Epp S F, Pechère J, Kok M
Department of Genetics and Microbiology, Centre Médical Universitaire (CMU), 9, ave de Champel, CH-1211 4, Geneva, Switzerland.
J Microbiol Methods. 2001 Jul 30;46(1):1-8. doi: 10.1016/s0167-7012(01)00236-6.
OprD is an outer membrane porin of Pseudomonas aeruginosa that mediates uptake of basic amino acids, peptides as well as carbapenem antibiotics. Polyclonal antibodies were raised against the OprD porin by creating protein fusions between the Escherichia coli maltose binding protein and four OprD fragments. These were expressed in E. coli and shown to be exported to the periplasm. The fusion proteins were purified by amylose affinity chromatography and used to immunize rabbits intramuscularly. We established that MalE fusions to OprD fragments retain maltose and amylose binding activities in vivo and in vitro, confirming proper folding of the MalE domain of hybrid proteins. Furthermore, we demonstrate that this strategy can be used to obtain specific antibodies against bacterial outer membrane proteins (OMPs).
OprD是铜绿假单胞菌的一种外膜孔蛋白，介导碱性氨基酸、肽以及碳青霉烯类抗生素的摄取。通过在大肠杆菌麦芽糖结合蛋白与四个OprD片段之间创建蛋白质融合体，制备了针对OprD孔蛋白的多克隆抗体。这些融合体在大肠杆菌中表达，并显示被转运至周质。融合蛋白通过直链淀粉亲和层析进行纯化，并用于肌肉注射免疫兔子。我们证实，与OprD片段融合的MalE在体内和体外均保留麦芽糖和直链淀粉结合活性，这证实了杂合蛋白MalE结构域的正确折叠。此外，我们证明该策略可用于获得针对细菌外膜蛋白（OMP）的特异性抗体。
