{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,25]],"date-time":"2026-09-25T17:53:09Z","timestamp":1790358789679,"version":"4.1.0"},"reference-count":45,"publisher":"National Academy of Sciences","issue":"15","content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2002,7,23]]},"abstract":"<jats:p>\n                    Tyrosyl\u2013tRNA synthetase (TyrRS) from\n                    <jats:italic>Escherichia coli<\/jats:italic>\n                    was engineered to preferentially recognize 3-iodo-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine rather than\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine for the site-specific incorporation of 3-iodo-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine into proteins in eukaryotic translation systems. The wild-type TyrRS does not recognize 3-iodo-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine, because of the bulky iodine substitution. On the basis of the reported crystal structure of\n                    <jats:italic>Bacillus stearothermophilus<\/jats:italic>\n                    TyrRS, three residues, Y37, Q179, and Q195, in the\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine-binding site were chosen for mutagenesis. Thirty-four single amino acid replacements and 16 of their combinations were screened by\n                    <jats:italic>in vitro<\/jats:italic>\n                    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-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine 10-fold more efficiently than\n                    <jats:sc>l<\/jats:sc>\n                    -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\n                    <jats:italic>E. coli<\/jats:italic>\n                    suppressor tRNA\n                    <jats:sup>Tyr<\/jats:sup>\n                    was not aminoacylated by the wheat germ enzymes, but was aminoacylated by the\n                    <jats:italic>E. coli<\/jats:italic>\n                    TyrRS(V37C195) variant with 3-iodo-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine. After the use of the 3-iodotyrosyl\u2013tRNA 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\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine in the translation. Therefore, the variant enzyme, 3-iodo-\n                    <jats:sc>l<\/jats:sc>\n                    -tyrosine, and the suppressor tRNA can serve as an additional set orthogonal to the 20 endogenous sets in eukaryotic\n                    <jats:italic>in vitro<\/jats:italic>\n                    translation systems.\n                  <\/jats:p>","DOI":"10.1073\/pnas.142220099","type":"journal-article","created":{"date-parts":[[2002,7,28]],"date-time":"2002-07-28T18:26:28Z","timestamp":1027880788000},"page":"9715-9720","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":156,"title":["An engineered\n                    <i>Escherichia coli<\/i>\n                    tyrosyl\u2013tRNA 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"],"prefix":"10.1073","volume":"99","author":[{"given":"Daisuke","family":"Kiga","sequence":"first","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kensaku","family":"Sakamoto","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koichiro","family":"Kodama","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takanori","family":"Kigawa","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takayoshi","family":"Matsuda","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Takashi","family":"Yabuki","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mikako","family":"Shirouzu","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoko","family":"Harada","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Nakayama","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koji","family":"Takio","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yoshinori","family":"Hasegawa","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology 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and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ichiro","family":"Hirao","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology Corporation c\/o RIKEN, Hirosawa, Wako-shi, Saitama 351-0198, Japan; Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Biomolecular Characterization Division, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan; and..."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shigeyuki","family":"Yokoyama","sequence":"additional","affiliation":[{"name":"RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan; Yokoyama CytoLogic Project, Exploratory Research for Advanced Technology (Japan), Japan Science and Technology 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