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

摘要

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