高山被孢霉ω-3脂肪酸脱饱和酶基因在麦胚无细胞蛋白质合成系统中的表达

Expression of the ω-3 Fatty Acid Desaturase from Mortierella alpina in Wheat-germ Cell-free Protein Expression System

无细胞蛋白质合成系统具有快速、方便等特点,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。以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——ω-3脂肪酸脱饱和酶为研究对象,构建了适宜体外表达ω-3脂肪酸脱饱和酶的表达载体p IVEX WG1.4-FADS15,并利用麦胚无细胞蛋白质合成系统实现了对该基因的高效表达。同时,通过在麦胚无细胞蛋白质合成过程中添加脂质体的方法,将所表达的膜蛋白正确定位至脂质体磷脂双分子层,以便目标蛋白质的正确折叠。研究结果显示,在此实验条件下目标蛋白质的表达量达1.8 mg/m L,经碘海醇密度梯度超速离心纯化后,该蛋白质的纯度可以达到90%以上。研究结果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。

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 to-3 fatty acid desaturase gene, which encoding the key enzyme involving in the polyunsaturated fatty acid biosynthesis pathways in the oleaginous fungi Mortierella alpina, was cloned into the in vitro expression vector pIVEX WGI.4, and expressed in the wheat-germ ceU-free protein expression system. To get the correctly folded ~o-3 fatty acid desaturase FADS15, liposomes that was able to relocate the expressed membrane protein into the phospholipid bilayer were also added in the process of cell-free protein synthesis, and 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 paved the way for the further study of catalytic property and crystal structure analysis of this enzyme.