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.
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.
Kiga
Daisuke
D
RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.
Sakamoto
Kensaku
K
Kodama
Koichiro
K
Kigawa
Takanori
T
Matsuda
Takayoshi
T
Yabuki
Takashi
T
Shirouzu
Mikako
M
Harada
Yoko
Y
Nakayama
Hiroshi
H
Takio
Koji
K
Hasegawa
Yoshinori
Y
Endo
Yaeta
Y
Hirao
Ichiro
I
Yokoyama
Shigeyuki
S
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
D000596:Amino Acids; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D014443:Tyrosine; D014448:Tyrosine-tRNA Ligase; D007470:Monoiodotyrosine