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Method for producing recombinant human type II collagen single chain by Pichia pastoris

一种胶原蛋白、毕赤酵母的技术，应用在基因工程领域，能够解决难以去除降解产物、无法满足纯度要求、翻译表达效率低下等问题，达到优秀工程菌种、杂质蛋白少、优化二级结构的效果

Active Publication Date: 2021-04-06
JIANGSU TRAUTEC MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no relevant research or patent on the use of Pichia pastoris to secrete and express the full-length α1 mature peptide chain of human type II collagen, and the recombinantly expressed human collagen currently used for production usually does not add an affinity tag or only adds an affinity tag at one end. However, the expressed peptide chain is easy to degrade, and the purity of the protein obtained by traditional non-specific purification methods or single affinity tag purification is not enough, and it is difficult to remove some degradation products, which cannot meet the requirements of high purity applications.
In previous studies or patents, the DNA sequences used to express collagen are natural coding sequences in the human genome (generally obtained by reverse transcription), but there are certain differences between yeast and humans in the process of gene transcription and translation. In particular, the codon bias is different. Using the natural coding DNA sequence of human collagen to express in Pichia pastoris will cause low translation and expression efficiency due to codon bias and the overall structure of the mRNA.

Method used

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  • Method for producing recombinant human type II collagen single chain by Pichia pastoris
  • Method for producing recombinant human type II collagen single chain by Pichia pastoris
  • Method for producing recombinant human type II collagen single chain by Pichia pastoris

Examples

Experimental program
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Effect test

Embodiment 1

[0064] The construction of embodiment 1 recombinant expression vector

[0065] The full length of the recombinant human type Ⅱ collagen single chain to be expressed is 1078 amino acids, the N-terminal is a Srtep-tagⅡ tag, and the C-terminal is a 6×His tag. This gene recombines the amino acids of the human type Ⅱ collagen single chain The sequence is shown in SEQ ID NO.2.

[0066] 1.1 Optimization and synthesis of gene sequence

[0067] Based on Genebank gene sequence NM_001844.4 and recombinant human type II collagen single-chain amino acid sequence SEQID NO.2, aiming at Pichia pastoris codon preference, GC content of DNA sequence, mRNA secondary structure, CpG island, repeat Various parameters such as sequence, PolyA site, and RNA unstable region were calculated and optimized systematically, and the synonymous conversion method was used to eliminate restriction sites such as EcoRI (GAATTC) and NotI (GCGGCCGC) in the sequence. Compared with the coding sequence of the natural...

Embodiment 2

[0244] Embodiment 2 Construction of recombinant Pichia pastoris engineering bacteria

[0245] 2.1 Linearization of expression vector pPIC9K-Strep-2A1-His

[0246] Restriction endonuclease SalI was used to digest overnight at 37°C, and the reaction system was as follows:

[0247]

[0248] Then 0.7% agarose gel electrophoresis was used to detect whether the cutting was complete. After the cutting was complete, the enzyme cutting solution was treated with a plasmid extraction kit, and the linearized plasmid was recovered, so that the volume was controlled at about 10 μL.

[0249] 2.2 Preparation of Pichia pastoris SMD1168 competent cells

[0250] 1) Pick a single colony of yeast SMD1168, inoculate it into a test tube containing 5mL of YPD liquid medium, and culture it overnight at 30°C and 220rpm with shaking;

[0251] 2) Inoculate 50 μL of the overnight culture into a 500 mL Erlenmeyer flask containing 50 mL of fresh YPD liquid medium, culture at 30°C and 220 rpm overnight ...

Embodiment 3

[0286] Example 3 Yeast Recombinant Collagen Expression, Purification and Preparation

[0287] Strep-Tactin affinity chromatography resin was purchased from IBA Life Science Company, and Ni-NTA His·Bands affinity chromatography resin was purchased from Merck Company.

[0288] 3.1 Fermentation-induced expression of yeast engineered bacteria

[0289] 1) Use YPD culture to cultivate Pichia engineered bacteria based on overnight activation at 30°C and 220rpm;

[0290] 2) Take the above-mentioned bacterial solution and transfer it to BMGY medium, and cultivate the engineering bacteria at 30°C and 220rpm for 16-18h, until OD600=2.0-6.0;

[0291] 3) Centrifuge at 1500g for 5min at room temperature, collect the bacteria, resuspend the bacteria with 10mL of BMMY medium to make OD600=1.0, put the obtained bacteria solution in a 100mL sterile Erlenmeyer flask, and continue shaking at 28°C and 220rpm nourish;

[0292] 4) adding methanol to the medium every 24 hours to a final concentrat...

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Abstract

The invention discloses a method for expressing and preparing recombinant human type II collagen single chain by Pichia pastoris. The present invention provides a nucleotide sequence encoding a single chain of recombinant human type II collagen, the nucleotide sequence is shown in SEQ ID NO.1, which is encoded by the nucleotide sequence SEQ ID NO.1 The amino acid sequence of the recombinant human type II collagen single chain is shown in SEQ ID NO.2. The recombinant human type II collagen single chain obtained according to the present invention contains the full-length amino acid sequence of the mature human type II collagen α1 chain, which can effectively support the realization of the biological functions of type II collagen and has good biological activity , and the amino terminus and carboxyl terminus contain different affinity purification tags, which can effectively purify and obtain full-length single-chain proteins, and are also conducive to the identification of collagen; at the same time, according to the expression characteristics of Pichia pastoris, the present invention encodes recombinant human II The gene sequence of type collagen single chain was systematically calculated and optimized to make it more suitable for expression in Pichia pastoris.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a nucleotide sequence encoding a recombinant human type II collagen single chain and a production method for the recombinant human type II collagen single chain. Background technique [0002] In mammals, collagen is rich in content, widely distributed, and various in variety. It is an essential and important component for cells, tissues and even organs to perform normal functions and repair body damage. Among the many members of the collagen family, type II collagen is mainly distributed in cartilage tissue, vitreous body, and cornea, accounting for more than 90% of the total collagen in adult cartilage matrix. In addition to the basic biological functions of collagen, such as hemostatic properties, biocompatibility, biodegradability, low immunogenicity, etc., type II collagen has some unique biological functions: (1) can form reticular fibers Structure, closely com...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/12C07K14/78C12N15/81C07K1/22
CPCC07K14/78C12N15/815
Inventor 钱松李佳佳
Owner JIANGSU TRAUTEC MEDICAL TECH CO LTD
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