Cloning and expression of CDK8 protein (human cyclin-dependent kinase)

• Grant number: 15/04161-4
• Support type: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): May 01, 2015
• Effective date (End): April 30, 2016
• Field of knowledge: Biological Sciences - Biochemistry - Molecular Biology
• Principal researcher: Fernanda Canduri
• Grantee: Danielle Karoline Silva Do Vale Castro
• Home Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil

Abstract

The human cyclin-dependent kinases (CDKs) comprise a family of protein serine/threonine kinases whose activity is dependent on association of the two subunits. One capable of regulating positively subunit (cyclin) or negatively (CDK inhibitor) and one catalytic subunit protein kinase regulated via site-specific phosphorylation and dephosphorylation reactions occurring in the threonine residue. The study of these proteins has become important because of the intense interest in the regulation of essential events for cell growth such as cell cycle and transcription. Thus, the regulation of the cell cycle is important in both cases, since the RNA polymerase II activity is modulated during the cell cycle where there are changes in phosphorylation state of the carboxy-terminal domain (CTD) of its larger subunit, and is closely involved in the development of diseases to cell division, such as cancer. CDKs regulate these cellular events through phosphorylation and dephosphorylation reactions, and the mechanisms by which the protein acts is the phosphorylation states of the kinase subunit, controlled degradation of cyclin subunit, gradual synthesis of cyclin and CDK inhibitory proteins and action of CDKs. CDKs 7-9 are important transcriptional regulators, the CDK8 is more complex, since it has been associated with positive and negative effects on transcription. The study of these proteins contributes to the understanding of the structure-function relationship of these and their relationship with oncogenes and tumor suppressors. This project aims to clone the cDNA of CDK8, express it in bacterial system and purify the protein by high-performance liquid chromatography. Additionally, essay should be performed to determine its secondary structure, such as essay to determine the enzyme activity in the presence of inhibitors.