Location: Commodity Utilization ResearchTitle: Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2) Author
|Howard Jr, Od|
|Klasson, K Thomas|
Submitted to: Applied Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/23/2011
Publication Date: 11/1/2012
Citation: Cao, H., Chapital, D.C., Howard Jr, O.D., Deterding, L.J., Mason, C.B., Shockey, J.M., Klasson, K.T. 2012. Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2. Applied Microbiology and Biotechnology. 96(3):711-727. Interpretive Summary: The genetic sequences of many organisms have been known for years, but the functions of many genes are unclear. The immediate challenge of post-genomic biology is to determine the biological functions of proteins coded for by the genes. However, it has been difficult to study many proteins because they occur in extremely low-abundance and are unstable in the native organisms. Genetically engineered proteins can be used as an alternative source to native proteins. Production of large quantities of proteins is necessary 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 associated with cellular membranes. This study reports the first description of a procedure for producing the complete DGAT2 protein from any species using E. coli, a workhorse for protein production. 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 in creating new oilseed crops that produce oils with value-added properties to replace petroleum-based compounds in products such as nylons, plastics, inks, and dyes.
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. Over-expression of DGATs increases TAG. DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. 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 the seeds. DGAT genes have been isolated from many organisms, but progress in characterization of the enzymes has been slow because DGATs are membrane-associated and difficult to express and purify. The objective of this study was to develop a procedure for full-length DGAT expression in E. coli and yeast. Expression plasmids were engineered to express tung DGAT2 fused to maltose binding protein (MBP), poly-histidine (His), and hemagglutinin (HA) affinity tags. Immunoblotting showed that MBP-DGAT2-His was expressed in E. coli. The recombinant protein was partially purified by batch and column methods with amylose resin, Ni-NTA beads, and tandem affinity beads. Multiple proteins co-purified with DGAT2 fusion protein. Size exclusion chromatography estimated the size of recombinant DGAT2 approximately to be eight times the monomer size. Affinity-purified DGAT2 fractions appeared light yellowish and contained fatty acids. Recombinant DGAT2 was also detected in membrane fractions. In contrast, HA-tagged DGAT2 expressed in yeast was localized exclusively in the microsomal membranes. This study describes a successful procedure for producing full-length recombinant DGAT2. The results suggest that recombinant DGAT2 is associated with lipids and other proteins.