A bilayer cell-free protein synthesis system for high-throughput screening of gene products

Sawasaki, Tatsuya; Hasegawa, Yoshinori; Tsuchimochi, Masateru; Kamura, Nami; Ogasawara, Tomio; Kuroita, Toshihiro; Endo, Yaeta

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Volume 514, Issue 1 , Pages 102-105, 6 March 2002

A bilayer cell-free protein synthesis system for high-throughput screening of gene products

Edited by Lev Kisselev

Tatsuya Sawasaki
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
Yoshinori Hasegawa
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
Masateru Tsuchimochi
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
Nami Kamura
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
Tomio Ogasawara
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
Toshihiro Kuroita
- Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., Toyo-cho, Tsuruga 914-0047, Japan
Yaeta Endo
- Department of Applied Chemistry, Faculty of Engineering, The Venture Business Laboratory, Ehime University, Matsuyama 790-8577, Japan
- Corresponding author. Fax: (81)-89-927 9941

Received 12 November 2001; accepted 14 November 2001.

Abstract

A high-throughput cell-free protein synthesis method has been described. The methodology is based on a bilayer diffusion system that enables the continuous supply of substrates, together with the continuous removal of small byproducts, through a phase between the translation mixture and substrate mixture. With the use of a multititer plate the system was functional for a prolonged time, and as a consequence yielded more than 10 times that of the similar batch-mode reaction. Combining this method with a wheat germ cell-free translation system developed by us, the system could produce a large amount of protein sufficient for carrying out functional analyses. This novel bilayer-based cell-free protein synthesis system with its simplicity, minimum time and low cost may be useful practical methodology in the post-genome era.

Keywords: Cell-free protein synthesis, Bilayer reaction method, Escherichia coli coupled transcription–translation, Wheat germ system, High-throughput screening

Abbreviations: CBB, Coomassie brilliant blue, FAD, flavin adenine dinucleotide, GFP, green fluorescent protein, KOD, archaeon Pyrococcus sp. strain KOD1 DNA polymerase, ER, estrogen receptor protein