Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2

• मुख्य लेखक: Wong, Glenda See Sing
• स्वरूप: थीसिस
• भाषा: अंग्रेज़ी
• प्रकाशित: 2010
• विषय: QR Microbiology
• ऑनलाइन पहुंच: https://eprints.ums.edu.my/id/eprint/8122/1/mt0000000283.pdf

Mycobacterium tuberculosis cause tuberculosis in human while Mycobacterium bovis cause tuberculosis in farm animals and milk contamination which is of major importance to cattle and dairy industry. Mycobacterium boviscan also cause human tuberculosis mostly by ingestion of contaminated unpasteurized cow's milk. On the other hand, the Mycobacterium bovis BCG Pasteur 1173P2 is attenuated strain from M. bovis which is widely used as vaccine. The mechanism contribute to the virulence of these bacteria is not fully known. However, reversible protein phosphorylation mediated by Signaling kinases and phosphatases is known to be involved. In this study, three genes encoding serine/ threonine protein kinases (pknfi pknJand pknL) from Mycobacterium bovis BCG Pasteur 1173P2 were cloned and characterized. Polymerase chain reaction was conducted using specific primers for amplification of serine/threonine kinase genes. Three DNA fragments at 1791bp, 1770 bp and 1200 bp were amplified, cloned and sequenced. The sequences were BLAST against the mycobacterium genome database and named pknH-BCG, pknJ-BCG and pknL-BCG respectively. Amplified pknH-BCG, pknJ-BCG and pknL-BCG were cloned into pET-16b and expressed as recombinant proteins in Escherichia coli BL21(DE3)pLysS. The proteins were purified from inclusion bodies using Ni²� column and analysed on SDS-PAGE. The sizes of the protein obtained were 66 kDa, 64 kDa and 45 kDa respectively as expected. Alignment of the PknH-BCG, PknJ-BCG and PknL -BCG protein sequences with other related species revealed 99% homology to transmembrane serine/ threonine protein kinase PknH, PknJ and PknL of Mycobacterium tuberculosis H37Rv. Bioinformatics studies showed that PknH-BCG, PknJ-BCG and PknL-BCG contained 11 Hanks kinase motifs, which is conserved in serine/threonine protein kinases. It revealed the presence of transmembrane region predicting the location of the protein in the mycobacterial cell wall. In order to examine their kinase activities, phosphorylation assay using [Y-³²P]ATP was performed. Phosphorylated bands were obtained indicating PknH-BCG and PknJ-BCG were able to autophosphorylate while PknL-BCG was weakly autophosphorylated. Further analyses using structural comparison with protein database and bioinformatics tools suggested that PknH-BCG might be involved in regulating cellular events to facilitate adaptation to the host environment. PknJ-BCG which situated directly downstream of several transposon genes still having unclear function however might act as receptor protein while PknL-BCG is predicted to be involved in transcriptional regulation and cell division