title: Expression and purification of functional human glycogen synthase-1 (hGYS1) in insect cells.
abstract: We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
authors.affiliation: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202-5126, USA.
authors.forename: May
authors.initials: M
authors.lastname: Khanna
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Imasaki
authors.forename: Vimbai M
authors.initials: VM
authors.lastname: Chikwana
authors.forename: Samantha
authors.initials: S
authors.lastname: Perez-Miller
authors.forename: Gerald O
authors.initials: GO
authors.lastname: Hunter
authors.forename: Amber
authors.initials: A
authors.lastname: Mosley
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Takagi
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Hurley
mesh_terms: D000818:Animals; D002460:Cell Line; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006006:Glycogen Synthase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007313:Insecta / Q000166:cytology; D010766:Phosphorylation; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D006023:Glycoproteins; D011994:Recombinant Proteins; C051887:glycogenin; D005964:Glucosyltransferases; D006006:Glycogen Synthase
keywords: Human glycogen synthase-1; Mass spectrometry; MultiBac baculovirus expression system; Phosphorylation