Cloning and expression of the gene coding for FtsH protease from Mycobacterium tuberculosis H37Rv.
This study was aimed at the molecular cloning and expression of the gene coding for FtsH protease of Mycobacterium tuberculosis H37Rv (virulent). PCR on the genomic DNA of M. tuberculosis H37Ra (non-virulent) using the oligodeoxynucleotide primers, which were designed based on the codon usage pattern of M. tuberculosis and against the nucleotide (nt) sequence corresponding to two conserved domains of the FtsH protein of Escherichia coli, yielded a 363-bp product. The amino-acid sequence, deduced from the nt sequence of the PCR product, revealed the presence of two ATP-binding motifs and the AAA Signature motif (Second Region of Homology) that are characteristic features found conserved in the FtsH molecules from eubacteria, archaebacteria, and eukaryotes. Southern hybridisation of the NheI digest of the cosmid SCY6F7 containing part of the genomic DNA of M. tuberculosis H37Rv using the PCR fragment as the probe identified the full-length ftsH gene in the 7.2-kb fragment. The gene was subcloned into pBS (SK+) vector, and the FtsH product that was expressed in E. coli transformed with the vector was identified as an 85-kDa protein localised in the membrane.
Anilkumar
G
G
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560 012, Karnataka, India.
Chauhan
M M
MM
Ajitkumar
P
P
D049069:ATP-Dependent Proteases; D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics* / Q000472:pathogenicity; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014774:Virulence / Q000235:genetics
D001426:Bacterial Proteins; D017931:DNA Primers; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D049069:ATP-Dependent Proteases; C513383:FtsH protein, E coli; D000251:Adenosine Triphosphatases