Ainol Azifa Bt Hj 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.
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 twocomponent 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 (pknI, pknK) and serine/threonine phosphatase (ppp) in Mycobacterium tuberculosis H37Rv were cloned from a less pathogenic bacteria, Mycobacterium bovis BCG (Pasteur 1173P2) obtained from Pasteur Institute. The pknI 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, pknI 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, PknI and PknK were 58.8 kDa, 94.4 kDa and 150.3 kDa respectively. Bioinformatics tools have suggested that PknI 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, PknI and Ppp also revealed the presence of a transmembrane region predicting the location of these proteins in mycobacterial cells. Ppp and PknI 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, PknI and PknK obtained after refolding were 0.110 mg/mL, 0.246 mg/mL and 0.463mg/mL respectively. Ppp was strictly dependent on MrI+ 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/JJg respectively. Kinetic analysis of the phosphatase activity of fixed amount of Ppp using threonine phosphopeptide resulted in a Km value of 1.34 i:. 0.704 mM and a Vmax value of 0.206 + 0.075 nmol/min/JJg. These results show that the Ppp enzyme was biologically active and successfully refolded.
|Item Type:||Thesis (Masters)|
|Uncontrolled Keywords:||Mycobacterium Genus, tuberculosis, protein phosphorylation, signal transduction, Polymerase Chain Reaction (PCR), DNA|
|Subjects:||?? QR75-99.5 ??|
Q Science > QR Microbiology
|Divisions:||SCHOOL > School of Science and Technology|
|Deposited By:||IR Admin|
|Deposited On:||18 Nov 2010 16:52|
|Last Modified:||12 Dec 2014 14:39|
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