Expression and characterisation of human glycerol kinase: the role of solubilising agents and molecular chaperones.

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 2 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 overexpressed bioactive His-GK was purified using coloumn chromatography and characterised using enzyme kinetics. RESULTS: The overexpressed 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. CONCLUSION: The present study demonstrates that co-expression of molecular chaperones assists with the expression of bioactive human GK for its characterisation.

Draft Genome of the Liver Fluke Fasciola gigantica.

Fascioliasis, a neglected foodborne disease caused by liver flukes (genus Fasciola), affects more than 200 million people worldwide. Despite technological advances, little is known about the molecular biology and biochemistry of these flukes. We present the draft genome of Fasciola gigantica for the first time. The assembled draft genome has a size of ∼1.04 Gb with an N50 and N90 of 129 and 149 kb, respectively. A total of 20 858 genes were predicted. The de novo repeats identified in the draft genome were 46.85%. The pathway included all of the genes of glycolysis, Krebs cycle, and fatty acid metabolism but lacked the key genes of the fatty acid biosynthesis pathway. This indicates that the fatty acid required for survival of the fluke may be acquired from the host bile. It may be hypothesized that the relatively larger F. gigantica genome did not evolve through genome duplications but rather is interspersed with many repetitive elements. The genomic information will provide a comprehensive resource to facilitate the development of novel interventions for fascioliasis control.