Purification and function analysis of the ?-17 fatty acid desaturase with or without transmembrane domain.

ABSTRACT: Fatty acid desaturation enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids. ?-3 desaturase has an important role in converting ?-6 fatty acids into ?-3 fatty acids. Although genes for this desaturase have been identified, the enzymatic activity of ?-17 with or without transmembrane domain, and the function of the ?-17 desaturase is poorly understood. In the present study, a transgenic microorganism was used to clone the ?-17 full length (?-17FL) and ?-17 without transmembrane domain (?-17NT), the expression efficiency was improved and western blotting was used to detect the protein expression level. The purification of ?-17 was precipitated using saturated ammonium sulfate solution, dissolved in phosphate buffered saline buffer, and then filtered using a 10 kDa ultrafiltration cube. Gas chromatography analysis was used to measure the effect of ?-17NT or ?-17FL expression on Pichia pastoris fatty acid composition. Furthermore, the function of ?-17NT in HepG2 cells was measured and the mechanism was explored. It was demonstrated that ?-17NT decreased cell growth and increased apoptosis in hepatocellular carcinoma cell lines in vitro. In conclusion, successful expression of high levels of recombinant ?-17NT represents a critical step towards the elucidation of the function of membrane fatty acid desaturases.

SUBMITTER: Zhou H 

PROVIDER: S-EPMC5609165 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature