1. Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells.
- Author
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Hunter RW, Zeqiraj E, Morrice N, Sicheri F, and Sakamoto K
- Subjects
- Animals, Humans, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Sf9 Cells, Spodoptera, Gene Expression, Glucosyltransferases biosynthesis, Glucosyltransferases genetics, Glucosyltransferases isolation & purification, Glycogen Synthase biosynthesis, Glycogen Synthase genetics, Glycogen Synthase isolation & purification, Glycoproteins biosynthesis, Glycoproteins genetics, Glycoproteins isolation & purification, Multienzyme Complexes biosynthesis, Multienzyme Complexes genetics, Multienzyme Complexes isolation & purification
- Abstract
We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2015
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