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CN-122011159-A - Yeast heterologously expressed III type recombinant human collagen peptide and preparation thereof

CN122011159ACN 122011159 ACN122011159 ACN 122011159ACN-122011159-A

Abstract

The invention discloses a yeast heterologous expression type III recombinant human collagen peptide and a preparation method thereof, wherein the recombinant human collagen peptide comprises an amino acid sequence shown as SEQ ID No. 1. The multicopy tandem repeat peptide comprises the basic repeat unit shown as SEQ ID No. 1. The preparation process includes the steps of constructing recombinant expression vector plasmid with nucleic acid molecule encoding multiple tandem repeat peptide connected to the vector, transferring the recombinant expression vector plasmid into host cell, culturing multiple positive transformant, inducing treatment, centrifuging, micro filtering, ultrafiltering and purifying the fermented liquid, ultrafiltering to collect and purify the multiple tandem repeat peptide after enzymolysis. The invention screens the amino acid sequence of the human collagen and replaces specific amino acid, thereby avoiding the influence caused by the combination of glycosylation and nonspecific of MMP-1 enzyme cutting site and lysine site, and remarkably improving the bioactivity and the utilization rate.

Inventors

  • RONG SHAOFENG
  • SHEN HANXIAO
  • LI QIANQIAN
  • GUAN SHIMIN

Assignees

  • 上海应用技术大学

Dates

Publication Date
20260512
Application Date
20260123

Claims (10)

  1. 1. The yeast heterologous expression type III recombinant human collagen peptide is characterized by comprising an amino acid sequence shown as SEQ ID No. 1.
  2. 2. A multi-copy tandem repeat peptide comprising a basic repeat unit as set forth in SEQ ID No. 1.
  3. 3. The multi-copy tandem repeat peptide of claim 2, wherein the number of basic repeat units is 5-15.
  4. 4. A multicopy tandem repeat peptide according to claim 3, wherein said multicopy tandem repeat peptide comprises the amino acid sequence shown in SEQ ID No. 2.
  5. 5. A nucleic acid molecule encoding a yeast heterologous expressed recombinant human collagen type III peptide according to claim 1 or a multicopy tandem repeat peptide according to any one of claims 2 to 4.
  6. 6. A nucleic acid molecule according to claim 5, comprising a nucleotide sequence as set forth in SEQ ID No. 3.
  7. 7. A recombinant expression vector plasmid comprising at least one nucleic acid molecule according to claim 5 or 6.
  8. 8. A host cell comprising at least one nucleic acid molecule according to claim 5 or 6 or a recombinant expression vector plasmid according to claim 7.
  9. 9. A method for preparing a yeast heterologous expressed type III recombinant human collagen peptide according to claim 1 or a multicopy tandem repeat peptide according to any one of claims 2 to 4, comprising the steps of: (1) Connecting the nucleic acid molecules for encoding the multi-copy tandem repeat peptide into a vector, constructing a recombinant expression vector plasmid, transferring the recombinant expression vector plasmid into a host cell, and screening to obtain a multi-copy positive transformant; (2) Culturing multiple copies of positive transformants, adding an inducer into the fermentation broth for induction treatment, and collecting the fermentation broth after induction expression; (3) Centrifuging the fermentation liquor, filtering with a microfiltration membrane, ultra-filtering for impurity removal and purifying to obtain the multi-copy tandem repeat peptide; (4) And (3) performing ultrafiltration collection and purification after enzymolysis on the multi-copy tandem repeat peptide obtained in the step (3) to obtain the type III recombinant human collagen peptide heterologously expressed by the yeast.
  10. 10. The method of claim 9, wherein the host cell is a pichia pastoris engineered strain; The procedure for the cultivation of the multicopy positive transformants was as follows: 1) Inoculating the multicopy positive transformant into YPD culture medium for culturing overnight; 2) Transferring the thalli cultivated in the step 1) into a BMGY culture medium, supplementing glycerol after the initial glycerol is exhausted in the cultivation process, stopping supplementing glycerol after the concentration of the thalli rises to reach an index, starving the thalli for a period of time, centrifuging, and collecting the thalli; The induction treatment comprises the steps of putting the thalli obtained in the step 2) into a BMMY culture medium for induction expression, and supplementing methanol for induction expression of multiple copies of tandem repeat peptide in the expression process; The purification is nickel column purification or alcohol precipitation purification or salting-out purification; the temperature of the culture and the induction treatment is 28-29 ℃; The step 2) is carried out in a fermentation tank, and the culture conditions are that the pH is 4.5-6.0, the rotation speed of a stirring paddle is 300-700 rpm, and the air flux is 1.0-1.5L/min; the YPD medium consists of 2wt% of tryptone, 1wt% of yeast extract, 2wt% of glucose and the balance of water; the BMGY culture medium comprises 2wt% of tryptone, 1wt% of yeast extract, 0.23wt% of dipotassium hydrogen phosphate, 1.18wt% of potassium dihydrogen phosphate, 1wt% of glycerol, 1.34wt% of YNB, 0.004wt% of biotin and the balance of water; the BMMY culture medium comprises 2wt% of tryptone, 1wt% of yeast extract, 0.23wt% of dipotassium hydrogen phosphate, 1.18wt% of potassium dihydrogen phosphate, 1wt% of methanol, 1.34wt% of YNB, 0.004wt% of biotin and the balance of water.

Description

Yeast heterologously expressed III type recombinant human collagen peptide and preparation thereof Technical Field The invention relates to the technical field of bioengineering, and relates to a type III recombinant human collagen peptide heterologously expressed by yeast and a preparation method thereof, in particular to a type III recombinant human collagen peptide heterologously expressed by yeast, a multi-copy tandem repeat peptide, a nucleic acid molecule, a recombinant expression vector plasmid containing at least one nucleic acid molecule, a host cell containing at least one nucleic acid molecule or the recombinant expression vector plasmid and a preparation method thereof. Background Collagen is a natural biopolymer that not only plays a vital role in the human body, but also has wide application in a variety of industries. As an important protein accounting for one third of the total protein of the human body, the collagen is responsible for maintaining the structural integrity of tissues and organs, and is a basic material for forming main organs such as skin, muscle, bone, blood vessels and the like. In addition, it plays an important role in providing proper elasticity and strength, and is an essential component for maintaining the normal function of the skin. In particular in the dermis, the protein fibers of collagen are extremely resistant to stretching and tearing. In the cosmetic industry, collagen is widely used for its biocompatibility with skin, which is part of natural skin ingredients. With age, the yield of natural collagen in the human body gradually decreases, which is also why cosmetic and cosmeceutical manufacturers develop products that can supplement collagen inside and outside. In addition, collagen also shows its importance in promoting wound healing, which makes collagen-based biomaterials, such as collagen sponges, a potential drug delivery system. Among the 29 known types of collagen, the protein encoded by the COL3A1 gene in humans is a protein molecule with a long triple-helical domain that requires three alpha 1 chains to form. Type III collagen is in a complex network distribution in the alveolar stroma, helping to maintain the flexibility and elasticity of pulmonary tissue. Meanwhile, the III type collagen has higher content in the blood vessel, maintains the strength and the tension of the blood vessel through the excellent mechanical property of the III type collagen, provides nutrition for cells to a certain extent, and promotes the formation of new blood vessels. Compared with other types, the III type collagen has outstanding aspects of tissue injury repair, activation of a coagulation mechanism, promotion of platelet aggregation and the like. Type III collagen is a natural protein of organisms, and is favored in the fields of biological medicine, food, daily chemicals and the like because of its strong biocompatibility, weak antigenicity, high affinity to skin and controllable biodegradability. However, collagen protein in the market is extracted from animal tissues at present, so that immune rejection reaction is easy to induce, and animal-derived pathogen pollution (such as prion, foot-and-mouth disease virus, HIV, rabies virus and the like) and the risk of exceeding heavy metal exist, so that the difficulty of product quality control is increased. With the development of genetic engineering and high-density fermentation technologies, the production of collagen by biosynthetic means has been the focus of attention of researchers. The method not only can effectively avoid the risks of virus infection and immune rejection existing in the traditional animal-derived collagen, but also has the advantages of simplified process, resource saving and easy mass production, and the obtained product generally shows excellent hydrophilicity and high safety. However, natural collagen is used as insoluble fibrin, and its unique (Gly-X-Y) n repetitive sequence and the characteristic of relying on complex post-translational modification and self-assembly processes make recombinant expression face challenges such as low expression level, poor secretion efficiency and difficult purification, and especially for type III collagen, these problems lead to scarcity of species and high cost. At the present stage, the development of collagen peptide with small molecular weight and easy transdermal is particularly critical, but the product is often more difficult to realize high-efficiency soluble expression in an expression system, and the subsequent purification process is complex, so that the application promotion of the product is restricted. In addition, the existing part of collagen peptide on the market does not effectively avoid the enzyme cleavage site of Matrix Metalloproteinases (MMPs) in the molecular design, so that the collagen peptide is easy to degrade in practical application, and the functional stability is affected. Therefore, the recombinant human-source III-type col