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Method for expression of fatty acid desaturase by acellular protein synthesis system

A saturase and fatty acid technology, applied in biochemical equipment and methods, enzymes, oxidoreductases, etc., can solve the problems of difficult purification, low expression of membrane-bound fatty acid desaturase, complicated expression steps, etc., to achieve simple expression. Effect

Active Publication Date: 2014-05-28
JIANGNAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is to solve the current problems such as low expression of membrane-bound fatty acid desaturase, difficulty in purification, complicated expression steps, etc., to efficiently express ω3 fatty acid desaturase through wheat germ cell-free protein synthesis system, including constructing Recombinant plasmid of desaturase gene, preparation of liposome, adding recombinant plasmid and liposome to wheat germ cell-free protein expression system for expression

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  • Method for expression of fatty acid desaturase by acellular protein synthesis system
  • Method for expression of fatty acid desaturase by acellular protein synthesis system
  • Method for expression of fatty acid desaturase by acellular protein synthesis system

Examples

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Embodiment 1

[0020] Example 1 Construction of a recombinant plasmid transformed into the ω3 fatty acid desaturase gene FADS15 in Mortierella alpina ATCC#32222

[0021]According to the sequence information of the ω3 fatty acid desaturase gene (FADS15) of Mortierella alpina ATCC#32222, primers P1 and P2 were designed, and the underlined parts are the enzyme cutting sites Not I and Xho I, respectively, to contain the ω3 desaturase gene (FADS15 ) plasmid pET19b-FADS15 as a template (this laboratory has published articles Haiqin Chen, Zhennan Gu, Hao Zhang, Mingxuan Wang, Wei Chen, W.Todd Lowther, Yong Q.Chen*.Expression and Purification of Integral Membrane Fatty Acid Desaturases .PLoS ONE.2013,8(3):e58139), with primer P 1 / P 2 , KOD high-fidelity polymerase, to amplify the ω3 desaturase gene (FADS15) by PCR. The nucleotide sequence of the obtained ω3 fatty acid desaturase gene is shown in SEQ ID NO.1. The PCR program was: 95°C for 30s, 60°C for 30s, 68°C for 90s, 30 cycles, and the PCR pr...

Embodiment 2

[0024] Example 2 Preparation of liposomes added to the cell-free protein synthesis system

[0025] Soybean Lipid Extract (Soybean Lipid Extract) purchased from Avanti Polar Lipids was used to prepare liposomes, which are different from drug embedding and available for membrane protein binding. The specific preparation method is as follows:

[0026] (1) Weigh 0.03g of soybean lipid extract (Soybean Lipid Extract) powder, dissolve it in an appropriate amount of chloroform, and after it is fully dissolved, remove the chloroform by vacuum rotary evaporation;

[0027] (2) Add 1ml of hydration buffer (containing 0.9% NaCl, 5% glucose, 10% sucrose) to the above mixture from which chloroform has been evaporated, and incubate at 55°C for 1 hour until the solution becomes turbid;

[0028] (3) Place the above turbid solution at 55°C for 5-10 minutes and sonicate it for 5-10 minutes. After the solution turns from turbid to clear, liposomes with a concentration of 30mg / ml are obtained, wh...

Embodiment 3

[0029] Example 3 Wheat germ cell-free protein synthesis system expresses ω3 fatty acid desaturase

[0030] (1) Preparation of expression vector: activate Escherichia coli DH5α transformed with pIVEX WG1.4-FADS15 recombinant plasmid, culture overnight at 37°C, take 20ml of bacterial liquid to extract the plasmid, and use plasmid medium extraction reagent purchased from Qiagen PlasmidMidiKit was used to extract the plasmid, and the obtained plasmid was further extracted with phenol chloroform (phenol: chloroform: isoamyl alcohol = 25:24:1) to remove the residual RNase, and finally the concentration and OD of the plasmid were measured 260 / 280 ratio.

[0031] (2) Protein expression: Using Roche’s Wheat Germ Extract Cell-Free Protein Synthesis Kit (RTS100Wheat Germ CECF kit), add about 1-2 μg of the above-mentioned purified plasmid into a 50 μl reaction system, and mix wheat germ extract, Mix the RNA polymerase, amino acid and other reaction mixtures and add them to the reaction t...

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Abstract

The invention discloses a method for expression of fatty acid desaturase by an acellular protein synthesis system, and belongs to the enzyme engineering field. The method uses the wheat germ acellular protein synthesis system for cloning and expression of omega 3 desaturase gene (FADS15) from mortierella alpine ATCC32222 (American type culture collection32222) without the need for preparation of mRNA, the expression amount is high, subsequent purification steps are simple, and the method lays the foundation for the next step of research of membrane protein crystal structures and functions.

Description

technical field [0001] The invention relates to a method for expressing fatty acid desaturase through a cell-free protein synthesis system, belonging to the technical field of enzyme engineering. Background technique [0002] Fatty acid desaturases are enzymes that convert single bonds between adjacent atoms into double bonds on fatty acyl carbon chains. They are present in most organisms such as bacteria, fungi, plants, and animals, and play an important role in maintaining the correct structure and function of the biofilms of these organisms. Depending on the intracellular localization of the protein, fatty acid desaturases can be divided into two classes, soluble or membrane-bound, which have evolved independently. Among them, acyl-ACP (Acyl-ACP) is a soluble desaturase, which mainly exists in the plastids of higher plants, and catalyzes the desaturation of fatty acids bound to acyl carrier proteins; and acyl-CoA (Acyl-CoA) is a membrane-bound protein , It is found that...

Claims

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Application Information

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IPC IPC(8): C12N9/02
CPCC12N9/0071C12N15/63
Inventor 陈海琴陈永泉陈卫陈思顾震南赵建新张灏杨芹王鸿超
Owner JIANGNAN UNIV
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