title: Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2).
abstract: In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.
authors.affiliation: Department of Microbiology and Immunology, University of California at Los Angeles, 90095, USA.
authors.forename: C I
authors.initials: CI
authors.lastname: Champion
authors.forename: D R
authors.initials: DR
authors.lastname: Blanco
authors.forename: M M
authors.initials: MM
authors.lastname: Exner
authors.forename: H
authors.initials: H
authors.lastname: Erdjument-Bromage
authors.forename: R E
authors.initials: RE
authors.lastname: Hancock
authors.forename: P
authors.initials: P
authors.lastname: Tempst
authors.forename: J N
authors.initials: JN
authors.lastname: Miller
authors.forename: M A
authors.initials: MA
authors.lastname: Lovett
mesh_terms: D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000032:analysis; D000954:Antigens, Surface / Q000032:analysis; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008051:Lipid Bilayers / Q000737:chemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry / Q000276:immunology; D017422:Sequence Analysis, DNA; D012995:Solubility; D014210:Treponema pallidum / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure
chemical_list: D000942:Antigens, Bacterial; D000954:Antigens, Surface; D001425:Bacterial Outer Membrane Proteins; D000939:Epitopes; D008051:Lipid Bilayers; D011994:Recombinant Proteins; C104240:Tromp2 protein, Treponema pallidum