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Journal of Biochemistry Advance Access originally published online on February 19, 2009
Journal of Biochemistry 2009 145(5):693-700; 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. Tel: +81-4-7136-3649, Fax: +81-4-7136-3648, E-mail: shimizu{at}k.u-tokyo.ac.jp

Received January 21, 2009; Accepted February 6, 2009


  Abstract

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 (mAbs). Using this technology, we selected peptides that specifically bind to three mAbs 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 (anti-β-Cat) mAbs, selected peptides had a homology for the partial peptide sequences of β-Cat. Western blot analysis showed that these putative epitopes had affinity for the corresponding mAbs and β-Cat mutants that lack these regions did not bind to the antibodies, indicating we correctly mapped the epitope for these mAbs. The study shown here provides a way for the quick identification of the epitope of mAbs.

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

Abbreviations: β-Cat, β-Catenin; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; pD, protein D; PURE system, protein synthesis using recombinant elements system; SD, Shine-Dalgarno; HRP, horseradish peroxidase; PBST, Phosphate Buffered Saline with Tween 2D


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