Single-Plasmid-Based System for Efficient Noncanonical Amino Acid Mutagenesis in Cultured Mammalian Cells

Chembiochem. 2016 Jun 2;17(11):1008-11. doi: 10.1002/cbic.201500681. Epub 2016 Apr 27.

Abstract

We describe a new expression system for efficient non-canonical amino acid mutagenesis in cultured mammalian cells by using the pyrrolysine tRNA synthetase/tRNACUA (Pyl) pair. A significant improvement in the incorporation of non-canonical amino acids into proteins was obtained by combining all the required genetic components into a single and compact vector that can be efficiently delivered to different mammalian cell lines by conventional transfection reagents.

Keywords: amber suppression; genetic code expansion; protein engineering; protein expression; pyrrolysine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Amino Acids / metabolism*
  • Amino Acyl-tRNA Synthetases / metabolism
  • Animals
  • Genetic Code
  • HCT116 Cells
  • HEK293 Cells
  • Humans
  • Lysine / analogs & derivatives
  • Lysine / metabolism
  • Mice
  • Mutagenesis
  • NIH 3T3 Cells
  • Plasmids / genetics
  • Plasmids / metabolism*
  • Protein Engineering
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism

Substances

  • Amino Acids
  • Tumor Suppressor Protein p53
  • Amino Acyl-tRNA Synthetases
  • pyrrolysine
  • Lysine