Method for efficiently preparing VEGF protein

Abstract

The invention discloses a method for efficiently preparing VEGF (vascular endothelial growth factor) protein. Relates to the technical field of biotechnology. Comprising the following steps: transforming a pET-21a-VEGF recombinant plasmid into an escherichia coli BL21 competent cell, and culturing; vEGF induced expression is carried out; washing and dissolving the inclusion body protein; carrying out Ni-IMAC gradient renaturation and purification; purifying by anion exchange chromatography; performing complete renaturation; and finally purifying by molecular exclusion chromatography. According to the method, the correct folding rate of the VEGF is remarkably improved, meanwhile, protein aggregation is reduced, and the purity reaches 95% or above.

Description

Technical Field

[0001] The present invention relates to the technical field of biotechnology, and more particularly to a method for efficiently preparing VEGF protein. Background Art

[0002] Vascular endothelial growth factor (VEGF), a key regulator of angiogenesis, plays a central role in promoting angiogenesis, tissue repair, and tumor angiogenesis. It is widely used in cardiovascular disease treatment, wound healing acceleration, and anti-tumor drug development. The VEGF family includes VEGF-A, VEGF-B, VEGF-C, and VEGF-D. VEGF-A has multiple isoforms, generated through alternative splicing, such as VEGF-A121, VEGF-A165, and VEGF-A189, each with distinct structures and functions depending on tissue and context. Among these isoforms, VEGF-A165 has become a core target in clinical research and therapeutic development due to its highly effective angiogenic and tissue repair abilities. To date, VEGF-A165 has a relatively mature delivery technology and a wide range of clinical indications, including ischemic diseases, tumors, wound repair, and neurological disorders. It has demonstrated significant translational potential in gene therapy, regenerative medicine, and precision medicine, making it the VEGF isoform with the greatest clinical potential.

[0003] The present invention selects VEGF-A165 as the research object, and the "VEGF" referred to in the text specifically refers to the VEGF-A165 subtype.

[0004] Naturally derived VEGF protein has numerous limitations: First, VEGF expression levels in vivo are extremely low, making it difficult to obtain sufficient protein through direct extraction. Second, the isolation and purification of VEGF from animal tissues or body fluids is complex, involving multiple chromatographic purification steps, resulting in low yields (typically less than 0.1%) and high costs (each milligram of purified protein can cost thousands of dollars). These factors have severely hampered the widespread use of VEGF protein in clinical treatment and scientific research.

[0005] To solve the above problems, researchers turned to recombinant DNA technology to produce VEGF protein. In the existing technology, eukaryotic expression systems (such as Chinese hamster ovary cells CHO, human embryonic kidney cells HEK293, etc.) are given priority because of their ability to modify proteins after translation. However, the obvious defects of CHO cell protein production are: long cell culture cycle, low expression level, high culture medium cost, resulting in high overall production costs. In addition, eukaryotic expression systems also have technical difficulties such as poor expression stability and large batch-to-batch differences. In contrast, prokaryotic expression systems have shown significant technical advantages: first, the bacteria proliferate quickly and can be fermented at high density in cheap culture medium; second, the expression level is high and the fermentation cycle is short; third, the culture conditions are simple and no expensive additives are required, which greatly reduces production costs. These characteristics make the E. coli system the preferred platform for industrial production of recombinant proteins.

[0006] However, the expression of VEGF protein in E. coli faces severe technical challenges: the expression product mostly exists in the form of insoluble inclusion bodies (accounting for more than 80%), and traditional inclusion body renaturation methods (such as dilution renaturation and on-column renaturation) are not effective for proteins such as VEGF that contain multiple disulfide bonds, and the renaturation efficiency is usually less than 20%. What is more serious is that intermolecular mismatched disulfide bonds are easily formed during the renaturation process, leading to the formation of protein aggregates (which can account for 30-50% of the total protein amount), which not only reduces the yield but also may cause immunogenicity risks. Izumi Kumagai et al. tried to use gradient dialysis combined with a redox buffer system to improve the renaturation effect, but the final active protein yield was still less than 30%, and the process stability was poor. These problems make it difficult for existing technologies to meet the demand for high-activity and high-purity VEGF proteins in clinical applications.

[0007] Therefore, it is an urgent problem for those skilled in the art to provide a method for efficiently preparing VEGF protein and solve the key problems of low refolding efficiency and multiple aggregates in the prior art. Summary of the Invention

[0008] In light of this, the present invention provides a method for efficiently preparing VEGF protein. Through an optimized inclusion body refolding process, a properly folded active protein can be obtained. This method features high refolding efficiency and simple purification steps. It provides a reliable and economical source of VEGF protein for clinical treatments such as ischemic diseases and wound repair, thereby promoting the development of related biopharmaceutical industries.

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] A method for efficiently preparing VEGF protein comprises the following steps:

[0011] (1) The pET-21a-VEGF recombinant plasmid was transformed into Escherichia coli BL21 competent cells. After initial culture, the cells were plated on LB solid medium plates containing ampicillin and cultured until single colonies were formed.

[0012] (2) VEGF-induced expression: positive clones were picked and inoculated into 2YT medium containing ampicillin, and cultured with shaking until OD 600 When the pH reached 0.6, IPTG was added to induce protein expression;

[0013] (3) Washing and solubilization of inclusion body protein: The cells were collected by centrifugation, washed with PBS buffer, and then treated with resuspension buffer, washing buffer, and solubilization buffer in sequence. The supernatant was collected by centrifugation to obtain a crude VEGF protein extract;

[0014] (4) Ni-IMAC gradient refolding and purification: The crude VEGF protein extract was purified by nickel ion immobilized metal ion affinity chromatography, washed with gradient refolding buffer, eluted with imidazole, and concentrated by ultrafiltration to obtain a crude protein solution;

[0015] (5) Purification by anion exchange chromatography: The crude protein solution obtained in step (4) is further purified by anion exchange chromatography, linear gradient elution, and ultrafiltration concentration to obtain a purified crude product;

[0016] (6) Complete renaturation: The purified crude product obtained in step (5) is completely renatured, concentrated by ultrafiltration, and the liquid is replaced;

[0017] (7) Final purification by size exclusion chromatography: The product obtained in step (6) is purified by size exclusion chromatography, concentrated by ultrafiltration, and freeze-dried to obtain VEGF protein.

[0018] Preferably: Step (1) initial culture: culture in LB liquid medium at 37° C. and 220 rpm with shaking for 1 hour.

[0019] Preferably: the final concentration of IPTG in step (2) is 1 mM; induction expression: induction expression at 37° C. for 16 hours.

[0020] Preferably: step (3) resuspension buffer: 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM DTT;

[0021] Wash buffer: 50 mM Tris-HCl, 50 mM NaCl, 10 mM DTT;

[0022] Lysis buffer: 50 mM Tris-HCl, 50 mM NaCl, 8 M urea.

[0023] Preferably, step (4) is specifically as follows: mixing the crude VEGF protein extract with the pre-equilibrated Ni-NTA agarose resin, gently suspending and incubating at 4°C for 2 hours; transferring the above mixture to a 12 mL chromatography column, and washing with the following gradient renaturation buffer in sequence, using 5 column volumes for each step:

[0024] Refolding buffer includes:

[0025] Refolding buffer 7M: 50 mM Tris-HCl, 50 mM NaCl, 7 M urea;

[0026] Refolding buffer 6M: 50 mM Tris-HCl, 50 mM NaCl, 6 M urea;

[0027] Refolding buffer 5M: 50 mM Tris-HCl, 50 mM NaCl, 5 M urea;

[0028] Refolding buffer 4M: 50 mM Tris-HCl, 50 mM NaCl, 4 M urea;

[0029] The resin was washed three times with Ni wash buffer containing 25 mM imidazole, each time for 10 CV; eluted with Ni elution buffer containing 500 mM imidazole, and the eluted fractions were collected; the eluate was placed in an ultrafiltration centrifuge tube with a 10 kDa molecular weight cutoff, and centrifuged at 4°C and 4500 rpm to obtain a crude protein solution;

[0030] Ni elution buffer containing 500 mM imidazole: contains 500 mM imidazole, 50 mM Tris-HCl, 50 mM NaCl, and 4 M urea.

[0031] Preferably, step (5) is specifically as follows: using a low-salt equilibration buffer to fully equilibrate the anion exchange chromatography column at a flow rate of 0.5 mL / min until the ultraviolet absorption at 280 nm and the conductivity signal are stable; loading the crude protein solution onto the equilibrated anion exchange chromatography column at a flow rate of 0.5 mL / min; adopting a linear gradient elution program: transitioning from 100% low-salt equilibration buffer to 100% high-salt equilibration buffer within 1 hour, and collecting the target protein elution fraction according to the peak shape; loading the eluate into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and centrifuging and concentrating at 4°C and 4500 rpm to obtain a purified crude product;

[0032] The low-salt equilibration buffer was 50 mM Tris-HCl, 50 mM NaCl, and 4 M urea;

[0033] The high salt equilibration buffer was 50 mM Tris-HCl, 1 M NaCl, and 4 M urea.

[0034] Preferably, step (6) is specifically as follows: placing the purified crude product in an ice bath, adding dropwise to a dilution renaturation buffer pre-cooled at 4°C in advance, controlling the dilution ratio to be 1:200, and the final protein concentration to be 2 μM; in a 4°C environment, stirring at a rate of 100 rpm, continuing the renaturation for 7 days to obtain a protein solution; loading the protein solution into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuging and concentrating at 4°C, 4500 rpm, and exchanging the solution to a molecular exclusion buffer;

[0035] Dilution refolding buffer: 50 mM Tris-HCl, 1 M arginine, 1 mM EDTA, 1 mM reduced glutathione;

[0036] Exclusion buffer: 50 mM Tris-HCl, 150 mM NaCl.

[0037] Preferably, step (7) is specifically as follows: using a molecular exclusion buffer to fully equilibrate a 26 / 600 Finedex 75pg chromatography column at a flow rate of 2.6 mL / min until the UV absorption at 280 nm and the conductivity signal are stable; loading the product obtained in step (6) onto the equilibrated chromatography column at a flow rate of 1 mL / min; eluting at a constant flow rate of 2.6 mL / min; monitoring the changes in UV absorption at 280 nm and conductivity, and collecting the target protein elution fractions according to the peak shape; loading the eluate into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kD, and centrifuging and concentrating at 4°C and 4500 rpm; exchanging the solution with pure water, and freeze-drying the sample to obtain VEGF protein;

[0038] Exclusion buffer: 50 mM Tris-HCl, 150 mM NaCl.

[0039] It can be seen from the above technical solutions that, compared with the prior art, the present invention discloses a method for efficiently preparing VEGF protein, and the technical effects achieved are:

[0040] The invention significantly improves the correct folding rate of VEGF and reduces protein aggregation, with a purity of more than 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0042] Figure 1 The accompanying drawing is a schematic diagram of the VEGF purification process provided by the present invention.

[0043] Figure 2 The accompanying drawings are SDS-page electrophoresis results of the induced expression of VEGF provided by the present invention, wherein lane 1: pre-stained protein molecular weight standard (unit: kD); lane 2: uninduced Escherichia coli BL21 (DE3) whole cell lysate (negative control); lane 3: IPTG-induced Escherichia coli BL21 (DE3) / pET-21a-VEGF whole cell lysate.

[0044] Figure 3 The accompanying figure is an SDS-page electrophoresis result diagram of VEGF provided by the present invention by gradient renaturation and Ni-NTA affinity chromatography purification, wherein lane 1: pre-stained protein molecular weight standard (unit: kD); lane 2: Ni-NTA column 25mM imidazole wash fraction; lane 3: Ni-NTA column 500mM imidazole elution fraction.

[0045] Figure 4 The accompanying drawing is a diagram showing the SDS-page electrophoresis results of VEGF provided by the present invention after anion exchange chromatography Q columnization, wherein lane 1: pre-stained protein molecular weight standard (unit: kD); lane 2: VEGF after Q column purification).

[0046] Figure 5 The accompanying drawing is an analysis diagram of the renaturation effect of VEGF provided by the present invention under non-denaturing alkaline PAGE, wherein lane 1: natural alkaline protein molecular weight standard (unit: kD); lane 2: active VEGF protein (dimer) after arginine-assisted renaturation.

[0047] Figure 6 The accompanying drawing is a diagram showing the alkaline non-denaturing PAGE electrophoresis results of VEGF provided by the present invention after purification by a molecular sieve Finex column, wherein lane 1: natural alkaline protein molecular weight standard (unit: kD); lane 2: active VEGF protein after molecular sieve column purification. DETAILED DESCRIPTION

[0048] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0049] The embodiment of the present invention discloses a method for efficiently preparing VEGF protein.

[0050] All raw materials and reagents not mentioned in the examples are conventional, and all experimental methods not mentioned are conventional methods and will not be described in detail here. For example, the pET-21a-VEGF recombinant plasmid is an existing plasmid, as shown in Expression, purification and functionality of bioactive recombinant hμMan vascular endothelial growth factor VEGF (165) in E. coli. For the process flow chart, see Figure 1 .

[0051] Example 1

[0052] A method for preparing recombinant human vascular endothelial growth factor (VEGF) comprises the following steps:

[0053] (1) Take 1 μL of pET-21a-VEGF recombinant plasmid (the recombinant plasmid contains a 6His tag at the C-terminus) and add it to 50 μL of BL21 (DE3) competent cells. Incubate the mixture on ice for 30 minutes. Heat shock the mixture in a 42°C thermostated metal block for 90 seconds and cool it on ice for 2 minutes. Add 500 μL of LB liquid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl) and culture it at 37°C, 220 rpm, and shake for 1 hour. Take 100 μL of the bacterial solution and evenly spread it on a LB solid medium (10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, 15 g / L agar) plate containing 100 μg / mL ampicillin. Incubate it at 37°C for about 12 hours until a single colony is formed.

[0054] (2) VEGF-induced expression: A single positive clone was picked from the LB solid plate and inoculated into 5 mL of 2YT medium (17 g / L tryptone, 10 g / L yeast extract, 5 g / L NaCl) containing 100 μg / mL ampicillin. The culture was shaken at 37°C and 220 rpm for 12 h. 1 mL of the bacterial solution was inoculated into 1 L of 2YT medium with the same antibiotic concentration and shaken at 37°C and 220 rpm until the OD 600 The expression level reached about 0.6. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM and the expression was induced at 37°C for 16 hours.

[0055] like Figure 2As shown, after IPTG induction, SDS-PAGE analysis showed that the protein band near 22 kD (indicated by the arrow) in lane 3 was consistent with the theoretical molecular weight of VEGF, indicating that the VEGF protein was successfully expressed in the BL21 (DE3) system.

[0056] (3) Washing and solubilization of inclusion body proteins: The induced cells were collected by centrifugation at 6000 rpm for 15 minutes at 4°C and washed twice with pre-cooled PBS buffer (pH 7.4). Resuspended with resuspension buffer (50mM Tris-HCl (pH 8.0), 50mM NaCl, 1mM EDTA, 1% Triton X-100, 10mM DTT). Repeat the washing three times with washing buffer (50mM Tris-HCl (pH 8.0), 50mM NaCl, 10mM DTT). After each wash, centrifuge at 10000 rpm for 15 minutes at 4°C. The final precipitate was resuspended in solubilization buffer (50mM Tris-HCl (pH 8.0), 50mM NaCl, 8M urea) at a ratio of 1:5 (w / v) and dissolved at 4°C for 12 hours. Centrifuge at 10,000 rpm for 30 minutes at 4°C, collect the supernatant as the crude VEGF protein extract, and store at -80°C for later use or use directly in the next purification step.

[0057] (4) Ni-IMAC gradient refolding and purification: The crude VEGF protein extract (containing the His tag) was purified by nickel ion immobilized metal ion affinity chromatography (Ni-IMAC). Specifically, the crude VEGF protein extract was mixed with pre-equilibrated Ni-NTA agarose resin and gently suspended and incubated at 4°C for 2 h to ensure that the His tag was reattached to the Ni on the resin. 2+ The ions are fully chelated. The above mixture is transferred to a 12 mL chromatography column and washed with the following gradient renaturation buffer in sequence, using 5 column volumes (CV) for each step:

[0058] Refolding buffer 7M: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 7 M urea;

[0059] Refolding buffer 6M: 50mM Tris-HCl (pH 8.0), 50mM NaCl, 6M urea;

[0060] Refolding buffer 5M: 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 5 M urea;

[0061] Refolding buffer 4M: 50mM Tris-HCl (pH 8.0), 50mM NaCl, 4M urea;

[0062] Wash the resin three times with 10 CV each time using Ni wash buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 4 M urea) containing 25 mM imidazole. Elute with Ni elution buffer (50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 4 M urea) containing 500 mM imidazole, and collect the eluted fractions. Transfer the eluate to a 10 kDa molecular weight cutoff ultrafiltration tube and concentrate by centrifugation at 4500 rpm at 4°C to obtain a crude protein solution. Store at -80°C until use or for further purification.

[0063] like Figure 3 As shown, after gradient renaturation and Ni-NTA affinity chromatography purification, SDS-PAGE analysis showed that lane 3 showed a major obvious band at approximately 22 kD (indicated by the arrow), which was consistent with the theoretical molecular weight of VEGF, indicating that the Ni-IMAC gradient renaturation and purification of the present invention can effectively purify VEGF inclusion bodies.

[0064] (5) Anion exchange chromatography purification: Use low salt equilibration buffer (50mM Tris-HCl (PH 8.0), 50mM NaCl, 4Murea) to fully equilibrate the anion exchange chromatography column (Q column) at a flow rate of 0.5mL / min until the UV absorption (280nm) and conductivity signals are stable. The crude protein solution is loaded onto the equilibrated Q column at an optimized flow rate of 0.5mL / min, so that the target protein is adsorbed on the quaternary ammonium salt ligand through electrostatic interaction. A linear gradient elution program is used, transitioning from 100% low salt equilibration buffer to 100% high salt equilibration buffer (50mM Tris-HCl (PH 8.0), 1M NaCl, 4Murea) within 1h to dissociate impurities and target proteins with different binding strengths. Monitor the changes in UV absorption (280nm) and conductivity, and collect the target protein elution fractions according to the peak shape. The eluate was placed into an ultrafiltration centrifuge tube with a molecular weight cut-off of 10 kDa, and concentrated by centrifugation at 4°C and 4500 rpm to obtain a purified crude product, which was stored at -80°C for later use or for further purification.

[0065] like Figure 4 As shown, after purification by anion exchange chromatography, SDS-PAGE analysis showed that lane 2 presented a single main band at approximately 22 kD (indicated by the arrow), which was consistent with the theoretical molecular weight of VEGF, indicating that anion exchange chromatography in the present invention can effectively purify VEGF inclusion bodies.

[0066] (6) Complete renaturation: Place the above-mentioned purified crude product in an ice bath and add dropwise to the dilution renaturation buffer (50mM Tris-HCl (PH8.0), 1M arginine, 1mM EDTA, 1mM reduced glutathione) pre-cooled at 4°C. Control the dilution ratio to 1:200 and the final protein concentration to 2μM. Use a low-shear magnetic stirrer at 4°C and set the stirring rate to 100rpm. Continue renaturation for 7 days to obtain a protein solution. The protein solution is placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kDa, centrifuged and concentrated at 4°C, 4500rpm, and exchanged into molecular exclusion buffer (50mM Tris-HCl (PH 8.0), 150mM NaCl). Store at -80°C for later use or for further purification.

[0067] like Figure 5 As shown, non-denaturing alkaline PAGE analysis showed that the protein sample after gradient renaturation presented a single main band at approximately 44 kD (indicated by the arrow), which was consistent with the theoretical molecular weight of VEGF protein, indicating that the renaturation process successfully obtained the correctly folded active protein (dimer).

[0068] (7) Final purification by size exclusion chromatography: Use size exclusion buffer (50mM Tris-HCl (pH8.0), 150mM NaCl) to fully equilibrate a 26 / 600 Finedex 75pg chromatography column at a flow rate of 2.6mL / min until the UV absorption (280nm) and conductivity signals are stable. Load the product obtained in step (6) onto the equilibrated chromatography column at an optimized flow rate (1mL / min). Elution is performed at a constant flow rate of 2.6mL / min. Monitor the changes in UV absorption (280nm) and conductivity, and collect the target protein elution fractions according to the peak shape. The eluate is placed in an ultrafiltration centrifuge tube with a molecular weight cutoff of 10kD and concentrated by centrifugation at 4°C and 4500rpm. Replace the solution with pure water, freeze-dry the sample, and obtain recombinant human vascular endothelial growth factor (VEGF) and store it at -80°C.

[0069] like Figure 6 As shown, non-denaturing alkaline PAGE analysis showed that the protein sample after molecular sieve purification showed a single uniform band at about 44kD, with a purity of more than 95% (Coomassie Brilliant Blue staining) and maintained its native conformation, proving that this purification method can obtain high-purity VEGF protein.

[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0071] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for efficiently preparing VEGF protein, characterized in that: The following steps are involved: (1) The pET-21a-VEGF recombinant plasmid was transformed into Escherichia coli BL21 competent cells. After initial culture, the cells were plated on LB solid medium containing ampicillin and cultured until single colonies were formed. (2) VEGF induced expression: positive clones were picked and inoculated into 2YT medium containing ampicillin and cultured with shaking until OD 600 When the pH reached 0.6, IPTG was added to induce protein expression; (3) Washing and solubilization of inclusion body protein: The cells were collected by centrifugation, washed with PBS buffer, and then treated with resuspension buffer, washing buffer, and solubilization buffer in sequence. The supernatant was collected by centrifugation to obtain a crude VEGF protein extract; (4) Ni-IMAC gradient refolding and purification: The crude VEGF protein extract was purified by nickel ion immobilized metal ion affinity chromatography, washed with gradient refolding buffer, eluted with imidazole, and concentrated by ultrafiltration to obtain a crude protein solution; (5) Purification by anion exchange chromatography: The crude protein solution obtained in step (4) is further purified by anion exchange chromatography, linear gradient elution, and ultrafiltration concentration to obtain a purified crude product; (6) Complete renaturation: The purified crude product obtained in step (5) is completely renatured, concentrated by ultrafiltration, and the liquid is replaced; (7) Final purification by size exclusion chromatography: The product obtained in step (6) is purified by size exclusion chromatography, concentrated by ultrafiltration, and freeze-dried to obtain VEGF protein.

2. The method for efficiently preparing VEGF protein according to claim 1, wherein The initial culture in step (1) was carried out in LB liquid medium at 37° C. and 220 rpm with shaking for 1 hour.

3. The method for efficiently preparing VEGF protein as claimed in claim 2, wherein The final concentration of IPTG in step (2) is 1 mM; induction expression: induction expression at 37° C. for 16 hours.

4. The method for efficiently preparing VEGF protein according to claim 3, wherein The resuspension buffer in step (3) includes 50 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 10 mM DTT. Wash buffer: 50 mM Tris-HCl, 50 mM NaCl, 10 mM DTT; Lysis buffer: 50 mM Tris-HCl, 50 mM NaCl, 8 M urea.

5. The method for efficiently preparing VEGF protein according to claim 4, wherein Step (4) is specifically as follows: the crude VEGF protein extract is mixed with the pre-equilibrated Ni-NTA agarose resin, and the mixture is gently suspended and incubated at 4°C for 2 hours; the mixture is transferred to a 12 mL chromatography column, and washed in sequence with the following gradient renaturation buffer, using 5 column volumes for each step: The renaturation buffer comprises: Refolding buffer 7M: 50 mM Tris-HCl, 50 mM NaCl, 7 M urea; Refolding buffer 6M: 50 mM Tris-HCl, 50 mM NaCl, 6 M urea; Refolding buffer 5M: 50 mM Tris-HCl, 50 mM NaCl, 5 M urea; Refolding buffer 4M: 50 mM Tris-HCl, 50 mM NaCl, 4 M urea; The resin was washed three times with Ni wash buffer containing 25 mM imidazole, each time for 10 CV; eluted with Ni elution buffer containing 500 mM imidazole, and the eluted fractions were collected; the eluate was placed in an ultrafiltration centrifuge tube with a 10 kDa molecular weight cutoff, and centrifuged at 4°C and 4500 rpm to obtain a crude protein solution; The Ni elution buffer containing 500 mM imidazole contains: 500 mM imidazole, 50 mM Tris-HCl, 50 mM NaCl, and 4 M urea.

6. The method for efficiently preparing VEGF protein according to claim 5, wherein Step (5) is specifically as follows: using a low-salt equilibration buffer to fully equilibrate the anion exchange chromatography column at a flow rate of 0.5 mL / min until the ultraviolet absorption at 280 nm and the conductivity signal are stable; loading the crude protein solution onto the equilibrated anion exchange chromatography column at a flow rate of 0.5 mL / min; using a linear gradient elution program: transitioning from 100% low-salt equilibration buffer to 100% high-salt equilibration buffer within 1 hour, and collecting the target protein elution fraction according to the peak shape; loading the eluate into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, and centrifuging and concentrating at 4°C and 4500 rpm to obtain a purified crude product; The low salt equilibration buffer is 50 mM Tris-HCl, 50 mM NaCl, and 4 M urea; The high salt equilibration buffer was 50 mM Tris-HCl, 1 M NaCl, and 4 M urea.

7. The method for efficiently preparing VEGF protein according to claim 6, wherein Step (6) is specifically as follows: placing the purified crude product in an ice bath, adding dropwise to a dilution renaturation buffer pre-cooled at 4°C, controlling the dilution ratio to be 1:200, and the final protein concentration to be 2 μM; in a 4°C environment, stirring at a rate of 100 rpm, continuing the renaturation for 7 days to obtain a protein solution; loading the protein solution into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kDa, centrifuging and concentrating at 4°C, 4500 rpm, and exchanging the solution to a molecular exclusion buffer; The dilution and renaturation buffer: 50 mM Tris-HCl, 1 M arginine, 1 mM EDTA, 1 mM reduced glutathione; The molecular exclusion buffer: 50 mM Tris-HCl, 150 mM NaCl.

8. The method for efficiently preparing VEGF protein according to claim 7, wherein Step (7) is specifically as follows: using molecular exclusion buffer to fully equilibrate a 26 / 600 Finedex 75pg chromatography column at a flow rate of 2.6 mL / min until the UV absorption at 280 nm and the conductivity signal are stable; loading the product obtained in step (6) onto the equilibrated chromatography column at a flow rate of 1 mL / min; eluting at a constant flow rate of 2.6 mL / min; monitoring the changes in UV absorption at 280 nm and conductivity, and collecting the target protein elution fractions according to the peak shape; loading the eluate into an ultrafiltration centrifuge tube with a molecular weight cutoff of 10 kD, and centrifuging and concentrating at 4°C and 4500 rpm; exchanging the solution with pure water, and freeze-drying the sample to obtain VEGF protein; The molecular exclusion buffer: 50 mM Tris-HCl, 150 mM NaCl.

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