Page Banner

United States Department of Agriculture

Agricultural Research Service

Research Project: Engineering Enzymatic Redirection of Natural Crop Oil Production to Industrial Oil Production Title: Purification of recombinant tung tree diacylglycerol acyltransferases from E. coli

Authors
item Cao, Heping
item Chapital, Dorselyn
item Howard Jr, Od
item Jiang, Xiangning -
item Shockey, Jay
item Klasson, K Thomas

Submitted to: Journal of Federation of American Societies for Experimental Biology
Publication Type: Abstract Only
Publication Acceptance Date: February 2, 2011
Publication Date: April 9, 2011
Citation: Cao, H., Chapital, D.C., Howard Jr, O.D., Jiang, X., Shockey, J.M., Klasson, K.T. 2011. Purification of recombinant tung tree diacylglycerol acyltransferases from E. coli (abstract). Journal of the Federation of American Societies for Experimental Biology. 25:765.8.

Technical Abstract: Understanding plant oil biosynthesis will help to create new oilseed crops with value-added properties to replace petroleum-based compounds. Diacylglycerol acyltransferases (DGATs) are key enzymes catalyzing the last step of triacylglycerol (TAG) biosynthesis in eukaryotes. Over-expression of DGATs increases TAG in seeds and other tissues. DGATs are membrane-associated proteins and difficult to express and purify. The objective of this study was to purify recombinant DGATs of tung tree (Vernicia fordii), which contains ~80% high-value eleostearic acid in the seeds. E. coli expression plasmids were engineered to express DGATs fused to maltose binding protein (MBP) at its N-terminus and poly-histidine (His) at its C-terminus. Recombinant DGATs were localized in the membranes and the cytosol of E. coli. The proteins were partially purified under native conditions by amylose resin, Ni-NTA beads, tandem affinity beads, size exclusion and Mono Q anion exchange chromatography. Recombinant DGATs were solubilized by 7 detergents (Brij 35, CHAPS, NP-40, SDS, Triton X-100, Tween 20 and Tween 80) with SDS being the most effective. The proteins were purified to near homogeneity by Ni-NTA affinity beads following SDS solubilization. This study describes a successful procedure for producing full-length recombinant DGATs from E. coli. The DGATs will be used for antibody production and biochemical characterization.

Last Modified: 10/21/2014
Footer Content Back to Top of Page