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

被引：12

作者：

Pinhero R. ^{[1
]}

Liaw P. ^{[1
]}

Yankulov K. ^{[1
]}

机构：

[1] Department of Molecular Biology and Genetics, University of Guelph, Guelph

来源：

Biological Procedures Online | 2004年 / 6卷 / 1期

关键词：

Baculoviridae; Cyclin-dependent kinases; Isolation and purification;

D O I：

10.1251/bpo86

中图分类号：

学科分类号：

摘要：

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. © 2004 by the author(s).

引用

页码：163 / 172

页数：9

共 30 条

[1]

Morgan D.O., Cyclin-dependent kinases: Engines, clocks, and microprocessors. The dynamics of cyclin dependent kinase structure, Annu. Rev. Cell Dev. Biol., 13, 6, pp. 261-291, (1997)

[2]

Kobor M.S., Greenblatt J., Regulation of transcription elongation by phosphorylation, Biochim. Biophys. Acta, 1577, 2, pp. 261-275, (2002)

[3]

Palancade B., Bensaude O., Investigating RNA polymerase II carboxyl-terminal domain (CTD) phosphorylation, Eur. J. Biochem., 270, 19, pp. 3859-3870, (2003)

[4]

Ramanathan Y.S., Rajpara M., Reza S.M., Lees E., Shuman S., Mathews M.B., Pe'ery T., Three RNA polymerase II carboxylterminal domain kinases display distinct substrate preferences, J. Biol. Chem., 276, 14, pp. 10913-10920, (2001)

[5]

Rickert P., Corden J.L., Lees E., Cyclin C/CDK8 and cyclin H/CDK7/p36 are biochemically distinct CTD kinases, Oncogene, 18, 4, pp. 1093-1102, (1999)

[6]

Price D.H., P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II, Mol. Cell Biol., 20, 8, pp. 2629-2634, (2000)

[7]

Ping Y.H., Rana T.M., Tat-associated kinase (P-TEFb): A component of transcription preinitiation and elongation complexes, J. Biol. Chem., 274, 11, pp. 7399-7404, (1999)

[8]

Nigg E.A., Cyclin-dependent kinase-7 - At the cross-roads of transcription, DNA-repair and cell-cycle control, Curr. Opin. Cell Bio., 8, 3, pp. 312-317, (1996)

[9]

Rochette-Egly C., Adam S., Rossignol M., Egly J.M., Chambon P., Stimulation of RAR alpha activation function AF-1 through binding to the general transcription factor TFIIH and phosphorylation by CDK7, Cell, 90, 1, pp. 97-107, (1997)

[10]

Bastien J., Adam-Stitah S., Riedl T., Egly J.M., Chambon P., Rochette-Egly C., TFIIH interacts with the retinoic acid receptor gamma and phosphorylates its AF-1-activating domain through cdk7, J. Biol. Chem., 275, 29, pp. 21896-21904, (2000)

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