The prokaryotic expression of cyclin-dependent kinase 2, and the establish of its inhibitor screening system

Abstract

Cyclin dependent kinases 2 (CDK2), a kind of serine/threonine protein kinase, can form a complex with Cyclin A, which is indispensible ingredient in co-regulating the cell cycle of the DNA synthesis phase (S phase). The disorder of CDK2 gives rise to abnormal cell cycle regulation and thus results in tumor progression. In recent years, the development of highly effective CDK2 inhibitors has become one of the most talked about topics in the field of antitumor drug research. Firstly, the recombinant plasmids of CDK2 and Cyclin A were constructed in this paper. Then the soluble expression protein of CDK2 and Cyclin A were induced in E. coli BL21 (DE3) Plyss cells. The proteins were purified by Ni-beads and eluted by imidazole to obtain recombinant CDK2 and Cyclin A respectively. Next, the in vitro CDK2 kinase activity test platform was constructed with purified CDK2 and Cyclin A. Roscovitine, a well-known CDK inhibitor, was used as a positive control in our screening system. The half of the inhibition concentration (IC50) of Roscovitine in the system was 1.87 μM, which is in agreement with the previous reported. In summary, we established a rapid and stable CDK2 expression system, which could be used for the inhibitor screening and lay a good foundation for the study of new CDK inhibitors in the future.