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Nucleic Acids Research, 2003, Vol. 31, No. 15 e78
© 2003 Oxford University Press

Highly stable and efficient mRNA templates for mRNA–protein fusions and C-terminally labeled proteins

Etsuko Miyamoto-Sato, Hideaki Takashima, Shinichiro Fuse, Kaori Sue, Masamichi Ishizaka, Seiji Tateyama, Kenichi Horisawa, Tatsuya Sawasaki1, Yaeta Endo1 and Hiroshi Yanagawa*

Department of Biosciences and Informatics, Faculty of Science and Technology, Keio University, 3-14-1, Hiyoshi, Kohoku-ku, Yokohama 223-8522, Japan and 1 Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama 790-8577, Japan

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

For high-throughput in vitro protein selection using genotype (mRNA)–phenotype (protein) fusion formation and C-terminal protein labeling as a post-selection analysis, it is important to improve the stability and efficiency of mRNA templates for both technologies. Here we describe an efficient single-strand ligation (90% of the input mRNAs) using a fluorescein-conjugated polyethylene glycol puromycin (Fluor-PEG Puro) spacer. This ligation provides a stable c-jun mRNA with a flexible Fluor-PEG Puro spacer for efficient fusion formation (70% of the input mRNA with the PEG spacer) in a cell-free wheat germ translation system. When using a 5' untranslated region including SP6 promoter and {Omega}29 enhancer (a part of tobacco mosaic virus {Omega}), an A8 sequence (eight consecutive adenylate residues) at the 3' end is suitable for fusion formation, while an XA8 sequence (XhoI and the A8 sequence) is suitable for C-terminal protein labeling. Further, we report that Fluor-PEG N-t-butyloxycarbonylpuromycin [Puro(Boc)] spacer enhances the stability and efficiency of c-jun mRNA template for C-terminal protein labeling. These mRNA templates should be useful for puromycin-based technologies (fusion formation and C-terminal protein labeling) to facilitate high-throughput in vitro protein selection for not only evolutionary protein engineering, but also proteome exploration.


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