Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi.
The surface-exposed antigens of Borrelia burgdorferi represent important targets for the development of a protective immune response. We have identified a proteinase K-accessible, 66-kDa protein from B. burgdorferi and have demonstrated that at least a portion of this protein is surface exposed. The 66-kDa protein was purified by sequential extraction of spirochetes with butanol and Triton X-114 followed by preparative gel electrophoresis. Polyclonal antibodies developed against the purified 66-kDa protein were Borrelia spp. specific, whereas a monoclonal antibody, Route 66, displayed a genospecies-specific pattern of recognition for the 66-kDa protein. N-terminal amino acid sequence was obtained from an internal fragment, a truncated version, and the full-length form of the 66-kDa protein. A search of protein and gene databases for homologous sequences yielded a match with the predicted amino acid sequence from a segment of B. burgdorferi chromosomal DNA (P. A. Rosa, D. Hogan, and T. G. Schwan, J. Clin. Microbiol. 29:524-532, 1991). The construction of primers based on this DNA sequence and the N-terminal amino acid sequence allowed the amplification and cloning of the 66-kDa-protein gene. The identity of the cloned gene was verified by the recognition of the expressed gene product by Route 66. Pulsed-field gel electrophoresis and Southern blot analysis were performed to confirm the chromosomal location of the 66-kDa-protein gene. This study describes the identification and cloning of the first chromosomally encoded, surface-exposed protein from B. burgdoferi.
Probert
W S
WS
Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis 95616, USA.
Allsup
K M
KM
LeFebvre
R B
RB
D000595:Amino Acid Sequence; D000907:Antibodies, Bacterial / Q000276:immunology; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D015748:Borrelia burgdorferi Group / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D002876:Chromosomes, Bacterial / Q000235:genetics; D017124:Conserved Sequence; D019286:Endopeptidase K; D005798:Genes, Bacterial / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
D000907:Antibodies, Bacterial; D000954:Antigens, Surface; D008565:Membrane Proteins; D012697:Serine Endopeptidases; D019286:Endopeptidase K