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bioRxiv · 10.1101/452888

Directed evolution of split APEX peroxidase

Han, Y.·Martell, J.·Branon, T.·Boassa, D.·Shechner, D.·Ellisman, M.·Ting, A.
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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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Han, Y., Martell, J., Branon, T., Boassa, D., Shechner, D., Ellisman, M., Ting, A.. 2018-10-25. Directed evolution of split APEX peroxidase. https://doi.org/10.1101/452888

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