ARS | Publication request: Expression and purification of diacylglycerol acyltransferases

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: December 20, 2012
Publication Date: N/A

Technical Abstract: Diacylglycerol acyltransferases (DGATs) are integral membrane proteins that catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG and over-expression of DGATs increases TAG. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. DGAT genes have been identified from at least 83 organisms, but none of the enzymes from any organism had been expressed and purified from bacterial expression system. We developed a procedure for full-length DGAT expression in E. coli by being fused to maltose binding protein and poly-histidine affinity tags. The recombinant DGATs were purified by affinity and conventional chromatography and used to raise high-titer antibodies. The results suggest that recombinant DGATs are part of a large complex associated with other proteins, lipids, and membranes. This study represents the first procedure for producing full-length recombinant DGAT from any species using an E. coli expression system.