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

FEBS Lett. 2012 Mar 23;586(6):729-33. doi: 10.1016/j.febslet.2012.01.029. Epub 2012 Jan 28.

Abstract

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 AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Directed Molecular Evolution*
  • Lysine / chemistry
  • Lysine / metabolism*
  • Lysine-tRNA Ligase / chemistry
  • Lysine-tRNA Ligase / genetics
  • Lysine-tRNA Ligase / metabolism*
  • Methanosarcina / genetics
  • Methanosarcina / metabolism
  • Protein Processing, Post-Translational
  • RNA, Transfer, Amino Acyl / chemistry
  • RNA, Transfer, Amino Acyl / genetics
  • RNA, Transfer, Amino Acyl / metabolism*
  • Substrate Specificity

Substances

  • Bacterial Proteins
  • CcdB protein, Plasmid F
  • RNA, Transfer, Amino Acyl
  • Lysine-tRNA Ligase
  • Lysine