Functional phosphoglucose isomerase from Mycobacterium tuberculosis H37Rv: Rapid purification with high yield and purity

Phosphoglucose isomerase (PGI) EC 5.3.1.9, is a housekeeping enzyme that catalyzes the reversible isomerization of d-glucopyranose-6-phosphate and d-fructofuranose-6-phosphate. We have previously reported expression and multistep purification of recombinant PGI from Mycobacterium tuberculosis using conventional methods. We now describe an improved and simplified single step approach for purification of functionally active mycobacterial rPGI. The gene encoding PGI from M. tuberculosis H37Rv was cloned in bacterial expression vector pET22b(+). Expression of recombinant PGI with six-histidine-tag protein was observed both in the soluble fraction and inclusion bodies. Approximately 116 mg of recombinant enzyme was purified to near homogeneity with ∼80% yield from the soluble fraction of 1 L culture at shake flask level using one step Ni–NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His6) was ∼800 U/mg of protein. The apparent Km value of the active recombinant protein followed Michaelis–Menten kinetics and was 0.27 ± 0.03 mM. Ki for the competitive inhibitor 6-phosphogluconate was 0.75 mM. The enzyme had pH optima in the range of pH 7.6–9.0 and was stable up to 55 °C. rPGI-His6 exhibited enzyme activity almost equal to that of enzyme without histidine tag.