Purification of CK1 by affinity chromatography on immobilised axin.
Members of the Casein Kinase 1 (CK1) family are implicated in the regulation of a variety of physiological processes like development and circadian rhythm, as well as in diseases like cancer and Alzheimer's disease. From that perspective, CK1 family members are interesting targets for potential chemotherapy. We describe here a rapid and efficient method for the purification of CK1 by affinity chromatography on an immobilised fragment of axin. Axin is a scaffolding protein that interacts with a multitude of proteins, amongst them APC, GSK-3, beta-catenin, CK1alpha, delta, and epsilon, and PP2A. A GST-tagged axin peptide (residues 495-684) was produced in Escherichia coli and either immobilised on glutathione agarose beads or purified and immobilised on CNBr-activated sepharose 4B. These "GST-axin" matrices were found to selectively bind native CK1alpha and CK1epsilon from porcine brain. The affinity-purified enzymes displayed high kinase activity. This single step purification method provides a convenient tool to efficiently purify large amounts of active native CK1 for screening purposes. This single step purification method also provides a convenient tool to follow the status of the axin-binding CK1 isoforms alpha, delta, and epsilon (protein levels, composition of isoforms, kinase activity) under different physiological settings.
Reinhardt
Jens
J
Amyloids and Cell Division Cycle, Station Biologique de Roscoff, CNRS, Place Georges Teissier, 29680 Roscoff, France.
Ferandin
Yoan
Y
Meijer
Laurent
L
D000818:Animals; D060466:Axin Protein; D001923:Brain Chemistry; D047389:Casein Kinase I / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D012097:Repressor Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013552:Swine / Q000378:metabolism
D060466:Axin Protein; D010455:Peptides; D020033:Protein Isoforms; D012097:Repressor Proteins; D047389:Casein Kinase I