Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/26/2011
Publication Date: 5/9/2011
Citation: Cao, H. 2011. Expression and purification of membrane protein diacylglycerol acyltransferase (abstract). Cambridge Healthtech Institute's Protein Engineering Summit 2011. p. 8.
Technical Abstract: Diacylglycerol acyltransferases (DGATs) catalyze the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG. Over-expression of DGATs increases TAG in seeds and other tissues. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. Therefore, understanding the roles of DGATs in plants will help to create new oilseed crops with value-added properties. Knowledge on the precise functions of DGATs in animals and human may provide clues for nutritional prevention and therapeutic intervention related to obesity and associated diseases. Database search has identified at least 115 DGAT sequences from 69 organisms including plants (such as Arabidopsis, barley, caster bean, cauliflower, corn, rape, rice, sorghum, soybean, tobacco, tung tree), animals (such as bird, chimpanzee, cow, dog, fish, fly, frog, monkey, mosquito, mouse, pig, rabbit, rat, sheep, worm), fungi (such as yeast), and human. However, only a few papers have been published in the last 28 years on the expression of the recombinant DGAT proteins in a bacterial expression system. No full-length DGAT1 or DGAT2 proteins had been expressed in E. coli. The difficulties in DGAT expression and purification are due to the facts that these proteins are integral membrane proteins and more than 40% of their amino acid residues are hydrophobic. Therefore, progress in characterization of the enzymes has been slow. We recently developed a procedure for full-length DGAT expression in E. coli. Expression plasmids were engineered to express tung DGATs fused to maltose binding protein and poly-histidine. The development of the technique should help to purify full-length DGATs for further studies such as raising high titer antibodies and studying the structure-function relationship.