Abstract
Antibodies induced by native antigens often crossreact with denatured antigen or with peptides that contain a segment of the antigen polypeptide chain. The epitopes recognized by these antibodies are linear or sequential, i.e., specified by the linear sequence of the amino acid residues rather than by the protein conformation. Presumably, linear epitopes correspond to flexible parts of the surface of the antigen (1). These epitopes can be localized by synthesis of peptides and testing their antigenicity, i.e., the binding by antibodies. However, this is not practical for antigens of several hundreds of residues. In this chapter, we describe a convenient alternative, the prokaryotic expression of fragments of the coding sequence. In most systems, these fragments are expressed as part of a fusion protein, i.e., coupled to a bacterial polypeptide, which serves as a convenient carrier or provides a tag for affinity purification.
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Van Regenmortel, M. H. V. (1989) Antigenic cross-reactivity between proteins and peptides: new insights and applications. Trends Biochem. Sci. 12, 237–240.
Lenstra, J. A., Kusters, J. G., and Van der Zeijst, B. A. M. (1990) Mapping of viral epitopes with prokaryotic expression systems (review). Arch. Viral 110, 1–24.
Nuijten, P. J., van der Zeijst, B. A. M., and Newell, D. G. (1991) Localization of immunogenic regions on the flagellin proteins of Cumpylobacter jejuni 81116. Infect. Immun. 59, 1100–1105.
Van Vliet, A. H. M., JongeJan, F., van Kleef, M., and van der Zeijst, B. A. M (1994) Molecular cloning, sequence analysis, and expression of the gene encoding the immunodominant 32-kilodalton protein of Cowdria ruminantium. Infect Immun. 62, 1451–1456.
Stanley, K. K. and Luzio, J. P. (1984) Construction of a new family of high efficiency bacterial expression vectors: identification of cDNA clones coding from human liver proteins. EMBO J. 3, 1429–1434.
Kusters, J. G., Jager, E. J., and Van der Zeijst, B. A. M. (1989) Improvement of the cloning linker of the bacterial expression vector pEX Nucleic Acids Res 17, 8007.
Smith, D. B. and Johnson, K. S. (1988) Single-step purification of polypeptides expressed in Escherichia coli. Gene 29, 263–269.
The world of Pharmacia Biotech’ 95-96, catalogue of Pharmacia Biotech.
Qtaexpress booklet (1992) of Diagen GmbH, Qiagen Inc.
Kusters, J. G., Jager, E. J., Lenstra, J. A., Koch., G., Posthumus, W. P. A., Meloen, R. H., and Van der Zeijst, B. A. M. (1989) Analysis of an immunodominant region in the peplomer protein of avian coronavirus infectious bronchitis virus. J. Immunol. 143, 2692–2698.
Lenstra, J. A., Kusters, J. G., Koch, G., and Van der Zeijst, B. A. M. (1988) Antigenicity of the peplomer protein of infectious bronchitis virus. Mol Immunol. 26, 7–15.
Lenstra, J. A., Erkens, J. H. F., Langeveld, J. P. M., Posthumus, W. P. A., Meloen, R. H., Gebauer, F., Correa, I., Enjuanes, L., and Stanley, K. K. (1992) Isolation of sequences from a random-sequence expression library that mimic viral epitopes. J. Immunol. Methods 152, 149–157.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Stanley, K. K. (1983) Solubilization and immune-detection of β-galactosidase hybrid proteins carrying foreign antigenic determinants. Nucleic Acids Res 11, 4077–4092.
Stanley, K. K. (1988). Expression screening of cDNA libraries in pEX. Methods Mol. Biol. 4, 329–342.
Van Vliet, A. H. M., Van der Zeijst, B. A. M., Camus, E., Mahan, S. M., Martinez, D., and Jongejan, F. (1995) Use of a specific immunogenic region on the Cowdria ruminantium MAP1 protein in a serological assay. J. Clin Microbiol. 33, 2405–2410.
Costa, G. L., Grafsky, A., and Weiner, M. P. (1994) Cloning and analysis of PCR-generated DNA fragments. PCR Methods Applic. 3, 338–345.
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Lenstra, J.A., Van Vliet, A.H.M. (1996). Epitope Mapping by Expression of Restriction Enzyme or PCR Fragments in Bacterial Plasmids. In: Morris, G.E. (eds) Epitope Mapping Protocols. Methods in Molecular Biology™, vol 66. Humana Press. https://doi.org/10.1385/0-89603-375-9:287
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DOI: https://doi.org/10.1385/0-89603-375-9:287
Publisher Name: Humana Press
Print ISBN: 978-0-89603-375-7
Online ISBN: 978-1-59259-552-5
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