Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv |
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Authors: | Nampoothiri K M Rubex R Patel A K Narayanan S S Krishna S Das S M Pandey A |
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Institution: | Biotechnology Division, National Institute for Interdisciplinary Science and Technology (Formerly Regional Research Laboratory), CSIR, Trivandrum, Kerala, India |
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Abstract: | Aim: Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity. Methods and Results: Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics. Conclusion: The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams. Significance and Impact of the Study: Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains. |
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Keywords: | β-lactamases β-lactam antibiotics Mycobacterium tuberculosis nitrocefin |
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