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

SUBMITTER: Hunter RW 

PROVIDER: S-EPMC4370744 | biostudies-literature | 2015 Apr

REPOSITORIES: biostudies-literature