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KMID : 0364820180540040420
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Korean Journal of Microbiology
2018 Volume.54 No. 4 p.420 ~ p.427
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Improving amber suppression activity of an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase and a variant of E. coli initiator tRNA, fMam tRNACUA, for the efficient incorporation of unnatural amino acids
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Tekalign Eyob
Oh Ju-Eon
Park Jung-Chan
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Abstract
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The orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNAsynthetase (Sc YRS) and a variant of E. coli initiator tRNA,fMam tRNACUA which recognizes the amber stop codon is aneffective tool for site-specific incorporation of unnatural aminoacids into the protein in E. coli. To evolve the amber suppressionactivity of the orthogonal pair, we generated a mutant library ofSc YRS by randomizing two amino acids at 320 and 321 whichinvolve recognition of the first base of anticodon in fMamtRNACUA. Two positive clones are selected from the libraryscreening with chloramphenicol resistance mediated by ambersuppression. They showed growth resistance against highconcentration of chloramphenicol and their IC50 values wereapproximately 1.7~2.3 fold higher than the wild type YRS. In vivoamber suppression assay reveals that mutant YRS-3 (mYRS-3)clone containing amino acid substitutions of P320A and D321Ashowed 6.5-fold higher activity of amber suppression comparedwith the wild type. In addition, in vitro aminoacylation kineticsof mYRS-3 also showed approximately 7-fold higher activitythan the wild type, and the enhancement was mainly due to theincrease of tRNA binding affinity. These results demonstratethat optimization of anticodon recognition by engineeredaminoacyl tRNA synthetase improves the efficiency of unnaturalamino acid incorporation in response to nonsense codon.
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KEYWORD
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amber suppression, aminoacyl-tRNA synthetase, anticodon recognition, genetic code expansion, unnatural amino acid
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