Rhee S D, Jung H H, Yang G H, Moon Y S, Yang K H
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Taejon, South Korea.
FEMS Microbiol Lett. 1997 May 15;150(2):203-8. doi: 10.1016/s0378-1097(97)00114-6.
The light chain of Clostridium botulinum type B toxin was expressed in Escherichia coli using the expression vector pEt-3a containing phage T7 promoter. The expressed protein was then purified by DEAE-cellulose and phosphocellulose chromatography and the proteolytic activity of the purified light chain was studied. The purified recombinant light chain cleaved synaptobrevin when mixed with the mouse brain microsome and the proteolytic activity of the light chain was inhibited if a metal chelating agent such as EDTA or 2,2'-dipyridyl was added. The recombinant light chain cleaved synaptobrevin more effectively than the native type B toxin. When the native toxin was trypinized and was reduced with DTT, its proteolytic activity was similar to that of the recombinant light chain.
使用含有噬菌体T7启动子的表达载体pEt-3a在大肠杆菌中表达B型肉毒杆菌毒素的轻链。然后通过DEAE-纤维素和磷酸纤维素色谱法纯化表达的蛋白质，并研究纯化的轻链的蛋白水解活性。纯化的重组轻链与小鼠脑微粒体混合时可切割突触结合蛋白，并且如果添加金属螯合剂如EDTA或2,2'-联吡啶，则轻链的蛋白水解活性会受到抑制。重组轻链比天然B型毒素更有效地切割突触结合蛋白。当天然毒素用胰蛋白酶处理并用DTT还原时，其蛋白水解活性与重组轻链相似。
