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Gene optimization of cholesterol dehydrogenase and high-efficiency expression thereof

A technology of cholesterol and dehydrogenase, which is applied in the field of protein production by recombinant DNA technology, can solve the problems of harsh reaction conditions in the production process, high requirements for production equipment and production conditions, and low protein expression

Inactive Publication Date: 2014-06-25
TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In summary, the current production of 7-dehydrocholesterol has the following deficiencies: 1. Part of the production process has harsh reaction conditions, which require high production equipment and production conditions, which increases production costs; 2. Some methods have low protein expression. Not suitable for industrial production

Method used

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  • Gene optimization of cholesterol dehydrogenase and high-efficiency expression thereof
  • Gene optimization of cholesterol dehydrogenase and high-efficiency expression thereof
  • Gene optimization of cholesterol dehydrogenase and high-efficiency expression thereof

Examples

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Embodiment 1

[0038] Example 1: Optimization of cholesterol 7,8-dehydrogenase gene and construction, screening and induced expression of recombinant Escherichia coli genetically engineered strains, the preparation steps are:

[0039] 1.1 Optimization design of cholesterol 7,8-dehydrogenase gene daf-36

[0040] (1) The sequence (SEQ ID NO. 1) of the original cholesterol 7,8-dehydrogenase gene (daf-36) cloned in the present invention from the nematode (Caenorhabditis elegans), the full length of the original gene is 1287bp, encoding a total of 428 amino acids and a stop codon;

[0041] (2) Through the analysis of http: / / people.mbi.ucla.edu / sumchan / caltor.html, under the premise of not changing the amino acid sequence of the protein, the preferred codons of Escherichia coli were selected;

[0042] (3) Considering factors such as the frequency of codon usage, adjustment of GC content, deletion of unstable sequences, stability of mRNA secondary structure, etc., according to the preferred codons...

Embodiment 2

[0051] Example 2: Optimization of cholesterol 7,8-dehydrogenase gene and construction and screening of recombinant Pichia pastoris genetically engineered strains, the preparation steps are:

[0052] 2.1 Optimization design of cholesterol 7,8-dehydrogenase gene daf-36

[0053] (1) The sequence of the original cholesterol 7,8-dehydrogenase gene (daf-36) cloned in the present invention from the nematode (Caenorhabditis elegans), the full length of the original gene is 1287 bp, encoding a total of 428 amino acids and a stop codon;

[0054] (2) Through the analysis of http: / / www.jcat.de / , on the premise of not changing the amino acid sequence of the protein, the preferred codons of Pichia pastoris were selected;

[0055] (3) Considering factors such as the frequency of codon usage, adjustment of GC content, deletion of unstable sequences, stability of mRNA secondary structure, etc., according to the preferred codons of Pichia pastoris, the method of systematic codon optimization wa...

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Abstract

The invention relates to gene optimization of cholesterol dehydrogenase and high-efficiency expression thereof, belonging to the field of production of protein by utilizing recombinant DNA (deoxyribonucleic acid) technology. Particularly, the invention provides an optimized DNA sequence of cholesterol 7,8-dehydrogenase, which is suitable for a Escherichia coli expression system or a pichia pastoris expression system, and research on construction of engineering bacteria and the expression optimization technology of the cholesterol 7,8-dehydrogenase. The optimized gene is transferred into Escherichia coli and pichia pastoris to be expressed, and compared with the expression quantity of a non-optimized gene, the expression quantity of the optimized gene in the Escherichia coli and pichia pastoris is obviously improved.

Description

technical field [0001] The present invention relates to the field of protein production using recombinant DNA technology. More specifically, the present invention relates to the DNA sequence of an optimized recombinant cholesterol 7,8-dehydrogenase based on E. coli codon preference design, and a method for secreting and expressing cholesterol 7,8-dehydrogenase in E. coli; and based on Pichia pastoris Codon bias design optimized DNA sequence of recombinant cholesterol 7,8-dehydrogenase, and method for secretory expression of cholesterol 7,8-dehydrogenase in Pichia pastoris. Background technique [0002] Vitamin D 3 It is an essential fat-soluble vitamin for the growth, development, reproduction, maintenance of life and health of humans and animals. Its main function is to regulate calcium and phosphorus metabolism, promote intestinal calcium and phosphorus absorption and bone calcification, and maintain the balance of blood calcium and blood phosphorus. Humans and animals ...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/04C12N15/70C12N1/21C12N1/19C12R1/19C12R1/84
Inventor 王敏申雁冰汤睿牛丹丹格日勒郑宇
Owner TIANJIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
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