J. Bacteriol., 12 1996, 6685-6692, Vol 178, No. 23
Copyright © 1996, American Society for Microbiology

Recombinant Treponema pallidum rare outer membrane protein 1 (Tromp1) expressed in Escherichia coli has porin activity and surface antigenic exposure

DR Blanco, CI Champion, MM Exner, ES Shang, JT Skare, RE Hancock, JN Miller and MA Lovett
Department of Microbiology and Immunology, School of Medicine, University of California at Los Angeles, 90095, USA. DBLANCO@microimmun.medsch.ucla.edu.

We recently reported the cloning and sequencing of the gene encoding a 31-kDa Treponema pallidum subsp. pallidum rare outer membrane porin protein, designated Tromp1 (D. R. Blanco, C. I. Champion, M. M. Exner, H. Erdjument-Bromage, R. E. W. Hancock, P. Tempst, J. N. Miller, and M. A. Lovett, J. Bacteriol. 177:3556-3562, 1995). Here, we report the stable expression of recombinant Tromp1 (rTromp1) in Escherichia coli. rTromp1 expressed without its signal peptide and containing a 22- residue N-terminal fusion resulted in high-level accumulation of a nonexported soluble protein that was purified to homogeneity by fast protein liquid chromatography (FPLC). Specific antiserum generated to the FPLC-purified rTromp1 fusion identified on immunoblots of T. pallidum the native 31-kDa Tromp1 protein and two higher-molecular-mass oligomeric forms of Tromp1 at 55 and 80 kDa. rTromp1 was also expressed with its native signal peptide by using an inducible T7 promoter. Under these conditions, rTromp1 fractionated predominantly with the E. coli soluble and outer membrane fractions, but not with the inner membrane fraction. rTromp1 isolated from the E. coli outer membrane and reconstituted into planar lipid bilayers showed porin activity based on average single-channel conductances of 0.4 and 0.8 nS in 1 M KCl. Whole- mount immunoelectron microscopy using infection-derived immune serum against T. pallidum indicated that rTromp1 was surface exposed when expressed in E. coli. These findings demonstrate that rTromp1 can be targeted to the E. coli outer membrane, where it has both porin activity and surface antigenic exposure.

This article has been cited by other articles:

• Deka, R. K., Brautigam, C. A., Yang, X. F., Blevins, J. S., Machius, M., Tomchick, D. R., Norgard, M. V. (2006). The PnrA (Tp0319; TmpC) Lipoprotein Represents a New Family of Bacterial Purine Nucleoside Receptor Encoded within an ATP-binding Cassette (ABC)-like Operon in Treponema pallidum. J. Biol. Chem. 281: 8072-8081 [Abstract] [Full Text]  
• Brinkman, M. B., McKevitt, M., McLoughlin, M., Perez, C., Howell, J., Weinstock, G. M., Norris, S. J., Palzkill, T. (2006). Reactivity of Antibodies from Syphilis Patients to a Protein Array Representing the Treponema pallidum Proteome.. J. Clin. Microbiol. 44: 888-891 [Abstract] [Full Text]  
• LaFond, R. E., Lukehart, S. A. (2006). Biological Basis for Syphilis. Clin. Microbiol. Rev. 19: 29-49 [Abstract] [Full Text]  
• McKevitt, M., Brinkman, M. B., McLoughlin, M., Perez, C., Howell, J. K., Weinstock, G. M., Norris, S. J., Palzkill, T. (2005). Genome Scale Identification of Treponema pallidum Antigens. Infect. Immun. 73: 4445-4450 [Abstract] [Full Text]  
• Lee, Y.-H., Dorwart, M. R., Hazlett, K. R. O., Deka, R. K., Norgard, M. V., Radolf, J. D., Hasemann, C. A. (2002). The Crystal Structure of Zn(II)-Free Treponema pallidum TroA, a Periplasmic Metal-Binding Protein, Reveals a Closed Conformation. J. Bacteriol. 184: 2300-2304 [Abstract] [Full Text]  
• Centurion-Lara, A., Godornes, C., Castro, C., Van Voorhis, W. C., Lukehart, S. A. (2000). The tprK Gene Is Heterogeneous among Treponema pallidum Strains and Has Multiple Alleles. Infect. Immun. 68: 824-831 [Abstract] [Full Text]  
• Haake, D. A., Mazel, M. K., McCoy, A. M., Milward, F., Chao, G., Matsunaga, J., Wagar, E. A. (1999). Leptospiral Outer Membrane Proteins OmpL1 and LipL41 Exhibit Synergistic Immunoprotection. Infect. Immun. 67: 6572-6582 [Abstract] [Full Text]  
• Zhang, H. H., Blanco, D. R., Exner, M. M., Shang, E. S., Champion, C. I., Phillips, M. L., Miller, J. N., Lovett, M. A. (1999). Renaturation of Recombinant Treponema pallidum Rare Outer Membrane Protein 1 into a Trimeric, Hydrophobic, and Porin-Active Conformation. J. Bacteriol. 181: 7168-7175 [Abstract] [Full Text]  
• Arroll, T. W., Centurion-Lara, A., Lukehart, S. A., Van Voorhis, W. C. (1999). T-Cell Responses to Treponema pallidum subsp. pallidum Antigens during the Course of Experimental Syphilis Infection. Infect. Immun. 67: 4757-4763 [Abstract] [Full Text]  
• Blanco, D. R., Champion, C. I., Lewinski, M. A., Shang, E. S., Simkins, S. G., Miller, J. N., Lovett, M. A. (1999). Immunization with Treponema pallidum Outer Membrane Vesicles Induces High-Titer Complement-Dependent Treponemicidal Activity and Aggregation of T. pallidum Rare Outer Membrane Proteins (TROMPs). J. Immunol. 163: 2741-2746 [Abstract] [Full Text]  
• Blanco, D. R., Whitelegge, J. P., Miller, J. N., Lovett, M. A. (1999). Demonstration by Mass Spectrometry that Purified Native Treponema pallidum Rare Outer Membrane Protein 1 (Tromp1) Has a Cleaved Signal Peptide. J. Bacteriol. 181: 5094-5098 [Abstract] [Full Text]  
• Deka, R. K., Lee, Y.-H., Hagman, K. E., Shevchenko, D., Lingwood, C. A., Hasemann, C. A., Norgard, M. V., Radolf, J. D. (1999). Physicochemical Evidence that Treponema pallidum TroA Is a Zinc-Containing Metalloprotein That Lacks Porin-Like Structure. J. Bacteriol. 181: 4420-4423 [Abstract] [Full Text]  
• Centurion-Lara, A., Castro, C., Barrett, L., Cameron, C., Mostowfi, M., Van Voorhis, W. C., Lukehart, S. A. (1999). Treponema pallidum Major Sheath Protein Homologue Tpr K Is a Target of Opsonic Antibody and the Protective Immune Response. J. Exp. Med. 189: 647-656 [Abstract] [Full Text]  
• Fraser, C. M., Norris, S. J., Weinstock, G. M., White, O., Sutton, G. G., Dodson, R., Gwinn, M., Hickey, E. K., Clayton, R., Ketchum, K. A., Sodergren, E., Hardham, J. M., McLeod, M. P., Salzberg, S., Peterson, J., Khalak, H., Richardson, D., Howell, J. K., Chidambaram, M., Utterback, T., McDonald, L., Artiach, P., Bowman, C., Cotton, M. D., Fujii, C., Garland, S., Hatch, B., Horst, K., Roberts, K., Sandusky, M., Weidman, J., Smith, H. O., Venter, J. C. (1998). Complete Genome Sequence of Treponema pallidum, the Syphilis Spirochete. Science 281: 375-388 [Abstract] [Full Text]