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Construction of human-like collagen recombinant pichia pastoris engineering bacteria and rapid protein purification method

A technology of human-like collagen and recombinant collagen, which is applied in the fields of biotechnology and genetic engineering, can solve the problems of long purification process, low purity of collagen, environmental pollution, etc., and achieve simple, fast and cost-effective results

Pending Publication Date: 2022-05-27
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Extract natural collagen and collagen peptides from livestock and poultry animal tissues and marine organisms (livestock and poultry bones, pig skin, Rana skin, skin scraps before tanning in tannery, sea cucumber, fish scale, fish skin), although the process is simple , but there are problems: 1) The extraction efficiency is low, and the purity of the extracted collagen is generally not high, and contains more impurities
2) Limited by the extraction process, the molecular weight of the extracted collagen is above 500Da, and it is difficult to extract even the smallest collagen
3) Different extraction processes, especially when extracting collagen by acid and alkali methods, will pollute the environment and cannot fully achieve green extraction
[0004] At present, most human-like collagens are expressed heterologously using Escherichia coli as the expression host, but their expression will be limited. Whether it is expressed intracellularly or secreted to extracellular expression, the protein components are complex and the purification process is long.

Method used

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  • Construction of human-like collagen recombinant pichia pastoris engineering bacteria and rapid protein purification method
  • Construction of human-like collagen recombinant pichia pastoris engineering bacteria and rapid protein purification method
  • Construction of human-like collagen recombinant pichia pastoris engineering bacteria and rapid protein purification method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of recombinant expression vector pPIC9k-hHLC

[0047] Codon optimization was performed based on the sequence of SEQ ID NO: 1 to obtain a human-like collagen coding gene sequence of SEQ ID NO: 2, avoiding the SacI and SalI restriction sites, so as not to affect the subsequent transformation into Pichia pastoris. The sequence contains too many repetitive sequences, and it is not easy to use PCR amplification. Therefore, two sets of restriction sites were designed at both ends of the sequence (N-terminal added EcoRI and SacII restriction sites, C-terminal added PacI and NotI enzymes cleavage site) to facilitate the construction of this sequence into different expression vectors. Suzhou Jinweizhi Biotechnology Co., Ltd. was entrusted to conduct whole gene synthesis for the sequence of SEQ ID NO: 2.

[0048] The synthetic full gene sequence SEQ ID NO:2 was integrated into the pPIC9k vector through the two restriction sites of EcoRI and NotI, transform...

Embodiment 2

[0049] Example 2 Construction of recombinant genetically engineered strains

[0050] The recombinant expression vector pPIC9k-hHLC in Example 1 was linearized by cutting the recombinant expression vector pPIC9k-hHLC in Example 1 by SacI restriction endonuclease at 37°C for 1 h, and the linearized fragment was purified and recovered using a PCR purification kit to obtain pPIC9k-hHLC linearized fragment . The purified pPIC9k-hHLC linearized fragment was transformed into Pichia pastoris GS115 competent cells, spread on MD plates, and cultured at 30°C for 3-5 days. Select positive clones on YPD plates containing different concentrations of G418, culture at 30°C for 3-5 days, select positive clones that can grow at different concentrations for induction expression, and obtain recombinant Pichia engineering bacteria with multiple copies. . The specific method is as follows:

[0051] 1) Inoculate the activated Pichia GS115 on the YPD plate in 25mLYPD medium / 250mL conical flask, an...

Embodiment 3

[0058] Example 3 Shake flask fermentation of recombinant Pichia pastoris

[0059] The high-copy transformant GS115 / pPIC9k-hHLC grown on the YPD plate of 4 mg / mL G418 was picked, inoculated into a 250 mL Erlenmeyer flask containing 50 mL of YPD, and cultured at 30 °C and 220 rpm for 16-20 h to obtain a strain activation solution. Add 500 μL of bacterial activation solution to 50 mL of BMGY medium, and cultivate it to the OD of the bacterial suspension at 30 °C and 220 rpm. 600 For 2-6, centrifuge at 5000rpm for 10min, collect the cells and resuspend in BMMY medium to dilute the OD to about 1. The resuspended bacterial suspension was placed at 28° C. and 220 rpm to induce expression, and methanol with a final concentration of 0.5% (V / V) was supplemented every 24 h for continuous induction. After 72h-96h of methanol-induced culture, centrifuge at 5000rpm for 10min, and collect the supernatant. The protein expression in the fermentation supernatant was detected by SDS-PAGE ( f...

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Abstract

The invention discloses a construction method of human-like collagen recombinant pichia pastoris engineering bacteria and a rapid protein purification method, and belongs to the field of biotechnology and genetic engineering. The strain for producing the recombinant collagen can achieve high-density fermentation, meanwhile, extracellular secretion of the recombinant collagen is achieved, and the yield of the recombinant collagen can reach 2.33 g / L. The method for preparing the recombinant collagen through rapid purification is simple, rapid and low in cost and plays an important role in maintaining the integrity of the collagen structure, the purity of the obtained protein reaches up to 95% or above, and the method has good application prospects.

Description

technical field [0001] The invention relates to a method for constructing a human-like collagen recombinant Pichia pastoris engineering bacteria and a method for rapid protein purification, belonging to the fields of biotechnology and genetic engineering. Background technique [0002] Collagen is an important structural protein widely distributed in animal tissues. It is widely distributed in connective tissues such as skin, bone, ligament, tendon and blood vessels of animals. It has the functions of supporting organs and protecting the body. Collagen is widely used in the fields of medicine, food, cosmetics, tissue engineering and material engineering due to its unique structural characteristics, good physical properties, biodegradability and biocompatibility. The collagen structure is a homogeneous trimer with a triple helical structure, of which 1 / 4 is in the form of heterotrimers and the rest is in the form of homotrimers. The main amino acids of its polypeptide chain a...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/81C12N1/19C07K14/78C07K1/30C07K1/34C07K1/36C12R1/84
CPCC07K14/78C12N15/815
Inventor 周景文马玲玲曾伟主徐沙余世琴陈坚
Owner JIANGNAN UNIV
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