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

Daisuke Kiga, Kensaku Sakamoto, Koichiro Kodama, Takanori Kigawa, Takayoshi Matsuda, Takashi Yabuki, Mikako Shirouzu, Yoko Harada, Hiroshi Nakayama, Koji Takio, Yoshinori Hasegawa, Yaeta Endo, Ichiro Hirao, Shigeyuki Yokoyama

Research output: Contribution to journalArticle

135 Citations (Scopus)

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 eukaryrotic 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 TyrR5 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 was 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 tRNATyr 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 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.

Original languageEnglish
Pages (from-to)9715-9720
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number15
DOIs
Publication statusPublished - 2002 Jul 23
Externally publishedYes

Fingerprint

Tyrosine-tRNA Ligase
Cell-Free System
Germ Cells
Triticum
Escherichia coli
Amino Acids
Tyrosine
Transfer RNA
Proteins
Enzymes
RNA, Transfer, Tyr
Monoiodotyrosine
Geobacillus stearothermophilus
Terminator Codon
Mutagenesis
Iodine
3-iodotyrosine
Binding Sites
Mutation

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

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. / Kiga, Daisuke; Sakamoto, Kensaku; Kodama, Koichiro; Kigawa, Takanori; Matsuda, Takayoshi; Yabuki, Takashi; Shirouzu, Mikako; Harada, Yoko; Nakayama, Hiroshi; Takio, Koji; Hasegawa, Yoshinori; Endo, Yaeta; Hirao, Ichiro; Yokoyama, Shigeyuki.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 99, No. 15, 23.07.2002, p. 9715-9720.

Research output: Contribution to journalArticle

Kiga, Daisuke ; Sakamoto, Kensaku ; Kodama, Koichiro ; Kigawa, Takanori ; Matsuda, Takayoshi ; Yabuki, Takashi ; Shirouzu, Mikako ; Harada, Yoko ; Nakayama, Hiroshi ; Takio, Koji ; Hasegawa, Yoshinori ; Endo, Yaeta ; Hirao, Ichiro ; Yokoyama, Shigeyuki. / 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. In: Proceedings of the National Academy of Sciences of the United States of America. 2002 ; Vol. 99, No. 15. pp. 9715-9720.
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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 eukaryrotic 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 TyrR5 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 was 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 tRNATyr 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 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.",
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AU - Kigawa, Takanori

AU - Matsuda, Takayoshi

AU - Yabuki, Takashi

AU - Shirouzu, Mikako

AU - Harada, Yoko

AU - Nakayama, Hiroshi

AU - Takio, Koji

AU - Hasegawa, Yoshinori

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