Pinhero Reena, Liaw Peter, Yankulov Krassimir
Department of Molecular Biology and Genetics, University of Guelph. Guelph, Ontario N1G 2W1. Canada.
Biol Proced Online. 2004;6:163-172. doi: 10.1251/bpo86. Epub 2004 Aug 18.
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.
我们已经建立了一套统一的程序，用于表达和纯化细胞周期蛋白依赖性激酶CDK7/CycH/MAT1、CDK8/CycC和CDK9/CycT1。我们在每个复合物的一个亚基上连接一个His(6)标签，然后在草地贪夜蛾昆虫细胞中与其他亚基一起共表达。随后通过Ni(2+)-NTA和Mono S色谱法纯化CDK复合物。这种方法产生了大量无污染激酶活性的活性重组激酶。重要的是，这些重组激酶的性质与其天然对应物相似（皮涅罗等人，2004年，《欧洲生物化学杂志》271:1004-14）。我们的方案为纯化这三种（以及可能的其他）重组CDK提供了一种新颖的系统方法。
