Preparation method and application of chaperonin-assisted recombinant human hFGF-2 protein

Abstract

The invention belongs to the technical field of globular protein synthesis, and particularly relates to a chaperonin-assisted recombinant human hFGF-2 protein preparation method and application thereof, the chaperonin-assisted recombinant human hFGF-2 protein preparation method comprises the following steps: S1, constructing a recombinant vector TrxA-6H-DDDDK-hFGF2 containing an hFGF-2 fusion protein coding sequence; s2, transforming the recombinant vector into an escherichia coli host cell for induced expression to obtain an expression thallus; s3, crushing the expression thalli to obtain a cell lysis supernatant; s4, taking the supernate, carrying out nickel column affinity chromatography, carrying out desalination treatment on the fusion protein by adopting gel chromatography, and collecting a fusion protein eluent; s5, performing enterokinase cutting treatment on the fusion protein to obtain an enzyme-digested product; s6, performing heparin affinity chromatography on the enzyme digestion product, and collecting hFGF-2 protein eluent; and S7, carrying out dialysis treatment on the protein eluent to obtain the recombinant hFGF-2 protein.

Description

Technical Field

[0001] This invention belongs to the field of globular protein synthesis technology, specifically relating to a method for preparing recombinant human hFGF-2 protein based on chaperone protein assistance and its application. Background Technology

[0002] Basic fibroblast growth factor (bFGF), also known as FGF-2 (fibroblast growth factor 2), is a member of the fibroblast growth factor (FGF) family. This family acts as a mitotic promoter to accelerate cell proliferation and plays a crucial role in embryonic development, maintenance of adult organ systems, tissue regeneration, wound repair, and hematopoiesis. hFGF-2 is a typical and most studied member of the FGF family and a key component of human embryonic stem cell culture media. Multiple studies have shown that hFGF-2 is an important regulator of cell growth and differentiation under physiological and pathological conditions, inducing angiogenesis and lymphatic vessel growth through the VEGF-C / VEGF-D / VEGFR-3 signaling axis. Furthermore, hFGF-2 participates in bone development, regulating chondrocyte differentiation and bone formation: it is stored in the extracellular matrix (ECM) of articular cartilage by binding to heparan sulfate proteoglycans; upon cartilage injury or degeneration, the ECM rapidly releases hFGF-2, thereby activating the ERK signaling pathway to drive cartilage regeneration. Furthermore, hFGF-2 is also a heparin-binding cationic protein involved in various pathological conditions, including angiogenesis and solid tumor growth. Therefore, hFGF-2 is considered a key component of cancer chemoprevention and treatment strategies.

[0003] hFGF-2 is a globular protein composed of a single-chain polypeptide with a molecular weight of 18 kDa, containing four cysteine ​​residues in the form of free sulfhydryl groups. Initially, FGF-2 was mainly extracted and purified from animal tissues and organs, but its extremely low content made it very expensive. With the cloning and characterization of the human gene-encoded FGF-2, heterologous expression became the main source of commercial hFGF-2. However, the FGF-2 protein obtained by traditional preparation methods exists in the form of inclusion bodies. Although denaturation and in vitro refolding processes can form complete hFGF-2 protein, there are obvious drawbacks: the process is complex, the process is lengthy, and the product contains a large number of non-hFGF-2 components, resulting in low purity and extremely low yield.

[0004] Therefore, overcoming the deficiency of insufficient purity of hFGF-2 in traditional preparation processes is a technical problem that urgently needs to be solved in this field.

[0005] It should be noted that the information disclosed in this background section is only for understanding the background technology of the present application concept, and therefore, the above description is not considered to constitute prior art information. Summary of the Invention

[0006] This disclosure provides at least one method for preparing recombinant human hFGF-2 protein based on chaperone protein-assisted folding and its application.

[0007] In a first aspect, embodiments of this disclosure provide a method for preparing recombinant human hFGF-2 protein based on chaperone protein assistance, comprising the following steps: S1, constructing a recombinant vector TrxA-6H-DDDDK-hFGF2 containing the encoding sequence of the hFGF-2 fusion protein; S2, transforming the recombinant vector into Escherichia coli host cells to induce expression, obtaining expression cells; S3, lysing the expression cells to obtain cell lysis supernatant; S4, taking the supernatant for nickel column affinity chromatography, and using gel chromatography to desalt the fusion protein, collecting the fusion protein eluent; S5, cleaving the fusion protein with enterokinase to obtain the enzyme digestion product; S6, performing heparin affinity chromatography on the enzyme digestion product, collecting the hFGF-2 protein eluent; S7, dialysis the protein eluent to obtain recombinant hFGF-2 protein.

[0008] In one optional embodiment, the amino acid sequence of the recombinant vector TrxA-6H-DDDDK-hFGF2 fusion protein is SEQ ID NO:2, the host cell is preferably ArcticExpress host cell, and the hFGF-2 nucleic acid sequence optimized for the ArcticExpress codon preference of the expression host is SEQ ID NO:3, and the TrxA-6H-DDDDK-hFGF2 nucleic acid sequence is SEQ ID NO:4.

[0009] In one optional embodiment, the recombinant vector includes the pET series, pGEX series, and pMAL series; the expression host includes ArcticExpress and BL21; and the fusion protein tag includes thioredoxin, glutathione S-transferase, maltose-binding protein, small ubiquitin protein modifier, and transcription anti-termination factor.

[0010] In an optional embodiment, the induction conditions in step S2 include: when the bacterial culture OD600 reaches 1.2-1.3, adding IPTG to make the final concentration 0.2-1.0 mM, and culturing at 16°C with shaking for 14-18 hours.

[0011] In one optional embodiment, the nickel column affinity chromatography includes: i. washing with Buffer A containing 10 mM imidazole for 1 CV; ii. washing with Buffer B containing 30 mM imidazole for 5 CV; iii. washing with Buffer C containing 110 mM imidazole for 12 CV; iv. eluting the target protein with Buffer D containing 250 mM imidazole; v. collecting the protein-containing eluent, adding Buffer F to bring the final EDTA concentration to 1-5 mM, and desalting the fusion protein using gel chromatography.

[0012] In one alternative implementation, the Buffer AD is based on 1×PBS buffer, pH 8.0, and contains 300 mM NaCl.

[0013] In one optional embodiment, the desalting treatment of the fusion protein includes: i. using a 300 mL dextran gel G-25 column, the dextran gel G-25 being pre-swollen with pure water for 24 h; ii. using wet packing, pre-equilibrating the packed gel column with 5 CV Buffer E; iii. loading the fusion protein onto the column and rinsing it with Buffer E, collecting and combining the protein-containing eluent; iv. adding recombinant enterokinase to the eluent at an enzyme-to-substrate mass ratio of 1:10000, and incubating at 26 °C for 16 h.

[0014] In one alternative embodiment, the equilibration buffer E is 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.8.

[0015] In one optional embodiment, the heparin affinity chromatography includes: i. using 50 mM...

[0016] ii. Equilibrate the chromatography column with Tris-HCl, 50 mM NaCl, and Buffer G, pH 7.8;

[0017] iii. Perform a 0-70% linear gradient elution with Tris-HCl, 2M NaCl, and Buffer H at pH 7.8; iii. Rinse with 70% Buffer H for 4-5 CV, dialyze to desalt in pre-cooled PBS buffer, and collect the target protein.

[0018] Secondly, embodiments of this disclosure also provide the application of chaperone-assisted recombinant human hFGF-2 protein in the preparation of cell culture medium additives and drugs that promote tissue regeneration.

[0019] The beneficial effects of this invention are that the method for preparing recombinant human hFGF-2 protein based on chaperone protein-assisted folding achieves large-scale preparation of high-purity target protein through the synergistic implementation of fusion expression and dual-affinity chromatography. Specifically, hFGF-2 is constructed into a TrxA-6H-DDDDK-hFGF2 fusion form, and the fusion protein is efficiently expressed in the supernatant in a soluble form using a low-temperature expression system. The purification process employs a three-step process: nickel column elution, enterokinase site-specific cleavage, and heparin chromatography linear elution. Finally, natural N-terminal hFGF-2 is obtained by dialysis, and the purity reaches 99.44% as verified by RP-HPLC. CCK-8 cell experiments confirmed that the proliferative activity of the obtained hFGF-2 on L929 fibroblasts is essentially no different from that of commercially available recombinant hFGF-2 (PeproTech#100-18B). Compared to traditional inclusion body extraction processes, this invention avoids the refolding step through fully soluble expression, significantly reducing the risk of activity loss and providing a highly efficient preparation route with both high purity and high activity for clinical applications.

[0020] Other features and advantages of the invention will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the invention. The objects and other advantages of the invention are realized and obtained through the structures particularly pointed out in the description and the drawings.

[0021] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, preferred embodiments are described below in detail with reference to the accompanying drawings. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 A graph showing the effect of nickel affinity chromatography purification of the fusion protein provided in the embodiments of this disclosure;

[0024] Figure 2 A diagram illustrating the enterokinase-specific cleavage of fusion proteins provided in embodiments of this disclosure;

[0025] Figure 3 Heparin affinity chromatography chromatograms provided in embodiments of this disclosure;

[0026] Figure 4 Electrophoresis image of purified hFGF-2 protein provided in the embodiments of this disclosure;

[0027] Figure 5 A chromatogram showing the RP-HPLC method for detecting hFGF-2 purity provided in this embodiment of the disclosure;

[0028] Figure 6 This is an hFGF-2 activity test diagram provided in an embodiment of this disclosure. Detailed Implementation

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0030] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0031] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1-20 should be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0032] As used herein, the term "comprising" or "including" means "including, but not limited to." This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "substantially consisting of." In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of."

[0033] As used in this article, "elution" refers to the desorption of molecules bound to the stationary phase by altering the solution conditions within the chromatographic column. This can be achieved by increasing the concentration of exchangeable counterions or by changing the pH to affect the binding affinity of the analyte. Molecules that lose their affinity for the stationary phase and enter the mobile phase are then "eluted" from the column.

[0034] As used herein, "buffer" refers to a reagent, typically a solution, used to alter the adsorption of an analyte (e.g., hFGF-2 protein) onto a stationary phase and / or to remove unbound material from the stationary phase. The elution properties of an eluent can depend on factors such as pH, ionic strength, and detergent strength.

[0035] As used herein, "elution buffer" refers to a solution (e.g., a wash solution or buffer solution) containing unbound material (including "eluted" or desorbed analyte molecules, such as hFGF-2 protein) that travels through the stationary phase and exits the column during chromatographic separation. As used herein, "an" and "a" are used to refer to one or more grammatical objects.

[0036] As used in this article, "high purity" means a purity of ≥99.0% (peak area percentage) as detected by RP-HPLC, and "virtually no difference" means a difference of ≤10% in proliferation activity as detected by CCK-8 method.

[0037] As used herein, the term "pH" is a numerical value indicating the degree of acidity or alkalinity of a solution and is an indicator of hydrogen ion concentration. Within the pH range of 0 to 14, a solution with a pH of 7 is neutral, a solution with a pH less than 7 is acidic, and a solution with a pH greater than 7 is alkaline. pH can be measured using a pH meter, and the pH of buffer solutions can be adjusted using acids or bases such as HCl or NaOH.

[0038] As used herein, the term "purification" refers to the operation of increasing purity by removing coexisting impurities from a substance, and in this specification, purification refers to the isolation of hFGF-2 protein from a culture of E. coli that is produced when overgrown hFGF-2 protein dies, and also refers to the process of increasing purity during the production of hFGF-2 protein.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. All reagents or instruments without a specified manufacturer are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0040] This disclosure provides a method for preparing recombinant human hFGF-2 protein based on chaperone protein assistance, comprising the following steps: S1, constructing a recombinant vector TrxA-6H-DDDDK-hFGF2 containing the encoding sequence of the hFGF-2 fusion protein; S2, transforming the recombinant vector into Escherichia coli host cells to induce expression, obtaining expression cells; S3, lysing the expression cells to obtain cell lysis supernatant; S4, performing nickel column affinity chromatography on the supernatant, and desalting the fusion protein using gel chromatography, collecting the fusion protein eluent; S5, cleaving the fusion protein with enterokinase to obtain the enzyme digestion product; S6, performing heparin affinity chromatography on the enzyme digestion product, collecting the hFGF-2 protein eluent; S7, dialysis the protein eluent to obtain recombinant hFGF-2 protein.

[0041] Specifically, step S1 includes (1) constructing a nucleic acid molecule encoding the recombinant basic fibroblast growth factor hFGF-2; and (2) constructing a vector containing the nucleic acid molecule.

[0042] Specifically, the target gene was designed and synthesized as follows: Based on the amino acid sequence of the hFGF-2 protein (SEQ ID NO:1), the nucleotide sequence of the target gene was designed and optimized according to the codons preferred by *E. coli* to determine the protein's nucleotide sequence. The sequence was synthesized and then constructed into the C-terminus of the enterokinase cleavage site of the prokaryotic expression plasmid pET32a using homologous recombination, ultimately forming the fusion protein TrxA-6H-DDDDK-hFGF2 (SEQ ID NO:2) in an open reading frame.

[0043] In some embodiments, specifically, the carrier in step 1 includes, but is not limited to, the pET series, pGEX series, and pMAL series.

[0044] In some embodiments, specifically, the expression host mentioned in step 1 includes, but is not limited to, ArcticExpress (DE3) and BL21 (DE3).

[0045] In some embodiments, specifically, the fusion protein tag in step 1 includes, but is not limited to, thioredoxin (TrxA), glutathione S-transferases (GSTs), maltose-binding protein (MBP), small ubiquitin protein modifier (SUMO), and transcription anti-termination factor (NusA).

[0046] Specifically, step S2 includes (1) converting the nucleic acid vector into a suitable expression host; and (2) culturing the host cells to allow or induce the host cells to express hFGF-2 encoded by the vector in vivo.

[0047] Specifically, the vector constructed in step S1 was transformed into the expression host using a heat shock method. A single transformant was picked and inoculated into LB medium, and Amp antibiotic was added. The culture was then shaken overnight to obtain the seed culture. Subsequently, the seed culture was inoculated into Good LB medium at a ratio of 1:100 (V:V) and shaken. The OD600 value of the bacterial culture was measured by spectrophotometry every 2 hours. When the OD600 of the bacterial culture obtained in step S2 reached 1.2-1.3, a final concentration of 0.5 mM IPTG was added to the bacterial culture to induce expression, and then the culture was continued at 16°C with shaking.

[0048] In some embodiments, specifically, the host cell of step S2 includes: an Escherichia coli host capable of fermenting and expressing hFGF-2, preferably Escherichia coli Arctic Express (DE3).

[0049] In some embodiments, specifically, step S3 includes: lysing cells expressing hFGF-2 to obtain cell lysates containing hFGF-2; and purifying and enriching the hFGF-2 protein in the lysates using a multiple affinity chromatography column.

[0050] Specifically, the induced bacterial suspension described in step S2 was centrifuged at low temperature, the supernatant was discarded, and the bacterial cells were resuspended in Buffer A. The suspension was then centrifuged again at low temperature, the supernatant was discarded, and the bacterial cells were resuspended in a small amount of Buffer A. The suspension was pre-cooled using a high-pressure homogenizer, and the suspension was broken up several times until the suspension was clear. The lysate was collected, centrifuged at low temperature and high speed, and the supernatant was collected and placed on ice for later use. The hFGF-2 protein in the lysate was purified and enriched sequentially using nickel column affinity chromatography and heparin affinity chromatography, as follows:

[0051] After equilibration with Buffer A, the nickel column was loaded with the sample. The sample bound to the packing material for 10 min, followed by washing with Buffers A, B, and C sequentially (1 / 5 / 12 CV), and elution with Buffer D. The protein eluent was collected, and Buffer F (final EDTA concentration 2 mM) was added. The eluent was then concentrated by ultrafiltration (molecular weight cutoff 5 kD). The G-25 gel column was swollen in pure water for 24 h, equilibrated with Buffer E, and then loaded with the sample for desalting. The protein eluent was collected, and enterokinase (1:10000 V:V) was added. The column was digested at 26°C for 16 h.

[0052] After equilibration with Buffer G, the heparin column was loaded with the sample and eluted with a gradient of Buffer H (0-70% linear gradient, maintain 70% wash for 4-5 CV). The protein eluent was collected and dialyzed with PBS to obtain native N-terminal hFGF-2.

[0053] The multiple chromatography columns used in step S3 are, in order, a nickel affinity chromatography column with NTA as the ligand type for chelating nickel ions; a dextran G-25 gel chromatography column; and a heparin affinity chromatography column.

[0054] Step S3 further includes: bioactivity assessment.

[0055] Specifically, the bioactivity of hFGF-2 was detected using the CCK-8 assay: L929 cells were seeded at 4000 cells / well in 96-well plates, treated with hFGF-2 for 48 hours after 24 hours, and then the cells were washed and the absorbance at 450 nm was measured using the CCK-8 assay to assess cell proliferation.

[0056] This disclosure also provides the application of chaperone-assisted recombinant human hFGF-2 protein in the preparation of cell culture medium additives and drugs that promote tissue regeneration.

[0057] Example 1: hFGF-2 sequence feature analysis and vector construction

[0058] The nucleotide sequence described in SEQ ID NO:3 (synthesized by GenScript Biotech) was cloned into the C-terminus of the pET32a enterokinase cleavage site via homologous recombination, ultimately forming the fusion protein TrxA-6H-DDDDK-hFGF2 in an open reading frame, thus obtaining the recombinant plasmid. The constructed vector was transformed into the prokaryotic expression strain ArcticExpress(DE3) using the heat shock method. After sequencing verification, it was stored for later use.

[0059] The recombinant plasmid was transformed into host cells, and after selection with ampicillin (Amp), single-clonal recombinant cells were obtained and expanded in LB medium to obtain seed cells. A single transformant was picked and inoculated into LB medium, and Amp antibiotic was added to a concentration of 100 μg / mL. The mixture was incubated overnight at 37°C with shaking at 200 rpm to obtain the seed culture.

[0060] Example 2: Induced expression of hFGF-2

[0061] The seed culture was inoculated into Good LB medium at a ratio of 1:100 (V:V) (the total volume of the medium should not exceed 1 / 4 of the volumetric flask). The culture was shaken at 37°C and 220 rpm. The OD600 value of the bacterial culture was measured by spectrophotometry every 2 hours. When the OD600 of the bacterial culture reached 1.2-1.3, 0.2-1.0 mM IPTG was added to the bacterial culture. The culture was then continued at 16°C and 150 rpm for 14-18 hours.

[0062] Example 3: Extraction and Purification of hFGF-2

[0063] Experiment 1: Acquisition of soluble supernatant

[0064] Take the induced bacterial suspension and centrifuge at 17700g, 4℃ for 8 min. Discard the supernatant and resuspend the bacterial cells in 1 volume of Buffer A. Centrifuge again at 17700g, 4℃ for 8 min, discard the supernatant, and resuspend the bacterial cells in 0.2-0.3 volumes of Buffer A. Pre-cool the suspension using a high-pressure homogenizer. Disrupt the bacterial suspension at 900-1000 bar for 3-5 min until the suspension is clear. Collect the disrupted liquid and centrifuge at 25000g, 4℃ for 60 min. Collect the supernatant and store it on ice for later use.

[0065] Experiment 2: Nickel column affinity chromatography

[0066] The nickel column (NTA) was pre-equilibrated with Buffer A. The supernatant was loaded into the nickel column at a low flow rate (ensuring a binding time between the supernatant and the packing material of at least 10 min). The column was washed with Buffer A for 1 CV, Buffer B for 5 CV, and Buffer C for 12 CV. Elution was then performed with Buffer D. The eluent containing protein was collected and combined, and Buffer F was rapidly added to bring the final EDTA concentration to 1-

[0067] The protein obtained after elution with a nickel column at 5 mM is as follows: Figure 1 The fusion protein was desalted using gel chromatography, as follows: G-25 dextran gel was pre-swollen in pure water for 24 hours, packed using a wet packing method, and pre-equilibrated with 5 CV Buffer E. After loading the fusion protein, the column was washed with Buffer E, and the protein-containing eluent was collected and combined. The total amount of desalted fusion protein was calculated using the Coomassie Brilliant Blue method. A certain proportion (W:W = 1:10000) of recombinant enterokinase was added, and the mixture was incubated at 26°C for 16 hours. The cleaved protein mixture is shown below. Figure 2 .

[0068] Experiment 3: Heparin Affinity Chromatography

[0069] The cleaved protein was further purified using the SCG-P protein purification system. Specifically, the heparin chromatography column was pre-equilibrated with Buffer G at 10 CV. After sample loading, Buffer H was increased to 70% at a linear flow rate. The rinsing was continued for 4-5 CV while maintaining the Buffer H ratio. The eluent containing protein was collected and combined, and then dialyzed to desalt in pre-chilled PBS buffer. Figure 3 As shown, hFGF-2 with a natural N-terminus was finally obtained. The SDS-PAGE electrophoresis results of the sample are as follows. Figure 4 .

[0070] Implementation Case 4: Purity Testing

[0071] The purity of hFGF-2 was determined by RP-HPLC. The chromatographic column was PLRP-S (150 × 4.6 μm, Agilent). The mobile phase was deionized water containing 0.1% TFA and acetonitrile. The flow rate was 0.5 mL / min, and the detection wavelength was 280 nm. Figure 5 As shown, after integration, the purity of hFGF-2 is 99.44%.

[0072] Implementation Case 5: Bioactivity Assessment

[0073] Using CCK-8(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid)

[0074] The bioactivity of hFGF-2 was tested using (benzene)-2H-tetrazole monosodium salt. Mouse fibroblast L929 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. Cells were seeded at 4000 cells per well in 96-well plates. The next day, after washing the cells with DPBS, hFGF-2 was added and the cells were cultured for another 48 hours. Cells were washed again, and cell viability was measured at 450 nm using a CCK-8 cell proliferation assay kit (Beyotime). Figure 6 As shown, the recombinant hFGF-2 obtained by this process can promote the proliferation of L929 cells, and its ability to promote mitosis is basically the same as that of commercially available samples.

[0075] In the above embodiments, specifically, SEQ ID NO:1 is the amino acid sequence of hFGF-2.

[0076] SEQ ID NO:2 is the amino acid sequence of the fusion protein (TrxA-6H-DDDDK-hFGF2) after adding the chaperone protein TrxA and the enterokinase cleavage sequence to the N-terminus of hFGF-2. SEQ ID NO:3 is the hFGF-2 nucleic acid sequence optimized for E. coli codon preference. SEQ ID NO:4 is the TrxA-6H-DDDDK-hFGF2 nucleic acid sequence optimized for E. coli codon preference. The underlined parts in the sequences represent the enterokinase cleavage motif.

[0077] Specifically, the culture medium and buffer formulations involved in the above embodiments are as follows:

[0078] LB medium: 10g NaCl, 5g yeast extract, 10g tryptone, diluted with pure water to 1L, adjust pH to 7.5, sterilize at 121℃ and use for later use;

[0079] Good LB medium: 10g NaCl, 24g yeast extract, 12g tryptone, 2.31g KH2PO4, 12.54g K2HPO4, 4g glycerol, diluted with pure water to 1L, adjust pH to 7.5, sterilize at 121℃ and use for later use;

[0080] Buffer A: 1×PBS buffer, 300mM NaCl, 10mM imidazole, pH 8.0;

[0081] Buffer B: 1×PBS buffer, 300mM NaCl, 30mM imidazole, pH 8.0;

[0082] Buffer C: 1×PBS buffer, 300mM NaCl, 110mM imidazole, pH 8.0;

[0083] Buffer D: 1×PBS buffer, 300mM NaCl, 250mM imidazole, pH 8.0;

[0084] Buffer E: 50mM Tris-HCl, 150mM NaCl, 0.05% Tween 20, pH 7.8;

[0085] Buffer F: 200mM EDTA, pH 7.8;

[0086] Buffer G: 50mM Tris-HCl, 50mM NaCl, pH 7.8;

[0087] Buffer H: 50mM Tris-HCl, 2M NaCl, pH 7.8;

[0088] Buffer I: 1×PBS buffer, pH 7.4.

[0089] In summary, this method for preparing recombinant human hFGF-2 protein based on chaperone protein-assisted folding achieves large-scale preparation of high-purity target protein through the synergistic implementation of fusion expression and dual-affinity chromatography. Specifically, hFGF-2 was constructed into a TrxA-6H-DDDDK-hFGF2 fusion form, and the fusion protein was efficiently expressed in soluble form in the supernatant using a low-temperature expression system. The purification process employed a three-step process: nickel column elution, enterokinase site-specific cleavage, and heparin chromatography linear elution. Finally, dialysis yielded native N-terminal hFGF-2, with a purity of 99.44% verified by RP-HPLC. CCK-8 cell experiments confirmed that the proliferative activity of the obtained hFGF-2 on L929 fibroblasts was essentially no different from that of commercially available recombinant hFGF-2 (PeproTech#100-18B). Compared to traditional inclusion body extraction processes, this invention avoids the refolding step through fully soluble expression, significantly reducing the risk of activity loss and providing a highly efficient preparation route with both high purity and high activity for clinical applications.

[0090] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification, but must be determined according to the scope of the claims.

Claims

1. A method for preparing recombinant human hFGF-2 protein based on chaperone protein assistance, characterized in that, Includes the following steps: S1, construct the recombinant vector TrxA-6H-DDDDK-hFGF2 containing the encoding sequence of the hFGF-2 fusion protein; S2, the recombinant vector is transformed into E. coli host cells and induced to express the expression cells; S3, lyse the expression cells to obtain cell lysis supernatant; S4, take the supernatant for nickel column affinity chromatography, and use gel chromatography to desalt the fusion protein, and collect the fusion protein eluent; S5, the fusion protein is cleaved by enterokinase to obtain the enzyme digestion product; S6, the enzyme digestion product was subjected to heparin affinity chromatography, and the hFGF-2 protein eluent was collected; S7. The protein eluent was dialyzed to obtain recombinant hFGF-2 protein.

2. The protein preparation method according to claim 1, characterized in that, The amino acid sequence of the recombinant vector TrxA-6H-DDDDK-hFGF2 fusion protein is SEQ ID NO:

2. The host cell is preferably ArcticExpress host cell, and the hFGF-2 nucleic acid sequence optimized for the codon preference of the ArcticExpress expression host is SEQ ID NO:3, and the TrxA-6H-DDDDK-hFGF2 nucleic acid sequence is SEQ ID NO:

4.

3. The protein preparation method according to claim 1, characterized in that, The recombinant vectors include the pET series, pGEX series, and pMAL series; the expression hosts include ArcticExpress and BL21; and the fusion protein tags include thioredoxin, glutathione S-transferase, maltose-binding protein, small ubiquitin protein modifier, and transcription anti-termination factor.

4. The protein preparation method according to claim 1, characterized in that, The induction conditions in step S2 include: when the bacterial culture OD600 reaches 1.2-1.3, add IPTG to make the final concentration 0.2-1.0 mM, and culture at 16℃ with shaking for 14-18 hours.

5. The protein preparation method according to claim 1, characterized in that, The nickel column affinity chromatography includes: i. Rinse 1CV with Buffer A containing 10mM imidazole; ii. Rinse 5 CV with Buffer B containing 30 mM imidazole; iii. Rinse 12 CV with Buffer C containing 110 mM imidazole; iv. Elute the target protein with Buffer D containing 250 mM imidazole. v. Collect the eluent containing protein, add Buffer F to make the final concentration of EDTA 1-5 mM, and desalt the fusion protein by gel chromatography.

6. The protein preparation method according to claim 5, characterized in that, All Buffer ADs are based on 1×PBS buffer, pH 8.0, and contain 300mM NaCl.

7. The protein preparation method according to claim 5, characterized in that, The desalting process of the fusion protein includes: i. Use a 300 mL dextran gel G-25 column, which is pre-swollen with pure water for 24 hours; ii. Use wet packing and pre-equilibrate the packed gel column with 5CV Buffer E; iii. Load the fusion protein onto the sample and rinse it with Buffer E. Collect and combine the effluent containing the protein. iv. Add recombinant enterokinase to the effluent at an enzyme-to-substrate mass ratio of 1:10000 and incubate at 26°C for 16 hours.

8. The protein preparation method according to claim 7, characterized in that, The equilibration buffer E is composed of 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, and pH 7.

8.

9. The protein preparation method according to claim 1, characterized in that, The heparin affinity chromatography includes: i. Equilibrate the chromatography column with 50 mM Tris-HCl, 50 mM NaCl in Buffer G, pH 7.8; ii. Elution was performed using a 0-70% linear gradient in Buffer H of 50 mM Tris-HCl, 2 M NaCl, pH 7.8; iii. Rinse with 70% Buffer H for 4-5 CV, dialyze to desalt in pre-cooled PBS buffer, and collect the target protein.

10. The application of a chaperone-based recombinant human hFGF-2 protein in the preparation of cell culture medium additives and drugs that promote tissue regeneration.

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