Affinity purification of Csk protein tyrosine kinase based on its catalytic requirement for divalent metal cations.
Protein tyrosine kinase Csk requires two Mg2+ ions for activity: one magnesium is part of the ATP-Mg complex, and the second free Mg2+ ion is required as an essential activator. Zn2+ can bind to this site to replace Mg2+, which inhibits Csk kinase activity. The binding is reversible and removal of Zn2+ results in an active Csk apoenzyme. In this communication, we report that this tight binding can be used as a mechanism for affinity purification of Csk. When bacterial cell lysate containing overexpressed GST-Csk was applied to a column of Zn2+-iminodiacetic acid immobilized to agarose, Csk was specifically retained by the column. Since the binding of Csk to Zn2+ is not affected by up to 200 mM NaCl, high ionic strength conditions were used in the purification procedure, minimizing nonspecific binding due to ionic interactions. Washing the column with 200 mM NaCl and 50 mM imidazole removed virtually all other proteins from the column while Csk remained bound. The retained Csk enzyme was eluted with 1 M imidazole. The 1 M imidazole-eluted fraction contained pure Csk that had a specific activity similar to the enzyme purified by a glutathione-agarose affinity column.
Sun
G
G
Department of Neuro-Oncology, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Budde
R J
RJ
D002384:Catalysis; D002413:Cations, Divalent / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004926:Escherichia coli; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002413:Cations, Divalent; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C086603:Matk protein, mouse; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D008274:Magnesium; D015032:Zinc