Monoclonal antibody that binds to the central loop of the Tn10-encoded metal tetracycline/H+ antiporter of Escherichia coli

被引:1
作者
Nada, S
Murakami, S
Okamoto, S
Kubo, Y
Yamaguchi, A
机构
[1] Osaka Univ, Inst Sci & Ind Res, Dept Cell Membrane Biol, Ibaraki, Osaka 5670047, Japan
[2] Osaka Univ, Fac Pharmaceut Sci, Suita, Osaka 5650871, Japan
[3] Japan Sci & Technol Corp, CREST, Yokohama, Kanagawa, Japan
关键词
drug resistance; epitope; exporter; monoclonal antibody; tetracycline;
D O I
10.1093/oxfordjournals.jbchem.a002840
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 (183)Lys-X-Thr-Arg-Asp-Asn(188) 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.
引用
收藏
页码:87 / 91
页数:5
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