Sequence analysis and recombinant expression of a 28-kilodalton Treponema pallidum subsp. pallidum rare outer membrane protein (Tromp2)
| Autor: | Cheryl I. Champion, R. E. W. Hancock, H Erdjument-Bromage, David R. Blanco, Michael A. Lovett, M. M. Exner, P Tempst, James N. Miller |
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| Rok vydání: | 1997 |
| Předmět: |
Signal peptide
Vesicle-associated membrane protein 8 Lipid Bilayers Molecular Sequence Data Biology Microbiology Protein Structure Secondary Epitopes Escherichia coli Amino Acid Sequence Treponema pallidum Cloning Molecular Microscopy Immunoelectron Molecular Biology Integral membrane protein Peptide sequence Signal peptidase Antigens Bacterial Base Sequence Cell Membrane Sequence Analysis DNA Molecular biology Transmembrane protein Recombinant Proteins Molecular Weight Biochemistry Membrane protein Solubility Genes Bacterial Antigens Surface Bacterial outer membrane Bacterial Outer Membrane Proteins Research Article |
| Zdroj: | Journal of bacteriology. 179(4) |
| ISSN: | 0021-9193 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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