title: Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2.
abstract: Diacylglycerol acyltransferases (DGATs) esterify sn-1,2-diacylglycerol with a long-chain fatty acyl-CoA, the last and rate-limiting step of triacylglycerol (TAG) biosynthesis in eukaryotic organisms. At least 74 DGAT2 sequences from 61 organisms have been identified, but the expression of any DGAT2 as a partial or full-length protein in Escherichia coli had not been reported. The main objective of this study was to express and purify recombinant DGAT2 (rDGAT2) from E. coli for antigen production with a minor objective to compare rDGAT2 expression in yeast. A plasmid was engineered to express tung tree DGAT2 fused to maltose binding protein and poly-histidine (His) affinity tags. Immunoblotting showed that rDGAT2 was detected in the soluble, insoluble, and membrane fractions. The rDGAT2 in the soluble fraction was partially purified by amylose resin, nickel-nitrilotriacetic agarose (Ni-NTA) beads, and tandem affinity chromatography. Multiple proteins co-purified with rDGAT2. Size exclusion chromatography estimated the size of the rDGAT2-enriched fraction to be approximately eight times the monomer size. Affinity-purified rDGAT2 fractions had a yellow tint and contained fatty acids. The rDGAT2 in the insoluble fraction was partially solubilized by seven detergents with SDS being the most effective. Recombinant DGAT2 was purified to near homogeneity by SDS solubilization and Ni-NTA affinity chromatography. Mass spectrometry identified rDGAT2 as a component in the bands corresponding to the monomer and dimer forms as observed by SDS-PAGE. Protein bands with monomer and dimer sizes were also observed in the microsomal membranes of Saccharomyces cerevisiae expressing hemagglutinin-tagged DGAT2. Nonradioactive assay showed TAG synthesis activity of DGAT2 from yeast but not E. coli. The results suggest that rDGAT2 is present as monomer and dimer forms on SDS-PAGE, associated with other proteins, lipids, and membranes, and that post-translational modification of rDGAT2 may be required for its enzymatic activity and/or the E. coli protein is misfolded.
authors.affiliation: Commodity Utilization Research Unit, Southern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 1100 Robert E. Lee Blvd, New Orleans, LA 70124, USA. Heping.Cao@ars.usda.gov
authors.forename: Heping
authors.initials: H
authors.lastname: Cao
authors.forename: Dorselyn C
authors.initials: DC
authors.lastname: Chapital
authors.forename: O D
authors.initials: OD
authors.lastname: Howard
authors.forename: Leesa J
authors.initials: LJ
authors.lastname: Deterding
authors.forename: Catherine B
authors.initials: CB
authors.lastname: Mason
authors.forename: Jay M
authors.initials: JM
authors.lastname: Shockey
authors.forename: K Thomas
authors.initials: KT
authors.lastname: Klasson
mesh_terms: D031284:Aleurites / Q000201:enzymology* / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D051048:Diacylglycerol O-Acyltransferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D010802:Phylogeny; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D017385:Sequence Homology
chemical_list: D011993:Recombinant Fusion Proteins; D051048:Diacylglycerol O-Acyltransferase