Large-Scale Protein Production and Activity Assay Protocols for Human Glycogen Synthase-Glycogenin Complex

Glycogen synthase (GS) is the rate-limiting enzyme for glycogen production and together with glycogenin (GN) and glycogen branching enzyme (GBE), can generate glycogen particles containing up to 50,000 glucose units. Dysregulation of glycogen synthesis, for example overproduction or accumulation of malformed glycogen, is the source of many glycogen storage diseases affecting glucose homeostasis and muscle and neuronal cell function. As such, GS is an attractive candidate enzyme for therapeutic targeting, which until recently, was hampered by difficulties in producing active human GS enzyme preparations. Here, we describe the large-scale production of GS in complex with GN, and assay conditions to measure enzyme activity in the absence and presence of the allosteric activator glucose-6-phosphate (G6P). These protocols, together with assays for quality control assessment of enzyme preparations, provide a useful resource for studying the biochemical, biophysical, and structural properties of the GS-GN complex, and facilitate drug discovery pipelines to develop therapeutics for glycogen storage diseases.