Journal
FEBS LETTERS
Volume 589, Issue 6, Pages 773-778Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.febslet.2015.02.008
Keywords
Triacylglycerol biosynthesis; Diacylglycerol acyltransferase 1; Membrane protein purification; Oilseed rape
Funding
- Alberta Innovates Bio Solutions
- Canada Research Chairs Program
- Natural Science and Engineering Research Council of Canada
- University of Alberta Recruitment Scholarship
- Alberta Canola Producers Graduate Award in Canola Production Research
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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-beta-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 alpha-linolenoyl-CoA > oleoyl-CoA = palmitoyl-CoA > linoleoyl-CoA > stearoyl-CoA. (C) 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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