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Yeast recombinant collagen

A technology for recombinant collagen and collagen, which is applied in the production field of yeast recombinant collagen, can solve the problem of low collagen expression, and achieve the effect of simple operation and product safety

Inactive Publication Date: 2017-08-25
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem solved by the present invention includes providing an optimized human type III collagen fragment gene sequence to solve the problem of low expression of collagen in Pichia pastoris caused by codon utilization efficiency in the prior art

Method used

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  • Yeast recombinant collagen
  • Yeast recombinant collagen
  • Yeast recombinant collagen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1 The acquisition of recombinant constitutive expression plasmids (such as figure 1 shown)

[0073] (1) Optimize gene synthesis

[0074] According to the amino acid sequence of collagen obtained by the method disclosed in Chinese patent 201310033299.6 and the gene sequence of type III collagen peptide registered in Genbank, referring to the preferred codons of Pichia pastoris, the DNASTAR software was used without changing the original amino acid sequence. Optimize the gene sequence, and then obtain the optimized gene sequence through whole gene synthesis, and introduce the coding sequence of Kex2 restriction site at the front end, and add TAA site and EcolRI restriction site at the 3′ end of the optimized gene. Synthesized by the method to obtain the plasmid pPIC9k-col3-col3-6his containing the target gene sequence.

[0075] (2) Construction of recombinant expression plasmids

[0076]In the related experiments of the present invention, the plasmid pPIC9k-col...

Embodiment 2

[0077] Example 2 The acquisition of recombinant Pichia pastoris engineering bacteria

[0078] (1) Linearization of recombinant constitutive expression plasmid pGHKα-col3-col3-6his

[0079] Extract the recombinant plasmid pGHKα-col3-col3-6his, digest it with restriction endonuclease SalI at 37°C, and then use 0.7% agarose gel electrophoresis to detect whether it is completely cut. After it is completely cut, use gel to recover The kit processes the enzyme cutting solution, recovers the linearized plasmid, and desalts it.

[0080] (2) Preparation of Pichia pastoris SMD1168 competent cells

[0081] ①Pick a single colony of yeast SMD1168, inoculate it into a Erlenmeyer flask containing 5mL of YPD liquid medium, and cultivate overnight at 30°C and 220rpm with shaking;

[0082] ② Take 50 μL of the overnight culture and inoculate it into a 500 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, culture at 30°C and 220 rpm with shaking overnight, until the OD 600 The v...

Embodiment 3

[0104] Example 3 Yeast recombinant collagen production

[0105] ⑴Use YPD to cultivate recombinant Pichia pastoris engineering bacteria based on overnight cultivation at 30°C and 220rpm;

[0106] (2) Take an appropriate amount of overnight culture and insert it into BMDY medium, so that the OD 600 =1.0-2.0, cultured with shaking at 28°C and 220 rpm for 4 days.

[0107] (3) After the fermentation culture, centrifuge at 5000×g for 20 min at 4°C, and collect the supernatant. Take a small amount of supernatant for SDS-PAGE analysis and detection, the results are as follows figure 2 As shown, according to the His tag, Western Blot analysis was performed to confirm that the main protein band was the target protein, and the results were as follows image 3 shown;

[0108] (4) Add 5 to 7 times the volume of pure water to the supernatant, and then concentrate it to 20% of the initial volume by ultrafiltration;

[0109] (5) Add 100 μL 1×Ni-NTA binding buffer to 1 mL of concentrated...

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Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a production method for a yeast recombinant collagen, which includes construction of recombinant pichia pastoris engineered strain and production of constitutively and secretively expressed recombinant collagen without methanol induction. The invention first utilizes conventional codons of pichia pastoris at the genetic level to optimize a collagen gene sequence and performs artificial total gene synthesis, the optimized collagen gene sequence is then integrated into yeast chromosome, and thereby a constitutively and secretively expressed recombinant pichia pastoris engineered strain is constructed. The fermentation process is easy to operate, yield is high, and the separation and purification of the product are simple; methanol induction is not needed, the fire-proof and explosion-proof indexes of production equipment and factory buildings are low, and the production environment is environmentally friendly and pollution-free; and no toxic and harmful substances are use, so the product is safer. A specific affinity purification label is carried, the product with higher purity can be easily obtained, and the product can be applied to biomedical materials.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for producing yeast recombinant collagen. Background technique [0002] Collagen is the most abundant protein family in animals. It is an important component of the connective tissue that supports and protects the body, and maintains a certain mechanical strength of the connective tissue. Collagen accounts for about 30% of the total protein in the animal body and mainly exists in the skin, bones, tendons, soft tissues, etc. It plays an important role in maintaining the normal physiological functions and repairing of cells, tissues, and organs. Collagen, as a natural biopolymer material, has good biocompatibility, biodegradability and absorbability, promotes cell formation and epithelial cell formation and other functions, and can be widely used in medicine, beauty, cosmetics, health care products, etc. In the field, it can maintain the elasticity of blood vessel ...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/78A61L26/00A61L15/32A61L15/28C12R1/84
CPCC07K14/78A61L15/28A61L15/325A61L26/0023A61L26/0033C12N15/815
Inventor 梁亮张永正
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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