Directed Evolution of Split APEX2 Peroxidase.
IF: 4.634
Cited by: 71


APEX is an engineered peroxidase that catalyzes the oxidation of a wide range of substrates, facilitating its use in a variety of applications from subcellular staining for electron microscopy to proximity biotinylation for spatial proteomics and transcriptomics. To further advance the capabilities of APEX, we used directed evolution to engineer a split APEX tool (sAPEX). A total of 20 rounds of fluorescence activated cell sorting (FACS)-based selections from yeast-displayed fragment libraries, using 3 different surface display configurations, produced a 200-amino-acid N-terminal fragment (with 9 mutations relative to APEX2) called "AP" and a 50-amino-acid C-terminal fragment called "EX". AP and EX fragments were each inactive on their own but were reconstituted to give peroxidase activity when driven together by a molecular interaction. We demonstrate sAPEX reconstitution in the mammalian cytosol, on engineered RNA motifs within a non-coding RNA scaffold, and at mitochondria-endoplasmic reticulum contact sites.


Spatial Proteomics

MeSH terms

Ascorbate Peroxidases
Cell Separation
Directed Molecular Evolution
Endoplasmic Reticulum
Flow Cytometry
HEK293 Cells
Peptide Library
Plant Proteins
Saccharomyces cerevisiae


Han, Yisu
Branon, Tess Caroline
Martell, Jeffrey D
Boassa, Daniela
Shechner, David
Ellisman, Mark H
Ting, Alice

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