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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

Location: Commodity Utilization Research

Title: Expression of tung tree diacylglycerol acyltransferase 1 in E. coli

Authors
item Cao, Heping
item Chapital, Dorselyn
item Shockey, Jay
item Klasson, K Thomas

Submitted to: BioMed Central (BMC)Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: July 11, 2011
Publication Date: July 11, 2011
Repository URL: http://hdl.handle.net/10113/49906
Citation: Cao, H., Chapital, D.C., Shockey, J.M., Klasson, K.T. 2011. Expression of tung tree diacylglycerol acyltransferase 1 in E. coli. BMC Biotechnology. 11:73 (13 pages).

Interpretive Summary: The genetic sequences of many organisms have been uncovered for years, but the functions of many individual genes are poorly understood. One of the major challenges in today’s research is to understand the biological functions of proteins coded for by the genes. Unfortunately, it has been difficult to learn the functions of many proteins because they occur in extremely low-abundance in the original organisms. Bioengineered proteins can be used as an alternative source to native proteins. Biotechnology methods can help to produce large quantities of proteins for studying proteins and producing antibodies and pharmaceutical reagents. Diacylglycerol acyltransferases (DGATs) are proteins that control the amounts of fats and oils in plants, animals, and human. Plants deficient in DGATs accumulate less oil, but more enzymes in transgenic plants increase oils in the seeds. The genetic knockout animals accumulate less fat and resist diet-induced obesity. Thus, DGAT proteins are potential targets for producing more useful oils and alleviating obesity and related health problems. The technical challenge is that DGAT enzymes are difficult to produce because they are integral membrane proteins in the cells. This study established a procedure for the first time to produce the complete DGAT1 protein from any species using a bacterial expression system. The ability to express the complete form of this important enzyme will help to raise antibodies and study the functions of the protein. Eventually, we can understand how plants make oils better. The new knowledge gained will assist to create new oilseed crops that produce oils with value-added properties.

Technical Abstract: Diacylglycerol acyltransferases (DGATs) catalyze the last step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. DGAT isoforms have nonredundant functions in TAG biosynthesis in species such as tung tree (Vernicia fordii) which contains 80% high-value eleostearic acid in its seed oils. DGATs are integral membrane proteins and difficult to express and purify. The objective of this study was to express full-length tung DGAT1 in E. coli. Previously, only the N-terminal regions of DGAT1 from Brassica napus and mice had been expressed. An expression plasmid containing the open reading frame for tung DGAT1 fused to maltose binding protein and poly-histidine affinity tags was constructed and expressed in E. coli BL21(DE3). Immunoblotting showed that the recombinant DGAT1 was expressed, but mostly targeted to the membranes and insoluble fractions. Extensive degradation also occurred. Nonetheless, the fusion protein was partially purified from the soluble fraction by Ni-NTA and amylose resin affinity chromatography. Multiple proteins co-purified with DGAT1 fusion protein. These fractions appeared yellow in color and contained fatty acids. The recombinant DGAT1 was solubilized from the insoluble fraction by seven detergents and urea, with SDS and Triton X-100 being the most effective detergents. The solubilized recombinant DGAT1 was partially purified by Ni-NTA affinity chromatography. This study reports the first procedure for expressing full-length DGAT1 from any species using a bacterial expression system.

Last Modified: 8/22/2014
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