Nature Biotechnology
20, 177 - 182 (2002)
doi:10.1038/nbt0202-177
An unnatural base pair for incorporating amino acid analogs into proteinsIchiro Hirao1, 2, Takashi Ohtsuki3, Tsuyoshi Fujiwara1, Tsuneo Mitsui1, 2, Tomoko Yokogawa1, Taeko Okuni1, Hiroshi Nakayama4, Koji Takio4, Takashi Yabuki3, Takanori Kigawa3, Koichiro Kodama3, 5, Takashi Yokogawa6, Kazuya Nishikawa6
& Shigeyuki Yokoyama1, 2, 3, 51
Yokoyama CytoLogic Project, ERATO, JST, c/o RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. 2
Current address: Protein Preparation/NMR Facilities, Genomic Sciences Center, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. 3
Genomic Sciences Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa 230-0045, Japan. 4
Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan. 5
Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. 6
Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagito, Gifu 501-1193, Japan.
Correspondence should be addressed to Shigeyuki Yokoyama yokoyama@biochem.s.u-tokyo.ac.jp or Ichiro Hirao ihirao@postman.riken.go.jpAn unnatural base pair of 2-amino-6-(2-thienyl)purine (denoted by s) and pyridin-2-one (denoted by y) was developed to expand the genetic code. The ribonucleoside triphosphate of y was site-specifically incorporated into RNA, opposite s in a template, by T7 RNA polymerase. This transcription was coupled with translation in an Escherichia coli cell-free system. The yAG codon in the transcribed ras mRNA was recognized by the CUs anticodon of a yeast tyrosine transfer RNA (tRNA) variant, which had been enzymatically aminoacylated with an unnatural amino acid, 3-chlorotyrosine. Site-specific incorporation of 3-chlorotyrosine into the Ras protein was demonstrated by liquid chromatography−mass spectrometry (LC-MS) analysis of the products. This coupled transcription−translation system will permit the efficient synthesis of proteins with a tyrosine analog at the desired position.
|
