Blanco D R, Champion C I, Exner M M, Shang E S, Skare J T, Hancock R E, Miller J N, Lovett M A
Department of Microbiology and Immunology, School of Medicine, University of California at Los Angeles, 90095, USA.
J Bacteriol. 1996 Dec;178(23):6685-92. doi: 10.1128/jb.178.23.6685-6692.1996.
We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22-residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole-mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.
我们最近报道了编码31 kDa梅毒螺旋体苍白亚种稀有外膜孔蛋白的基因的克隆和测序，该蛋白被命名为Tromp1（D. R. Blanco、C. I. Champion、M. M. Exner、H. Erdjument-Bromage、R. E. W. Hancock、P. Tempst、J. N. Miller和M. A. Lovett，《细菌学杂志》177:3556 - 3562，1995年）。在此，我们报道重组Tromp1（rTromp1）在大肠杆菌中的稳定表达。rTromp1在没有其信号肽的情况下表达，并含有一个22个残基的N端融合序列，导致一种非分泌性可溶性蛋白的高水平积累，该蛋白通过快速蛋白质液相色谱（FPLC）纯化至同质。针对FPLC纯化的rTromp1融合蛋白产生的特异性抗血清在梅毒螺旋体的免疫印迹中鉴定出天然的31 kDa Tromp1蛋白以及两种分子量更高的Tromp1寡聚体形式，分别为55 kDa和80 kDa。rTromp1还通过使用可诱导的T7启动子与其天然信号肽一起表达。在这些条件下，rTromp1主要分布在大肠杆菌的可溶性和外膜部分，而不在内膜部分。从大肠杆菌外膜分离并重组到平面脂质双分子层中的rTromp1基于在1 M KCl中的平均单通道电导为0.4和0.8 nS显示出孔蛋白活性。使用针对梅毒螺旋体的感染源免疫血清进行的整装免疫电子显微镜检查表明，rTromp1在大肠杆菌中表达时位于表面。这些发现表明rTromp1可以靶向到大肠杆菌外膜，在那里它既具有孔蛋白活性又具有表面抗原暴露。
