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Journal of Biochemistry Advance Access published online on December 5, 2006
Journal of Biochemistry, doi:10.1093/jb/mvm003
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© 2006 The Japanese Biochemical Society

High-Throughput Fluorescence Labeling of Full-Length cDNA Products based on a Reconstituted Translation System

Yuko Kawahashi1,2, Nobuhide Doi1, Yo Oishi1, Chizuru Tsuda1, Hideaki Takashima1, Tomoya Baba3, Hirotada Mori3, Takashi Ito4 and Hiroshi Yanagawa1,*

1Department of Biosciences and Informatics, Keio University, Yokohama 223-8522; 2Tsukuba Technical Center, Ikedarika Scientific Corporation, Tsukuba 305-0062; 3Graduate School of Biological Sciences, Nara Institute of Science and Technology, Nara 636-0101; 4Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa 277-8583

* To whom correspondence should be addressed. Tel: +81-45-566-1775, Fax: +81-45-566-1440, E-mail: hyana{at}bio.keio.ac.jp


  Abstract

Although recent advances in fluorescence-based technologies, such as protein microarrays, have made it possible to analyze more than 10,000 proteins at once, there is a bottleneck in the step of preparation of large numbers of fluorescently-labeled proteins for the comprehensive analysis of protein-protein interactions. Here we describe two independent methods for high-throughput fluorescence-labeling of full-length cDNA products at their C-termini using a reconstituted translation system containing fluorescent puromycin. For the first method, release factor-free systems were used. For the second method, stop codons were excluded from cDNAs by using a common mismatch primer in mutagenic PCR. These methods yielded large numbers of labeled proteins from cDNA sets of various organisms, such as mouse, yeast and Escherichia coli.

Key Words: cell-free protein synthesis, fluorescence labeling, proteomics, puromycin, release factor


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