A full-length Caenorhabditis elegans cDNA that encodes the α subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCKα. Escherichia coli DE21 lysozyme S that was transformed with pCKα expressed soluble, catalytically active casein kinase IIα upon induction with isopropyl β-D-thiogalactopyranoside. The expressed α subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose. The M(r) values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M(r) = 42,000) or calculated from hydrodynamic measurements (S(20,w) = 3.3 S, Stokes radius = 2.8 nm, M(r) = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric. The native holoenzyme and the expressed α subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin. However, the k(cat) for E. coli-derived α was only 9% of the k(cat) for the native holoenzyme, and catalytic activity was not stimulated by polyamines. Recombinant casein kinase IIα aggregates at low ionic strength, and the aggregation is partially reversible. A mutant α subunit in wich Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis. The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g. IC50 = 0.3 μg/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased ~ 70-fold. Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain. The successful expression of casein kinase IIα in E. coli will facilitate the analysis of the structural basis for functional domains in this enzyme.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1990|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology