Related Experiment Video
Updated: Apr 10, 2026

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening
Published on: April 1, 2016
Directed evolution of APOX for proximity labeling using phenols with high redox potentials
Sifei Fang1, Leslie D Acevedo1, Alexander J Solivais1
1Department of Chemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
Abstract:
Proximity labeling (PL) can identify endogenous proteins in specific subcellular regions. APEX2 (enhanced ascorbate peroxidase 2) enables PL with high temporal resolution by oxidizing biotin phenol (BP) into a radical that promiscuously tags nearby proteins, followed by the isolation and identification of the tagged biomolecules. However, the BP radical has a relatively large diffusion radius, limiting spatial resolution. Replacing the phenol in BP with nitrophenol (NP) could shorten the labeling radius by shortening the radical lifetime, but existing PL heme peroxidases cannot efficiently oxidize substrates with high redox potentials. We report the ultrahigh-throughput directed evolution of APOX (APEX with high redOX potential), a quadruple mutant of APEX2 with a higher reduction potential. Using APOX with a membrane-permeable alkyne-NP probe, we demonstrate PL in living mammalian cells, including proteomic mapping with excellent subcellular compartment specificity.
Related Concept Videos
Oxidation of Phenols to Quinones
o-hydroxy phenols are oxidized to o-quinones and p-hydroxy phenols to p-quinones. Such redox reactions involve the transfer of two electrons and two protons. The reversible redox...
Redox Reactions
Redox Reactions
Oxidation of Alkenes: Anti Dihydroxylation with Peroxy Acids
Vicinal Diols via Reductive Coupling of Aldehydes or Ketones: Pinacol Coupling Overview
Labeling DNA Probes
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...

