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

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 unpasteurize...

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Main Author:	Wong, Glenda See Sing
Format:	Thesis
Language:	English
Published:	2010
Subjects:	QR Microbiology
Online Access:	https://eprints.ums.edu.my/id/eprint/8122/1/mt0000000283.pdf
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spelling	my-ums-ep.81222017-10-12T03:09:12Z Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2 2010 Wong, Glenda See Sing QR Microbiology 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 NiÂ²â�º 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-Â³Â²P]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 2010 Thesis https://eprints.ums.edu.my/id/eprint/8122/ https://eprints.ums.edu.my/id/eprint/8122/1/mt0000000283.pdf text en public masters Universiti Malaysia Sabah School of Science and Technology
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English
topic	QR Microbiology
spellingShingle	QR Microbiology Wong, Glenda See Sing Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
description	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 NiÂ²â�º 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-Â³Â²P]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
format	Thesis
qualification_level	Master's degree
author	Wong, Glenda See Sing
author_facet	Wong, Glenda See Sing
author_sort	Wong, Glenda See Sing
title	Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
title_short	Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
title_full	Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
title_fullStr	Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
title_full_unstemmed	Serine/threonine protein kinases in Mycobacterium bovis BCG Pasteur 1173P2
title_sort	serine/threonine protein kinases in mycobacterium bovis bcg pasteur 1173p2
granting_institution	Universiti Malaysia Sabah
granting_department	School of Science and Technology
publishDate	2010
url	https://eprints.ums.edu.my/id/eprint/8122/1/mt0000000283.pdf
_version_	1747836350421270528

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

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