title: Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli.
abstract: Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tetracycline/H+ antiporter [TetA(B)His] as an antigen. From them, those reacting equally with His-tagged and wild-type TetA(B) were selected and named TCL-1. Cysteine-scanning mutants were used to determine the TCL-1 binding site on the TetA(B) protein. First, 12 Cys mutants of TetA(B) in which one residue in a protruding loop region was replaced by cysteine were constructed. Western blot analysis revealed the binding of TCL-1 to all of these Cys-mutants except for R186C. Then, we constructed 13 cysteine-scanning mutants, F179C to T191C. Among them, eight mutants, F179C to T182C, N184C, and T189C to T191C, exhibited TCL-1 binding, whereas the other five, K183C, T185C, R186C, D187C, and N188C, exhibited no or lower TCL-1 binding. These results clearly indicate that the sequence recognized by TCL-1 is 183Lys-X-Thr-Arg-Asp-Asn188 in the central loop region of TetA(B). TCL-1 is the first reported antibody that binds to a region other than the C-terminus of TetA(B), and the recognized amino acid sequence was identified.
authors.affiliation: Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University, Ibaraki, Osaka 567-0047, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Nada
authors.forename: S
authors.initials: S
authors.lastname: Murakami
authors.forename: S
authors.initials: S
authors.lastname: Okamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Kubo
authors.forename: A
authors.initials: A
authors.lastname: Yamaguchi
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D017920:Antiporters / Q000737:chemistry / Q000276:immunology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology*; D001666:Binding Sites, Antibody; D003545:Cysteine / Q000737:chemistry; D000939:Epitopes; D004926:Escherichia coli / Q000276:immunology*; D006639:Histidine / Q000737:chemistry; D008969:Molecular Sequence Data; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; D017920:Antiporters; D001426:Bacterial Proteins; D000939:Epitopes; C079051:tetA protein, Bacteria; D006639:Histidine; D003545:Cysteine