A heat-stable hbFGF mutant and a method for high-efficiency expression thereof in escherichia coli

Abstract

This invention provides a thermostable hbFGF mutant and a method for its efficient expression in Escherichia coli. By mutating five amino acid sites of wild-type hbFGF (D15I / C78S / C96Y / S137P / K128N) to improve thermostability, optimizing molecular design, constructing an expression vector, and transforming the expression strain of Escherichia coli, efficient and soluble expression of hbFGF in Escherichia coli is achieved.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more particularly to a thermostable hbFGF mutant and a method for its efficient expression in Escherichia coli. Background Technology

[0002] Human basic fibroblast growth factor (hbFGF), also known as fibroblast growth factor 2 (FGF2), is a non-glycosylated single-chain protein involved in a variety of biological processes (Powers et al., 2000; Barrientos et al., 2008; Beenken and Mohammadi, 2009; Korc and Friesel, 2009; Kuo et al., 2015).

[0003] To date, hbFGF has been approved in China for skin wound repair and in Japan for the treatment of periodontitis, pressure ulcers, and skin ulcers (Hui et al., 2018a). Furthermore, increasing evidence suggests that hbFGF is a potential therapeutic agent for a variety of diseases, such as cardiac repair (Li et al., 2021), nerve injury (Li et al., 2017), bone regeneration (Novais et al., 2021), asthma, and chronic obstructive pulmonary disease (Tan et al., 2020), and is also a potential predictive biomarker for hematologic malignancies and solid tumors (Akl et al., 2016).

[0004] In recent decades, hbFGF (especially the 146-amino acid form) has been successfully expressed in a variety of host systems, including *Escherichia coli* (Feng et al., 2004; Chen et al., 2012; Rassouli et al., 2013; Soleyman et al., 2016), *Bacillus subtilis* (Kwong et al., 2013; Hu et al., 2018), yeast (Le et al., 2020), silkworm (Masuda et al., 2018), and plants (Ding et al., 2006; Yang et al., 2018). Among these, the *Escherichia coli* expression system has become the preferred host for the industrial-scale production of hbFGF due to its rapid growth, ease of operation, and low cost.

[0005] To improve the soluble expression of hbFGF in prokaryotic systems, researchers have employed various fusion tag strategies, including glutathione S-transferase (GST) (Sheng et al., 2003; Sekiguchi et al., 2018), thioredoxin (Trx) (Imsoonthornruksa et al., 2015; Soleyman et al., 2016), maltose-binding protein (MBP) (Lemaitre et al., 1995), and collagen-like protein (Scl2) (Rahman et al., 2020). These fusion tags significantly improved the soluble expression levels of the protein.

[0006] Although bacterial expression systems remain the mainstream route for the preparation and purification of hbFGF, the lack of complex post-translational modification mechanisms in prokaryotes often results in expression products existing as inactive inclusion bodies. While strategies such as low-temperature induction and denaturation-renaturation optimization can partially alleviate this, these methods struggle to simultaneously achieve high bioactivity and high recovery yields. This leads to persistently high costs for preparing recombinant hbFGF with complete bioactivity, severely hindering its large-scale clinical application and basic research.

[0007] In addition, early production of similar growth factors such as hbFGF also faced similar challenges: although eukaryotic expression platforms can achieve soluble expression, they have low yields and are prone to problems such as excessive glycosylation or N-terminal degradation; although E. coli expression is highly efficient, it often forms inclusion bodies, requiring a time-consuming denaturation-renaturation process; although fusion tag strategies are effective, bottlenecks such as incomplete tag cleavage, high protease costs, and long processing cycles further limit their application in large-scale industrial production. Summary of the Invention

[0008] To address the shortcomings of existing technologies, this invention provides a thermostable hbFGF mutant and a method for its efficient expression in *E. coli*. This invention enhances thermostability by mutating five amino acid sites of hbFGF (D15I / C78S / C96Y / S137P / K128N), optimizes molecular design, constructs an expression vector, and transforms *E. coli* expression strains, achieving efficient and soluble expression of FGF-2 in *E. coli*.

[0009] In one aspect, the present invention provides a mutant human basic fibroblast growth factor (hbFGF) protein, wherein the amino acid sequence of the mutant hbFGF protein is mutated at at least one amino acid site selected from positions 15, 78, 96, 137 and 128 relative to wild-type hbFGF (SEQ ID NO:1) or a functional fragment thereof.

[0010] In one embodiment of the present invention, the mutant hbFGF protein is mutated at two, three, four, or all five sites selected from positions 15, 78, 96, 137, and 128.

[0011] In one embodiment of the present invention, the mutation is selected from one or more of substitution, deletion or insertion, preferably amino acid substitution.

[0012] In one embodiment of the present invention, the mutant hbFGF protein includes one, several, or all of the following mutations: D15I, C78S, C96Y, S137P, and K128N.

[0013] In one embodiment of the present invention, the amino acid sequence of the mutant hbFGF protein is as shown in SEQ ID NO:2 or SEQ ID NO:3, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:2 or SEQ ID NO:3.

[0014] In one embodiment of the present invention, the mutant hbFGF protein has improved thermal stability, solubility, and expression level in Escherichia coli compared to wild-type hbFGF.

[0015] In a second aspect, the present invention provides a recombinant fusion protein comprising the aforementioned mutant hbFGF protein.

[0016] In one embodiment of the present invention, the recombinant fusion protein comprises, from the N-terminus to the C-terminus, the following: a solubilization tag, an affinity purification tag, a linker sequence that can be recognized and cleaved by a protease, and the mutant hbFGF protein.

[0017] In one embodiment of the present invention, the solubilizing tag is selected from VNP6 tag, GB1 tag, MBP tag, GST tag, SUMO tag, NusA tag or other functionally equivalent solubilizing sequences, or combinations thereof.

[0018] In one embodiment of the present invention, the affinity purification tag is selected from polyhistidine tags (His tags), FLAG tags, Strep tags, or other affinity tags.

[0019] In one embodiment of the present invention, the linker sequence that can be recognized and cleaved by proteases is selected from the TEV protease cleavage site, the coagulation factor Xa cleavage site, the thrombin cleavage site, or other protease cleavage sites.

[0020] In one embodiment of the present invention, the amino acid sequence of the recombinant fusion protein is as shown in SEQ ID NO:4, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:4.

[0021] In a third aspect, the present invention provides an isolated nucleic acid molecule encoding the mutant hbFGF protein or the recombinant fusion protein described herein.

[0022] In one embodiment of the present invention, the nucleic acid molecule is codon-optimized to be efficiently expressed in Escherichia coli or other prokaryotic host cells.

[0023] In one embodiment of the present invention, the sequence of the nucleic acid molecule is as shown in SEQ ID NO:5, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:5.

[0024] The present invention also provides an expression vector comprising the aforementioned nucleic acid molecule.

[0025] In one embodiment of the present invention, the expression vector is selected from pET series vectors (such as pET28a), pQE series, pGEX series, pBac series, or other prokaryotic, eukaryotic, or viral vectors that can be induced to express. Preferably, the nucleic acid molecule is inserted via NcoI, XhoI, BamHI, EcoRI, or other restriction endonuclease sites.

[0026] The present invention also provides a host cell comprising the expression vector described above, or expressing the mutant hbFGF protein or recombinant fusion protein described above.

[0027] In one embodiment of the present invention, the host cell is selected from Escherichia coli (such as BL21(DE3)), Bacillus subtilis, yeast (such as Pichia pastoris), mammalian cells (such as CHO, HEK293) or other prokaryotic / eukaryotic cells.

[0028] In a fourth aspect, the present invention provides a method for producing mutant hbFGF protein or recombinant fusion protein, comprising the following steps: (a) Provide the host cell; (b) Culture the host cells and induce the expression of the mutant hbFGF protein or the recombinant fusion protein; (c) Collect the expression products.

[0029] In one embodiment of the present invention, the induced expression is induced by IPTG at a concentration of 0.05-1 mM, preferably about 0.1 mM; the induction temperature is 15-37°C, preferably about 37°C; the induction time is 2-24 hours, preferably about 5 hours; the culture is carried out using LB medium, TB medium, SOC medium or other nutrient medium, the medium containing corresponding antibiotics such as kanamycin, ampicillin or chloramphenicol at a concentration of 10-200 mg / L.

[0030] In one embodiment of the present invention, the method results in the expression level of the mutant hbFGF protein or recombinant fusion protein accounting for at least 40% of the total bacterial protein, preferably at least 44%, more preferably at least 45%, and even more preferably at least 46%.

[0031] In one embodiment of the present invention, the method further includes step (d): using a protease to cleave the fusion tag to obtain purified mutant hbFGF protein.

[0032] In one embodiment of the present invention, the purification is performed using affinity chromatography, ion exchange chromatography, size exclusion chromatography, or other purification techniques.

[0033] The present invention also provides the use of the mutant hbFGF protein, recombinant fusion protein, nucleic acid molecule, expression vector, host cell, or the method thereof in the preparation of therapeutic agents or products.

[0034] In one embodiment of the present invention, the therapeutic agent or product is used to promote cell proliferation, tissue regeneration and repair, including promoting wound healing, tissue repair, angiogenesis, neuroprotection, bone repair, cartilage regeneration or stem cell culture; preferably used to treat or prevent burns, trauma, diabetic ulcers, myocardial infarction, stroke, fractures, arthritis and other diseases related to tissue damage or regenerative disorders.

[0035] In one embodiment of the present invention, the use includes formulating the mutant hbFGF protein or recombinant fusion protein into injections, gels, sprays, implants, cell culture additives or other dosage forms.

[0036] The present invention also provides a pharmaceutical composition comprising the mutant hbFGF protein, recombinant fusion protein, nucleic acid molecule, expression vector, host cell or combination thereof, and pharmaceutically acceptable carrier, excipient or stabilizer.

[0037] In one embodiment of the present invention, the composition is used to treat or prevent burns, trauma, diabetic ulcers, myocardial infarction, stroke, fractures, arthritis, or other diseases related to tissue damage or regenerative disorders.

[0038] The present invention also provides a kit comprising the mutant hbFGF protein, recombinant fusion protein, nucleic acid molecule, expression vector, host cell, pharmaceutical composition or combination thereof.

[0039] Compared with the prior art, the present invention has the following beneficial effects: This invention fuses a highly efficient soluble tag (such as VNP6+GB1) to the N-terminus and combines it with a specific site mutation, enabling ultra-efficient soluble expression of the recombinant fusion protein in common engineered strains such as BL21(DE3). The expression level reaches 44.5%–46.6% or more of the total protein, far exceeding the level of existing recombinant hbFGF expression systems. This high expression characteristic significantly reduces downstream purification costs and improves the economics of large-scale industrial production. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of the hbFGF recombinant fusion protein; Figure 2 The results are shown in the SDS-PAGE assay for three strains. Detailed Implementation

[0041] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0042] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0043] Terminology Definition hbFGF (human basic fibroblast growth factor) refers to human basic fibroblast growth factor, also known as FGF2 (fibroblast growth factor 2). In this invention, unless otherwise stated, the term "hbFGF" encompasses wild-type hbFGF and its functional variants (including fragments, variants containing amino acid substitutions, or fusion proteins). Its wild-type reference polypeptide sequence is shown in SEQ ID NO: 1, a 18kDa protein of 155 amino acids in length.

[0044] The amino acid sequence of wild-type hbFGF (SEQ ID NO:1): MAAGSITTLPALPEDGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTYRSRKYTSWYVALKRTGQYKLGSKTGPGQKAILFLPMSAKS Fragment or functional fragment: refers to a polypeptide moiety in which the N-terminus and / or C-terminus of the wild-type hbFGF polypeptide is deleted (truncated), but still retains the biological activity of FGF2 (such as cell-binding activity or heparin-binding activity). Preferably, the fragment contains the cell-binding domain and the heparin-binding domain of the FGF2 polypeptide.

[0045] In this invention, the functional fragment also includes a sequence having at least 85%, 90%, 95% or more sequence identity with the corresponding region of SEQ ID NO: 1, and the sequence may contain one or more amino acid substitutions, additions or deletions, provided that it retains the biological activity of FGF2.

[0046] Those skilled in the art will understand that the FGF2 protein has structural flexibility at its N-terminus, and truncation at the N-terminus (e.g., deletion of 1-9 amino acids) generally does not affect its core structure and biological activity. Therefore, the 'mutant hbFGF' described in this invention includes not only full-length (positions 1-155) mutants but also explicitly covers N-terminal truncated mutant forms, particularly the 151-amino acid form (deletion of positions 1-4) and the 146-amino acid form (deletion of positions 1-9). In some embodiments, the N-terminus of the polypeptide may optionally contain one or more residual amino acids (e.g., glycine) derived from protease cleavage sites.

[0047] Mutated human basic fibroblast growth factor (hbFGF): refers to a protein variant in which the amino acid sequence of wild-type human basic fibroblast growth factor (hbFGF, as shown in SEQ ID NO: 1) or a fragment thereof is altered at at least one specified site. In this invention, the mutations mainly include positions 15, 78, 96, 137, and 128 (using SEQ ID NO: 1 as the reference sequence; that is, the amino acid site numbers described in this invention are all based on the wild-type full-length hbFGF sequence (SEQ ID NO: 1)). The five site mutants include D15I / C78S / C96Y / S137P / K128N.

[0048] The hbFGF sequence containing 5 mutation sites in the form of 151 amino acids is shown in SEQ ID NO:2: SITTLPALPEiGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVsANRYLAMKEDGRLLASKyVTDECFFFERLESNNYNTYRSRKYTSWYVALnRTGQYKLGpKTGPGQKAILFLPMSAKS The 146-amino acid form of hbFGF containing 5 mutation sites is shown in SEQ ID NO:3: PALPEiGGSGAFPPGHFKDPKRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVsANRYLAMKEDGRLLASKyVTDECFFFERLESNNYNTYRSRKYTSWYVALnRTGQYKLGpKTGPGQKAILFLPMSAKS Recombinant fusion proteins: These are single polypeptide chains formed by fusing multiple functional domains or tag sequences through genetic engineering, where each domain can function independently or synergistically. In this invention, from the N-terminus to the C-terminus are, in sequence, a lysin expression tag (such as VNP6 and GB1, used to enhance soluble expression), a His tag (used for Ni-NTA affinity purification), a TEV restriction enzyme sequence (used for protease cleavage to remove the tag and obtain purified mutant hbFGF), and a mutant hbFGF sequence. The expression level after fusion reaches 44.5%-46.6% (quantitatively determined by SDS-PAGE).

[0049] Solubilizing tags are short peptide or protein domain sequences fused to the N-terminus or C-terminus of a target protein to enhance the soluble expression level of recombinant proteins in host cells and reduce inclusion body formation. This invention uses VNP6 (a nanoscale solubilizing tag) and GB1 (from the B1 domain of protein G) as a solubilizing tag combination, placed at the N-terminus of the fusion protein. This tag promotes the correct folding of mutant hbFGF by increasing hydrophilicity and chaperone-like interactions. In experiments, this design, through binding site mutation, achieved high expression and is suitable for industrial-scale production.

[0050] Affinity tags: Short peptide sequences that specifically bind to specific ligands (such as metal ions or antibodies) for protein purification. This invention uses a His tag (typically 6-10 consecutive histidine residues) inserted after the solubilization tag and before the TEV sequence. (The last sentence appears to be incomplete and possibly refers to a different technology or process.) 2+ or Co 2+ Affinity chromatography is used to purify the fusion protein in one step, achieving a purity >90%. This tag does not affect downstream TEV cleavage and can be removed after cleavage to obtain tag-free hbFGF.

[0051] TEV restriction enzyme sequences refer to amino acid sequences (such as ENLYFQG) that can be specifically recognized and cleaved by tobacco etch virus (TEV) proteases, used to remove the fusion tag after purification. In this invention, the sequence is placed after the His tag and before the mutant hbFGF, with precise sequence design to ensure efficient cleavage (cleavage efficiency >95%). After cleavage, the remaining mutant hbFGF is close to its natural form, retaining complete biological activity, thus avoiding tag interference in downstream applications such as cell therapy.

[0052] Codon optimization refers to the DNA sequence modification process that adjusts gene sequences to improve translation efficiency and protein expression levels based on host cell codon usage preferences. This invention optimizes the codons of the mutant hbFGF gene (including the fusion portion) using molecular biology software (such as Codon Optimization Tool or GeneOptimizer), and commissioned Sangon Biotech (Shanghai) Co., Ltd. to chemically synthesize the optimized sequence. The optimized sequence was inserted into the pET28a vector (NcoI+XhoI site), significantly improving expression efficiency in *E. coli* BL21(DE3). At OD600 = 0.8-1.0, expression levels reached 44.5%-46.6% after 5 hours of induction with 0.1 mM IPTG.

[0053] Expression vectors: These are plasmids or vectors containing a promoter, origin of replication, multiple cloning site, and selection marker, used to express recombinant genes in host cells. This invention uses the pET28a vector (T7 promoter, KanR resistance), with optimized sequence insertion via NcoI+XhoI double digestion. This vector supports IPTG induction and exhibits high-efficiency expression in BL21(DE3). Other equivalent vectors (such as pET series variants) are also applicable.

[0054] Host cells: These refer to microbial, yeast, or mammalian cells that are transformed or transfected with the expression vector for the production of recombinant proteins. This invention primarily uses *E. coli* BL21(DE3). After transformation, single clones are selected, cultured overnight at 37°C, and then transferred to 50 ml of LB (50 mg / L Kan) until OD600 = 0.8-1.0 for induction. This system is simple, economical, and produces high expression levels; it can be extended to other hosts such as *Bacillus subtilis*.

[0055] Induced expression: This refers to the process of activating the promoter by adding an inducer to initiate the expression of recombinant proteins. In this invention, 0.1 mM IPTG was used to induce expression for 5 h (37°C, 200 rpm), and the expression levels of the two monoclonal antibodies were 44.5% and 46.6% (SDS-PAGE), respectively. These conditions optimized the balance between expression and growth, avoiding the accumulation of toxicity.

[0056] Expression level: refers to the proportion of recombinant protein in the total host cell protein, usually quantified by SDS-PAGE, Western blot, or densitometer. The mutant fusion protein of this invention exhibits an expression level ≥44%, far exceeding the low expression of wild-type hbFGF. This indicator was obtained through cell centrifugation and SDS-PAGE detection, supporting high-yield production.

[0057] Site: refers to the specific amino acid residue position in the wild-type hbFGF sequence (SEQ ID NO: 1), for example, "15th position" refers to the 15th amino acid residue in the sequence (e.g., aspartic acid, D15).

[0058] Purification refers to the process of separating and obtaining high-purity (>95%) mutant hbFGF through steps such as affinity chromatography (Ni-NTA), TEV enzyme digestion, and ultrafiltration. The purified protein of this invention does not contain the fusion tag and is suitable for clinical or research applications.

[0059] Example 1: Construction and Expression of Mutants First, the structure of hbFGF was analyzed and the stability of its mutations was predicted, resulting in a five-amino acid mutant (D15I / C78S / C96Y / S137P / K128N). To achieve highly efficient expression of hbFGF, a lysosomal expression tag was added to its N-terminus, such as... Figure 1 As shown, starting from the N-terminus, the sequences are VNP6, GB1, His tag, TEV restriction enzyme sequence, and finally the five-mutated hbFGF sequence.

[0060] The DNA codon sequence encoding the protein was optimized using molecular biology software. Then, the optimized hbFGF gene sequence was chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd., and inserted into the pET28a vector (NcoI+XhoI). The designed sequence is as follows: The amino acid sequence of the fusion protein (SEQ ID NO:4): MDVFKKGFSIADEGVVGAVEKTDQGVTEAAEKTKEGVMGGSGYKLILNGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEHHHHHHssgENLYFQ SITTLPALPEiGGSGAFPPGHFKDPKRLYCKNG GFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVsANRYLAMKEDGRLLASKyVTDECFFFERLESNNYN TYRSRKYTSWYVALnRTGQYKLGpKTGPGQKAILFLPMSAKS The DNA sequence of the fusion protein is (SEQ ID NO:5): ATGGATGTTTTTAAAAAGGGTTTCAGCATTGCTGACGAAGGCGTAGTGGGCGCCGTGGAAAAGACAGATCAGGGCGTGACTGAAGCCGCAGAGAAGACCAAGGAAGGTGTGATGGGCGGCAGCGGCTATAAGCTGATTCTTAATGGTAAAACGCTGAAGGGTGAAACAACCACTGAAGCCGTGGATGCCGCGACTGCCGAGAAAGTATTTAAACAATATGCTAATGATAACGGAGTTGACGGCGAATGGACCTATGACGATGCTACCAAAACCTTTACCGTCACAGAGCATCACCATCATCATCACTCTTCGGGCGAGAATCTTTACTTTCAGGGCAGCATTACCACCCTGCCGGCATTACCCGAAATCGGGGGATCGGGAGCGTTCCCGCCGGGCCATTTCAAGGACCCGAAGCGCCTGTATTGCAAGAACGGGGGCTTCTTCCTGCGTATCCACCCGGATGGGCGCGTTGATGGGGTCCGCGAGAAATCCGATCCTCATATTAAGTTACAGTTACAGGCAGAAGAACGTGGTGTGGTCAGCATTAAAGGTGTGTCGGCGAATCGTTATTTAGCGATGAAAGAAGATGGCCGCCTGCTGGCATCTAAATACGTGACGGATGAATGCTTTTTTTTCGAGCGCTTGGAATCGAATAACTACAATACCTATCGTAGCCGTAAGTATACCTCCTGGTATGTCGCACTGAATCGCACGGGGCAGTATAAACTGGGTCCGAAAACAGGCCCTGGTCAGAAGGCCATTCTGTTTCTGCCGATGAGTGCGAAATCGTGA The synthesized plasmid was transformed into *E. coli* BL21(DE3). Three single clones were then picked and cultured overnight at 37°C in 10 ml Erlenmeyer flasks containing LB medium (50 mg / L kanamycin) with shaking. The next day, the clones were transferred at a 1:50 ratio to 50 ml of fresh LB medium (50 mg / L kanamycin) and cultured at 37°C and 200 rpm for approximately 2 hours until OD600 = 0.8–1.0. Then, 0.1 mM IPTG was added to induce expression. After 5 hours, the bacterial culture was collected, centrifuged, and the cells were analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown in the figure. According to the test results, the expression levels of the three strains were 44.5%, 46.6%, and 42.6%, respectively.

[0061] Example 2 Stability Testing To investigate the stability of mutant hbFGF, pure mutant hbFGF protein was purified by chromatographic chromatography. The protein was placed in an environment with a temperature of 2℃-8℃ and a relative humidity not exceeding 60%. The residual protein content in the supernatant was measured after 0, 1, 2, and 3 weeks (Table 1). Simultaneously, the protein was prepared into a gel formulation and placed at 25℃ for 0, 3, 7, 14, and 28 days, and the protein content in the supernatant was measured. The results showed that mutant hbFGF was stable for 3 weeks under 2-8℃ conditions, while wild-type hbFGF showed 69% protein aggregation and precipitation in the first week. After preparation into a gel formulation, mutant hbFGF remained stable for more than 28 days at 25℃, while wild-type hbFGF showed only 31% concentration after 7 days (Table 2). Therefore, the stability of mutant hbFGF is significantly greater than that of wild-type hbFGF.

[0062] Table 1. Stability study of protein solutions at 2℃-8℃

[0063] Table 2. Stability study of protein gel formulation at 25℃

[0064] Example 3 Activity Test hbFGF activity was detected using the cell proliferation method / MTT colorimetric method as described in the pharmacopoeia. The detection method is as follows: 1. Preparation of protein solutions 1.1 Preparation of standard solutions The national standard for recombinant human basic fibroblast growth factor activity assay was reconstituted according to the instructions and diluted with maintenance culture medium to a concentration of 40 IU per ml. Serial dilutions of 4-fold were performed in 96-well cell culture plates, resulting in 8 dilutions, with 2 wells for each dilution. All procedures were performed under aseptic conditions.

[0065] 1.2 Preparation of hbFGF solution Take wild-type hbFGF solution and mutant hbFGF solution, and continue to dilute with maintenance culture medium. Perform 4-fold serial dilutions in 96-well cell culture plates, for a total of 8 dilutions, with 2 wells for each dilution. All the above operations are performed under aseptic conditions.

[0066] 2. Activity Assay 2.1 BALB / c 3T3 cell line was cultured in complete culture medium at 37℃ and 5% carbon dioxide, with the cell concentration controlled at 1.0 × 10⁻⁶ cells per ml. 5 ~5.0×10 5 Cells were passaged and used for biological activity assays 24–36 hours later.

[0067] 2.2 Discard the culture medium in the culture flask, digest and collect the cells, and prepare a solution containing 5.0 × 10⁶ cells per ml using complete culture medium. 4 ~1.0×10 5 Cell suspensions of 100 μl were seeded into 96-well cell culture plates and cultured at 37°C with 5% carbon dioxide.

[0068] 2.3 After 24 hours, replace with maintenance medium and incubate at 37°C and 5% CO2 for 24 hours. Discard the maintenance medium from the prepared cell culture plates, add 100 μl of standard solution and test solution to each well, and incubate at 37°C and 5% CO2 for 64–72 hours.

[0069] 2.4 Add 20 μl of MTT solution to each well and incubate at 37°C and 5% carbon dioxide for 5 hours.

[0070] 2.5 The above steps shall be performed under aseptic conditions.

[0071] 2.6 After discarding the liquid in the culture plate, add 100 μl of dimethyl sulfoxide to each well, mix well, place in a microplate reader, use 630 nm as the reference wavelength, and measure the absorbance at a wavelength of 570 nm. Record the measurement results.

[0072] Experimental data were processed using a computer program or a four-parameter regression method, and the results were calculated using the following formula:

[0073] In the formula, P represents the biological activity of the standard, IU / ml; D represents the pre-dilution factor of the test sample; D represents the pre-dilution factor of the standard; E represents the dilution factor of the test sample equivalent to half the effective amount of the standard; and E represents the dilution factor of half the effective amount of the standard.

[0074] The activity of wild-type hbFGF was measured to be 1.7 × 10⁻⁶. 5 IU / ml, the activity of mutant hbFGF is 2.5×10 5 IU / ml.

[0075] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A mutant human basic fibroblast growth factor (hbFGF) protein, characterized in that, The amino acid sequence of the mutant hbFGF protein is mutated at at least one amino acid site selected from positions 15, 78, 96, 137, and 128 relative to wild-type hbFGF (SEQ ID NO:1) or a functional fragment thereof.

2. The mutant human basic fibroblast growth factor (hbFGF) protein as described in claim 1, characterized in that, The mutant hbFGF protein is mutated at two, three, four, or all five sites selected from positions 15, 78, 96, 137, and 128. Preferably, the mutation is selected from one or more of substitution, deletion or insertion, and more preferably amino acid substitution; Preferably, the mutant hbFGF protein includes one, several, or all of the following mutations: D15I, C78S, C96Y, S137P, and K128N. Preferably, the amino acid sequence of the mutant hbFGF protein is as shown in SEQ ID NO:2 or SEQ ID NO:3, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:2 or SEQ ID NO:

3.

3. A recombinant fusion protein, characterized in that, The recombinant fusion protein includes the mutated hbFGF protein as described in claim 1 or 2; Preferably, the recombinant fusion protein comprises, from the N-terminus to the C-terminus, the following: a solubilization tag, an affinity purification tag, a linker sequence that can be recognized and cleaved by proteases, and the mutated hbFGF protein; Preferably, the solubilizing tag is selected from VNP6 tag, GB1 tag, MBP tag, GST tag, SUMO tag, NusA tag or other functionally equivalent solubilizing sequences, or combinations thereof; Preferably, the affinity purification tag is selected from polyhistidine tags (His tags), FLAG tags, Strep tags, or other affinity tags; Preferably, the linker sequence that can be recognized and cleaved by proteases is selected from TEV protease cleavage sites, coagulation factor Xa cleavage sites, thrombin cleavage sites, or other protease cleavage sites; Preferably, the amino acid sequence of the recombinant fusion protein is as shown in SEQ ID NO:4, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

4.

4. An isolated nucleic acid molecule encoding the mutant hbFGF protein of claim 1 or 2 or the recombinant fusion protein of claim 3; Preferably, the nucleic acid molecules are codon-optimized to be efficiently expressed in Escherichia coli or other prokaryotic host cells; Preferably, the sequence of the nucleic acid molecule is as shown in SEQ ID NO:5, or has at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the sequence shown in SEQ ID NO:

5.

5. An expression vector comprising the nucleic acid molecule of claim 4.

6. A host cell comprising the expression vector of claim 5, or expressing the mutant hbFGF protein of claim 1 or 2, or the recombinant fusion protein of claim 3.

7. A method for producing mutant hbFGF protein or recombinant fusion protein, comprising the following steps: (a) Providing the host cell as described in claim 6; (b) Culturing the host cells and inducing the expression of the mutant hbFGF protein or the recombinant fusion protein; (c) Collect the expression products; Preferably, the induced expression is induced by IPTG at a concentration of 0.05-1 mM, preferably about 0.1 mM; the induction temperature is 15-37℃, preferably about 37℃; the induction time is 2-24 hours, preferably about 5 hours; the culture is carried out using LB medium, TB medium, SOC medium or other nutrient medium containing antibiotics such as kanamycin, ampicillin or chloramphenicol at a concentration of 10-200 mg / L. Preferably, the method further includes step (d): using a protease to cleave the fusion tag to obtain purified mutant hbFGF protein.

8. Use of the mutant hbFGF protein of claim 1 or 2, the recombinant fusion protein of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6, or the method of claim 7 in the preparation of a therapeutic agent or product; The therapeutic agents or products are preferably used to promote cell proliferation, tissue regeneration and repair, including promoting wound healing, tissue repair, angiogenesis, neuroprotection, bone repair, cartilage regeneration or stem cell culture; preferably for the treatment or prevention of burns, trauma, diabetic ulcers, myocardial infarction, stroke, fractures, arthritis and other diseases related to tissue damage or regenerative disorders. Preferred uses include formulating the mutant hbFGF protein or recombinant fusion protein into injections, gels, sprays, implants, cell culture additives or other dosage forms.

9. A pharmaceutical composition, characterized in that, The composition comprises the mutant hbFGF protein of claim 1 or 2, the recombinant fusion protein of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6 or a combination thereof, and a pharmaceutically acceptable vector, excipient or stabilizer. The composition is preferably used for the treatment or prevention of burns, trauma, diabetic ulcers, myocardial infarction, stroke, fractures, arthritis, or other diseases related to tissue damage or regenerative disorders.

10. A reagent kit, characterized in that, The kit comprises the mutant hbFGF protein of claim 1 or 2, the recombinant fusion protein of claim 3, the nucleic acid molecule of claim 4, the expression vector of claim 5, and the host cell of claim 6 or a combination thereof.

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