Hoenger A, Ghosh R, Schoenenberger C A, Aebi U, Engel A
M. E. Müller-Institute for Microscopic Structural Biology, University of Basel, Switzerland.
J Struct Biol. 1993 Nov-Dec;111(3):212-21. doi: 10.1006/jsbi.1993.1051.
An OmpA-deficient mutant of an OmpF/OmpC-free Escherichia coli B strain was selected using phage K3. The mutant strain was characterized by SDS-gel electrophoresis, immunoblotting, and electron microscopy. All major outer membrane proteins, including OmpA, were absent. This strain was then transformed with the plasmid pMY222 encoding the K12 OmpF porin or with pBlue-script-derived plasmids, encoding the porins OmpC, PhoE, and maltoporin, respectively. Following SDS extraction of outer membrane sacculi from strains expressing individual porins, crystalline porin arrays that allowed in situ structural analysis to be performed were observed. Furthermore, the absence of endogenous major outer membrane proteins facilitated the purification of native porin-lipopolysaccharide complexes, the functionally active channels, from the sacculi of transformed strains.
使用噬菌体K3筛选出了一种不含OmpF/OmpC的大肠杆菌B菌株的OmpA缺陷型突变体。通过SDS凝胶电泳、免疫印迹和电子显微镜对该突变株进行了表征。包括OmpA在内的所有主要外膜蛋白均缺失。然后用编码K12 OmpF孔蛋白的质粒pMY222或分别编码孔蛋白OmpC、PhoE和麦芽糖孔蛋白的pBlue-script衍生质粒转化该菌株。在用SDS从表达单个孔蛋白的菌株中提取外膜囊泡后，观察到了允许进行原位结构分析的结晶孔蛋白阵列。此外，内源性主要外膜蛋白的缺失有助于从转化菌株的囊泡中纯化天然孔蛋白-脂多糖复合物，即功能活性通道。
