Cover Image

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...

Full description

Saved in:
Bibliographic Details
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
https://eprints.ums.edu.my/id/eprint/38490/
Tags: Add Tag
No Tags, Be the first to tag this record!
id my.ums.eprints.38490
record_format eprints
spelling my.ums.eprints.384902024-04-15T07:20:26Z https://eprints.ums.edu.my/id/eprint/38490/ Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2) 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 NonPeerReviewed text en https://eprints.ums.edu.my/id/eprint/38490/1/24%20PAGES.pdf text en https://eprints.ums.edu.my/id/eprint/38490/2/FULLTEXT.pdf Ainol Azifa Mohd Faik (2007) Cloning, expression and characterization of serine/threonine protein phosphatase and kinases of Mycobacterium bovis BCG (Pasteur 1173P2). Masters thesis, Universiti Malaysia Sabah.
institution Universiti Malaysia Sabah
building UMS Library
collection Institutional Repository
continent Asia
country Malaysia
content_provider Universiti Malaysia Sabah
content_source UMS Institutional Repository
url_provider http://eprints.ums.edu.my/
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
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)
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
https://eprints.ums.edu.my/id/eprint/38490/
_version_ 1797908633959268352
score 13.211869

Similar Items