CLONING THE MUTATED PYRAZINAMIDE ENZYME OF MYCOBACTERIUM TUBERCULOSIS IN ESCHERICHIA COLI BL21

Background and Aim:Pyrazinamide (PZA) is one of first line drugs for tuberculosis treatment. It should be metabolized into its active form by the M. tuberculosis pyrazinamidase (PZase). It has been shown that resistance to PZA could be related to mutations in the PZase enzyme. Therefore the understanding of the effect of mutations on the structure of PZase and its relation with the function of the enzyme can be useful in designing better anti tuberculosis drugs. In this study one of the most important mutation which has reported in most studies were selected for study.Methods:Using gene runner software and designing related primers and performing PCR, V155G mutation was developed in the sequence of PZase gene. Then the gene was cloned in expression vector pET21a (+). For the recombinant enzyme overexpression, the Escherichia coli BL21 (DE3) cells were transformed by the constructed vector. After induction, the recombinant PZase was purified using the Ni_NTA sepharose column and confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blotting.Results:After cloning, expression and purification process the band of purified protein was observed with a molecular mass of 20 kDa in SDS-PAGE and in western blotting the band of enzyme was confirmed using Anti his tag antibody.Conclusion:Using expression vector pET21a (+) and Escherichia coli BL21 (DE3) is a suitable cloning system for cloning and production of recombinant PZase and preparing the enzyme for further structural and functional studies.

کلیدواژه ها:

Pyrazinamidase enzyme ، Mycobacterium tuberculosis ، Cloning ، mutation