Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)

Pathogenesis of most bacteria is connected to its survival within the host by adaptive regulation of gene expression in response to alterations of the environment Before completion of the bacterial genome sequencing data, it was thought that protein phosphorylation/dephosphorylation only involves th...

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Main Author:	Ainol Azifa Mohd Faik
Format:	Thesis
Language:	English English
Published:	2007
Subjects:	QR75-99.5 Bacteria
Online Access:	https://eprints.ums.edu.my/id/eprint/38490/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/38490/2/FULLTEXT.pdf
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spelling	my-ums-ep.384902024-04-15T07:20:26Z Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2) 2007 Ainol Azifa Mohd Faik QR75-99.5 Bacteria Pathogenesis of most bacteria is connected to its survival within the host by adaptive regulation of gene expression in response to alterations of the environment Before completion of the bacterial genome sequencing data, it was thought that protein phosphorylation/dephosphorylation only involves the so-called two component system consisting of histidine kinase sensors and their associated response regulators. Recent evidence revealed that some prokaryotes contain protein kinases and phosphatases. In this study, three genes with sequence homology to those encoding serine/threonine kinases (pknl, pknK) and serine/threonine phosphatase (ppp) in Mycobacterium tuberculosis HxJ?v were cloned from a less pathogenic bacteria, Mmcobacterium bovis BCG {Pasteur 1173P2} obtained from Pasteur Institute. The pknl and ppp genes were proposed to be involved in regulation of cell division and elongation while pknK gene might regulate the production of secondary metabolite in Mycobacterium. Amplified ppp, pknl and pknK genes were cloned and expressed as a recombinant proteins in pTrcHis and pET42-a(+). The calculated molecular masses of these proteins designated as Ppp, Pknl and PknK were 58.8 kDa, 94. 4 kDa and 150.3 kDa respectively. Bioinformatics tools have suggested that Pknl and PknK contain 12 Hanks kinase motifs in contrast with Ppp which has 11 motifs that are universally conserved and characteristic of PP2C phosphatases. In addition, Pknl and Ppp also revealed the presence of a transmembrane region predicting the location of these proteins in mycobacterial cells. Ppp and Pknl were expressed predominantly as inclusion bodies while PknK was found to have partial solubility. Therefore, these proteins were purified as inclusion bodies, solubilized using high concentration of urea and refolded using dialysis by decreasing the urea concentration gradually. Protein concentrations of Ppp, Pknl and PknK obtained after refolding were 0.110 mg/ml, 0.246 mg/ml and 0.463mg/ml respectively. Ppp was strictly dependent on Mrl+ in vitro and the activity was highest at 55°C Ppp was not inhibited by okadaic acid, sodium orthovanadate and low concentration of EDTA and NaF but showed a substantially decreased activity when incubated at high concentration of EDTA and NaF. Km and Vmax values of the phosphatase activity using pNPP as a substrate and a fixed amount of Ppp were determined as 0.83 ± 0.07 mM and 1.49 ± 0.02 nmol/min/μg respectively. Kinetic analysis of the phosphatase activity of fixed amount of Ppp using threonine phosphopeptide resulted in a Km value of 1.34 ± 0.704 mM and a Vmax value of 0.206 ± 0. 075 nmol/min/μg. These results show that the Ppp enzyme was biologically active and successfully refolded. 2007 Thesis https://eprints.ums.edu.my/id/eprint/38490/ https://eprints.ums.edu.my/id/eprint/38490/1/24%20PAGES.pdf text en public https://eprints.ums.edu.my/id/eprint/38490/2/FULLTEXT.pdf text en validuser masters Universiti Malaysia Sabah Sekolah Sains dan Teknologi
institution	Universiti Malaysia Sabah
collection	UMS Institutional Repository
language	English English
topic	QR75-99.5 Bacteria
spellingShingle	QR75-99.5 Bacteria Ainol Azifa Mohd Faik Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
description	Pathogenesis of most bacteria is connected to its survival within the host by adaptive regulation of gene expression in response to alterations of the environment Before completion of the bacterial genome sequencing data, it was thought that protein phosphorylation/dephosphorylation only involves the so-called two component system consisting of histidine kinase sensors and their associated response regulators. Recent evidence revealed that some prokaryotes contain protein kinases and phosphatases. In this study, three genes with sequence homology to those encoding serine/threonine kinases (pknl, pknK) and serine/threonine phosphatase (ppp) in Mycobacterium tuberculosis HxJ?v were cloned from a less pathogenic bacteria, Mmcobacterium bovis BCG {Pasteur 1173P2} obtained from Pasteur Institute. The pknl and ppp genes were proposed to be involved in regulation of cell division and elongation while pknK gene might regulate the production of secondary metabolite in Mycobacterium. Amplified ppp, pknl and pknK genes were cloned and expressed as a recombinant proteins in pTrcHis and pET42-a(+). The calculated molecular masses of these proteins designated as Ppp, Pknl and PknK were 58.8 kDa, 94. 4 kDa and 150.3 kDa respectively. Bioinformatics tools have suggested that Pknl and PknK contain 12 Hanks kinase motifs in contrast with Ppp which has 11 motifs that are universally conserved and characteristic of PP2C phosphatases. In addition, Pknl and Ppp also revealed the presence of a transmembrane region predicting the location of these proteins in mycobacterial cells. Ppp and Pknl were expressed predominantly as inclusion bodies while PknK was found to have partial solubility. Therefore, these proteins were purified as inclusion bodies, solubilized using high concentration of urea and refolded using dialysis by decreasing the urea concentration gradually. Protein concentrations of Ppp, Pknl and PknK obtained after refolding were 0.110 mg/ml, 0.246 mg/ml and 0.463mg/ml respectively. Ppp was strictly dependent on Mrl+ in vitro and the activity was highest at 55°C Ppp was not inhibited by okadaic acid, sodium orthovanadate and low concentration of EDTA and NaF but showed a substantially decreased activity when incubated at high concentration of EDTA and NaF. Km and Vmax values of the phosphatase activity using pNPP as a substrate and a fixed amount of Ppp were determined as 0.83 ± 0.07 mM and 1.49 ± 0.02 nmol/min/μg respectively. Kinetic analysis of the phosphatase activity of fixed amount of Ppp using threonine phosphopeptide resulted in a Km value of 1.34 ± 0.704 mM and a Vmax value of 0.206 ± 0. 075 nmol/min/μg. These results show that the Ppp enzyme was biologically active and successfully refolded.
format	Thesis
qualification_level	Master's degree
author	Ainol Azifa Mohd Faik
author_facet	Ainol Azifa Mohd Faik
author_sort	Ainol Azifa Mohd Faik
title	Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
title_short	Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
title_full	Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
title_fullStr	Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
title_full_unstemmed	Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)
title_sort	cloning, expression and characterization of serine/threonine protein phosphatase and kinases of mycobacterium bovis bcg (pasteur 1173p2)
granting_institution	Universiti Malaysia Sabah
granting_department	Sekolah Sains dan Teknologi
publishDate	2007
url	https://eprints.ums.edu.my/id/eprint/38490/1/24%20PAGES.pdf https://eprints.ums.edu.my/id/eprint/38490/2/FULLTEXT.pdf
_version_	1804890315551670272

Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2)

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