Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi

W. S. Probert, K. M. Allsup, Rance B Lefebvre

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Abstract

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. burgdorferi.

Original languageEnglish (US)
Pages (from-to)1933-1939
Number of pages7
JournalInfection and Immunity
Volume63
Issue number5
StatePublished - 1995

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Borrelia burgdorferi
Amino Acid Sequence
Proteins
Organism Cloning
Membrane Proteins
Borrelia
Endopeptidase K
Spirochaetales
Protein Databases
Butanols
Pulsed Field Gel Electrophoresis
Surface Antigens
Sequence Homology
Southern Blotting
Genes
Electrophoresis
Gels
Monoclonal Antibodies
Antibodies
DNA

ASJC Scopus subject areas

  • Immunology

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Identification and characterization of a surface-exposed, 66-kilodalton protein from Borrelia burgdorferi. / Probert, W. S.; Allsup, K. M.; Lefebvre, Rance B.

In: Infection and Immunity, Vol. 63, No. 5, 1995, p. 1933-1939.

Research output: Contribution to journalArticle

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