Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2) - PubMed (original) (raw)

Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2)

C I Champion et al. J Bacteriol. 1997 Feb.

Abstract

In this study, we report the cloning, sequencing, and expression of the gene encoding a 28-kDa Treponema pallidum subsp. pallidum rare outer membrane protein (TROMP), designated Tromp2. The tromp2 gene encodes a precursor protein of 242 amino acids including a putative signal peptide of 24 amino acids ending in a type I signal peptidase cleavage site of Leu-Ala-Ala. The mature protein of 218 amino acids has a calculated molecular weight of 24,759 and a calculated pI of 7.3. The predicted secondary structure of Tromp2 shows nine transmembrane segments of amphipathic beta-sheets typical of outer membrane proteins. Recombinant Tromp2 (rTromp2) was expressed with its native signal peptide, using a tightly regulated T7 RNA polymerase expression vector. Under high-level expression conditions, rTromp2 fractionated exclusively with the Escherichia coli outer membrane. Antiserum raised against rTromp2 was generated and used to identify native Tromp2 in cellular fractionations. Following Triton X-114 extraction and phase separation of T. pallidum, the 28-kDa Tromp2 protein was detected prominently in the detergent phase. Alkali and high-salt treatment of purified outer membrane from T. pallidum, conditions which remove peripherally associated membrane proteins, demonstrated that Tromp2 is an integral membrane protein. Whole-mount immunoelectron microscopy of E. coli cells expressing rTromp2 showed specific surface antibody binding. These findings demonstrate that Tromp2 is a membrane-spanning outer membrane protein, the second such protein to be identified for T. pallidum.

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References

    1. J Bacteriol. 1988 Dec;170(12):5789-96 -PubMed
    1. Nucleic Acids Res. 1988 Mar 11;16(5):1829-36 -PubMed
    1. J Bacteriol. 1989 Sep;171(9):5005-11 -PubMed
    1. J Immunol. 1990 Mar 1;144(5):1914-21 -PubMed
    1. Infect Immun. 1990 Jun;58(6):1697-704 -PubMed

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