Studies on glucokinase regulatory protein (GCKR)

Abstract

The glucokinase regulatory protein (GCKR) regulates the activity of hepatic and pancreatic glucokinase by acting as a competitive inhibitor of glucose, thereby facilitating the control of glucokinase activity. At physiologically low glucose levels, GCKR binds to glucokinase, causing the complex to translocate into the nucleus, where glucokinase is inactivated. When glucose levels rise, glucokinase is released from GCKR and moves into the cytosol, where it becomes active. In the pancreas, glucokinase functions as a glucose sensor, triggering the release of insulin. Mutations in either glucokinase or GCKR have been implicated in Maturity Onset Diabetes of the Young (MODY), making it crucial to understand how these mutations affect the physiochemical properties of both proteins. This study focuses on the production of human GCKR protein, as most previous research has utilized the most stable mouse protein. Oligonucleotides were designed to amplify the human GCKR gene from a cDNA clone, which was purified and cloned into an expression vector designed in our laboratory to express the novel histidine-tagged SUMO-GCKR fusion protein. The expression conditions were optimized using various E.coli cell types, including BL-21 (DE3), Rosetta 2, and SHuffle cells. Different concentrations of the inducer IPTG (ranging from 0.1 to 2.0 mM) were tested, and cultured induced cells were grown at different temperatures (18oC or 37oC) and harvested at various times post-induction (18 or 43 hours). Protein expression was analysed using SDS-PAGE and confirmed through immunoblotting. The SUMO-GCKR fusion protein was then purified using metal chelating affinity chromatography.