A Pichia pastoris strain capable of stably expressing recombinant collagen and its applications

Abstract

This invention provides a Pichia pastoris strain capable of stably expressing recombinant collagen and its applications. The strain uses a Pichia pastoris strain as the starting strain, expresses the recombinant collagen-encoding gene, and knocks out the YPS1 gene, a protein-degrading enzyme in Pichia pastoris. By optimizing the Pichia pastoris strain and knocking out its protein-degrading enzyme-related genes, the resulting mutant GS115-Col-yps1 expresses recombinant type III collagen with greater stability and higher purity. This provides a research basis and new approach for improving the expression and stability of recombinant collagen in Pichia pastoris.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a Pichia pastoris strain that can stably express recombinant collagen and its applications. Background Technology

[0002] Collagen is a biological macromolecule, a major component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals. It is an important bioactive material widely used in cosmetics, medicine, and other fields.

[0003] In existing technologies, collagen is obtained through animal tissue extraction and microbial fermentation. Compared to natural collagen, collagen derived through microbial fermentation overcomes the risk of viral contamination inherent in traditional extraction methods. Currently, host bacteria used for fermentation include Escherichia coli and yeast.

[0004] However, the Pichia pastoris strains currently used for recombinant type III collagen expression have been found to suffer from problems such as easy degradation and instability of the fermentation product, recombinant type III collagen, during actual production. Therefore, there is an urgent need to provide a recombinant yeast strain with more stable collagen expression and higher activity. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a Pichia pastoris strain capable of stably expressing recombinant collagen and its applications. This invention modifies a Pichia pastoris strain to express the recombinant collagen-encoding gene and further optimizes it by knocking out genes related to secretory protein-degrading enzymes. The resulting mutant expresses recombinant type III collagen with greater stability and higher purity. This invention provides a research basis and new approach for improving the expression level and stability of recombinant collagen in Pichia pastoris.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a Pichia pastoris strain that can stably express recombinant collagen, wherein the strain is based on a Pichia pastoris strain, expresses a recombinant collagen encoding gene, and knocks out the gene YPS1, a secretory protein degrading enzyme in Pichia pastoris.

[0008] This invention constructs a recombinant yeast strain with more stable collagen expression and higher activity. This provides a research basis and new approach for improving the expression and stability of recombinant collagen in Pichia pastoris.

[0009] YPS1 is the gene encoding the protease YAP3. The action site of YAP3 is located at the C-terminus of a single or paired basic amino acid. Since such sites are extremely common in proteins and polypeptides, most foreign proteins are subjected to degradation by the protease YAP3 when expressed in yeast cells. In this invention, knocking out the YPS1 gene can significantly increase the expression level of foreign proteins without significantly affecting the growth rate of the host.

[0010] Preferably, the Pichia pastoris strain is Pichia pastoris strain GS115.

[0011] In this invention, Pichia pastoris strain GS115 is used as the starting strain. GS115 is a histidine-deficient strain (His-), which is suitable for screening expression vectors carrying the his4 gene.

[0012] Preferably, the recombinant collagen encoding gene includes the one shown in SEQ ID NO.1.

[0013] In this invention, the gene encoding the recombinant collagen is optimized, and the optimized sequence is more suitable for expression in Pichia pastoris strain.

[0014] Preferably, the recombinant collagen-encoding gene is expressed by plasmid or chromosome integration.

[0015] Preferably, the plasmid includes pPIC9K, pGAPZa, or pPICZa, etc.

[0016] In this invention, the pPIC9K vector contains an alpha factor secretion signal peptide sequence, which can secrete and express exogenous protein genes; the pPIC9K vector also contains the promoter and transcription terminator of the alcohol oxidase-1 (AOX1) gene, which can be induced by methanol, making the extraction and purification process of recombinant proteins simpler and more convenient.

[0017] Preferably, the method for knocking out the gene YPS1, a secretory protein degrading enzyme, in Pichia pastoris includes:

[0018] A YPS1 gene editing vector was constructed using the CRISPR / Cas9 gene editing method targeting the YPS1 gene sequence. The YPS1 gene editing vector was then transformed into a Pichia pastoris expression strain containing a recombinant collagen-encoding gene, and the YPS1 gene in the strain was knocked out.

[0019] Preferably, the YPS1 gene editing vector includes gRNA and hsCas9.

[0020] Preferably, the gRNA targets the YPS1 gene; the DNA sequence corresponding to the gRNA includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0021] In this invention, the gRNA knockout effect is very good, and it can cause frameshift mutations in the target gene, thus having a good targeting effect.

[0022] Secondly, the present invention provides a YPS1 gene editing vector, the vector comprising: gRNA and hsCas9; the gRNA targets the YPS1 gene; the DNA sequence corresponding to the gRNA includes the sequences shown in SEQ ID NO.3 and SEQ ID NO.4.

[0023] Thirdly, the present invention provides a vector for expressing recombinant collagen, wherein the vector includes a recombinant collagen encoding gene.

[0024] Fourthly, the present invention provides a method for stably expressing recombinant collagen, the method comprising:

[0025] The Pichia pastoris strain, which can stably express recombinant collagen as described in the first aspect, was used for fermentation. The fermentation product was purified by a four-step hydrophobic chromatography method to obtain pure collagen.

[0026] Preferably, the four-step purification method of hydrophobic chromatography includes the following steps:

[0027] (1) Equilibration: The chromatography column was flushed with equilibration buffer at a linear flow rate until the baselines of pH, UV and Cond detection were stable and consistent with the equilibration buffer.

[0028] (2) Sample loading: Load the sample and collect the flow-through components during the loading process; after the sample loading is completed, rinse with equilibration buffer to wash out unbound material until the UV detection value drops to the initial value;

[0029] (3) Washing: Use elution buffer and equilibration buffer to wash away impurities with weak binding force;

[0030] (4) Elution: Elution is performed using elution buffer and equilibration buffer to elute the target protein and collect the elution fraction.

[0031] Preferably, in step (1), the equilibrium solution comprises, by concentration: 19-20 mM PB, 0.9-1 M ammonium sulfate, and pH 7.9-8.0. The 19-20 mM PB can be, for example, 19 mM, 19.5 mM, or 20 mM; the 0.9-1 M ammonium sulfate can be, for example, 0.9 M, 0.95 M, or 1 M; and the pH 7.9-8.0 can be, for example, 7.9 or 8.0.

[0032] Preferably, in step (1), the linear flow rate is 20-30 mL / min, for example, it can be 20 mL / min, 23 mL / min, 25 mL / min, 27 mL / min, 29 mL / min or 30 mL / min, etc., and the amount of equilibration solution is 2.5-3.5 times the column volume, for example, it can be 2.5, 2.7, 2.9, 3.0, 3.1, 3.3 or 3.5, etc.

[0033] Preferably, in step (2), the sample loading volume is 1.25-1.5 times the column volume, for example, it can be 1.25, 1.3, 1.35, 1.4, 1.45 or 1.5, etc.; the sample loading flow rate is 20-30 mL / min, for example, it can be 20 mL / min, 22 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 28 mL / min or 30 mL / min, etc.

[0034] In one specific embodiment, the 1.25-1.5 column volume is 500-600 mL.

[0035] Preferably, in step (2), the sample loaded is a mixture of fermentation broth supernatant and equilibration buffer of equal volume.

[0036] Preferably, in step (2), the rinsing flow rate is 20-30 mL / min, for example, it can be 20 mL / min, 22 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 28 mL / min or 30 mL / min, etc., and the amount of the equilibration buffer is 2-3 times the column volume, for example, it can be 2, 2.5 or 3, etc.

[0037] Preferably, in step (3), the volume ratio of the elution buffer to the equilibration buffer is (3-3.5):(6.5-7), for example, it can be 3:6.5, 3:7, 3.5:6.5 or 3.5:7, etc.

[0038] Preferably, the elution buffer comprises, by concentration: 19-20 mM PB, for example, 19 mM, 19.5 mM or 20 mM, etc., and pH 7.9-8.0, for example, 7.9 or 8.0, etc.

[0039] Preferably, in step (3), the flow rate of the washing process is 20-30 mL / min, for example, it can be 20 mL / min, 22 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 28 mL / min or 30 mL / min, etc.

[0040] Preferably, in step (4), the volume ratio of the elution buffer to the equilibration buffer is (6-6.5):(3.5-4), for example, it can be 6:3.5, 6:4, 6.5:3.5 or 6.5:4, etc.

[0041] Preferably, in step (4), the elution flow rate is 20-30 mL / min, for example, it can be 20 mL / min, 22 mL / min, 24 mL / min, 25 mL / min, 26 mL / min, 28 mL / min or 30 mL / min, etc.

[0042] In this invention, the above four-step purification method is supplemented with sample pretreatment, using the premixer of the purification instrument to mix the sample with the equilibration solution, which improves the stability of the target protein; the washing and elution process is simplified, and a small amount of equilibration solution and elution solution are used to successfully wash and elute.

[0043] In this invention, after eluting proteins, the chromatography column is further washed and stored. The column is washed with at least two column volumes of 1-1.2M NaOH at a rate of 15-20 mL / min to remove residual contaminants such as lipids, endotoxins, and nucleic acids. The column is then washed with 5-10 CV of distilled water at a rate of 15-20 mL / min until the effluent pH is neutral and Cond is close to 0, to remove NaOH. Finally, the column is washed with at least 3 CV of 20% ethanol for storage to prevent microbial growth.

[0044] Fifthly, the present invention provides the application of the Pichia pastoris strain capable of stably expressing recombinant collagen as described in the first aspect, the YPS1 gene editing vector as described in the second aspect, the vector for expressing recombinant collagen as described in the third aspect, or the method for stably expressing recombinant collagen as described in the fourth aspect in the preparation of recombinant collagen.

[0045] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0046] Compared with the prior art, the present invention has the following beneficial effects:

[0047] This invention constructs a strain with significantly improved recombinant collagen yield and stability; and optimizes the hydrophobic chromatography purification process, simplifying the process, shortening the cycle, and improving the collagen recovery rate. Attached Figure Description

[0048] Figure 1 This is the identification result of the Yps1-1 transformant.

[0049] Figure 2 This is the identification result of the Yps1-2 transformant.

[0050] Figure 3 This is a schematic diagram of a recombinant plasmid.

[0051] Figure 4 These are growth curves for different transformants.

[0052] Figure 5 This is the result of electrophoresis detection of the fermentation broth supernatant.

[0053] Figure 6 This is a chromatographic diagram of the starting strain GS115-Col.

[0054] Figure 7 This is a chromatographic diagram of the mutant GS115-Col-yps1-1.

[0055] Figure 8 This is a chromatographic diagram of the mutant GS115-Col-yps1-2.

[0056] Figure 9 This is the quantitative detection result of BCA protein in Example 1.

[0057] Figure 10 This is the result of quantitative detection of BCA protein in COL2 in Comparative Example 1. Detailed Implementation

[0058] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0059] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0060] Example 1

[0061] 1. Design gRNA sequences

[0062] The YPS1 gene encoding the Pichia pastoris GS115 aspartic protease was located in the NCBI database. Based on its nucleotide sequence, the corresponding gRNA sequence was designed using the CHOPCHOP website (https: / / chopchop.cbu.uib.no / ), and the corresponding gRNA expression cassettes were obtained.

[0063] The DNA sequence of the YPS1 gene is shown in SEQ ID NO.2.

[0064] A gRNA targeting the YPS1 gene, the DNA sequence of which is shown in SEQ ID NO.3 and SEQ ID NO.4:

[0065] SEQ ID NO. 3: AGTTTGTGCTCGGTGATACCTT.

[0066] SEQ ID NO. 4: TATTCAATTTTGGGGGTGCCTAC.

[0067] 2. Construction of recombinant plasmid vectors

[0068] The recombinant vector pPpT4_pHTX1-PARS1-YPS1-gRNA-hsCas9 was synthesized and constructed by Suzhou Biotechnology Co., Ltd. The Pichia pastoris gene editing plasmid vector pPpT4_pHTX1-PARS1-hsCas9 was digested with NotI and then ligated to two gRNA expression cassette clones of the YPS1 gene to obtain pPpT4_pHTX1-PARS1-YPS1-1-gRNA-hsCas9 and pPpT4_pHTX1-PARS1-YPS1-2-gRNA-hsCas9, respectively.

[0069] 3. Transfection of the recombinant plasmid into Pichia pastoris strain GS115-Col

[0070] Using the pPpT4_pHTX1-PARS1-gRNA-hsCas9 plasmid vector, hsCas9 and gRNA synergistically target and cleave the yeast genome. Following repair via non-homologous end ligation, knockout mutants of the corresponding genes can be easily obtained through simple transformation and screening. A simplified method for preparing Pichia pastoris electrocompetent cells and the preparation of electroporation-transformed Pichia pastoris electrocompetent cells followed the method described in (Lin-Cereghino J, 2005). Specific steps included:

[0071] (1) Pichia pastoris strains that need to be prepared into competent states were streaked on YPD plates and incubated at 30°C for two days.

[0072] (2) Pick a healthy single colony and incubate it overnight in a shaker at 30°C.

[0073] (3) Transfer an appropriate amount of overnight culture to a 1L Erlenmeyer flask containing 100mL of liquid YPD, and control the initial OD. 600 Incubate at 0.15-0.2°C in a shaker at 30°C.

[0074] (4) To OD 600 Cells were collected by centrifugation at room temperature for 5 minutes at a concentration of 0.8-1.0 g.

[0075] (5) Resuspend the cells in 9 mL of BEDS solution pre-cooled on ice, and add 1 mL of 1 M DTT solution dropwise. Incubate at 30 °C on a shaker (100 rpm) for 5 min.

[0076] (6) Collect cells by centrifugation at room temperature for 5 minutes at 500g, and resuspend the cells in 2 mL of BEDS pre-cooled on ice.

[0077] (7) Dispense the resuspended cells into pre-cooled 1.5mL centrifuge tubes, 50μL per tube, and quickly store the prepared competent cells in a -80℃ freezer.

[0078] (8) Mix 100 ng circular or 2 μg linear plasmid DNA with 50 μL competent cells and incubate the mixture on ice for 2 min.

[0079] (9) Take the mixture of competent cells and plasmids into an electroporation cup pre-cooled on ice at 2 mm, wipe the water off the cup, and then place it in the groove of the electroporation instrument. After a 1.5 kV 5.0 s pulse.

[0080] (10) Immediately add 1 mL of a 1:1 mixture of YPD and 1 M sorbitol. After the cells in the electroporation cup are thoroughly mixed by pipetting, the solution is dried and placed in a 1.5 mL sterile centrifuge tube. Incubate at 30°C on a shaker for 1 h. Spread the solution onto MD or YPD (with a final concentration of 100 μg / mL Zeocin) plates.

[0081] 4. Screening and identification of transformants

[0082] The plates were incubated at 30°C for 3 days, and single colonies grew on all plates. Ten single colonies were picked from each transformed plate and cultured in liquid YPD medium containing 100 μg / mL Zeocin for 16 h. Then, an appropriate amount of bacterial culture was taken to extract genomic DNA, and the nucleotide sequence near the gene mutation site was sequenced. Finally, the Cas9 plasmid in the successfully identified strains was eliminated by subculturing in antibiotic-free YPD medium, and Zeocin-free Pichia pastoris strains were screened. Since the pPpT4_pHTX1-PARS1-hsCas9 plasmid carries a Pichia pastoris autonomous replication sequence (PARS1), the PARS1 sequence is easily lost under antibiotic-free conditions. Therefore, after screening for the correct knockout strain, only subculturing in antibiotic-free medium is needed to obtain the CRISPR / Cas9 plasmid-eliminated knockout mutant strain.

[0083] Pichia pastoris genome extraction reference The method of Fungal DNA Mini Kit (OMEGA):

[0084] (1) Take 10-50 mg of yeast cells into a 2 mL centrifuge tube, add 600 μL of CSPL buffer, and vortex vigorously.

[0085] (2) Incubate in a 65℃ water bath for 30 minutes. During the incubation process, turn the centrifuge tube several times to ensure that the sample is thoroughly mixed.

[0086] (3) Add 600 μL of chloroform:isoamyl alcohol (24:1), vortex vigorously, and centrifuge at 12000 rpm for 10 min.

[0087] (4) Carefully aspirate 300 μL of the supernatant into a new 1.5 mL centrifuge tube.

[0088] (5) Add 150 μL of CXD buffer and 300 μL of anhydrous ethanol to the centrifuge tube and vortex until homogeneous.

[0089] (6) Add the vortexed solution, including the precipitate, to the container fitted with the Collection Tube. Centrifuge at 12000 rpm for 1 min in the DNAMini column.

[0090] (7) Discard the supernatant, add 650 μL of DNA elution buffer, and centrifuge at 12000 rpm for 1 min.

[0091] (8) Repeat step 7 to wash the adsorbed DNA again.

[0092] (9) Centrifuge the empty adsorption column at the highest speed for 2 minutes.

[0093] (10) The DNAMini Column was transferred to a clean 1.5 mL centrifuge tube.

[0094] (11) Add 100 μL of elution buffer or deionized water heated to 65°C, and centrifuge at the highest speed for 1 min.

[0095] (12) Aspirate the collected solution containing genomic DNA into a DNA Mini Column and centrifuge at the highest speed for 1 min.

[0096] (13) Store the obtained genomic DNA at -20℃ for later use.

[0097] PCR detection was performed using detection primers. The PCR reaction conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 63℃ annealing for 30 s, 72℃ extension for 2 min, 35 cycles; 72℃ extension for 5 min. The PCR products were sequenced by Beijing Qingke Biotechnology Co., Ltd. The sequencing results were compared with the original genome sequence to screen for the correct knockout mutant.

[0098] The sequence of the upstream primer was detected as: 5'-GCTTTGATAGCGAGCGTACC. (SEQ ID NO.5)

[0099] The sequence of the downstream primer was detected as: 3'-CTGCTTCTTCCTCCCTCGAG. (SEQ ID NO.6)

[0100] Transformant sequencing alignment results are as follows Figure 1 and Figure 2 As shown, Figure 1 The identification results of the Yps1-1 transformant. Figure 2 The identification results for the Yps1-2 transformant.

[0101] 5. Comparison between the original strain and the recombinant strain, including expression level, yield, and stability.

[0102] The originating strain's secretory expression vector pPIC9K contains an alpha factor secretion signal peptide sequence, enabling the secretion and expression of exogenous protein genes. This vector also contains the promoter and transcription terminator of the alcohol oxidase-1 (AOX1) gene, separated by a multiple cloning site (MCS), where the coding sequence for recombinant collagen is inserted. Figure 3 As shown.

[0103] The sequence of the alpha factor secretion signal peptide is shown in SEQ ID NO.7. The sequence of the promoter-coding sequence of recombinant collagen-1 (AOX1) gene-transcription terminator is shown in SEQ ID NO.8. The sequence of the expression vector pPIC9K (empty vector) is shown in SEQ ID NO.9.

[0104] The secretory expression of recombinant collagen is controlled by the AOX1 promoter and can be induced by methanol. The secretory expression method is as follows:

[0105] Prepare YPD, BMGY, and BMMY culture media respectively.

[0106] BMGY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 1% glycerol and 10% 1M PBS (pH=6.0).

[0107] BMMY medium: 1% yeast extract, 2% peptone, 1.34% YNB, 1% methanol and 10% 1M PBS (pH=6.0).

[0108] YPD solid medium: 1% yeast extract, 2% peptone, 2% glucose and 2% agar powder.

[0109] First, the recombinant collagen expression strain was streaked on YPD solid medium and incubated at 30℃ for 20-24 hours to activate the strain. Single colonies were picked and transferred to 10 mL of BMGY medium and incubated in a shaker at 30℃ for 24 hours. The OD value of the bacterial solution was measured, and an appropriate amount of the bacterial solution was centrifuged at 5000 rpm for 5 minutes. The bacterial cells were resuspended in BMMY medium and washed once. The washed bacterial cells were transferred to 50 mL of BMMY medium and 1% methanol was added. The culture was incubated in a shaker at 30℃ for 5 days to start fermentation, with 1% methanol added every 24 hours. The fermentation broth was centrifuged at high speed, and the supernatant was collected for electrophoresis. The recombinant collagen was then purified by hydrophobic chromatography and other methods to obtain pure recombinant collagen.

[0110] Detection of bacterial OD in BMGY culture medium during fermentation 600 Plot the growth curve ( Figure 4 The study found no significant difference in growth rate between the knockout mutant and the starting strain.

[0111] Electrophoresis was used to detect the protein expression in the supernatant of each fermentation broth. Figure 5 The results showed that the content of the target protein in the two knockout mutants was significantly higher than that in the original strain.

[0112] 6. Comparison of recombinant collagen yields after hydrophobic chromatography purification

[0113] Both the knockout mutant and the original strain were fermented in 600 mL of water. After centrifugation, the supernatant was purified by hydrophobic chromatography. The purification process is as follows:

[0114] Sample: Supernatant of recombinant collagen fermentation broth

[0115] Hydrophobic chromatography column: PrePack 50×200-Phenyl Purose 6FF, purchased from Qianchun Biotechnology Co., Ltd.

[0116] Protein purification instrument from Saipu Biotechnology Co., Ltd.

[0117] Experimental materials: distilled water;

[0118] Fermentation broth sample: 300 mL fermentation broth supernatant.

[0119] Equilibration buffer: 20mM PB, 1M ammonium sulfate, pH 8.0.

[0120] Elution buffer: 20 mM PB, pH 8.0.

[0121] Washing buffer: 1M NaOH.

[0122] The above solution must be filtered using a 0.45μm filter.

[0123] 7. Experimental steps:

[0124] (1) Wash the chromatography column with equilibration buffer at a linear flow rate of 30 mL / min for 3CV (3 column volumes, 1CV = 400 mL) until the pH, UV, and Cond detection baselines are stable and consistent with the equilibration buffer.

[0125] (2) Load the sample and equilibration buffer at a ratio of 1:1 at a speed of 20 mL / min, collect the flow-through components during the loading process, and detect the purity and content of the target protein.

[0126] (3) After the sample loading is completed, rinse 2CV with equilibration buffer at a speed of 30 mL / min to wash out unbound material until the UV detection value drops to the initial value.

[0127] (4) Wash the elution buffer and equilibration buffer at a ratio of 3:7 at a rate of 30 mL / min for 2CV to remove impurities with weak binding force.

[0128] (5) Elute the target protein by mixing the elution buffer and equilibration buffer at a ratio of 6:4 at a rate of 30 mL / min for 2CV, and collect the elution fraction.

[0129] (6) Wash the chromatographic column with at least 2 column volumes of 1M NaOH at a rate of 20 mL / min to remove residual contaminants in the packed column, such as lipids, endotoxins, nucleic acids, etc.

[0130] (7) Wash the chromatography column with 5-10CV of distilled water at a rate of 20mL / min until the pH of the effluent is neutral and Cond is close to 0, in order to remove 1M NaOH.

[0131] (8) Wash and store with 20% ethanol in 3CV to prevent microbial growth.

[0132] Figure 6 This is a chromatographic diagram of the starting strain GS115-Col; Figure 7 Chromatographic diagram of the mutant GS115-Col-yps1-1; Figure 8 This is a chromatographic diagram of the mutant GS115-Col-yps1-2.

[0133] Chromatographic process diagram: The vast majority of recombinant collagen was eluted by 60% elution buffer and 40% equilibration buffer. Among them, the elution peak area of ​​the recombinant protein obtained by fermentation of mutant was significantly larger than that of the starting strain, indicating that the yield of recombinant collagen from mutant was significantly greater than that from the starting strain.

[0134] 8. Electrophoretic detection of hydrophobic chromatography samples

[0135] Electrophoresis results are shown Figure 5 Electrophoresis results showed that the target protein band of the mutant was significantly more concentrated than that of the original strain, indicating that the target protein yield of the mutant was higher than that of the original strain.

[0136] 9. Comparison of sample concentrations after hydrophobic chromatography purification

[0137] After chromatography, the protein concentrations of the eluted fractions of each sample were compared. The protein samples were quantified according to the instructions of the BCA protein quantification kit, and statistical analysis was performed using Prism7 software.

[0138] Figure 9 The results of BCA protein quantification showed that the content of recombinant collagen produced by the mutant fermentation was significantly higher than that of the starting strain, indicating that the knockout of the YPS1 gene significantly improved the stability of the recombinant collagen produced by secretion.

[0139] The plasmid vector used for knockout is shown in SEQ ID NO.10. The recombinant collagen-encoding gene is shown in SEQ ID NO.1.

[0140] Comparative Example 1

[0141] This embodiment provides a method for preparing recombinant collagen. The difference between this method and the method in Example 1 is that the recombinant collagen encoding gene COL2 expressed is shown in SEQ ID NO.11.

[0142] The final target protein content before and after YPS1 knockout was determined using the BCA method. A statistical chart of the target protein content is shown below. Figure 10 ,from Figure 10 It can be seen that the content of recombinant collagen produced by the mutant fermentation is not much different from that of the starting strain, and the yield of the above protein is lower than that of the protein shown in SEQ ID NO.1 in this application, indicating that the Pichia pastoris strain that stably expresses recombinant collagen constructed in this application has a better expression effect.

[0143] Comparative Example 2

[0144] This comparative example provides a method for purifying recombinant collagen, including the following steps:

[0145] 1) Wash the column with equilibration buffer at a linear flow rate of 60 cm / h (20 mL / min) for 1-2 CV until the pH, UV, and Cond detection baselines are stable and consistent with the equilibration buffer.

[0146] 2) Load the sample at a speed of 60 cm / h, collect the flow-through components during the loading process, and detect the purity and content of the target protein.

[0147] 3) After loading the sample, rinse the 3CV with equilibration buffer at a speed of 60 cm / h to wash out unbound material until the UV detection value drops to the initial value.

[0148] 4) Elute with elution buffer and equilibration buffer at a rate of 60 cm / h for 5 CV according to a gradient of 100%-0% equilibration buffer, and collect the eluent.

[0149] 5) Wash the chromatographic column with at least 2 column volumes of 1M NaOH at a speed of 60 cm / h to remove residual contaminants in the packed column, such as lipids, endotoxins, nucleic acids, etc.

[0150] 6) Wash the chromatography column with 5-10 CV of distilled water at a rate of 60 cm / h until the pH of the effluent is neutral and Cond is close to 0, in order to remove 1M NaOH;

[0151] 7) Wash and store with 20% ethanol in 3CV to prevent microbial growth.

[0152] In step 2) of the aforementioned comparative method, no sample pretreatment was added, and the sample was not diluted with equilibration buffer, which affected the stability of the sample during purification. In step 4) of the aforementioned comparative method, the elution buffer and equilibration buffer were eluted at a rate of 60 cm / h according to a 100%-0% equilibration buffer gradient of 5 CV, and the eluent was collected. In this invention, however, a fixed ratio of elution buffer and equilibration buffer of 2 CV each is used for elution, and the flow rate is increased to 90 cm / h. The elution method of this invention has better elution effect and yields higher protein purity.

[0153] In summary, this invention has constructed a Pichia pastoris strain that can stably and highly express recombinant collagen. The above strain expresses recombinant collagen more stably and with higher purity, providing a research basis and new approach for improving the expression and stability of recombinant collagen in Pichia pastoris.

[0154] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A Pichia pastoris strain capable of stably expressing recombinant collagen, characterized in that, The strain takes Pichia pastoris strain GS115 as a starting strain, and expresses a recombinant collagen coding gene, and the recombinant collagen coding gene is shown as SEQ ID NO.

1. A YPS1 gene editing vector is constructed by using a CRISPR / Cas9 gene editing method to target a target sequence of the YPS1 gene, the YPS1 gene editing vector is transformed into the Pichia pastoris expression strain containing the recombinant collagen coding gene, and the YPS1 gene in the strain is knocked out; the YPS1 gene editing vector comprises: gRNA and hsCas9; the gRNA targets a site of the YPS1 gene; the DNA sequence corresponding to the gRNA is shown as SEQ ID NO. 3 and SEQ ID NO.

4.

2. A method of stably expressing a recombinant collagen, characterized by, The method comprises: The Pichia pastoris strain capable of stably expressing the recombinant collagen in claim 1 is used for fermentation, and the fermentation product is purified by a four-step method of hydrophobic chromatography to obtain pure collagen.

3. The method for stably expressing recombinant collagen according to claim 2, characterized in that, The four-step method of hydrophobic chromatography comprises the following steps: (1) Equilibrium: linear flow rate of the equilibrium liquid is used to flush the chromatography column until the pH, UV and Cond detection baseline is stable and consistent with the equilibrium buffer; (2) Sample loading: sample loading and flow-through components during sample loading are collected; after sample loading is completed, the equilibrium buffer is used for flushing to wash out the unbound materials until the ultraviolet detection value decreases to the initial value; (3) Impurity washing: elution buffer and equilibrium buffer are used for impurity washing to remove impurities with weak binding force; (4) Elution: elution buffer and equilibrium buffer are used for elution to elute the target protein, and elution components are collected.

4. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (1), the equilibrium liquid comprises, in terms of concentration: 19-20 mM PB, 0.9-1 M ammonium sulfate, pH 7.9-8.

0.

5. The method of claim 3, wherein the recombinant collagen is stably expressed. In step (1), the linear flow rate is 20-30 mL / min, and the amount of the equilibrium liquid is 2.5-3.5 times the column volume.

6. The method of claim 3, wherein the recombinant collagen is stably expressed. In step (2), the sample loading amount is 1.25-1.5 times the column volume; and the flow rate of sample loading is 20-30 mL / min.

7. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (2), the sample for sample loading is an equal-volume mixture of fermentation liquid supernatant and equilibrium buffer.

8. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (2), the flow rate of flushing is 20-30 mL / min, and the amount of the equilibrium buffer is 2-3 times the column volume.

9. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (3), the volume ratio of the elution buffer to the equilibrium buffer is (3-3.5):(6.5-7).

10. The method for stably expressing recombinant collagen according to claim 9, characterized in that, The elution buffer comprises, in terms of concentration: 19-20 mM PB, pH 7.9-8.

0.

11. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (3), the flow rate of impurity washing is 20-30 mL / min.

12. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (4), the volume ratio of the elution buffer to the equilibrium buffer is (6-6.5):(3.5-4).

13. The method for stably expressing recombinant collagen according to claim 3, characterized in that, In step (4), the flow rate of elution is 20-30 mL / min.

14. Use of the Pichia pastoris strain capable of stably expressing the recombinant collagen in claim 1 or the method for stably expressing the recombinant collagen in any one of claims 2-13 in the preparation of a recombinant collagen.

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