Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins

ACS Chem Biol. 2018 Nov 16;13(11):3049-3053. doi: 10.1021/acschembio.8b00594. Epub 2018 Oct 17.

Abstract

We here present a method that combines genetic code expansion with CRISPR/Cas9 genome engineering to label endogenously expressed proteins with high spatiotemporal resolution. The method exploits the use of an orthogonal tRNA/tRNA synthetase pair in conjugation with noncanonical amino acids to create stop codon read through events. To demonstrate the functionality of the method, we pulse labeled endogenous β-actin and tumor protein p53 with a minimally invasive HA tag at their C-termini. Targeting the protein label with a proximity ligation assay plus real time imaging facilitates seamless quantification of the protein synthesis rate and spatial localization at the single cell level. The presented approach does not interfere with any physiological control of cellular expression, nor did we observe any perturbation of endogenous protein functions.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Actins / genetics
  • Actins / metabolism*
  • CRISPR-Cas Systems / genetics*
  • Codon, Terminator*
  • Genetic Engineering / methods
  • HEK293 Cells
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Humans
  • Luminescent Proteins / genetics
  • Lysine / analogs & derivatives
  • Lysine / genetics
  • Lysine-tRNA Ligase / genetics
  • Lysine-tRNA Ligase / metabolism*
  • Methanosarcina barkeri / enzymology
  • Microscopy, Fluorescence
  • Peptide Fragments / genetics
  • Protein Engineering / methods*
  • RNA, Transfer / genetics
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*

Substances

  • Actins
  • Codon, Terminator
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Luminescent Proteins
  • Peptide Fragments
  • Tumor Suppressor Protein p53
  • red fluorescent protein
  • epsilon-tert-butyloxycarbonyl-lysine
  • RNA, Transfer
  • Lysine-tRNA Ligase
  • Lysine