This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Qi, H L Articles by Blake, M S Search for Related Content PubMed PubMed Citation Articles by Qi, H L Articles by Blake, M S

research-article

Expression of large amounts of neisserial porin proteins in Escherichia coli and refolding of the proteins into native trimers.

Laboratory of Bacterial Pathogenesis and Immunology, Rockefeller University, New York 10021.

ABSTRACT

Porins from different neisserial strains and species have been shown to have differences in both primary amino acid sequence and biophysical characteristics as observed by functional assays. A closer examination of how the changes in the primary amino acid sequence of Neisseria porin molecules correlate with these observed biophysical changes has been impeded by the inability to easily manipulate the cloned porin genes by modern molecular techniques and then obtain enough of the expressed modified porin protein to purify and use in these biophysical functional assays. In this report, we describe a method by which the genes encoding three different porin proteins, lacking their neisserial promoter and signal sequences, were cloned into an expression plasmid and transformed into Escherichia coli. Upon induction, large amounts of the porin proteins were produced. The expressed porin proteins were then manipulated to regenerate their native trimer structure and purified by standard protein chemistry. Sufficient purified recombinant porin protein was obtained for further antigenic as well as biophysical characterization. This sets the stage for the biophysical characterization of these neisserial porin proteins in more detail.

This article has been cited by other articles:

• Olesky, M., Zhao, S., Rosenberg, R. L., Nicholas, R. A. (2006). Porin-Mediated Antibiotic Resistance in Neisseria gonorrhoeae: Ion, Solute, and Antibiotic Permeation through PIB Proteins with penB Mutations.. J. Bacteriol. 188: 2300-2308 [Abstract] [Full Text]  
• Leduc, I., Richards, P., Davis, C., Schilling, B., Elkins, C. (2004). A Novel Lectin, DltA, Is Required for Expression of a Full Serum Resistance Phenotype in Haemophilus ducreyi. Infect. Immun. 72: 3418-3428 [Abstract] [Full Text]  
• Van Voorhis, W. C., Barrett, L. K., Lukehart, S. A., Schmidt, B., Schriefer, M., Cameron, C. E. (2003). Serodiagnosis of Syphilis: Antibodies to Recombinant Tp0453, Tp92, and Gpd Proteins Are Sensitive and Specific Indicators of Infection by Treponema pallidum. J. Clin. Microbiol. 41: 3668-3674 [Abstract] [Full Text]  
• Cameron, C. E. (2003). Identification of a Treponema pallidum Laminin-Binding Protein. Infect. Immun. 71: 2525-2533 [Abstract] [Full Text]  
• Paquet, J.-Y., Diaz, M. A., Genevrois, S., Grayon, M., Verger, J.-M., De Bolle, X., Lakey, J. H., Letesson, J.-J., Cloeckaert, A. (2001). Molecular, Antigenic, and Functional Analyses of Omp2b Porin Size Variants of Brucella spp.. J. Bacteriol. 183: 4839-4847 [Abstract] [Full Text]  
• Thomas, K. L., Leduc, I., Olsen, B., Thomas, C. E., Cameron, D. W., Elkins, C. (2001). Cloning, Overexpression, Purification, and Immunobiology of an 85-Kilodalton Outer Membrane Protein from Haemophilus ducreyi. Infect. Immun. 69: 4438-4446 [Abstract] [Full Text]  
• Jolley, K. A., Appleby, L., Wright, J. C., Christodoulides, M., Heckels, J. E. (2001). Immunization with Recombinant Opc Outer Membrane Protein from Neisseria meningitidis: Influence of Sequence Variation and Levels of Expression on the Bactericidal Immune Response against Meningococci. Infect. Immun. 69: 3809-3816 [Abstract] [Full Text]  
• Turner, P. C., Thomas, C. E., Stojiljkovic, I., Elkins, C., Kizel, G., Ala’Aldeen, D. A. A., Sparling, P. F. (2001). Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences. Microbiology 147: 1277-1290 [Abstract] [Full Text]  
• Elkins, C., Yi, K., Olsen, B., Thomas, C., Thomas, K., Morse, S. (2000). Development of a Serological Test for Haemophilus ducreyi for Seroprevalence Studies. J. Clin. Microbiol. 38: 1520-1526 [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]  
• Minetti, C. A. S. A., Blake, M. S., Remeta, D. P. (1998). Characterization of the Structure, Function, and Conformational Stability of PorB Class 3 Protein from Neisseria meningitidis. A PORIN WITH UNUSUAL PHYSICOCHEMICAL PROPERTIES. J. Biol. Chem. 273: 25329-25338 [Abstract] [Full Text]  
• Ram, S., McQuillen, D. P., Gulati, S., Elkins, C., Pangburn, M. K., Rice, P. A. (1998). Binding of Complement Factor H to Loop 5 of Porin Protein 1A: A Molecular Mechanism of Serum Resistance of Nonsialylated Neisseria gonorrhoeae. J. Exp. Med. 188: 671-680 [Abstract] [Full Text]  
• Minetti, C. A.S.A., Tai, J. Y., Blake, M. S., Pullen, J. K., Liang, S.-M., Remeta, D. P. (1997). Structural and Functional Characterization of a Recombinant PorB Class 2Protein from Neisseria meningitidis. CONFORMATIONAL STABILITY AND PORIN ACTIVITY. J. Biol. Chem. 272: 10710-10720 [Abstract] [Full Text]  
• Flachmann, R., Kuhlbrandt, W. (1996). Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants. Proc. Natl. Acad. Sci. USA 93: 14966-14971 [Abstract] [Full Text]