Background: Glycerol kinase (GK; EC 2.7.1.30) facilitates the entry of glycerol into pathways of glucose and triglyceride metabolism and may play a potential role in type II diabetes mellitus (T2DM). However, the detailed regulatory mechanisms and structure of the human GK are unknown.  Methods: The human GK gene was cloned into the pET-24a(+) vector and over-expressed in Escherichia coli BL21 (DE3). Since the protein was expressed as inclusion bodies (IBs), various culture parameters and solubilising agents were used but they did not produce bioactive His-GK; however, co-expression of His-GK with molecular chaperones, specifically pKJE7, achieved expression of bioactive His-GK. The over-expressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics.</p>  Results: The over-expressed bioactive His-GK was purified apparently to homogeneity (~295 fold) and characterised. The native His-GK was a dimer with a monomeric molecular weight of ~55 kDa. Optimal enzyme activity was observed in TEA buffer (50 mM) at 7.5 pH. K+ (40 mM) and Mg2+ (2.0 mM) emerged as prefered metal ions for His-GK activity with specific activity 0.780 U/mg protein. The purified His-GK obeyed standard Michaelis-Menten kinetics with Km value of 5.022 µM (R2 = 0.927) for its substrate glycerol; whereas, that for ATP and PEP was 0.767 mM (R2 = 0.928) and 0.223 mM (R2 = 0.967), respectively. Other optimal parameters for the substrate and co-factors were also determined.</p>  Conclusion: This study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.</p>
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