N-Acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo

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. (C) 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.