Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum

R. D. Isaacs, J. H. Hanke, L. M. Guzman-Verduzco, G. Newport, N. Agabian, M. V. Norgard, S. A. Lukehart, J. D. Radolf

Research output: Contribution to journalArticle

27 Scopus citations

Abstract

We have used a combination of nucleotide and N-terminal-amino-acid-sequence analyses to determine the primary structure of the 37-kilodalton (kDa) endoflagellar outer layer, or sheath, protein. Initially, a λgt11 clone (designated λA34) expressing a portion of the 37-kDa protein was selected from a Treponema pallidum genomic library with a murine monoclonal antibody (H9-2) directed against an epitope of the 37-kDa protein. The insert from λA34 provided a probe with which a chimeric plasmid (pRI4) encoding all but the nine N-terminal amino acids from the entire protein was selected from a T. pallidum (pBR322) genomic library. The nine N-terminal amino acids determined by amino acid sequencing were combined with the DNA sequence encoded by pRI4 to determine the primary structure of the entire 37-kDa protein; the combined sequence made up a polypeptide with a calculated molecular mass of 36,948 Da. Approximately one-third of the deduced sequence was confirmed by N-terminal amino acid analysis of tryptic peptides from the purified 37-kDa protein. Repeated attempts to clone upstream portions of the gene (flaA) by using a variety of strategies were unsuccessful, suggesting that unregulated expression of the intact sheath protein or of its most amino-terminal portions is toxic in Escherichia coli. These studies should provide the basis for further molecular investigations of the endoflagellar apparatus and of treponemal motility.

Original languageEnglish (US)
Pages (from-to)3403-3411
Number of pages9
JournalInfection and immunity
Volume57
Issue number11
StatePublished - 1989

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Infectious Diseases

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