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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Publications at this Location » Publication #254150
Title: Expression of tung seed diacylglycerol acyltransferases (DGAT) in E. coli and yeast

Author
item Cao, Heping
item Chapital, Dorselyn
item Mason, Catherine
item Shockey, Jay
item Klasson, K Thomas

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 5/12/2010
Publication Date: 7/30/2010
Citation: Cao, H., Chapital, D.C., Mason, C.B., Shockey, J.M., Klasson, K.T. 2010. Expression of tung seed diacylglycerol acyltransferases (DGAT) in E. coli and yeast (abstract). Plant Biology 2010, July 31-August 4, Montreal, Canada.

Interpretive Summary:

Technical Abstract: Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. Plants and animals deficient in DGATs accumulate less TAG, resist obesity, and/or lack milk secretion. Over-expression of the DGATs increases TAG content in seeds and other tissues. DGATs have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii), which contains 80% high-value eleostearic acid (18:3 delta 9cis,11trans,13trans) in the TAG fraction of the seeds. DGAT genes have been isolated from many organisms, but progress has been slow in the characterization of the enzymes because DGATs are membrane-associated and difficult to express and purify. The objective of this study was to express tung DGATs in E. coli and yeast for antibody production and biochemical characterization. Expression plasmids were constructed to utilize various tags including maltose binding protein (MBP), poly histidine (His), and hemagglutinin (HA). Immunoblotting showed that MBP-DGAT1-His was expressed in E. coli, but little full-length protein was detected due to extensive degradation. Anti-MBP antibodies detected various fragments in the soluble fraction and anti-His antibodies detected fragments of DGAT1 in the insoluble fraction. The c-terminal portion of DGAT2 was predicted to be soluble. Anti-His antibodies detected it in the soluble fraction of E. coli. In contrast, full-length DGAT2 protein was detected in yeast cells and localized exclusively in the microsomal membranes. These results suggest that recombinant DGAT proteins are unstable in E. coli but stable in yeast. We speculate that the environment in the microsomal membranes may help to stabilize DGAT proteins in intact cells.