Ahmed Abo Bakr F, Noguchi Kanako, Asami Yasuo, Nomura Kazutaka, Fujii Hiroya, Sakata Minoru, Tokita Akihiko, Noda Kenichi, Kuroda Akio
Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima, Japan.
J Biosci Bioeng. 2007 Jul;104(1):55-61. doi: 10.1263/jbb.104.55.
We evaluated the cell wall binding (CWB) domain of Staphylococcus aureus autolysin as an affinity reagent for bacteria. A fusion of CWB domain and green fluorescent protein (CWB-GFP) bound to S. aureus with a dissociation constant of 15 nM. CWB-GFP bound to a wide range of gram-positive bacteria, but not to most gram-negative bacteria. We suspected that the outer membrane of gram-negative bacteria inhibits the access of CWB-GFP to peptidoglycan layer. Indeed, CWB-GFP bound to gram-negative bacteria when they were treated with benzalkonium chloride. Because CWB-GFP bound to the bacterial peptidoglycan layer, it appeared to be an effective affinity reagent for bacteria and CWB fusion with reporter proteins could be applied to detect bacteria. We also constructed a fusion of CWB and luciferase, which can be used for the rapid detection of bacteria.
我们评估了金黄色葡萄球菌自溶素的细胞壁结合（CWB）结构域作为细菌亲和试剂的性能。CWB结构域与绿色荧光蛋白的融合体（CWB-GFP）以15 nM的解离常数与金黄色葡萄球菌结合。CWB-GFP能与多种革兰氏阳性菌结合，但不能与大多数革兰氏阴性菌结合。我们推测革兰氏阴性菌的外膜会抑制CWB-GFP与肽聚糖层的结合。事实上，当用苯扎氯铵处理革兰氏阴性菌时，CWB-GFP能与之结合。由于CWB-GFP能与细菌肽聚糖层结合，它似乎是一种有效的细菌亲和试剂，并且CWB与报告蛋白的融合体可用于检测细菌。我们还构建了CWB与荧光素酶的融合体，可用于细菌的快速检测。
