%0 Journal Article
%T Directed Evolution of Split APEX2 Peroxidase
%J -
%D 2019
%R https://doi.org/10.1021/acschembio.8b00919
%X High Resolution Image Download MS PowerPoint Slide 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 ˇ°APˇ± and a 50-amino-acid C-terminal fragment called ˇ°EXˇ±. 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¨Cendoplasmic reticulum contact sites
%U https://pubs.acs.org/doi/10.1021/acschembio.8b00919