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标题：Cloning, Enzyme Characterization of Recombinant Human Eg5 and the Development of a New Inhibitor
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作者：Lei Yang ; Cheng Jiang ; Fei Liu 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2008
卷号：31
期号：7
页码：1397-1402
DOI：10.1248/bpb.31.1397
出版社：The Pharmaceutical Society of Japan
摘要：The microtubule-dependent motor protein Eg5 is essential for the development and function of the mitotic spindle. Now it has become an anti-mitotic drug target in high throughput screening for anticancer dugs in vitro . Here is a protocol for cloning, expression and purification of a human Eg5 that codes for motor and linker domain in Escherichia coli BL21 (DE3) cells. The effects of temperature, pH, metal ions and DMSO on ATPase activity were investigated. A new compound CPUYL064 showed good inhibitory effect against Eg5 (IC₅₀ value, 100 n M ). It inhibited the proliferation of human hepatocellular liver carcinoma cell line HepG2 in a dose- and time-dependent manner. CPUYL064 induced a clear G₂/M phase arrest and caused the monastral spindle in HepG2 cells. Induction of apoptosis was further confirmed by changes in membrane phospholipids, changes in mitochondrial membrane potential and by detection of DNA fragmentation. These results indicate that CPUYL064 could be developed as a new, potent mitotic arrest compound.
关键词：Eg5;molecular cloning;characterization;inhibitor;G2/M arrest;apoptosis