A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1.
We have established a uniform procedure for the expression and purification of the cyclin-dependent kinases CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. We attach a His(6)-tag to one of the subunits of each complex and then co-express it together with the other subunits in Spodoptera frugiperda insect cells. The CDK complexes are subsequently purified by Ni(2+)-NTA and Mono S chromatography. This approach generates large amounts of active recombinant kinases that are devoid of contaminating kinase activities. Importantly, the properties of these recombinant kinases are similar to their natural counterparts (Pinhero et al. 2004, Eur J Biochem 271:1004-14). Our protocol provides a novel systematic approach for the purification of these three (and possibly other) recombinant CDKs.
Department of Molecular Biology and Genetics, University of Guelph. Guelph, Ontario N1G 2W1. Canada.
Reena
R
Pinhero
Peter
P
Liaw
Krassimir
K
Yankulov