Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof

A collagen and repeating sequence technology, applied in the field of genetic engineering, can solve the problems affecting the application of proteins, affecting the immunogenicity of proteins, etc., and achieve the effects of good biocompatibility, fast production method and high stability

Active Publication Date: 2021-03-26
西安德诺海思医疗科技有限公司
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Problems solved by technology

At the same time, although the target protein with higher purity can be obtained by using specific purification tags, the purification tags will affect the immunogenicity of the protein, thereby affecting the application of the protein

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  • Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof
  • Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof
  • Recombinant human III-type collagen and pichia pastoris recombinant expression system thereof

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Embodiment Construction

[0033] The invention will be described in further detail below in conjunction with the accompanying drawings and embodiments, which are only used to explain the present invention rather than limit the protection scope of the present invention.

[0034] (1) Structural design of human collagen

[0035] According to the amino acid sequence of natural human type III collagen (reference sequence Genebank accession number: NM_000090.3), the present invention selects a peptide segment consisting of 169 amino acids, and the peptide segment contains valine (V), phenylpropanoid The Gly-X-Y structural peptides of amino acid (F), leucine (L), methionine (M) or alanine (A) are all optimized. The main optimization method is: using natural human type III collagen The helical region is replaced by a Gly-X-Y structural peptide that does not contain the above amino acids with a high frequency (≥3 times).

[0036] The 169AA sequence (position 537-705 in the reference sequence) selected by the p...

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Abstract

The invention discloses a recombinant human III-type collagen and a pichia pastoris recombinant expression system thereof. According to the invention, a Gly-X-Y structural peptide containing valine, phenylalanine, leucine, methionine and alanine in a part of peptide fragments of human III-type collagen is replaced with a Gly-X-Y structural peptide which is high in use frequency and does not contain the amino acid, and a gene sequence for encoding the peptide fragments is obtained according to the peptide fragments after the replacement. And then gene expression vectors for tandem expression ofpeptide fragments with different repeat numbers are constructed and transformed into pichia pastoris host bacteria, and a strain for high expression of the recombinant human III-type collagen is obtained through screening. Through artificial design optimization of a protein structure, the problem that natural collagen is degraded in fermentation is effectively solved, and an expression product has high stability and good biocompatibility and can be used for large-scale production.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to the expression of recombinant human type III collagen by Pichia pastoris. Background technique [0002] Collagen is the most abundant protein in the body and the main component of the extracellular matrix (ECM). It plays an important role in maintaining the normal physiological functions and repairing damage of cells, tissues and organs. It has been widely used in medicine, Health products and cosmetics industry. In terms of molecular structure, each collagen peptide chain is mainly composed of Gly-X-Y structural peptides (X, Y are any amino acid residues other than Gly), this peptide chain structure is necessary for the formation of collagen fiber advanced structure, determines Collagen has excellent biocompatibility and low immunogenicity. [0003] The main source of collagen is extracted from animal tissues. With the development of biotechnology, recombinant co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/81C12N15/12C12N1/19A61L27/24A61K8/65A61Q19/00C12R1/84
CPCC07K14/78C12N15/815A61L27/24A61K8/65A61Q19/00A61K2800/10A61K2800/805
Inventor 杨鹭黄文涛
Owner 西安德诺海思医疗科技有限公司
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