Molecular cloning and biochemical characterization of a serine threonine protein kinase, PknL, from Mycobacterium tuberculosis.
PknL, a eukaryotic like serine threonine protein kinase from Mycobacterium tuberculosis, is predicted to be involved in transcriptional regulation and cell division. Attempts to clone and over-express the protein in Escherichia coli using pET43.1c as the vector were unsuccessful. The fusion protein was expressed as a truncated product and showed feeble autokinase activity. To overcome this technical glitch, the pknL ORF was cloned into a mycobacterial expression vector, pALACE and the purified His-tagged protein was evaluated for autokinase activity. Phosphorylation experiments with exogenous substrates like myelin basic protein (MBP) were performed. For the fast identification of protein phosphorylation sites, chromatographic methods of separating the [gamma-32P]phosphate radio labeled amino acids using thin-layer chromatography (TLC) on cellulose sheets was carried out. Thus, the activity of PknL was demonstrated using autophosphorylation and substrate phosphorylation experiments. Phospho amino acid determinations revealed that PknL was phosphorylated predominantly on serine and also on threonine residues. A single amino acid substitution of lysine to methionine in the active site completely abolished enzymatic action, thereby confirming the authenticity of the kinase function of the expressed protein.
Lakshminarayan
Harini
H
Department of Immunology, Tuberculosis Research Centre (ICMR), Mayor V.R. Ramanathan Road, Chetput, Chennai 600031, India.
Narayanan
Sujatha
S
Bach
Horacio
H
Sundaram
K G Papavinasa
KG
Av-Gay
Yossef
Y
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020102:Mycobacterium smegmatis / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism
D017346:Protein Serine-Threonine Kinases