Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells
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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.
Original language | English |
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Journal | Protein Expression and Purification |
Volume | 108 |
Pages (from-to) | 23-29 |
Number of pages | 7 |
ISSN | 1046-5928 |
DOIs | |
Publication status | Published - Apr 2015 |
Externally published | Yes |
- Energy metabolism, Glucosyltransferase, Glycogenesis, Phosphorylation
Research areas
ID: 239212865