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WO2023221787A1 - Pichia pastoris engineering strain for recombinant type i collagen, construction method therefor and use thereof - Google Patents

Pichia pastoris engineering strain for recombinant type i collagen, construction method therefor and use thereof Download PDF

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WO2023221787A1
WO2023221787A1 PCT/CN2023/092412 CN2023092412W WO2023221787A1 WO 2023221787 A1 WO2023221787 A1 WO 2023221787A1 CN 2023092412 W CN2023092412 W CN 2023092412W WO 2023221787 A1 WO2023221787 A1 WO 2023221787A1
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collagen
type
recombinant
pichia pastoris
methanol
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PCT/CN2023/092412
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French (fr)
Chinese (zh)
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郑海洲
蒋艳
魏敬双
沈旭东
潘永刚
何丽
常亮
王鑫
刘振
何庆生
陈丽华
高健
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华北制药集团新药研究开发有限责任公司
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Publication of WO2023221787A1 publication Critical patent/WO2023221787A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the present invention relates to the technical field of genetic engineering. More specifically, the present invention relates to a recombinant type I collagen Pichia pastoris engineering strain and its construction method and application.
  • Collagen is the most abundant and widely distributed functional protein in mammals, accounting for approximately 25% to 30% of the total protein in the animal body.
  • type I collagen has the highest content among the 28 types of collagen that have been discovered so far, accounting for about 80% to 90% of the total collagen content. It is mainly distributed in skin, cornea, tendon and other parts, and is important for maintaining the normal function of cells, tissues, etc. Physiological functions and injury repair play an important role.
  • type I collagen is the collagen with the most widespread tissue distribution and the most thorough research. It has been used in fields such as beauty and cosmetics, health care, medical devices, and biomedical materials.
  • the traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to process animal-derived tissues such as pigs, cattle, and fish to extract mixed collagen peptides of varying lengths, which have poor biological activity and carry potential risks such as viral infection and immune rejection.
  • Recombinant collagen expressed in host cells using genetic engineering technology has the advantages of consistent composition, stable quality, and good safety, and is especially suitable for applications in high-end fields such as medical devices.
  • the Escherichia coli expression system generally has the following shortcomings: the target protein is generally expressed in the form of inclusion bodies, which makes purification and removal of impurities more difficult; residual endotoxins in the product can produce pyrogens that can easily trigger allergic reactions; and the post-translational processing and modification system is imperfect, making the expression product more difficult to purify and remove impurities. Lower biological activity.
  • Pichia pastoris system to recombinantly express type I humanized collagen fragments
  • Shaanxi Huikangsheng Biotechnology Co., Ltd. discloses the use of Pichia pastoris to recombinantly express type I humanized collagen fragments in patent CN106554410A
  • Xi'an Huaao Likang Bioengineering Co., Ltd. discloses in patent CN102020716B the use of Pichia pastoris SMD1168 to express a full-length 839 amino acid collagen.
  • a combination of protein fragments, the N-terminal is a human type III collagen peptide, the C-terminal is a human type I collagen peptide, and they are connected with glutamic acid and phenylalanine.
  • patent CN113667709A Xi'an Denohisi Medical Technology Co., Ltd. disclosed a fermentation method for expressing the 66.2KDa fragment of type I collagen in Pichia pastoris, which can reduce the degradation of recombinant humanized collagen during the fermentation process and stabilize it. Industrial production.
  • the dual-specific affinity purification tags designed at both ends are beneficial to the purification, detection and full-length identification of the recombinant protein. If the product does not remove the purification label, there is a potential risk of allergic reactions in medical applications; adding an enzyme digestion process to remove the purification label will complicate the production process and increase production costs.
  • the mature peptide of type I humanized collagen ⁇ 1 chain contains 1057 amino acids, has a higher molecular weight, has unique physiological and biochemical properties, and is a promising medical regenerative material.
  • Using Pichia pastoris to secrete and express a recombinant type I humanized collagen mature peptide that is 100% identical to the corresponding part of the amino acid sequence of human type I collagen can fill the current domestic technology gap.
  • the present invention aims to provide a Pichia pastoris engineered strain that recombinant type I collagen mature peptide and its construction method and application, realize efficient production of type I collagen mature peptide in Pichia pastoris, and obtain purified products.
  • the present invention provides the following technical solutions:
  • a recombinant type I collagen Pichia pastoris strain the type I collagen gene is connected in series to the downstream of the ⁇ -factor signal peptide Kex2 protease recognition site of pPICZalphaA, and the target protein is secreted out of the cell.
  • type I collagen is type I humanized collagen ⁇ 1 chain mature peptide.
  • amino acid sequence of the mature peptide of the type I humanized collagen ⁇ 1 chain is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2.
  • the target protein secreted and expressed by Pichia pastoris is 100% identical to the corresponding part of the amino acid sequence of human type I collagen, and does not contain purified tag proteins and other redundant amino acids.
  • the present invention provides a Pichia pastoris engineering strain of recombinant type I humanized collagen ⁇ 1 chain mature peptide.
  • the strain is deposited in the General Microorganism Center of the China Microbial Culture Collection Committee, and the deposit number is CGMCC No. 24534.
  • the invention also provides a method for constructing a Pichia pastoris engineering strain that secretes and expresses the mature peptide of the ⁇ 1 chain of recombinant type I humanized collagen: the synthesized type I humanized collagen gene is connected in series to the ⁇ -factor signal peptide Kex2 protease of pPICZalphaA downstream of the recognition site.
  • amino acid sequence of the mature peptide of the ⁇ 1 chain of recombinant type I humanized collagen is SEQ ID NO.1.
  • nucleotide sequence of the mature peptide of the ⁇ 1 chain of recombinant humanized type I collagen was optimized according to the preferred codons of Pichia pastoris, and the gene sequence of the mature peptide of the ⁇ 1 chain of humanized type I collagen was synthesized through the whole gene.
  • the nucleotide sequence is shown in SEQ ID NO.2.
  • the recombinant plasmid pPICZalphaA-col(I) ⁇ 1 was linearized with restriction endonuclease PmeI, transformed into Pichia pastoris X-33 competent cells, and positive recombinants were screened for Zeocin resistance to obtain recombinant type I Humanized collagen ⁇ 1 chain mature peptide Pichia pastoris engineered strain.
  • the transformation can be carried out by electroporation.
  • the recombinant type I humanized collagen ⁇ 1 chain mature peptide Pichia pastoris engineering strain obtained by the above construction steps can use Pichia pastoris' own Kex2 protease to excise the N-terminal signal peptide of the recombinant target protein and secrete and express the complete recombinant type I human collagen. Sourced mature collagen peptides.
  • the invention also provides a method for preparing type I collagen using the above-mentioned recombinant type I humanized collagen ⁇ 1 chain mature peptide Pichia pastoris engineering strain, including a fermentation culture method and a protein purification method:
  • the invention provides an improved fermentation medium, which contains components at the following concentrations: 85% phosphoric acid 24-28ml/L, calcium sulfate dihydrate 0.65-1.15g/L, potassium sulfate 16.2-20.2g/L, dihydrate Magnesium sulfate 12.9 ⁇ 16.9g/L, potassium hydroxide 3.53 ⁇ 4.95g/L, glycerin 30 ⁇ 50g/L, PTM 13.5 ⁇ 5.5ml/L, ascorbic acid 50 ⁇ 200mg/L, lysine 50 ⁇ 300mg/L , sorbitol 20 ⁇ 100g/L, inositol 5 ⁇ 20mg/L, folic acid 5 ⁇ 30mg/L, calcium pantothenate 2 ⁇ 20mg/L, PH5.0 ⁇ 7.0.
  • a modified fermentation medium contains components at the following concentrations: 85% phosphoric acid 26.7ml/L, calcium sulfate dihydrate 0.93g/L, potassium sulfate 18.2g/L, magnesium sulfate dihydrate 14.9g/L, Potassium hydroxide 4.13g/L, glycerol 40g/L, PMT 14.0ml/L, Ascorbic acid 20mg/L, lysine 150mg/L, sorbitol 36.4g/L, inositol 10mg/L, folic acid 20mg/L, calcium pantothenate 10mg/L, pH 6.0.
  • the invention also provides a high-density fermentation method: when the wet weight of the bacterial cell reaches 200-300g/L, it enters the methanol induction stage, starts to add methanol (100ml methanol plus 1.2ml PMT1), adjusts the pH to 5.5 ⁇ 0.3, and adjusts the temperature to 5.5 ⁇ 0.3. The temperature is 27 ⁇ 2°C. Control the methanol replenishment rate at 2-4ml/h/L for the first 12 hours, then adjust the methanol replenishment speed and flow rate to 5-7ml/h/L and maintain it for 10-14 hours. Finally, keep the methanol replenishment rate at 8-12ml/L. h/L continued induction for 70-74 hours.
  • the wet weight of the bacterial cells reaches 200-300g/L
  • start adding methanol (100ml methanol plus 1.2ml PMT1) control the supplemental acceleration rate for the first 12 hours to 3ml/h/L, and then adjust the flow rate to 6ml/h/L to maintain 12 hours, and finally 10ml/h/L to continue the induction for 72 hours.
  • high-density fermentation includes the following steps:
  • Glycerol culture stage Transfer the seed liquid to a fermentation tank containing 20L of modified culture medium, set the initial fermentation temperature to 30°C, PH 5.0, rotation speed 500rpm, ventilation 20L/min, and culture for about 20 hours.
  • Glycerin feeding stage The glycerol feeding stage starts about 20 hours after culturing, and 50% (w/v) glycerol is added at a rate of 18ml/h/L.
  • the trace element PTM1 is added to the glycerol (concentration is 12ml/L). Until the wet weight of the bacteria reaches 220-260g/L.
  • Methanol induction phase Start adding methanol (100ml methanol plus 1.2ml PMT1) 30 minutes after the end of the glycerol flow. Control the supplementary acceleration rate at 3ml/h/L in the first 12h of the methanol induction phase, and then adjust the flow rate to 6ml/h/L to maintain 12h, finally adjusted to 8 ⁇ 9ml/h/L and induced for 48h to end the fermentation.
  • the present invention uses the above-mentioned improved inorganic culture medium to carry out high-density fermentation of recombinant engineering strains, which can significantly increase the expression level of the target protein and enable large-scale production of type I humanized collagen.
  • the protein purification method of the mature peptide of the ⁇ 1 chain of recombinant type I collagen includes the following steps: centrifuge to collect the fermentation supernatant, add saturated ammonium sulfate solution to obtain a precipitate, dissolve the precipitate with a citric acid buffer, and perform cation exchange chromatography to obtain type I collagen. .
  • the protein purification method includes the following steps:
  • the type I humanized collagen prepared by the present invention is a complete recombinant collagen, which is 100% identical to the corresponding part of the amino acid sequence of type I human collagen without any redundant amino acids. Compared with gelatin and conventional recombinant collagen fragments, it has With more complete structure and biological properties, it can be widely used in cosmetics, medical devices, medical materials, tissue engineering, nutritional health care, cell culture and other fields.
  • the present invention is optimized based on the preferred codons of Pichia pastoris, eliminates uncommon codons and hairpin structures, and synthesizes the gene sequence of the mature peptide of the ⁇ 1 chain of type I humanized collagen through the whole gene, which is more suitable for use in Stably expressed in Pichia pastoris.
  • the present invention creatively designs and constructs a recombinant plasmid.
  • the target gene is inserted downstream of the Kex2 protease recognition site of the ⁇ -factor secretion signal peptide, and the N-terminal signal peptide of the recombinant target protein is excised using Pichia pastoris' own Kex2 protease to obtain a complete recombination.
  • Collagen The obtained recombinant collagen is 100% identical to the corresponding part of the amino acid sequence of type I human collagen, without any redundant amino acids. Compared with gelatin and conventional recombinant collagen fragments, it has a more complete structure and biological properties, and has broad application prospects. biomedical materials.
  • the present invention uses the eukaryotic expression system Pichia pastoris to secrete and express recombinant type I humanized collagen, which has no virus risks and has higher biocompatibility and biosafety. It overcomes the potential risks of viral infection and immune rejection of traditional animal-derived collagen, and can also avoid the disadvantages of the E. coli expression system, such as difficulty in purification and easy generation of pyrogens.
  • the present invention uses an improved culture medium for high-density fermentation to overcome the shortcomings of standard inorganic culture media.
  • the fermentation cycle is short, the operation is simple and convenient, and the expression level of the mature peptide of the ⁇ 1 chain of recombinant type I collagen can be significantly increased to more than 6g/L. .
  • the purification method of the present invention for preparing recombinant type I humanized collagen mature peptide has simple steps, the product purity reaches more than 98%, and can be safely used in biomedical materials, medical devices and other fields.
  • Figure 1 shows the map of recombinant plasmid pPICZalphaA-col(I) ⁇ 1.
  • Figure 2 is an agarose gel electrophoresis pattern of the recombinant plasmid pPICZalphaA-col(I) ⁇ 1 identified by PCR.
  • Lane M is DL5000DNA Marker, and lanes 1 and 2 are PCR amplification products of different recombinant plasmids.
  • Figure 3 is an agarose gel electrophoresis pattern of the PCR-verified recombinant pPICZalphaA-col(I) ⁇ 1/X-33.
  • Lane M is the DL5000DNA Marker, and lanes 1 and 2 are the PCR amplification products of different recombinants.
  • Figure 4 is an SDS-PAGE electrophoresis pattern of recombinant strains screened in shake flasks, in which lane M is PageRuler TM Prestained Protein Ladder, and lanes 1 to 3 are different recombinant colonies.
  • Figure 5 is a comparison of SDS-PAGE profiles of shake flask fermentation samples with different media.
  • Lane M is PageRuler TM Prestained Protein Ladder
  • lanes 1 to 3 are modified culture medium fermentation broth samples
  • lanes 4 to 6 are standard inorganic salt culture media fermentation samples. liquid sample.
  • Figure 6 is the SDS-PAGE electrophoresis detection pattern of the fermentation broth purified sample, in which lane M is PageRuler TM Prestained Protein Ladder, and lane 1 is the purified sample.
  • the experimental methods in the following examples are all conventional methods unless otherwise specified.
  • the Pichia pastoris X-33 strain and expression vector pPICZalphaA selected for this invention were purchased from Beijing Jinsaishi Biopharmaceutical Technology Development Co., Ltd.
  • the culture medium formula used in the present invention is as follows:
  • LLB medium 5g yeast extract, 10g peptone, 10g sodium chloride (solid medium contains 2% agar powder).
  • YPDS medium (1L): 10g of yeast extract, 20g of peptone, 20g of glucose, 1M sorbitol (solid medium contains 2% agar powder).
  • an engineering strain of Pichia pastoris (Pichia pastoris) of recombinant type I humanized collagen ⁇ 1 chain mature peptide is provided.
  • the strain is deposited in the General Microbiology Center of the China Microbial Culture Collection Committee, with the deposit number It is CGMCC No. 24534, preservation date: March 15, 2022, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, classification name: Pichia pastoris.
  • the following is the specific process of constructing, fermenting and purifying the genetically engineered strain of Pichia pastoris to prepare recombinant type I collagen.
  • Gene sequence of artificially synthesized type I humanized collagen ⁇ 1 chain mature peptide without changing the amino acid sequence (SEQ ID NO) of type I humanized collagen ⁇ 1 chain mature peptide (NCBI Reference Sequence: NP_000079.2) .1), according to the preferred codons of Pichia pastoris, factors such as GC content, codon usage frequency, RNase splicing site, and RNA stabilizing trans-acting elements were weighed, and BglII, BamHI, and Not were eliminated.
  • the kit extracts the recombinant plasmid pPICZalphaA-col(I) ⁇ 1 and linearizes it with restriction endonuclease PmeI.
  • the PCR reaction system is 50 ⁇ l, and the reaction components include: 5 ⁇ l of 10 ⁇ Taq buffer, 4 ⁇ l of dNTPs, and upstream and downstream primers. 1 ⁇ l, template 2 ⁇ l, Taq polymerase 1 ⁇ l, sterile double-distilled water 36 ⁇ l.
  • PCR amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min.
  • the agarose gel electrophoresis results are shown in Figure 3.
  • the band containing the target gene is approximately 3800 bp
  • the host bacterial AOX1 gene band is approximately 2200 bp in length.
  • the preserved glycerol bacterial solution is inoculated into 50 mL YPD medium, and cultured overnight with shaking at 30°C and 200 rpm for activation.
  • the above-mentioned improved fermentation medium contains components at the following concentrations: 85% phosphoric acid 26.7ml/L, calcium sulfate dihydrate 0.93g/L, potassium sulfate 18.2g/L, magnesium sulfate dihydrate 14.9g/L, potassium hydroxide 4.13 g/L, glycerol 40g/L, trace element PMT 14.0ml/L, ascorbic acid 100mg/L, lysine 150mg/L, sorbitol 36.4g/L, inositol 10mg/L, folic acid 20mg/L, calcium pantothenate 10mg /L, pH 6.0.
  • the fermentation supernatant was collected by centrifugation and subjected to SDS-PAGE electrophoresis.
  • the results in Figure 5 show that the expression of the target protein in the modified fermentation medium was significantly increased.
  • the grayscale analysis method was used to analyze the SDS-PAGE result spectrum.
  • the average grayscale of the fermentation broth of the three modified fermentation medium shake flasks was 3.18 times that of the standard inorganic salt medium.
  • the fermentation harvest liquid is centrifuged to remove bacterial cells to obtain the supernatant. Add saturated ammonium sulfate solution to a saturation level of 25%. After stirring thoroughly, let it stand for 30 minutes and centrifuge to collect the precipitate.
  • Balance solution 25mM citrate buffer, 50mM sodium chloride, pH 6.2, balance 4-5 column volumes (CV);

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Abstract

Disclosed are a pichia pastoris engineering strain for a recombinant type I collagen, a construction method therefor and use thereof. A gene sequence for a type I humanized collagen α1 chain mature peptide is synthesized and inserted downstream of a Kex2 protease recognition site in an α-factor secretion signal peptide of a vector to construct a recombinant plasmid pPICZalphaA-col(I)α1. The plasmid is introduced into pichia pastoris X-3, and screening is performed to obtain a recombinant strain. The present invention further provides a method for preparing a type I humanized collagen using the recombinant strain. An improved fermentation culture medium and a high-density fermentation method are provided, which allow for an expression level of up to 6 g/L or above. The purified recombinant I-type collagen has a purity of up to 98% or above, and is 100% identical to the corresponding part of an amino acid sequence of a human type I collagen. The production process is green and environment-friendly. The product has relatively high biocompatibility and bio-safety. The product can be applied to a wide range of fields such as cosmetic medicine, medical instruments, and medical biological materials.

Description

重组I型胶原蛋白毕赤酵母工程菌及其构建方法与应用Recombinant type I collagen Pichia pastoris engineering strain and its construction method and application 技术领域Technical field
本发明涉及基因工程技术领域，更具体地，本发明涉及一种重组I型胶原蛋白毕赤酵母工程菌及其构建方法与应用。The present invention relates to the technical field of genetic engineering. More specifically, the present invention relates to a recombinant type I collagen Pichia pastoris engineering strain and its construction method and application.
背景技术Background technique
胶原蛋白是哺乳动物体内含量最多、分布最广的功能性蛋白，约占动物机体蛋白质总量的25％～30％。其中I型胶原蛋白在目前已发现的28中胶原蛋白中含量最高，占全部胶原蛋白含量的80％～90％左右，主要分布于皮肤、角膜、肌腱等部位，对维护细胞、组织等的正常生理功能和损伤修复起着重要作用。在胶原蛋白家族的众多成员中，I型胶原蛋白是组织分布最为广泛和研究最为透彻的胶原蛋白，目前已应用于美容化妆、保健、医疗器械和生物医学材料等领域。Collagen is the most abundant and widely distributed functional protein in mammals, accounting for approximately 25% to 30% of the total protein in the animal body. Among them, type I collagen has the highest content among the 28 types of collagen that have been discovered so far, accounting for about 80% to 90% of the total collagen content. It is mainly distributed in skin, cornea, tendon and other parts, and is important for maintaining the normal function of cells, tissues, etc. Physiological functions and injury repair play an important role. Among the many members of the collagen family, type I collagen is the collagen with the most widespread tissue distribution and the most thorough research. It has been used in fields such as beauty and cosmetics, health care, medical devices, and biomedical materials.
传统生产胶原蛋白的方法是利用酸、碱、酶解法处理猪、牛、鱼等动物来源组织，提取长度不等的混合胶原肽段，生物活性差，并且有病毒感染和免疫排斥等潜在风险。而利用基因工程技术在宿主细胞表达的重组胶原蛋白具有组分一致、质量稳定、安全性好的优点，尤其适合在医疗器械领域等高端领域的应用。The traditional method of producing collagen is to use acid, alkali, and enzymatic hydrolysis to process animal-derived tissues such as pigs, cattle, and fish to extract mixed collagen peptides of varying lengths, which have poor biological activity and carry potential risks such as viral infection and immune rejection. Recombinant collagen expressed in host cells using genetic engineering technology has the advantages of consistent composition, stable quality, and good safety, and is especially suitable for applications in high-end fields such as medical devices.
国内外科研人员利用毕赤酵母系统分泌表达重组I型胶原蛋白进行了大量研究，Myllyharju等(2000年)开展了甲基酵母(Pichiapastoris)胞内表达人I、Ⅱ、Ⅲ型胶原蛋白的研究。美国Fibrogen公司与日本国家传染病研究中心(2005年)合作，利用毕赤酵母X-33表达长度101aa的重组I型人源化胶原蛋白。Takahiro等(2020年)根据人类I型胶原蛋白α1链716到779的蛋白质序列，融合4个重叠胶原片段构建胶原蛋白多肽(RCPhC1)。Pia等(2019年)在Ⅰ型人类胶原蛋白α链的541至940aa两侧分别连接(Pro-Gly-Pro)9序列构建GelMP，其与动物明胶有相似的生物相容性和细胞粘附性。Tsuneyuki等(2019年)在毕赤酵母表达人类I型胶原α1链的(RGD)12基序分子。Domestic and foreign researchers have conducted a large number of studies using the Pichia pastoris system to secrete and express recombinant type I collagen. Myllyharju et al. (2000) conducted research on the intracellular expression of human type I, II, and III collagen in Pichia pastoris. The American company Fibrogen cooperated with Japan's National Center for Infectious Disease Research (2005) to use Pichia pastoris X-33 to express recombinant type I humanized collagen with a length of 101aa. Takahiro et al. (2020) based on the protein sequence of human type I collagen α1 chain 716 to 779, fused four overlapping collagen fragments to construct a collagen polypeptide (RCPhC1). Pia et al. (2019) constructed GelMP by connecting (Pro-Gly-Pro) 9 sequences to both sides of 541 to 940aa of the α chain of type I human collagen, which has similar biocompatibility and cell adhesion to animal gelatin. . Tsuneyuki et al. (2019) expressed the (RGD) 12 motif molecule of human type I collagen α1 chain in Pichia pastoris.
国内胶原蛋白厂商大多采用大肠杆菌发酵表达胶原蛋白，山西锦波生物医药股份有限公司在专利CN109293783B公开利用大肠杆菌表达系统表达I型人源化胶原蛋白片段，国内专利CN106046151A公开一种大肠杆菌表达人源化可溶高活性I型胶原蛋白片段及其制备方法。大肠杆菌表达系统普遍存在以下缺点：目标蛋白一般以包涵体形式表达增加纯化及除去杂质的难度；产品中残留的内毒素会产生致热源容易引发过敏反应、翻译后加工修饰体系不完善使得表达产物生物活性较低等。Most domestic collagen manufacturers use Escherichia coli fermentation to express collagen. Shanxi Jinbo Biopharmaceutical Co., Ltd. disclosed the use of Escherichia coli expression system to express type I humanized collagen fragments in patent CN109293783B. Domestic patent CN106046151A disclosed an Escherichia coli expression system for humanized collagen. Sourced soluble highly active type I collagen fragment and preparation method thereof. The Escherichia coli expression system generally has the following shortcomings: the target protein is generally expressed in the form of inclusion bodies, which makes purification and removal of impurities more difficult; residual endotoxins in the product can produce pyrogens that can easily trigger allergic reactions; and the post-translational processing and modification system is imperfect, making the expression product more difficult to purify and remove impurities. Lower biological activity.
国内利用毕赤酵母系统重组表达I型人源化胶原蛋白片段的研究逐年增多，陕西慧康生 物科技有限责任公司在专利CN106554410A公开利用毕赤酵母重组表达I型人源化胶原蛋白片段；西安华澳丽康生物工程有限公司在专利CN102020716B公开在毕赤酵母SMD1168表达全长839个氨基酸的胶原蛋白片段组合，其N端为人Ⅲ型胶原蛋白肽段，C端为人I型胶原蛋白肽段，并用谷氨酸和苯丙氨酸连接起来。西安德诺海思医疗科技有限公司在专利CN113667709A公开了一种在毕赤酵母表达I型胶原蛋白66.2KDa片段的发酵方法，可以减少重组人源化化胶原蛋白在发酵过程发生降解，进行稳定的工业化生产。Domestic research on the use of Pichia pastoris system to recombinantly express type I humanized collagen fragments is increasing year by year. Shaanxi Huikangsheng Biotechnology Co., Ltd. discloses the use of Pichia pastoris to recombinantly express type I humanized collagen fragments in patent CN106554410A; Xi'an Huaao Likang Bioengineering Co., Ltd. discloses in patent CN102020716B the use of Pichia pastoris SMD1168 to express a full-length 839 amino acid collagen. A combination of protein fragments, the N-terminal is a human type III collagen peptide, the C-terminal is a human type I collagen peptide, and they are connected with glutamic acid and phenylalanine. In patent CN113667709A, Xi'an Denohisi Medical Technology Co., Ltd. disclosed a fermentation method for expressing the 66.2KDa fragment of type I collagen in Pichia pastoris, which can reduce the degradation of recombinant humanized collagen during the fermentation process and stabilize it. Industrial production.
迄今，有2家国内企业报道重组表达I型人源化胶原蛋白α1链成熟肽全长序列。其中西安巨子生物基因技术股份有限公司在专利CN103725622A公开毕赤酵母表达改造的I型胶原蛋白α1链成熟肽。江苏悦智生物医药有限公司在专利CN109988234A公开了一种酵母重组I型人源化胶原α1链蛋白、合成方法及其应用，从氨基端依次包含：氨基端亲和纯化标签、I型人源化胶原蛋白α1链成熟肽链以及羧基端亲和纯化标签。两端设计的双特异性亲和纯化标记虽然有利于该重组蛋白的纯化、检测及全长鉴定。如果产品不去除纯化标记，在医学领域应用中存在引发过敏反应的潜在风险；而增加酶切工序除去纯化标记则会使生产工艺复杂化，造成生产成本的提高。So far, two domestic companies have reported recombinant expression of the full-length mature peptide sequence of type I humanized collagen α1 chain. Among them, Xi'an Juzi Biogene Technology Co., Ltd. disclosed in patent CN103725622A the mature peptide of type I collagen α1 chain expressed and modified by Pichia pastoris. Jiangsu Yuezhi Biopharmaceutical Co., Ltd. discloses a yeast recombinant type I humanized collagen α1 chain protein, a synthesis method and its application in patent CN109988234A. From the amino end, it includes: amino end affinity purification tag, type I humanized collagen Collagen α1 chain mature peptide chain and carboxyl terminus affinity purification tag. The dual-specific affinity purification tags designed at both ends are beneficial to the purification, detection and full-length identification of the recombinant protein. If the product does not remove the purification label, there is a potential risk of allergic reactions in medical applications; adding an enzyme digestion process to remove the purification label will complicate the production process and increase production costs.
I型人源化胶原蛋白α1链成熟肽含有1057个氨基酸，具有更高的分子量，拥有独特的生理生化特性，是一种前景广阔的医学再生材料。利用毕赤酵母分泌表达与人I型胶原蛋白氨基酸序列相应部分100％相同的重组I型人源化胶原蛋白成熟肽，可以填补目前的国内技术空白。The mature peptide of type I humanized collagen α1 chain contains 1057 amino acids, has a higher molecular weight, has unique physiological and biochemical properties, and is a promising medical regenerative material. Using Pichia pastoris to secrete and express a recombinant type I humanized collagen mature peptide that is 100% identical to the corresponding part of the amino acid sequence of human type I collagen can fill the current domestic technology gap.
发明内容Contents of the invention
本发明旨在提供一种重组I型胶原蛋白成熟肽的毕赤酵母毕赤酵母工程菌及其构建方法与应用，实现I型胶原蛋白成熟肽在毕赤酵母的高效生产，并获得纯化产品。The present invention aims to provide a Pichia pastoris engineered strain that recombinant type I collagen mature peptide and its construction method and application, realize efficient production of type I collagen mature peptide in Pichia pastoris, and obtain purified products.
为解决上述技术问题，本发明提供如下技术方案：In order to solve the above technical problems, the present invention provides the following technical solutions:
一种重组I型胶原蛋白毕赤酵母菌株：将I型胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游，目标蛋白分泌至细胞外。A recombinant type I collagen Pichia pastoris strain: the type I collagen gene is connected in series to the downstream of the α-factor signal peptide Kex2 protease recognition site of pPICZalphaA, and the target protein is secreted out of the cell.
特别的，所述Ⅰ型胶原蛋白为Ⅰ型人源化胶原蛋白α1链成熟肽。In particular, the type I collagen is type I humanized collagen α1 chain mature peptide.
特别的，所述Ⅰ型人源化胶原蛋白α1链成熟肽的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示。利用毕赤酵母分泌表达的目标蛋白与人I型胶原蛋白氨基酸序列相应部分100％相同，不含纯化标签蛋白和其它多余的氨基酸。 In particular, the amino acid sequence of the mature peptide of the type I humanized collagen α1 chain is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2. The target protein secreted and expressed by Pichia pastoris is 100% identical to the corresponding part of the amino acid sequence of human type I collagen, and does not contain purified tag proteins and other redundant amino acids.
特别的，本发明提供了一种重组I型人源化胶原蛋白α1链成熟肽的毕赤酵母工程菌株，所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是CGMCC No.24534。In particular, the present invention provides a Pichia pastoris engineering strain of recombinant type I humanized collagen α1 chain mature peptide. The strain is deposited in the General Microorganism Center of the China Microbial Culture Collection Committee, and the deposit number is CGMCC No. 24534.
本发明还提供一种分泌表达重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌的构建方法：合成的I型人源化胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游。The invention also provides a method for constructing a Pichia pastoris engineering strain that secretes and expresses the mature peptide of the α1 chain of recombinant type I humanized collagen: the synthesized type I humanized collagen gene is connected in series to the α-factor signal peptide Kex2 protease of pPICZalphaA downstream of the recognition site.
特别地，上述构建方法由以下步骤实现：In particular, the above construction method is implemented by the following steps:
(1)重组I型人源化胶原蛋白α1链成熟肽的氨基酸序列为SEQ ID NO.1。(1) The amino acid sequence of the mature peptide of the α1 chain of recombinant type I humanized collagen is SEQ ID NO.1.
(2)重组I型人源化胶原蛋白α1链成熟肽的核苷酸序列根据毕赤酵母偏好性密码子进行优化，通过全基因合成I型人源化胶原蛋白α1链成熟肽的基因序列，核苷酸序列如SEQ ID NO.2所示。(2) The nucleotide sequence of the mature peptide of the α1 chain of recombinant humanized type I collagen was optimized according to the preferred codons of Pichia pastoris, and the gene sequence of the mature peptide of the α1 chain of humanized type I collagen was synthesized through the whole gene. The nucleotide sequence is shown in SEQ ID NO.2.
(3)双酶切改造载体pPICZalphaA，将目的基因串联至载体α-factor signal的Kex2蛋白酶识别位点下游，构建重组质粒pPICZalphaA-col(Ⅰ)α1。(3) Transform the vector pPICZalphaA by double enzyme digestion, connect the target gene downstream of the Kex2 protease recognition site of the vector α-factor signal, and construct the recombinant plasmid pPICZalphaA-col(Ⅰ)α1.
(4)重组质粒pPICZalphaA-col(Ⅰ)α1用限制性内切酶PmeI线性化处理，将其转化入毕赤酵母X-33感受态细胞中，Zeocin抗性筛选阳性重组子，得到重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株。所述转化可以选电击转化。(4) The recombinant plasmid pPICZalphaA-col(Ⅰ)α1 was linearized with restriction endonuclease PmeI, transformed into Pichia pastoris X-33 competent cells, and positive recombinants were screened for Zeocin resistance to obtain recombinant type I Humanized collagen α1 chain mature peptide Pichia pastoris engineered strain. The transformation can be carried out by electroporation.
上述构建步骤获得的重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株，能利用毕赤酵母自身的Kex2蛋白酶切除重组目标蛋白N-端信号肽，分泌表达完整的重组I型人源化胶原蛋白成熟肽。The recombinant type I humanized collagen α1 chain mature peptide Pichia pastoris engineering strain obtained by the above construction steps can use Pichia pastoris' own Kex2 protease to excise the N-terminal signal peptide of the recombinant target protein and secrete and express the complete recombinant type I human collagen. Sourced mature collagen peptides.
本发明还提供一种应用上述重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株制备I型胶原蛋白的方法，包括发酵培养方法和蛋白纯化方法：The invention also provides a method for preparing type I collagen using the above-mentioned recombinant type I humanized collagen α1 chain mature peptide Pichia pastoris engineering strain, including a fermentation culture method and a protein purification method:
1.发酵培养方法：1. Fermentation culture method:
本发明提供了一种改良型发酵培养基，包含以下浓度的组分：85％磷酸24～28ml/L，二水合硫酸钙0.65～1.15g/L，硫酸钾16.2～20.2g/L，二水合硫酸镁12.9～16.9g/L，氢氧化钾3.53～4.95g/L，甘油30～50g/L，PTM13.5～5.5ml/L，抗坏血酸50～200mg/L，赖氨酸50～300mg/L，山梨醇20～100g/L，肌醇5～20mg/L，叶酸5～30mg/L，泛酸钙2～20mg/L，PH5.0～7.0。The invention provides an improved fermentation medium, which contains components at the following concentrations: 85% phosphoric acid 24-28ml/L, calcium sulfate dihydrate 0.65-1.15g/L, potassium sulfate 16.2-20.2g/L, dihydrate Magnesium sulfate 12.9~16.9g/L, potassium hydroxide 3.53~4.95g/L, glycerin 30~50g/L, PTM 13.5~5.5ml/L, ascorbic acid 50~200mg/L, lysine 50~300mg/L , sorbitol 20~100g/L, inositol 5~20mg/L, folic acid 5~30mg/L, calcium pantothenate 2~20mg/L, PH5.0~7.0.
优选的，一种改良型发酵培养基包含以下浓度的组分：85％磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，PMT14.0ml/L， 抗坏血酸20mg/L，赖氨酸150mg/L，山梨醇36.4g/L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。Preferably, a modified fermentation medium contains components at the following concentrations: 85% phosphoric acid 26.7ml/L, calcium sulfate dihydrate 0.93g/L, potassium sulfate 18.2g/L, magnesium sulfate dihydrate 14.9g/L, Potassium hydroxide 4.13g/L, glycerol 40g/L, PMT 14.0ml/L, Ascorbic acid 20mg/L, lysine 150mg/L, sorbitol 36.4g/L, inositol 10mg/L, folic acid 20mg/L, calcium pantothenate 10mg/L, pH 6.0.
本发明还提供了一种高密度发酵方法：菌体湿重达到200-300g/L时，进入甲醇诱导阶段，开始补加甲醇(100ml甲醇加1.2ml PMT1)，调节PH为5.5±0.3，温度为27±2℃，前12小时控制甲醇补加速度2～4ml/h/L，然后调整甲醇补加速度流速至5～7ml/h/L保持10-14小时，最后甲醇补加速度保持8～12ml/h/L继续诱导70-74小时。The invention also provides a high-density fermentation method: when the wet weight of the bacterial cell reaches 200-300g/L, it enters the methanol induction stage, starts to add methanol (100ml methanol plus 1.2ml PMT1), adjusts the pH to 5.5±0.3, and adjusts the temperature to 5.5±0.3. The temperature is 27±2°C. Control the methanol replenishment rate at 2-4ml/h/L for the first 12 hours, then adjust the methanol replenishment speed and flow rate to 5-7ml/h/L and maintain it for 10-14 hours. Finally, keep the methanol replenishment rate at 8-12ml/L. h/L continued induction for 70-74 hours.
优选的，菌体湿重达到200-300g/L时，开始补加甲醇(100ml甲醇加1.2ml PMT1)，前12小时控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12小时，最后10ml/h/L继续诱导72小时。Preferably, when the wet weight of the bacterial cells reaches 200-300g/L, start adding methanol (100ml methanol plus 1.2ml PMT1), control the supplemental acceleration rate for the first 12 hours to 3ml/h/L, and then adjust the flow rate to 6ml/h/L to maintain 12 hours, and finally 10ml/h/L to continue the induction for 72 hours.
优选的，高密度发酵包括以下步骤：Preferably, high-density fermentation includes the following steps:
(1)将存的甘油菌液接种于1500ml YPD培养基，30℃，200rpm培养至OD＝4.0～6.0制备一级种子。(1) Inoculate the stored glycerol bacterial liquid into 1500ml YPD medium, culture it at 30°C and 200rpm until OD=4.0~6.0 to prepare first-level seeds.
(2)甘油培养阶段：将种子液转接至装有20L改良培养基的发酵罐，设定发酵初始温度30℃，PH5.0，转速500rpm，通气20L/min，培养20h左右。(2) Glycerol culture stage: Transfer the seed liquid to a fermentation tank containing 20L of modified culture medium, set the initial fermentation temperature to 30°C, PH 5.0, rotation speed 500rpm, ventilation 20L/min, and culture for about 20 hours.
(3)甘油流加阶段：培养20h左右开始甘油流加阶段，以18ml/h/L的速率补加50％(w/v)甘油，甘油中添加微量元素PTM1(浓度为12ml/L)，直到菌体湿重达到220-260g/L。(3) Glycerin feeding stage: The glycerol feeding stage starts about 20 hours after culturing, and 50% (w/v) glycerol is added at a rate of 18ml/h/L. The trace element PTM1 is added to the glycerol (concentration is 12ml/L). Until the wet weight of the bacteria reaches 220-260g/L.
(4)甲醇诱导阶段：甘油流加结束30min开始补加甲醇(100ml甲醇加1.2ml PMT1)，甲醇诱导阶段的前12h控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12h，最后调整至8～9ml/h/L诱导48h结束发酵。(4) Methanol induction phase: Start adding methanol (100ml methanol plus 1.2ml PMT1) 30 minutes after the end of the glycerol flow. Control the supplementary acceleration rate at 3ml/h/L in the first 12h of the methanol induction phase, and then adjust the flow rate to 6ml/h/L to maintain 12h, finally adjusted to 8~9ml/h/L and induced for 48h to end the fermentation.
进一步，本发明利用上述改良无机培养基进行重组工程菌株的高密度发酵，能显著提高目标蛋白表达水平，能够实现I型人源化胶原蛋白规模化生产。Furthermore, the present invention uses the above-mentioned improved inorganic culture medium to carry out high-density fermentation of recombinant engineering strains, which can significantly increase the expression level of the target protein and enable large-scale production of type I humanized collagen.
2.蛋白纯化方法：2.Protein purification method:
重组I型胶原蛋白α1链成熟肽的蛋白纯化方法包括以下步骤：离心收集发酵上清液，加入饱和硫酸铵溶液获得沉淀，柠檬酸缓冲液溶解沉淀，进行阳离子交换层析，得到I型胶原蛋白。The protein purification method of the mature peptide of the α1 chain of recombinant type I collagen includes the following steps: centrifuge to collect the fermentation supernatant, add saturated ammonium sulfate solution to obtain a precipitate, dissolve the precipitate with a citric acid buffer, and perform cation exchange chromatography to obtain type I collagen. .
优选的，蛋白纯化方法包括以下步骤：Preferably, the protein purification method includes the following steps:
(1)离心收集发酵上清液，加入饱和硫酸铵溶液至饱和度为30％，室温静置30min，5000rpm，离心10min，收集沉淀。 (1) Collect the fermentation supernatant by centrifugation, add saturated ammonium sulfate solution until the saturation is 30%, let it stand at room temperature for 30 minutes, centrifuge at 5000 rpm for 10 minutes, and collect the precipitate.
(2)25mM柠檬酸盐、50mM氯化钠、pH6.2的缓冲液溶解沉淀，利用Capto SP ImpRes(Cytiva公司产品)进行阳离子交换层析，收集的洗脱组分经30KD超滤膜包置换到20mM PBS(PH7.0)缓冲液，得到所述重组I型胶原蛋白α1链成熟肽。(2) Dissolve the precipitate in a buffer of 25mM citrate, 50mM sodium chloride, and pH 6.2, and perform cation exchange chromatography using Capto SP ImpRes (product of Cytiva). The collected elution fractions are replaced by a 30KD ultrafiltration membrane bag. to 20mM PBS (PH7.0) buffer to obtain the mature peptide of the recombinant type I collagen α1 chain.
本发明制备的I型人源化胶原蛋白为完整的重组胶原蛋白，与I型人胶原蛋白氨基酸序列相应部分100％相同，没有任何冗余氨基酸，与明胶和常规的重组胶原片段相比，拥有更完整的结构和生物学性能，可以广泛应用于化妆品、医疗器械、医学材料、组织工程、营养保健和细胞培养等领域。The type I humanized collagen prepared by the present invention is a complete recombinant collagen, which is 100% identical to the corresponding part of the amino acid sequence of type I human collagen without any redundant amino acids. Compared with gelatin and conventional recombinant collagen fragments, it has With more complete structure and biological properties, it can be widely used in cosmetics, medical devices, medical materials, tissue engineering, nutritional health care, cell culture and other fields.
与现有技术相比，本发明的有益效果是：Compared with the prior art, the beneficial effects of the present invention are:
(1)本发明根据毕赤酵母偏好性密码子进行优化，消除了不常用密码子及发夹结构，通过全基因合成I型人源化胶原蛋白α1链成熟肽的基因序列，更适于在毕赤酵母中稳定表达。(1) The present invention is optimized based on the preferred codons of Pichia pastoris, eliminates uncommon codons and hairpin structures, and synthesizes the gene sequence of the mature peptide of the α1 chain of type I humanized collagen through the whole gene, which is more suitable for use in Stably expressed in Pichia pastoris.
(2)本发明独创性设计构建重组质粒，目的基因插入α-factor分泌信号肽Kex2蛋白酶识别位点下游，利用毕赤酵母自身的Kex2蛋白酶切除重组目标蛋白N-端信号肽，得到完整的重组胶原蛋白。所得重组胶原蛋白与I型人胶原蛋白氨基酸序列相应部分100％相同，没有任何冗余氨基酸，与明胶和常规的重组胶原片段相比，拥有更完整的结构和生物学性能，是有广阔应用前景的生物医用材料。(2) The present invention creatively designs and constructs a recombinant plasmid. The target gene is inserted downstream of the Kex2 protease recognition site of the α-factor secretion signal peptide, and the N-terminal signal peptide of the recombinant target protein is excised using Pichia pastoris' own Kex2 protease to obtain a complete recombination. Collagen. The obtained recombinant collagen is 100% identical to the corresponding part of the amino acid sequence of type I human collagen, without any redundant amino acids. Compared with gelatin and conventional recombinant collagen fragments, it has a more complete structure and biological properties, and has broad application prospects. biomedical materials.
(3)本发明利用真核表达系统毕赤酵母分泌表达重组I型人源化胶原蛋白，无病毒隐患，具有更高的生物相容性和生物安全性。克服了传统动物源性胶原蛋白的病毒感染、免疫排斥等潜在风险，还可以避免大肠杆菌表达系统的纯化困难、易产生致热源等缺点。(3) The present invention uses the eukaryotic expression system Pichia pastoris to secrete and express recombinant type I humanized collagen, which has no virus risks and has higher biocompatibility and biosafety. It overcomes the potential risks of viral infection and immune rejection of traditional animal-derived collagen, and can also avoid the disadvantages of the E. coli expression system, such as difficulty in purification and easy generation of pyrogens.
(4)本发明利用改良培养基进行高密度发酵，克服标准无机培养基的不足，发酵周期短，操作简单方便，能够显著提高重组I型胶原蛋白α1链成熟肽的表达水平到6g/L以上。(4) The present invention uses an improved culture medium for high-density fermentation to overcome the shortcomings of standard inorganic culture media. The fermentation cycle is short, the operation is simple and convenient, and the expression level of the mature peptide of the α1 chain of recombinant type I collagen can be significantly increased to more than 6g/L. .
(5)本发明制备重组I型人源化胶原蛋白成熟肽的纯化方法步骤简单，产品纯度达98％以上，可以安全应用于生物医学材料、医疗器械等领域。(5) The purification method of the present invention for preparing recombinant type I humanized collagen mature peptide has simple steps, the product purity reaches more than 98%, and can be safely used in biomedical materials, medical devices and other fields.
附图说明Description of the drawings
图1为重组质粒pPICZalphaA-col(Ⅰ)α1图谱。Figure 1 shows the map of recombinant plasmid pPICZalphaA-col(Ⅰ)α1.
图2是PCR鉴定重组质粒pPICZalphaA-col(Ⅰ)α1的琼脂糖凝胶电泳图谱，其中泳道M为DL5000DNA Marker，泳道1、2为不同重组质粒的PCR扩增产物。Figure 2 is an agarose gel electrophoresis pattern of the recombinant plasmid pPICZalphaA-col(I)α1 identified by PCR. Lane M is DL5000DNA Marker, and lanes 1 and 2 are PCR amplification products of different recombinant plasmids.
图3是PCR验证重组子pPICZalphaA-col(Ⅰ)α1/X-33的琼脂糖凝胶电泳图谱。其中泳道M为DL5000DNA Marker，泳道1、2为不同重组子的PCR扩增产物。Figure 3 is an agarose gel electrophoresis pattern of the PCR-verified recombinant pPICZalphaA-col(Ⅰ)α1/X-33. Lane M is the DL5000DNA Marker, and lanes 1 and 2 are the PCR amplification products of different recombinants.
图4是摇瓶筛选重组菌株的SDS-PAGE电泳图谱，其中泳道M为PageRulerTM Prestained Protein Ladder，泳道1～3为不同的重组菌落。 Figure 4 is an SDS-PAGE electrophoresis pattern of recombinant strains screened in shake flasks, in which lane M is PageRuler TM Prestained Protein Ladder, and lanes 1 to 3 are different recombinant colonies.
图5是不同培养基摇瓶发酵样品对比的SDS-PAGE图谱，其中泳道M为PageRulerTMPrestained Protein Ladder，泳道1～3为改良培养基发酵液样品，泳道4～6为标准无机盐培养基发酵液样品。Figure 5 is a comparison of SDS-PAGE profiles of shake flask fermentation samples with different media. Lane M is PageRuler TM Prestained Protein Ladder, lanes 1 to 3 are modified culture medium fermentation broth samples, and lanes 4 to 6 are standard inorganic salt culture media fermentation samples. liquid sample.
图6是发酵液纯化样品的SDS-PAGE电泳检测图谱，其中泳道M为PageRulerTM Prestained Protein Ladder，泳道1为纯化样品。Figure 6 is the SDS-PAGE electrophoresis detection pattern of the fermentation broth purified sample, in which lane M is PageRuler TM Prestained Protein Ladder, and lane 1 is the purified sample.
具体实施方式Detailed ways
下面结合具体实施方式解释本发明，参考附图描述的实施例是示例性的，不能理解为对本发明的限制。The present invention will be explained below in conjunction with specific embodiments. The embodiments described with reference to the drawings are illustrative and should not be construed as limiting the present invention.
以下实施例中的实验方法，如无特别说明，均为常规方法。其中，本发明所选用的毕赤酵母(Pichia pastoris)X-33菌株、表达载体pPICZalphaA均购自北京金赛狮生物制药技术开发有限责任公司。本发明应用的培养基配方如下：The experimental methods in the following examples are all conventional methods unless otherwise specified. Among them, the Pichia pastoris X-33 strain and expression vector pPICZalphaA selected for this invention were purchased from Beijing Jinsaishi Biopharmaceutical Technology Development Co., Ltd. The culture medium formula used in the present invention is as follows:
LLB培养基(1L)：酵母提取物5g，蛋白胨10g，氯化钠10g(固体培养基含2％琼脂粉)。LLB medium (1L): 5g yeast extract, 10g peptone, 10g sodium chloride (solid medium contains 2% agar powder).
YPD培养基(1L)：酵母提取物10g，蛋白胨20g，葡萄糖20g。YPD medium (1L): 10g yeast extract, 20g peptone, 20g glucose.
YPDS培养基(1L)：酵母提取物10g，蛋白胨20g，葡萄糖20g，1M山梨醇(固体培养基含2％琼脂粉)。YPDS medium (1L): 10g of yeast extract, 20g of peptone, 20g of glucose, 1M sorbitol (solid medium contains 2% agar powder).
BSM无机培养基配方(1L)：85％磷酸26.7ml，二水合硫酸钙0.93g，硫酸钾18.2g，七水合硫酸镁14.9g，氢氧化钾4.13g，甘油40g，PMT14.35ml。BSM inorganic medium formula (1L): 85% phosphoric acid 26.7ml, calcium sulfate dihydrate 0.93g, potassium sulfate 18.2g, magnesium sulfate heptahydrate 14.9g, potassium hydroxide 4.13g, glycerol 40g, PMT 14.35ml.
PMT1培养基配方(1L)：五水合硫酸铜6.0g，碘化钾0.088g，一水合硫酸锰3.0g，二水合钼酸钠0.2g，硼酸0.02g，六水合氯化钴0.5g，氯化锌20.0g，七水合硫酸亚铁65.0，生物素0.2g，浓硫酸5.0ml。PMT1 medium formula (1L): 6.0g copper sulfate pentahydrate, 0.088g potassium iodide, 3.0g manganese sulfate monohydrate, 0.2g sodium molybdate dihydrate, 0.02g boric acid, 0.5g cobalt chloride hexahydrate, 20.0 zinc chloride g, ferrous sulfate heptahydrate 65.0, biotin 0.2g, concentrated sulfuric acid 5.0ml.
在本发明中，提供了一种重组I型人源化胶原蛋白α1链成熟肽的毕赤酵母工程菌株(Pichia pastoris)，所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是CGMCC No.24534，保藏日期：2022年3月15日，地址朝阳区北辰西路1号院3号中国科学院微生物研究所，分类命名：巴斯德毕赤酵母(Pichia pastoris)。In the present invention, an engineering strain of Pichia pastoris (Pichia pastoris) of recombinant type I humanized collagen α1 chain mature peptide is provided. The strain is deposited in the General Microbiology Center of the China Microbial Culture Collection Committee, with the deposit number It is CGMCC No. 24534, preservation date: March 15, 2022, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1, Beichen West Road, Chaoyang District, classification name: Pichia pastoris.
以下为毕赤酵母基因工程菌构建、发酵及纯化制备重组I型胶原蛋白的具体过程。The following is the specific process of constructing, fermenting and purifying the genetically engineered strain of Pichia pastoris to prepare recombinant type I collagen.
1、基因合成及重组质粒的构建1. Gene synthesis and construction of recombinant plasmids
(1)人工合成I型人源化胶原蛋白α1链成熟肽的基因序列：在不改变I型人源化胶原蛋白α1链成熟肽(NCBI Reference Sequence:NP_000079.2)的氨基酸序列(SEQ ID NO.1所示)情况下，根据毕赤酵母偏好性密码子，权衡GC含量、密码子使用频率和RNA酶的剪接位点、RNA稳定反式作用元件等因素，并剔除了BglⅡ、BamHI、Not I、PmeI、Sac I、Stu I等限制 性内切酶位点，由南京金斯瑞生物科技有限公司全合成I型人源化胶原蛋白α1链成熟肽的基因序列col(Ⅰ)α1，核苷酸序列如SEQ ID NO.2所示。(1) Gene sequence of artificially synthesized type I humanized collagen α1 chain mature peptide: without changing the amino acid sequence (SEQ ID NO) of type I humanized collagen α1 chain mature peptide (NCBI Reference Sequence: NP_000079.2) .1), according to the preferred codons of Pichia pastoris, factors such as GC content, codon usage frequency, RNase splicing site, and RNA stabilizing trans-acting elements were weighed, and BglⅡ, BamHI, and Not were eliminated. I, PmeI, Sac I, Stu I and other restrictions Sexual endonuclease site, the gene sequence col(Ⅰ)α1 of the mature peptide of type I humanized collagen α1 chain was fully synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the nucleotide sequence is shown in SEQ ID NO.2 .
(2)扩增目的基因：以合成基因col(Ⅰ)α1为模板，利用引物P1、P2进行PCR扩增，其中P1的基因序列如SEQ ID NO.3所示，P2的基因序列如SEQ ID NO.4，胶回收目的基因片段。(2) Amplify the target gene: Use the synthetic gene col(Ⅰ)α1 as a template and use primers P1 and P2 to perform PCR amplification. The gene sequence of P1 is as shown in SEQ ID NO.3, and the gene sequence of P2 is as SEQ ID NO.4, gel to recover the target gene fragment.
(3)构建重组质粒：分别用XhoI和XbaI双酶切PCR扩增产物col(Ⅰ)α1和载体质粒pPICZalphaA，经T4DNA连接酶于16℃连接4h，连接产物热击转化至感受态细胞DH10b，涂布于Zeocin抗性的低盐LLB平板。挑取单菌落提取质粒进行PCR鉴定，如图2所示，扩增条带与I型胶原蛋白α1链成熟肽基因(3196bp)大小相符，得到阳性重组质粒pPICZalphaA-col(Ⅰ)α1。(3) Construct the recombinant plasmid: double-digest the PCR amplification product col(Ⅰ)α1 and the vector plasmid pPICZalphaA with XhoI and Spread on Zeocin-resistant low-salt LLB plates. A single colony was picked to extract the plasmid for PCR identification. As shown in Figure 2, the amplified band was consistent in size with the type I collagen α1 chain mature peptide gene (3196 bp), and the positive recombinant plasmid pPICZalphaA-col(Ⅰ)α1 was obtained.
2、制备毕赤酵母X-33感受态细胞2. Preparation of Pichia pastoris X-33 competent cells
(1)挑取酵母X-33单菌落，接种至含有5mL的YPD液体培养基试管，30℃、200rpm振荡培养过夜。(1) Pick a single colony of yeast X-33, inoculate it into a test tube containing 5 mL of YPD liquid medium, and culture overnight at 30°C and 200 rpm with shaking.
(2)取500μL的过夜培养物接种至含有50mL新鲜YPD液体培养基的三角瓶中，30℃、200rpm振荡培养过夜。(2) Take 500 μL of the overnight culture and inoculate it into an Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, and culture overnight at 30°C and 200 rpm with shaking.
(3)将培养物于4℃，1500×g离心5min，用50mL预冷的无菌双蒸水重悬菌体后离心。(3) Centrifuge the culture at 1500 × g for 5 minutes at 4°C, resuspend the cells in 50 mL of pre-cooled sterile double-distilled water, and centrifuge.
(4)用25mL预冷的无菌双蒸水重悬菌体后离心。(4) Resuspend the cells in 25 mL of pre-cooled sterile double-distilled water and centrifuge.
(5)用20mL预冷的1M山梨醇溶液重悬菌体后离心。(5) Resuspend the cells in 20 mL of pre-cooled 1M sorbitol solution and centrifuge.
(6)用0.5mL预冷的1M山梨醇溶液重悬菌体即为制备的感受态细胞。(6) Resuspend the bacterial cells in 0.5 mL of pre-cooled 1M sorbitol solution to prepare the competent cells.
3、线性化重组质粒及电转化毕赤酵母X-333. Linearized recombinant plasmid and electrotransformation into Pichia pastoris X-33
(1)试剂盒提取重组质粒pPICZalphaA-col(Ⅰ)α1，限制性内切酶PmeI进行线性化处理。(1) The kit extracts the recombinant plasmid pPICZalphaA-col(Ⅰ)α1 and linearizes it with restriction endonuclease PmeI.
(2)取1-10μg线性化质粒加入80μl毕赤酵母X-33感受态细胞中，混合均匀转至2mm电转化杯中，冰浴5min。(2) Add 1-10 μg of linearized plasmid to 80 μl of Pichia pastoris X-33 competent cells, mix evenly, transfer to a 2 mm electrotransformation cup, and incubate on ice for 5 minutes.
(3)电转仪预热后设置参数(1.5KV，200Ω，25μF)进行电转。(3) After preheating the electroporation instrument, set the parameters (1.5KV, 200Ω, 25μF) for electroporation.
(4)电转结束立即往转化杯中加入1ml预冷的1M山梨醇溶液混匀，30℃静置1h。(4) After electroporation, immediately add 1 ml of pre-cooled 1M sorbitol solution to the transformation cup, mix well, and let stand at 30°C for 1 hour.
(5)取200μl菌液涂布于Zeocin抗性(100μg/ml)的YPDS平板，30℃培养2-5天，直到有单菌落出现。(5) Take 200 μl of bacterial solution and spread it on a Zeocin-resistant (100 μg/ml) YPDS plate, and culture it at 30°C for 2-5 days until a single colony appears.
4、重组子的PCR验证4. PCR verification of recombinants
挑取YPDS平板生长出的单菌落提取基因组DNA，利用引物5′AOX1和3′AOX1进行PCR验证。PCR反应体系为50μl，反应组分包括：10×Taq buffer 5μl，dNTPs 4μl，上下游引物各 1μl，模板2μl，Taq聚合酶1μl，灭菌双蒸水36μl。PCR扩增条件为：95℃预变性5min；95℃变性30s，56℃退火30s，72℃延伸3min，30个循环；72℃延伸10min。琼脂糖凝胶电泳结果如图3所示，其中包含目的基因的条带约是3800bp，宿主菌AOX1基因条带长度是2200bp左右。Single colonies grown on YPDS plates were picked to extract genomic DNA, and primers 5′AOX1 and 3′AOX1 were used for PCR verification. The PCR reaction system is 50 μl, and the reaction components include: 5 μl of 10× Taq buffer, 4 μl of dNTPs, and upstream and downstream primers. 1 μl, template 2 μl, Taq polymerase 1 μl, sterile double-distilled water 36 μl. PCR amplification conditions were: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 3 min, 30 cycles; extension at 72°C for 10 min. The agarose gel electrophoresis results are shown in Figure 3. The band containing the target gene is approximately 3800 bp, and the host bacterial AOX1 gene band is approximately 2200 bp in length.
5、基因工程菌株的诱导表达5. Induced expression of genetically engineered strains
(1)挑取阳性重组子单菌落于50mL YPD培养基中，30℃、200rpm振摇过夜培养进行活化。(1) Pick a single colony of the positive recombinant in 50 mL YPD medium, and culture it overnight with shaking at 30°C and 200 rpm for activation.
(2)1500g离心5min收集菌体，重悬菌体于50mL BMGY培养基中，使起始浓度OD600＝1.0左右，30℃、200rpm下培养18～20h。(2) Centrifuge at 1500g for 5 minutes to collect the cells, resuspend the cells in 50mL BMGY medium to make the initial concentration OD600 = about 1.0, and culture at 30°C and 200rpm for 18 to 20 hours.
(3)调节温度为28℃，每隔24小时向培养基中添加甲醇至终浓度为1％进行诱导表达，甲醇诱导96h。离心收集发酵上清液进行SDS-PAGE电泳检测，图4结果显示3个重组菌落都有与预期分子量130KDa相符的表达条带，但是表达量有所不同。经筛选获得高表达量的重组菌株。(3) Adjust the temperature to 28°C, add methanol to the culture medium every 24 hours to a final concentration of 1% to induce expression, and induce expression with methanol for 96 hours. The fermentation supernatant was collected by centrifugation and subjected to SDS-PAGE electrophoresis. The results in Figure 4 showed that the three recombinant colonies all had expression bands consistent with the expected molecular weight of 130KDa, but the expression levels were different. Recombinant strains with high expression levels were obtained through screening.
6、不同发酵培养基摇瓶对比实验6. Comparative experiment of shake flasks with different fermentation media
(1)保存的甘油菌液接种于50mL YPD培养基中，30℃、200rpm振摇过夜培养进行活化。(1) The preserved glycerol bacterial solution is inoculated into 50 mL YPD medium, and cultured overnight with shaking at 30°C and 200 rpm for activation.
(2)1500g离心5min收集菌体，分别重悬菌体于一种改良型发酵培养基和标准无机盐培养基(BSM无机培养基)中，PH 5.0，使起始浓度OD600＝1.0左右，各做3个重复实验。30℃、200rpm下培养18～20h。(2) Centrifuge at 1500g for 5 minutes to collect the cells, and resuspend them in a modified fermentation medium and a standard inorganic salt medium (BSM inorganic medium), pH 5.0, so that the starting concentration OD600=about 1.0, each Do 3 replicate experiments. Cultivate for 18 to 20 hours at 30°C and 200 rpm.
(3)调节温度为28℃，每隔24小时向培养基中添加甲醇(100ml甲醇加1.2ml PMT1)至终浓度为1％进行诱导表达，甲醇诱导96h。上述改良型发酵培养基包含以下浓度的组分：85％磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，微量元素PMT14.0ml/L，抗坏血酸100mg/L，赖氨酸150mg/L，山梨醇36.4g/L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。(3) Adjust the temperature to 28°C, add methanol (100ml methanol plus 1.2ml PMT1) to the culture medium every 24 hours to a final concentration of 1% to induce expression, and induce expression with methanol for 96h. The above-mentioned improved fermentation medium contains components at the following concentrations: 85% phosphoric acid 26.7ml/L, calcium sulfate dihydrate 0.93g/L, potassium sulfate 18.2g/L, magnesium sulfate dihydrate 14.9g/L, potassium hydroxide 4.13 g/L, glycerol 40g/L, trace element PMT 14.0ml/L, ascorbic acid 100mg/L, lysine 150mg/L, sorbitol 36.4g/L, inositol 10mg/L, folic acid 20mg/L, calcium pantothenate 10mg /L, pH 6.0.
离心收集发酵上清液进行SDS-PAGE电泳检测，图5结果显示改良型发酵培养基的目标蛋白表达量明显提高。采用灰度分析法对SDS-PAGE结果图谱进行分析，3个改良型发酵培养基摇瓶发酵液的灰度平均值是标准无机盐培养基的3.18倍。The fermentation supernatant was collected by centrifugation and subjected to SDS-PAGE electrophoresis. The results in Figure 5 show that the expression of the target protein in the modified fermentation medium was significantly increased. The grayscale analysis method was used to analyze the SDS-PAGE result spectrum. The average grayscale of the fermentation broth of the three modified fermentation medium shake flasks was 3.18 times that of the standard inorganic salt medium.
7、重组菌株的高密度发酵培养7. High-density fermentation culture of recombinant strains
(1)将保存的甘油菌液接种于1000ml YPD培养基，30℃，200rpm培养至OD＝2～6制备一级种子。 (1) Inoculate the preserved glycerol bacterial liquid into 1000 ml YPD medium, and culture it at 30°C and 200 rpm until OD=2~6 to prepare first-level seeds.
(2)按5％接种量将一级种子液转接至装有20L培养基的的发酵罐，设定发酵初始温度30℃，PH5.0，转速500rpm，通气20L/min。(2) Transfer the first-level seed liquid to a fermentation tank containing 20L culture medium according to an inoculation amount of 5%. Set the initial fermentation temperature to 30°C, pH 5.0, rotation speed 500rpm, and ventilation 20L/min.
(3)培养20h左右开始甘油流加阶段，以18ml/h/L的速率补加50％(w/v)甘油，甘油中添加微量元素PTM1(浓度为12ml/L)，直到菌体湿重达到220～260g/L。(3) Start the glycerol feeding phase after about 20 hours of culture, add 50% (w/v) glycerol at a rate of 18ml/h/L, and add trace element PTM1 (concentration: 12ml/L) to the glycerin until the wet weight of the cells Reach 220~260g/L.
(4)甘油流加结束30分钟开始补加甲醇(100ml甲醇加1.2ml PMT1)，甲醇诱导阶段的前12h控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12h，最后调整至8～9ml/h/L诱导48h结束发酵，离心收集发酵上清液。利用Bradford方法测定使用改良型培养基的重组蛋白表达水平达到6.25g/L，明显高于标准培养基的发酵水平。(4) Start adding methanol (100ml methanol plus 1.2ml PMT1) 30 minutes after the end of the glycerol flow. Control the supplementation rate at 3ml/h/L in the first 12h of the methanol induction phase, then adjust the flow rate to 6ml/h/L and maintain it for 12h. Adjust to 8~9ml/h/L and induce for 48h to end the fermentation, and centrifuge to collect the fermentation supernatant. The Bradford method was used to determine that the expression level of the recombinant protein using the modified medium reached 6.25g/L, which was significantly higher than the fermentation level of the standard medium.
8、重组I型胶原蛋白的纯化8. Purification of recombinant type I collagen
(1)发酵收获液经离心去除菌体得到上清液，加入饱和硫酸铵溶液至饱和度25％，搅拌充分混匀后，静置30min，离心收集沉淀。(1) The fermentation harvest liquid is centrifuged to remove bacterial cells to obtain the supernatant. Add saturated ammonium sulfate solution to a saturation level of 25%. After stirring thoroughly, let it stand for 30 minutes and centrifuge to collect the precipitate.
(2)用超纯水将沉淀稀释后，用30KD超滤膜包进行换液，置换到25mM柠檬酸盐、50mM氯化钠、pH6.2的柠檬酸缓冲液中，得到层析上样液。(2) After diluting the precipitate with ultrapure water, use a 30KD ultrafiltration membrane bag to replace the liquid with 25mM citrate, 50mM sodium chloride, and pH 6.2 citric acid buffer to obtain a chromatography loading solution. .
(3)进行Capto SP ImpRes(Cytiva公司)层析：(3) Perform Capto SP ImpRes (Cytiva) chromatography:
平衡：平衡液25mM柠檬酸盐缓冲液、50mM氯化钠、pH6.2，平衡4-5柱体积(CV)；Balance: Balance solution: 25mM citrate buffer, 50mM sodium chloride, pH 6.2, balance 4-5 column volumes (CV);
上样：上述超滤置换液，30-40g/L resin；洗涤：25mM柠檬酸缓冲液盐、50mM氯化钠、pH6.2，2CV；Loading: the above ultrafiltration displacement solution, 30-40g/L resin; washing: 25mM citrate buffer salt, 50mM sodium chloride, pH6.2, 2CV;
洗脱：25mM柠檬酸缓冲液盐、250mM氯化钠、pH6.2，洗脱4CV得目标蛋白组分。Elution: 25mM citrate buffer salt, 250mM sodium chloride, pH 6.2, elute 4CV to obtain the target protein component.
(4)收集的洗脱组分经过30KD超滤膜包换液，目标蛋白置换到20mM PBS(PH7.0)缓冲液中，图6的SDS-PAGE电泳检测结果显示得到的重组I型胶原蛋白α1链成熟肽，与预期分子量130KDa大小相符，纯度达到98％以上。并且该纯化组分可以被胶原蛋白水解酶(Sigma，C5138-25MG)专一性水解。(4) The collected elution fractions were exchanged with 30KD ultrafiltration membrane, and the target protein was replaced into 20mM PBS (PH7.0) buffer. The SDS-PAGE electrophoresis detection results in Figure 6 show the obtained recombinant type I collagen. The mature α1 chain peptide is consistent with the expected molecular weight of 130KDa and has a purity of over 98%. And the purified component can be specifically hydrolyzed by collagen hydrolase (Sigma, C5138-25MG).
本发明的内容不限于实施例所列举，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。 The content of the present invention is not limited to the examples. Any equivalent transformation of the technical solution of the present invention by those of ordinary skill in the art after reading the description of the present invention will be covered by the claims of the present invention.

Claims (10)

  1. 一种重组I型胶原蛋白毕赤酵母工程菌，其特征在于：将I型胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游，目标蛋白分泌至细胞外。A recombinant type I collagen Pichia pastoris engineering strain is characterized in that: the type I collagen gene is connected in series to the downstream of the α-factor signal peptide Kex2 protease recognition site of pPICZalphaA, and the target protein is secreted out of the cell.
  2. 根据权利要求1所述的重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述Ⅰ型胶原蛋白为Ⅰ型人源化胶原蛋白α1链成熟肽。The recombinant type I collagen Pichia pastoris engineering strain according to claim 1, characterized in that: the type I collagen is a type I humanized collagen α1 chain mature peptide.
  3. 根据权利要求2所述的重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述Ⅰ型人源化胶原蛋白α1链成熟肽的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示。The recombinant type I collagen Pichia pastoris engineering strain according to claim 2, characterized in that: the amino acid sequence of the mature peptide of the α1 chain of the type I humanized collagen is as shown in SEQ ID NO.1, and the nucleotide The sequence is shown as SEQ ID NO.2.
  4. 根据权利要求1-3任一所述重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是CGMCC No.24534。The recombinant type I collagen Pichia pastoris engineering strain according to any one of claims 1-3 is characterized in that: the strain is deposited in the General Microbiology Center of the China Microbial Culture Collection Committee, and the deposit number is CGMCC No. 24534.
  5. 一种权利要求1所述重组I型胶原蛋白毕赤酵母工程菌的构建方法，其特征在于：合成的I型人源化胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游。A method for constructing recombinant type I collagen Pichia pastoris engineering strain according to claim 1, characterized in that: the synthesized type I humanized collagen gene is connected in series to the downstream of the α-factor signal peptide Kex2 protease recognition site of pPICZalphaA .
  6. 一种应用权利要求1所述工程菌制备I型胶原蛋白的方法，包括发酵培养和蛋白纯化，其特征在于，发酵培养基包含以下浓度的组分：85％磷酸24～28ml/L，二水合硫酸钙0.65～1.15g/L，硫酸钾16.2～20.2g/L，二水合硫酸镁12.9～16.9g/L，氢氧化钾3.53～4.95g/L，甘油30～50g/L，PTM1 3.5～5.5ml/L，抗坏血酸50～200mg/L，赖氨酸50～300mg/L，山梨醇20～100g/L，肌醇5～20mg/L，叶酸5～30mg/L，泛酸钙2～20mg/L，PH 5.0～7.0。A method for preparing type I collagen using the engineered bacteria described in claim 1, including fermentation culture and protein purification, characterized in that the fermentation medium contains components at the following concentrations: 85% phosphoric acid 24-28ml/L, dihydrate Calcium sulfate 0.65~1.15g/L, potassium sulfate 16.2~20.2g/L, magnesium sulfate dihydrate 12.9~16.9g/L, potassium hydroxide 3.53~4.95g/L, glycerin 30~50g/L, PTM1 3.5~5.5 ml/L, ascorbic acid 50~200mg/L, lysine 50~300mg/L, sorbitol 20~100g/L, inositol 5~20mg/L, folic acid 5~30mg/L, calcium pantothenate 2~20mg/L , PH 5.0～7.0.
  7. 根据权利要求6所述的方法，其特征在于，发酵培养基包含以下浓度的组分：85％磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，PMT1 4.0ml/L，抗坏血酸100mg/L，赖氨酸150mg/L，山梨醇36.4g/L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。The method according to claim 6, wherein the fermentation medium contains components at the following concentrations: 85% phosphoric acid 26.7ml/L, calcium sulfate dihydrate 0.93g/L, potassium sulfate 18.2g/L, sulfuric acid dihydrate Magnesium 14.9g/L, potassium hydroxide 4.13g/L, glycerol 40g/L, PMT1 4.0ml/L, ascorbic acid 100mg/L, lysine 150mg/L, sorbitol 36.4g/L, inositol 10mg/L, Folic acid 20mg/L, calcium pantothenate 10mg/L, PH 6.0.
  8. 根据权利要求6所述的方法，其特征在于，所述发酵培养包括以下步骤：菌体湿重达到220～260g/L时开始补加甲醇，100ml甲醇加1.2ml PMT1，调节PH为5.5±0.3，温度为27±2℃，前12小时甲醇补加速度为2～4ml/h/L，然后为5～7ml/h/L保持10-14小时，最后为8～12ml/h/L保持70-74小时。The method according to claim 6, characterized in that the fermentation culture includes the following steps: when the wet weight of the bacterial cells reaches 220-260g/L, start adding methanol, add 1.2ml PMT1 to 100ml methanol, and adjust the pH to 5.5±0.3 , the temperature is 27±2℃, the methanol supplement rate is 2~4ml/h/L for the first 12 hours, then 5~7ml/h/L for 10-14 hours, and finally 8~12ml/h/L for 70- 74 hours.
  9. 根据权利要求8所述的方法，其特征在于，所述发酵培养包括以下步骤：菌体湿重达到220～260g/L时开始补加甲醇，100ml甲醇加1.2ml PMT1，前12小时甲醇补加速度3ml/h/L，然后为6ml/h/L保持12小时，最后为10ml/h/L保持72小时。The method according to claim 8, characterized in that the fermentation culture includes the following steps: starting to add methanol when the wet weight of the bacterial cells reaches 220-260g/L, adding 1.2ml PMT1 to 100ml methanol, and the methanol supplement speed is increased in the first 12 hours. 3ml/h/L, then 6ml/h/L for 12 hours, and finally 10ml/h/L for 72 hours.
  10. 根据权利要求6～9任一所述的方法，其特征在于，所述蛋白纯化包括以下步骤：离心收集发酵上清液,加入饱和硫酸铵溶液获得沉淀，柠檬酸缓冲液溶解沉淀，进行阳离子交换层析，得到I型胶原蛋白。 The method according to any one of claims 6 to 9, characterized in that the protein purification includes the following steps: centrifuge to collect fermentation supernatant, add saturated ammonium sulfate solution to obtain precipitate, dissolve the precipitate with citric acid buffer, and perform cation exchange Chromatography to obtain type I collagen.
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