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J. Biochem, 2001, Vol. 129, No. 1 87-91
© 2001 Japanese Biochemical Society

other

Monoclonal Antibody That Binds to the Central Loop of the Tn10-Encoded Metal Tetracycline/H+ Antiporter of Escherichia coli1

Shigeyuki Nada*,{ddagger}, Satoshi Murakami*,{ddagger}, Shizuka Okamoto*,{dagger}, Yoshiyuki Kubo*,{dagger} and Akihito Yamaguchi*,{dagger},{ddagger},2

*Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka University Ibaraki, Osaka 567-0047
{dagger}Faculty of Pharmaceutical Science, Osaka University Suita, Osaka 565-0871
{ddagger}CREST, Japan Science and Technology Corporation Japan

2To whom correspondence should be addressed at: Institute of Science and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567-0047. Phone: +81-6-6879-8545, Fax: +81-6-6879-8549, E-mail: akihito{at}sanken.osaka-u.acjp

Mouse monoclonal antibodies were prepared using His-tagged Tn10-encoded metal-tet-racycline/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.

1This work was supported by Grants-in-Aid from the Ministry of Education, and the Ministry of Science and Technology of Japan.


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L. M. McMurry, M. L. Aldema-Ramos, and S. B. Levy
Fe2+-Tetracycline-Mediated Cleavage of the Tn10 Tetracycline Efflux Protein TetA Reveals a Substrate Binding Site near Glutamine 225 in Transmembrane Helix 7
J. Bacteriol., September 15, 2002; 184(18): 5113 - 5120.
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