Zaman S, Richards H, Ward J
Department of Biology, University College, London, UK.
Nucleic Acids Res. 1992 Jul 25;20(14):3693-700. doi: 10.1093/nar/20.14.3693.
A 0.5kb Spel-BclI fragment containing the pIJ101 korB ORF was cloned into pUC8 under the control of the lacZ promoter, creating pQR206. In vitro coupled transcription-translation of pQR206 identified a protein product of approximately 10kDa, which corresponds to the predicted molecular weight deduced from the korB sequence. pQR206 was used to express the 10kDa KorB protein in vivo in E. coli. Crude E. coli protein extracts containing KorB were shown to bind to a 0.8kb kilB fragment and a 0.5kb korB fragment in gel retardation assays. DNasel footprinting indicated that the DNA recognition sequence of the KorB protein lies within a 60bp protected region encompassing the kilB promoter and a 36bp region encompassing the korB promoter.
将包含pIJ101 korB开放阅读框的0.5kb Spel - BclI片段在lacZ启动子的控制下克隆到pUC8中，构建成pQR206。对pQR206进行体外偶联转录 - 翻译，鉴定出一种约10kDa的蛋白质产物，这与从korB序列推导的预测分子量相符。pQR206用于在大肠杆菌体内表达10kDa的KorB蛋白。在凝胶阻滞试验中，含有KorB的大肠杆菌粗蛋白提取物显示能与一个0.8kb的kilB片段和一个0.5kb的korB片段结合。DNasel足迹分析表明，KorB蛋白的DNA识别序列位于一个包含kilB启动子的60bp保护区域内以及一个包含korB启动子的36bp区域内。
