[KSCI] Korea Science Citation Index Service

http://dx.doi.org/10.7845/kjm.2018.8101

Improving amber suppression activity of an orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase and a variant of E. coli initiator tRNA, fMam tRNA_CUA, for the efficient incorporation of unnatural amino acids

Tekalign, Eyob (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Oh, Ju-Eon (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)
Park, Jungchan (Department of Bioscience and Biotechnology, Hankuk University of Foreign Studies)

Publication Information

Korean Journal of Microbiology / v.54, no.4, 2018 , pp. 420-427 More about this Journal

Abstract

The orthogonal pair of Saccharomyces cerevisiae tyrosyl-tRNA synthetase (Sc YRS) and a variant of E. coli initiator tRNA, fMam $tRNA_{CUA}$ which recognizes the amber stop codon is an effective tool for site-specific incorporation of unnatural amino acids into the protein in E. coli. To evolve the amber suppression activity of the orthogonal pair, we generated a mutant library of Sc YRS by randomizing two amino acids at 320 and 321 which involve recognition of the first base of anticodon in fMam $tRNA_{CUA}$ . Two positive clones are selected from the library screening with chloramphenicol resistance mediated by amber suppression. They showed growth resistance against high concentration of chloramphenicol and their $IC_{50}$ values were approximately 1.7~2.3 fold higher than the wild type YRS. In vivo amber suppression assay reveals that mutant YRS-3 (mYRS-3) clone containing amino acid substitutions of P320A and D321A showed 6.5-fold higher activity of amber suppression compared with the wild type. In addition, in vitro aminoacylation kinetics of mYRS-3 also showed approximately 7-fold higher activity than the wild type, and the enhancement was mainly due to the increase of tRNA binding affinity. These results demonstrate that optimization of anticodon recognition by engineered aminoacyl tRNA synthetase improves the efficiency of unnatural amino acid incorporation in response to nonsense codon.

Keywords

amber suppression; aminoacyl-tRNA synthetase; anticodon recognition; genetic code expansion; unnatural amino acid;

Citations & Related Records

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