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Yeast recombined human-source I-type collagen alpha1 chain protein, synthesis method and application thereof

A collagen and chain protein technology, applied in the field of bioengineering, can solve the problems of complicated purification process operation, difficult to improve the purity, difficult to stabilize the purification process, etc., and achieve the effect of simple fermentation process, short fermentation period and improved cutting efficiency.

Inactive Publication Date: 2019-07-09
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, molecular sieves and ion exchange resins are mostly used for the separation and purification of collagen, but collagen is difficult to separate due to its unique G-X-Y structure, especially with the degradation fragments of the same collagen, and the purification process is complicated and different. The purification process between batches is difficult to stabilize, resulting in the collagen short peptide products on the market are mostly mixtures of short peptides and their degradation products; and affinity purification technology can obtain products with higher purity due to its specific identification, and is easy to operate. It is expected to solve the problems that its purity is difficult to improve and the purification process is difficult to stabilize

Method used

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  • Yeast recombined human-source I-type collagen alpha1 chain protein, synthesis method and application thereof
  • Yeast recombined human-source I-type collagen alpha1 chain protein, synthesis method and application thereof
  • Yeast recombined human-source I-type collagen alpha1 chain protein, synthesis method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] In Example 1, the preparation of recombinant human type I collagen α1 chain protein was carried out, and the experimental steps were as follows:

[0062] 1. Synthesize the gene sequence of human type I collagen α1 chain mature peptide: under the condition of not changing the amino acid sequence of human type I collagen α1 chain mature peptide, the corresponding gene sequence corresponding to Pichia pastoris is not commonly used The codons were optimized to the preferred codons of Pichia pastoris, and DNASTAR software was used to optimize the gene sequence, and the restriction endonuclease sites such as ApaI, Bgl II, BamHI, EcoR I, Not I, Sac I, Sal I were removed, The gene sequence of human type I collagen α1 chain mature peptide was synthesized, see SEQ ID NO.3;

[0063]2. Prepare the modified plasmid pPIC9ks containing the Strep-Tag II coding sequence: first use the pPIC9k plasmid as a template, and use primers P1 and P2 to perform PCR amplification, and the product l...

Embodiment 2

[0141] An example of the use of yeast recombinant human type I collagen α1 chain protein in cosmetic and skin care products for external use is now given:

[0142] According to the following mass ratio, use purified water as a solvent to dissolve all raw materials, and stir well to form a colorless, odorless and transparent moisturizing skin care essence;

[0143] Among them, glycerin 2%, butanediol 2%, sodium hyaluronate 0.5%, recombinant human type I collagen α1 chain protein 0.5%, vitamin C ethyl ether 0.5%, vitamin C sodium phosphate 0.5%, dipotassium glycyrrhizinate 0.3%, EDTA·Na 2 0.5%, silk peptide 0.5%, β-glucan 0.3%, Luba oil 0.3%, water-soluble azone 0.5%, and the rest is purified water.

[0144] How to use: After cleansing in the morning and evening, apply directly to the face, gently pat until fully absorbed.

Embodiment 3

[0146] Now provide the embodiment that the recombinant human source type I collagen α1 chain protein is used in cosmetic and skin care products for external use:

[0147] According to the following mass ratio, use purified water as a solvent to dissolve all raw materials, and stir well to form a colorless, odorless and transparent moisturizing skin care essence;

[0148] Glycerin 1%, Butylene Glycol 1%, Sodium Hyaluronate 0.01%, Recombinant Human Type I Collagen α1 Chain Protein 0.01%, Vitamin C Ethyl Ether 0.01%, Vitamin C Sodium Phosphate 0.01%, Dipotassium Glycyrrhizinate 0.1%, EDTA·Na 2 0.01%, silk peptide 0.01%, β-glucan 0.1%, Luba oil 0.1%, water-soluble azone 0.1%, and the rest is purified water.

[0149] How to use: After cleansing in the morning and evening, apply directly to the face, gently pat until fully absorbed.

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Abstract

The invention discloses yeast recombined human-source I-type collagen alpha1 chain protein, a synthesis method and an application thereof. The recombinant human-source I-type collagen alpha1 chain protein is composed of N-terminal affinity purification marker, human-source I-type collagen alpha1 chain mature peptide sequence and C-terminal affinity purification marker, and the double specific affinity purification markers are designed at two ends of the protein, so that purification of the recombinant protein is facilitated, and detection and full-length identification of the recombinant protein are facilitated. The synthesis method includes utilizing pichia pastoris idiomatic codon to optimize a collagen gene sequence and performing artificial whole-gene synthesis on a gene level; building a recombinant vector through PCR, enzyme digestion and connection, and integrating the recombinant vector into yeast chromosome to build a recombinant pichia pastoris engineered strain secreting andexpressing the recombined human-source I-type collagen alpha1 chain protein. The double specific affinity purification markers carried by the protein is supportive of two-step specific purification,and easiness in acquiring a high-purity product is realized.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a yeast recombinant human type I collagen α1 chain protein, a synthesis method and an application thereof. Background technique [0002] Collagen is the most abundant protein family in animals, accounting for about 30% of the total protein in animals. Among the 29 collagens found so far, type I collagen has the highest content, accounting for about 90%. In the morphogenesis and cell metabolism of new tissues, it endows new tissues with mechanical strength and biochemical properties; it is mainly distributed in skin, cornea, tendon and other parts, and it plays an important role in maintaining the normal physiological functions and repairing of cells and tissues effect. [0003] Type I collagen has good biocompatibility, biodegradability, non-cytotoxicity, anti-oxidation and other functions, and can promote cell adhesion and cell proliferation, and can be widely used in m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/78C12N15/81A61L15/28A61L15/32A61L26/00A61L27/20A61L27/24A61L27/54A61K8/34A61K8/44A61K8/49A61K8/63A61K8/64A61K8/65A61K8/67A61K8/73A61K8/92A61Q19/00
CPCA61K8/345A61K8/44A61K8/4906A61K8/63A61K8/64A61K8/65A61K8/676A61K8/73A61K8/735A61K8/922A61L15/28A61L15/325A61L26/0033A61L26/008A61L27/20A61L27/24A61L27/54A61L2300/252A61Q19/00C07K14/78C12N15/815C08L5/00C08L5/08
Inventor 梁亮张永正
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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