title: Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure.
abstract: 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.
authors.affiliation: Department of Microbiology and Immunology, School of Medicine, University of California at Los Angeles, 90095, USA. DBLANCO@microimmun.medsch.ucla.edu.
authors.forename: D R
authors.initials: DR
authors.lastname: Blanco
authors.forename: C I
authors.initials: CI
authors.lastname: Champion
authors.forename: M M
authors.initials: MM
authors.lastname: Exner
authors.forename: E S
authors.initials: ES
authors.lastname: Shang
authors.forename: J T
authors.initials: JT
authors.lastname: Skare
authors.forename: R E
authors.initials: RE
authors.lastname: Hancock
authors.forename: J N
authors.initials: JN
authors.lastname: Miller
authors.forename: M A
authors.initials: MA
authors.lastname: Lovett
mesh_terms: D000942:Antigens, Bacterial / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D001426:Bacterial Proteins; D002462:Cell Membrane / Q000378:metabolism*; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D008051:Lipid Bilayers; D016253:Microscopy, Immunoelectron; D018272:Porins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D014210:Treponema pallidum / Q000737:chemistry*
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001426:Bacterial Proteins; D000939:Epitopes; D008051:Lipid Bilayers; D018272:Porins; D011993:Recombinant Fusion Proteins; C093836:Tromp1 protein, Treponema pallidum