Abstract
The glpX gene (Rv1099c) of Mycobacterium tuberculosis (Mtb) encodes Fructose 1,6-bisphosphatase II (FBPase II; EC 3.1.3.11); a key gluconeogenic enzyme. Mtb possesses glpX homologue as the major known FBPase. This study explored the expression, purification and enzymatic characterization of functionally active FBPase II from Mtb. The glpX gene was cloned, expressed and purified using a two step purification strategy including affinity and size exclusion chromatography. The specific activity of Mtb FBPase II is 1.3 U/mg. The enzyme is oligomeric, followed Michaelis-Menten kinetics with an apparent km = 44 μM. Enzyme activity is dependent on bivalent metal ions and is inhibited by lithium and inorganic phosphate. The pH optimum and thermostability of the enzyme have been determined. The robust expression, purification and assay protocols ensure sufficient production of this protein for structural biology and screening of inhibitors against this enzyme.
Highlights
Tuberculosis (TB) is a global public health problem, with 2 billion people, equal to about onethird of the world’s population, infected with Mycobacterium tuberculosis (Mtb), the microbe that causes TB
Cloning, expression and purification of MtFBPase In order to produce recombinant His-tagged MtFBPase protein in E. coli, glpX gene encoding mycobacterial FBPase was cloned into pET15b, a T7 promoter based inducible bacterial expression vector
Significant expression of MtFBPase was observed upon induction with 0.5mM IPTG in the E. coli cell cultures transformed with pET15b vector construct harboring the glpX gene
Summary
Tuberculosis (TB) is a global public health problem, with 2 billion people, equal to about onethird of the world’s population, infected with Mycobacterium tuberculosis (Mtb), the microbe that causes TB. Standard assays were performed in a 96 well plate format in 100 μl reaction mixture aliquots containing 50 mM KCl, 20 mM tricine pH 7.70, 8 mM MgCl2, 1.0 mM NADP+, yeast glucose-6-phosphate dehydrogenase G6PDH (1.0 U/ ml), yeast phosphoglucoisomerase PGI(2.5 U/ml) and purified MtFBPase at a concentration of 1 μg/ml (10 μl of 1:100 dilution of 1mg/ml concentration of the protein added to a final volume of 100 μl reaction mixture).
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