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.
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.
authors.affiliation: RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Kiga
authors.forename: Kensaku
authors.initials: K
authors.lastname: Sakamoto
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kodama
authors.forename: Takanori
authors.initials: T
authors.lastname: Kigawa
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Matsuda
authors.forename: Takashi
authors.initials: T
authors.lastname: Yabuki
authors.forename: Mikako
authors.initials: M
authors.lastname: Shirouzu
authors.forename: Yoko
authors.initials: Y
authors.lastname: Harada
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Nakayama
authors.forename: Koji
authors.initials: K
authors.lastname: Takio
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hasegawa
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Ichiro
authors.initials: I
authors.lastname: Hirao
authors.forename: Shigeyuki
authors.initials: S
authors.lastname: Yokoyama
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000378:metabolism*; D002474:Cell-Free System; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D014644:Genetic Variation; D007700:Kinetics; D007470:Monoiodotyrosine / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000378:metabolism; D012639:Seeds / Q000378:metabolism; D014908:Triticum / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D014448:Tyrosine-tRNA Ligase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
chemical_list: D000596:Amino Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase; D007470:Monoiodotyrosine