Nucleic Acids Research Advance Access originally published online on October 24, 2006
Nucleic Acids Research 2006 34(20):e140; doi:10.1093/nar/gkl771
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Nucleic Acids Research, 2006, Vol. 34, No. 20 e140
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Methods Online |
Solid-phase translation and RNA–protein fusion: a novel approach for folding quality control and direct immobilization of proteins using anchored mRNA
1 Rational Evolutionary Design of Advanced Biomolecules, Saitama Small Enterprise Promotion Corporation SKIP city, 3-12-18 Kamiaoki, Kawaguchi, Saitama 333-0844, Japan 2 Department of Functional Materials Science, Saitama University 255 Shimo-Okubo, Sakura-ku, Saitama-shi, Saitama 338-8570, Japan 3 Innovation center for start-ups, National Institute of Advanced Industrial Science and Technology 2-2-2, Marunouchi, Chiyoda-ku, Tokyo, Japan
*To whom correspondence should be addressed at Janusys Corporation, #655, Saitama Industrial Technology Center, SKIP City, 3-12-18, Kami-Aoki. Kawaguchi City, Saitama 338-0824, Japan. Tel: +81 48 262 1247; Fax: +81 48 262 1248; Email: nemoto{at}janusys.co.jp
*Correspondence may also be addressed to Manish Biyani. Tel: +81 48 263 0738; Fax: +81 48 263 0938; Email: biyanijp{at}yahoo.co.in
Received July 26, 2006. Revised September 21, 2006. Accepted September 28, 2006.
A novel cell-free translation system is described in which template-mRNA molecules were captured onto solid surfaces to simultaneously synthesize and immobilize proteins in a more native-state form. This technology comprises a novel solid-phase approach to cell-free translation and RNA–protein fusion techniques. A newly constructed biotinylated linker-DNA which enables puromycin-assisted RNA–protein fusion is ligated to the 3' ends of the mRNA molecules to attach the mRNA-template on a streptavidin-coated surface and further to enable the subsequent reactions of translation and RNA–protein fusion on surface. The protein products are therefore directly immobilized onto solid surfaces and furthermore were discovered to adopt a more native state with proper protein folding and superior biological activity compared with conventional liquid-phase approaches. We further validate this approach via the production of immobilized green fluorescent protein (GFP) on microbeads and by the production and assay of aldehyde reductase (ALR) enzyme with 4-fold or more activity. The approach developed in this study may enable to embrace the concept of the transformation of ‘RNA chip-to-protein chip’ using a solid-phase cell-free translation system and thus to the development of high-throughput microarray platform in the field of functional genomics and in vitro evolution.