ArticleApplied microbiology and biotechnology2012
Expression and purification of recombinant tung tree diacylglycerol acyltransferase 2.
Article in Applied microbiology and biotechnology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 21 citations in OpenAlex.
- A rice SOUL family heme-binding protein REAC1 enhances the antioxidative capacity of C. elegans through modulation of ROS-related gene expression.Scientific reports · 2025Article
- Oil-Producing Metabolons Containing DGAT1 Use Separate Substrate Pools from those Containing DGAT2 or PDAT.Plant physiology · 2020Article
- Tung Tree (Vernicia fordii) Genome Provides A Resource for Understanding Genome Evolution and Improved Oil Production.Genomics, proteomics & bioinformatics · 2019Article
- Identification of the major diacylglycerol acyltransferase mRNA in mouse adipocytes and macrophages.BMC biochemistry · 2018Article
- Article
- Characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia).PloS one · 2014Article
- Fatty acid profile and unigene-derived simple sequence repeat markers in tung tree (Vernicia fordii).PloS one · 2014Article
- Identification, classification and differential expression of oleosin genes in tung tree (Vernicia fordii).PloS one · 2014Article
- Developmental regulation of diacylglycerol acyltransferase family gene expression in tung tree tissues.PloS one · 2013Article
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Authors and funding
7 authors at 4 institutions in 1 country.
Funding
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.
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