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Kinetic Characterization of Thrombin Cleavable Procaspase-3

2008년 8월 9일 14시 28분 26초
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목 <발표Ⅰ>
저자 및
강효진, 이영미1, 정상전2
과학기술연합대학원대학교 나노바이오공학, Korea
1한국생명공학연구원 바이오나노연구단, Korea
2한국생명공학연구원 바이오나노연구단, Korea
Caspase-3 which is also known as CED-3, murine ICE and a protease resembling ICE/CPP32 in human is the first reported apoptotic effector as well as the main downstream effector that cleaves the majority of cellular substrates in apoptotic cells. A large scale preparation of caspase-3 is required for screening chemical library, structural study and kinetic analysis. Since expression of the full-length caspase-3 gene in E. coli resulted in a fully activated enzyme with a low yield, probably due to the cytotoxicity of the active caspase-3, the present method includes a separated expression of N- and C-terminal domains of caspase-3 in E. coli and subsequent refolding of the two domains for activation. Reported here is a novel method for a high level expression and purification of active caspase-3 in E. coli. The method includes engineering the cleavage (activation) sites of the precursor protein to a sequence susceptible to thrombin hydrolysis. The engineered caspase-3 precursors were highly overexpressed in E. coli and successfully purified by affinity column, resulting in 10~15 mg of proteins from 1 L culture. By thrombin digestion, the purified 30 kDa precursors were easily converted to active proteins, consisting of 12 and 17 kDa peptides on PAGE analysis. As expected, the digestion products showed roughly two orders of magnitudes-increased catalytic activities compared to those of the corresponding precursors.