Use of a series of ompF-ompC chimeric proteins for locating antigenic determinants recognized by monoclonal antibodies against the ompC and ompF proteins of the Escherichia coli outer membrane.
A method is presented for the efficient location of antigenic determinants using a series of chimeric proteins. By means of in vivo homologous recombination between the ompC and ompF genes coding for OmpC and OmpF, homologous proteins of the Escherichia coli outer membrane, a series of ompF-ompC chimeric genes was constructed (Nogami, T., Mizuno, T., & Mizushima, S. (1985) J. Bacteriol. 164, 797-801, and this work). The OmpF-OmpC chimeric proteins expressed by these genes were successfully used to locate antigenic determinants recognized by monoclonal antibodies, which specifically react with either the OmpC or OmpF protein. Interaction between monoclonal antibodies and the chimeric proteins was examined by means of either enzyme-linked immunosorbent assay or immunoblot analysis. The antigenic determinants recognized by three anti-OmpC antibodies and one anti-OmpF antibody were thus located. Finally, the polypeptides covering these regions were chemically synthesized for two of them and then tested as to their reactivity with the antibodies. The peptides reacted with the corresponding antibodies when the former were chemically coupled with bovine serum albumin. Most of the monoclonal antibodies isolated in this work were highly specific to the unfolded monomer of the protein against which the antibody was raised. But they did not react with the trimer, the native form. These results are discussed in relation to the structures and functions of the OmpC and OmpF proteins. The use of a series of monoclonal antibodies for studying the mechanism of protein translocation across the cytoplasmic membrane is also discussed.
D000907:Antibodies, Bacterial / Q000302:isolation & purification; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D002352:Carrier Proteins / Q000032:analysis; D002678:Chimera; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology
D000907:Antibodies, Bacterial; D000911:Antibodies, Monoclonal; D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D000939:Epitopes; D010455:Peptides; D011994:Recombinant Proteins