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.
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Malumbres M (2014) Cyclin-dependent kinases. Chem Rev 101(8):2511–2526
Mascarenhas NM, Ghoshal N (2008) An efficient tool for identifying inhibitors based on 3D-QSAR and docking using feature-shape pharmacophore of biologically active conformation—a case study with CDK2/CyclinA. Eur J Med Chem 43(12):2807–2818
Malumbres M, Barbacid M (2005) Mammalian cyclin-dependent kinases. Trends Biochem Sci 30(11):630–641
Camalier CE et al (2013) An integrated understanding of the physiological response to elevated extracellular phosphate. J Cell Physiol 228(7):1536–1550
Takuwa N, Takuwa Y (2001) Regulation of cell cycle molecules by the Ras effector system. Mol Cell Endocrinol 177(1–2):25–33
Roskoski R (2016) Cyclin-dependent protein kinase inhibitors including palbociclib as anticancer drugs. Pharmacol Res 107:249–275
Cicenas J, Valius M (2011) The CDK inhibitors in cancer research and therapy. J Cancer Res Clin Oncol 137(10):1409–1418
Cheng PH et al (2013) Molecular basis for viral selective replication in cancer cells: Activation of CDK2 by adenovirus-induced cyclin E. PLoS ONE 8(2):e57340
Hongo F et al (2014) CDK1 and CDK2 activity is a strong predictor of renal cell carcinoma recurrence. Urol Oncol 32(8):1240–1246
Duca JS (2009) Recent advances on structure-informed drug discovery of cyclin-dependent kinase-2 inhibitors. Fut Med Chem 1(8):1453–1466
Dölker N et al (2014) The SH2 domain regulates c-Abl kinase activation by a cyclin-like mechanism and remodulation of the hinge motion. PLoS Comput Biol 10(10):e1003863–e1003863
Schulman BA, Harlow E (1998) Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on Cyclin A. Proc Natl Acad Sci USA 95(95):10453–10458
Wang Lixing et al (2014) Mini review for the inhibitors of cyclin dependent kinase(CDK). Chin Med Sci J 13:44–47
Shwrr CJ, Beach D (2016) Targeting CDK4 and CDK6: from discovery to therapy. Cancer Discov 6(4):353–367
Meijer L, Meijer L, Borgne A, Mulner O, Chong JP, Blow JJ, Inagaki N et al (1997) Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases CDC2, CDK2 and CDK5. Eur J Biochem 243(1–2):527–536
Koresawa M, Okabe T (2004) High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Dev Technol 2(2):153–160
Peyressatre M et al (2015) Targeting cyclin-dependent kinases in human cancers: from small molecules to peptide inhibitors. Cancers 7(1):179–237
Lim S, Kaldis P (2013) Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development 140(15):3079–3093
Malumbres M, Barbacid M (2009) Cell cycle, CDKs and cancer: a changing paradigm. Nat Rev Cancer 9(3):153–166
Malumbres M (2011) Physiological relevance of cell cycle kinases. Physiol Rev 91(3):973–1007
Acknowledgements
The authors sincerely thank the financial support from the financial support from National Natural Science Foundation of China (31301142), Tianjin Natural Science Foundation of China (12JCYBJC31600 and 11JGYBJC14300) and International Science & Technology Cooperation Program of China (2013DFA31160).
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Yuan, Y. et al. (2018). The Prokaryotic Expression of Cyclin-Dependent Kinase 2, and the Establish of Its Inhibitor Screening System. In: Liu, H., Song, C., Ram, A. (eds) Advances in Applied Biotechnology. ICAB 2016. Lecture Notes in Electrical Engineering, vol 444. Springer, Singapore. https://doi.org/10.1007/978-981-10-4801-2_13
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DOI: https://doi.org/10.1007/978-981-10-4801-2_13
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