A recombinant Pichia pastoris strain, recombinant protein and method

Abstract

This invention relates to a recombinant Pichia pastoris strain, recombinant protein, and method, belonging to the field of bioengineering technology. The strain provided by this invention can stably express the recombinant protein, which has a structure close to that of natural collagen and the biological activity of type I collagen, and has high cross-linking, viscosity, and stability, providing better assurance for the application of subsequent products. At the same time, the recombinant protein has excellent cell adhesion activity and has broad market application prospects.

Description

Technical Field

[0001] This invention relates to a recombinant Pichia pastoris strain, recombinant protein, and method, belonging to the field of bioengineering technology. Background Technology

[0002] Collagen is the main component of connective tissue and the most abundant and widely distributed functional protein in mammals. Collagen provides support and strength to the skin, keeping it firm and elastic. In bones, collagen combines with minerals such as hydroxyapatite to form a hard bone matrix, giving bones strength and toughness. In tendons and ligaments, collagen fibers are tightly packed and withstand enormous tensile forces, ensuring joint stability and movement function.

[0003] Collagen is widely used due to its excellent biocompatibility, bioactivity, and biodegradability. Currently, the main methods for preparing collagen include traditional animal tissue extraction and emerging genetic engineering methods. To overcome the bottlenecks of traditional processes, modern industry also introduces membrane separation technology as an efficient auxiliary purification method. Animal tissue extraction is currently the most mature and widely used method. Simply put, it involves extracting collagen from the skin, bones, and scales of animals such as cattle, pigs, and fish. As the current mainstay of the market, it is advantageous due to its low cost and simple process, but its disadvantages are equally obvious, including the risk of xenogeneic rejection, pathogen safety risks, and high-temperature denaturation and inactivation. Genetic engineering methods suffer from the inherent difficulty of forming a natural triple helix structure. Current microbial fermentation technology struggles to perfectly replicate this process, resulting in recombinant collagen often being single-chain fragments, which still raises questions about its supportive properties (such as in cosmetic fillers) and its ability to promote advanced bioactivity such as bone / skin repair.

[0004] Among various recombinant collagen expression methods, Pichia pastoris, being a eukaryotic microorganism, can perform certain post-translational modifications on the translated protein (especially glycosylation), strongly supporting the realization of protein biological functions. Although the Pichia pastoris expression system is an ideal method for the large-scale industrial expression and production of recombinant human collagen, not all collagen can be produced using Pichia pastoris expression, or it may have defects such as low expression levels, low cross-linking of the expressed protein, low viscosity, and poor stability.

[0005] Existing reports on collagen synthesis and expression primarily focus on the biological activity or hydrophilicity of expressed humanized collagen sequences, often neglecting the stability of the expressed product itself. This leads to significant degradation of unstable collagen during expression, making subsequent purification difficult and reducing yield. Even when high-purity lyophilized sponges are obtained from unstable recombinant collagen through purification processes, substantial degradation occurs upon remelting, increasing the risk to subsequent use and significantly limiting its applications. Furthermore, there are currently almost no reports or studies on the cross-linking properties and viscosity of recombinant collagen itself. Summary of the Invention

[0006] This invention addresses some shortcomings in existing technologies by providing a recombinant Pichia pastoris strain, recombinant protein, and method. The strain provided by this invention can stably and efficiently express the recombinant protein, which has a structure close to that of natural collagen, exhibits type I collagen biological activity, and has high cross-linking properties, viscosity, and stability, thus providing better assurance for the application of subsequent products.

[0007] In the sequence design of this invention, potential restriction enzyme sites and unstable sequence fragments are avoided by analyzing existing amino acid sequences, so that it can be efficiently expressed in Pichia pastoris and the collagen obtained after purification still has high stability; while taking into account relevant biological activity sites and hydrophilicity, it still has relevant biological activities while maintaining high stability.

[0008] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0009] This invention first provides a recombinant Pichia pastoris strain, the strain containing a nucleotide encoding an amino acid fragment A, wherein the amino acid fragment A includes any of the following:

[0010] (1) The amino acid sequence shown in SEQ ID No:1;

[0011] (2) An amino acid sequence modified by a certain degree of amino acid substitution, insertion, substitution, addition, or deletion based on the amino acid sequence shown in SEQ ID No:1;

[0012] (3) An amino acid sequence that has greater than 80% identity with the amino acid sequence shown in SEQ ID No:1.

[0013] Furthermore, the recombinant Pichia pastoris strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38641.

[0014] More preferably, the nucleotide sequence is as shown in SEQ ID No: 2.

[0015] The present invention also provides a method for preparing a recombinant protein, the method comprising: fermenting and culturing the recombinant Pichia pastoris strain to express the recombinant protein.

[0016] The present invention also provides a recombinant protein, characterized in that the recombinant protein comprises any one of the following:

[0017] (1) The amino acid sequence shown in SEQ ID No:1;

[0018] (2) An amino acid sequence modified by a certain degree of amino acid substitution, insertion, substitution, addition, or deletion based on the amino acid sequence shown in SEQ ID No:1;

[0019] (3) An amino acid sequence that has greater than 80% identity with the amino acid sequence shown in SEQ ID No:1.

[0020] The present invention also provides a polynucleotide that encodes a recombinant protein expressed by the recombinant Pichia pastoris strain, or a recombinant protein prepared by the method, or the recombinant protein; preferably, the polynucleotide sequence is shown in SEQ ID No: 2.

[0021] The present invention also provides a recombinant expression vector containing the aforementioned polynucleotide.

[0022] The present invention also provides a composition comprising the recombinant protein, or the recombinant protein expressed by the recombinant Pichia pastoris strain, or the recombinant protein prepared by the method, or the recombinant expression vector, or the recombinant Pichia pastoris strain.

[0023] The present invention also provides an article comprising the recombinant protein, or the recombinant protein expressed by the recombinant Pichia pastoris strain, or the recombinant protein prepared by the method, or the recombinant expression vector, or the recombinant Pichia pastoris strain, or the composition thereof; preferably, the article is selected from pharmaceuticals, pharmaceutical compositions, medical devices, biomaterials, tissue engineering products, cosmetics, or health products.

[0024] The present invention also provides the use of the recombinant protein, or the recombinant protein expressed by the recombinant Pichia pastoris strain, or the recombinant protein prepared by the method, or the recombinant expression vector, or the recombinant Pichia pastoris strain, or the composition, or the product thereof in the preparation of pharmaceuticals, pharmaceutical compositions, medical devices, biomaterials, tissue-engineered products, cosmetics, and health products.

[0025] Furthermore, the applications include the preparation of highly cross-linked, high-viscosity products.

[0026] The beneficial effects of this invention are as follows:

[0027] This invention successfully constructed a recombinant strain that stably expresses TTC-2 recombinant collagen, and successfully and stably expressed the target protein without protein degradation during the expression process, demonstrating stable expression. The recombinant protein provided by this invention has been verified to have high stability, exhibiting a single band during fermentation, making it easy to purify. Compared with the control protein, the obtained protein has higher purity and is more suitable for application in medical devices. The purified TTC-2 recombinant collagen sample of this invention performed better than the control protein in viscosity testing experiments, providing better assurance for subsequent product applications. The recombinant collagen of this invention achieves higher expression efficiency and high stability while maintaining high biological activity, and contains no exogenous amino acids such as tags, exhibiting high biocompatibility and low risk of use. Compared to some methods that add chemical cross-linking agents such as formaldehyde and glutaraldehyde during production to achieve high cross-linking and viscosity characteristics in collagen, which carries the risk of side effects, the recombinant collagen provided by this invention is prepared through strain fermentation during production, with no chemical residues and no risk of exogenous tags. Attached Figure Description

[0028] Figure 1 The results of SDS-PAGE analysis of the supernatant of TTC-2 recombinant collagen expression in shake flasks (induction for 48 h).

[0029] Figure 2 SDS-PAGE analysis results of the supernatant from the 5L fermentation tank for expressing TTC01 and TTC-2 recombinant collagen.

[0030] Figure 3 Image of a small-scale test sponge sample of TTC-2 recombinant collagen.

[0031] Figure 4 This is the result of mass spectrometry analysis of the TTC-2 recombinant collagen sequence.

[0032] Figure 5 Diagram showing the preparation of TTC01 protein sponge solution.

[0033] Figure 6 Diagram showing the preparation of TTC-2 protein sponge solution.

[0034] Figure 7 This is a diagram showing the self-crosslinking results of the TTC01 protein.

[0035] Figure 8 This is a diagram showing the self-crosslinking results of the TTC-2 protein. Detailed Implementation

[0036] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. The technical solutions of the embodiments of the present invention are clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise defined, the technical or scientific terms used herein shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0038] The culture medium and formulation involved in the embodiments of the present invention are as follows:

[0039] YPG seed culture medium: yeast extract 10 g / L, peptone 20 g / L, glycerol 10 g / L;

[0040] Fermentation medium: NH4H2PO4 190.4g / L, KH2PO4 10.06g / L, CaSO4•2H2O 1.18g / L, K2SO4 18.2g / L, MgSO4•7H2O 14.9g / L, glycerol 40g / L; After the fermentation medium is sterilized at high temperature, wait for the temperature to drop to room temperature, add 4mL of PTM1 trace element per liter, and adjust the pH to 5 with ammonia water.

[0041] Feeding medium: 50% w / v glycerol, with 12 mL PTM1 trace element per liter;

[0042] Induction medium: 100% methanol, with 12 mL of trace elements added per liter;

[0043] PTM1 Trace Elements: Sterilize by filtration through a 0.22μm filter membrane and store at 4℃.

[0044] Example 1: Construction of recombinant strains and acquisition of recombinant protein sequences

[0045] The amino acid sequence of natural human type I collagen was obtained from the UniProt protein database (https: / / www.uniprot.org / uniprotkb / P02452). The biological information of the obtained sequence was deeply mined and analyzed. Protein fragments suitable for expression in Pichia pastoris, characterized by high stability and resistance to enzymatic cleavage during expression, were selected. Based on the characteristics of the amino acids, regions rich in large R groups were selected. Further optimization was performed according to the characteristics of exogenous proteins expressed in Pichia pastoris, resulting in the recombinant protein TTC-2. The sequence of TTC-2 is shown in SEQ ID No:1.

[0046] GAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQ GAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQGAPGPSGARGERGAPGDKGESGPSGPAGPTGARGAPGARGPAGPQGPRGDKGETGEQ

[0047] The nucleotides provided in this embodiment include fragments that encode amino acids suitable for expression in Pichia pastoris, which are not easily cleaved by enzymes during expression, have high stability, and are rich in R groups. Preferably, the nucleic acid molecule encodes the recombinant protein TTC-2.

[0048] The following work was commissioned to Suzhou Hongxun Biotechnology Co., Ltd.: Codon optimization was performed based on the preferences of Pichia pastoris, and the coding gene fragment for TTC-2 was synthesized. The synthesized gene fragment was cloned into the EcoRI and NotI spaces of the pPIC9K empty vector (purchased from Thermo Fisher Scientific), ensuring that the target fragment was accurately inserted into the reading frame of the secretory vector containing the secretion signal α-factor, thus obtaining the recombinant plasmid pPIC9K-TTC-2 expressing TTC-2. The nucleotide sequence of the coding gene for TTC-2 is shown in SEQ ID No:2.

[0049] SEQ ID No:2:

[0050]

[0051] Take 10 μg of the recombinant plasmid pPIC9K-TTC-2 expressing TTC-2 and digest it overnight at 37°C with SalI (purchased from TaKaRa, Dalian; specific procedures follow the kit instructions) to linearize it. Then, recover the linearized plasmid using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.), maintaining a volume of approximately 10 μL. Electroporate the linearized plasmid into competent Pichia pastoris GS115 cells (purchased from the China Industrial Microbial Culture Collection Center). Spread the electroporated bacterial suspension onto MD plates, 100 μL–200 μL per plate, incubate at room temperature for 10 min, and then incubate at 30°C upside down for 2–5 days until single colonies (positive transformants) appear. Add 2 mL of sterile double-distilled water to the surface of the MD plate, then gently scrape off the His+ transformants using a sterile triangular platter and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water. 5 One cell was spread on a YPD plate containing 0.5 mg / mL G418 and incubated upside down at 30°C for 3-4 days until a single colony was obtained, thus obtaining the Pichia pastoris engineered strain expressing TTC-2.

[0052] Example 2: Induced Expression and Identification of Recombinant Protein

[0053] The engineered Pichia pastoris strain expressing TTC-2 was placed in a 100mL Erlenmeyer flask containing 10mL of BMGY medium and cultured at 28-30℃ and 220rpm until the OD600 reached 10-15 (approximately 18-24 hours). The cells were centrifuged at 1500-3000g for 5 minutes at room temperature, and the cells were collected. The cells were resuspended in 10mL of BSM medium to an OD600 of approximately 2, and the culture was placed on a shaker at 28-30℃ and 220rpm for further growth and induction for 2 days. Every 24 hours, 100% methanol was added to the medium until the final concentration reached 1.0%. After 48 hours of methanol induction, a 1mL sample of the bacterial culture was collected and placed in a 1.5mL EP tube. The sample was centrifuged at 12000g for 5 minutes at 4℃, and the supernatant was collected. The sample to be tested was stored at -80℃ for later use.

[0054] The collected expression supernatant was added to 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), and heated in a 100°C metal bath for 10 min. SDS-PAGE analysis was then performed, with sequential loading of 5 μL, 10 μL, 15 μL, and 20 μL respectively. The detection results are as follows: Figure 1As shown. Analysis was performed using software (Image Lab) to calculate the proportion of the main band in each lane. The results showed that for different loading volumes of 5 μL, 10 μL, 15 μL, and 20 μL, the proportion of detected bands was 100%. Figure 1 The results, as well as the calculation results from the software, show that TTC-2 can be expressed efficiently and with a single band in shake-flask culture based on BSM medium.

[0055] The obtained strain, which stably and efficiently expresses the protein, was sent to the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 38641. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; accession date: May 11, 2026. Classification and nomenclature: *Pichia pastoris* Komagataella phaffii.

[0056] Example 3: Fermentation purification pilot test

[0057] The recombinant strain that stably and efficiently expressed TTC-2 recombinant type I collagen in Example 2 was tested in a 5L fermenter, and lyophilized sponge samples of TTC-2 recombinant type I collagen were obtained for further verification. At the same time, the expression strain in our patent CN201911135958.0 (full-length human type I α1 chain sequence, hereinafter referred to as TTC01) (deposited at the China General Microbiological Culture Collection Center, accession number CGMCC NO.17150) was compared, and the fermentation conditions of the two expression strains were consistent.

[0058] A fed-batch culture method was used, with the culture temperature set at 30℃. The recombinant strain expressing TTC-2 recombinant type I collagen was inoculated into a 1L shake flask containing 200mL of YPG seed culture medium and cultured at 220rpm and 30℃ for 18-20 hours until OD600 = 2~10. A 5L fermenter (Baoxing Biotechnology) was used, filled with 2L of fermentation medium. 2% glycerol was sterilized separately. Before inoculation, the fermentation speed was adjusted to 300rpm, aeration rate to 4L / min, and temperature to 30℃. The pH was adjusted to 4.5 using a concentrated ammonia solution. Then, 0.9mL of PTM1 was added to the fermenter, followed by 200mL of seed culture (inoculated using a flame ring). After calibrating with the dissolved oxygen electrode, fermentation began. When dissolved oxygen first drops to 30%, use the dissolved oxygen cascade stirring function to maintain dissolved oxygen at 30%. Wait for glycerol to deplete, and for dissolved oxygen to rebound to above 70% (OD600 value approximately 20). Then, cancel the dissolved oxygen cascade stirring and increase the stirring speed to 650 rpm. Use a 30% cascade feeding method, adding 80 mL of glycerol. Stop feeding glycerol. Once dissolved oxygen rebounds to above 70%, set the pH to 5 and the temperature to 29℃. Induce culture using a mixed carbon source of methanol and glycerol (methanol:50% glycerol = 7:3). Manually add 5 mL of the mixed carbon source (methanol:50% glycerol = 7:3). Once dissolved oxygen rebounds to above 70%, set the feeding rate to 8 mL / h. After one hour, increase to 10 mL / h, and then increase again to 20 mL / h after another hour. When dissolved oxygen drops below 30%, stop feeding and wait for dissolved oxygen to rebound. Resume feeding once dissolved oxygen returns to 30%. Induction lasts 40-60 hours. Once the protein concentration shows no significant increase or decrease as measured by UV, the culture vessel can be removed.

[0059] UV protein quantification formula: C(mg / mL)=0.144*(A215-A225), A215<1.5.

[0060] The fermentation supernatant was collected and analyzed by SDS-PAGE electrophoresis. 5 μL of sample was loaded, and the results are as follows: Figure 2 As shown, there are two groups for both TTC01 and TTC-2, representing two replicates; the results show that each strain can express the target protein efficiently; and it can be seen that TTC01 is severely degraded, while TTC-2 has no degradation band.

[0061] The fermentation supernatant obtained from the fermentation of TTC-2 recombinant type I collagen in a 5L fermenter was purified to obtain freeze-dried sponges of TTC-2 recombinant type I collagen. Based on the properties of TTC-2 recombinant type I collagen, ion exchange purification was performed. The raw materials, excipients, and purification equipment used are shown below:

[0062] Raw materials and excipients include: TTC-2 protein supernatant: obtained by fermentation in a 5L fermenter; phosphate (AR grade): Sinopharm Group; potassium dihydrogen phosphate (AR grade): Sinopharm Group; sodium chloride (AR grade): Sinopharm Group; UniGel-80SP: 500mL column volume, Nanomicro Technology.

[0063] The purification equipment involved includes: a chromatography system: model Bio-Lab100, purchased from Hanbang; an ultrafiltration system: model BONA-GM-18, purchased from Bona; and a freeze dryer: model Pilot2-4MC, purchased from Boyikang.

[0064] The purification steps are as follows:

[0065] Prepare the chromatography buffers: Buffer A: 20mM KH2PO4, pH 4.0; Buffer B: 20mM KH2PO4, 1M NaCl, pH 4.0.

[0066] Process flow: Fermentation supernatant ultrafiltration desalting → chromatography → eluent ultrafiltration desalting → freeze-drying into sponge; Fermentation supernatant pretreatment: ultrafiltration desalting of fermentation broth to a conductivity of 7 mS / cm, pH adjusted to 4.0. The chromatography process includes:

[0067] Column equilibration: Manual mode, flow rate set to 35 mL / min, inlet A1 (Buffer A), column position valve positive flush, continuously monitor the Cond conductivity curve and pH curve on the spectrum interface until the conductivity curve drops to its lowest point and flattens out (about 3 column volumes). After the pH curve flattens out, it means that the column has been equilibrated. Click pause.

[0068] Sample loading: Manual mode, flow rate set to 30 mL / min, inlet A2 (sample pH value, 1000 mL), click pause when the injection volume reaches the required volume.

[0069] Rebalancing: Manual mode, flow rate set to 35 mL / min, inlet A1 (Buffer A), click continue, run the program to continuously monitor the chromatogram curves, and continue balancing after the UV curve, conductivity curve and pH curve have stabilized, then click pause.

[0070] Elution: Manual mode, flow rate set to 35 mL / min, inlet B1 (Buffer B), set 25% B and 100% B for elution and collection respectively, click Continue. When the A215 spectrum curve rises, click Outlet1, click Continue to start eluting and collecting the target component until UV215 drops to its lowest point, at which point collection ends. Observe the eluent volume and detect the concentration.

[0071] Eluent treatment:

[0072] The collected eluent was ultrafiltered and desalted until the conductivity was below 1 ms, then lyophilized. The sponge was collected, and the sponge sample was as follows: Figure 3 As shown.

[0073] Through the above purification experiments, TTC-2 recombinant type I collagen lyophilized sponge was successfully obtained.

[0074] The TTC-2 recombinant type I collagen sponge obtained above was sent to Beijing Biotech Biotechnology Co., Ltd. for full-sequence sequencing. The test results are as follows: Figure 4 As shown in the figure, it is clear that the obtained TTC-2 recombinant type I collagen sequence is completely consistent with the theoretical sequence SEQ ID No:1. This invention successfully constructed a recombinant strain that stably expresses TTC-2 recombinant collagen and successfully and stably expressed the target protein. No protein degradation occurred during the expression process, and the expression process was stable.

[0075] Example 4: Self-crosslinking and viscosity test

[0076] The TTC-2 recombinant type I collagen sponge obtained in Example 3 above through small-scale testing was subjected to a self-crosslinking experiment with our existing product TTC01, and the viscosity of the crosslinked samples was compared.

[0077] Recombinant type I collagen sponges TTC01 and TTC-2 were prepared into 5 mL solutions with concentrations of 200 mg / mL, 250 mg / mL, 300 mg / mL, and 500 mg / mL, respectively, using ddH2O. The solutions were sterilized by 0.22 μm filtration. See [link to prepared solutions]. Figure 5 and Figure 6 The TTC01 and TTC-2 recombinant type I collagen sponges at various concentrations were completely dissolved, and the solutions were placed at 4°C.

[0078] After 16 hours of self-crosslinking, the solution was removed, and the centrifuge tubes were placed upside down on a table. The viscosity of TTC01 and TTC-2 at various concentrations was observed. Higher viscosity indicates that self-crosslinking is more likely to occur. The results for TTC01 are shown below. Figure 7 It can be seen that TTC01 has no obvious viscosity at various concentrations; the results for TTC-2 are shown in [reference needed]. Figure 8 It can be seen that as the concentration increases, TTC-2 exhibits significant viscosity at concentrations of 300 mg / mL and 500 mg / mL, enabling it to adhere to the wall.

[0079] Samples of TTC01 and TTC-2 after self-crosslinking were taken, and solutions of 200 mg / mL and 500 mg / mL were subjected to viscosity testing using a rheometer (manufacturer: TA Instruments, model: Discovery HR). The test results are shown in Table 1. As can be seen from the results in Table 1, the viscosity of TTC-2 after self-crosslinking, as measured by the rheometer, is significantly greater than that of TTC01.

[0080] Table 1. Viscosity test results for TTC-2 and TTC01

[0081]

[0082] In summary, the strain provided by this invention can stably express recombinant proteins. These recombinant proteins have a structure close to that of natural collagen, possess type I collagen biological activity, and exhibit high cross-linking properties, viscosity, and stability, providing better assurance for subsequent product applications. The recombinant collagen of this invention also has excellent cell adhesion activity and broad market application prospects.

Claims

1. A recombinant Pichia pastoris strain, characterized in that, The strain contains nucleotides that encode a recombinant protein, the amino acid sequence of which is shown in SEQ ID No:1; The recombinant Pichia pastoris strain is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38641.

2. The recombinant Pichia pastoris strain according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID No:

2.

3. A method for preparing a recombinant protein, characterized in that, The preparation method includes: fermenting and culturing the recombinant Pichia pastoris strain according to any one of claims 1-2 to express the recombinant protein.

4. A recombinant protein, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID No:

1.

5. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant protein expressed by the recombinant Pichia pastoris strain according to any one of claims 1-2, or the recombinant protein prepared by the method according to claim 3, or the recombinant protein according to claim 4.

6. The polynucleotide according to claim 5, characterized in that, The polynucleotide sequence is shown in SEQ ID No:

2.

7. A recombinant expression vector, characterized in that, The recombinant expression vector contains the polynucleotide as described in claim 5 or 6.

8. A composition, characterized in that, The composition comprises the recombinant protein expressed by the recombinant Pichia pastoris strain according to any one of claims 1-2, or the recombinant protein prepared by the method according to claim 3, or the recombinant protein according to claim 4, or the recombinant expression vector according to claim 7, or the recombinant Pichia pastoris strain according to any one of claims 1-2.

9. The product, characterized in that, The product comprises a recombinant protein expressed by the recombinant Pichia pastoris strain according to any one of claims 1-2, or a recombinant protein prepared by the method according to claim 3, or a recombinant protein according to claim 4, or a recombinant expression vector according to claim 7, or a recombinant Pichia pastoris strain according to any one of claims 1-2, or a composition according to claim 8; the product is selected from pharmaceuticals, medical devices, tissue-engineered products, cosmetics, or health products.

10. Use of the recombinant protein expressed by the recombinant Pichia pastoris strain according to any one of claims 1-2, or the recombinant protein prepared by the method according to claim 3, or the recombinant protein according to claim 4, or the recombinant expression vector according to claim 7, or the recombinant Pichia pastoris strain according to any one of claims 1-2, or the composition according to claim 8, or the article according to claim 9 in the preparation of pharmaceuticals, medical devices, tissue-engineered products, cosmetics, and health products.

11. The use according to claim 10, characterized in that, The applications include the preparation of highly cross-linked, high-viscosity products.

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