RU2488635C1 - Method to produce recombinant anti-angiogenic polypeptide - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: method consists in expression of a gene of a human plasminogen fragment from 453 to 543 amino acid within E.coli cells with subsequent extraction and treatment of a finished product from cell periplasm. Expression is carried out in cells E.coli BL21 (DE3), transformed by plasmid DNA pEK5 or pEK5H with physial maps represented in figure 2, containing a gene of target polypeptide fused with a gene of signal peptide OmpA, origin of replication pUC ori, a gene of resistance to kanamycin and a gene coding lac-repressor under control of T7-promotor. At the same time the recombinant plasmid DNA pEK5H additionally contains between genes OmpA and target polypeptide the sections coding amino acid sequences HHHHHH and DDDDK.

EFFECT: invention provides for secretion of target polypeptide into cell periplasm with high yield.

4 cl, 5 dwg, 6 ex

Description

The invention relates to the field of biotechnology, in particular to genetic engineering, and is a method for producing a recombinant polypeptide, which is a fragment of a human plasminogen from 453 to 543 amino acids, its isolation and purification.

The invention can be used in the medical industry to create new drugs with anti-angiogenic therapeutic effect.

One of the most important tasks facing medicine is the search for effective methods for treating patients with malignant diseases. One of the key achievements of 20th century science in this area is the proof of the necessity of the angiogenesis process for the growth of solid malignant tumors, as well as the creation of the concept of antitumor therapy based on the suppression of angiogenesis developed by J. Folkman [Folkman J., Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med., 1: 27-31, 1995].

Angiogenesis is the process of capillary growth from blood vessels, resulting in the formation of new vascular networks. In a healthy adult organism, angiogenesis does not occur in most organs and tissues, or its intensity is negligible, except for the processes of tissue regeneration, as well as the formation of the corpus luteum, endometrium and placenta. However, when expressing growth factors (such as vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF (s)), etc.), vascular endothelial cells that are at rest can enter the cell cycle, proliferate, migrate, and form new blood vessels. Moreover, the pathological growth of new vessels causes the progression of many diseases, especially the growth and metastasis of solid tumors [Carmeliet P. and Jain R.K. Angiogenesis in cancer and other diseases. Nature, 407: 249-257, 2000].

Under normal physiological conditions, the intensity of angiogenesis depends not only on the expression level of these and some other growth factors, but also on the expression level of angiogenesis inhibitors. The low intensity of angiogenesis is ensured by parity in the expression of both growth factors that stimulate angiogenesis and inhibitors of the latter. Among the specific angiogenesis inhibitors that act on proliferating vascular endothelial cells, one of the most potent is angiostatin - a protein with a molecular weight of 38-40 kDa, isolated by M. O'Reilly et al. [O 'Reilly M.S. et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell, 79: 315-328, 1994] from the blood and serum of mice with solid tumors in the body.

Specifically inhibiting angiogenesis, it is possible to treat malignant neoplasms and diseases associated with neovascularization, for example, retina, such as diabetic and sickle cell retinopathy, etc. A number of antiangiogenic drugs, for example, angiostatin, squolamine, 2-methoxyestradiol, etc., are currently present. time at the stage of clinical trials in Western Europe and the United States [Moschetta M. et al. Angiogenesis inhibitors: implications for combination with conventional therapies, Curr. Pharm. Des., 16 (35): 3921-39 31, 2010]. In this regard, the search for new angiogenesis inhibitors and the development of methods for their preparation in quantities sufficient for preclinical studies is becoming an important scientific and applied task of modern medicine.

When studying angiostatin, one of the most powerful inhibitors of endothelial cell proliferation in vitro and in vivo angiogenesis, it was found that this protein is a fragment of one of the components of the blood coagulation system, namely plasminogen. Angiostatin is formed in an organism carrying a cancer tumor by proteolysis of plasminogen by a number of enzymes that are specifically expressed in tumor tissue. In addition to angiostatin, during the proteolysis of plasminogen by various enzymes, a number of other polypeptides are formed that also have antiangiogenic activity.

Human plasminogen, a protein with a molecular weight of 90 kDa, contains five so-called kringle domains (denoted by numbers from 1 to 5), which are a special rigid structure of the polypeptide chain, folded into two rings and supported by three disulfide bonds; the chain length of each kringle domain is approximately 80 amino acid residues. Angiostatin is a structure corresponding to the kringle domains 1-3 (K1-3) or the kringle domains 1-4 (K1-4) of plasminogen. In studies of plasminogen fragments (angiostatin, K1, K3, K2-3, etc.), it was shown that the kringle domain 5 (K5) has the highest inhibitory activity [Cao Y. et al. Kringle 5 of plasminogen is a novel inhibitor of endothelial cell growth. J Biol Chem, 272: 22924-22928, 1997]. Its antiproliferative effect is several times greater than that of angiostatin, as well as any individual kringle domain. This can be explained by the fact that the anti-endothelial effect of K5 and other kringle domains is realized through different mechanisms. So, the K5 receptor on the surface of endothelial cells is a special protein - an electrically dependent anion channel (VDAC1) [Gonzalez-Grono-w M. et al. The voltage-dependent anion channel is a receptor for plasminogen kringle 5 on human endothelial cells, J Biol Chem, 278: 27312-27318, 2003], while for angiostatin, the receptors are ATP synthase associated with the cytoplasmic membrane and integrin α _v β ₃ [Tarui T. et al. Specific interaction of angiostatin with integrin α _v β ₃ in endothelial cells. J Biol Chem, 276: 39562-29567, 2001].

In addition to its high antiproliferative activity, other advantages of the kringle domain 5 of human plasminogen as an antiangiogenic drug are obvious. First, K5 exhibits its inhibitory activity specifically, only on proliferating blood vessel endotheliocytes, and is therefore non-toxic to other cell types, including previous normal endothelial cells [Zhang D. et al. Intravitreal injection of plasminogen kringle 5, an endogenous angiogenic inhibitor, arrests retinal neovascularization in rats. Diabetologia, 44: 757-765, 2001]). Secondly, plasminogen is an endogenous protein of human tissues, and therefore K5 will not cause an immune response. Thirdly, K5, as a low molecular weight polypeptide (12-13 kDa), can be easily obtained as a recombinant protein in E. coli cells, and in addition, as a stable protein, it can be used in pharmacokinetic systems of targeted delivery and delayed release .

The kringle domain 5 of human plasminogen is a protein with a molecular weight of 12.8 kDa. To date, its inhibitory effect on the migration and proliferation of endothelial cells in vitro and in vivo has been established, and, therefore, K5 is a promising therapeutic agent for cancer growth and metastasis. Its therapeutic effect is due to the suppressive effect on the cell cycle of endothelial cells and the launch of apoptosis in the latter [Lu H. et al. Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells. Biochem Biophys Res Communic, 258: 668-673, 1999}. The prospect of the successful use of K5 as a therapeutic agent in pathological processes associated with impaired regulation of angiogenesis makes it necessary to develop methods for its production as a genetically engineered product, which will provide researchers with sufficient quantities of this protein.

To date, recombinant K5 expression systems in Pichia pastoris yeast have been described [Zhu M. et al. Expression of kringle 5 domain of human plasminogen in Pichia pastoris. Prep Biochem Biotechnol. 2003 Nov; 33 (4): 2 69-81] and E. coli (E. coli).

Closest to the technical details of our invention is recombinant plasmid DNA and created as a result of its transfection, the producer strain E. coli, which provides for the production of biologically active mouse K5 [Lu H. et al. Kringle 5 causes cell cycle arrest and apoptosis of endothelial cells. Biochem Biophys Res Communic, 258: 668-673, 1999]. Recombinant DNA (plasmid pET-22b (+), Novagen, Germany) contains the mouse plasminogen kringle gene 5 (amino acid residues Cys48i-Ala563) fused to the nucleotide sequence of 6 histidine residues (His-tag) and the signal sequence (peIB leader), which provides secretion of the target polypeptide into the periplasm of the bacterial cell. Recombinant K5 was expressed in E. coli and then purified using an Ni-NTA agarose sorbent. However, using this technique, only about 15% of the protein was expressed in a form secreted both in periplasm and partially in the culture medium; the rest (about 85% of total K5) after biosynthesis formed insoluble inclusion bodies in E. coli cells. For its purification, denaturing conditions had to be applied, at a urea concentration of up to 8 M. This led to a decrease in the yield of the target recombinant product; in addition, it is quite difficult to ensure renaturation, given the multi-stage process when working with inclusion bodies, as well as the K5 kringle structure.

The objective of the invention is to develop a method for producing a recombinant polypeptide, which is a fragment of a human plasminogen from 453 to 543 amino acids, including the kringle domain 5 of human plasminogen, in the cells of the producer strain E. coli, providing secretion of the target polypeptide into the periplasm of the cell in high yield.

In general, the technical result is achieved as a result of expression of the target protein gene in the composition of the created expression vectors. The methods for culturing bacterial cells in this case follow from the features of the obtained strain based on E. coli BL21 cells (DE3), and the methods of isolation and purification of the final product from the features of its expression (in periplasm, as well as in the case of the pEC5H vector containing additional sequences introduced in order to facilitate and increase the efficiency of the cleaning procedure).

The problem is solved by the method of producing this polypeptide as a part of E. coli cells, followed by isolation and purification of the final product from cell periplasm, in which expression is carried out in E. coli BL21 (DE3) cells transformed with recombinant plasmid DNA pЕК5 or рЕК5Н. The recombinant plasmid DNA pEK5 has a size of 3592 base pairs and includes the following main elements: the target polypeptide gene fused to the OTPA signal peptide gene, pUC ori replication origin, the kanamycin resistance gene, and the gene encoding the lac repressor under the control of the T7 promoter. The recombinant plasmid DNA pEK5H has a size of 3625 base pairs and consists of the following basic elements: the target polypeptide gene fused to the OmpA signal peptide gene, regions encoding the amino acid sequences of IUUHH and DDDDK, the pUC ori replication origin, the kanamycin resistance gene, and the lac coding gene -pressor under the control of the T7 promoter.

A method of producing a recombinant polypeptide is that gene expression is carried out by culturing cells in LB medium containing 40-50 μg / ml kanamycin to a density level of 0.4-0.6 OD, then cultivation is continued in the presence of 0.1-0, 3 mM isopropylthio-β-D-galactopyranoside prior to product accumulation, and the target polypeptide was isolated from the periplasm of E. coli cells by various methods, depending on the used recombinant plasmid DNA. Purification of the target product obtained in strains transfected with recombinant plasmid pEK5 from a solution of periplasm proteins is carried out using the method of reverse phase liquid chromatography using the HPLC system. Purification of the target product obtained in strains transformed with recombinant plasmid DNA pEK5H is carried out using sequentially applied stages of cation exchange chromatography on an agarose carrier, enzymatic digestion with enterokinase and ion exchange chromatography on a DEAE-Sepharose sorbent.

The developed expression vectors carry the gene encoding the target polypeptide, the amino acid sequence of which is shown in FIG. 1 and includes the human plasminogen kringle domain 5 with several additional amino acid residues — 9 residues from the N-terminus and 2 residues from the C-terminus (in FIG. 1 underlined). This sequence exactly matches the amino acid sequence of human plasminogen, limited by residues Asp 453 and Ala 543 (SEQ ID NO 1). In the sites of Leu 451-Pro 452 and Ala 543-Pro 544 of the plasminogen molecule, there are sites of the proteolytic action of the elastase enzyme, as a result of which native K5 is formed by limited proteolysis; in addition, this choice of the primary sequence of the target polypeptide is due to the fact that with the remaining amino acid “tips” (ie amino acid sequences that do not form the structure of the kringle domain), various chemical modifications can be made: covalent attachment of antibodies to tumor-specific antigens, conjugation with antitumor antibiotics etc. By incorporating into this genetic construct a sequence linked to the kringle domain gene 5 and encoding an additional six amino acid residues of histidine in the target protein molecule (not shown in FIG. 1), we also obtained recombinant plasmid DNA pEK5H. Such a modification of the target polypeptide allows to facilitate its further purification using ion-exchange chromatography. At the same time, the indicated genetic construct provides for the coding of the amino acid sequence Asp-Asp-Asp-Asp-Lys in the target protein (not shown in Fig. 1), which is the site of proteolytic cleavage by endonuclease enterokinase, which allows cleavage from purified by ion exchange chromatography the polypeptide obtained using the expression vector pEK5H, the amino acid sequence of six histidine residues, thus obtaining a polypeptide whose structure corresponds to the native one. The genetic constructs of pEC5 and pEC5H are shown in FIG. 2.

The invention is illustrated by the following examples:

Example 1. The creation of genetic constructs that ensure biosynthesis and secretion of the target polypeptide in E. coli cells.

A) Synthesis of a polynucleotide sequence containing the 3'-part of the human plasminogen K5 gene, stop codon and restriction enzyme sites SacII and HmdIII (K5 B).

Primers for synthesis:

K5 V 5 ': ATCTA CCGCGG CAAGAGGGCGACCACTGTTA

K5B-R 5 ': ACAAAGCTTATTATCAGGCCGCACACTGAGGGACAT

Using primers K5 B 5 '(SEQ ID NO 2), K5B-R 5' (SEQ ID NO 3) and the cDNA of the human gene library synthesized K5 B DNA sequence (SEQ ID NO 4) containing the 3'-part of the K5 gene, stop codon and restriction enzyme sites SacII and HindIII:

ATCTA CCGCGG CAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGAC

TGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAAT

CCACGGGCGGOTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAG

GTGGTCCCTGGTGCTACACGACAAATCCAAGAAAACTTTACGACTACTGTGA

TGTCCCTCAGTGTGCGGCCTGATAATAAGCTTTGT,

the underlined fragment is the recognition site of the restriction enzyme SacII;

the highlighted fragment is the Hind III restriction enzyme recognition site.

B) Synthesis of a polynucleotide sequence containing the 5'-part of the OTPA signal peptide with restriction enzyme sites BamHI and NruI (OMP5).

Primers for synthesis:

OMR5 5 ': ACTATA GGATCC ATGAAAAAGACAGCTATCGCAATTGCAGT

OMP5-R 5 ': ACTATATCGCGAAACCAGCCAAGGCCACTGCAATTGCGATA

For the synthesis of OMP5, the OMP5 5 ′ primers (SEQ ID NO 5) and OMP5-R 5 ′ (SEQ ID NO 6) were mixed and amplified to obtain the following OMP5 oligonucleotide (SEQ ID NO 7):

ACTT GGATCC ATGAAAAAGACAGCTATCGCAATTGCAGTGGCCTTGOCTGGT

TCGCGATATAGT,

the underlined fragment is the BamHI restriction enzyme recognition site;

the highlighted fragment is the recognition site for the Nml restriction enzyme.

C) Synthesis of a polynucleotide sequence encoding the 3'-part of the OTPA signal peptide with the restriction enzyme Nml and containing the 5'-part of the K5 gene with the restriction site of the SacII enzyme (OMDK5).

Primers for synthesis:

OMDK5 5 ':

ACTATA TCGCGA CCGTAGCGCAAGCCCAGATGTAGAGACTCCTTCCGAAGAAGACTGT

OMDK5-R 5 ':

ACTATACCGCGGTATCCTTTCCCATTCCCAAACATACAGTCTTCTTCGGAAGGAGTCT

For the synthesis of OMDK5, the primers OMDK5 5 ′ (SEQ ID NO 8) and OMDK5-R 5 ′ (SEQ ID NO 9) were mixed and amplified to give the following oligonucleotide OMDK5 (SEQ ID NO 10):

ACTAT ATCGCGA CCGTAGCGCAAGCCCAGATGTAGAGACTCCTTCCGAAGA

AGACTGTATGTTTGGGAATGGGAAAGGATACCGCGGTATAGT,

the underlined fragment is the recognition site of the restriction enzyme NruI;

the selected fragment is the recognition site of the restriction enzyme SacII.

D) Synthesis of a polynucleotide sequence encoding a signal peptide gene with a restriction site by the BamHI enzyme and a start codon, and containing a 5 ′ portion of the K5 gene with a restriction site by the SacII enzyme (SP5K5).

To obtain SP5K5, the oligonucleotides OMP5 (SEQ ID NO 7) and OMDK5 (SEQ ID NO 10) were treated with restriction enzyme Nml and ligated to each other using ligase of phage T4. As a result, the following sequence of SP5K5 (SEQ ID NO 11) was obtained:

ACTT GGATCC ATGAAAAAGACAGCTATCGCAATTGCAGTGGCCTTGGCTGGT

TTCGCGACCGTAGCGCAAGCCCAGATGTAGAGACTCCTTCCGAAGAAGACT

GTATGTTTGGGAATGGGAAAGGATACCGCGGTATAGT,

the underlined fragment is the BamHI restriction enzyme recognition site;

the selected fragment is the recognition site of the restriction enzyme SacII.

E) Synthesis of a polynucleotide sequence containing a signal peptide gene with a restriction site by the BamHI enzyme and a start codon, and a K5 gene with a stop codon and a restriction site by the HindIII enzyme (SPAK5).

To obtain SPAK5, the oligonucleotides SP5K5 (SEQ ID NO 11) and K5 B (SEQ ID NO 4) were treated with SacII restriction enzyme and ligated to each other using T4 phage ligase. As a result, the following sequence of SPAK5 (SEQ ID NO 12) was obtained:

ACTT GGATCC ATGAAAAAGACAGCTATCGCAATTGCAGTGGCCTTGGCTGGT

TTCGCGACCGTAGCGCAAGCCCAGATGTAGAGACTCCTTCCGAAGAAGACT

GTATGTTTGGGAATGGGAAAGGATACCGCGGCAAGAGGGCGACCACTGTTA

CTGGGACGCCATGCCAGGACTGGGCTGCCCAGGAGCCCCATAGACACAGCA

TTTTCACTCCAGAGACAAATCCACGGGCGGGTCTGGAAAAAAATTACTGCCG

TAACCCTGATGGTGATGTAGGTGGTCCCTGGTGCTACACGACAAATCCAAGA

AAACTTTACGACTACTGTGATGTCCCTCAGTGTGCGGCCTGATAATAAGCTT

TGT,

the underlined fragment is the BamHI restriction enzyme recognition site;

the selected fragment is the recognition site of the HindIII restriction enzyme.

E) Synthesis of a polynucleotide sequence encoding the 3 ′ part of the OmpA signal peptide with the restriction site of the NruI enzyme, and the peptide of six histidine residues with the restriction site of the RcaI enzyme (3O6H).

Primers for synthesis:

3O6H 5 ': ACTATA TCGCGA CCGTAGCGCAAGCCATCATCAT

3O6H-R 5 ': ACTATATCATGATTGGTGATGATGATGGCTTGCGCTA

For the synthesis of 3O6H, the primers 3O6H 5 ′ (SEQ ID NO 13) and 3O6H-R 5 ′ (SEQ ID NO 14) were mixed and amplified to give the following 3O6H oligonucleotide (SEQ ID NO 15):

ACTATA TCGCGA CCGTAGCGCAAGCCATCATCATCACCATCATGATATAGT

the underlined fragment is the recognition site of the Nrul restriction enzyme;

the selected fragment is the recognition site of the RcaI restriction enzyme.

G) Synthesis of a polynucleotide sequence that is a DNA fragment encoding the amino acid sequence of an enterokinase cleavage site with a restriction site by the Rcal enzyme and a 5 ′ portion of the K5 gene with a restriction site by the SacII enzyme (E5K5). Primers for synthesis:

E5K5 5 ':

ACTATA TCATGAT GATGACGATAAACCAGATGTAGAGACTCCTTCCGAAGA

BUT

E5K5-R 5 ':

ACTATACCGCGGTATCCTTTCCCATTCCCAAACATACAGTCTTCTTCGGAAG

GT

For the synthesis of E5K5, primers E5K5 5 '(SEQ ID NO 16) and E5K5-R 5' (SEQ ID NO

17) were mixed and amplified, with the following

E5K5 oligonucleotide (SEQ ID NO 18):

ACTATA TCATGA TGATGACGATAAACCAGATGTAGAGACTCCTTCCGAAGA AGACTGTATGTTTGGGAATGGGAAAGGATACCGCGGTATAGT,

the underlined fragment is the recognition site of the restriction enzyme RcaI;

the selected fragment is the recognition site of the restriction enzyme SacII.

3) Synthesis of a polynucleotide sequence encoding a signal peptide with a restriction site by the BamHI enzyme and a start codon, a peptide of six histidine residues, the amino acid sequence of the enterokinase cleavage site and containing the 5 ′ part of the K5 gene with the restriction site of the SacII enzyme (SPH5K5).

To obtain SPH5K5, the oligonucleotides OMP5 (SEQ ID NO 7), 3O6H (SEQ ID NO 15), and E5K5 (SEQ ID NO 16) were digested with restriction enzymes Nrul and RcaI and were led together using T4 phage ligase. The following sequence of SPH5K5 (SEQ ID NO 19) was obtained:

ACTT GGATCCA TGAAAAAGACAGCTATCGCAATTGCAGTGGCCTTGGCTGGT

TTCGCGACCGTAGCGCAAGCCATCATCATCACCATCATGATGATGACGATAA

ACCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA

GGATACCGCGGTATAGT,

the underlined fragment is the BamHI restriction enzyme recognition site;

the selected fragment is the recognition site of the restriction enzyme SacII.

I) Synthesis of a polynucleotide sequence encoding a signal peptide with a restriction site by the BamHI enzyme and a start codon, a peptide of six histidine residues, the amino acid sequence of the enterokinase cleavage site and containing the K5 gene with a stop codon and the restriction site by the HindIII enzyme (SPHisK5).

To obtain SPHisK5, the oligonucleotides SPH5K5 (SEQ ID NO 19) and K5 B (SEQ ID NO 4) were treated with SacII restriction enzyme and ligated to each other using phage T4 ligase. The following sequence of SPHisK5 (SEQ ID NO 20) was obtained:

ACTT GGATCC ATGAAAAAGACAGCTATCGCAATTGCAGTGGCCTTGGCTGGT

TTCGCGACCGTAGCGCAAGCCATCATCATCACCATCATGATGATGACGATAA

ACCAGATGTAGAGACTCCTTCCGAAGAAGACTGTATGTTTGGGAATGGGAAA

GOATACCGCGGCAAGAGGGCGACCACTGTTACTGGGACGCCATGCCAGGAC

TGGGCTGCCCAGGAGCCCCATAGACACAGCATTTTCACTCCAGAGACAAAT

CCACGGGCGGGTCTGGAAAAAAATTACTGCCGTAACCCTGATGGTGATGTAG

GTGOTCCCTGGTGCTACACGACAAATATAAGAAAACTTTACGACTACTGTGA

TGTCCCTCAGTGTGCGGCCTGATAATAAGCTTTGT,

the underlined fragment is the BamHI restriction enzyme recognition site;

the selected fragment is the recognition site of the HindIII restriction enzyme.

To obtain expression vectors, the SPAK5 (SEQ ID NO 12) and SPHisK5 (SEQ ID NO 20) sequences were treated with the BamHI and Hind III restriction enzymes and inserted at the BamHI and Hind III restriction sites into the pBSH2 vector. The resulting vectors were called pEK5 and pEK5H, respectively. A schematic representation of the structure of the expression vectors pEK5 and pEK5H is shown in FIG.

Example 2. Production of the target polypeptide in E. coli strains.

The expression vector pEK5, or pEK5H, obtained as described in example 1, was incorporated into Escherichia coli BL-21 / DE3 Escherichia coli strain. To realize the expression of the target protein, 30 ml of the transfected strain was kept shaking for 2-3 h in LB medium at 37 ° С with the addition of kanamycin (50 μg / ml) and glucose (0.2%). Then, this culture was diluted 20 times with fresh LB medium containing kanamycin (40 μg / ml) and increased for 2-3 hours with shaking. When the culture medium reached a density of OD _{280 nm} = 0.5, isopropylthio-β-D-galactopyranoside (IPTG) was added to it to a concentration of 0.5 mM to induce the biosynthesis of the target polypeptide. Then this culture was incubated for about 5 hours at 25 ° C with shaking, centrifuged at 10,000 g for 15 minutes, the supernatant was discarded.

Example 3. The selection of the target polypeptide from the producer strain of E. coli containing recombinant plasmid DNA pEK5.

The cell pellet obtained as described in Example 2 was resuspended in buffer A (20 mM Tris, pH 8.0, 15% galactose and 1 mM EDTA) for 10 min at 20 ° C. The cell suspension was then centrifuged at 10,000 g for 15 min; the supernatant was removed and the cells were resuspended in a 7 mM solution of magnesium (II) sulfate at 0 ° C for 20 min. The resulting periplasm proteins were then separated from the cells by centrifugation at 10,000 g for 20 min at 4 ° C; the precipitate was discarded. The resulting solution of periplasm proteins was concentrated by ultrafiltration in a concentration cell and 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 strains transfected with recombinant plasmid pEK5 was carried out by reverse phase liquid chromatography using the HPLC system. The periplasm protein solution was passed through a Brownlee Aquapore C4 chromatography column (Applied Biosystems, USA); A 0-60% gradient of acetonitrile containing 0.05% trifluoroacetic acid was used as an eluent. The eupture profile of the target polypeptide obtained in a strain containing the pEK5 expression vector when performing reverse phase high performance liquid chromatography is shown in FIG. 3.

Example 4. Isolation of the desired polypeptide from a producer strain of E. coli containing recombinant plasmid DNA pEK5H.

The cell pellet obtained as described in Example 2 was resuspended in buffer A (20 mM Tris, pH 8.0, 15% galactose and 1 mM EDTA) for 10 min at 20 ° C. The cell suspension was then centrifuged at 10,000 g for 15 min; the supernatant was removed and the cells were resuspended in a 7 mM solution of magnesium (II) sulfate at 0 ° C for 20 min. As a result, the proteins of the periplasmic space that passed into the solution were then separated from the cells by centrifugation at 10,000 g for 20 min at 4 ° C; the precipitate was discarded. The resulting solution of periplasm proteins was concentrated by ultrafiltration in a concentration cell and sodium dihydrogen phosphate was added to it to a final concentration of 20 mM, sodium chloride to a final concentration of 200 mM, and imidazole to a final concentration of 10 mM. Purification of the target polypeptide was carried out on a chromatographic sorbent Ni-NTA-agarose. The sorbent placed in the column with a volume of 7 ml was previously balanced with 10 volumes of buffer B (30 mM Tris, pH 8.0, 20 mM sodium dihydrogen phosphate, 30 mM imidazole). A solution of periplasm proteins was applied to the column and buffer B was passed through it until the baseline was reached, after which the column was washed with buffer G (30 mM Tris, pH 8.0, 20 mM sodium dihydrogen phosphate, 150 mM imidazole), also until the baseline was reached. The target protein was eluted with buffer D (30 mM Tris, pH 8.0, 20 mM sodium dihydrogen phosphate, 300 mM imidazole). The protein eluted from the column was dialyzed three times against 100 volumes of buffer E (10 mM sodium dihydrogen phosphate, 3 mM potassium chloride, 120 mM sodium chloride, pH 7.4) at 4 ° C for 12 hours. The elution profile of the target polypeptide obtained in the strain containing the expression vector pEK5H, when conducting ion-exchange chromatography on Ni-NTA-agarose is presented in figure 4.

Then, the cleavage of the amino acid sequence of six histidine residues from the target polypeptide obtained from the producer strain containing the expression vector pEK5H was carried out. For this, a solution with a protein concentration was incubated for 6 h with enterokinase in a mass ratio of protein: enzyme = 100000: 1. Then, the target polypeptide without 6 × His was isolated from the reaction mixture using ion exchange chromatography on a DEAE-Sepharose sorbent.

Example 5. Determination of the level of endotoxin in the preparation of the target polypeptide.

Given the future use of the target polypeptide obtained using the described method, mainly as a therapeutic agent, administered parenterally, the resulting product was tested for the content of bacterial endotoxins (lipolysaccharides). Analysis of the qualitative content of bacterial endotoxins (lipolysaccharides) (LAL-test) was carried out in pyrogen-free sodium glass tubes 10 × 75 mm in size. A reaction mixture was prepared consisting of 0.1 ml of a solution of LAL reagent (Limulus Amebocyte Lysate, Pyrotell, USA) and 0.1 ml of a solution of the target polypeptide. After the reaction mixture was added to the tubes and mixed, the tubes were incubated at 37 ° C for 60 min. Then the tubes were removed from the incubator and turned over 180 °. If a solid gel formed at the bottom of the tube that did not break when turned 180 °, this was considered as a positive result for the presence of bacterial endotoxin in the analyzed solution; in the case of destruction of the gel upon inversion by 180 ° or its absence, a conclusion was drawn about the pyrogen-free nature of the analyzed solution. All samples of the target polypeptide obtained using the above method showed a negative reaction to the presence of bacterial endotoxins.

Example 6. Determination of the biological activity of the target polypeptide.

The influence of the studied drugs on the proliferation of endothelial cells was studied by the method described previously (O'Reilly M. et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell, 88: 277-285, 1997). HUVEC cells (human umbilical vein endothelial cells) from the monolayer were trypsinized and resuspended in M199 medium containing 5% fetal bovine serum. The cells were then scattered into 96-well gelatin coated plates at a density of 5000 cells per well. After 24 hours, a solution of the desired polypeptide at various concentrations was added. After 20 minutes, the cell cultures were treated with 5 ng / ml BFGF solution (Life Technilogies Inc., USA) in the presence of heparin (1 μg / ml). Cell survival in the control and in the groups treated with the solutions of the target polypeptide was determined using the MTT test after 72 hours of incubation. The MTT test effectively determines the metabolic activity of mitochondria, and its result directly correlates with the number of living cells. Figure 5 shows the dependence of the proliferation of HUVEC cells on the concentration of the target polypeptide. As can be seen in figure 5, the preparations of the target polypeptide caused the death of human endothelial cells in a dose-dependent manner.

Claims (4)

1. A method of producing a recombinant polypeptide, which is a fragment of a human plasminogen from 453 to 543 amino acids (SEQ ID NO 1), which consists in expressing its gene in E. coli cells, followed by isolation and purification of the final product from cell periplasm, in which expression is carried out in E. coli BL21 (DE3) cells transformed with pEK5 or pEK5H recombinant plasmid DNA according to FIG. 2 containing the target polypeptide gene fused to the OmpA signal peptide gene, pUC ori replication origin, kanamycin resistance gene and gene encoding a lac-repressor is under the control of the T7 promoter, the recombinant plasmid further comprises a DNA rEK5N between OmpA genes and the desired polypeptide coding regions and amino acid sequence NNNNNN DDDDK. 2. The method for producing the recombinant polypeptide according to claim 1, wherein the gene expression is carried out by culturing cells in LB medium containing 40-50 μg / ml kanamycin to a density level of 0.4-0.6 OD, then cultivation is continued in the presence of 0.1-0.3 mm isopropylthio-β-D-galactopyranoside prior to product accumulation, and the target polypeptide is isolated from the periplasm of E. coli cells by various methods, depending on the used recombinant plasmid DNA. 3. The production method according to claims 1 and 2, which consists in the fact that the selection of the target polypeptide obtained by culturing cells transformed with recombinant plasmid DNA pEK5 is carried out using reverse phase chromatography. 4. The production method according to claims 1 and 2, which consists in the fact that the isolation and further purification of the target polypeptide obtained by culturing cells transformed with recombinant plasmid DNA pEK5H is carried out by cation exchange chromatography on an agarose carrier, enzymatic digestion with enterokinase and ion exchange chromatography on sorbent DEA -Sepharose.

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