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Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum.
Title | Molecular cloning and DNA sequence analysis of the 37-kilodalton endoflagellar sheath protein gene of Treponema pallidum. |
Publication Type | Journal Article |
Year of Publication | 1989 |
Authors | Isaacs, RD, Hanke, JH, Guzman-Verduzco, LM, Newport, G, Agabian, N, Norgard, MV, Lukehart, SA, Radolf, JD |
Journal | Infect Immun |
Volume | 57 |
Issue | 11 |
Pagination | 3403-11 |
Date Published | 1989 Nov |
ISSN | 0019-9567 |
Keywords | Amino Acid Sequence, Antibodies, Monoclonal, Antigens, Bacterial, Bacterial Proteins, Base Sequence, Cloning, Molecular, DNA, Bacterial, Electrophoresis, Gel, Two-Dimensional, Flagella, Genes, Bacterial, Molecular Sequence Data, Recombinant Fusion Proteins, Regulatory Sequences, Nucleic Acid, Restriction Mapping, Solubility, Treponema pallidum |
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 lambda gt11 clone (designated lambda A34) 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 lambda A34 provided a probe with which a chimeric plasmid (pR14) encoding all but the nine N-terminal amino acids of 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 pR14 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. |
Alternate Journal | Infect. Immun. |
PubMed ID | 2680972 |
PubMed Central ID | PMC259836 |
Grant List | AI-16692 / AI / NIAID NIH HHS / United States AI-17366 / AI / NIAID NIH HHS / United States AI-26756 / AI / NIAID NIH HHS / United States |