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 116mg of recombinant enzyme was purified to near homogeneity with approximately 80% yield from the soluble fraction of 1L culture at shake flask level using one step Ni-NTA affinity chromatography. The specific activity of the purified six-histidine-tagged recombinant PGI (rPGI-His(6)) was approximately 800U/mg of protein. The apparent K(m) value of the active recombinant protein followed Michaelis-Menten kinetics and was 0.27+/-0.03mM. K(i) for the competitive inhibitor 6-phosphogluconate was 0.75mM. The enzyme had pH optima in the range of pH 7.6-9.0 and was stable up to 55 degrees C. rPGI-His(6) exhibited enzyme activity almost equal to that of enzyme without histidine tag.
Mathur
Divya
D
Gene Regulation Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Garg
Lalit C
LC
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D011994:Recombinant Proteins; D005956:Glucose-6-Phosphate Isomerase