Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 23, 2011
Publication Date: July 24, 2011
Citation: Cao, Heping, Chapital, Dorselyn C., Howard, Jr., O.D., Shockey, Jay M., Klasson, K. Thomas. 2011. Bioengineering recombinant tung tree diacylglycerol acyltransferases (abstract). Presented at the Society for Industrial Microbiology and Biotechnology Annual Meeting and Exhibition in New Orleans, Louisiana, July 24-28. http://sim.confex.com/sim/2011/webprogram/Paper19213.html. 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. Plants and animals deficient in DGATs accumulate less TAG. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. Over-expression of DGATs increases TAG in seeds and other tissues. None of the full-length DGATs from any species was heterogeneous produced because they are integral membrane proteins and difficult to express and purify. The objective of this study was to bioengineer recombinant DGATs of tung tree (Vernicia fordii), which contains ~80% high-value eleostearic acid in the seed oils. 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 agarose, 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 and Triton X-100 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 raising antibodies and studying structure-function relationship.