Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis

Gopalan, Akilandeswari and Bhagavat, Raghu and Chandra, Nagasuma and Subbarao, Savithri Handanahal and Raja, Alamelu and Bethunaickan, Ramalingam (2018) Biophysical and biochemical characterization of Rv3405c, a tetracycline repressor protein from Mycobacterium tuberculosis. In: BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 496 (3). pp. 799-805.

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Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, is one among the deadliest pathogens in the world. Due to long treatment regimen, HIV co-infection, persistence of bacilli in latent form and development of XDR and TDR strains of Mtb, tuberculosis has posed serious concerns for managing the disease, and calls for discovery of new drugs and drug targets. Using a computational pipeline involving analysis of the structural models of the Mtb proteome and an analysis of the ATPome, followed by a series of filters to identify druggable proteins, solubility and length of the protein, several candidate proteins were shortlisted. From this, Rv3405c, a tetR family of DNA binding protein involved in antibiotic resistance, was identified as one of the good drug targets. Rv3405c binds to the upstream non coding region of Rv3406 and causes repression of Rv3406 activity there by affecting the downstream processes involved in antibiotic resistance was further characterized. The Rv3405c gene was cloned; the gene product was over-expressed in E. coli and purified by Ni NTA chromatography. DNA binding studies by EMSA showed that the recombinant Rv3405c protein binds to the DNA sequence corresponding to the promoter region of Rv3406 and upon addition of tetracycline, the DNA binding activity was lost. P-galactosidase reporter assay in E. coli using both wild type and a DNA binding defective mutant protein indeed proved that Rv3405c acts as a repressor. (C) 2018 Elsevier Inc. All rights reserved.

Item Type:	Journal Article
Publication:	BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Publisher:	ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA
Additional Information:	Copy right for the article belong to ACADEMIC PRESS INC ELSEVIER SCIENCE, 525 B ST, STE 1900, SAN DIEGO, CA 92101-4495 USA
Department/Centre:	Division of Biological Sciences > Biochemistry
Date Deposited:	27 Mar 2018 18:27
Last Modified:	27 Mar 2018 18:27
URI:	http://eprints.iisc.ac.in/id/eprint/59393

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