Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells

Kensaku Sakamoto, Akiko Hayashi, Ayako Sakamoto, Daisuke Kiga, Hiroshi Nakayama, Akiko Soma, Takatsugu Kobayashi, Makoto Kitabatake, Koji Takio, Kazuki Saito, Mikako Shirouzu, Ichiro Hirao, Shigeyuki Yokoyama

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176 Citations (Scopus)

Abstract

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-lodo-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.

Original languageEnglish
Pages (from-to)4692-4699
Number of pages8
JournalNucleic acids research
Volume30
Issue number21
DOIs
Publication statusPublished - 2002 Nov 1
Externally publishedYes

ASJC Scopus subject areas

  • Genetics

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    Sakamoto, K., Hayashi, A., Sakamoto, A., Kiga, D., Nakayama, H., Soma, A., Kobayashi, T., Kitabatake, M., Takio, K., Saito, K., Shirouzu, M., Hirao, I., & Yokoyama, S. (2002). Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells. Nucleic acids research, 30(21), 4692-4699. https://doi.org/10.1093/nar/gkf589