RU2395568C2 - RECOMBINANT VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) SECRETING Escherichia coli BL21 (pVEGF-A165) CELL STRAIN - Google Patents

Abstract

FIELD: chemistry; biochemistry.

SUBSTANCE: invention pertains to biotechnology. In particular, the invention relates to an Escherichia coli BL21 (pVEGF-A165) strain and can be used to produce a vascular endothelial growth factor - GST-VEGF-A165 protein. A novel Escherichia coli BL21 (pVEGF-A165) cell strain is obtained, which is transformed by the pGEX-VEGF-A165 plasmid. This strain produces a recombinant GST-VEGF-A165 protein.

EFFECT: invention enables to obtain a Escherichia coli BL21 (pVEGF-A165) strain which is stably transformed by plasmid which codes VEGF, and which secrete this factor in extracellular space when cultured in vitro.

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Description

The technical field of the present invention

The invention relates to the field of microbiology, molecular biology and biochemistry, in particular to the production of a strain of a genetically modified microorganism used to produce recombinant vascular endothelial growth factor (VEGF).

BACKGROUND OF THE INVENTION

The presence of factors stimulating the growth of blood vessels and thereby attracting newly formed vessels to nourish a growing tumor was postulated several decades ago, although the identification and isolation of these factors remained unattainable for a long time. Currently, vascular endothelial growth factor (VEGF), which was identified in the 1980s, is considered a key regulator of normal and abnormal blood vessel growth. In 1993, it was shown that monoclonal antibodies against VEGF cause a sharp inhibition of tumor growth in vivo. This observation was the basis for the development of the anticancer agent bevacizumab (Avastin (Avastin; Genentech)), a humanized variant of this anti-VEGF antibody. The recent approval by the US FDA of bevacizumab as a first-line therapeutic for metastatic colorectal cancer confirms the notion that VEGF is a key mediator of tumor angiogenesis and that blocking angiogenesis is an effective treatment strategy for malignant tumors in humans.

The observation that tumor growth may be accompanied by increased vascularization was published over a century ago (the review can be found in reference (1). However, only in 1939 Ide et al. First postulated the presence of a tumor factor that stimulates the growth of blood vessels, which helps to attract blood supply to a growing tumor (2) Several years later, based on the observation that rapid growth of the tumor is preceded by a local increase in vascular network density, Algire et al. suggested that the rapid growth of tumor transporters lanthates depends on the formation of a branched vasculature (3) .Then new messages in this field of technology did not appear until the 1960s, when in the experiments of Greenblatt and Shubik (4) and Ehrmann and Knoth (5) the first evidence was obtained that tumor angiogenesis is mediated by diffusible factors produced by tumor cells.

In 1971, Folkman suggested that suppressing angiogenesis could be an effective anti-cancer strategy (6). Based on this revolutionary hypothesis in the early 1970s, Folkman et al. efforts have been made to isolate the “tumor angiogenesis factor” from human and animal tumors (7). In 1978, Gullino also suggested that blocking angiogenesis can prevent the development of a malignant tumor (8). Subsequently, the angiogenic effects of many factors have been described (e.g., epidermal growth factor (EGF), transforming growth factor (TGF) -α, TGF-β, tumor necrosis factor-α (TNF-α) and angiogenin) (9). However, although these factors enhanced angiogenesis in the course of some biological analyzes, none of them acted physiologically (10).

The lion's share of attention was paid to two widespread and highly effective endotheliocyte mitogens and angiogenic factors - acidic and basic fibroblast growth factors (aFGF and bFGF). In the mid-1980s, homogeneity was purified, cDNA sequencing and cloning of these FGFs was carried out (11). An unexpected finding was that both the aFGF and bFGF genes do not encode a standard secretory signal peptide. However, as indicated above, earlier studies suggested that tumor angiogenesis is mediated by diffusible molecules (4, 5). In addition, some studies indicated that FGF immuno-neutralization had no effect or had a weak effect on tumor angiogenesis (12, 13), suggesting that key regulators of angiogenesis have not yet been identified.

It is now known that one such key regulator of angiogenesis is vascular endothelial growth factor (VEGF, also called VEGF-A), and the role of the VEGF gene family in the regulation of angiogenesis has been intensively studied for over a decade (1). The VEGF family includes the prototype member of VEGF-A, placental growth factor (P1GF) (14), VEGF-B (15), VEGF-C (16) and VEGF-D (17). Convincing evidence suggests that while the construction and maturation of the vascular wall are very complex processes that require the combined effects of angiopoietins, platelet-derived growth factor B (PDGF-B) and other factors (18), the action of VEGF-A is a rate-limiting stage of normal and abnormal growth of blood vessels (19). It is important that VEGF-C and VEGF-D regulate angiogenesis of lymphatic vessels (20), which emphasizes the unique role of this family of genes in controlling the growth and differentiation of some anatomical components of the vascular system.

In 1983, Senger et al. reported partial isolation of vascular permeability factor (VPF), a protein that induced an increase in vascular permeability of the skin, from a medium conditioned by a guinea pig tumor cell line (21). However, since VPF was not isolated and sequenced, at that time the molecular structure of the factor remained unknown. In 1989, there was a report on the isolation of vascular endothelial growth factor (VEGF) specific for mitogen endotheliocytes from the medium conditioned by follicular cells of the pituitary of the cow (22). The amino terminal amino acid sequence of VEGF did not match any known protein in the available databases (22). Then, the work of Connolly et al., Based on the work of Senger et al., Independently led to the isolation and sequencing of VPF (23). Cloning of VEGF (24) and VPF (25) cDNAs showed that VEGF and VPF are the same molecule. This result was unexpected, given that other mitogen endotheliocytes, such as FGF, do not increase vascular permeability.

VEGF is characterized by significant homology with chains A and B of PDGF (24). The gene encoding human VEGF-A consists of eight exons separated by seven introns (26, 27). Alternative exon splicing leads to the formation of four main isoforms - VEGF121, VEGF165, VEGF189, and VEGF206 - consisting of 121, 165, 189 and 206 amino acids, respectively, after cleavage of the signal (24). Alternative splicing regulates the bioavailability of VEGF (28, 29). To date, there is ample evidence that VEGF165 is the most physiologically competent isoform (30, 31). Extracellular proteolysis also plays an important role in the regulation of the bioavailability of VEGF. Plasmin is able to cleave VEGF165 and VEGF 189 and release a biologically active product consisting of the first 110 amino-terminal amino acids (32). Given the importance of plasminogen activation during physiological and pathological angiogenesis (33), this mechanism may be of particular importance for regulating the activity and bioavailability of VEGF during the development of remodeling and in response to microenvironment signals. In addition, in some tumors, VEGF proteolysis mediated by mtrix metalloproteinase-9 (MMP9) may be responsible for triggering angiogenesis (34).

A well-established action of VEGF is to accelerate the growth of endotheliocytes in arteries, veins and lymph vessels (for a review, see reference 35). VEGF induces a potent angiogenic response in many in vivo models (24, 36). In addition, as indicated above, VEGF increases vascular permeability, and this property underlies the important role of this molecule in inflammation and other pathological processes (37). In this context, VEGF also induces endothelial expression of certain adhesion molecules that regulate the adhesion of leukocytes during inflammation (38).

In vitro, VEGF prevents serum depletion induced apoptosis of endotheliocytes, which is mediated by a cascade of phosphatidylinositol-3'-kinase (PI3K) / Akt (39). VEGF also induces the expression of antiapoptotic proteins BCL2 and A1 in endotheliocytes (40). Dependence on VEGF was shown on the endothelial cells of newly formed, but not completely formed tumor vessels (41, 42). Pericyte lining is presumably one of the key events leading to endothelial cell loss of dependence on VEGF (42).

It should be emphasized that although endotheliocytes are the main targets of VEGF, several studies have also shown its effects on the mitotic activity / survival of some nonendothelial cell types, including neurons (43).

VEGF is fundamentally important for normal embryonic vasculogenesis and angiogenesis. Inactivation of a single VEGF allele in mice leads to embryonic mortality (44, 45). Inhibition of VEGF in the early neonatal period leads to stunting, apoptosis of endotheliocytes and death, mainly as a result of renal failure (46, 47). VEGF is also required for intracranial ossification, a fundamental mechanism for the growth of tubular bones. VEGF inhibitors inhibit these processes in rodents and primates (48, 49). It is important to note that this effect is completely reversible upon termination of anti-VEGF processing (48, 49). Angiogenesis is a key aspect of the normal ovarian-menstrual cycle and the functioning of the endometrium (50). The expression of VEGF mRNA in time and space is associated with the proliferation of blood vessels in the ovaries of many species (51, 52). The introduction of VEGF inhibitors slows the development of follicles (53) and inhibits angiogenesis in the corpus luteum of rodents (54) and primates (49, 55, 56).

In situ hybridization studies indicate the expression of VEGF mRNA in many human tumors, including lung carcinomas (57), breast (58), gastrointestinal tract (59), kidney (60) and ovary (61). However, VEGF expression varies not only between different types of tumors, but also within the same tumor. In the case of polymorphic glioblastoma and other tumors with a significant necrotic component, the expression of VEGF mRNA is most intense in hypoxic tumor cells adjacent to necrosis zones (62, 63). A tumor with particularly strong VEGF expression is renal cell carcinoma. Upregulation of VEGF expression can be associated with a number of factors, among which hypoxia and some mutations can be noted.

The clinical applicability of VEGF inhibition in oncology is not limited to solid tumors. There is increasing evidence that VEGF and its receptors are expressed by a variety of malignant tumors of the blood system, suggesting the promise of inhibition of VEGF or VEGFR signal transmission for the treatment of such conditions (64).

Disclosure of the present invention

The essence of the present invention is the creation of a new strain of Escherichia coli cells BL21 (pVEGF-A165) producing a recombinant vascular endothelial growth factor - protein GST-VEGF-A165. The ability of Escherichia coli BL21 cells (pVEGF-A165) to secrete recombinant VEGF is achieved by gene modification of Escherichia coli cells with plasmid pGEX-VEGF-A165 (SEQ ID NO: 1).

The technical result consists in obtaining a strain of Escherichia coli cells BL21 (pVEGF-A165) stably transfected with a plasmid encoding VEGF and secreting this factor into the extracellular space during in vitro cultivation.

The aim of the present invention is to expand the collection of unique cell strains that can be used to obtain VEGF. This task is especially relevant for modern pharmacological biotechnology. The VEGF obtained by this strain can be used in many fields of biological and medical research, in particular in the development and study of drugs and other compounds that are VEGF agonists or antagonists.

The problem is solved in that a new strain of Escherichia coli cells BL21 (pVEGF-A165) was obtained, transformed with the plasmid pGEX-VEGF-A165 (SEQ ID NO: 1) and producing recombinant protein GST-VEGF-A165 in an amount of at least 2 μg of purified recombinant protein per 1 ml of growth medium in 24 hours (according to ELISA). The resulting strain has stable cultural and morphological properties and, as a result of gene modification, has the ability to secrete the recombinant vascular endothelial growth factor. The strain of cells Escherichia coli BL21 (pVEGF-A165) was deposited in the All-Russian collection of industrial microorganisms (VKPM) FSUE GosNIIGenetika under the number VKPM B-10042.

Brief Description of the Drawings

Figure 1 shows the genetic map of the plasmid pGEX-VEGF-A165. Figure 2 shows the result of the analysis of fractions collected during the purification of the recombinant protein GST-VEGF-A165. Proportional amounts of protein from each fraction were analyzed using SDS-PAGE in a 10% acrylamide gel, followed by gel staining with Coomassie Brilliant Blue (Serva). The molecular weight marker Unstained Protein Molecular Weight Marker (Fermentas), extreme lane to the right, was used to evaluate the molecular weight of the proteins. The arrow indicates the position in the gel of the recombinant protein GST-VEGF-A165. The apparent molecular weight of the GST-VEGF-A165 protein is about 45 kDa and corresponds to the theoretically calculated (45526 Da).

Figure 3 shows the result of stability analysis of the recombinant protein GST-VEGF-A165 during dialysis and freezing-thawing. Proportional amounts of protein from each fraction were analyzed using SDS-PAGE in a 10% acrylamide gel, followed by gel staining with Coomassie Brilliant Blue (Serva). The molecular weight marker Unstained Protein Molecular Weight Marker (Fermentas), left lane, was used to evaluate the molecular weight of the proteins. The arrow indicates the position in the gel of the recombinant protein GST-VEGF-A165. Protein GST-VEGF-A165 is stable during dialysis and can withstand freezing in liquid nitrogen and subsequent thawing.

Examples of the implementation of the present invention

Example 1. The procedure for obtaining recombinant plasmid DNA encoding the cDNA of the VEGF-A165 protein, fused to the protein glutathione-8-transferase (GST-VEGF-A165)

Human VEGF cDNAs were amplified by PCR using first strand cDNA from the human placenta as a template. The primers were selected so that the signal peptide (amino acids 1-26) was removed from the VEGF cDNA, and the 5'-primer [5'-CAT GGA TCC GCT GCA CCC ATG GCA GAA GGA-3 '] and 3'-primer [5 The '-GTC ACC CGG GTC ACC GCC TCG GCT-3'] contained BamHI and SmaI restriction endonuclease recognition sites, respectively. Next, the BamHI-SmaI fragment was cloned at the appropriate sites into the pGEX-4T2 plasmid vector (Amersham) so that the VEGF-A165 protein cDNA was in the same reading frame with the GST protein cDNA.

The resulting plasmid construct was 5462 bp in size. (figure 1). Recombinant protein GST-VEGF-A165 encoded by this plasmid had a size of 392 amino acids and a calculated molecular weight of 45526 Da.

Example 2. The procedure for obtaining a strain of bacteria E. coli BL21, producing recombinant protein GST-VEGF-A165

The plasmid vector pGEX-VEGF-A165 was transformed into E. coli strain BL21 using a standard bacterial transformation technique (65) and plated on LB agar (0.171 M NaCl, 1% tryptone, 0.5% yeast extract, 1.5% agar) containing a selective antibiotic ampicillin at a concentration of 50 μg / ml, and grown for 16-18 hours at a temperature of 37 ° C. Single colonies of bacteria grown on selective medium were reseeded on a fresh selective plate and grown for 16-18 hours at a temperature of 37 ° C. A single colony of bacteria was taken from a Petri dish with a bacterial loop and 10 ml of LB liquid growth medium (0.171 M NaCl, 1% tryptone, 0.5% yeast extract) containing a selective antibiotic ampicillin at a concentration of 50 μg / ml was inoculated and grown for 16 -18 hours at a temperature of 37 ° C with constant stirring. 2 ml of the bacterial suspension were taken, the cells were pelleted by centrifugation, the supernatant was removed, and the pellet was resuspended in 1.5 ml of freezing medium (LB liquid medium containing 15% glycerol). The resulting producer strain was stored at a temperature of -80 ° C.

Example 3. The procedure for obtaining recombinant protein GST-VEGF-A165 in a bacterial expression system

The recombinant protein producer strain GST-VEGF-A165 was streaked onto a Petri dish with LB agar containing a selective antibiotic ampicillin at a concentration of 50 μg / ml and grown for 16-18 hours at 37 ° C. A single colony of bacteria was taken from a Petri dish, 10 ml of LB liquid medium containing 50 μg / ml ampicillin were inoculated, and grown with constant stirring at 37 ° C for 16-18 hours.

A 5 ml overnight culture of the producer strain was transferred with a sterile pipette into two 2-liter flasks containing 200 ml of LB liquid medium and grown with constant stirring at 37 ° C for 2.5 hours, after which expression of the recombinant protein was induced by adding isopropyl-β-D-thiogalactoside (Fermentas) to a final concentration of 0.02 mm and bacteria were grown at a temperature of 28 ° C for 1.5 hours.

Cells were pelleted by centrifugation at 4000 rpm and a temperature of 4 ° C for 10 minutes, the supernatant was removed, and the pellet was washed using 40 ml of phosphate-buffered saline (0.01 M sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, pH 7.4), pre-cooled to 4 ° C, and then the cells were besieged by centrifugation at 4000 rpm and a temperature of 4 ° C for 10 minutes. The supernatant was removed and the cell pellet was resuspended in 10 ml of buffer (0.01 M sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, 2.5 mm MgCl ₂ , 1 mm EDTA, 0.05% NP-40, pH 7 , 4) and crystalline lysozyme (Sigma) was added to a final concentration of 10 μg / ml. Next, the suspension of bacterial cells was frozen in liquid nitrogen and stored at a temperature of -80 ° C until the protein purification stage.

The suspension of bacterial cells was slowly thawed on ice and subjected to ultrasound at + 0 ° C using a Vibracell 72434 instrument (Bioblock Scientific) until the suspension was clarified and the viscosity disappeared (the duration of the ultrasonic pulse was 60 seconds, breaks were made between pulses at least 60 seconds; spent at least 3 pulses).

The bacterial lysate was transferred to 1.5 ml chilled Microcentrifuge tubes (Costar) and centrifuged in a benchtop microcentrifuge at 13,000 rpm and a temperature of 4 ° C. for 15 minutes. The clarified lysate was collected and mixed with 500 μl of 4Fast Flow glutathione sepharose (Amersham Biosciences), previously equilibrated with chilled phosphate-buffered saline.

Then, for 1.5 h at 4 ° С with constant stirring, the binding of the recombinant protein GST-VEGF-A165 with glutathione sepharose was carried out. After binding, the lysate at 4 ° C was applied to a column mechanically inhibiting glutathione-sepharose, and the lysate was allowed to flow through the column under gravity. Then, at a temperature of 4 ° C, glutathione-sepharose was washed once with 10 ml of buffer (0.01 M sodium phosphate, 0.138 M NaCl, 0.0027 M KCl, 2.5 mm MgCl ₂ , 1 mm EDTA, 0.05% NP- 40, pH 7.4) and twice with 5 ml of phosphate-buffered saline.

GST-VEGF-A165 recombinant protein bound to glutathione-sepharose was eluted three times with 500 μl of elution buffer (50 mM Tris, pH 8.0, 10 mM reduced glutathione), incubating glutathione-sepharose in the elution buffer for 10 minutes at room temperature and stirring constantly with rotomix (10 rpm).

At all stages of protein purification, fractions were collected and analyzed using SDS-PAGE in a 10% acrylamide gel (66). The result of the analysis of fractions collected during the purification of the recombinant protein GST-VEGF-A165, shown in figure 2.

The eluted recombinant protein was dialyzed at 4 ° C against phosphate-buffered saline, frozen in liquid nitrogen, and stored at -80 ° C in aliquots. To verify the stability of the GST-VEGF-A165 protein, an aliquot was thawed and the concentration of the recombinant protein was analyzed by SDS-PAGE. The result of the stability analysis of the recombinant protein GST-VEGF-A165 during dialysis and freeze-thaw is shown in Fig.3.

Example 4. The procedure for determining the concentration of recombinant protein GST-VEGF-A165.

The concentration of recombinant protein GST-VEGF-A165 was determined by enzyme-linked immunosorbent assay (ELISA) using the ELISA kit for determining the protein VEGF Human VEGF ELISA Development kit manufactured by PeproTech in accordance with the manufacturer's protocol.

Antibodies for binding were diluted in phosphate-buffered saline to a final concentration of 0.5 μg / ml, 100 μl of this solution was added to the wells of a 96-well ELISA plate and incubated at room temperature for 16-18 hours. The antibody solution was removed and the wells were washed four times with 300 μl wash buffer (phosphate-buffered saline containing 0.05% Tween-20). The residual wash buffer was carefully removed, 300 μl block buffer (1% BSA in phosphate buffered saline) was added to the wells, and the plate was incubated at room temperature for 1 hour. The blocking solution was removed and the wells were washed four times with 300 μl of washing buffer.

Protein standards (at a concentration of 0 to 4 ng / ml) and samples in a volume of 100 μl were added to the wells of the tablet, 3 samples in parallel and incubated at room temperature for 2 hours.

Next, the samples were removed, and the wells were washed four times with 300 μl of washing buffer. Detection antibodies were diluted in dilution buffer (phosphate buffered saline containing 0.05% Tween-20 and 0.1% BSA) to a final concentration of 0.25 μg / ml, 100 μl was added to the well and incubated at room temperature in within 2 hours.

The antibody solution was removed, and the wells were washed four times with 300 μl of wash buffer. 6 μl of avidin peroxidase was diluted in a dilution buffer at the rate of 1: 2000, 100 μl of this solution was added to each well and incubated at room temperature for 30 minutes.

The avidin peroxidase solution was removed and the wells washed four times with 300 μl wash buffer. 100 μl of ABTS liquid substrate was added to the wells and incubated at room temperature until color development.

The optical density of the samples in the wells was measured at a wavelength of 405 nm and correction of a wavelength of 650 nm using a Multiscan EX microplate reader (Labsystems).

The main characteristics of the cells (according to the strain passport)

1. Generic and species name of the host strain (recipient): Escherichia coli

2. Number or name of strain: BL21 (pVEGF-A165)

3. A method of obtaining a strain: obtained by transformation.

4. The product synthesized by the strain: GST-VEGF-A165.

5. The activity (productivity) of the strain: 2 μg of purified recombinant protein per 1 ml of growth medium according to ELISA.

6. The method, conditions and composition of the media for long-term storage of the strain: LB medium + 15% glycerol in ampoules at -80 ° C.

7. The method, conditions and composition of the media for the propagation of the strain: LB medium containing 50 μg / ml ampicillin).

8. The risk level group of the strain: I

9. Genetic features of the strain:

but. The genotype of the host strain (mutations, deletions, inversions, the presence of plasmids or prophages, resistance (sensitivity) to antibiotics, phages, etc., other genetic features):

[F-, ompT, hsdSB (r-B, m-B), dcm, gal].

b. Description of the recombinant plasmid:

Name of the plasmid: pGEX-VEGF-A165.

Plasmid size: 5462 nucleotide pairs.

at. Information about the vector on the basis of which the plasmid was constructed:

Vector Name: pGEX-4T2.

Vector Size: 4970 bp

The complete nucleotide sequence of the vector pGEX-4T2: SEQ ID NO: 2.

The origin of the vector and its basic genetic elements:

vector contains

- ampicillin resistance gene (bla);

- a gene encoding the enzyme glutathione S-transferase from Schistosoma japonicum,

- lad gene repressor from E. coli;

- the origin of replication from plasmid pBR322 from E. coli.

Information about cloned DNA:

Species of the donor organism: Homo sapiens

The size of the cloned fragment and the genes included in its composition: 501 bp;

VEGF cDNA fragment (GenBank No. NM_003376).

Literature

Claims (1)

The strain of Escherichia coli cells BL21 (pVEGF-A165), transformed with the plasmid pGEX-VEGF-A165 (SEQ ID NO: 1) and producing recombinant protein GST-VEGF-A165, deposited in the All-Russian collection of industrial microorganisms (VKPM) under the number VKPM B-10042 .

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