Nucleic Acids Research, 2002, Vol. 30, No. 21 4692-4699
© 2002 Oxford University Press
Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells
1 Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, 2 Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), 4-1-8 Hou-cho, Kawaguchi-shi, Saitama 332-0012, Japan, 3 RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan and 4 Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
*To whom correspondence should be addressed at Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel: +81 3 5841 4395; Fax: +81 3 5841 8057; Email: yokoyama{at}biochem.s.u-tokyo.ac.jp
Present addresses:
Akiko Hayashi, Laboratory of Public Health, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Akiko Soma, Department of Chemistry, College of Science, Rikkyo (St Pauls) University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
Ichiro Hirao, Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan
A suppressor tRNATyr and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNATyr gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNATyr, which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNATyr, while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.
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