Cloning and Heterologous Expression of the Head Domain Vimentin

• Main Authors: Huang, Huei-Luen, 黃慧倫
• Other Authors: Margaret Dah-Tsyr Chang
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/91934755909716226667

碩士 === 國立清華大學 === 生命科學系 === 85 === The head domains of cytoskeletal intermediate filaments (IFs) are considered to play an important role in IF assembly and network formation. Previously, it has been shown that the head domain of vimentin is the major target site of phosphorylation, and the degree of phosphorylation is increased in cells experiencing heat shock. In order to investigate the conformation of the head domain of vimentin and obtain firmly evidence of the temperature-related conformation change, the head domain has been cloned and heterologously expressed in E. coli by pET32/BL21(DE3) expression system. The resulting protein contains a thioredoxin fusion which can increase the solubility of the target protein and has a calculated molecular weight 33,279 dalton. The cloned protein has been purified by His-Bond affinity column and identified by Western blotting. Protein degradation during the purification process made the desired pure head domain difficult to achieve. The conformation of the fusion protein containing thioredoxin with head domain vimentin, instead of the pure head domain, was analyzed by circular dichorism. However, the expected temperature-related conformation change was not observed clearly in the CD spectrum of the fusion protein. The problem of proteolysis should still be resolved to achieve the exact conformation of the head domain.