title: Systematic cloning of Treponema pallidum open reading frames for protein expression and antigen discovery.
abstract: A topoisomerase-based method was used to clone PCR products encoding 991 of the 1041 open reading frames identified in the genome sequence of the bacterium that causes syphilis, Treponema pallidum subsp. pallidum. Cloning the open reading frames into the univector plasmid system permitted the rapid conversion of the original clone set to other functional vectors containing a variety of promoters or tag sequences. A computational prediction of signal sequences identified 248 T. pallidum proteins that are potentially secreted from the cell. These clones were systematically converted into vectors designed to express the encoded proteins as glutathione-S-transferase fusion proteins. To test the potential of the clone set for novel antigen discovery, 85 of these fusion proteins were expressed from Escherichia coli, partially purified, and tested for antigenicity by using sera from rabbits infected with T. pallidum. Twelve of the 85 proteins bound significant levels of antibody. Of these 12 proteins, seven had previously been identified as T. pallidum antigens, and the remaining five represent novel antigens. These results demonstrate the potential of the T. pallidum clone set for antigen discovery and, more generally, for advancing the biology of this enigmatic spirochete.
authors.affiliation: Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: McKevitt
authors.forename: Krupa
authors.initials: K
authors.lastname: Patel
authors.forename: David
authors.initials: D
authors.lastname: Smajs
authors.forename: Michael
authors.initials: M
authors.lastname: Marsh
authors.forename: Melanie
authors.initials: M
authors.lastname: McLoughlin
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Norris
authors.forename: George M
authors.initials: GM
authors.lastname: Weinstock
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D000942:Antigens, Bacterial / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D005822:Genetic Vectors / Q000096:biosynthesis / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014210:Treponema pallidum / Q000235:genetics* / Q000276:immunology*
chemical_list: D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005982:Glutathione Transferase