Site-specific incorporation of an unnatural amino acid into proteins in mammalian cells.
A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter.
Sakamoto
Kensaku
K
Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Hayashi
Akiko
A
Sakamoto
Ayako
A
Kiga
Daisuke
D
Nakayama
Hiroshi
H
Soma
Akiko
A
Kobayashi
Takatsugu
T
Kitabatake
Makoto
M
Takio
Koji
K
Saito
Kazuki
K
Shirouzu
Mikako
M
Hirao
Ichiro
I
Yokoyama
Shigeyuki
S
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D003062:Codon / Q000235:genetics; D006224:Cricetinae; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D005798:Genes, Bacterial / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics; D016153:Genes, Suppressor; D001411:Geobacillus stearothermophilus / Q000235:genetics; D006801:Humans; D008322:Mammals; D013058:Mass Spectrometry; D007470:Monoiodotyrosine / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D012365:RNA, Transfer, Tyr / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics; D013752:Tetracycline / Q000494:pharmacology; D014448:Tyrosine-tRNA Ligase / Q000235:genetics / Q000378:metabolism
D003062:Codon; D011506:Proteins; D012365:RNA, Transfer, Tyr; D014448:Tyrosine-tRNA Ligase; D013752:Tetracycline; D007470:Monoiodotyrosine