关键词: 高山被孢霉, &omega, -3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

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 ω-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 WG1.4, and expressed in the wheat-germ cell-free protein expression system. To get the correctly folded ω-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.

Key words: Mortierella alpina, ω-3 fatty acid desaturase, wheat-germ cell-free protein expression system, liposome, protein purification