title: Purification and properties of recombinant Brassica napus diacylglycerol acyltransferase 1.
abstract: Diacylglycerol acyltransferase 1 (DGAT1) catalyzes the final step in the acyl-CoA-dependent triacylglycerol biosynthesis. Although the first DGAT1 gene was identified many years ago and the encoded enzyme catalyzes a key step in lipid biosynthesis, no detailed structure-function information is available on the enzyme due to difficulties associated with its purification. This study describes the purification of recombinant Brassica napus DGAT1 (BnaC.DGAT1.a) in active form through solubilization in n-dodecyl-β-D-maltopyranoside, cobalt affinity chromatography, and size-exclusion chromatography. Different BnaC.DGAT1.a oligomers in detergent micelles were resolved during the size-exclusion process. BnaC.DGAT1.a was purified 126-fold over the solubilized fraction and exhibited a specific activity of 26 nmol TAG/min/mg protein. The purified enzyme exhibited substrate preference for α-linolenoyl-CoA>oleoyl-CoA=palmitoyl-CoA>linoleoyl-CoA>stearoyl-CoA.
authors: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada.
authors: Kristian Mark P
authors: KM
authors: Caldo
authors: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada.
authors: Michael S
authors: MS
authors: Greer
authors: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada.
authors: Guanqun
authors: G
authors: Chen
authors: Membrane Protein Disease Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
authors: M Joanne
authors: MJ
authors: Lemieux
authors: Alberta Innovates Phytola Centre, Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada. Electronic address: randall.weselake@ualberta.ca.
authors: Randall J
authors: RJ
authors: Weselake
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry; D000595:Amino Acid Sequence; D029688:Brassica napus / Q000201:enzymology*; D051048:Diacylglycerol O-Acyltransferase / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae; D013379:Substrate Specificity
chemical_list: D000214:Acyl Coenzyme A; D010940:Plant Proteins; D051048:Diacylglycerol O-Acyltransferase
keywords: Diacylglycerol acyltransferase 1; Membrane protein purification; Oilseed rape; Triacylglycerol biosynthesis