title: Cloning, expression, purification and characterization of the stress kinase YeaG from Escherichia coli.
abstract: We cloned, overexpressed and purified the Escherichia coli yeaG gene product, whose amino acid sequence displays homology to prokaryotic serine protein kinases. The gene coding for YeaG was generated by amplifying the yeaG gene from E. coli by polymerase chain reaction. It was inserted into the expression plasmid pET-21a, under the transcriptional control of the bacteriophage T7 promoter and lac operator. A BL21(DE3) E. coli strain transformed with the YeaG-expression vector pET-21a-yeaG accumulates large amounts of a soluble protein with a molecular mass of 76kDa in SDS-PAGE, which matches the expected YeaG molecular weight of 74.5kDa. YeaG, although soluble, has a marked tendency to aggregate in the absence of detergents, so that it was purified in the presence of 0.1% Triton X-100, by ion exchange chromatography and hydroxyapatite chromatography. The purified protein is monomeric and displays autokinase and casein kinase activities which are optimal in the presence of 10mM Mn(2+). The purification of the active protein kinase YeaG described in this study should allow us to characterize its biochemical target(s) in E. coli extracts.
authors.affiliation: Stress Molecules, Institut Jacques Monod, CNRS, Universite Paris 7, 2 place Jussieu, 75005 Paris, France.
authors.forename: Jihen
authors.initials: J
authors.lastname: Tagourti
authors.forename: Ahmed
authors.initials: A
authors.lastname: Landoulsi
authors.forename: Gilbert
authors.initials: G
authors.lastname: Richarme
mesh_terms: D002364:Caseins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008970:Molecular Weight; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D002364:Caseins; D029968:Escherichia coli Proteins; D017346:Protein Serine-Threonine Kinases; C528225:YeaG protein, E coli