高山被孢霉ω3脂肪酸脱饱和酶基因体外表达载体的构建及其在无细胞蛋白质合成系统中的表达
首发时间:2014-03-26
摘要:无细胞蛋白质合成系统具有快速、方便等特点,且能表达对活细胞具有一定毒性的膜蛋白和抗菌肽,近年来被广泛应用。本文选取来自产油丝状真菌高山被孢霉多不饱和脂肪酸合成途径中的关键膜结合酶,ω3脂肪酸脱饱和酶基因为研究对象,构建了适宜于体外表达ω3脂肪酸脱饱和酶基因的表达载体pIVEX WG1.4-FADS15,并利用麦胚无细胞蛋白质合成系统实现了高效表达。同时在麦胚无细胞蛋白质合成过程中添加脂质体,以使表达的膜蛋白正确定位至脂质体磷脂双分子层,便于目标蛋白质的正确折叠。研究结果显示,添加脂质体条件下目标蛋白质的表达量达1.8 mg/mL,经碘海醇(Accudenz)密度梯度离心纯化,目标蛋白质纯度可达到90%以上,这为后续对该酶进行催化特性研究和蛋白质晶体结构解析奠定了基础。
关键词: 高山被孢霉 ω3脂肪酸脱饱和酶 麦胚无细胞蛋白质合成系统 脂质体 蛋白纯化
For information in English, please click here
Cloning and expression of the ω3 fatty acid desaturase from Mortierella alpina in the cell-free protein expression system
Abstract:Cell-free protein expression system as a tool of rapid and convenient protein expression has been widely used in producing membrane proteins and antibacterial peptides that have certain toxicity to living cells. In this paper, the membrane-bound ω3 fatty acid desaturase gene ,which encoding the key enzyme involves in the polyunsaturated fatty acid biosynthesis pathways in the oleaginous fungi Mortierella alpina ,was cloned into the in vitro expression vector pIVEX WG1.4 and expressed in the Wheat-germ cell-free protein expression system. To get the correctly folded ω3 fatty acid desaturase FADS15, liposomes that is able to relocate the expressed membrane protein into the phospholipid bilayer were also added in the process of cell-free protein synthesis .Our results indicated that, in the presence of liposomes, the production of FADS15 reached 1.8 mg/mL, after accudenz density gradient centrifugation, the purity of FADS15 was higher than 90%.This research work paves the way for the further study of catalytic property and crystal structure analysis of this enzyme.
Keywords: Mortierella alpina ω3 fatty acid desaturase Wheat-germ cell-free expression system Liposome Protein purification
论文图表:
引用
No.4590494964117139****
同行评议
共计0人参与
勘误表
高山被孢霉ω3脂肪酸脱饱和酶基因体外表达载体的构建及其在无细胞蛋白质合成系统中的表达
评论
全部评论0/1000