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N ‐Acetyl lysyl‐tRNA synthetases evolved by a CcdB‐based selection possess N ‐acetyl lysine specificity in vitro and in vivo
Author(s) -
Umehara Takuya,
Kim Jihyo,
Lee Sangsik,
Guo Li-Tao,
Söll Dieter,
Park Hee-Sung
Publication year - 2012
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2012.01.029
Subject(s) - biochemistry , lysine , amino acid , biology , transfer rna , enzyme , acetylation , in vitro , aminoacyl trna synthetase , in vivo , gene , genetic code , chemistry , genetics , rna
Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl‐tRNA synthetase (PylRS) from Methanosarcina mazei to create N ‐acetyl lysyl‐tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published [1,2] AckRSs was tested in vitro and in vivo, and the enzyme‐kinetic properties of the AckRSs were evaluated for the first time.