title: Genetic and physicochemical characterization of the recombinant DNA-derived 47-kilodalton surface immunogen of Treponema pallidum subsp. pallidum.
abstract: Previous work has established the importance of the 47-kilodalton (kDa) surface immunogen of Treponema pallidum subsp. pallidum (T. pallidum) in the immunopathogenesis of syphilis; the 47-kDa immunogen gene was cloned and expressed in Escherichia coli (M. V. Norgard, N. R. Chamberlain, M. A. Swancutt, and M. S. Goldberg, Infect. Immun. 54:500-506, 1986). To facilitate additional structural-functional analysis of this protein for immunopathogenesis studies, the recombinant DNA-derived molecule was examined with respect to its genetic expression and physicochemical properties. Subcloning of partial PstI digests of the original 47-kDa antigen-encoding DNA segment localized the 47-kDa antigen gene to a 1.3-kilobase (kb) T. pallidum DNA fragment. A 20- to 100-fold enhanced expression of the 47-kDa antigen was obtained when a 2.85-kb DNA insert containing the entire 1.3-kb structural gene was subcloned into a T7 RNA polymerase-dependent expression vector system. Under these conditions, several derivatives of the recombinant 47-kDa protein possessing different molecular masses were observed that were identical to those previously detected on Western blots of native T. pallidum antigens with monoclonal antibodies. Sarkosyl extraction of E. coli recombinant cell envelopes localized the 47-kDa protein to both the inner and outer membranes of E. coli. The absolute requirement of detergents (N-lauroylsarcosine, 3-[(3-chloramidopropyl)dimethylammonio]-1-propane sulfonate, N-octyl-beta-D-glucopyranoside, or Nonidet P-40) for solubilization of the antigen from E. coli cell envelopes and the observation that the recombinant protein partitioned into the detergent phase on Triton X-114 solubilization were consistent with the fact that it is a hydrophobic, integral membrane protein. Western blots of the 47-kDa antigen purified by immunoaffinity chromatography supported results of previous reports that the 47-kDa protein is specific to pathogenic treponemes.
authors.affiliation: Department of Microbiology, University of Texas Health Science Center, Dallas 75235.
authors.forename: N R
authors.initials: NR
authors.lastname: Chamberlain
authors.forename: J D
authors.initials: JD
authors.lastname: Radolf
authors.forename: P L
authors.initials: PL
authors.lastname: Hsu
authors.forename: S
authors.initials: S
authors.lastname: Sell
authors.forename: M V
authors.initials: MV
authors.lastname: Norgard
mesh_terms: D000954:Antigens, Surface / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification*; D013045:Species Specificity; D014210:Treponema pallidum / Q000235:genetics / Q000276:immunology* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D000954:Antigens, Surface; D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D017830:Octoxynol; C010615:Nonidet P-40