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

Genetic Code Expansion Method for Temporal Labeling of Endogenously Expressed Proteins.

Author information

1
Microfluidic and Biological Engineering, IMTEK - Department of Microsystems Engineering , University of Freiburg , Georges-Koehler-Allee 103 , 79110 Freiburg , Germany.
2
BIOSS - Centre for Biological Signalling Studies , University of Freiburg , Schänzlestrasse 18 , 79104 Freiburg , Germany.
3
Helmholtz Pioneer Campus , Helmholtz Centrum Munich , Ingolstaedter Landstr. 1 , 85764 Neuherberg , Germany.
4
Faculty of Biology , University of Freiburg , Schänzlestrasse 1 , 79104 Freiburg , Germany.

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.

PMID:
30335949
DOI:
10.1021/acschembio.8b00594

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