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Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv

Gopalaswamy, Radha and Narayanan, P R and Narayanan, Sujatha (2004) Cloning, overexpression, and characterization of a serine/threonine protein kinase pknI from Mycobacterium tuberculosis H37Rv. Protein Expression and Purification , 36 (1). pp. 82-89. ISSN Print: 1046-5928; Online: 1096-027

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Protein phosphorylation-dephosphorylation is the principal mechanism for translation of external signals into cellular responses. Eukaryotic-like serine/threonine kinases have been reported to play important roles in bacterial development and/or virulence. The PknI protein is one of the 11 eukaryotic-like serine/threonine kinases in Mycobacterium tuberculosis H37Rv. From the bioinformatic studies, PknI protein has been shown to have an N-terminal cytoplasmic domain followed by a transmembrane region and an extracellular C-terminus suggestive of a sensor molecule. In this study, we have cloned, overexpressed, and characterized the entire coding region and the cytoplasmic domain of PknI as a fusion protein with an N-terminal histidine tag, and used immobilized metal aYnity chromatography for puriWcation of recombinant proteins. The puriWed recombinant proteins were found to be functionally active through in vitro phosphorylation assay and phosphoamino acid analysis. In vitro kinase assay of both proteins revealed that PknI is capable of autophosphorylation and showed manganese-dependent activity. Phosphoamino acid analysis indicated phosphorylation at serine and threonine residues. Southern blot analysis with genomic DNA highlighted the conserved nature of pknI among the various mycobacterial species. In silico analysis revealed a close homology of PknI to Stk1 from Streptococcus agalactiae, shown to have a role in virulence and cell segregation of the organism.

Item Type: Article
Uncontrolled Keywords: Serine/threonine kinase; Mycobacterium tuberculosis; IPTG induction; NaCl induction; Kinase assay; Phosphoamino acid analysis
Subjects: Tuberculosis > Laboratory Research > Immunological
Divisions: Basic Science Research > Immunology
Depositing User: Dr. Rathinasabapati R
Date Deposited: 19 Nov 2013 10:58
Last Modified: 09 Mar 2016 10:12

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