期刊
ACS CHEMICAL BIOLOGY
卷 14, 期 4, 页码 619-635出版社
AMER CHEMICAL SOC
DOI: 10.1021/acschembio.8b00919
关键词
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资金
- NIH [R01-CA186568, R01-GM086197, P41-GM103412]
- HHMI/Jane Coffin Childs Fellowship
- Dow Graduate Research fellowship
- Lester Wolfe fellowship
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.
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