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A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1

Biological Procedures Online volume 6, pages 163–172 (2004)Cite this article

Abstract

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 His6-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 Ni2+-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.

Abbreviations

CDK:

cyclin-dependent kinase

CTD:

carboxyl-terminal domain of RNA polymerase II

Cyc:

cyclin

MBP:

myelin basic protein

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Authors and Affiliations

  1. Department of Molecular Biology and Genetics, University of Guelph, N1G 2W1, Guelph, Ontario, Canada

    Reena Pinhero, Peter Liaw & Krassimir Yankulov

Authors
  1. Reena Pinhero
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  2. Peter Liaw
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  3. Krassimir Yankulov
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Correspondence to Reena Pinhero.

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Published: August 18, 2004.

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Pinhero, R., Liaw, P. & Yankulov, K. A uniform procedure for the purification of CDK7/CycH/MAT1, CDK8/CycC and CDK9/CycT1. Biol. Proced. Online 6, 163–172 (2004). https://doi.org/10.1251/bpo86

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  • DOI: https://doi.org/10.1251/bpo86

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Biological Procedures Online

ISSN: 1480-9222

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