Directed evolution of split APEX peroxidase

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 spatially restricted proteomics and transcriptomics. While this strategy has provided access to many cellular regions and organelles, there are still many compartments and structures that cannot be accessed; this strategy is limited by the specificity of genetic targeting; there are cellular regions that cannot be exclusively targeted by a single genetic tag (Chapter 1). To further advance the capabilities of APEX and address the need for an interaction-dependent proximity labeling tool, this thesis describes the development of a split APEX2 system. Short enzymatic reconstitution times are also desired, to further ensures both organelles' morphological integrity. Thus, it is critical that split APEX2 reconstitute peroxidase activity both rapidly and robustly. We first performed two subsequent rounds of structure-guided screening to determine the most optimal cut site (Chapter 2). We then used directed evolution on the top candidate pair to engineer a split APEX tool (sAPEX). Selections were performed via FACS on yeast-displayed fragment libraries, and 20 rounds of evolution produced a 200-amino acid Nterminal fragment (with 9 mutations relative to APEX2) called "AP" and a 50-amino acid Cterminal 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 (Chapter 3). Our resulting split APEX2 fragment pair has significantly diverged from its parental sequence and shows interaction-dependent reconstitution in multiple contexts in living mammalian cells (Chapter 4). Our split APEX tool adds to the proximity labeling toolkit (Chapter 5 and 6), and in the future, should extend the utility of APEX-based approaches to new areas of biology at higher spatiotemporal resolution.