Published online 20 March 2006
Methods Online |
In vitro selection of tRNAs for efficient four-base decoding to incorporate non-natural amino acids into proteins in an Escherichia coli cell-free translation system
1 Department of Bioscience and Bioengineering, Okayama University Tsushimanaka, Okayama 700-8530, Japan 2 School of Materials Science, Japan Advanced Institute of Science and Technology 1-1 Asahidai, Nomi, Ishikawa 923-1292, Japan 3 PRESTO, Japan Science and Technology Agency 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
*To whom correspondence should be addressed. Tel: +81 761 51 1681; Fax: +81 761 51 1683; Email: hohsaka{at}jaist.ac.jp
Received September 7, 2005. Revised September 26, 2005. Accepted March 7, 2006.
Position-specific incorporation of non-natural amino acids into proteins is a useful technique in protein engineering. In this study, we established a novel selection system to obtain tRNAs that show high decoding activity, from a tRNA library in a cell-free translation system to improve the efficiency of incorporation of non-natural amino acids into proteins. In this system, a puromycin–tRNA conjugate, in which the 3'-terminal A unit was replaced by puromycin, was used. The puromycin–tRNA conjugate was fused to a C-terminus of streptavidin through the puromycin moiety in the ribosome. The streptavidin–puromycin–tRNA fusion molecule was collected and brought to the next round after amplification of the tRNA sequence. We applied this system to select efficient frameshift suppressor tRNAs from a tRNA library with a randomly mutated anticodon loop derived from yeast 
. After three rounds of the selection, we obtained novel frameshift suppressor tRNAs which had high decoding activity and good orthogonality against endogenous aminoacyl-tRNA synthetases. These results demonstrate that the in vitro selection system developed here is useful to obtain highly active tRNAs for the incorporation of non-natural amino acid from a tRNA library.