An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system
10.1073/pnas.142220099
Crossref journal-article
Proceedings of the National Academy of Sciences
Proceedings of the National Academy of Sciences (341)
Abstract

Tyrosyl–tRNA synthetase (TyrRS) from Escherichia coli was engineered to preferentially recognize 3-iodo- l -tyrosine rather than l -tyrosine for the site-specific incorporation of 3-iodo- l -tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo- l -tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of Bacillus stearothermophilus TyrRS, three residues, Y37, Q179, and Q195, in the l -tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by in vitro biochemical assays. A combination of the Y37V and Q195C mutations changed the amino acid specificity in such a way that the variant TyrRS activates 3-iodo- l -tyrosine 10-fold more efficiently than l -tyrosine. This engineered enzyme, TyrRS(V37C195), was tested for use in the wheat germ cell-free translation system, which has recently been significantly improved, and is now as productive as conventional recombinant systems. During the translation in the wheat germ system, an E. coli suppressor tRNA Tyr was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the E. coli TyrRS(V37C195) variant with 3-iodo- l -tyrosine. After the use of the 3-iodotyrosyl–tRNA in translation, the resultant uncharged tRNA could be aminoacylated again in the system. A mass spectrometric analysis of the produced protein revealed that more than 95% of the amino acids incorporated for an amber codon were iodotyrosine, whose concentration was only twice that of l -tyrosine in the translation. Therefore, the variant enzyme, 3-iodo- l -tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic in vitro translation systems.

Bibliography

Kiga, D., Sakamoto, K., Kodama, K., Kigawa, T., Matsuda, T., Yabuki, T., Shirouzu, M., Harada, Y., Nakayama, H., Takio, K., Hasegawa, Y., Endo, Y., Hirao, I., & Yokoyama, S. (2002). An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system. Proceedings of the National Academy of Sciences, 99(15), 9715–9720.

Authors 14
  1. Daisuke Kiga (first)
  2. Kensaku Sakamoto (additional)
  3. Koichiro Kodama (additional)
  4. Takanori Kigawa (additional)
  5. Takayoshi Matsuda (additional)
  6. Takashi Yabuki (additional)
  7. Mikako Shirouzu (additional)
  8. Yoko Harada (additional)
  9. Hiroshi Nakayama (additional)
  10. Koji Takio (additional)
  11. Yoshinori Hasegawa (additional)
  12. Yaeta Endo (additional)
  13. Ichiro Hirao (additional)
  14. Shigeyuki Yokoyama (additional)
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Dates
Type When
Created 23 years, 1 month ago (July 28, 2002, 6:26 p.m.)
Deposited 3 years, 4 months ago (April 13, 2022, 12:13 a.m.)
Indexed 1 month, 4 weeks ago (July 7, 2025, 8:04 a.m.)
Issued 23 years, 2 months ago (July 3, 2002)
Published 23 years, 2 months ago (July 3, 2002)
Published Online 23 years, 2 months ago (July 3, 2002)
Published Print 23 years, 1 month ago (July 23, 2002)
Funders 0

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@article{Kiga_2002, title={An engineered Escherichia coli tyrosyl–tRNA synthetase for site-specific incorporation of an unnatural amino acid into proteins in eukaryotic translation and its application in a wheat germ cell-free system}, volume={99}, ISSN={1091-6490}, url={http://dx.doi.org/10.1073/pnas.142220099}, DOI={10.1073/pnas.142220099}, number={15}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Kiga, Daisuke and Sakamoto, Kensaku and Kodama, Koichiro and Kigawa, Takanori and Matsuda, Takayoshi and Yabuki, Takashi and Shirouzu, Mikako and Harada, Yoko and Nakayama, Hiroshi and Takio, Koji and Hasegawa, Yoshinori and Endo, Yaeta and Hirao, Ichiro and Yokoyama, Shigeyuki}, year={2002}, month=jul, pages={9715–9720} }