Objective:To clone,express and purify 2—methylcitrale synthase(Rv1131) gene of Mycobacterium tuberculosis(M.tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 ...Objective:To clone,express and purify 2—methylcitrale synthase(Rv1131) gene of Mycobacterium tuberculosis(M.tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified In polymerase chain reaction using M.tuberculosis H37 Rv genomic DNA and cloned into pGEM-T easy vector and sequenced.The gene was sub-cloned in pET28 c vector,expressed in Escherichia coli RL22(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 bioinformaties tools.Tertiary structure of the protein and phylogenetic analysis was aiso established by in silico analysis.Results:The 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 aspartie acid residues at 242.281 & 332 positions respectively.Phylogenetic analysis showed100%homology with major mycobacterial species.Secondary structure predicts 2-methyleitrate synthase contain 51.9%;alpha-helk.8.7%extended strand and 39.4%random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme 2-methylcitrate synthase from M.tuberculosis H37 Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods lor screening new inhibitors.展开更多
基金supported by funding from Council of Scicntific and Industrial Researd(CSIR),India
文摘Objective:To clone,express and purify 2—methylcitrale synthase(Rv1131) gene of Mycobacterium tuberculosis(M.tuberculosis) and to study its structural characteristics using various bioinformatics tools.Methods:Rv1131 gene was amplified In polymerase chain reaction using M.tuberculosis H37 Rv genomic DNA and cloned into pGEM-T easy vector and sequenced.The gene was sub-cloned in pET28 c vector,expressed in Escherichia coli RL22(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 bioinformaties tools.Tertiary structure of the protein and phylogenetic analysis was aiso established by in silico analysis.Results:The 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 aspartie acid residues at 242.281 & 332 positions respectively.Phylogenetic analysis showed100%homology with major mycobacterial species.Secondary structure predicts 2-methyleitrate synthase contain 51.9%;alpha-helk.8.7%extended strand and 39.4%random coils.Tertiary structure of the protein was also established.Conclusions:The enzyme 2-methylcitrate synthase from M.tuberculosis H37 Rv has been successfully expressed and purified.The purified protein will further be utilized to develop assay methods lor screening new inhibitors.
