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Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer
C I Champion1, D R Blanco, M M Exner
1Department of Microbiology and Immunology, University of California at Los Angeles, 90095, USA.
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.
Insights
Researchers cloned and expressed the Treponema pallidum rare outer membrane protein (Tromp2). This integral membrane protein is crucial for understanding syphilis pathogenesis and potential diagnostics.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- Treponema pallidum subsp. pallidum is the causative agent of syphilis.
- Outer membrane proteins (OMPs) are crucial for bacterial interactions and potential vaccine targets.
- Identifying novel OMPs in T. pallidum is essential for understanding its biology.
Purpose of the Study:
- To clone, sequence, and express the gene for a rare outer membrane protein (TROMP) of T. pallidum, designated Tromp2.
- To characterize the localization and membrane association of Tromp2.
- To identify Tromp2 as a potential integral membrane protein.
Main Methods:
- Gene cloning, sequencing, and recombinant protein expression in E. coli.
- Biochemical fractionation (Triton X-114 extraction, alkali/high-salt treatment) of T. pallidum.
- Immunological detection using antiserum against recombinant Tromp2 (rTromp2).
- Immunoelectron microscopy for surface localization.
Main Results:
- The tromp2 gene encodes a 242-amino acid precursor protein with a signal peptide.
- Mature Tromp2 (218 amino acids) is a 28-kDa protein with a predicted pI of 7.3 and nine transmembrane beta-sheets.
- Recombinant Tromp2 expressed in E. coli localized to the outer membrane.
- Native Tromp2 was detected in the detergent phase after Triton X-114 extraction and identified as an integral membrane protein.
Conclusions:
- Tromp2 is a novel, integral, membrane-spanning outer membrane protein of T. pallidum.
- This finding expands the known repertoire of T. pallidum outer membrane proteins.
- Tromp2 represents a potential target for future diagnostic or therapeutic strategies against syphilis.