Cloning, expression, purification and bioinformatic analysis of 2–methylcitrate synthase from Mycobacterium tuberculosis

AbstractObjectiveTo clone, express and purify 2–methylcitrate synthase (Rv1131) gene of Mycobacterium tuberculosis (M. tuberculosis) and to study its structural characteristics using various bioinformatics tools.MethodsRv1131 gene was amplified by polymerase chain reaction using M. tuberculosis H37Rv genomic DNA and cloned into pGEM–T easy vector and sequenced. The gene was sub–cloned in pET28c vector, expressed in Escherichia coli BL21 (E. coli BL21) (DE3) cells and the recombinant protein was identified by Western blotting. The protein was purified using Nickel affinity chromatography and the structural characteristics like sub–cellular localization, presence of transmembrane helices and secondary structure of the protein were predicted by bioinformatics tools. Tertiary structure of the protein and phylogenetic analysis was also established by in silico analysis.ResultsThe expression of the recombinant protein (Rv1131) was confirmed by western blotting using anti–HIS antibodies and the protein was purified from the soluble fraction. In silico analysis showed that the protein contains no signal peptide and transmembrane helices. Active site prediction showed that the protein has histidine and aspartic acid residues at 242, 281 & 332 positions respectively. Phylogenetic analysis showed 100% homology with major mycobacterial species. Secondary structure predicts 2–methylcitrate synthase contain 51.9% alpha–helix, 8.7% extended strand and 39.4% random coils. Tertiary structure of the protein was also established.ConclusionsThe enzyme 2–methylcitrate synthase from M. tuberculosis H37Rv has been successfully expressed and purified. The purified protein will further be utilized to develop assay methods for screening new inhibitors

Isolation and characterization of bacteriophages from India, with lytic activity against Mycobacterium tuberculosis.

Bacteriophages are being considered as a promising natural resource for the development of alternative strategies against mycobacterial diseases, especially in the context of the wide spread occurrence of drug-resistance amongst the clinical isolates of M. tuberculosis. However, there isnâ t much information documented on mycobacteriophages from India. Here, we report the isolation of 17 mycobacteriophages using M. smegmatis as the bacterial host where 9 phages also lyse M. tuberculosis H37Rv. We present detailed analysis of one of these mycobacteriophage (PDRPv). TEM and PCR analysis (of a conserved region within the TMP gene) shows PDRPv to belong to Siphoviridae family and B1 sub-cluster, respectively. The genome (69110 bp) of PDRPv is circularly permuted double-stranded DNA with ~66% GC content and has 106 open reading frames (ORFs). On the basis of sequence similarity and conserved domains, we have assigned function to 28 ORFs and have broadly categorized them into six groups that are related to replication genome maintenance, DNA packaging, virion release, structural proteins, lysogeny related genes and endolysins. The present study reports the occurrence of novel anti-mycobacterial phages in India and highlights their potential to contribute to our understanding of these phages and their gene products as potential antimicrobial agents.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author