Production method of recombinant collagen in pichia pastoris and pichia pastoris chassis cell

Abstract

The invention discloses a production method of recombinant collagen in pichia pastoris and a pichia pastoris chassis cell, and the production method comprises the following steps: 1, codon optimization: adjusting the codon sequence of an exogenous gene COL fragment to be matched with the most common codon of pichia pastoris so as to improve the translation efficiency and the protein expression level; 2, in order to improve the stability of a collagen COL fragment, the fragment is repeated for four times, an enterokinase cleavage site is fused to the N end of a collagen COL structural domain so that a label can be conveniently removed, pichia pastoris is used for successfully secreting and expressing the recombinant collagen variant C109, purification is convenient, the cost is low, and the method has the basis for large-scale production; the expressed collagen is confirmed to be C109 through identification, has a triple helix structure like traditional collagen, and has good characteristics of forming hydrogel at low temperature.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, in particular to a method for producing recombinant collagen in Pichia pastoris and Pichia pastoris chassis cells. Background Art

[0002] Collagen is an extracellular matrix protein present throughout the human body and has multiple important biological functions in normal physiology. Collagen can self-assemble into striated fibrils, which not only provide support for cell growth but are also responsible for the mechanical elasticity of connective tissue. Based on differences in amino acid sequence, function, and protein structure, 29 different collagens (I–XXIX) have been identified. Despite the expanding market demand for collagen in the fields of biomedicine, cosmetics, and health foods, the traditional method of extracting collagen from animals is expensive, inefficient, and unsuitable to meet the growing industrial demand. Therefore, various expression systems (including insects, animal cells, plants, bacteria, and yeast) have been developed to synthesize recombinant collagen. With increasing usage, the collagen market is expected to reach US$6.63 billion by 20254. Although the synthesis of recombinant collagen has achieved good commercialization, further research and expansion are needed to improve biosafety, yield, and functionality. In addition, full-length collagen is poorly soluble and has low yields, so it is necessary to identify new functional collagen fragments that can be commercially produced for use as biomaterials.

[0003] Pichia pastoris is a type of methanol-trophic yeast that can use methanol as the sole carbon source and energy source. Like other yeasts, it mainly exists in a haploid form during the asexual growth period. When the environment is nutrient-limited, it often induces two mating haploid cells of different physiological types to mate and fuse into a diploid.

[0004] The existing recombinant collagen variants have a low production speed and high cost, and therefore cannot be promoted for large-scale production. During the research and development process, the inventors found that Pichia pastoris can be used to secrete and express recombinant collagen variants, and the purification cost is low. Therefore, a method for producing recombinant collagen in Pichia pastoris is proposed to reduce the production cost of recombinant collagen variants. Summary of the invention

[0005] The object of the present invention is to provide a method for producing recombinant collagen in Pichia pastoris and Pichia pastoris chassis cells to solve the problems raised in the above background technology.

[0006] In order to solve the above technical problems, the present invention provides the following technical solution: a method for producing recombinant collagen in Pichia pastoris, comprising the following steps: Step 1: Codon optimization: adjust the codon sequence of the exogenous gene COL fragment to match the most commonly used codons in Pichia pastoris to improve translation efficiency and protein expression level.

[0007] Step 2: To improve the stability of the collagen COL fragment, the fragment was repeated four times, and the enterokinase cleavage site was fused to the N-terminus of the collagen COL domain to facilitate the removal of the tag; Step three: Optimize the expression frame for expression verification, try different promoters and signal peptide elements to express the collagen. Since the promoter plays a key role in the expression of heterologous proteins, the promoter and signal peptide of the pPIC9K plasmid are experimentally compared to find that the combination of AOX1 and a-factor is most beneficial for expressing C109. Finally, the expression frame with AOX1 as the promoter and a-factor as the signal peptide is determined. The present invention uses Pichia pastoris to successfully secrete and express the recombinant collagen variant C109, and the purification is convenient and low-cost, making it a basis for large-scale production. The expressed collagen is confirmed to be C109 by identification, and has a triple helix structure like traditional collagen, and has good properties of forming hydrogels at low temperatures.

[0008] Step 4: construct an extracellular enterokinase expression cassette and integrate it into the Pichia pastoris genome to facilitate the extracellular secretion of collagen with high purity and without traces; Step 5: Knock out the main protease Pep4; Step 6: Fed-batch culture in 5L tank to confirm the suitable fermentation process for the chassis cells.

[0009] According to the above technical scheme, the enterokinase tag is a commonly used biotechnology tool, usually used to obtain traceless proteins. It is an enzyme widely used in molecular biology and protein purification, and has a specific cleavage site. The characteristic of enterokinase cleavage is that it recognizes and cleaves a specific amino acid sequence, usually "DDDDK*", where "*" represents the cleavage site. In the expression and purification of recombinant proteins, the enterokinase cleavage site is often inserted between the tag and the target protein, and the amino acids remaining after the secretion of the signal peptide are removed by enterokinase cleavage to obtain a purified target protein.

[0010] According to the above technical scheme, the AOX1, AOX2, FLD1, FLD2, THI11, DC, DI1, FDH, THI1, ADS1, and ADS2 promoters in Pichia pastoris are inducible promoters that can be used for the expression of recombinant proteins and can be efficiently activated in the presence of methanol, thereby promoting the expression of target proteins. The chassis cells are constructed by screening the most suitable promoters.

[0011] According to the above technical scheme, a-factor, a-MF, INUa-F86, INUa-Q86, PHO1, Kex2, HSS, nSB, Fre2, and AF-27 signal peptides in Pichia pastoris are common signal peptides for protein secretion, and are mainly used to promote the secretion of recombinant proteins to the extracellular space.

[0012] According to the above technical scheme, the SDS-PAGE (polyacrylamide gel electrophoresis) is a commonly used biochemical technique, in which a sample is loaded into a gel and an electric field is applied to cause the protein to migrate in the gel. Proteins with small molecular weight migrate quickly, while proteins with large molecular weight migrate slowly, thereby achieving separation. After the electrophoresis is completed, the gel is stained with a dye (such as Coomassie Brilliant Blue) to visualize the separated protein bands.

[0013] According to the above technical scheme, the Maldi-tof (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry) is a powerful mass spectrometry analysis technology that can quickly identify proteins in a sample by comparing the mass spectra of known proteins.

[0014] According to the above technical scheme, a PGAP-afactor-enterokinase-TAOX1 expression frame was constructed and integrated into the Pichia pastoris Int23 site, so that Pichia pastoris could express enterokinase and achieve seamless separation from the signal peptide when collagen was secreted into the extracellular space, which was confirmed by N-terminal sequencing.

[0015] According to the above technical scheme, in order to avoid the degradation of collagen by its endogenous protease during the expansion culture and fermentation, the main protease Pep4 was knocked out; According to the above technical scheme, the constructed Pichia chassis strain producing C109 collagen variant was fermented in a 5L fermenter in a fed-batch manner. First, a single colony was fermented in YPD medium for 24 h to prepare the seed solution, and a fully fed batch fermentation was carried out in a 5L fermenter (T&J Bioengineering, Shanghai, China) containing 1.8 LBSM medium and 10% (v / v) inoculum. The temperature was set to 30 °C, and the pH was adjusted to 5.5 with 50% (v / v) ammonia. After the dissolved oxygen returned to 55%, 50% (v / v) glycerol and 1.2% (v / v) PTM1 were added at a rate of 6 mL / L / h. When the wet cell weight reached 200 g / L, the pH was adjusted to 5.0, and the temperature was maintained at 30°C while supplementing the carbon source. During the entire fermentation process, the dissolved oxygen was maintained at 20-30% under controlled stirring and aeration. Beneficial Effects

[0016] The present invention successfully secretes and expresses the recombinant collagen variant C109 using Pichia pastoris, and the purification is convenient and low-cost, making it a basis for large-scale production; the expressed collagen protein is confirmed to be C109 by identification, and has a triple helix structure like traditional collagen, and has a good property of forming a hydrogel at low temperature. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation of the present invention. In the accompanying drawings: Figure 1 The schematic diagram of the expression frame for optimizing the recombinant collagen protein, replacing different promoters and signal peptides, and finally determining the AOX1 promoter and a-factor signal peptide as the final expression frame element for expressing C109 in Pichia pastoris; Figure 2 is an SDS-PAGE analysis of the C109 secretory COL variant; Figure 3 It is a schematic diagram of time-of-flight mass spectrometry identification; Figure 4 It is the components of the expression cassette: gel map and bar graph of the promoter; Figure 5 It is the components of the expression cassette: gel image and bar graph of the signal peptide; Figure 6 is a schematic diagram of extracellular Pep4 knockout; Figure 7 Schematic diagram of the enterokinase expression cassette integrated into the Int23 site of the Pichia pastoris genome; Figure 8 It is a line chart of the tank loading output of the present invention; Fig. 9 The reliability of protein identification was verified by MaldiT of MS of the present invention, and the schematic diagram of the score of sample C109 was 116. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Example

[0019] A method for efficiently producing recombinant collagen in Pichia pastoris chassis cells comprises the following steps: Step 1: To improve the stability of the collagen COL fragment, C109 was codon-optimized for Pichia pastoris and the fragment was repeated four times, and the thrombin cleavage site was fused to the N-terminus of the collagen COL domain to facilitate the removal of residual signal peptide amino acids; Step 2: Optimize the expression frame for expression verification. Finally, the expression frame with AOX1 as the promoter and a-factor as the signal peptide was determined. Since the promoter plays a key role in the expression of heterologous proteins, the promoter and signal peptide of the pPIC9K plasmid were compared experimentally and it was concluded that the combination of AOX1 and a-factor was most beneficial for the expression of C109. Step 3: Integrate the enterokinase expression cassette into the Pichia pastoris genome to facilitate the extracellular secretion of collagen with high purity and without traces; Step 4: Knock out the endogenous protease Pep4 in Pichia pastoris to avoid severe collagen degradation during expansion culture; Step 5: Verification was performed using SDS-PAGE and Maldi-tof; Example

[0020] Recombinant expression of recombinant collagen variant C109 in Pichia pastoris: (1) Recombinant expression of recombinant collagen variant C109 in Pichia pastoris: Integration was performed using plasmid pPIC9k in P. pastoris GS115. The gene of recombinant collagen variant C109 was optimized and synthesized by Genscript (Nanjing, China). In Pichia pastoris, the amino acid fragments were replicated multiple times to reach a length of 570-650 amino acids to facilitate recombinant expression. The gene was cloned into pPIC9k, and the plasmid was linearized through the SaI I site and integrated into the Pichia pastoris genome before transformation. The single colony with the correct colony PCR was inoculated into YPD medium as a seed culture, cultured in a shaker at 30°C and 220 rpm for 24 h, and then inoculated into BMMY medium, 1% methanol was added to induce protein expression, and cultured in a shaker at 30°C and 220 rpm for 48 h, of which 1% methanol was supplemented once every 24 h, and the fermentation supernatant was collected.

[0021] (2) Extraction and purification of recombinant collagen variant C109 from Pichia pastoris: The fermentation broth obtained by the above culture was centrifuged to collect the supernatant. Protein purification was performed using molecular chromatography using an ion exchange column Q Purose 6 (Tofflon). Recombinant collagen variant C109 was eluted by linear gradient elution using 20 mM Tris-HCl and 0.5M NaCl (pH 7.0). The collagen solution can restore its native structure after high temperature induced unwinding, so the purified sample was subjected to protease cleavage to remove residual amino acids, and then heated to 85°C, cooled, centrifuged and dialyzed to remove contaminating proteins and cleaved tags to obtain highly pure recombinant collagen variant C109. Example

[0022] The samples obtained by fermentation of Pichia pastoris in Example 2 were subjected to SDS-PAGE analysis. The results showed that the recombinant collagen variant C109 recombinantly expressed in Pichia pastoris was secreted in a soluble form, showing obvious bands. Maldi-Tof confirmed that the protein was indeed the target protein. N-terminal sequencing confirmed that the collagen was traceless. Example

[0023] The strain was cultured in a 5L fermenter in a fed-batch manner to efficiently produce collagen variant C109: Fully supplemented batch fermentation was performed in a 5 L fermentor (T&J Bioengineering, Shanghai, China) containing 1.8 L BSM medium and 10% (v / v) inoculum. The temperature was set to 30 °C, and the pH was adjusted to 5.5 using 50% (v / v) ammonia. After the dissolved oxygen recovered to 55%, 50% (v / v) glycerol and 1.2% (v / v) PTM1 were fed at a rate of 6 mL / L / h. When the wet cell weight reached 200 g / L, the pH was adjusted to 5.0, and the temperature was maintained at 30 °C while supplementing the carbon source. During the entire fermentation, the dissolved oxygen was maintained at 20-30% under controlled agitation and aeration. The culture was induced under this condition for 84 h.

[0024] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for producing recombinant collagen in Pichia pastoris, characterized in that: The following steps are involved: Step 1: Codon optimization of collagen variants; Step 2: Repeat the fragment four times and fuse the enterokinase cleavage site to the N-terminus of the collagen COL domain to facilitate obtaining traceless collagen and improve the stability of the collagen COL fragment; Step 3: Optimize the expression frame, verify the expression by replacing different promoters and signal peptides, and determine the expression frame with the AOX1 promoter in Pichia pastoris as the promoter and the a-factor signal peptide in Pichia pastoris as the signal peptide; Step 4: construct the enterokinase expression cassette; Step 5: Verify using SDS-PAGE and Maldi-tof; Step 6: Knockout the protease Pep4; Step 7: Fed-batch culture in a 5 L fermenter.

2. The method for producing recombinant collagen in Pichia pastoris according to claim 1, characterized in that Add enterokinase tag, which is added to the N-terminus of the target protein during genetic engineering.

3. The method for producing recombinant collagen in Pichia pastoris according to claim 2, characterized in that: The SDS-PAGE is polyacrylamide gel electrophoresis, and the specific steps are: S1, loading the sample into the gel and applying an electric field to cause the protein to migrate in the gel; S2. Small molecular weight proteins migrate quickly, while large molecular weight proteins migrate slowly, thus achieving separation; S3. After electrophoresis, stain the gel with dye to visualize the separated protein bands.

4. The method for producing recombinant collagen in Pichia pastoris according to claim 3, characterized in that: The Maldi-tof is a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer, which can quickly identify proteins in a sample by comparing the mass spectra of known proteins.

5. A Pichia pastoris chassis cell for efficiently synthesizing collagen variants, characterized in that: Used for producing the recombinant collagen according to any one of claims 1 to 4.

6. The fed-batch culture method of Pichia pastoris chassis cells for efficiently synthesizing collagen variants according to claim 5, characterized in that: include: Step 1: Fully supplemented batch fermentation was performed in a 5-L fermenter containing 1.8 L BSM medium and 10% inoculum; The temperature was set to 30°C and the pH was adjusted to 5.5 using 50% ammonia; After the dissolved oxygen returned to 55%, 50% glycerol and 1.2% PTM1 were added at a rate of 6 mL / L / h; When the wet cell weight reached 200 g / L, the pH was adjusted to 5.0, and the temperature was maintained at 30°C while supplementing the carbon source; During the whole fermentation process, the dissolved oxygen was maintained at 20-30% under controlled stirring and aeration, and the culture was induced under this condition for 84 h.

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