A structural and functional study on Cot (MAPKKK8)

• Main Author: Wu, Binhui
• Other Authors: Rupert Charles Wilmouth
• Format: Theses and Dissertations
• Language: English
• Published: 2008
• Subjects: DRNTU::Science::Biological sciences
• Online Access: https://hdl.handle.net/10356/13576
• Institution: Nanyang Technological University

Cot (cancer Osaka thyroid, also known as Tpl-2), a proto-oncogenic kinase, is a member of the MAPKKK family and plays a key role in the regulation of the immune response to pro-inflammatory stimuli such as LPS (lipopolysaccharide) and TNF- (tumor necrosis factor-). After LPS-stimulation, Cot activates the MEK1/2-ERK1/2 pathway, which is parallel to Raf, to regulate gene transcription, cell growth, differentiation and survival. In this study, the cot gene was cloned and expressed in E. coli, insect cells and mammalian cells. Cot constructs which contained the last 30 bp of cotfull-length did not express well in E. coli. However, C-terminally truncated constructs of Cot can express well. The 6×His tag could not be used for purification as it was inaccessible. The GST tag was useful for purification, but cleavage between GST and the Cot protein was difficult. Purified Cot proteins were used for crystallization screening. Several crystals were observed with Cot1-388 (refolded protein), GST-Cot1-388 and GST-Cot1-388 (selenomethionine-substituted). However, the quality of the crystals was inadequate to obtain good X-ray diffraction.