title: Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells.
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.
authors.affiliation: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G, 1015 Lausanne, Switzerland.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Hunter
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada.
authors.forename: Elton
authors.initials: E
authors.lastname: Zeqiraj
authors.affiliation: Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Morrice
authors.affiliation: Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, Ontario M5G 1X5, Canada; Departments of Biochemistry and Molecular Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Frank
authors.initials: F
authors.lastname: Sicheri
authors.affiliation: Nestlé Institute of Health Sciences SA, EPFL Innovation Park, bâtiment G, 1015 Lausanne, Switzerland. Electronic address: Kei.Sakamoto@rd.nestle.com.
authors.forename: Kei
authors.initials: K
authors.lastname: Sakamoto
mesh_terms: D000818:Animals; D015870:Gene Expression; D005964:Glucosyltransferases / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006006:Glycogen Synthase / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D009097:Multienzyme Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D061987:Sf9 Cells; D018411:Spodoptera
chemical_list: D006023:Glycoproteins; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C051887:glycogenin; D005964:Glucosyltransferases; D006006:Glycogen Synthase
keywords: Energy metabolism; Glucosyltransferase; Glycogenesis; Phosphorylation