Recombinant human-derived fibronectin mutant with enhanced protein stability and expression strain thereof

Abstract

The application discloses a recombinant human fibronectin mutant with enhanced protein stability and an expression strain thereof. The recombinant human fibronectin mutant is a mutant formed by mutating the 28th or 78th amino acid in the recombinant human fibronectin into isoleucine or proline. Compared with the original recombinant human fibronectin, the mutant of the application can be stably expressed in Pichia pastoris and has increased yield, has excellent performance of promoting cell proliferation, migration and adhesion of human fibroblasts, and shows excellent cell biology efficacy, and has wide application prospects in the fields of skin care, beauty and tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and relates to a recombinant human fibronectin mutant with enhanced protein stability and its expression strain. Background Technology

[0002] Fibronectin (FN) is a large glycoprotein found in the extracellular matrix and basement membrane of various animal cells. It plays a crucial role in cell adhesion and is also involved in cell proliferation, differentiation, hemostasis, and wound repair. It has broad application prospects in cancer diagnosis, premature birth prediction, animal cell culture, and cosmetic skincare. Fibronectin consists of two subunits cross-linked at the carboxyl terminus by disulfide bonds to form a V-shaped structure. Each subunit has a molecular weight of up to 200 kDa and contains various repeating modules: 12 FN type I, 2 FN type II, 15-17 FN type III, 2 alternative splicing sites (EIIIA and EIIIB), and 1 variable region. The FN type I and FN type II modules are β-sheets fixed by disulfide bonds; the FN type III module is a 7-strand β-barrel structure, highly susceptible to mechanical denaturation, and can generate EDA and EDB after alternative splicing at the EIIIA and EIIIB sites; while the variable region is present in almost all types of fibronectin. The FN III10 region contains the minimal functional sequence RGD, which recognizes and binds to integrin heterodimers. This is a core functional domain of fibronectin responsible for cell adhesion, migration, proliferation, and wound repair. In addition, the FN III9 region contains a co-functional domain that promotes the formation of the core functional domain and maintains the correct active conformation. Therefore, rationally combining the FN III9 and FN III10 regions will further enhance the binding performance of recombinant human fibronectin to cells, helping to promote the proliferation, migration, and adhesion of human fibroblasts.

[0003] Traditional natural fibronectin is mainly obtained by isolation and extraction from human or animal plasma. This process is complex, resulting in extremely low yields, and animal-derived raw materials pose certain safety risks. With the rapid development of synthetic biology, technologies such as DNA recombination, de novo protein design, and structure prediction have greatly promoted the rational design and efficient industrial production of functional proteins. The Pichia pastoris expression system, a eukaryotic expression system developed in the last decade, offers significant advantages over prokaryotic expression systems reliant on E. coli in terms of post-translational modification, glycosylation, and extracellular secretion of proteins. It has already achieved heterologous production of thousands of proteins in laboratory and industrial-scale operations. Furthermore, Pichia pastoris is recognized by the US FDA as a GRAS (Generally Recognized As Safe) microorganism. Therefore, utilizing Pichia pastoris secretion to express recombinant human fibronectin fragments is an effective strategy to overcome the limitations of traditional extraction methods. Summary of the Invention

[0004] This invention provides a recombinant human fibronectin mutant with enhanced protein stability and its expression strain.

[0005] The technical solution of the present invention is as follows:

[0006] Based on the key functional domains FN III9-10 of human fibronectin, this invention mutates the 28th or 78th amino acid in the recombinant human fibronectin shown in SEQ ID No. 1 to form a recombinant human fibronectin mutant with enhanced protein stability.

[0007] Furthermore, the recombinant human fibronectin mutant with enhanced protein stability described in this invention is either the recombinant human fibronectin mutant 1 with the amino acid sequence shown in SEQ ID No. 1, formed by mutating the 28th amino acid of the recombinant human fibronectin to isoleucine, or the recombinant human fibronectin mutant 2 with the amino acid sequence shown in SEQ ID No. 3, formed by mutating the 78th amino acid of the recombinant human fibronectin to proline.

[0008] Based on the codon preference of Pichia pastoris, this invention optimizes the codons of recombinant human fibronectin and its mutants. The nucleotide sequence of the recombinant human fibronectin is shown in SEQ ID No. 4, the nucleotide sequence of the recombinant human fibronectin mutant 1 with enhanced protein stability is shown in SEQ ID No. 5, and the nucleotide sequence of the recombinant human fibronectin mutant 2 with enhanced protein stability is shown in SEQ ID No. 6.

[0009] Furthermore, this invention constructs strains that efficiently express recombinant human fibronectin mutant 1 or mutant 2. This involves synthesizing the nucleotide sequence of recombinant human fibronectin mutant 1 or mutant 2, amplifying and seamlessly cloning it into the pPIC9K empty vector, and then transforming the correctly sequenced recombinant plasmid into Pichia pastoris after enrichment and SalI restriction enzyme linearization. The resulting Pichia pastoris genetically engineered strains that efficiently secrete and express recombinant human fibronectin mutant 1 or mutant 2 are obtained through G418 resistance gradient screening.

[0010] Furthermore, the present invention provides a method for expressing the above-mentioned recombinant human fibronectin mutant 1 or mutant 2, specifically: Pichia pastoris genetically engineered bacteria that efficiently express recombinant human fibronectin mutant 1 or mutant 2 are inoculated in BMMY medium, and expressed by methanol at 30°C for 72-120 hours. The supernatant is collected and purified to obtain recombinant human fibronectin mutant 1 or mutant 2.

[0011] The recombinant human fibronectin sequence contains the core cytoin-binding sequence and cooperating sequence of the human fibronectin III region. However, the inventors' previous experiments found that the recombinant human fibronectin was highly susceptible to degradation during Pichia pastoris secretory expression, with a yield of only 0.1 g / L after 48 hours of shake-flask fermentation. Furthermore, the protein was completely degraded after 72 hours of fermentation, making it undetectable and uncollectible, significantly impacting the yield and purity of the target protein. The inventors unexpectedly discovered that mutating the 28th or 78th amino acid in the recombinant human fibronectin to isoleucine or proline significantly improved the stability of mutant 1 and mutant 2 during Pichia pastoris secretory expression. These mutants remained stable in the fermentation broth after 72 hours of fermentation, and the yield was significantly higher than the original recombinant human fibronectin, reaching 0.2-0.3 g / L after 72 hours of shake-flask fermentation. In addition, compared with the original recombinant human fibronectin, mutants 1 and 2 can further promote the cell proliferation, migration and adhesion of human fibroblasts, showing excellent cell biological efficacy and broad application prospects in the fields of skin care, beauty and tissue engineering. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of a recombinant plasmid containing recombinant human fibronectin mutant 1.

[0013] Figure 2 This is a schematic diagram of a recombinant plasmid containing recombinant human fibronectin mutant 2.

[0014] Figure 3 The images show the nucleic acid electrophoresis patterns of the proteins and recombinant plasmids. Lane 1: 1kb DNA marker; Lane 2: pPIC9K+ original recombinant human fibronectin gene fragment; Lane 3: original recombinant human fibronectin; Lane 4: pPIC9K+ recombinant human fibronectin mutant 1 gene fragment; Lane 5: recombinant human fibronectin mutant 1 gene fragment; Lane 6: pPIC9K+ recombinant human fibronectin mutant 2 gene fragment; Lane 7: recombinant human fibronectin mutant 2 gene fragment.

[0015] Figure 4 SDS-PAGE images of the supernatant (induction time 72 hours) of the culture medium of Wild type Pichia pastoris genetically engineered strain expressing recombinant human fibronectin and Mutant 1.1 Pichia pastoris genetically engineered strain expressing recombinant human fibronectin mutant 1.

[0016] Figure 5SDS-PAGE images of the supernatant (induced for 72 hours) of the culture medium of Wild type Pichia pastoris genetically engineered strain expressing recombinant human fibronectin and Mutant 2.1 Pichia pastoris genetically engineered strain expressing recombinant human fibronectin mutant 2.

[0017] Figure 6 SDS-PAGE image of the fermentation supernatant of a Pichia pastoris genetically engineered strain expressing the original recombinant human fibronectin (induced for 48 hours).

[0018] Figure 7 Figure showing the effects of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 on the proliferation of human fibroblasts.

[0019] Figure 8 Figure showing the effects of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 on the migration rate of human fibroblasts.

[0020] Figure 9 Figure showing the effect of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 on the relative adhesion rate of human fibroblasts. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments and accompanying drawings, but this does not limit the invention to the scope of the embodiments described. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0022] In the following examples, Pichia pastoris GS115 was used as the starting strain. The expression vectors for recombinant human fibronectin, mutant 1, and mutant 2 were pPIC9K as the integration vector. The nucleotide sequences of SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6, synthesized by Shanghai Qingke Biotechnology Co., Ltd., were amplified and purified by PCR and seamlessly cloned into the pPIC9K empty plasmid.

[0023] Example 1

[0024] Construction of high-copy Pichia pastoris genetically engineered strains:

[0025] (1) The sequence selected in this invention is from the cytotegrin (α5β1, α) in the human fibronectin III region. VThe optimized sequence (amino acid sequence such as SEQ ID No. 1) of the β3 binding domain and its related structural domains not only contains basic peptides that can specifically bind to cytokines, but also contains cooperating peptides that help the above-mentioned basic peptides form and maintain the correct functional structure. It has biological activities such as promoting cell proliferation, migration and adhesion. The optimized nucleotide sequence SEQ ID No. 4 was obtained based on the codon preference of Pichia pastoris.

[0026] (2) To improve the stability of the amino acid sequence of SEQ ID No. 1 in Pichia pastoris, the present invention mutates the 28th amino acid position of SEQ ID No. 1 to isoleucine, or mutates the 78th amino acid position of SEQ ID No. 1 to proline, to obtain the amino acid sequence of mutant 1 with enhanced protein stability, SEQ ID No. 2, and the amino acid sequence of mutant 2, SEQ ID No. 3. Based on the codon bias of Pichia pastoris, the optimized nucleotide sequence of mutant 1, SEQ ID No. 5, and the optimized nucleotide sequence of mutant 2, SEQ ID No. 6, are obtained respectively.

[0027] (3) The nucleotide sequences SEQ ID No. 4, SEQ ID No. 5, and SEQ ID No. 6 were synthesized by Shanghai Qingke Biotechnology Co., Ltd. The target gene fragment was amplified using primer pairs P1 (nucleotide sequence as shown in SEQ ID No. 7) and P2 (nucleotide sequence as shown in SEQ ID No. 8), and then seamlessly cloned into the pPIC9K empty vector (purchased from Thermo Fisher Scientific). The resulting recombinant plasmid containing recombinant human fibronectin mutant 1 is shown below. Figure 1 As shown, the recombinant plasmid containing recombinant human fibronectin mutant 2 is as follows: Figure 2 As shown in the figure. After successful sequencing verification, the correct recombinant plasmids were enriched. The recombinant plasmids were linearized using SalI single-restriction digestion, electroporated into Pichia pastoris, and finally screened using a G418 antibiotic resistance gradient to obtain three high-copy Pichia pastoris genetically engineered strains: Wild type expressing recombinant human fibronectin, Mutant 1.1 expressing recombinant human fibronectin mutant 1, and Mutant 2.1 expressing recombinant human fibronectin mutant 2. The nucleic acid electrophoresis images of each protein and recombinant plasmid are shown in the figure. Figure 3As shown, lane 1: 1kb DNA marker; lane 2: pPIC9K+ original recombinant human fibronectin gene fragment; lane 3: original recombinant human fibronectin; lane 4: pPIC9K+ recombinant human fibronectin mutant 1 gene fragment; lane 5: recombinant human fibronectin mutant 1 gene fragment; lane 6: pPIC9K+ recombinant human fibronectin mutant 2 gene fragment; lane 7: recombinant human fibronectin mutant 2 gene fragment.

[0028] Example 2

[0029] Fermentation of high-copy Pichia pastoris genetically engineered strains and expression of mutants 1 and 2:

[0030] High-copy-ratio Pichia pastoris strain Wild type expressing recombinant human fibronectin, high-copy-ratio Pichia pastoris strain Mutant 1.1 expressing recombinant human fibronectin mutant 1, and high-copy-ratio Pichia pastoris strain Mutant 2.1 expressing recombinant human fibronectin mutant 2 were inoculated into 30 mL of BMGY medium (composed of 20 g / L peptone, 10 g / L yeast extract, 10 × YNB 100 mL, 10 × potassium phosphate buffer pH 6.0 100 mL, 10 × glycerol 100 mL, with the remainder being water; all percentages are by mass percentages) and cultured for 16–20 hours. Then, the initial OD was used as the starting point. 600 The inoculum was 1 inoculated into 30 mL of BMMY medium (which consists of 20 g / L peptone, 10 g / L yeast extract, 100 mL of 10×YNB, 100 mL of 10×potassium phosphate buffer pH 6.0, with the remainder being water; the percentages are by mass percentages) and cultured. Every 24 hours, 300 μL of filtered sterilized methanol was added, and 1 mL of fermentation broth was collected after 72 hours. HPLC analysis showed that mutant 1 expression was 0.2 g / L and mutant 2 expression was 0.3 g / L. 500 μL of supernatant was collected after centrifugation at 12000 rpm for 10 min. This supernatant was then sterilized by passing it through a 0.22 μm sterile filter membrane. The supernatant was then concentrated 10-fold by centrifugation at 5000 rpm and 4 °C using a 1.5 mL ultrafiltration tube (10 kDa). 24 μL of the concentrated supernatant was mixed with 6 μL of 5× loading buffer and heated at 100 °C for 10 min. 15 μL of the mixture was then loaded onto an SDS-PAGE gel for protein electrophoresis. The results are shown below. Figure 4 and 5 .

[0031] from Figure 4 and 5It can be seen that the original recombinant human fibronectin (Wild Type) is highly susceptible to degradation during Pichia pastoris secretory expression. Therefore, only the corresponding degradation band, rather than the target protein band, was detected in the 72-hour fermentation broth. Figure 6 SDS-PAGE of the fermentation supernatant of a Pichia pastoris genetically engineered strain expressing the original recombinant human fibronectin (induced for 48 hours) shows that the band of the undegraded original recombinant human fibronectin is located at 20 kDa. The stability of recombinant human fibronectin mutant 1 (Mutant 1.1), formed by mutating amino acid 28 (arginine) to isoleucine, and recombinant human fibronectin mutant 2 (Mutant 2.1), formed by mutating amino acid 78 (leucine) to proline, in Pichia pastoris was significantly improved, and the degradation of Mutant 2.1 in Pichia pastoris was significantly weaker than that of Mutant 1.1. These results indicate that the mutation of amino acid 28 or 78 of the original recombinant human fibronectin can significantly improve the stability of recombinant human fibronectin expression in Pichia pastoris, and the mutation effect at amino acid 78 is significantly better than that at amino acid 28.

[0032] Example 3

[0033] Since the original recombinant human fibronectin (Wild Type) was almost undetectable after 72 hours of induced fermentation, the fermentation supernatant after 48 hours of induced fermentation was selected for cytological experiments to extract and purify the original recombinant human fibronectin. The content of the original recombinant human fibronectin was 0.1 g / L by HPLC. For recombinant human fibronectin mutants 1 and 2, the fermentation supernatant after 72 hours of induced fermentation was selected.

[0034] 1. Cell proliferation assay

[0035] The MTT assay was used to detect the ability of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 to promote cell proliferation, using human fibroblasts as experimental cells. The results are as follows: Figure 7 As shown, at concentrations of 0.01%–0.1%, the cell viability of the wild type, mutant 1.1, and mutant 2.1 groups increased slowly with decreasing concentration, among which mutant 2.1 showed the strongest ability to promote human fibroblast proliferation.

[0036] 2. Cell migration assay

[0037] The cell scratch method was used to determine cell migration. Human fibroblasts were used as experimental cells to detect the ability of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 to promote cell migration. The results are as Figure 8 shown. At concentrations of 0.01% - 0.1%, the cell migration rates of the wild type, mutant 1.1, and mutant 2.1 groups increased with increasing concentration. At the test concentrations, mutant 2.1 had the strongest ability to promote the migration of human fibroblasts.

[0038] 3. Cell adhesion assay

[0039] The centrifugation method was used. Human fibroblasts were used as experimental cells to detect the ability of recombinant human fibronectin wild type, mutant 1.1, and mutant 2.1 to promote cell adhesion. The results are as Figure 9 shown. At concentrations of 0.01% - 0.1%, the cell adhesiveness of the wild type, mutant 1.1, and mutant 2.1 groups increased with increasing concentration. At the test concentrations, mutant 2.1 had a relatively strong ability to promote the adhesion of human fibroblasts.

[0040] Based on the above cell proliferation, cell migration, and cell adhesion assays, it can be determined that the order of the promoting effects on cells is: recombinant human fibronectin wild type < mutant 1.1 < mutant 2.1. The results show that mutant 2.1 has a more excellent effect on promoting cell proliferation, cell migration, and cell adhesion.

[0041] The above is only the preferred embodiment of the present invention. It should be noted that the above preferred embodiment should not be regarded as a limitation of the present invention. The protection scope of the present invention should be determined by the scope defined in the claims. For those of ordinary skill in the art, without departing from the spirit and scope of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A recombinant human-derived fibronectin mutant with enhanced protein stability, characterized in that, The amino acid sequence formed by the mutation of amino acid position 28 of the recombinant human fibronectin shown in SEQ ID No. 1 to isoleucine is the recombinant human fibronectin mutant 1 shown in SEQ ID No. 2, or the amino acid sequence formed by the mutation of amino acid position 78 of the recombinant human fibronectin shown in SEQ ID No. 1 to proline is the recombinant human fibronectin mutant 2 shown in SEQ ID No.

3.

2. The recombinant human-derived fibronectin mutant according to claim 1, characterized in that, The nucleotide sequence of recombinant human fibronectin is shown in SEQ ID No. 4, the nucleotide sequence of recombinant human fibronectin mutant 1 with enhanced protein stability is shown in SEQ ID No. 5, and the nucleotide sequence of recombinant human fibronectin mutant 2 with enhanced protein stability is shown in SEQ ID No.

6.

3. A strain efficiently expressing the recombinant human-derived fibronectin mutant of claim 1, characterized in that, To synthesize the nucleotide sequence of recombinant human fibronectin mutant 1 as shown in SEQ ID No. 5 or recombinant human fibronectin mutant 2 as shown in SEQ ID No. 6, the sequence was then amplified and seamlessly cloned into the pPIC9K empty vector. The correctly sequenced recombinant plasmids were enriched and... Sal After linearization by I enzyme digestion, the Pichia pastoris was transformed into Pichia pastoris and the highly efficient Pichia pastoris genetically engineered strains expressing recombinant human fibronectin mutant 1 or mutant 2 were obtained through G418 resistance gradient screening.

4. A method for expressing a recombinant human-derived fibronectin mutant, characterized by, Specifically, the Pichia pastoris genetically engineered strain that efficiently expresses recombinant human fibronectin mutant 1 or mutant 2 as described in claim 3 is inoculated into BMMY medium and induced to express with methanol at 30°C for 72-120 hours. The supernatant is collected and purified to obtain recombinant human fibronectin mutant 1 or mutant 2.

Images