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United States Department of Agriculture

Agricultural Research Service

Research Project: Engineering Enzymatic Redirection of Natural Crop Oil Production to Industrial Oil Production Title: Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2

Authors
item Howard, O.D. -
item Chapital, Dorselyn
item Shockey, Jay
item Cao, Heping

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 30, 2011
Publication Date: April 9, 2011
Citation: Howard, O., Chapital, D.C., Shockey, J.M., Cao, H. 2011. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2 (abstract). Southern Section of the American Society of Plant Biologists. p. 5.

Technical Abstract: Diacylglycerol acyltransferases (DGATs) are responsible for the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Different forms of DGATs have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii), which contains approximately 80% high-value eleostearic acid in its seed oils. DGATs are integral membrane proteins and difficult to express and purify. The expression of DGAT2 as a full-length or partial protein in E. coli was not reported previously. The objective of this study was to develop a procedure for full-length DGAT2 expression and purification in E. coli. Expression plasmid was engineered to express tung DGAT2 fused to maltose binding protein and poly-histidine affinity tags. Immunoblotting showed that recombinant DGAT2 was expressed and localized in both soluble and insoluble fractions of E. coli. DGAT2 fusion protein from the insoluble fraction was partially solubilized by 7 detergents (Brij 35, CHAPS, NP-40, SDS, Triton X-100, Tween 20 and Tween 80) with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. This study represents the first description of a procedure for producing full-length DGAT2 fusion protein from any species using a bacterial expression system. Production of recombinant DGAT2 should help to understand plant oil biosynthesis and create new oilseed crops with value-added properties.

Last Modified: 10/25/2014
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