Oct 25, 2018

Directed evolution of split APEX peroxidase

BioRxiv : the Preprint Server for Biology
Yisu HanAlice Y Ting

Abstract

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). Twenty rounds of FACS-based selections from yeast-displayed fragment libraries, using three different yeast 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 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 telomerase noncoding RNA, and at mitochondria-endoplasmic reticulum contact sites.

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Mentioned in this Paper

Directed Evolution
Carboxy-Terminal Amino Acid
Protein Biotinylation
RNA, Untranslated
Spatial Distribution
Fluorescence-Activated Cell Sorting
Peroxidases
Yeasts
Peroxidase Activity
Selenium and Vitamin E Efficacy Trial

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