of the IC receiving hybrid polynucleotide DNA between the DNA coding for the UK, and fibrillazione domains fibronectin person and its cloning and expression in E. coli explain in detail in US Patent No. 7,163,817. Briefly fibrillazione domains attached to the streptokinase N-end-or-end, or both the N-and C-end for various covalently modified forms of streptokinase.
The choice of residues or regions of the protein to obtain cysteine variants of streptokinase
The choice of balance for a replacement or deletion is key to preserve the functionality of the modified polypeptides. Therefore, the plan cysteine mutagenesis requires structural information available in the crystal structure and functional representations received from the data for the study solutions. Detailed structural and functional studies for many years collected in a volume of information on the roles of different regions of streptokinase in the activation of plasminogen. For decision-making regarding residues or regions in which the natural amino acids can be preferably substituted by cysteine, we used the information contained in the three-dimensional structure of the IC or its individual domains along with their functional is important. The selection of residues for cysteine mutagenesis is based in part on surface accessibility of residues. The introduction of cysteine is also limited flexible regions of streptokinase. To determine the surface of the ledge using the program DSSP. Code DSSP is often used to describe the secondary structure of a protein with a single letter code. DSSP stands for "Dictionary of Secondary structure of the Protein". The program DSSP (Kabsch and Sander, 1983) defines the secondary structure, geometrical parameters and the availability of proteins in relation to solvents set of atomic coordinates in the format of the Bank the Protein Data. DSSP defines the accessibility of each residue in square angstroms. Once the program DSSP on this PDB file leads to reduced DSSP output format information. You can get the amount of surface availability under the Acc output format DSSP. To determine the surface accessibility of the use permitted crystal structure of streptokinase (Wang et al., 1998, PDB ID 1BML) in complex with microplasmin. To determine the surface accessibility of areas missed in this structure (175-181 and 252-262), use a crystalline structure selected beta domain (Wang et al., 1999, PDB ID 1c4p). Some of the loops is missed in the crystal structure, increased in experimentally received the Noah structure and it was determined the most preferred conformation using molecular models. Residues that are not necessarily found in the structure, but are defined as highly accessible because they are located in the flexible region, also selected for cysteine mutagenesis. Table 1 presents the magnitude of the surface availability for different cysteine variants of SK (SEQ ID no: 1), which is replaced by cysteine. This list, however, does not limit the scope cysteine replacement for other natural amino acids in SC. The value of the distance calculated by the DSSP program, selected directly as a measure of surface availability. The DSSP program includes many surface-accessible residues. However, selecting residues for cysteine substitutions conducted a careful selection. This experiment involves mutations are evenly distributed along the three different domains, namely alpha-, beta - and gamma-streptokinase. In addition, select mutations that fall in the secondary structural region. The selection includes a replacement for several cysteine residues, which have a particularly low surface accessibility, only in order to illustrate the fact that essentially all without exception remains of streptokinase can be replaced by cysteine and successfully modified reagents that interact with Tilney group.
Despite the variety nucleotide the th and polypeptide sequences there is considerable structural similarity among different bacterial plasminogen activators. Single-domain staphylokinase has structural homology with alpha-domain of streptokinase. Also dual-domain bullish plasminogen activator derived from Streptococcus uberis is characterized by its structural similarity to alpha - and beta-domains streptokinase. The evolutionary persistence of the three-dimensional structure of the protein among various bacterial plasminogen activators gives an opportunity to plan cysteine modification other variants of streptokinase, isolated from different bacterial species.
Genetic engineering of cysteine variants of streptokinase
All genetic constructs for ekspressirovali of streptokinase usually designed using conventional approaches known in the art. Describes how the manipulation of DNA to include mutations, for example, in PCR Protocols: A Guide to Methods and Applications, edited by Innis, M. A. et at, 1990, Academic Press Inc., San Diego, Calif. and PCR Protocols: Current Methods and Applications edited by B. A. White, 1993., Hurriana Press, Inc., Totowa, N. J., USA. Bacterial and yeast expression cassette is produced by introduction of a DNA molecule that encodes a desired streptokinase in an appropriate vector (or the introduction of the original matrix DNA in the vector and mutagenesis of sequences it is optional), after the existing transformation of host cells using expression cassettes using conventional methods, known in the art. Specific mutations introduced into the desired design using different procedures, for example, the technique of PCR-mutagenesis (Innis et al., 1990), set for mutagenesis, such that sells Stratagene ("Quick-Change Mutagenesis kit, San Diego, Calif.) or Promega (Gene Editor Kit, Madison Wis.). Typically, the oligonucleotides designed to include nucleotide changes in the coding sequence of streptokinase, which leads to the substitution, deletion or adding balance natural balance. Also design of mutagenic primers to add a cysteine at the beginning of the molecules of the Mature protein, i.e. in the position nearest to the N-terminal amino acid, or following the last amino acid in the Mature protein, i.e., after the C-terminal amino acids of streptokinase and its truncated constructs. A similar strategy is used for the introduction of cysteine residues between any two selected amino acids streptokinase or any of its forms, represented by SEQ ID NO: listed in table 2. Using standard methods, generate appropriate cysteinaemia mutants on various forms of IC. This was followed by the screening of the transformed clones for different mutants and fix the result of automated DNA sequencing using fluorescent dyes on 16 capillary sequestered Applied Biosystems 3130×1 16 Genetic Analyzer.
Table 2 lists the different polypeptide constructs expressing the following substances: streptokinase; it mutiny; species derived; or covalently modified forms. The sequence of the full-sized native streptokinase polypeptide defined by SEQ ID NO: 1. A shortened form of the SC, which remains To-end 31 removed, reflected in SEQ ID NO: 2. Shortened form of IC, where the remains of the N-end 15 removed, reflected in SEQ ID NO: 3. The polypeptide containing a deletion from both the N-terminal residues, and the C-terminal 31 residues of the SC is presented in SEQ ID NO: 4. Functional fragment of the IC, which removed the remains of the N-end 49 is depicted in SEQ ID NO: 5. Design streptokinase, in which remnants as the N-end 59 and the end 31 represented by SEQ ID NO: 6. Full sized polypeptide SC (residues 1-414), which contains a substitution of alanine for asparagine 90 in the alpha domain represented by SEQ ID NO: 7. Beta-domain mutant full-length polypeptide IC, in which alanine is substituted by tyrosine presented in SEQ ID NO: 8. Similarly, SEQ ID NO: 9 represents the beta-domain of the mutant SC, where the aspartate residue at position 238 substituted by alanine. SEQ ID NO: 10 defines the beta-domain of the mutant streptokinase, in which the glutamate at position 240 substituted by alanine. SEQ ID NO: 11 and SEQ ID NO: 12 represent the mutation of arginine to alanine and lysine to alanine at 244 and 246 residues, respectively, for p is llerasmuney SC. Mutant loop 250 beta-domain of the IC, in which a leucine residue 260 position substituted by alanine represented by SEQ ID NO: 13. Gamma-domain mutant SC, in which the aspartate residue in 359 position is substituted by an arginine represented by SEQ ID NO: 14. Double mutant SK, where as histidine 92 and serine 93 substituted by alanine represented by SEQ ID NO: 15. Another double mutant IC, in which two consecutive lysine residue at 278 and 279 position substituted by alanine represented by SEQ ID NO: 16. Mutants IC, in which asparagine at 90 position in the alpha domain of the aspartate in the 22nd position in the beta-domain or aspartate in 359 the position of the gamma-domain deleted represented by SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19, respectively. Mature and active form of the SC can be obtained from the derivatives of species and subspecies of the species Streptococcus. To evaluate the possibility of implementing cysteine mutagenesis and subsequent paglierani for all different forms of IC, were also selected derivatives IC, obtained from a species of Streptococcus, namely pyogenes and dysgalactiae. Species derived SC obtained from Streptococcus pyogenes represented by SEQ ID NO: 20 and one derived from Streptococcus dysgalactiae represented by SEQ ID NO: 21. Covalently modified form of IC, in which fibrillazione domains located at the N-end IC, defined by SEQ ID NO: 22. Product fitting C-terminal fibrinovogo domain SK represented by SEQ ID NO: 23. Hybrid polypeptide, provided fibrinolitikami domain as N-, and With the end of the IC represented by SEQ ID NO: 24. Genetic structure merged with fibrin domain forms IC are described in detail in US Patent No.. 7163817. A variety of polypeptides, including native full size IC, it mutiny, species variants and covalently modified forms additionally used to produce cysteine variants.
Cysteine variants derived from various forms of IC, referred to in table 2, presents a unique SEQ ID NO:. In table 3-28 lists derived from their unique SEQ ID NO:.
Table 3: options, designed on the basis of the native full-ck (SEQ ID NO: 1)
Table 4: options, designed on the basis of shortened SC 1-383 (SEQ ID NO: 2)
Table 5: options, designed on the basis of shortened SC 16-414 (SEQ ID NO: 3)
Table 6: options, designed on the basis of shortened SC 16-383 (SEQ ID NO: 4)
Table 7: options, designed on the basis of shortened SC 50-414 (SEQ ID NO: 5)
Table 8: options, designed on the basis of shortened SC 60-383 (SEQ ID NO: 6)
Table 9: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 7)
Table 10: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 8)
Table 11: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 9)
Table 12: options skonstruiroval the data on the basis of the polypeptide mutant SC (SEQ ID NO: 10)
Table 13: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 11)
Table 14: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 12)
Table 15: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 13)
Table 16: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 14)
Table 17: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 15)
Table 18: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 16)
Table 19: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 17)
Table 20: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 18)
Table 21: options, designed on the basis of the polypeptide mutant SC (SEQ ID NO: 19)
Table 22: cysteine variants of Streptococcus pyogenes MGASI0270 (SEQ ID NO: 20)
Table 23: cysteine variants of Streptococcus dysgalactiae subsp. equisimilis (SEQ ID NO: 21)
Table 24: cysteine variants of SK containing attached to the N-end fibrinolitikami domain (SEQ ID NO: 22)
Table 25: cysteine variants of SK containing attached to the C-end fibrinolitikami domain (SEQ ID NO: 23)
Table 26: cysteine variants of SK containing fibrillazione domains attached to both the N-and C-end (SEQ ID NO: 24)
Table 27: mutants SK, received inser what their cysteine
Table 28: options for the UK, in which cysteine is located at the N - or C-end, accompanied or not accompanied by the elongation of the peptide.
These examples show that it is possible to obtain cysteine variants of almost all forms of IC, for example, the native full-length, truncated, elongated N - or C-end or merged with another polypeptide sequence. We also got cysteine variants, obtained by replacement, insertion or deletion mutants SC. This proves the applicability of the present invention to any form SC for cysteine mutagenesis and subsequent modification agents that interact with Tilney group.
Designs obtained in example 2 are used in all further experiments, carried out in order to successfully implement the present invention. However, it should be understood that the list of cysteine variants of streptokinase is just a typical, but not exclusive. Design and synthesis of alternative and additional cysteine variants of streptokinase according to the present invention can be easily doable by specialists in this field. The synthesis of such derivatives can be successfully performed by conventional means and methods.
Overexpression and purification of biologically active streptokinase
All substances native protein streptokinase, NSC(nCK), her mutant and further cysteine mutants that need to be cleared, grown from a single colony, do the sowing stroke on a bowl of LB-Amp from their mother solutions BL21 (DE3) in glycerol. Primary cultures get inocula pET-23d-CK or variants SC in 10 ml of LB medium containing 100 μg/ml ampicilin environment (LB-Amp) and incubated for 8-16 hours at 30-37°C with shaking (180-280 rpm). This reinoculated used for inoculation of 500 ml of medium LB-Amp at a concentration of 2-10%.about. and give to grow at 30-37°C, at a speed of 180-280 rpm to O. D600 nm(optical density measured at 600 nm) of 0.5 to 1.0. At this stage it induce IPTG (final concentration of 0.5-1.0 mm) (Director et al., 1999; Dhar et al., 2002) and further grown at 40°C for 6-12 hours with shaking. The cells are then harvested by centrifugation at 6000-7000 g for 10 min. the Precipitate is then washed twice with ice buffer (final concentration of 100-150 mm NaCl, 10-50 mm Tris-HCl, pH 8.0 and 1-5 mm EDTA) and subjected to sonication (Heat System, New York) at 4°C, the duration of the sound pulse, equal to 30 seconds, alternating with equal periods of rest. Then cell lysate centrifuged at high speeds turnover (10000-14000 g) for 15 min SDS-PAGE analysis shows that more than 90% of the desired protein goes into Taurus on (IB). Then IB solubilizer in 8 M urea at room temperature is over 45 min with constant gentle shaking. Protein in the supernatant folded after 10-36-fold dilution (Sundram et al., 2003) in the buffer for the application (0.4 M NaCl in 20 mm PB). The sample was then chromatografic on the granules penyagolosa 6XL and elute with water. The thus obtained protein is then subjected to additional purification by anion-exchange chromatography on a column with DEAE-Separati® (GE-Amersham Biosciences). Protein fractions after HIC combine and add Tris-HCl buffer at pH 7.5 to a final concentration of 20 mm Tris-HCl, after which it put on a column of DEAE-Separate (Fast Flow), pre-equilibrated to 20 mm Tris-HCl (pH 7.5). After washing with buffer containing 20 mm Tris-HCl (pH 7.5) associated protein elute using a linear salt gradient (0-0 .5 M NaCl) at 20-25 mm Tris-HCl. Bweremana proteins IC usually have more than 95% purity, as shown SDS-PAGE. The amount of protein in each fraction was measured by the method of Bradford determination of proteins (Bradford., 1976) and confirmed by absorption measurement at 280 nm. All chromatographic stage is carried out at 4°C. Fractions containing protein, analyze together with standard IC and molecular weight standards for SDS-PAGE. The right faction of the conservative combine to obtain a homogeneous preparation of IC or mutants SC.
Overexpression and purification of various covalently modified structures formed by ck and fibrillazione domains (FBDs) in coli, and their refolding in vitro is described in US Patent No.. 7163817, where downregulation of proteins subjected to refolding in vitro and purified column chromatography. Briefly, solubilization bullock enable diluted to a final protein concentration of 1 mg/ml using distilled water with the addition of the following additional components (see final concentration in the diluted mixture): Tris-HCl, pH 8.0, 50 mm; NaCl, 100 to 150 mm; EDTA 1-5 mm; a mixture of reduced and oxidized glutathione (1.25 mm : 0.5 mm. Denaturirovannogo fraction is separated and purified on a column filled with a fibrin-sepharose granules. For a detailed description of refolding, purification and properties denaturirovannogo protein, please refer to multi-stories et al.; 2007 (US Patent no. 7,163,817).
Covalent conjugation of cysteine variants of streptokinase with polyethylene glycol
Tirinya group in cysteine variants of streptokinase selectively pagealert when using maleimide-activated linear methoxide different lengths, for example, 5 kDa, 20 kDa and 40 kDa (JenKem Technology, USA). For the reaction of paglierani polypeptide to be pegylation stand in 50-100 mm Tris-HCl buffer at pH 8.0 containing 100 to 150 mm NaCl. To it add 5 molar excess of PEG-reagent. The molar excess is calculated from the number of free tylnej groups that interact with the EH-reagent, and not just from polyarnosti protein. The reaction mixture is left under stirring at room temperature for 1.5-4 hours, and then the reaction stopped by the addition of 1 mm DTT. Targeted protein from the free and unreacted PEG SK purified by anion-exchange chromatography on a column with DEAE separate (GE-Amersham Biosciences). The reaction mixture was diluted with 10-15 times 25 mm buffer phosphate at pH of 7.4, and then it is placed on a column filled DEAE-separate (Fast Flow), previously calibrated with the same buffer. After washing with buffer containing 25 mm sodium phosphate associated protein elute using a linear salt gradient (0-0 .5 M NaCl in 25 mm sodium phosphate. Alternatively, if some of paglierani derivatives cannot purely be separated from unreacted PEG ion exchange chromatography, these reaction mixture is subjected to gel chromatography on Sephadex 75 (Amersham Biosciences), using a buffer at neutral pH and the final concentration of NaCl equal to 100-150 mm. In addition, in some cases, when peeled containergrown protein samples that contain dimers, linked by disulfide bonds, first restore the addition of 10 mm dithiothreitol. Samples treated with dithiothreitol, absoluut on a column Packed granules Sephadex G-25 (fine) and now sportsouth La conjugation with PEG. Homodimer KS, linked by disulfide bonds, can also be separated using gel chromatography on Sephadex G-75 and can be used instead of the Monomeric streptokinase for therapeutic purposes, because of their large size and slow clearance.
Attaching PEG in all cases confirmed by SDS PAGE. Gel electrophoresis shows that >95% paglinawan protein in the fractions obtained after the removal of unreacted protein and free PEG-reagent.
Layering casein-plasminogen to quickly identify the ability to activate plasminogen
The activity of different cysteine variants of SK and chimeric derivatives IC is determined by the layering of casein and HPG in soft agar. The initial way Malke and Ferretti, 1984 modified so that put a sample purified SK (0.1 to 0.5 micrograms) marked directly on the notch on plates with LB-Amp-agar. Then the plates are incubated at 37°C for 10 minutes; then carefully layer the mixture of casein-HPG the agarose careful pouring the mixture of solutions a and b on the surface of the Cup containing spots. A solution obtained by heating 1 g of skim milk in 15 ml of 50 mm Tris-HCl buffer (pH 7.5), and then kept at 37°C in a water bath until the next use. Rest the R obtained by heating of 0.38 g of agarose in 15 ml of 50 mM HCl buffer (pH 7.5) at 50°C. After keeping the solution at 37°C add 3 ál of Triton X-100 (0,04% vol./about.) and 100-200 μg HPG and mix carefully without foaming. The cap is then incubated at 37°C for 1-4 hours, and observing the formation of zones of enlightenment (education halos), due to the hydrolysis of casein, with subsequent activation of the HPG. You should take into account the size of the zone of lysis (halo) surrounding the hole in agarose medium, for comparing the ability to activate plasminogen streptokinase and its covalently modified derivatives. All cysteine variants of streptokinase retain significant ability to activate plasminogen, as shown by the test for casein-plasminogen overlay. Cysteine variants of streptokinase then used for conjugation with samples of PEG of different molecular masses in the range from 5,000 Da to 40,000 Da, reacting with thiol groups, and their activity determined using test layering casein-HPG control activity of the conjugated PEG against the loss of its ability to activate plasminogen. All streptokinase show a significant ability to activate plasminogen, as shown caseinolytic test, and therapeutically applicable. However, the desirable combination of a suitable time-life, which is required for clinical practice, as well as ponie the fair immunoreactivity, makes some derivatives are more applicable than others. Relatively lower activity may in some cases be well offset by increased proteolytic stability and half-life of PEG-protein adducts in vivo.
Alternative ability to activate plasminogen was also determined, as explained previously. Table 29 presents the activator activity of the HPG and the kinetic constants for several typical representatives paglierani derivatives IC. Table 30 summarizes the range of activity of the various paglierani derivatives covalently modified streptokinase containing attached fibrin domain.
Measurement of the activity of shortened forms SK (50-414 and 60-383) and their paglierani derivatives require the addition of synthetic peptides 1-49 for the manifestation of optimal amylolyticus and plazmogeneratora abilities. In the literature it has been shown that truncated derivatives IC, lacking the N-terminal peptide corresponding to a region 1-59 are poor activators of plasminogen and show increased activity when added to the reaction mixture or peptide SC 1-59, or fibrin for the manifestation of optimal amylolyticus ability and capacity to activate plasminogen (Nihalani et al., 1998 and Sazonova et al., 2004). To activate p is Aminogen paglierani derivatives IC, not containing peptides 1-49 or shorter N-terminal peptides, the results show a dependence on the presence of fibrin; their actions are limited by a blood clot, so they become transpacificus.
Proteolytic stability paglierani cysteine variants of streptokinase
To determine the proteolytic stability of 50 micrograms each paglinawan derived and native SK as a control incubated with 50 Microlitre 50 mm Tris-HCl buffer and 100 mm NaCl. To this mixture, add trypsin to obtain the final correlation of targeted protein:trypsin, 1000:1 (wt./wt.). The reaction is carried out with shaking at 25°C for two to four hours. An equal number trypsinization protein is applied to plates with LB-Amp-agar and measure the residual activity as described in example 5 for the determination of ck activity. Equal aliquots applied from the control reactions in which the reaction mixture was added only trypsin or only SC or in the reaction mixture was added a hybrid IC. In this case, for each trypsinization paglierani streptokinase or covalently modified derivatives also determine the zone of lysis in the layering agarose. The area of the zone of lysis gives a direct measurement of the residual capacity to activate plasminogen is protected streptokinase or its variants. This test shows significant protection for the various study derived the UK, when they are conjugated with one, two or three residues of PEG and incubated with trypsin or plasmin. Found a many fold increase in proteolytic stability when attaching more than one group of PEG derivatives IC.
An alternative measurement of the residual activity is also confirmed for trypsinization paglierani streptokinase or covalently modified derivatives by measuring their ability to activate plasminogen using spectrophotometric one-step test described previously using a chromogenic substrate. Trypsinization SC and paglierani derivatives IC in various concentrations (1-10 nm) is added to a final volume of 100 microliters in multilauncher tablet containing 1-2 μm HPG in the buffer for testing (50 mm Tris-HCl buffer, pH 7.5, containing 0.5-1 mm chromogenic substrate and 0.1 M NaCl). Aliquots of protein add after adding in the hole all the other components. Then measure the change in absorption at 405 nm as a function of time in a custom tablet reader model Versa-Max from Molecular Devices Inc., USA. The results obtained in this way are consistent with the results obtained caseinolytic test. It is shown that when incubate is with trypsin in all paglierani forms of streptokinase and its covalently modified forms remains a significant functional activity. Naegeliana streptokinase falling in such conditions are hardly detektiruya activity and prone to digestion by trypsin.
The samples subjected to trypsinization, as taught in pampering SDS-PAGE in order to physically observe proteolytic stability and the generation of truncated fragments as a result of proteolysis. This gives a qualitative assessment of bypass protein, subjected trypsinization. With this purpose from the reaction mixture are selected aliquots at different time intervals and inhibit the reaction by adding 20-molar excess of soybean trypsin inhibitor (GE-Amersham Biosciences) in order to suppress further trypticase activity. Samples collected at different points in time (5-180 min), subjected to electrophoresis on 10% SBS-PAGE and analyze the content of protected intact protein. The results obtained in this experiment also confirm the correctness of the conclusions obtained in our functional study trypsinization proteins. Large residual capacity to activate plasminogen in the experiment is also reflected in a greater degree of protection protein when considering physical integrity by SDS-PAGE.
Extra long N - and C-ends of options SC and PEG-modified form.
To establish that an arbitrary extension number and amino acids as N-, and with the end of the streptokinase molecule or its variants inevitably leads to the same result that was obtained with their newlistname counterparts; SK or its cysteine variants modify or N-end, either on the C-end with a small amino acid extension.
N-terminal extended form get by using two different strategies, leading to the polypeptides of two different sizes, namely, one with 6 amino acid elongation and another with 20 amino acid extension. Using the strategy of overlapping movements encoding 18 nucleotide elongation 6 his-tag residues placed before the N-terminal amino acid of the Mature streptokinase. The product of this modification is extended from the side of the N-end of the protein, optionally containing six amino acids. To enable extensions in the 20 amino acids cassette encoding the SC or its variants suffer from pet 23d in pet 15b (Cat. No.. 69661-3, Novagen, Inc. US). The tape cartridge in the pet 15b gives the N-terminal extension of 20 amino acids, including the lengthening of the six his-tag residues and the site of cleavage by thrombin. Cleavage of N-terminal elongation of the polypeptide can be carried out by thrombin. This leads to elongation with his-tag marker and to receive processioning polypeptide with the amino acid sequence, which is peculiar to the UK. SEQ ID NO: 496 dem will strirred amino acid sequence SK, obtained by cloning into pet 15b.
To obtain C-terminal elongated product lengthening of the six his-tag residues are added at once after the last amino acid of the IC or its derivatives by using the strategy of overlapping movements. This leads to the introduction of six additional residues at the C-end. The samples are cleaned by use of either a metal-affinity chromatography, or the application of cleaning methods described in example 2 to obtain homogeneity of the pure product. Further, these purified N - or C-terminal elongated derivatives IC or its derivatives modify the PEG, using the chemical reaction described in example 4. Biochemical study of the functional activity and proteolytic stability gives a result similar to that obtained for the corresponding elongated counterparts. This is a good reason to conclude that other N - or C-terminal elongated derivatives IC or its derivatives will lead to similar results. Specialists in this field can think of numerous possibilities extend from both the N-and C-end IC or its derivatives to obtain the functional forms of streptokinase, which can be used to replace the cysteine, insertions or additions of cysteine and their subsequent modification by thio who inim groups of PEG or other reagents for sulfhydryl group.
The acquisition of new functional properties derived SC, where the PEG is attached on two ends, namely, to the N - and C-end IC or its truncated variants.
It is shown that the attachment of PEG groups as both N-and C-ends of the molecule streptokinase and any of its shortened study makes it functional activity dependent on the presence of plasmin. These new functional properties can be defined, when we observe that the profile of plasminogen activation biegeleben derived IC detects a lag-period of several minutes. To our surprise, when was studied activation of plasminogen biegeleben derived SC after it formed a complex with plasmin, it showed a normal (i.e., similar to the native SK) kinetics. The inability to nezamechennoi someactivity (as in the case of native, unmodified IC, which activates PG almost in contact) due to dependent plasmin nature of its actions. In contrast, native of SC does not require to activate any of plasmin and activated almost as soon as it forms a complex with PG. In experiments where samples are taken from the reaction mixture at different points in time on the kinetic curves of plasminogen activation, i.e., at an early stage (during the l the g-period), in the fast phase activation, etc., and analyzed using SDS-PAGE to monitor the type of products, it was found that activation is closely related to the formation of truncated fragments, in which the peptide segments containing PEG at the ends of the polypeptide, hatshepsuts under the action of plasmin. Similar results were obtained mass spectroscopic analysis. These results clearly establish the correlation between the activation of plasminogen PEG-modified IC and deleting groups PEG, indicating mediated plasmin activation mechanism, according to which? once removed a bulky group of the PEG, the fragments SC the opportunity to interact with plasminogen, as well as the NSC, and quickly to activate it. Therefore, it is clear that the presence of PEG groups on the ends, besides giving a stabilizing effect on proteolysis, immunoreactivity, and so on, expected regardless of the position also leads to unexpected emergence of a plasmin-dependent "switch", which has a strong beneficial effect on thrombolytic therapy.
Functional dependency property of plasmin, i.e., the built-in "plastikowy switch"? were also found in other biegeleben cysteine variants shortened the UK, where additional cysteine are two extreme positions of the polypeptide, and the name is about, the N - and C-ends that are unexpected ability in respect of activation of human plasminogen, when it occurs with a significantly lower initial rate of activation of plasminogen (PG) compared to unmodified SK, but acquires full capacity to activate plasminogen as well as unmodified IC after an initial lag period, which lasts several minutes, when determining the activation of PEG in vitro. Table 4 presents the stationary kinetic parameters for HPG activation by streptokinase and two different pipelinename derivatives IC. NC 1-414 refers to a derivative of the IC, in which cysteine is administered before the naturally occurring N-terminal amino acid, and after the C-terminal amino acid to obtain a double cysteine mutant SC. NC 1-383 refers to a shortened version of the IC, in which one cysteine is administered before the naturally occurring N-terminal amino acid, and after three consecutive glycine residues, which are located at 383 amino acid. The data show that both biegeleben derivatives are characterized by a pronounced lag period before they become fully functional. Kinetic parameters calculated by linear plots of the kinetic curves after a lag-period, show that immediately after that is about, as biegeleben derived fully activated after an initial lag period, have significant activity in terms of the ability to activate the PG compared to SC. Similar results were obtained with two pipelinename derivatives IC, in which PEG is attached at the ends, show that the manifestation of this new functional properties depends on the position and almost does not depend on the presence of a double modification of PEG in the same molecule. Thus, the dependence on plasmin reported the molecule, when one or two ends at N - and C-ends, in any functional fragment SC paglierani.
In addition, for specialists in this area it is obvious that such a functional property that can be communicated to the molecule in any modification inside and near the two ends (e.g., in a suitable side chain of lysine) may also lead to the emergence of dependent plasmin lag period when the plasminogen activation through proteolytic processing of streptokinase in which such "blocking" groups, which may be the remains of PEG, other protein domains, intact proteins, e.g. albumin, etc. are removed during proteolysis, allowing the remaining polypeptide to become functionally active against activation of plasminogen. Thus, it is possible what to expect similar effects, when groups of PEG attached to the ends, using any available chemical reaction. One such chemical approaches uses the difference in the values PKandbetween the alpha amino group at the N-Terminus and is usually involved in conjugation with PEG-reactive alpha-amino group. Similar properties in different shortened combinations of end groups indicate that as only two groups of PEG fixed in any position within or near the two end groups of the molecule may purchase dependent on plasmin.
Thus, after introduction into the body is derived IC starts its movement through the vasculature, while still being in an inactive or partially active state. However, it starts preferably be activated in the immediate vicinity of the thrombus, in the moment of contact with the blood clot, which is known to be enriched by plasmin, while the overall flow no (free plasmin is rapidly inactivated in the "open" blood flow due to the presence plastisizing of Sechenov (serine proteinase inhibitors), for example, alpha-2-antiplasmin and alpha-2-macroglobulin), which thus eliminates or significantly reduces systemic activation of GHGs occurring synchronously with the introduction of natural SC, which nezamecheno activates the PG with the introduction involving side effe is Tami, for example, bleeding and large-scale destruction of various protein components of the cardiovascular system. This property, i.e., placentalia activation with increased half-life and low immunogenicity and antigenicity should not only lead to overall reduced formation of free plasmin in the overall flow, but also to the ability to thrombolytic to be re-introduced in various diseases of the circulatory system at relatively low doses, which avoids unwanted immune reactions. The total result is longer lasting and more effective fibrinolysis in the target assumes a much lower therapeutically effective dose of thrombolytic agent by minimizing side effects.
Further improvement of this property is to give biegeleben molecule based on fibrin introduction of PEG groups in the two extreme positions derivatives IC 50-414 or SC 60-383. This result is possible because the deletion of the "catalytic switch" (the remnants of SC 1-59) changes the conformation of the alpha-domain SC and turns these truncated fragments in firesafety plasminogen activator, as shown by Reed et al., 1999 and Sazonova et al., 2004. The expected result of constructing such biegeleben derivative is C the dependence of plasmin and improved the ratio of fibrin dependence/affinity. These two properties, i.e., platinova dependence and the ratio of fibrin affinity/selectivity makes the molecule activator of PG, which is directed exclusively against fibrin clots. Such molecules can effectively resolve the problem of systemic activation of PG and to allow for a strict PG activation only in the immediate vicinity of the fibrin clot.
Pharmacokinetic analysis paglierani cysteine variants of streptokinase
All the proteins that are used for the introduction of animals, carefully process for removing endotoxin by passing through a column containing Agarose gel-Polymyxin B (BioRad Inc., Palo Alto, CA, USA). Various derivatives monopegylated SC, representing the inclusion of cysteine in each domain, and native SK (as control) radioactively mark125I when using Imogene (Fraker & Speck, 1978) and separated from free radioactive iodine by passing through a desalting column with Sephadex G25. CDI mice (23-25 g) anaesthetize 3% isofluorane and induce a weak dilation of blood vessels, substituting the tail under the fluorescent lamp 100 watts. Then mouse injected approximately 7 micrograms labeled with radioactive iodine protein in sterile saline via the tail vein and collect all blood samples of approximately 50 MK is in a period of time necessary for blood sampling from the tail or from retroorbital cavity, and stored in heparinized appendrows tubes. Samples processed for plasma and determine the activity of the125I scintillation counter, Perkin-Elmer. After determining the activity125I to each aliquot of plasma, add an equal volume of 20% THU to determine the number of125I, remaining associated with the intact protein. Samples quickly stirred on the vortex and place on ice for 15 min Aliquots centrifuged at approximately 3000 g for 10 min, and the supernatant, containing free label or the label associated with fragmented protein, is sucked off from each sample using an aspirator. Formed after deposition THU upsetting determine the activity of the125I. Usually handle duplicate samples and average values.
Residual radioactivity different plasma samples, obtained by precipitation with acid, after administration paglierani cysteine variants of streptokinase is used to determine the half-life in the experiments in vivo. The results of determination of time-life show varying degrees of retention in vivo of various derivatives of PEG, when they are conjugated with one, or two, or three residues of PEG. These results show that with the introduction of more than one group PEG recip is different as a result, the half-life is also increased. The half-life for several selected paglierani derivatives IC are summarized in table 32, which shows that, on average, is 5-120mm-fold longer half-life for various mono-, bi - and trippelironi cysteine variants of streptokinase. It is shown that the retention time paglierani cysteine variants of SK depend on the provisions of the attached PEG. Paglierani derivatives in the alpha loop 88-97 are characterized by a maximum increase (~15-20-fold) retention time in vivo, while paglierani derivatives IC in the beta-domain characterized by intermediate increase (~10-12-fold) retention time in vivo. Paglierani derivatives in the gamma domain are characterized by approximately 4-5-fold longer half-life in vivo. In General, all paglierani streptokinase are characterized by multiple time increase the half-life compared to native SK (12-15 minutes). This means that paglierani cysteine variants are characterized by a significant increase in the time half-life in vivo, and demonstrates increased deceleration steps paglierani SC. Similar results were obtained with other containername variants of SK after paglierani. It is well known that increased slow steps paglinawan derived due to the balance of PEG and does not depend on the position of the polymer PEG molecule SC. Thus, joining the rest of the PEG through the cysteine residue leads to paglierani IC or hybrid derived SC, having increased the inhibition of determining the possibility of the introduction of smaller paglinawan connections while maintaining a high level connections in the blood for a long time.
Additivity in the elimination of time-life when joining two groups of PEG shows that can be manipulated as desired or to provide in accordance with the requirements of the elimination of time-life by conjugation with PEG groups in the right quantity, position and also by varying the length of the polymer molecules of PEG.
Immunoreactivity paglierani cysteine variants of streptokinase
The reactivity of the NSC and paglierani cysteine variants of SK and its covalently modified forms in respect of polyclonal antisera IC induced in the rat, studied by the method based on the method of enzyme-linked immunosorbent assay (ELISA). The ELISA procedure is as follows.
1. SC and paglierani options SK first dissolved in 0.2 M bicarbonate buffer, pH of 9.2 to obtain 100 microliter solution containing from 0.75 to 1.5 micrograms micrograms of protein, and the solution was added to each well of microtiter planches the TA (Nunc 96-Well Microplates, Cole-Parmer, USA).
2. Tablet sensitized antigen, cover with paraffin and incubated in a cold room over night in a humid box containing a wet paper towel or at room temperature and humidity for two hours when lightly shaken.
3. The tablet is empty and unoccupied space block 200 μl of blocking buffer containing 5-10% skim milk in phosphate-buffered saline (PBS) for one hour at room temperature.
4. The tablet is empty and washed four times proryvnym buffer obtained from PBS.
5. A solution of primary antibody is first diluted with PBS to obtain the dilution factor equal to 50000. To each well add 100 ál of diluted antibody. The tablet then incubated at room temperature for 45-60 minutes when lightly shaken.
6. Again empty tablet and washed four times proryvnym buffer.
7. Labeled with the enzyme horseradish peroxidase antibody against the antigen dissolved in PBS. 100 μl of this solution was added to each well and incubated at room temperature for 1 hour.
8. Again empty tablet and washed six times in 1 X PBS.
9. To each well add 100 ál of TMB liquid substrate, tetramethylbenzidine, Sigma-Aldrich, USA) and the plate is left for 10 minutes at room temperature the re.
To stop the reaction in each well add 50 μl of 1 N sulfuric acid and the development of color fix spectrophotometrically at 450 nm.
The absorption values at 450 nm in ELISA obtained for unmodified NSC and various paglierani options IC, is used to estimate the relative degrees of their immunoreactivity against the polyclonal anticigarette IC induced in rats. A study using ELISA show that conjugation with one group of PEG molecular weight of 20 kDa in one of three domains of IC reduces its reactivity in relation to the contents of the wells are less than 20% against polyclonal antisera SC. Conjugation with two groups of PEG molecular weight of 20 kDa, i.e., one with N-and the other with C-end IC reduces its reactivity in relation to the contents of the wells are less than 20% against polyclonal antisera. Conjugation with two groups of PEG molecular weight of 20 kDa, i.e., one in each domain SC brings reactivity almost to the detected level. Therefore, it is clear that conjugation of molecules(s) PEG with different areas of the IC significantly reduces their reactivity against polyclonal serum. In vitro tests showing low reactivity of the antibodies, prove that the reduced induction of the immune response occurs as soon as Mahilyow the config protein is administered to the living animal.
Table 33 lists the data immunoreactivity in percent remaining in paglierani versions of the IC, if the reactivity of unmodified SK wild-type is taken as 100%. Thus, the present invention discloses paglierani variants of streptokinase with significantly reduced immunoreactivity, but with intact thrombolytic activity.
The ability to activate plasminogen in different Paglierani mutant polypeptides of streptokinase.
In tables 35 and 36 below summarizes the capacity to activate plasminogen in different variants of SEQ ID NO:4 (16-383) that contain the replacement of a particular amino acid to cysteine. The analysis was carried out according to the technique disclosed in the description of this application.
Found that the profile activation Paglierani the cysteine variants of SEQ ID NO:4 have approximately a 10-minute lag period. This phenomenon was also observed for mutants derived from SEQ ID NO:491 (see example 8). However, it is noteworthy that when tested the ability to activate plasminogen these options after they formed a complex with equimolar amounts of plasmin, the profile of activation was the same as that of the natural streptokinase, i.e. without significant lag in the activation of plasminogen.
The results also absurd is defended in the example 8 of the present application, indicate dependent plasmin mechanism of action. Obviously, mediated plasmin activation of plasminogen is a significant advantage in thrombolytic therapy because of the blood clots enriched by plasmin, whereas in free flow, as a rule, there is no free active plasmin. Thus, activation of plasminogen, which depends on plasmin is an effective transpacificus mechanism of action inherent in variants of streptokinase according to the invention.
Advantages of the invention
The advantages of the present invention consist in solving design cysteine variants of streptokinase, Malinov, species variants and covalently modified forms. Sitespecifically conjugation of PEG to cysteine variants disclosed in this invention, according to the streptokinase molecule useful therapeutic properties, such as increased proteolytic stability, improved half-life in vivo and reduced immunoreactivity. More specifically, the present invention relates to the production of engineered variants of streptokinase for use in pharmaceutical compositions for the treatment of circulatory disorders.
|Quantities characterizing the availability of the solvent for different amino acids, selected to replace the cysteine|
|SC or variant||Localization of cysteine mutations||Availability|
|188||loop 88-97 alpha-domain||30|
|S 93||loop 88-97 alpha-domain||42|
|D 95||loop 88-97 alpha-domain||132|
|D 96||loop 88-97 alpha-domain||58|
|D 102||β4 alpha-domain||58|
|S 105||prior β4||123|
|D 120||the area between β5 and β6||65|
|K 121||the area between β5 and β6 domain||199|
|D 122||the area between β5 and β6 alpha-domain||101|
|E 148||the linker region between alpha - and beta-domains||118|
|K 156||the linker region between alpha - and beta-domains||109|
|D 173||the loop between β1 and β2 beta-domain||145|
|D 174||the loop between β1 and β2 beta-domain||111|
|L 179||β2 beta-domain||54|
|D 181||at the end of β2 beta-domain||36|
|S 205||α-helix (α3,4) beta-domain||62|
|N 255||loop 250 beta-domain||41|
|K 256||loop 250 beta-domain||174|
|K 257||loop 250 beta-domain||189|
|S 258||loop 250 beta-domain||79|
|L 260||ETS 250 beta-domain||112|
|K 282||the linker region between beta - and gamma-domains||154|
|F 287||the linker region between beta - and gamma-domains||161|
|D 303||β1 gamma-domain||134|
|L 321||The region of the double helix gamma-domain||47|
|L 326||the region of the double helix gamma-domain||12|
|And 333||the region of the double helix gamma-domain||24|
|D 347||β4 gamma-domain||53|
|R 372||β7 gamma-domain||234|
|Various designs of IC, its maleinos and fused polypeptides, used for cysteine mutagenesis|
|SEQ ID NO:1||NSC (1-414) ATS 12449|
|SEQ ID NO:2||SC (1-383)|
|SEQ ID N0:3||SC (16-414)|
|SEQ ID NO:4||SC (16-383)|
|SEQ ID NO:5||SC (50-414)|
|SEQ ID NO:6||SC (60-383)|
|SEQ ID NO:7||SC Asn 90 Ala (1-414)|
|SEQ ID NO:8||IC Asp Tyr 227 (1-414)|
|SEQ ID NO:9||IC Asp 2~8 Ala (1-414)|
|SEQ ID NO:10||SC 240 Glu Ala (1-414)|
|SEQ ID NO:11||SK Arg 244 Ala (1-414)|
|SEQ ID NO:12||SC Lys 246 Ala (1-414)|
|SEQ ID NO:13||SC Leu Ala 260 (1-414)|
|SEQ ID NO:14||IC Asp 359 Arg (1-414)|
|SEQ ID NO:15||SC His, Ser 92, 93 Ala, Ala (1-414)|
|SEQ ID NO:16||SC Lys, Lys 278, 279 Ala, Ala (1-414)|
|SEQ ID NO:17||SC Asn 90 del (1-413)|
|SEQ ID NO:18||IC Asp 227 del (1-413)|
|SEQ ID NO:19||IC Asp 359 del (1-413)|
|SEQ ID NO:20||SC (1-406) Streptococcus pyogenes MGAS10270, ABF348181|
|SEQ ID NO:21||SC (1-414) Streptococcus dysgalactiae subsp. Equisimilis, AAC60418|
|SEQ ID NO:22||Fn SK (1-531)|
|SEQ ID NO:23||SK Fn (1-502)|
|SEQ ID NO:24||Fn SK Fn (t-619)|
|Replacement of the cysteine in the polypeptide SC (SEQ ID NO:1): 1-414|
|SEQ ID NO:25||Gly 49 Cys|
|SEQ ID NO:26||Ser 57 Cys|
|SEQ ID NO:27||Ala 64 Cys|
|SEQ ID NO:28||lle 88 Cys|
|SEQ ID NO:29||Ser Cys 93|
|SEQ ID NO:30||Asp 95 Cys||SEQ ID NO:31||Asp 96 Cys|
|SEQ ID NO:32||Asp 102 Cys|
|SEQ ID NO:33||Ser 105 Cys|
|SEQ ID NO:34||Asp 120 Cys|
|SEQ ID NO:35||Lys 121 Cys|
|SEQ ID NO:36||Asp 122 Cys|
|SEQ ID NO:37||Glu Cys 148|
|SEQ ID NO:38||Lys Cys 156|
|SEQ ID NO:39||Asp Cys 173|
|SEQ ID NO:40||Asp Cys 174|
|SEQ ID NO:41||Leu Cys 179|
|SEQ ID NO:42||Asp Cys 181|
|SEQ ID NO:43||Ser 205 Cys|
|SEQ ID NO:44||Ala 251 Cys|
|SEQ ID NO:45||lle Cys 254|
|SEQ ID NO:46||Asn 255 Cys|
|SEQ ID NO:47||Lys 256 Cys|
|SEQ ID NO:48||Lys 257 Cys|
|SEQ ID NO:49||Ser 258 Cys|
|SEQ ID NO:50||Leu Cys 260|
|SEQ ID NO:51||Glu Cys 281|
|SEQ ID NO:52||Lys Cys 282|
|SEQ ID NO:53||Phe 287 Cys|
|SEQ ID NO:54||Asp Cys 303|
|SEQ ID NO:55||Leu 321 Cys|
|SEQ ID NO:56||Leu 326 Cys|
|SEQ ID NO:57||Ala 333 Cys|
|SEQ ID NO:58||Asp 347 Cys|
|SEQ ID NO:59||Asp 360 Cys|
|SEQ ID NO:60||Arg 372 Cys|
|SEQ ID NO:61||Ile 88 Cys, Ser 205 Cys|
|SEQ ID NO:62||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:63||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:64||Ser 105 Cys and Phe 287 Cys|
|SEQ ID NO:65||Lys 121 Cys, Asp 360 Cys/td>|
|SEQ ID NO:66||lle 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:67||Ser Cys 93, 255 Asn Cys, Asp 347 Cys|
|SEQ ID NO:68||Ser Cys 93, 255 Asn Cys, Phe 287 Cys|
|SEQ ID NO:69||Asp 102 Cys, Leu 260 Cys, Arg·372 C~|
|SEQ ID NO:70||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Single replacement of cysteine in a truncated polypeptide SC (SEQ ID NO:2): 1-383|
|SEQ ID NO:71||Gly 49 Cys|
|SEQ ID NO:72||Ser 57 Cys|
|SEQ ID NO:73||Ala 64 Cys|
|SEQ ID NO:74||Ile 88 Cys|
|SEQ ID NO:75||Ser Cys 93|
|SEQ ID NO:76||Asp 95 Cys|
|SEQ ID NO:77||Asp 96 Cys|
|SEQ ID NO:78||Asp 102 Cys||SEQ ID NO:79||Ser 105 Cys|
|SEQ ID NO:80||Asp 120 Cys|
|SEQ ID NO:81||Lys 121 Cys|
|SEQ ID NO:82||Asp 122 Cys|
|SEQ ID NO:83||Glu Cys 148|
|SEQ ID NO:84||Lys Cys 156|
|SEQ ID NO:85||Asp Cys 173|
|SEQ ID NO:86||Asp Cys 174|
|SEQ ID NO:87||Leu Cys 179|
|SEQ ID NO:88||Asp Cys 181|
|SEQ ID NO:89||Ser 205 Cys|
|SEQ ID NO:90||Ala 251 Cys|
|SEQ ID NO:91||Ile Cys 254|
|SEQ ID NO:92||Asn 255 Cys|
|SEQ ID NO:93||Lys 256 Cys|
|SEQ ID NO:94||Lys 257 Cys|
|SEQ ID NO:95||Ser 258 Cys|
|SEQ ID NO:96||Leu Cys 260|
|SEQ ID NO:97||Glu Cys 281|
|SEQ ID NO:98||Lys Cys 282|
|SEQ ID NO:99||Phe 287 Cys|
|SEQ ID NO:100||Asp Cys 303|
|SEQ ID NO:101||Leu 321 Cys|
|SEQ ID NO:102||Leu 326 Cys|
|SEQ ID NO:103||Ala 333 Cys|
|SEQ ID NO:104||Asp 347 Cys|
|SEQ ID NO:105||Asp 360 Cys|
|SEQ ID NO:106||Arg 372 Cys|
|SEQ ID NO:107||Il 88 Cys, Ser 205 Cys|
|SEQ ID NO:108||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:109||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:110||Ser 105 Cys and Phe 287 Cys|
|SEQ ID NO:111||Lys 121 Cys, Asp 360 Cys|
|SEQ ID NO:112||Ile 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:113||Ser Cys 93, 255 Asn Cys, Asp 347 Cys|
|SEQ ID NO:114||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:115||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:116||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Single replacement of cysteine in a truncated polypeptide SC (SEQ ID NO:3): 16-414|
|SEQ ID NO:117||Gly 49 Cys|
|SEQ ID NO:118||Ser 57 Cys|
|SEQ ID NO:119||Ala 64 Cys|
|SEQ ID NO:120||Ile 88 Cys|
|SEQ ID NO:121||Ser Cys 93|
|SEQ ID NO:122||Asp 95 Cys|
|SEQ ID NO:123||Asp 96 Cys|
|SEQ ID NO:124||Asp 102 Cys|
|SEQ ID NO:125||Ser 105 Cys|
|SEQ ID NO:126||Asp 120 Cys|
|SEQ ID NO:127||Lys 121 Cys|
|SEQ ID NO:128||Asp 122 Cys|
|SEQ ID NO:129||Glu Cys 148|
|SEQ ID NO:130||Lys Cys 156|
|SEQ ID NO:131||Asp Cys 173|
|SEQ ID NO:132||Asp Cys 174|
|SEQ ID NO:133||Leu Cys 179|
|SEQ ID NO:134||Asp Cys 181|
|SEQ ID NO:135||Ser 205 Cys|
|SEQ ID NO:136||Ala 251 Cys|
|SEQ ID NO:137||Ile Cys 254|
|SEQ ID NO:138||Asn 255 Cys|
|SEQ ID NO:139||Lys 256 Cys|
|SEQ ID NO:140||Lys 257 Cys|
|SEQ ID NO:141||Ser 258 Cys|
|SEQ ID NO:142||Leu Cys 260|
|SEQ ID NO:143||Glu Cys 281|
|SEQ IDNO:144||Lys Cys 282|
|SEQ ID NO:145||Pile 287 Cys|
|SEQ ID NO:146||Asp Cys 303|
|SEQ ID NO:147||Leu 321 Cys|
|SEQ ID NO:148||Leu 326 Cys|
|SEQ ID NO:149||Ala 333 Cys|
|SEQ ID NO:150||Asp 347 Cys|
|SEQ ID NO:151||Asp 360 Cys|
|SEQ ID NO:152||Arg 372 Cys|
|SEQ ID NO:153||Ile 88 Cys, Ser 205 Cys|
|SEQ ID NO:154||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:155||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:156||Ser 105 Cys and Phe 287 Cys|
|SEQ ID NO:157||Lys 121 Cys, Asp 360 Cys|
|SEQ ID NO:158||Ile 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:159||Ser Cys 93, 255 Asn Cys, Asp347 Cys|
|SEQ ID NO:160||Ser Cys 93, 255 Asn Cys, Phe 287 Cys|
|SEQ ID NO:161||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:162||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Single replacement of cysteine in a truncated polypeptide (SEQ ID NO:4): 16-383|
|SEQ ID NO:163||Gly 49 Cys|
|SEQ ID NO:164||Ser 57 Cys|
|SEQ ID NO:165||Ala 64 Cys|
|SEQ ID NO:166||Ile 88 Cys|
|SEQ ID NO:167||Ser Cys 93|
|SEQ ID NO:168||Asp 95 Cys|
|SEQ ID NO:169||Asp 96 Cys|
|SEQ ID NO:170||Asp 102 Cys|
|SEQ ID NO:171||Ser 105 Cys|
|SEQ ID NO:172||Asp 120 Cys|
|SEQ ID NO:173||Lys 121 Cys|
|SEQ ID NO:174||Asp 122 Cys|
|SEQ ID NO:175||Glu Cys 148|
|SEQ ID NO:176||Lys Cys 156|
|SEQ ID NO:177||Asp Cys 173|
|SEQ 10 no 178||Asp Cys 174|
|SEQ ID NO:179||Leu Cys 179|
|SEQ ID NO:180||Asp Cys 181|
|SEQ ID NO:181||Ser 205 Cys|
|SEQ ID NO:182||Ala 251 Cys|
|SEQ ID NO:183||Ile Cys 254|
|SEQ ID NO:184||Asn 255 Cys|
|SEQ ID NO:185||Lys 256 Cys|
|SEQ ID NO:186||Lys 257 Cys|
|SEQ ID NO:187||Ser 258 Cys|
|SEQ ID NO:188||Leu Cys 260|
|SEQ ID NO:189||Glu Cys 281|
|SEQ ID NO:190||Lys Cys 282|
|SEQ I NO:191||Phe 287 Cys|
|SEQ ID NO:192||Asp Cys 303|
|SEQ ID NO:193||Leu 321 Cys|
|SEQ ID NO:194||Leu 326 Cys|
|SEQ ID NO:195||Ala 333 Cys|
|SEQ ID NO:196||Asp 347 Cys|
|SEQ ID NO:197||Asp 360 Cys|
|SEQ ID NO:198||Arg 372 Cys|
|SEQ ID NO:199||Ile 88 Cys, Ser 205 Cys|
|SEQ ID NO:200||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:201||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:202||Ser 105 Cys. and Phe 287 Cys|
|SEQ ID NO:203||Lys 121 Cys, Asp 360 Cys|
|SEQ ID NO:204||Ile 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:205||Ser Cys 93, 255 Asn Cys, Asp 347 Cys|
|SEQ ID NO:206||Ser93 Cys, Asn 255 Cys, Phe 287 Cys|
|SEQ ID NO:207||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:208||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Single replacement of cysteine in a truncated polypeptide (SEQ ID NO:5): 50-414|
|SEQ ID NO:209||Ala 64 Cys|
|SEQ ID NO:210||Ile 88 Cys|
|SEQ ID NO:211||Ser Cys 93|
|SEQ ID NO:212||Asp 95 Cys|
|SEQ ID NO:213||Asp 96 Cys|
|SEQ ID NO:214||Asp t02 Cys|
|SEQ ID NO:215||Ser 105 Cys|
|SEQ ID NO:216||Asp 120 Cys|
|SEQ ID NO:217||Lys 121 Cys|
|SEQ ID NO:218||Asp 122 Cys|
|SEQ ID NO:219||Glu Cys 148|
|SEQ ID NO:220||Lys Cys 156|
|SEQ ID NO:221||Asp Cys 173|
|SEQ ID NO:222||Asp Cys 174|
|SEQ ID NO:223||Leu Cys 179|
|SEQ ID NO:224||Asp Cys 181|
|SEQ ID NO:225||Ser 205 Cys|
|SEQ ID NO:226||Ala 251 Cys|
|SEQ ID NO:227||Ile Cys 254|
|SEQ ID NO:228||Asn 255 Cys|
|SEQ ID NO:229||Lys 256 Cys|
|SEQ ID NO:230||Lys 257 Cys|
|SEQ ID NO:231||Ser 258 Cys|
|SEQ ID NO:232||Leu Cys 260|
|SEQ ID NO:233||Glu Cys 281|
|SEQ ID NO:234||Lys Cys 282|
|SEQ ID NO:235||Phe 287 Cys|
|SEQ ID NO:236||Asp Cys 303|
|SEQ ID NO:237||Leu 321 Cys|
|SEQ IDNO:238||Leu 326 Cys|
|SEQ ID NO:239||Ala 333 Cys|
|SEQ ID NO:240||Asp 347 Cys|
|SEQ ID NO:241||Asp 360 Cys|
|SEQ ID NO:242||Arg 372 Cys|
|SEQ ID NO:243||Ile 88 Cys, Ser 205 Cys|
|SEQ ID NO:244||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:245||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:246||Ser 105 Cys and Phe 287 Cys|
|SEQ ID NO:247||Lys 121 Cys, Asp 360 Cys|
|SEQ ID NO:248||Ile 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:249||Ser Cys 93, 255 Asn Cys, Asp 347 Cys|
|SEQ ID NO:250||Ser Cys 93, 255 Asn Cys, Phe 287 Cys|
|SEQ ID NO:251||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:252||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Single replacement of cysteine in a truncated polypeptide (SEQ ID NO:6): 60-383|
|SEQ ID NO:253||Ala 64 Cys|
|SEQ ID NO:254||Ile 88 Cys|
|SEQ ID NO:255||Ser Cys 93|
|SEQ ID NO:256||Asp 95 Cys|
|SEQ ID NO:257||Asp 96 Cys|
|SEQ ID NO:258||Asp 102 Cys|
|SEQ ID NO:259||Ser 105 Cys|
|SEQ ID NO:260||Asp 120 Cys|
|SEQ ID NO:261||Lys 121 Cys|
|SEQ ID NO:262||Asp 122 Cys|
|SEQ ID NO:263||Glu Cys 148|
|SEQ ID NO:264||Lys Cys 156|
|SEQ ID NO:265||Asp Cys 173|
|SEQ ID NO:266||Asp Cys 174|
|SEQ ID NO:267||Leu Cys 179|
|SEQ ID NO:268||Asp Cys 181|
|SEQ ID NO:269||Ser 205 Cys|
|SEQ ID NO:270||Ala 251 Cys|
|SEQ ID NO:271||Ile Cys 254|
|SEQ ID NO:272||Asn 255 Cys|
|SEQ ID NO:273||Lys 256 Cys|
|SEQ ID NO:274||Lys 257 Cys|
|SEQ ID NO:275||Ser 258 Cys|
|SEQ ID NO:276||Leu Cys 260|
|SEQ ID NO:277||Glu Cys 281|
|SEQ ID NO:278||Lys Cys 282|
|SEQ ID NO:279||Phe 287 Cys|
|SEQ ID NO:280||Asp Cys 303|
|SEQ ID NO:281||Leu 321 Cys|
|SEQ ID NO:282||Leu 326 Cys|
|SEQ ID NO:283||Ala 333 Cys|
|Asp 347 Cys|
|SEQ ID NO:285||Asp 360 Cys|
|SEQ ID NO:286||Arg 372 Cys|
|SEQ ID NO:287||Ile 88 Cys, Ser 205 Cys|
|SEQ ID NO:288||Ser Cys 93, 255 Asn Cys|
|SEQ ID NO:289||Asp 102 Cys, Arg 372 Cys|
|SEQ ID NO:290||Ser 105 Cys and Phe 287 Cys|
|SEQ ID NO:291||Lys 121 Cys, Asp 360 Cys|
|SEQ ID NO:292||Ile 88 Cys, Ser 205 Cys, Arg 372 Cys|
|SEQ ID NO:293||Ser Cys 93, 255 Asn Cys, Asp 347 Cys|
|SEQ ID NO:294||Ser Cys 93, 255 Asn Cys, Phe 287 Cys|
|SEQ ID NO:295||Asp 102 Cys, Leu 260 Cys, Arg 372 Cys|
|SEQ ID NO:296||Ser 105 Cys, Leu 260 Cys, Phe 287 Cys|
|Cysteine variants mutein SC 1-414, Asn 90 Ala: (SEQ ID NO:7)|
|Asp 102 Cys|
|SEQ ID NO:298||Leu Cys 260|
|SEQ ID NO:299||Asp 347 Cys|
|Cysteine variants mutein SC 1-414, Asp Tyr 227: (SEQ ID NO:8)|
|SEQ ID NO:297||Asp 102 Cys|
|SEQ ID NO:298||Leu Cys 260|
|SEQ ID NO:299||Asp 347 Cys|
|SEQ ID NO:300||Asp 102 Cys|
|SEQ ID NO:301||Leu Cys 260|
|SEQ ID NO:302||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Asp 238 Ala: (SEQ ID NO:9)|
|SEQ ID NO:303||Asp 102 Cys|
|SEQ ID NO:304|
|SEQ ID NO:305||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), 240 Glu Ala: (SEQ ID NO:10)|
|SEQ ID NO:306||Asp 102 Cys|
|SEQ ID NO:307||Leu Cys 260|
|SEQ ID NO:308||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Arg 244 Ala: (SEQ ID NO:11)|
|SEQ ID NO:309||Asp 102 Cys|
|SEQ ID NO:310||Leu Cys 260|
|SEQ ID NO:311||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Lys 246 Ala: (SEQ ID NO:12)|
|SEQ ID NO:312||Asp 102 Cys|
|SEQ ID NO:313||Leu Cys 260|
|SEQ ID NO:314||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Leu Ala 260: (SEQ ID NO:13)|
|SEQ ID NO:315||Asp 102 Cys|
|SEQ ID NO:316||Asn 255 Cys|
|SEQ ID NO:317||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Asp 359 Arg: (SEQ ID NO:14)|
|SEQ ID NO:318||Asp 102 Cys|
|SEQ ID NO:319||Leu Cys 260|
|SEQ ID NO:320||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), His, Ser, 92,93 Ala, Ala: (SEQ ID NO:15)|
|SEQ ID NO:321||Asp 102 Cys|
|SEQ ID NO:322||Leu Cys 260|
|SEQ ID NO:323||Asp 347 Cys|
|Cysteine variants mutein SC (1-414), Lys, Lys 278,279 Ala, Ala: (SEQ ID NO:16)|
|SEQ ID NO:324||Asp 102 Cys|
|SEQ ID NO:325||Leu Cys 260|
|SEQ ID NO:326||Asp 347 Cys|
|Cysteine variants mutein SC (1-413), Asn 90 cases.: (SEO ID NO:17)|
|SEQ ID NO:327||Asp Cys 101|
|SEQ ID NO:328||Leu 259 Cys|
|SEQ ID NO:329||Asp 346 Cys|
|Cysteine variants mutein SC (1-413), Asp 227 Affairs.: (SEO ID NO:18)|
|SEQ ID NO:330||Asp 102 Cys|
|SEQ ID NO:331||Leu 259 Cys|
|SEQ ID NO:332||Asp 346 Cys|
|Cysteine variants mutein SC (1-413), Asp 359 Affairs.: (SEO ID NO:19)|
|SEQ ID NO:333||Asp 102 Cys|
|SEQ ID NO:334||Leu Cys 260|
|SEQ ID NO:335||Asp 347 Cys|
|Cysteine variants of Streptococcus pyogenes MGAS 10270 (SEQ ID NO:20)|
|SEQ ID NO:336||Ile 80 Cys|
|SEQ ID NO:337||Ser Cys 85|
|SEQ ID NO:338||Asp 94 Cys|
|SEQ ID NO:339||Ile Cys 246|
|SEQ ID NO:340||Asp 339 Cys|
|SEQ ID NO:341||Arg 364 Cys|
|Cysteine variants of Streptococcus dysgalactiae subsp. equisimilis (SEQ ID NO:21)|
|SEQ ID NO:342||Ile 88 Cys|
|SEQ ID NO:343||Ser Cys 93|
|SEQ ID NO:344||Asp 102 Cys|
|SEQ ID NO:345||Leu Cys 260|
|SEQ ID NO:346||Asp 347 Cys|
|SEQ ID NO:347||Arg 372 Cys|
|Cysteine variants of the polypeptide, in which fibrin domain attached to the N-end IC (SEQ ID NO:22): 1-531|
|SEQ ID NO:348||His 16 Cys|
|SEQ ID NO:349||Ala 17 Cys|
|SEQ ID NO:350||Asp 62 Cys|
|SEQ ID NO:351||Gly 80 Cys|
|SEQ ID NO:352||Gly Cys 166|
|SEQ ID NO:353||Ser Cys 174|
|SEQ ID NO:354||Ala Cys 181|
|SEQ ID NO:355||Ile 205 Cys|
|SEQ ID NO:356||Ser Cys 210|
|SEQ ID NO:357||Asp 212 Cys|
|SEQ ID NO:358||Asp Cys 213|
|SEQ ID NO:359||Asp 219 Cys|
|SEQ ID NO:360||Asp 222 Cys|
|SEQ ID NO:361||Asp 237 Cys|
|SEQ ID NO:362||Lys 238 Cys|
|SEQ ID NO:363||Asp Cys 239|
|SEQ ID NO:364||Glu Cys 265|
|SEQ ID NO:365||Lys Cys 273|
|SEQ ID NO:366||Asp Cys 290|
|SEQ ID NO:367||Asp 291 Cys|
|SEQ ID NO:368||Leu 296 Cys|
|SEQ ID NO:369||Asp Cys 298|
|SEQ ID NO:370||Ser Cys 322|
|SEQ ID NO:371||Ile 371 Cys|
|SEQ ID NO:372||Asn 372 Cys|
|SEQ ID NO:373||Lys 373 Cys|
|SEQ ID NO:374||Lys Cys 374|
|SEQ ID NO:375||Ser Cys 375|
|SEQ ID NO:376||Leu 377 Cys|
|SEQ ID NO:377||Glu 398 Cys|
|SEQ ID NO:378||Lys Cys 399|
|SEQ ID NO:379||Phe 404 Cys|
|SEQ IDNO:380||Asp Cys 420|
|SEQ ID NO:381||Leu-438 Cys|
|SEQ ID NO:382||Leu Cys 443|
|SEQ ID NO:383||Ala Cys 450|
|SEQ ID NO:384||Asp 464 Cys|
|SEQ ID NO:385||Asp 477 Cys|
|SEQ ID NO:386||Arg 489 Cys|
|SEQ ID NO:387||His 16 Cys, Ile 205 Cys|
|SEQ ID NO:388||His 16 Cys, Ser Cys 322|
|SEQ ID NO:389||His 16 Cys, Leu 377 Cys|
|SEQ ID NO:390||His 16 Cys, Arg 489 Cys|
|Cysteine variants of the polypeptide, in which fibrin domain attached to the C-end IC (SEQ ID NO:23): 1-502|
|SEQ ID NO:391||Gly 49 Cys|
|SEQ ID NO:392||Ser 57 Cys|
|SEQ ID NO:393||Ala 64 Cys|
|SEQ ID NO:394||Ile 88 Cys|
|SEQ ID NO:395||Ser Cys 93|
|SEQ ID NO:396||Asp 95 Cys|
|SEQ ID NO:397||Asp 96 Cys|
|SEQ ID NO:398||Asp 102 Cys|
|SEQ ID NO:399||Ser 105 Cys|
|SEQ ID NO:400||Asp 120 Cys|
|SEQ ID NO:401||Lys 121 Cys|
|SEQ ID NO:402||Asp 122 Cys|
|SEQ ID NO:403||Glu Cys 148|
|SEQ ID NO:404||Lys Cys 156|
|SEQ ID NO:405||Asp Cys 173|
|SEQ ID NO:406||Asp Cys 174|
|SEQ ID NO:407||Leu Cys 179|
|SEQ ID NO:408||Asp Cys 181|
|SEQ ID NO:409||Ser 205 Cys|
|SEQ ID NO:410||Ala 251 Cys|
|SEQ ID NO:411||Ile Cys 254/td>|
|SEQ ID NO:412||Asn 255 Cys|
|SEQ ID NO:413||Lys 256 Cys|
|SEQ ID NO:414||Lys 257 Cys|
|SEQ ID NO:415||Ser 258 Cys|
|SEQ ID NO:416||Leu Cys 260|
|SEQ ID NO:417||Glu Cys 281|
|SEQ ID NO:418||Lys Cys 282|
|SEQ ID NO:419||Phe 287 Cys|
|SEQ ID NO:420||Asp Cys 303|
|SEQ ID NO:421||Leu 321 Cys|
|SEQ ID NO:422||Leu 326 Cys|
|SEQ ID NO:423||Ala 333 Cys|
|SEQ ID NO:424||Asp 347 Cys|
|SEQ ID NO:425||Asp 360 Cys|
|SEQ ID NO:426||Arg 372 Cys|
|SEQ ID NO:427||His Cys 401|
|SEQ ID NO:428||Ala 402 Cys|
|SEQ ID NO:429||Asp 447 Cys||SEQ ID NO:430||Gly 465 Cys|
|SEQ ID NO:431||Ile 88 Cys, His, Cys 401|
|SEQ ID NO:432||Ser 205 Cys, His, Cys 401|
|SEQ ID NO:433||Leu 260 Cys, His, Cys 401|
|SEQ ID NO:434||Arg 372 Cys, His, Cys 401|
|Cysteine variants of the polypeptide, in which fibrin domain is connected as N-,|
and C-end IC (SEQ ID NO:24): 1-619
|SEQ ID NO:435||His 16 Cys|
|SEQ ID NO:436||Ala 17 Cys|
|SEQ ID NO:437||Asp 62 Cys|
|SEQ ID NO:438||Gly 80 Cys|
|SEQ ID NO:439||Gly Cys 166|
|SEQ ID NO:440||Ser174 Cys|
|SEQ ID NO:441||Ala Cys 181|
|SEQ ID NO:442||Ile 205 Cys/tr>|
|SEQ ID NO:443||Ser Cys 210|
|SEQ ID NO:444||Asp 212 Cys|
|SEQ ID NO:445||Asp Cys 213|
|SEQ ID NO:446||Asp 219 Cys|
|SEQ ID NO:447||Ser 222 Cys|
|SEQ ID NO:448||Asp 237 Cys|
|SEQ ID NO:449||Lys 238 Cys|
|SEQ ID NO:450||Asp Cys 239|
|SEQ ID NO:451||Glu Cys 265|
|SEQ ID NO:452||Lys Cys 273|
|SEQ ID NO:453||Asp Cys 290|
|SEQ ID NO:454||Asp 291 Cys|
|SEQ ID NO:455||Leu 296 Cys|
|SEQ ID NO:456||Asp Cys 298|
|SEQ ID NO:457||Ser Cys 322|
|SEQ ID NO:458||Ile 371 Cys|
|SEQ ID NO:459||Asn 372 Cys|
|SEQ ID NO:460||Lys 373 Cys|
|Lys Cys 374|
|SEQ ID NO:462||Ser Cys 375|
|SEQ ID NO:463||Leu 377 Cys|
|SEQ ID NO:464||Glu 398 Cys|
|SEQ ID NO:465||Lys Cys 399|
|SEQ ID NO:466||Phe 404 Cys|
|SEQ ID NO:467||Asp Cys 420|
|SEQ ID NO:468||Leu 438 Cys|
|SEQ ID NO:469||Leu Cys 443|
|SEQ ID NO:470||Ala-Cys 450|
|SEQ ID NO:471||Asp 464 Cys|
|SEQ ID NO:472||Asp 477 Cys|
|SEQ ID NO:473||Arg 489 Cys|
|SEQ ID NO:474||His 518 Cys|
|SEQ ID NO:475||Ala 519 Cys|
|SEQ ID NO:476||Asp 564 Cys|
|SEQ ID NO:477||Gly 582 Cys|
|SEQ ID NO:478||His 16 Cys, Leu 377 Cys|
|SEQ ID NO:479||His 16 Cys, Ser Cys 322|
|SEQ ID NO:480||His 16 Cys, His, 518 Cys|
|SEQ ID NO:481||Ala 17 Cys, Ala 519 Cys|
|SEQ ID NO:482||Asp 62 Cys, Asp 564 Cys|
|SEQ ID NO:483||Gly 80 Cys, Gly 582 Cys|
|SEQ ID NO:484||His 16 Cys, Leu 377 Cys, Arg 489 Cys|
|SEQ ID NO:485||His 16 Cys, Leu 377 Cys, His, 518 Cys|
|Mutants SC with insertion of cysteine|
|SEQ ID NO:486||Cys between Il 88 and Ala 89 SK|
|SEQ ID NO:487||Cys between Lys 256 and Lys 257 SK|
|SEQ ID NO:488||Between Asp Cys 347 and Tyr 348 SK|
|Table 28||Cysteine variants of SK, where Cys is located at the ends of the SC or its functional fragment, peptide elongation or without him|
|SEQ ID NO:489||Cys at position N-1 in SC|
|SEQ ID NO:490||Cys at position C+1 in SK|
|SEQ ID NO:491||Cys at position N-1 and+1 in SK|
|SEQ ID NO:492||Cys at position C+1 SEQ 1D (1-383, G3 of SC)|
|SEQ ID NO:493||Cys at position N-1 and C+1 in SK (1-383, G3)|
|SEQ ID NO:494||Cys at the N-terminal SK with his-tag labeled|
|SEQ ID NO:495||Cys at the C-terminal IC with a his-tag labeled|
|SEQ ID NO:496||Cys with an additional 20 AA label pEt 15b before Il|
|The parameters of the stationary kinetics of some typical paglierani cysteine variants paglierani of streptokinase. Act what you want to make native SK is taken for 100% relative activity options|
|SEQ ID NO:||Activator protein %||% activation||The plasminogen Km(µm)|
|SEQ ID NO:1||NSC||100||0,4±0,1|
|SEQ ID NO:489||N-Cys||88±5||0,4±0,05|
|SEQ ID NO:30||SC D95C||96±6||0,5±0,05|
|SEQ ID NO:31||SC D96C||98±4||0,5±0,05|
|SEQ ID NO:32||SC D102C||74±4||0,5±0,1|
|SEQ ID NO:33||SC S105C||76±5||0,5±0,12|
|SEQ ID NO:35||SC KS||95±5||0,4±0,11|
|SEQ ID NO:39||SC D173C||72±4||0,4±0,11|
|SEQ ID NO:41||SC L179C||22±3||0,5±0,12|
|SEQ ID NO:46||SC N255C||98±3||0,4±0,13|
|SEQ ID NO:48||SC N257C||92±4||0,4±0,10|
|SEQ ID NO:49||L258C||100±6||0,5±0,11|
|SEQ ID NO:50||L260C||100±6||0,6±0,12|
|SEQ ID NO:492||C-383 Cys||92±4||0,4±0,05|
|SEQ ID NO:490||C-Cys||95±5||0,4±0,06|
|The parameters of the stationary kinetics of activation of the HPG paglierani cysteine variant form IC*, merged with fibrin domain|
|Paglierani variants of SEQ ID NO:||Molecule||The lag-period (min)||Km (µm)||% Activation|
|SEQ ID NO:22||CKFn||10||0,25-0,6||80-100|
|SEQ ID NO:23||Fn CK||08||of 0.24 to 0.63||60-100|
|SEQ ID NO:24||Fn SK Fn||18||0,48±1,0||60-80|
|*Parameters calculated from the linear plots of the reaction kinetic curves after removing the lag-period.|
#Defined relative to the activity of native SK from streptococcus sp. (ATCC 12,499), which is taken as 100%.
|Stationary kinetic parameters for HPG activation by streptokinase and pipelinename derivatives IC. The activity of native SK is taken as 100% for comparison with the activity of derivatives|
|SEQ ID NO:||Molecule||The lag-period (min)||Km (µm)||% Akti is then|
|SEQ ID NO:1||NSC||1||0,45±0,02||100||100|
|SEQ ID NO:491||biegeleben NC 1-414||14||0,42±0,03||<5||88±4|
|SEQ ID NO:493||biegeleben NC 1-383||12||0,48±0,02||<5||92±6|
|*ability to activate plasminogen, when the company or its derivatives pre-formed complex with equimolar amounts of plasmin to obtain enzymatic complex SC-GON.|
|The half-life in vivo of various paglierani variants of the SC and the thrombus-specific IC in mice|
|SEQ ID NO:||Molecule||Site mutations||The half-life (t1/2)|
|SEQ ID NO:1||NSC||-||<15 min|
|SEQ ID NO:489||N-cys||immediately after methionine||>3 hours|
|SEQ ID NO:30||D 95C||loop 88-97 alpha-domain||>4 h|
|SEQ ID NO:31||D 96C||loop 88-97 alpha-domain||>4 h|
|SEQ ID NO:48||K 257||loop 250 beta-domain||>2 hours|
|SEQ ID NO:49||S 258C||loop 250 beta-domain||>2 hours|
|SEQ ID NO:50||L 260C||loop 250 beta-domain||>2 hours|
|SEQ ID NO:487||QC 256, 257 KSK||embedded between Lys 256 and Lys 257||>2 hours|
|loop 250 beta-domain|
|SEQ ID NO:55||L 321||double-stranded region of the gamma-domain||>1 hour|
|SEQ ID NO:58||D 347C||β4 gamma-domain||>1 hour|
|SEQ ID NO:492||C-382-cys||C-terminal shortening in position 383 and cysteine located after three glycine residues||>1 hour|
|SEQ ID NO:490||C-cys||cysteine after C-terminal amino acids||>1 hour|
|SEQ ID NO:491||biegeleben NC 1-414||cysteine at both N - and C-ends||>6 h|
|SEQ ID NO:493||biegeleben NC 1-383||the cysteine at the N-end and C-end, where the cysteine is located after three Gly after truncating the ri 383||>6 h|
|Immunoreactivity paglierani cysteine variants of streptokinase|
|SEQ ID NO:||Paglierani derived||Immunoreactivity*|
|SEQ ID NO:30||D95C||15±2|
|SEQ ID NO:31||D96C||14±2,5|
|SEQ ID NO:37||E148C||16±5|
|SEQ ID NO:38||K156C||22±4|
|SEQ ID NO:41||L179C||17±5|
|SEQ ID NO:43||S205C||19±3|
|SEQ ID NO:50||L260C||17±2|
|SEQ ID NO:490||C-Cys||19±2|
|SEQ ID NO:491||N Cys, Cys||7±1|
|SEQ ID NO:62||S93C, N255C||2±02|
|SEQ ID NO:63||D102C,R372C||2,5±02|
|SEQ ID NO:61||I88C, S205C||2±05|
|SEQ ID NO:67||S93C,N255C,D347C||<1|
|SEQ ID NO:68||S93C, N255C, F287C||<1|
|*The immunoreactivity determined against Antis antibodies induced in rats. Values are in % of residual reactivity, if you take the reactivity of the protein wild type for 100%.|
|Deeply hidden remains of streptokinase, not suitable for replacement and subsequent modifications (SEQ ID NQ:1)|
|no residue in SEQ ID NO:||Amino acid||Surface accessibility|
|no residue in SEQ ID NO:||Amino acid||Surface accessibility|
|The parameters of the stationary kinetics of activation of the HPG shortened Paglierani cysteine variants of SK (16-383).|
|Paglierani variants of SEQ ID||The lag-period (min)||Km (µm)||% Of plasminogen activation||Numbering AK|
|SEQ ID NO 163||~10 min||0.4±0.07||84±9||G34C|
|SEQ ID N0164||~10 m is n||0.42±0.1||82±7||S42C|
|SEQ ID NO 165||~10 min||0.45±0.1||76±8||AS|
|SEQ ID NO 166||~10 min||0.4±0.04||72±5||S|
|SEQ ID NO 167||~10 min||0.4±0.1||76±6||S78C|
|SEQ ID NO 168||~10 min||0.4±0.1||75±7||D80C|
|SEQ ID NO 169||~10 min||0.4±0.1||64±5||D81C|
|SEQ ID NO 170||~10 min||0.4±0.1||68±8||D87C|
|SEQ ID NO 171||~10 min||0.5±0.1||72±6||S90C|
|SEQ ID NO 172||~0 min||0.4±0.05||66±5||D105C|
|SEQ ID NO 173||~10 min||0.4±0.05||78±6||KS|
|SEQ ID NO 174||~10 min||0.4±0.07||62±7||D107C|
|SEQ ID NO 175||~10 min||0.4±0.1||66±9||ES|
|SEQ ID NO 176||~10 min||0.4±0.05||77±6||KS|
|SEQ ID NO 177||~10 min||0.45±0.1||67±3||D158C|
|SEQ ID NO 178||~10 min||0.43±0.1||80±6||D159C|
|SEQ ID NO 179||~10 min||0.4±0.05||17±8||L164C|
|SEQ ID NO 180||~10 min||0.4±0.1||67±8||D166C|
|SEQ ID NO 181||~10 min||0.4±0.07||69±5||S190C|
|SEQ ID NO 182||~10 min||0.48±0.03||78±7||AS|
|SEQ ID NO 183||~10 min||0.4±0.05||83±8||I239C|
|SEQ ID NO 184||~10 min||0.4±0.1||84±4||N240C|
|SEQ ID NO 185||~10 min||0.4±0.5||66±8||KS|
|SEQ ID NO 186||~10 min||0.38±0.05||83±6||KS|
|SEQ ID NO 187||~10 min||0.4±0.1||75±5||S243C|
|SEQ ID NO 188||0.39±0.1||77±7||L245C|
|SEQ ID NO 189||~10 min||0.4±0.2||63±8||ES|
|SEQ ID NO 190||~10 min||0.4±0.05||66±5||L267C|
|SEQ ID NO 191||~10 min||0.4±0.2||79±8||F272C|
|SEQ ID NO 192||~10 min||0.4±0.1||81±5||D288C|
|SEQ ID NO 193||~10 min||0.4±0.1||82±6||L306C|
|SEQ ID NO 194||~10 min||0.4±0.05||85±8||L311C|
|SEQ ID NO 195||~10 min||0.4±0.08||79±8||AS|
|SEQ ID NO 196||~10 min||0.4±0.03||76±5||D332C|
|SEQ ID NO 197||~10 min||0.4±0.1||78±5||D345C|
|SEQ ID NO 198||~10 min||0.4±0.05||73±9||R357C|
|*Ability to activate plasminogen was measured when SK or its shortened version of a pre-formed complex with equimolar amounts of plasmin - enzyme complex SK:PN, which was subsequently used to activate the substrate plasminogen.TOn the contrary, showed characteristic lag-the period when the ability to activate the substrate - plasminogen was studied in option by itself (without prior formation of a complex with plasmin).|
|The parameters of the stationary kinetics of activation of the HPG shortened Paglierani cysteine variants of SK (16-383).|
|The position of the two/three simultaneous mutations in the sequence (16-383)||Paglierani variants of SEQ ID||The lag-period (Min)||Km (µm)||% Activation|
|Il 73 and Ser 190||SEQ ID NO 199||~10 min||0.4±0.1||66±7|
|Ser 78 and Asn 240||SEQ ID NO 200||~10 min||0.45±0.1||62±5|
|Ile 73, Ser 190 and Arg 357||SEQ ID NO 204||~10 min||0.5±0.1||53±5|
|*Activation of plasminogen, when SK or its shortened version of a pre-formed complex with equimolar amounts of plasmin - enzyme complex SK:PN was observed almost immediately. On the contrary, showed characteristic lag period, when to activate plasminogen used option by itself (without prior formation of a complex with plasmin).|
|The parameters of the stationary kinetics of activation of the HPG Paglierani sistei the new variants of natural SK. % Of plasminogen activation|
|The position of the two/three simultaneous mutations in the sequence of the original sequence SK(1-414).||Paglierani variants of SEQ ID||Km (µm)||% Of plasminogen activation|
|Il 88 and Ser 205||SEQ ID NO 61||0.4±0.1||88±6|
|Ser 93 and Asn 255||SEQ ID NO 62||0.4±0.1||90±7|
|Ile 88, Ser 205 and Arg 372||SEQ ID NO 66||0.5±0.1||85±5|
In the kinetics of plasminogen activation lag period was not observed
Abuchowski, A., Kazo, G. M., Verhoest, P. R., van Es, T., Kafkewitz, D., Nucci, M. L. Viau, A. T. and Davis, F. F. Cancer Biochem. Biophys. 1984; 7: 175-186.
Adams D. S, Griffin L. A, Nachajko W. R, Reddy V. B, Wei C. M. A synthetic DNA encoding a modified human urokinase resistant to inhibition by serum plasminogen activator inhibitor. J. Biol. Chem. 1991; 266 (13): 8476-8482.
Alien, T. M. Liposomes: opportunities in drug development. Drugs 1997; 54: Suppl. 4, 8-14.
Baker D. P E. Lin Y, Lin K, Pellegrini M, Petter R. C, Chen L, R. Arduini M, Brickelmaier M, Wen D, Hess D. M, Chen L, Grant D, Whitty A, Gill A, O. Lindner J, Pepinsky, R. B. N-terminally PEGylated human interferon-beta-la with. improved pharmacokinetic properties and in vivo efficacy in a melanoma angiogenesis model. Bioconjug Chem. 2006; 17 (1): 179-188.
Banerjee, A., Chisti y, Banerjee U. C. Streptokinase-a clinically usful thrombolytic agent. Biotechnol. Adv. 2004; 22 (4): 287-307.
Basu, A., Yang, K., Wang M., Liu, S., R. Chintala, Palm, T., Zhao, H., Peng, P., O. Wu, Z. Zhang, Hua J., Hsieh, M. C., J. Zhou, G. Petti, X. Li, A. Janjua, Mendez M., Liu J., Longley, C., Zhang Z.; Mehlig, M., Borowski V., Viswanathan M., Filpula D. Structure-function engineering of interferon-beta-lb for improving stability, solubility, potency, immunogenicity, and pharmacokinetic properties by site-selective monoPEGylation. Bioconjug Chem. 2006; 17 (3): 618-630.
Castellino F. J., A unique enzyme-protein substrate reaction modifier plasmin/streptokinase interaction. Trends Biochem. Sci. 1979, 4: 1-5.
Castelino, F. J. Recent advances in the chemistry of the fibrinolytic system. Chem. Rev. 1981; 81: 431-446.
Director A., Vasudha S., Rajagopal K., Komath, S. S., Garg N., Yadav M., Mande, S. C., G. multi-stories Function of the central domain of streptokinase in. substrate plasminogen docking and processing revealed by site-directed mutagenesis. Protein Sci 1999; 8: 2791-2805.
COLLEN D, STUMP D. C., GOLD, H. K., THROMBOLYTIC THERAPY. ANN. REV. MED., 1988, - 39: 405-423.
COLLEN D. CORONARY THROMBOLYSIS: STREPTOKINASE OR RECOMBINANT TISSUE-TYPE PLASMNOGEN ACTIVATOR? ANN INTERN MED. 1990; 112 (7): 529-538.
Deutsch D. G, Mertz, E. T. Plasminogen: purification from human plasma by affinity chromatography. Science. 1970; 170 (962): 1095-1096.
Dhar J, Pande A. H, V Sundram, J. Nanda, S., Mande, S. C., multi-stories G. Involvement of a nine-residue loop of streptokinase in the generation of macromolecular substrate specificity by the activator complex through interaction with substrate kringle domains. J. BioI. Chem. 2002; 277, 13257-13267.
Doherty, D. H., Rosendahl, M. S., Smith D. J., Hughes J. M., Chlipala, E. A., Cox, G. N. Site-specific PEGylation of engineered cysteine analogues of recombinant human granulocyte-macrophage colony-stimulating factor. Bioconjug Chem. 2005; 16 (5): 1291-1298.
Esmon C. T., T. Mather Switching serine protease specificity. Nat Struct Biol. 1998; 5 (11): 933-937.
Fraker, P. J., Speck J, C., Jr. Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem. Biophys. Res. Comm. 1978; 80 (4): 849-857.
Francis C. W., Marder, V. J. Fibmolytic therapy for venous thrombosis. Prog. Cardiovasc. Dis 1991; 34 (3); 193-204.
Grafe, S., Ellinger So, Maike H. Structural dissection and functional analysis of the promoter complex of the streptokinase gene from Streptococcus equisimilis H46A.
Med Immunol Environ. 1996; 185 (1): 11-17.
Hershfield, M. S., Buckley, R. H., Greenberg, M. L., Melton A. L., R. Schiff, Hatem C, Kurtzberg J., Markert, M. L., R. H. Kobayashi, A. Kobayashi L. Treatment of adenosine deaminase deficiency = MKD with. polyethylene glycol-modified adenosine deaminase. N. Engl. J. Med. 1987; 316 (10): 589-596;
Huang I. T., H. Malke, Ferretti J. J. Heterogeneity of the streptokinase gene in group A streptococci. Infect Immun. 1989; 57 (2): 502-506.
ISIS-3: a randomised comparison of streptokinase vs tissue plasmnogen activator vs anistreplase and of aspirin plus heparin vs aspirin alone among 41 299 cases of suspected acute myocardial infarction. ISIS-3 (Third International Study of infarct dementia Survival) Collaborative Group. Lancet. 1992; 339 (8796): 753-770.
Innis M. A., Gelfand D. A., J. J. Sninsky, T. J. White (1990); PCR. protocols. Academic Press, Inc, San Diego
Jackson K. W., Tang J. Complete aminoacid sequence of streptokinase and its homology with serine proteases. Biochemistry. 1982; 21 (26): 6620-6625.
Jalihal, S., Morris G. K. Antistreptokinase titres after intravenous streptokinase. Lancet. 1990; 335 (8688): 534.
Kabsch W, Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogenbonded and geometrical features. Biopolymers. 1983; 22 (12): 2577-2637.
Katre N. V, Knauf, M. J, Laird W. J. Chemical modification of recombinant interleukin 2 by polyethylene glycol increases its potency in the murine Meth A sarcoma model. Proc. Natl. Acad. Sci. USA. 1987; 84 (6): 1487-1491.
Katre, N. V. Immunogenicity of recombinant IL-2 modified by covalent attachment of polyethylene glycol. J Immunol. 1990; 144 (1): 209-213.
Kurflirst M. M. Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal. Biochem. 1992; 200 (2): 244-248.
Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227 (5259): 680-685.
Lahteenmaki, K., Kuusela, P., Korhonen, T. K. Bacterial plasminogen activators and receptos. FEMS Environ. Rev. 2001; 25 (5): 531-552. Review.
Lee H. S, Cross S, Davidson R, Reid T, Jennings K. Raised levels of antistreptokinase antibody and neutralization titres from 4 days to 54 months after administration of streptokinase or anistreplase. Eur. Heart J. 1993; 14 (1): 84-89.
Lijnen h R, J. Stassen M, Vanlinthout I., Fukao, H., Okada K., Matsuo O., Collen D. Comparative fibrinolytic properties of staphylokinase and streptokinase in animal models of ve. No. us thrombosis. Thromb. Haemost. 1991; 66 (4): 468-473.
Lyczak, J. B. & Morrison, S. L. Biological and pharmacokinetic properties of a novel immunoglobulin-CD4 fusion protein. Arch. Virol. 1994; 139, 189-196.
Malke, H., Ferretti J. J. Streptokinase: cloning, expression, and excretion by Escherichia coli. Proc. Natl. Acad. Sci. USA. 1984; 81 (11): 3557-3561.
Malke, H., J. J. Ferretti Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184. J Basic Environ. 1985; 25 (6): 393-400.
Malke H. Polymorphism of the streptokinase gene: implications for the pathogenesis of poststreptococcal glomerulonephritis. Zentralbl. Bakteriol. 1993; 278 (2-3): 246-257.
Marder VJ. Recombinant streptokinase: opportunity for an improved agent. Blood Coagul. Fibrinolysis. 1993; 4 (6): 1039-1040.
Mateo C., J. Lombardero, Moreno E., Morales, A., Bombino G., J. Coloma, Wims L., Morrison, S. L., Perez R. Removal of amphipathic epitopes from genetically-engineered antibodies: production of modified immmunoglobulins with reduced immunogenicity. Hybridoma. 2000; 19, 436-471.
McGrath K. G., Patterson R. Anaphylactic reactivity to streptokinase. JAMA. 1984; 252 (10): 1314-1317.
McGrath K, Patterson R. Immunology of streptokinase in human subjects. Clin. Exp. Immunol 1985; 62 (2): 421-426.
Meyers F. J, Paradise, C., Scudder S. A, Goodman G., Konrad M. A phase I study including pharmacokinetics of polyethylene glycol conjugated interleukin-2. Clin. Phannacol Ther. 1991; 49 (3): 307-313.
Monfardini, C., et al. A branched monomethoxypolyethylene glycol for protein modification. Bioconjug. Chem. 1995; 6: 62-69.
Moreadith, R. W., Collen D. Clinical development of PEGylated recombinant staphylokinase (PEG-Sak) for bolus thrombolytic treatment of patients with acute myocrdial infarction. Adv. Drug Deliv Rev. 2003; 55 (10): 1337-45.
Nihalani, D., Kumar R., Rajagopal K., multi-stories G. Role of the amino-terminal region of streptokinase in the generation of a fully functional plasminogen activator complex probed with synthetic peptides. Protein Sci 1998; 7, 637-648.
Nicolini F. A, Nichols W. W., Saldeen T. G., Khan, S., Mehta J, L. Adjunctive therapy with low molecular weight heparin with recombinant tissue-type plasminogen activator causes sustained reflow in canine coronary thrombosis. Am. Heart J. 1992; 124 (2): 280-288.
Osbom B. L., Olsen, H. S., Nardelli Century, Murray J. H., Zhou J. X., Garcia, A., G. Moody, Zaritskaya L. S., Sung C. Pharmacokinetic and pharmacodynamic studies of a human serum albumin-interferonalpha fusion protein in cynomolgus monkeys. J. Phannacol Exp. Ther. 2002; 303 (2): 540-548.
Ouriel K. Comparison of safety and efficacy of the various thrombolytic agents. Rev. Cardiovasc. Med. 2002; 3 Supp. 12: S 17-24. Review.
Pratap J., Kaur J., G. RajaMohan, D. Singh, K. L. Dikshit Role of N-terminal domain of streptokinase in protein transport. Biochem. Biophys. Res. Commun. 1996; 227, 303-310.
Ms. Rajagopalan, S., and S. L. Gonias, S. V. Pizzo A nonantigenic covalent streptokinase-polyethylene glycol complex with plasminogen activator function. J Clin Invest. 1985; 75 (2): 413-9.
Rabijns, A., Hendrik, L., De Bondt, H. L. and De Ranter, C. Three dimensional structure of staphylokinase, a plasminogen activator with therapeutic potential. Nat. Struct. Biol. 1997; 4, 357-360.
Reed G. L., Houng, A. K., Liu L., Parhami-Seren Century, L. Matsueda H., Wang, S., Hedstrom L. A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator. Proc. Natl. Acad. Sci. USA. 1999; 96 (16): 8879-83.
Roberts, M. J., Bentley, M. D. & Harris, J. M. Chemistry for peptide and protein PEGylation. Adv. Drug Delivery Rev. 2002; 54, 459-476.
Ross A. M. New plasminogen activators: a clinical review. Clin. Cardiol. 1999; 22 (3): 165-171. Review.
Sazo no va I. Y., Robinson B. R., Gladysheva I. P., Castellino F. J., Reed, G. L. Alpha-domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator. J. Biol. Chem. 2004; 279 (24): 24994-5001.
Schweitzer D. H., van der Wall, E. E., Bosker h A, Scheffer E., Macfarlane I. D. Serum-like illness as a complication after streptokinase therapy for acute myocardial infarction. Cardiology, 1991; 78 (1): 68-71.
Sherry S., Marder V J.. Thrombosis, fibrinolysis, and thrombolytic therapy: a perspective. Prog Cardiovasc. Dis. 1991; 34 (2): 89-100.
Shi, G. Y., Chang. B. I., Chen S. M., Wu D. H., Wu H. L. Function of streptokinase fragments in plasminogen activation. Biochem J. 1994; 304, 235-241.
Spotti F., Kaiser R. Rapid detection and quantitation of precipitating streptokinase-antibodies. Thromb Diath. Haemorrh. 1974; 32 (2-3): 608-616.
Studier F. W., Moffatt, B. A. Use of bacteriophage T7 RNA polymerase to direct selective high level expression of cloned genes. J. Mol. Biol. 1986; 189, 113-130.
Studier F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol. 1990; 185: 60-89.
Sundram V., J. Nanda, S., Rajagopal K, J. Dhar, Director A., G. multi-stories Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover. J. Biol. Chem. 2003; 278 (33): 30569-30577.
Syed, S. et al. Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin. Blood, 1997; 89 (9): 3243-3252.
Tillet, W. S., R. L. Garner The fibrinolytic activity of hemolytic streptococci. J. Exp. Med. 1933, 68: 485-488.
Wang X., Lin X., Loy J. A., Tang, J., Zhang X. C. Crystal structure of the catalytic domain of human plasmin complexed with streptokinase. Science. 1998; 281 (5383): 1662-1665.
Wang X., Tang J., Hunter, C., Zhang X. C. Crystal structure of streptokinase beta-domain. FEBS Lett. 1999; 459 (1): 85-89.
Wohl, R. C., L. Summaria, Robbins, K. C. Kinetics of activation of human plasminogen by different activator species at pH 7.4 and 37 degrees C. J. Biol. Chem. 1980; 255, 2005-2013.
Wu H. L., Shi, G. Y., Bender M. L. Preparation and purification of microplasmin. Proc. Nat. Acad. Sci. USA. 1987; 84 (23): 8292-8295.
Wu X. C., Ye, R., Duan Y., Wong, S. L. Engineering of plasmin-resistant forms of streptokinase and their production in Bacillus subtilis: streptokinase with longer functional half-life. Appi Enviro. Environ. 1998; 64 (3):824-829.
1. Purified mutant streptokinase polypeptide containing a substitution of one to three amino acid residues of the sequence SEQ ID NO:4 by a cysteine residue, in which any replaceable residue selected from the group consisting of: G34; S42; A; I73; S78; D80; D81; D87; S90; D105; K106; D107; E133; K141; D158; D159; L164; D166; S190; A; I239; N240; K241; K242; S243; L245; E; K267; F272; D288; L306; L311; a; D332; D345 and R357.
2. Purified mutant streptokinase polypeptide under item 1, in which the substituted residues selected from the group I73 and S190; S78 and N240; D87 and R357; S78, N240 and D272; S78, N240 and F272.
3. Fused polypeptide having the activity of streptokinase and the ability to activate plasminogen to thrombolyse containing mutant streptokinase polypeptide under item 1, with the lengthening of the N-end C-end or N - and C-ends of the polypeptide, and any extension includes fibrinolitikami domain.
4. Fused polypeptide under item 3, wherein the mutant polypeptide of streptokinase is mutein streptokinase, which contained the amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24.
5. Fused polypeptide under item 3, containing the amino acid sequence selected from the group of SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24, where from one to three amino acid residues is replaced by a cysteine residue, where the substituted amino acid corresponds to the amino acid selected from the group, with Toyama of G34; S42; A; I73; S78; D80; D81; D87; S90; D105; K106; D107; E133; K141; D158; D159; L164; D166; S190; A; I239; N240; K241; K242; S243; L245; E; K267; F272; D288; L306; L311; a; D332; D345 and R357 according to SEQ ID NO:4.
6. Fused polypeptide under item 4, where the mutant streptokinase polypeptide is mutein streptokinase, which contained the amino acid sequence of SEQ ID NO:24.
7. Covalently modified mutant streptokinase polypeptide containing a substitution of one to three amino acid residues of the sequence SEQ ID NO:4 by a cysteine residue, in which any replaceable residue selected from the group consisting of: G34; S42; A; I73; S78; D80; D81; D87; S90; D105; K106; D107; E133; K141; D158; D159; L164; D166; S190; A; I239; N240; K241; K242; S243; L245; E; K267; F272; D288; L306; L311; A; D332; D345; R357; I73 and S190; S78 and N240; D87 and R357; S78, N240 and D272; S78, N240 and F272, in which any substitute cysteine residue modified reactive towards cysteine group containing polyethylene glycol.
8. Covalently modified mutant streptokinase polypeptide under item 7, where the polyethylene glycol is a linear or branched polymer with a molecular weight in the range from about 5000 daltons to about 40,000 daltons.
9. Purified fused polypeptide having the activity of streptokinase and the ability to activate plasminogen to thrombolyse, which contains a substitution of one to three amino acid residues after the outermost SEQ ID NO:22 cysteine residue, in which any replaceable residue selected from the group consisting of: G166; S174; A; I205; S210; D212; D213; D219; S222; D237; K238; D239; E; K273; D290; D291; L296; D298; S322; A; I371; N372; K373; K374; S375; L377; E; K399; F404; D420; L438; L443; A450; D464; D477; R489; I205 and S322; S210 and N372; D219 and R489; S210, N372 and D464; S210, N372 and F404.
10. Purified fused polypeptide having the activity of streptokinase and the ability to activate plasminogen to thrombolyse, which contains a substitution of one to three amino acid residues of the sequence SEQ ID NO:23 a cysteine residue, in which any replaceable residue selected from the group consisting of: G49; S57; A; I88; S93; D95; D96; D102; S105; D120; K121; D122; E; K156; D173; D174; L179; D181; S205; A; I254; N255; K256; K257; S258; L260; E; K282; F287; D303; L321; L326; A; D347; D360; R372; I88 and S205; S93 and N255; D102 and R372; S93, N255 and D347; S93, N255 and F287.
11. Purified fused polypeptide having the activity of streptokinase and the ability to activate plasminogen to thrombolyse, which contains a substitution of one to three amino acid residues of the sequence SEQ ID NO:24 cysteine residue, in which any replaceable residue selected from the group consisting of: G166; S174; A; I205; S210; D212; D213; D219; S222; D237; K238; D239; E; K273; D290; D291; L296; D298; S322; A; I371; N372; K343; K374; S375; L377; E; K399; F404; D420; L438; L443; A450; D464; D477; R489; I205 and S322; S210 and N372; D 189 and R489; S210, N372 and D464; S210, N372 and F404.
12. Covalently modified mutant streptokinase polypeptide containing a sequence selected from the group consisting of SQ ID NO:22, 23, 24, 489, 490, 491 and 492, in which from one to three amino acid residues is replaced by a cysteine residue, in which any replaceable residue corresponds to the amino acid selected from the group consisting of: G34; S42; A; I73; S78; D80; D81; D87; S90; D105; K106; D107; E133; K141; D158; D159; L164; D166; S190; A; I239; N240; K241; K242; S243; L245; E; K267; F272; D288; L306; L311; A318; D332; D345 and R357 according to SEQ ID NO:4, in which any substitute cysteine residue modified reactive towards cysteine group containing polyethylene glycol.
13. Pharmaceutical composition for treatment of circulatory disorders, containing a modified mutant streptokinase according to any one of paragraphs.7 or 8 and a pharmaceutically acceptable filler.
14. Pharmaceutical composition for the treatment of thrombosis, containing a modified mutant streptokinase according to any one of paragraphs.7 or 8 and a pharmaceutically acceptable filler.
15. Pharmaceutical composition for the treatment of thrombosis, containing the protein according to any one of paragraphs.3, 4, 5 or 6 and a pharmaceutically acceptable filler.
16. Pharmaceutical composition for treating thrombosis containing protein under item 10 or 11, and a pharmaceutically acceptable filler.
17. Pharmaceutical composition for the treatment of thrombosis, containing a modified mutant streptokinase on p. 12 and a pharmaceutically acceptable filler.
18.Mutant polypeptide streptokinase, containing the amino acid sequence of SEQ ID NO:163.
19. Pharmaceutical composition for treatment of diseases of the circulatory containing polypeptide under item 18 and a pharmaceutically acceptable filler.
20. Pharmaceutical composition for the treatment of thrombosis, containing polypeptide under item 18 and a pharmaceutically acceptable excipient.
SUBSTANCE: protease is presented which has enhanced milk clotting activity, containing amino acid sequence at least 80 % identical to SEQ ID NO: 3, where the said protease has at least one mutation selected from the group consisting of: (a) substitution of glutamine, corresponding to glutamine at a position of 265 in SEQ ID NO: 3, with amino acid; and (b) replacement of glutamine, corresponding to glutamine at a position of 266 in SEQ ID NO: 3, with amino acid. DNA is described which encodes the said protease, the expression vector containing the said DNA, and the cell transformed with the said vector, designed for expression of the said protease. The method of production of protease having enhanced milk clotting activity is proposed, comprising culturing the said transformed cell in the cultural medium and isolation of protease from the cultural medium.
EFFECT: invention enables to obtain the protease with enhanced milk clotting activity.
16 cl, 2 dwg, 4 tbl
SUBSTANCE: synthetic oligonucleotide primers and probes are disclosed to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus. Also the method is described to detect genomes of 1st, 4th and 16th serotypes of the bluetongue disease virus using such synthetic oligonucleotide primers and probes.
EFFECT: method makes it possible to perform determination of a serotype of the bluetongue disease virus with the help of PCR with a stage of reverse transcription in real time mode, and also to increase specificity and sensitivity, to reduce time to perform work aimed at determination of a serotype of the bluetongue disease virus in samples of an investigated biological material.
4 cl, 4 tbl
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biotechnology and deals with recombinant plasmid DNA, which contains sequence of gene of mature staphylokinase Staphylococcus aureus with replacement of codons K74, E75 and R77 with triplets, which code Ala, of strain Escherichia coli MZ09 and method of obtaining recombinant protein, which contains sequence of gene of mature staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets. Essence of method includes recombinant plasmid DNA, which codes sequence of gene of mature protein of staphylokinase from Staphylococcus aureus staphylokinase with replacement of codons K74, E75 and R77 with Ala-coding triplets, which has nucleotide sequence, given in dwg.1. Invention also includes strain Esherichia coli MZ09, producent of recombinant staphylokinase, with sequence, given in dwg.1, as well as method of its obtaining.
EFFECT: invention makes it possible to obtain staphylokinase protein which possesses high fibrinogenic activity, with output of recombinant protein to 20% from total amount.
3 cl, 2 dwg
SUBSTANCE: invention relates to new staphylokinase derivatives which represent expression products in heterogeneous system of mutant genes obtained by local gene PCR-mutagenesis ← wild type enzyme and differ from respective native staphylokinase form in one or more amino acid substitutions between 104 and 113 amino acid residues that leads to formation of RGD or KGD sequence in said site. Obtained recombinant staphylokinase derivatives (RGD/KGD-Sak), as well as variants thereof having deletions of 1-16 amino acids from NH2-terminal combine properties of thrombolitic and antocoagulating agents and are characterized by decreased polymerization ability and immunogenicity in contrast to wild type enzyme.
EFFECT: new effective agents for thrombosis preventing and treatment.
25 cl, 3 dwg, 3 tbl, 2 ex
FIELD: molecular biology, medicinal industry.
SUBSTANCE: invention relates to a method for preparing recombinant double-stranded enzyme urokinase (ds-uAP). Method involves culturing cells CHO-Meissi subjected for genetic manipulations that are transfected stably with cDNA encoding pre-prourokinase in culturing medium containing alkanoic acids or their derivatives at temperature from 30°C to 37°C and the following separation the prepared ds-uAP for low-molecular and high-molecular forms. Also, invention involves recombinant ds-uAP, high-molecular ds-uAP and low-molecular ds-uAP in different stages of their preparing. Advantage of proposed method involves the development of method for preparing recombinant forms of mature double-stranded protein ds-uAP and its low-molecular and high-molecular forms.
EFFECT: improved preparing method.
19 cl, 4 tbl, 5 ex
FIELD: genetic and protein engineering, medicine, molecular biology, pharmacy.
SUBSTANCE: invention proposes a modified form of plasminogen urokinase type activator (activator) wherein amino acid sequence differs from that in the natural activator as result of replacing the sequence Arg-Arg-His-Arg-Gly-Gly-Ser in the composition of inhibitory loop for the sequence Arg-His-His-Ala-Gly-Gly-Ser and by replacing 24 N-terminal amino acids for the foreign sequence consisting of 16 amino acid residues. Invention proposes the constructed recombinant plasmid (pUABC 34) comprising DNA fragment that encodes a new activator. As result of transformation of E. coli K-12 JM109 cells with plasmid pUABC 34 the recombinant strain E. coli VKPM-8145 as a producer of a modified form of activator is obtained. This polypeptide is characterized by reduced sensitivity to effect of inhibitor PAI-1 and absence of some by-side effects in the complete retention of biological activity of the natural activator produced by the recombinant. This provides effective applying a new activator as a component of pharmaceutical compositions eliciting thrombolytic effect.
EFFECT: valuable medicinal properties of activator and pharmaceutical composition.
6 cl, 6 dwg, 8 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to field of biochemistry, in particular to single variable domain, aimed against IL-6R, to polypeptide and construction, directed against IL-6R, containing said single variable domain, as well as to methods of obtaining them. Disclosed are nucleic acids, coding said single variable domain, polypeptide and construction, as well as genetic constructions, containing said nucleic acids. Described are host cells and host organisms, containing said nucleic acids. Invention also deals with composition for blocking interaction of IL-6/IL-6R, containing effective quantity of described single variable domain, polypeptide, construction, nucleic acid or genetic construction. Also disclosed is method of prevention and/or treatment of at least one of diseases or disorders, associated with IL-6, IL-6R, complex IL-6/IL-6R and/or signal pathways, in which IL-6, IL-6R or complex IL-6/IL-6R is involved and/or biological functions and reactions, win which IL-6, IL-6R or complex IL-6/IL-6R takes part with application of described single variable domain, polypeptide, construction or composition.
EFFECT: invention makes it possible to block interaction of IL-6/IL-6R effectively with increased affinity and biological activity.
25 cl, 70 dwg, 56 tbl, 61 ex
SUBSTANCE: invention represents a combined recombinant protein of the formula: S-L-R, including SR10, SR13, SR15, SdR10, SdR13 or SdR15, which specifically recognises melanoma cells, where S - streptavidin monomer, L - linker having amino-acid sequence Ser-Arg-Asp-Asp-Asp-Asp-Lys containing a restriction site with enteropeptidase and marked as "d", or amino-acid sequence Ser-Arg-Ala-Gly-Ala,R - melanoma-addressing oligopeptide representing R10 having amino-acid sequence Asp-Gly-Ala-Arg-Tyr-Cys-Arg-Gly-Asp-Cys-Phe-Asp-Gly, or R13 having amino-acid sequence Leu-Ser-Gly-Cys-Arg-Gly-Asp-Cys-Phe-Glu-Glu, or R15 having amino-acid sequence Asp-Gly-Phe-Pro-Gly-Cys-Arg-Gly-Asp-Cys-Ser-Gln-Glu. This invention also describes recombinant plasmid DNAs pSR and pSdR for expression of the specified combined proteins, bacterial strains Escherichia coli MG1655/pSR and MG1655/pSdR, producers of the specified combined proteins and a producing method of melanoma-addressing oligopeptide R from combined recombinant proteins SdR10, SdR13 or SdR15.
EFFECT: invention allows producing combined proteins that provide selective and effective binding to receptors on the surface of melanoma cells and can be used in diagnostics and therapy of cancer of a human being.
9 cl, 7 dwg, 5 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.
EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.
11 cl, 36 dwg, 3 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention refers to biotechnology and medicine, and concerns an vaccine against influenza caused by known viral strains of influenza A and B, as well as potential reassortants. The presented polyvalent influenza vaccine is based on a hybrid protein containing N1, N3 and N5 protein fragments of influenza A virus, a haemagglutinin fragment of influenza B virus and FliC1 and FliC2 flagellin components (SEQ ID NO:1) bridged flexibly. The protein-coding nucleotide sequence (SEQ ID NO:2) is optimised for the high expression in Escherichia coli cells.
EFFECT: using the characterised vaccine enables providing the general protection against influenza.
3 cl, 4 dwg, 1 tbl, 13 ex
FIELD: medicine, pharmaceutics.
SUBSTANCE: invention relates to the field of biochemistry, in particular to a polypeptide, which is capable, when fused with a biologically active polypeptide, of increasing its half-life time in serum, as well as to its application for increasing the half-life time in the circulation of erythropoietin, the stimulation factor of granulocyte colony, p40 and a receptor of the tumour necrosis factor. Disclosed are a molecule of nucleic acid, coding the said polypeptide, containing it expression vector, as well as a method of obtaining the said polypeptide by an introduction of a nucleic acid molecule, which codes it, into a mammalian host cell, its growth, as well as collection of the expressed polypeptide.
EFFECT: invention makes it possible to increase the time of half-life of the biologically active polypeptide in serum in an efficient way.
15 cl, 9 dwg, 1 tbl, 8 ex
SUBSTANCE: invention refers to biotechnology, more specifically to modified von Willebrand factor (VWF), and can be used in medicine. A recombinant method is used to preparing modified VWF fused in C-terminal of its primary translation product with N-terminal of albumin by the linker SSGGSGGSGGSGGSGGSGGSGGSGGSGGSGS. The prepared modified VWF is used as a part of the pharmaceutical composition for treating or preventing coagulation failure.
EFFECT: invention enables preparing the modified VWF which maintains its ability to N-terminal dimerisation and C-terminal multimerisation with a prolonged half-period of functional blood plasma occurrence as compared to the half-period of functional VWF occurrence.
17 cl, 5 dwg, 4 tbl, 11 ex
SUBSTANCE: inventions refer to biotechnology and concern a fused protein for the specific inhibition of blood coagulation, an expression plasmid DNA coding this fused protein, a bacterium of the genus Escherichia transformed by this DNA, and to a method for preparing the fused protein. The presented fused protein contains thioredoxin I E.coli and infestine-4 and is characterised by the sequence SEQ ID NO:2. The plasmid DNA contains the sequence SEQ ID NO:1 coding the presented fused protein and controlled by a promoter functioning in a bacterial cell. The method for preparing the above fused protein involves culturing the above bacteria in a nutrient medium, breaking the bacterial cells and purifying the above fused protein with using a metal chelate chromatography and an anion-exchange chromatography.
EFFECT: characterised solutions enables preparing the protein providing the above specificity to be used in blocking the contact activation of blood coagulation by inhibition of the XIIa factor and the absence of inhibition of the Xa factor.
4 cl, 10 dwg, 7 ex
SUBSTANCE: invention refers to genetic engineering and can be used for methane-producing cell permeability control. What is prepared is a polypeptide able to permeate into a methane-producing cell and to increase its permeability, characterised by an amino acid sequence SEQ ID NO:117, 118 or 119 or being at least 90% identical to the above sequence, or at least 15 sequential amino acids of the above sequence. What is also prepared is a polynucleotide coding the above polypeptide cloning and expressing vectors used for producing host cells producing the polypeptide or used for the vector replication. The polypeptide can contain a fluorescent tag on an N-terminal amino acid residue.
EFFECT: invention enables providing higher methane-producing cell permeability.
18 cl, 35 dwg, 3 ex
SUBSTANCE: claimed invention relates to biotechnology and represents a polypeptide construction for treatment, prevention and relief of disorders, associated with an adhesion of platelets and platelet-mediated aggregation or its dysfunction, which includes one or more single-domain antibodies, aimed against the von Willebrand factor (vWF), and one or more single-domain antibodies aimed against serum albumen (SA). The invention also relates to nucleic acid, coding such polypeptide construction, to compositions, containing the said construction, and to its application for obtaining medications for prevention, treatment and relief of the said disorders.
EFFECT: claimed invention makes it possible to extend an assortment of medications for treatment, prevention and relief of disorders, associated with the platelet adhesion and platelet-associated aggregation or its dysfunction.
15 cl, 30 dwg, 32 tbl, 69 ex
SUBSTANCE: invention refers to biochemistry, particularly to artificial immunogenic proteins having the properties of melanoma antigens. What is declared is an artificial gene coding MEL-TCI-A0201 polyepitope immunogenic protein containing multiple cytotoxic restricted HLA-A*0201 and T-helper epitopes of NY-ESO-1, MART1, MAGE-A1, MAGE-A3, MAGE-A11, MAGE-C1 melanoma antigens, having a sequence of 1,535 base pairs presented in Fig. 3. There are also declared a recombinant plasmid DNA containing the above artificial gene, and MEL-TCI-A0201 immunogenic protein with the properties of the melanoma antigens.
EFFECT: invention enables providing higher immunogenicity of the artificial polyepitope T-cell immunogen inducing a higher level of cytotoxic T-lymphocyte response.
3 cl, 11 dwg, 3 tbl, 2 ex
FIELD: genetic and tissue engineering, biotechnology, medicine, agriculture.
SUBSTANCE: invention relates to the development of simple with constructive relation peptide vector (PGE-κ) consisting of polypeptide sequence of epidermal growth factor (EGF) and modified sequence of signal peptide T-antigen SV-40. New vector PGE-κ is able to provide the selective delivery of genetic material in target-cell cytoplasm carrying external receptors to EGF and the following its transport across nuclear membrane. Also, invention proposes a method for preparing peptide vector PGE-κ involving its expression as a fused protein "mutant thioredoxine-linker-vector" and cleavage of product expressed in E. coli in the linker region with specific protease. Invention provides preparing the recombinant strain E. coli B-8389 VKPM as a producer of the fused protein comprising PGE-κ. Proposed vector shows simple structure, absence of toxicity and immunogenicity and these properties provide its usefulness for the directed genetic modification of epithelial, embryonic and tumor cells in vivo.
EFFECT: improved preparing method, valuable medicinal properties of vector, improved genetic modification.
7 cl, 12 dwg, 4 tbl, 16 ex