Biological method for large-scale preparation of collagen having 164.88° triple-helix structure

Abstract

Provided is a biological method for large-scale preparation of a collagen having a 164.88° triple-helix structure. Provided is a method for constructing a yeast cell expressing a collagen, comprising introducing a recombinant expression vector into a yeast cell, the recombinant expression vector containing a polynucleotide encoding a collagen, the collagen comprising repeat units represented by the sequence of SEQ ID No.1, the number of the repeat units being 2-32, and the repeat units being directly linked. The collagen is a recombinant type III humanized collagen, preferably the collagen having a 164.88° triple-helix structure. Modifying a ''chassis bacterium'' enables large-scale production of the recombinant humanized collagen having the 164.88° triple-helix structure which is a type A collagen. The large-scale production can satisfy market demands.

Description

Large-scale biological method for preparing 164.88° triple helical collagen

[0001] This application claims the priority benefit of the Chinese invention patent application with application date: September 28, 2023, application number: 202311272338.8, and invention name: "Biomethod for large-scale preparation of 164.88° triple helix structure collagen". Technical Field

[0002] The invention relates to a method for preparing collagen with a 164.88° triple helix structure by utilizing large-scale biological fermentation, belonging to the technical field of synthetic biology. Background Art

[0003] Humans have 28 different types of collagen. Collagen is the most abundant protein in humans and animals, accounting for 25-30% of the total body protein and approximately 6% of body weight. Collagen is a crucial structural protein found in skin, bones, teeth, corneas, tendons, ligaments, and various tissues and organs, supporting and protecting tissues. Collagen is a major component of the extracellular matrix, and its biological activities are primarily manifested in promoting cell adhesion, growth and differentiation, cell signaling responses, platelet aggregation, maintenance of cell and tissue function, and repair of injuries. Collagen exhibits a triple helical structure, with three independent collagen α-peptide chains primarily forming the helical structure through hydrogen bonds. In the body, collagen binds to tissues and cells in this triple helical structure to exert its biological effects. Therefore, collagen's biological activity is largely dependent on its natural triple helical structure. Collagen's excellent biological activity holds immense application value and potential in biomedical materials, medical aesthetics, and cosmetics.

[0004] The main collagen products currently on the market are derived from animal tissue extraction. This method destroys the natural triple helix structure during the extraction process, affecting the biological activity of collagen. Furthermore, collagen extracted using animal-derived extraction processes has uneven peptide molecular weights and poor processability. Due to differences in amino acid sequences between animal and human collagen, these products may pose potential immunogenicity and poor safety in clinical settings. Furthermore, due to the source of animal tissue, the safety risk of animal viruses must be addressed before they can enter the market. As market demands for the safety and efficacy of collagen products increase, traditional methods of extracting collagen from animal tissue are no longer able to meet current market demand. Furthermore, the demand for products using collagen as a biomaterial for medical applications is also increasing, necessitating alternative approaches to address current market challenges. Producing recombinant collagen with a natural triple helix structure through molecular biology techniques such as genetic engineering holds great potential for addressing these issues.

[0005] There are already companies on the market that produce type III collagen products through genetic recombination, but it is difficult to achieve a triple helix structure. Using synthetic organisms to ferment collagen with the correct triple helix structure has high technical barriers.

[0006] There is a need in the art for methods of synthetically fermenting collagen having the correct triple helical structure.

[0007] Summary of the Invention

[0008] The inventors have discovered a human type III collagen, and structural studies have confirmed that the core functional region of human type III collagen has a 164.88° triple helix structure (CN109593126A). The inventors have successfully prepared recombinant type III humanized collagen using an Escherichia coli expression system through genetic recombination. However, the inventors found that the Escherichia coli expression system has shortcomings such as limited protein expression and limited process amplification. Therefore, the inventors attempted to express human type III collagen in a eukaryotic expression system. After a lot of preliminary screening work, the inventors found that when human type III collagen was expressed in Pichia pastoris, a large amount of collagen with a 164.88° triple helix structure could be synthesized in vitro. In a previous application, the inventors also found that human type III collagen contains bacterial endotoxins when expressed in Escherichia coli, which increases the complexity and difficulty of collagen purification. The inventors eliminated this technical problem by expressing collagen in a yeast expression system.

[0009] The present invention achieves large-scale production by transforming the "chassis bacteria". The recombinant humanized collagen with a 164.88° triple helix structure belongs to type A and can meet market demand through large-scale production.

[0010] In one aspect, a method for constructing a yeast cell expressing collagen is provided, comprising introducing a recombinant expression vector into the yeast cell, wherein the recombinant expression vector comprises a polynucleotide encoding the collagen, wherein the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units being 2-32, and the repeating units being directly linked. In one embodiment, the collagen is recombinant humanized type III collagen. In one embodiment, the collagen is 164.88° triple helical collagen.

[0011] In one embodiment, the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3. In one embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 4 or 5. The polynucleotide of the present invention can effectively express collagen in yeast host cells.

[0012] In one embodiment, the recombinant expression vector is a yeast system expression vector. In one embodiment, the yeast system expression vector is pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector.

[0013] In one embodiment, the yeast is Pichia pastoris. In one embodiment, the Pichia pastoris is a Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain.

[0014] In one embodiment, the method comprises one or more of the following steps:

[0015] (1) inserting the polynucleotide into an expression vector, preferably between the NotI and XhoI restriction sites of the pPiczalαA expression vector, to obtain a recombinant expression plasmid;

[0016] (2) digesting the recombinant expression plasmid with enzymes, preferably using sac1;

[0017] (3) Purifying the recombinant expression plasmid after enzyme digestion;

[0018] (4) The recombinant expression plasmid after enzyme digestion is introduced into yeast cells through electroporation.

[0019] In another aspect, a yeast cell expressing collagen is provided, wherein the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly linked; and the yeast cell is Pichia pastoris.

[0020] In one embodiment, the Pichia pastoris is a Pichia pastoris strain X33, GS115, SMD1168, KM71, or KM71H. In one embodiment, the collagen comprises the amino acid sequence set forth in SEQ ID No. 2 or 3. In one embodiment, the collagen is recombinant humanized type III collagen. In one embodiment, the collagen is a 164.88° triple helical collagen.

[0021] In one embodiment, the yeast cell comprises a yeast system expression vector. In one embodiment, the yeast system expression vector is pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector. In one embodiment, the expression vector comprises a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 2 or 3.

[0022] In another aspect, there is provided use of the yeast cell described herein for producing collagen.

[0023] In one embodiment, the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly linked. In one embodiment, the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3. In one embodiment, the collagen is recombinant humanized type III collagen. In one embodiment, the collagen is 164.88° triple helical collagen.

[0024] In another aspect, a method for producing collagen by fermentation is provided, which comprises culturing the yeast cells described herein under suitable conditions, adding methanol to the yeast cells to induce expression, and harvesting the culture supernatant to obtain collagen.

[0025] In one embodiment, the culture medium is BMMY medium supplemented with an amino-free yeast nitrogen source and biotin.

[0026] In one embodiment, the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly linked. In one embodiment, the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3. In one embodiment, the collagen is recombinant humanized type III collagen, preferably collagen with a 164.88° triple helical structure.

[0027] Advantages of the present invention include:

[0028] 1. The inventors have discovered yeast cells suitable for expressing recombinant humanized type III collagen, as well as methods for their construction and production;

[0029] 2. The yeast cell of the present invention is Pichia pastoris, which has the advantages of high expression yield and scalable process compared to prokaryotic expression systems such as Escherichia coli;

[0030] 3. The method of the present invention synthesizes collagen with a 164.88° triple helix structure in vitro, achieving large-scale mass production at the ton level, which has important industrial significance;

[0031] 4. The present invention does not require the step of eliminating endotoxins. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 shows the electrophoresis of high molecular weight P-012 induced expression.

[0033] FIG2 shows the electrophoresis diagram of the induced expression of medium molecular weight P-C3T8.

[0034] Figure 3 shows the cell adhesion activity of high molecular weight P-012.

[0035] FIG4 shows the adhesion activity of middle molecular weight P-C3T8 cells.

[0036] Figure 5 shows the circular dichroism spectrum of high molecular weight P-012.

[0037] Figure 6 shows the circular dichroism results of medium molecular weight P-C3T8. DETAILED DESCRIPTION

[0038] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are 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 making creative efforts shall fall within the scope of protection of the present invention.

[0039] As used in this article, recombinant collagen is a new type of biomaterial that is screened and prepared using cutting-edge structural biology, genetic engineering and other technologies, using the gene encoding the functional region of a specific type of human collagen as a template to obtain the same or similar amino acid sequence as human collagen.

[0040] As used herein, "human recombinant type III collagen" refers to a recombinant protein consisting of or essentially consisting of a sequence derived from human type III collagen. In this article, human recombinant type III collagen may consist of or essentially consist of a fragment or multiple repeats of a fragment derived from human type III collagen. As used herein, "yeast" includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).

[0041] As used herein, yeast host cell means any yeast host cell that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or recombinant expression vector comprising a polynucleotide of the present invention. The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces or Yarrowia cell, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis or Yarrowia lipolytica cell. In this article, the preferred yeast cell is Pichia pastoris. More preferably, the Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain.

[0042] As used herein, the term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0043] As used herein, the term "expression vector" refers to a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide and operably linked to a control sequence for its expression. In this context, the expression vector is a yeast system expression vector, preferably pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector.

[0044] As used herein, the term "recombinant expression vector" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not originally found in nature to contain a nucleic acid segment, or is synthetic, and that contains one or more control sequences.

[0045] As used herein, the term "control sequence" means the nucleic acid sequence necessary for expressing the polynucleotide encoding the mature polypeptide of the present invention. Each control sequence can be natural (i.e., from the same gene) or exogenous (i.e., from different genes) for the polynucleotide encoding the polypeptide, or natural or exogenous relative to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the control sequence includes a promoter, and transcription and translation termination signals. For the purpose of introducing specific restriction sites that are conducive to connecting the control sequence to the coding region of the polynucleotide encoding the polypeptide, these control sequences can be provided with multiple linkers.

[0046] collagen

[0047] The present invention provides collagen. The collagen may comprise a sequence shown in SEQ ID No. 1 or a sequence thereof having 80-100% (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity. The number of repeating units is 2-32, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32. Each repeating unit may be directly linked. The collagen is a recombinant humanized type III collagen, preferably a 164.88° triple helical structure collagen. The collagen may comprise an amino acid sequence as shown in SEQ ID No. 2 or 3, or an amino acid sequence with 80-100% (e.g., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity thereto. The collagen described herein may have a triple helical structure or three identical chains (i.e., in the form of a trimer). The sequence of each chain may be a sequence described herein.

[0048] Recombinant expression vector

[0049] The present invention also relates to a recombinant expression vector comprising a nucleic acid of the present invention operably linked to one or more control sequences that direct expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. The vector may comprise a recombinant expression vector. The nucleic acid of the present invention may comprise the nucleotide sequence of SEQ ID NO. 4 or 5.

[0050] Nucleic acids can be manipulated in a variety of ways to provide for the expression of collagen. Depending on the expression vector, it may be desirable or necessary to manipulate the nucleic acid prior to its insertion into the vector. Techniques for modifying nucleic acids using recombinant DNA methods are well known in the art.

[0051] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell for expression of the collagen protein or polypeptide encoding the present invention. The promoter comprises transcriptional control sequences that mediate the expression of the collagen protein or polypeptide. The promoter can be any nucleic acid that exhibits transcriptional activity in the host cell, including variants, truncated forms, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular collagen protein or polypeptide homologous or heterologous to the host cell.

[0052] Examples of suitable promoters for directing transcription of the vector or recombinant expression vector of the present invention in yeast host cells are not particularly limited. In yeast hosts, useful promoters are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP), Saccharomyces cerevisiae triosephosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase.

[0053] The control sequence can also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator can be operably linked to the 3' end of the polynucleotide encoding the collagen or polypeptide. Any terminator that is functional in the host cell can be used in the present invention. Preferred terminators for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. The control sequence can also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

[0054] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0055] The control sequence can also be a leader sequence, i.e., a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the polynucleotide encoding the collagen or polypeptide. Any leader sequence that is functional in the host cell can be used. Suitable leader sequences for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0056] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenylic acid residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the host cell may be used. Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0057] The control sequence can also be a signal peptide coding region that encodes a signal peptide connected to the N-terminus of the collagen protein and directs the collagen protein to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide itself can contain a signal peptide coding sequence that is naturally connected to the coding sequence segment of the collagen protein in the translation reading frame. Alternatively, the 5'-end of the coding sequence can contain a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally contain a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, the exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the collagen protein or polypeptide. However, any signal peptide coding sequence that directs the expressed collagen protein to enter the secretory pathway of the host cell can be used. Useful signal peptides for yeast host cells are obtained from the following genes: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Yeast cells can be applied to the collagen protein for the preparation of this article.

[0058] host cells

[0059] The present invention also relates to recombinant host cells comprising polynucleotides of the present invention operably linked to one or more control sequences that direct the production of the collagen protein of the present invention. The construct comprising the polynucleotide is introduced into the host cell so that the construct is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector. The term "host cell" encompasses any parent cell progeny that is not identical to the parent cell due to mutations that occur during replication. The selection of the host cell will depend largely on the gene encoding the collagen protein or polypeptide and its source.

[0060] Herein, the host cell may be a yeast cell, including ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell. In particular, the present inventors have found that Pichia pastoris is suitable for producing the collagen described herein. Yeast cells can express the collagen described herein. Preferably, the collagen is recombinant humanized type III collagen, preferably 164.88° triple-helical collagen. The yeast cell is Pichia pastoris; preferably, Pichia pastoris X33, GS115, SMD1168, KM71, or KM71H strains. The yeast cell may contain a yeast system expression vector, preferably pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P. The expression vector may contain a polynucleotide comprising the nucleotide sequence set forth in SEQ ID No. 4 or 5.

[0061] Construction method

[0062] The present invention provides a method for constructing a yeast cell expressing collagen, comprising introducing a recombinant expression vector into a yeast cell, wherein the recombinant expression vector comprises a polynucleotide encoding the collagen described herein. The recombinant expression vector may comprise a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 4 or 5. The recombinant expression vector may be a yeast system expression vector, preferably a pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector. The yeast may be Pichia pastoris, preferably a Pichia pastoris X33, GS115, SMD1168, KM71, or KM71H strain.

[0063] The method may include one or more of the following steps:

[0064] (1) inserting the polynucleotide into an expression vector, preferably between the NotI and XhoI restriction sites of the pPiczalαA expression vector, to obtain a recombinant expression plasmid;

[0065] (2) digesting the recombinant expression plasmid with enzymes, preferably using sac1;

[0066] (3) Purifying the recombinant expression plasmid after enzyme digestion;

[0067] (4) The recombinant expression plasmid after enzyme digestion is introduced into yeast cells through electroporation.

[0068] Fermentation method

[0069] Provided herein are methods for producing collagen by fermentation. The methods may include culturing yeast cells as described herein under suitable conditions, adding methanol to the yeast cells to induce expression, and harvesting the culture supernatant to obtain the collagen. The conditions and culture medium for culturing the yeast cells are known to those skilled in the art and are not particularly limited. For example, the culture medium may be BMMY medium supplemented with an amino-free yeast nitrogen source and biotin.

[0070] The fermentation method may include (1) seed liquid culture, wherein the seed liquid is the seed liquid of the yeast cells described herein; (2) fermentation culture: inoculating the seed liquid into a fermentation medium, culturing in a fermenter, and starting to feed methanol at a suitable growth stage to induce expression; (3) preparing a fermentation supernatant: culturing for a suitable time, terminating the culture, removing the bacterial cells from the fermentation liquid, and filtering to obtain a fermentation supernatant;

[0071] The fermentation method may further comprise a step of purifying the collagen. The purification step is known to those skilled in the art and is not particularly limited. For example, the purification method may employ the purification step described in CN109593126A.

[0072] The present invention is further illustrated by the following examples. It should be understood that the scope of the present invention should be based on the appended claims, and the examples should not be interpreted as limiting the scope of the present invention.

[0073] Example

[0074] The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims. In conjunction with this specification and common knowledge in the art, a person of ordinary skill in the art will clearly understand the scope defined by the claims. Without departing from the spirit and scope of the present invention, those skilled in the art may make any modifications or changes to the technical solution of the present invention, and such modifications and changes are also included in the scope of the present invention.

[0075] General methods of PCR, cloning, ligation, etc. of nucleotide sequences are well known to those skilled in the art and can be found, for example, in the following references: "Molecular cloning: A laboratory manual", Sambrook et al. (1989), Cold Spring Harbor lab., Cold Spring Harbor, NY; Ausubel, FM et al. (eds.); "Current protocols in Molecular Biology", John Wiley and Sons (1995); Harwood, CR and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", DN Glover, ed. (1985); "Oligonucleotide Synthesis", MJ Gait, ed. (1984); "Nucleic Acid Hybridization", BD Hames & S.J. Higgins, eds. (1985); "A Practical Guide To Molecular Biology", John Wiley and Sons (1995); Harwood, CR and Cutting, SM (eds.); "DNA Cloning: A Practical Approach, Volumes I and II", DN Glover, ed. (1985); "Oligonucleotide Synthesis", MJ Gait, ed. (1984); "Nucleic Acid Hybridization", BD Hames & S.J. Higgins, eds. (1985); "A Practical Guide To Molecular Cloning [A Practical Guide to Molecular Cloning]", B. Perbal (1984).

[0076] Example 1: Construction, expression and screening of protein fragments

[0077] 1) Amino acid sequence of high molecular weight recombinant humanized type III collagen:

[0078] (SEQ ID No. 2; repeating unit: gergapgfrgpagpngipgekgpagergap; SEQ ID No. 1).

[0079] High molecular weight recombinant humanized type III collagen protein nucleotide sequence:

[0080] 2) Amino acid sequence of medium molecular weight recombinant humanized type III collagen:

[0081] Nucleotide sequence of medium molecular weight recombinant humanized type III collagen protein:

[0082] The above coding nucleotide sequence was commercially synthesized. The above coding nucleotide sequence was inserted between the NotI and XhoI restriction sites of the pPiczalαA expression vector, with the target gene at the end of the signal cleavage, to obtain recombinant expression plasmids P-012 (high molecular weight) and P-C3T8 (medium molecular weight).

[0083] Example 2: Yeast transformation of recombinant humanized type III collagen

[0084] 1. Plasmid digestion

[0085] Take 20-40ug each of the successfully constructed expression plasmids P-012 and P-C3T8, use sac1 for enzyme digestion, and perform enzyme digestion at 37°C for 2-3h according to the restriction enzyme digestion ratio. After the enzyme digestion is completed, perform nucleic acid gel electrophoresis to detect the plasmid before and after enzyme digestion to verify whether all enzyme digestion is complete and that the position of the band after enzyme digestion is larger than that before enzyme digestion.

[0086] 2. Plasmid Purification

[0087] The plasmid after enzyme digestion was purified using a plasmid purification kit to remove impurities such as ions, and dissolved in 20-25ul sterilized purified water. The plasmid concentration was tested and the plasmid was placed at 4°C for use.

[0088] 3 Yeast transformation

[0089] Take out the yeast competent cells (X33) and perform the following operations under a biosafety cabinet:

[0090] a. Take a 0.2 cm electric rotating cup, soak it in 75% alcohol for 10 minutes to disinfect it, rinse it repeatedly with sterile water for 3 times and dry it;

[0091] b. Place the electroporation cup in an ice bath for 20 minutes and turn on the electroporator;

[0092] c. Add 80ul of competent cells and 5-15ug of enzyme-digested plasmid to a sterilized 1.5ml EP tube and mix repeatedly using a pipette;

[0093] d. Transfer 100 μl of the mixed solution to an electroporation cup. After ice bathing for 5 minutes, wipe the outer surface of the electroporation cup and transfer the cup to the electroporation instrument tank. Click Start to start electroporation.

[0094] e. After electroporation, add 1 ml of 1 M sterile sorbitol solution to the electroporation cup, seal it with parafilm, and incubate at 30°C for 2 h.

[0095] f. Transfer the mixed solution in the electroporation cup to a sterilized 10 ml EP tube, then add 1 ml of YPD liquid medium (0.2 g / L yeast peptone, 0.1 g / L yeast extract powder) to the EP tube and culture at 30°C, 300 rpm for 1 h.

[0096] g. Plate spreading: Under a biosafety cabinet, take 200 μl of the transformed and incubated bacterial solution and add it to the poured plates 1-3. Plate 4 is used as a control. Use a sterile spreader to evenly spread the plate. After completion, incubate the plate at 30°C for about 2-5 days.

[0097] h. Bacterial Culture: Culture on plates until plaques form. Add 10 ml of anhydrous glucose solution to 40 ml of YPD liquid medium. When the medium cools to room temperature, dispense 50 ml of the medium into 10 50 ml bioreactor tubes at a rate of 5 ml / tube. Add 2000 mg / L bleomycin solution. Pick 10 single colonies from the plate and inoculate them into 10 small tubes for overnight culture at 30°C and 300 rpm. Prepare 200 ml of BMGY medium. After sterilization, add 20 ml of 10*YNB stock solution and 200 μl of 500*biotin stock solution to a shake flask and mix thoroughly. Divide the 200 ml medium into 10 50 ml centrifuge tubes at a rate of 20 ml / tube. Then, take 4 ml of the cultured bacterial solution and inoculate 10 20 ml / tubes. Culture overnight until the bacterial solution has an OD value between 3 and 10.

[0098] i. Induction of expression: Prepare 500 ml of BMMY medium. After sterilization, add 50 ml of 10*YNB stock solution and 500 μl of 500*biotin stock solution to a shake flask and mix well. Divide the 500 ml medium into 10 250 ml shake flasks, 50 ml per flask. Centrifuge the cultured bacterial solution at 1500 rpm for 10 min and discard the supernatant. Resuspend the bacterial pellets in 10 centrifuge tubes with the medium from each of the 10 shake flasks and transfer them to their own shake flasks for shake flask culture at 30°C and 300 rpm. Every 24 h, sample 1 ml from each of the 10 shake flasks and transfer them to 1.5 ml EP tubes, which are stored at -20°C. Add 1% methanol (filtered using a 0.22 μm filter) to the shake flasks to induce expression until the end of the 5th day. Centrifuge the sample in the 1.5 ml EP tube at 12000 rpm for 5 min, remove the supernatant for electrophoresis, and observe the expression of each of the 10 clones.

[0099] 4 Electrophoresis detection

[0100] The specific procedure was as follows: 40 μl of sample solution was added to 10 μl of 5× protein loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 5% β-mercaptoethanol). The sample was then boiled in 100°C water for 10 minutes. Then, 10 μl per well was added to an SDS-PAGE gel and run at 80 V for 2 hours. Proteins were then stained with Coomassie Brilliant Blue (0.1% Coomassie Brilliant Blue R-250, 25% isopropanol, and 10% glacial acetic acid) for 20 minutes and destained with protein destaining buffer (10% acetic acid and 5% ethanol). The electrophoresis results are shown in Figures 1 and 2: the P-012 target protein has a theoretical molecular weight of 44.75 kD and an apparent molecular weight of 50 kD; the P-C3T8 target protein has a theoretical molecular weight of 22.39 kD and an apparent molecular weight of 28 kD. The electrophoresis test results showed that the yeast transformation was successful.

[0101] Example 3: Mass spectrometry detection of recombinant humanized type III collagen

[0102] 1. Experimental instruments:

[0103] 1) High-resolution mass spectrometer: XevoG2-XS QTof (Waters)

[0104] 2) Ultra-high performance liquid chromatography: UPLC (Acquity UPLC I-Class) (Waters)

[0105] 2 Materials and reagents

[0106] 1) Guanidine HCl (Sigma)

[0107] 2) Urea (Bio-Rad)

[0108] 3) Tris-base (Bio-Rad)

[0109] 4) DTT (Bio-Rad)

[0110] 5) IAM (Sigma)

[0111] 6)Zeba Spin column(Pierce)

[0112] 7)ACQUITY UPLC Peptide BEH C18 Column, 1.7μm, 2.1mm X150mm (Waters)

[0113] 8) UNIFI (Waters)

[0114] 9) Trypsin (Promega)

[0115] 10) Chymotrypsin (Sigma)

[0116] 11) Glu-C (Wako)

[0117] 12) LysC (Wako)

[0118] 3 Experimental methods

[0119] 1) Enzymatic digestion of trypsin, chymotrypsin, and Glu-C: Take an appropriate amount of the test sample and pre-treat it before adding trypsin, chymotrypsin, and Glu-C. Digest the sample at 37°C for 20 hours.

[0120] 2) High-Performance Liquid Chromatography: After enzymatic hydrolysis, the sample was separated using an Acquity UPLC I-Class system. Liquid A consisted of 0.1% FA in water, and Liquid B consisted of 0.1% FA in acetonitrile. The sample was loaded onto the column via an autosampler and separated by chromatography. The column temperature was 55°C, the flow rate was 300 μl / min, and the TUV detector wavelength was 214 nm. The relevant liquid gradients were as follows:

[0121] 3) Mass Spectrometry: The samples were desalted and separated by ultra-high performance liquid chromatography (UPLC), followed by mass spectrometry analysis using a Xevo G2-XS QTof mass spectrometer (Waters). Analysis time: 63 min. Detection method: positive ion MS, scan range (m / z): 300-2000.

[0122] 4) Mass spectrometry data processing: The original database was checked using UNIFI (1.8.2, Waters) software. The main parameters are as shown in (Table 1):

[0123] 4 Experimental results and analysis

[0124] After using Trypsin, Chymotrypsin and Glu-C to perform in-solution enzymatic hydrolysis on the test samples, the obtained peptide samples were analyzed by LC-MS / MS equipment, and the obtained raw data were checked in the library using UNIFI software.

[0125] 1) Mass spectrometry detected the molecular weight of P-012 and the corresponding peptide

[0126] The coverage of the detected peptide fragments was 100%.

[0127] 2) Mass spectrometry detection of the molecular weight of P-C3T8 and the corresponding peptide

[0128] The coverage of the detected peptide fragments was 100%.

[0129] The enzymatically hydrolyzed samples were analyzed by LC-MS / MS and the results were integrated into the database. The final peptide coverage of the samples was 100%, and the test results were very reliable.

[0130] Example 4: Cell Adhesion Activity Detection of Recombinant Humanized Type III Collagen

[0131] The method for detecting collagen activity can be found in the literature Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura, Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643-649 (2004). The specific implementation method is as follows:

[0132] (1) The concentration of the protein sample to be tested is detected by ultraviolet absorption method, including bovine type I collagen (China Food and Drug Inspection Institute, No.: 380002) and the recombinant humanized protein provided by the present invention. Specifically, the ultraviolet absorption of the sample at 215nm and 225nm is measured respectively, and the protein concentration is calculated using the empirical formula C (μg / mL) = 144×(A215-A225). Note that the test must be performed when A215 < 1.5. The principle of this method is: to measure the characteristic absorption of the peptide bond under far ultraviolet light, it is not affected by the chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop color with Coomassie Brilliant Blue. (Reference is Walker JM. The Protein Protocols Handbook, second edition. Humana Press. 43-45.). After the protein concentration is detected, the concentration of all the proteins to be tested is adjusted to 0.5 mg / mL with PBS.

[0133] (2) Add 100 μL of various protein solutions and a blank PBS solution as a control to a 96-well plate and let it stand at room temperature for 60 min.

[0134] (3) Add 105 well-cultured NIH / 3T3 cells to each well and incubate at 37°C for 60 min.

[0135] (4) Wash each well 4 times with PBS.

[0136] (5) Use the LDH detection kit (Roche, 04744926001) to measure the absorbance at OD492nm. Based on the blank control value, the cell adhesion rate can be calculated. The calculation formula is as follows: Cell adhesion rate = × 100%. The cell adhesion rate can reflect the activity of collagen. The higher the activity of the protein, the better the external environment can be provided to the cells in a short time, helping the cells to adhere. The test results are shown in Figures 3 and 4 below.

[0137] Example 5: UV-scanning analysis of recombinant humanized type III collagen by circular dichroism

[0138] Experimental methods

[0139] (1) Instrument parameter setting

[0140] (2) Far and near UV scanning of standard products

[0141] The scanning wavelength was set to 180-340 nm for background test and blank buffer test, and then the near- and far-UV circular dichroism absorption of 1 mg / mL CSA standard solution in the range of 180-340 nm was collected.

[0142] (3) Sample processing

[0143] P-012 and P-C3T8 protein samples were collected and concentrated to a protein concentration of 1 mg / ml using 10KD ultrafiltration concentrators (Millipore).

[0144] (4) Sample far-UV scanning

[0145] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry, collect the background first, then collect the blank buffer, then add an appropriate amount of test sample to the cuvette, perform far-UV scanning at 190-260nm according to the above parameters and collect data.

[0146] (5) Sample near-UV scanning

[0147] Soak the cuvette in 2M HNO3 overnight, rinse with deionized water and dry it. Collect the background first, then collect the blank buffer solution, then add an appropriate amount of test sample to the cuvette, perform near-UV scanning at 250-340nm according to the above parameters and collect data.

[0148] (6) Scanning spectrum processing

[0149] All scanned images were processed by subtracting baseline and smoothing using the software Pro-Data Viewer.

[0150] Experimental results and analysis

[0151] The results showed that both P-012 and P-C3T8 had positive peaks at 221 nm, indicating that the proteins had triple helical structures. The results are shown in Figures 5 and 6.

Claims

1. A method for constructing a yeast cell expressing collagen, comprising introducing a recombinant expression vector into a yeast cell, wherein the recombinant expression vector comprises a polynucleotide encoding collagen, wherein the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly connected; preferably, the collagen is recombinant type III humanized collagen, preferably 164.88° triple helical structure collagen.

2. The method according to claim 1, wherein the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3; preferably, the polynucleotide comprises the nucleotide sequence shown in SEQ ID No. 4 or 5.

3. The method according to claim 1 or 2, wherein the recombinant expression vector is a yeast system expression vector, preferably pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector.

4. The method according to any one of claims 1 to 3, wherein the yeast is Pichia pastoris; preferably Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strains.

5. The method according to any one of claims 1 to 4, comprising one or more of the following steps: (1) inserting the polynucleotide into an expression vector, preferably between the NotI and XhoI restriction sites of the pPiczalαA expression vector, to obtain a recombinant expression plasmid; (2) digesting the recombinant expression plasmid with enzymes, preferably using sac1; (3) Purifying the recombinant expression plasmid after enzyme digestion; (4) The recombinant expression plasmid after enzyme digestion is introduced into yeast cells through electroporation.

6. A yeast cell expressing collagen, wherein the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly connected; the yeast cell is Pichia pastoris; preferably, it is Pichia pastoris X33, GS115, SMD1168, KM71 or KM71H strain; preferably, the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3; preferably, the collagen is recombinant type III humanized collagen, preferably 164.88° triple helical structure collagen.

7. The yeast cell according to claim 6, comprising a yeast system expression vector, preferably a pPICZαA, pPIC9, pPIC9K, pHIL-S1, or pYAM75P vector; the expression vector comprises a polynucleotide comprising the nucleotide sequence shown in SEQ ID No. 4 or 5.

8. Use of the yeast cell according to claim 6 or 7 for preparing collagen, preferably, the collagen comprises repeating units of the sequence shown in SEQ ID No.1, the number of repeating units is 2-32, and the repeating units are directly connected; preferably, the collagen comprises the amino acid sequence shown in SEQ ID No.2 or 3; preferably, the collagen is recombinant type III humanized collagen, preferably 164.88° triple helical structure collagen.

9. A method for producing collagen by fermentation, comprising culturing the yeast cells according to claim 6 or 7 under suitable conditions, adding methanol to the yeast cells to induce expression, and harvesting the culture supernatant to obtain collagen; preferably, the culture medium is a BMMY medium supplemented with an amino-free yeast nitrogen source and biotin.

10. The method according to claim 9, comprising: (1) seed liquid culture, wherein the seed liquid is the seed liquid of the yeast cell of claim 6 or 7; (2) Fermentation culture: inoculating the seed liquid into the fermentation medium, culturing in a fermenter, and starting to feed methanol at a suitable growth stage to induce expression; (3) Preparation of fermentation supernatant: culturing until a suitable time is reached, the fermentation liquid is taken out to remove the bacterial cells, and filtered to obtain the fermentation supernatant; Preferably, the collagen comprises repeating units of the sequence shown in SEQ ID No. 1, the number of repeating units is 2-32, and the repeating units are directly connected; preferably, the collagen comprises the amino acid sequence shown in SEQ ID No. 2 or 3; preferably, the collagen is recombinant type III humanized collagen, preferably 164.88° triple helix structure collagen.

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