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Journal of Biochemistry Advance Access published online on February 19, 2009
Journal of Biochemistry, doi:10.1093/jb/mvp027
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© The authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Epitope mapping using ribosome display in a reconstituted cell-free protein synthesis system

Eriko Osada, Yoshihiro Shimizu*, Bintang K. Akbar, Takashi Kanamori and Takuya Ueda

The Department of Medical Genome Sciences, Graduate School of Frontier Sciences, the University of Tokyo, FSB-401, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan

*To whom correspondence should be addressed: Yoshihiro Shimizu: Tel: +81(0)4-7136-3649, Fax: +81(0)4-7136-3648, E-mail: shimizu{at}k.u-tokyo.ac.jp

Received January 21, 2009; Accepted February 6, 2009


  Abstract

Summary Ribosome display is a powerful technology for selecting ligand-binding peptides or proteins. We demonstrate here that the ribosome display using the reconstituted cell-free protein synthesis system can be applied for the epitope mapping of monoclonal antibodies. Using this technology, we selected peptides that specifically bind to three monoclonal antibodies from random peptide library. When selection was performed against the anti-FLAG M2 antibody, selected peptides contained previously characterized consensus epitope, indicating that the methodology can be applied for the epitope mapping. When the selection was carried out against two anti-β-Catenin monoclonal antibodies, selected peptides had a homology for the partial peptide sequences of β-Catenin. Western blot analysis showed that these putative epitopes had affinity for the corresponding monoclonal antibodies and β-Catenin mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these monoclonal antibodies. The study shown here provides a way for the quick identification of the epitope of monoclonal antibodies

Key Words: ribosome display, epitope mapping, antibody, cell-free protein synthesis system, in vitro selection


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