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