Recombinant hybrid inhibitor of angiogenesis, and its production method

FIELD: biotechnologies.

SUBSTANCE: recombinant hybrid inhibitor of angiogenesis represents a protein shown in dwg. 1. This protein includes amino acid sequence of plasminogen of a human being from amino acid 82 to 341 and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, which are covalently connected to each other. An inhibitor production method involves expression of its gene in cells of E. coli producer strain, which are transfected with recombinant plasmid DNA pBSRK13 with a physical map presented in dwg. 2, which has the size of 4155 pairs of bases. This plasmid includes a gene coding the recombinant hybrid inhibitor of angiogenesis, as well as a gene of signal peptide OmpA, lac-operator, a gene of stability to kanamycin, replicative origin pUC ori and a gene coding the lac-operator under control of promoter T7. A target protein is extracted from periplasmatic area of bacterial cells by affine and gel-filtration chromatography. The invention can also be used in medicine for creation of new medicinal agents with antiangiogenic therapeutical effect.

EFFECT: invention allows producing a new protein having antiangiogenic activity and increased selectivity of action in relation of tumoral endothelium.

2 cl, 3 dwg, 1 tbl, 5 ex

 

The invention relates to the field of biomolecular pharmacology, biotechnology and genetic engineering, in particular, to the development of a new recombinant hybrid inhibitor of angiogenesis, with specific antiproliferation activity against endothelial cells of blood vessels. The invention relates also to the development of effective way of getting this drug by the expression of its gene in E. coli as part of plasmid DNA, followed by purification. The invention can be used in medicine and in the medical industry to create new drugs with antiangiogenic therapeutic effect.

One of the unresolved problems of modern medicine remains the problem of uncontrolled cell growth leading to the development of malignant tumors. Despite intensive research in the field of anticancer chemotherapy has not yet been achieved successes. An important achievement of recent years, however, was the discovery of the ability of tumors to the induction of specific inhibitors with antiangiogenic activity and effectively suppressing vascularization secondary tumors, thus preventing their rapid development and death of the organism-ofwholesale. Such inhibitors, in particular, are products limited proteoly is and enzymes, sekretiruemyi tumor tissue, physiological protein plasminogen [Y. Cao et al. Kringle structures and antiangiogenesis; Curr. Med. Chem. - Anti-Cancer Agents, 2: 667-681 (2002); S. Gately et al. The mechanism of cancer-mediated conversion of plasminogen to the angiogenesis inhibitor angiostatin; Proc. Natl. Acad. Sci. USA, 94: 10868-10872 (1997)]. The plasminogen has a structure that includes five so-called Kringle domains, special structures of polypeptide chain consisting of about 80 amino acid residues and supported by three disulfide bonds. In the plasminogen molecule has five Kringle domains, arranged in series and denoted by numbers from 1 to 5. Protein angiostatin, being a fragment of plasminogen, consists of four Kringle domains (K1-4); molecular mass ranges 37-42 kDa depending on length outside the Kringle-domain polypeptide groups [M.S. O'reilly et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma, Cell, 79: 315-328 (1994), Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease; Nat Med 1: 27-31 (1995)]. Proven potent inhibiting activity of angiostatin on the processes modulating tumor progression, and several types of malignant tumors. In vitro angiostatin inhibits proliferation of endothelial cells, and showing specificity, acting on the endothelial cells of blood vessels, infiltrating the tumor tissue, and inhibits the migration of endothelial cells, and education is the W blood vessels [Moser T.L. et al. Angiostatin binds ATP synthase on the surface of human endothelial cells; Proc Natl Acad Sci USA, 96(6): 2811-6 (1999)].

When proteolysis of plasminogen enzymes expressed in normal and especially in neoplastically tissues, except angiostatin are formed and other polypeptide fragments. When you research their inhibitory potential on the cultures of endothelial cells of the capillaries of the greatest bull inhibiting activity was found not angiostatin (K1-4), and fragments K1-3 and C5 [Y. Cao and L. Xue Angiostatin; Semin Thromb Hemostas, 30(1): 83-93 (2004)].

The described method of obtaining recombinant K1-3 with the introduction of the expression vector pMETK1 in producing strains of E. coli [Young-Ae Joe et al. Inhibition of human malignant glioma growth in vivo by recombinant human plasminogen kringles 1-3; Int J Cancer: 82: 694-699 (1999)]. When using this technique were recorded relatively low yield of the target protein, and in addition, the method provides renaturation only a fraction of a product, which ultimately leads to a very low yield of biologically active K1-3 and makes use of this technique is complicated and uneconomical.

It is known that the Association of many members of the family of integrins, cell adhesion molecules that connect the cytoskeleton with the extracellular matrix of other cells and mediating these cellular functions like move, invasion, proliferation, and others, with pathophysiological picture of the development of many diseases, including numbers and cancerous tumors makes the study of integrins attractive challenge for research in the field of anticancer therapy [Mousa S.A. Anti-integrin as novel drug discovery targets: Potential therapeutic and diagnostic implications; Curr Opin Chem Biol, 6(4): 534-541 (2002)]. Part of integrins include, in particular, the so-called RGD receptors recognizing short amino acid sequence RGD (Arg-Gly-Asp), the presence of which is detected in a number of ligands of integrins. RGD receptors, in particular, are part of the receptors mediating the process of angiogenesis, such as αv-integrins. Antagonists of αvβ3-integrins with different mechanisms of action are currently under clinical trials in the countries of Western Europe (e.g., Vitaxin-1 and -2, Cilengitide, ATN-161) [J.S. Kerr et al. The αv integrin antagonists as novel anticancer agents: An update; Expert Opin Investig Drugs, 11(12): 1765-1774 (2002)]. Oligopeptide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys was used for targeted transport of drugs, such as doxorubicin and theplain in tumor tissue [Arap, W. et al. Cancer treatment by targeted drug delivery to tumor vasculature in a mouse model; Science 279 (5349): 377-380 (1998)]. When this has been achieved as increasing the selectivity of action of drugs and their accumulation in the pathogenicity of the hearth and the potentiation of the effect due to specific inhibition of integrin-mediated processes in the tumor tissue.

The invention was the creation of the drug, which combines both antiangiogenic properties of the Kringle domains of plasminogen, and selectivity of action made RGD-component, and the development of the method of obtaining the proposed drug.

For the solution of inventive problems we propose a new hybrid inhibitor of angiogenesis, representing a protein comprising covalently linked amino acid sequence of human plasminogen 82 to 341 amino acid and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys with antiangiogenic activity and high selectivity of action in relation to tumor endothelium. It is also proposed a method of obtaining a hybrid inhibitor as a recombinant product, where the inhibitor is produced by the expression of its gene in the cells of the producer strain E. coli transfected with a recombinant plasmid DNA pBSRK13 sized 4155 base pairs and containing this gene and the gene signal peptide OmpA, lac operator, a resistance gene for kanamycin, replicative origin pUC ori and the gene encoding lac-repressor under control of the T7 promoter, the target protein is recovered from periplasmatic the field of bacterial cells affine and gelfiltration chromatography.

Developed expression vector carries a gene that encodes a protein, which is a Kringle domains 1, 2 and 3 of human plasminogen with several additional aminokislot the mi residues: 11 - from N-Terminus and 8 - to C-Terminus. Amino acid sequence of the proposed inhibitor is shown in figure 1. This polypeptide sequence corresponds exactly to the amino acid residues Ser 82-Glu 341 plasminogen man who from N-Terminus covalently attached integrin-inhibiting sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys (SEQ ID NO 1). The scheme of the genetic constructs are presented in figure 2.

In the proposed method, the expression of the gene in the cells of the producer strain is carried out in recombinant plasmid DNA pBSRK13 in the environment for the growth of E. coli, first in the absence of the inducer of the lac-promoter, isopropylthio-β-D-galactopyranoside (IPTG), and on reaching the turbidity of the environment of 0.4-0.6 OD - adding the inducer IPTG to a concentration of 0.1-0.3 mm and continuing expression under the control of induced lac promoter in recombinant plasmids. Target protein accumulated in the bacterial cells in periplasmatic area, allocate using consistently applied stages affine and gelfiltration chromatography.

The proposed method can achieve a high level of expression (about 60 mg of the target protein from 1 l of culture medium) recombinant hybrid inhibitor in the form of secreted into periplasm the cells of E. coli naturally aceleration protein to isolate and purify the product.

We offer inhibitor can be used to treat cancer and other diseases, involving inappropriate vascularization of tissues, both in monotherapy and in combination therapy in combination with other chemotherapy drugs or non-drug methods of treatment, for example, surgical manipulation and beam therapy.

The invention is illustrated by the following examples:

Example 1. The design of genetic constructs, encoding the biosynthesis of recombinant hybrid inhibitor in E. coli cells.

A) Synthesis of the sequence encoding the 3'portion of the gene K1-3 (3K13).

selection - site recognition by the restriction enzyme SacII, underline - site recognition by the restriction enzyme HindIII.

We have also been obtained by synthetic DNA sequence containing the restriction site of the enzyme BamHI, the start codon, the gene encoding the signal peptide, the sequence encoding the sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, and the fragment encoding the 5'portion of the gene K1-3 with a site restriction enzyme SacII (SSR5K13, SEQ ID NO 5).

The sequence SSR5K13:

the underlined fragment - site recognition by the restriction enzyme BamHI, selected site recognition by the restriction enzyme SacII.

B) Synthesis of the DNA sequence containing the restriction site of the enzyme BamHI, the start codon, GE is, encoding the signal peptide OmpA sequence encoding a hybrid protein consisting of the peptide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys and K1-3, as well as a stop codon and a restriction site of the enzyme HindIII (SSRK13, SEQ ID NO 6).

To create SSRK13 fragments SSR5K13 and C were treated with restriction enzyme SacII and ligated together using ligase of phage T4.

The sequence SSRK13:

the underlined fragment - site recognition by the restriction enzyme BamHI, discharge - site recognition by the restriction enzyme HindIII.

To create the expression vector sequence SSRK13 were treated with restriction enzymes BamHI and HindIII and ligated with the vector pBSH2, who had previously been treated the same restrictases. The resulting vector was named pBSRK13 (figure 2).

Example 2. The production of the target protein in the cells of the producer strain E. coli.

Expression vector pBSRK13, obtained as described in example 1 was incorporated into the E. coli strain BL21/DE3. To implement the expression of a target protein 30 ml transfected strain kept shaking for 2-3 h in LB medium at 37°C with the addition of kanamycin (50 μg/ml) and glucose (0.2 percent). Then this culture is diluted 20-fold with fresh LB medium containing kanamycin (40 μg/ml) and increased 2-3 h with rocking. Upon reaching the culture medium density OD280 nm=0.5 made in her isopro is ilti-β-D galactopyranoside (IPTG) to a concentration of 0.5 mm for induction of biosynthesis of K1-3. Then incubated the culture for about 5 h at 37°C with shaking, centrifuged at 10,000 g for 15 min, the supernatant was discarded.

Example 3. Isolation and purification of the target protein.

Sediment cells, obtained as described in example 2, was treated by ultrasound using a slotted probe in buffer A (50 mm Tris, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 0,11% sodium N-laurylsarcosine). To the resulting suspension were added to the lysozyme concentrations up to 30 μg/ml, kept for 25 min at room temperature and centrifuged at 10,000 g for 15 min at 4°C, the precipitate was discarded. The resulting solution then was applied to a chromatographic column Packed with a sorbent lysine-separate 4B (Amersham Biosciences, USA), pre-equilibrated with buffer B (50 mm sodium phosphate, pH 7,4). After applying the solution containing the target protein, the column was washed with two volumes of buffer B, and then with buffer B (50 mm sodium phosphate, 0.15 M sodium chloride, pH 7,4) prior to registration of the baseline. Then suirable target protein 0.2 M solution of ε-aminocaproic acid in buffer C. Fractions containing the target recombinant protein was collected and concentrated by membrane ultrafiltration (MW=10 kDa). Further purification was performed by gelfiltration chromatography, causing concentrated fractions on a chromatographic column filled sorbent the Sephacryl S-300 (Amersham Biosciences, USA), pre-equilibrated with buffer D (10 mm sodium phosphate, 2.7 mm potassium chloride, 0,137 M sodium chloride, pH 7,4) (the procedure was carried out at 4°C). Collected fractions containing purified target protein.

Combined fractions containing the target protein were placed in dialysis bags with the permeability of the pores of the molecular weight of 8 kDa and were dialyzed at 4°C for 8 h against buffer D (10 mm sodium phosphate, 15 mm sodium chloride, 0.02% sodium azide, pH 7.0), and then against buffer G for 5 o'clock the Final product was a recombinant hybrid polypeptide with a molecular mass of 42 kDa and a purity not less than 99% according to LTOs electrophoresis.

Example 4. Determination of biological activity of recombinant hybrid inhibitor of angiogenesis in vitro.

Determination of biological activity of the resulting inhibitor was performed by assessing its ability to inhibit the proliferation of human vascular endothelial cells in vitro. The study of the proliferation of endothelial cells was previously described method [M. O'reilly et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth; Cell 88: 277-285 (1997)]. Cells HUVEC (endothelial cells of the umbilical vein of a person) of the monolayer was trypsinization and resuspendable in M199 medium containing 5% fetal bovine serum. The cells are then dissipated in 96-well plates, coated with gelatin, in a density of 5000 cells per well. After 24 hours was added to the solution of the inhibitor at various concentrations. After 20 min of culture cells were treated with 5 ng/ml solution of basic fibroblast growth factor (Life Technilogies Inc., USA) in the presence of heparin (1 μg/ml). Cell survival in the control and in the treated solution By 1-3 groups was determined using the MTT test after 72 h of incubation. MTT-test effectively detects metabolities activity of mitochondria, and its result directly correlates with the number of living cells. According to the results of the analysis of the data were obtained, confirming the high antiendothelial the proposed activity inhibitor (figure 3).

Example 5. Determination of antitumor activity of recombinant hybrid inhibitor of angiogenesis in vivo.

Melanoma cells lines B16 maintained by weekly intraperitoneal inoculation of mice S B1/6. For the induction of solid tumors the mice were injected subcutaneously cell suspension (2·105cells in 0.1 ml of physiological solution). Inhibitor in a physiological solution was injected into experimental animals intravenously. As an additional control drugs used native angiostatin K1-3, isolated from human blood plasma, and the peptide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys obtained by the method of solid-phase peptide synthesis. The size of the solid tumors were measured once in 2-3 days. The relative size of the tumors (ORO) was determined by the formula:O PO=PaboutpPK100%where Popthe average size of tumors in the experimental group, Ptothe average size of tumors in untreated control animals. The increase in life span of treated animals compared to the control was determined by the formula:YWith aPW=(TWith a-1)100%where T is the average life expectancy (ALE) of immunized animals, days, C - ALE control animals, days.

The results of the experiment are shown in table. Compared to the mild effect of peptide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys and the lack of effect of the use of native angiostatin K1-3, treatment of the claimed hybrid inhibitor resulted in significant inhibition of tumor growth both during treatment and after its cancellation, and the increase in the average life span of the treated animals.

Table
The group of animalsORO, %USPS, %
Control (untreated)100-
Angiostatin K1-363,723,5
The peptide Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cysto 78.38,6
Hybrid inhibitorof 31.865,2

Thus, the proposed recombinant hybrid angiogenesis inhibitor can effectively suppress the development of malignant tumors.

1. Recombinant hybrid inhibitor of angiogenesis, representing a protein of figure 1, comprising covalently linked amino acid sequence of human plasminogen 82 to 341 amino acid and sequence Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys with antiangiogenic activity and high selectivity of action in relation to tumor endothelium.

2. The method of obtaining the inhibitor according to claim 1, which consists in the fact that it is produced by the expression of its gene in the cells of the producer strain E. coli transfected with a recombinant plasmid DNA pBSRK13 with physical map presented in figure 2, having a size 4155 base pairs and containing this gene and the gene signal peptide OmpA, lc-operator; the gene for resistance to kanamycin, replicative origin pUC ori and the gene encoding lac-repressor under control of the T7 promoter, the target protein is recovered from periplasmatic the field of bacterial cell affinity and gel filtration chromatography.



 

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Vns-met-histones // 2498997

FIELD: biotechnologies.

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19 cl, 4 ex, 2 tbl, 78 ex

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6 cl, 1 tbl, 5 dwg, 7 ex

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7 cl, 15 dwg, 5 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: obtained is protein complex, possessing GPCRα1L affinity to ligand, which includes GPCRα1A and polypeptide with sequence of amino acids SEQ ID NO:1. Binding of said G-protein-conjugated receptor with polypeptide alters ligand affinity of the receptor. Also claimed are methods of screening agonists or antagonists of G-protein-conjugated receptor with application of transformant, in which said altered G-protein-conjugated receptor is expressed.

EFFECT: carrying out analysis of many supposed G-protein-conjugated receptors with still unknown structure.

12 cl, 3 dwg, 2 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: method involves introduction to a plant, some part of the plant or a plant cell of nucleotide sequence for 80-100% of identical nucleotide sequence determined in SEQ ID NO: 17, and coding a composite protein containing a cytoplasmic end segment, a transmembrane domain, a steam area (CTS domain) of N-acetylglucosaminyl transferase (GNT1), which is merged with catalytic domain of beta-1,4-galactosyl transferase (GalT); with that, the above first nucleotide sequence is functionally connected to the first regulatory area being active in the plant; and the second nucleotide sequence for coding of a target protein; with that, the above second nucleotide sequence is functionally connected to the second regulatory area being active in the plant, as well as transient co-expression of the first and the second nucleotide sequences with synthesis of the target protein containing glycans, with reduced xylosylation, reduced fucosylation or their combination at comparison to the same target protein obtained from a wild plant. The invention described nucleic acid coding the protein that modifies glycosylation of target protein, a composite protein for modification of glycosylation of target protein; nucleic acid that codes it, as well as a plant, a plant cell and a seed, which contain the above nucleic acid or the above composite protein.

EFFECT: invention allows effective production of a target protein with reduced xylosylation, reduced fucosylation or their combination.

20 cl, 7 dwg, 9 ex

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