Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

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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.

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APA

Hunter, R. W., Zeqiraj, E., Morrice, N., Sicheri, F., & Sakamoto, K. (2015). Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells. Protein Expression and Purification, 108, 23–29. https://doi.org/10.1016/j.pep.2014.12.007

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