Recombinant hybrid polypeptide, capable of inhibiting human endothelial cell proliferation in vitro, and method for production thereof

FIELD: chemistry.

SUBSTANCE: hybrid polypeptide is a polypeptide 1 to which a polypeptide 2 is covalently bonded, where polypeptide 1 is a human endostatin sequence with 135-184 amino acid residues, wherein at position 173, Cis is replaced with Ala with a relatively native endostatin sequence, and polypeptide 2 is a human plasminogen sequence with 82-341 or 463-511 amino acid residues. Also disclosed is a method of obtaining the hybrid polypeptide using an E.coli producer, which involves methods for extraction and purification thereof.

EFFECT: polypeptide is capable of inhibiting human endothelial cell proliferation in vitro and can be used when producing nontoxic preparations for inhibiting angiogenesis.

4 cl, 4 dwg, 6 ex

 

The invention relates to the field of biomolecular pharmacology, biotechnology and genetic engineering and is a recombinant hybrid polypeptide drug, including fragments of plasminogen human and human endostatin with specific antiproliferation activity against endothelial cells of blood vessels, and the method of obtaining 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 major problems facing medicine, is the search for effective methods for the treatment of patients with malignant diseases. One of the key scientific achievements of the twentieth century in this area is evidence of the need for the process of angiogenesis for growth of malignant solid tumors, as well as the creation of the concept of anticancer therapy based on inhibition of angiogenesis, developed by Soros. Volkmann [Folkman J. Nat. Med., 1:27-31, 1995].

Angiogenesis - the growth of the capillaries of the blood vessels, resulting in the formation of new vascular network. In a healthy adult human body angiogenesis does not occur in most the ve organs and tissues, or its intensity is negligible, except for the processes of tissue regeneration, as well as education luteum, endometrium and placenta. However, the expression of growth factors (such as growth factor vascular endothelial (VEGF), fibroblast growth factors (FGF), and others), endothelial cells of blood vessels in dormant condition, can enter into the cell cycle, proliferate, migrate and form new blood vessels. This abnormal growth of new blood vessels leads to the progression of many diseases, especially the growth and metastasis of solid tumors [Carmeliet, P. and Jain, R. K. Nature, 407:249-257, 2000].

Under normal physiological conditions, the intensity of angiogenesis depends not only on the level of expression of these and other growth factors, but also on the level of expression of angiogenesis inhibitors. Low intensity of angiogenesis is provided by parity in the expression as growth factors that stimulate angiogenesis and inhibitors of the latter. Among specific inhibitors of angiogenesis affecting proliferating endothelial cells of blood vessels, one of the most powerful is the angiostatin protein with molecular weight of 38-40 kDa allocated M. O'reilly et al. from the blood and serum of mice with solid tumors in the body [O'reilly M. S., Holmgren L, et al. Cell, 79:315-328, 1994].

By specific what about the inhibition of angiogenesis can be therapy of malignant neoplasms and diseases associated with neovascularization of the retina, such as diabetes and sickle cell retinopathy and other a Number of antiangiogenic agents, such as angiostatin, endostatin, squalamine, 2-methoxyestradiol and others, are currently undergoing clinical trials in the countries of Western Europe and the USA [Ziche M. et al. Development of new drags in angiogenesis, Curr Drag Targ, 5:389-406, 2004]. In this regard, the search for new inhibitors of angiogenesis and the development of methods of obtaining them in quantities sufficient for preclinical studies, it becomes important scientific and applied problems of modern medicine.

In the study of angiostatin, one of the most potent inhibitors of proliferation of endothelial cells in vitro and angiogenesis in vivo, it was found that this protein is a fragment of one of the components of the blood coagulation system, namely plasminogen. Angiostatin is produced in the body that carries a cancerous tumor by proteolysis of plasmin by a number of enzymes, specifically expressed in tumor tissue, for example, the matrix metalloproteinases (gelatinase and stromelysin and others), prostatespecific the antigen and also as a result of autolysis of plasmin. Because proteolysis of plasmin different enzymes formed a number of different polypeptides having antiangiogenic activity, the attention of researchers is she attracted the question of the comparative studies of antiproliferative activity against endothelial cells of various fragments of plasminogen molecule.

The human plasminogen protein with a molecular mass of 90 kDa, which contains five Kringle domains (denoted by numbers from 1 to 5), which is a special rigid structure of polypeptide chains folded in two rings and supported by three disulfide bonds. Chain length of each Kringle domain of approximately 80 amino acid residues. Angiostatin is a structure corresponding Kringle domains 1-4 of plasminogen (K1-3). For studies of fragments of plasminogen (angiostatin, a Kringle-domain 1 (CH1), a Kringle-domain 3 (K3), Kringle domains 2-3 (K2-3) and so on) it was shown that the greatest inhibitory activity have a Kringle-domain 5 (K5) [Cao Y. Chen, A. et al. J Biol Chem, 272:22924-22928, 1997] and Kringle domains 1-3.

In addition to the high antiproliferative activity, other benefits are obvious and Kringle-domain 5 and K1-3 of human plasminogen as antiangiogenic agents. First, they show inhibitory activity specific, acting only on proliferating endothelial cells of blood vessels, and therefore non-toxic for other types of cells [Zhang D., Kaufman, P. et al. Diabetologia, 44:757-765, 2001]. Secondly, the plasminogen is an endogenous protein of human tissue, and therefore its peptide fragments K5 and K1-3 do not cause an immune response. Thirdly, a fragment of C5 as a polypeptide with a small molecular weight (12-13 kDa), can vitiligo obtained in the form of recombinant protein in the cells of E. coli.

Another potent inhibitor of angiogenesis is a fragment of collagen XVIII, endostatin representing a protein with a molecular mass of 20-22 kDa [M.S. O'reilly et al. Cell 88:277-285, 1997]. Fragments of endostatin have antiangiogenic and other biological properties characteristic of endostatin. Some fragments have a higher angiogenic activity compared with native endostatin [Cattaneo M.G. et al. Exp Cell Res, 283:230-236 (2003)].

In the currently described method of treatment of some diseases associated with corneal neovascularization, using gene therapy lentiviruses design, expressing a hybrid protein consisting of endostatin and K5 [R.C. Murthy et al. Investigative Ophthalmology & Visual Science, 44(5): 1837-1842, (2003)]. However, gene therapy, lying in essence described R.C. Murthy et al. the method, known for its disadvantages, primarily the unexamined consequences of implementing xenogenous and low efficiency.

The invention was the creation of new highly effective non-toxic polypeptide drugs with antiendothelial activity.

For solving inventive tasks proposed remedies, which represents a hybrid proteins containing different variants of fragments of endostatin (polypeptide 1) and plasminogen (2 polypeptide), covalently linked. In th is supplied with antiangiogenic drugs, as compared with the structure of native endostatin human polypeptide 1 replacement amino acid residue cysteine at position 173 to alanine (figa), that facilitates a drug genetic engineering methods, but does not affect the manifestation of them antiangiogenic activity. Polypeptide 2 in the proposed hybrid drugs is a sequence of human plasminogen 82 to 341 amino acid (figa) or 463 on 511 amino acid (figb).

In the framework of the invention are also ways of getting offered drugs as recombinant products using a producer E. coli, including methods for their isolation and purification. Recombinant hybrid polypeptide produced by the expression of their genes in the composition of the recombinant plasmid DNA pCOHRl (4378 base pairs) and pCOHR2 (3679 base pairs) in the cells of E. coli, and the expression is carried out in the environment for the growth of E. coli at first, in the absence of inducer 1 ° C-promoter of 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. The drug, derived from periplasmic the cells of E. coli transformed with recombinant plasmid DNA pCOHRl, purified using methods affine and gelfiltration chromatography, and the drug is excreted in the form of Taurus transmissions from the cells of E. coli transformed with recombinant plasmids the th DNA pCOHR2, centurybut and purified using high-performance liquid chromatography.

With getting offered drugs open up the possibility of their application in therapy of cancer and other diseases involving unbalanced neovascularization, such as diabetic retinopathy, Crohn's disease and other

The invention is illustrated by the following examples:

Example 1. The synthesis of the DNA sequence containing the restriction site of the enzyme BamHI, the start codon, the gene encoding the signal peptide sequence encoding a protein consisting of a fragment of endostatin produced with replacement (figb) and a fragment of plasminogen 82 at a 341 amino acid and the stop codon and a restriction site of the enzyme Hind III (sequence called SPEK13).

In the first stage, carried out the synthesis sequence that encodes a 3 bar part of the gene fragment of plasminogen (82-341) (sequence called K). Synthesis C was performed using primers C and C - R and cDNA library man:

The DNA sequence, called SPE that contains the restriction site of the enzyme BamHI (underlined), the start codon, the gene encoding the signal peptide sequence listed on fehb and called Vox, and a fragment encoding the 5 bar part of the gene fragment of plasminogen (82-341) (separation of the config) with the site of the restriction enzyme SacII (dotted underline), were obtained using a synthesizer oligonucleotides:

Example 2. The synthesis of the DNA sequence containing the restriction site of the enzyme BamHI, the start codon, the section that encodes a fusion protein consisting of a fragment of plasminogen 463 on 511 amino acid and a fragment of endostatin produced with replacement (figb), as well as a stop codon and a restriction site of the enzyme Hind III (sequence called PK5Vox).

Using synthesizer oligonucleotides received fragments, called RK and 3K5Vox:

RC - DNA sequence containing the restriction site of the enzyme BamHI, the start codon, patch, encoding the 5' portion of the fragment of plasminogen 463 on 511 amino acid with a restriction site of the enzyme AvaII.

Example 3. Production of hybrid polypetides in strains of E. coli

To create expression vectors sequence SPEK13 or PK5Vox was treated with restriction endonucleases BamHI and Hindlll and ligated with the vector pBSH2, who had previously been treated the same restrictases. The resulting vectors were named pCOHRl (figa) and pCOHR2(figb).

Expression vector pCOHRl or pCOHR2 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 CH the goats (0,2%). 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 ODNm=0.5 made in her isopropylthio-β-D galactopyranoside (IPTG) to a concentration of 0.5 mm for induction of biosynthesis. 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 4. Purification of the expression product of the vector pCOHRl

Sediment cells, obtained as described in example 3, resuspendable in buffer A (20 mm Tris, pH 8.0, 15% galactose and 1 mm EDTA) for 10 min at 20°C. the cell Suspension then was centrifuged at 10000 g for 15 min; the supernatant was removed and resuspendable cells in a 7 mm solution of magnesium (II) sulfate at 0°C for 20 minutes resulting proteins periplasmatic space was then separated from the cells by centrifugation at 10,000 g for 20 min at 4°C, the precipitate was discarded. The resulting solution proteins periplasmic concentrated using ultrafiltration cell for concentration and were dialyzed against buffer B (12 mm Tris, pH 7.5) for 5 h at 4°C. Then the purification of the target product, obtained in cultures transfected with recombinant plasmid pCOHRl, carried out by the method of affinity chromatography. The solution, provided the target product, was applied to the chromatographic column Packed with a sorbent lysine-separate, pre-equilibrated with buffer B (50 mm sodium phosphate, rn,4). After applying the solution of a target protein, the column was washed with two volumes of buffer, then the buffer G (50 mm sodium phosphate, 0.15 M sodium chloride, pH 7.4) prior to registration of the baseline. Then suirable target product of 0.2 m solution of ε-aminocaproic acid in the buffer, the Fractions containing the desired product were collected and concentrated by membrane ultrafiltration (MW=10 kDa). Further purification was performed by way gelfiltration chromatography. When cleaning the product obtained from the producer strain transfected with the expression vector pCOHRl concentrated fraction was applied to a chromatographic column Packed with a sorbent 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 product.

Example 5. Purification of the expression product of the vector pCOHR2

Sediment cells, obtained as described in example 3, was treated with ultrasound using a slotted probe in buffer A (50 mm Tris, pH 8.0, 1 mm EDTA). The resulting suspension was centrifuged at 10000 g for 15 min, supernatant was discarded is. The precipitate is then resuspendable in buffer B (50 mm Tris, pH 8.0, 1 mm EDTA, 1 mm dithiothreitol, 1% sodium N-laurylsarcosine) and centrifuged at 10,000 g for 15 min, the supernatant was discarded. Sediment resuspendable in buffer (20 mm Tris, pH 8.0, 2% sodium deoxycholate) and centrifuged at 10,000 g for 15 min, the supernatant was discarded; this procedure was repeated three times. Then the precipitate Taurus inclusion containing the target component, was dissolved in buffer G (50 mm Tris, pH 8.0, 8 M guanidine hydrochloride, 70 mm 2-mercaptoethanol). After complete dissolution of the precipitate obtained solution was diluted with buffer D (50 mm Tris, pH 8.0, 2 M urea,

2 mm restored glutathione, 300 μm oxidized glutathione, 5 mm L-lysine hydrochloride) in a ratio of 1:30. The resulting solution was left for the passage of renaturation of recombinant protein at 4°C for 24 h Then the solution was placed in dialysis bags with the permeability of the pores of the molecular weight of 8 kDa and were dialyzed for 8 h against buffer E (10 mm sodium phosphate, 15 mm sodium chloride, 0.02% sodium azide, pH 7.0); the procedure was repeated, replacing the buffer E. the Solution was then removed from dialysis system and further purification was performed by high performance liquid chromatography.

Example 6. Determination of biological activity of the resulting hybrid antiendothelial drugs

Definition biologist the political activity of the obtained preparations were performed by assessing their ability to inhibit the proliferation of human vascular endothelial cells in vitro. The study of the proliferation of endothelial cells was previously described method [O'reilly M, et al. 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 h the solution was added hybrid polypeptides in 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 treated preparations 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 drugs presented in figure 4, defined their IC50: the product obtained by transferowy strains of E. coli with plasmid pCOHR1, had IC50=190 nm; the product obtained by transferowy strains of E. coli with plasmid pCOHR2, had IC50=400nm. Thus, the present invention provides a high output to get a new hybrid polypeptide drug manifesting antiendothelial activity.

1. Recombi is based hybrid polypeptide, able to inhibit the proliferation of human vascular endothelial cells in vitro, representing polypeptide 1, which is covalently attached 2 polypeptide, where the polypeptide 1 is a sequence of endostatin person with 135 on a 184 amino acid residue, and at position 173 replacement of Cys to Ala compared with the native sequence of endostatin and the polypeptide 2 is a sequence of human plasminogen 82 to 341 or 463 on 511 amino acid residue.

2. The method of producing the polypeptide according to claim 1, characterized in that the polypeptide is produced by expression of its gene in cells of E. coli BL21 (DE3)transformed with recombinant plasmid DNA pCOHR1 (4378 gel) or pCOHR2 (3679 P.N.), and cells incubated in the growth medium to achieve a turbidity of 0.4-0.6 OD, then continue incubation in the presence of isopropylthio-β-D-galactopyranoside in a concentration of 0.1-0.3 mm, and the target polypeptide separated from periplasm cells or from Taurus enabled.

3. The method according to claim 2, characterized in that the polypeptide is recovered from periplasmic the cells of E. coli transformed with recombinant plasmid DNA pCOHR1, and purified using affinity chromatography and gelfiltration chromatography.

4. The method according to claim 2, characterized in that the polypeptide is recovered from the cells of E. coli, transformed recombinant plasma the Neu DNA pCOHR2, in Taurus on and carry out his renaturation, followed by purification by high-performance liquid chromatography.



 

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