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Nat Methods. 2015 Jan;12(1):51-4. doi: 10.1038/nmeth.3179. Epub 2014 Nov 24.

Directed evolution of APEX2 for electron microscopy and proximity labeling.

Author information

1
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.
2
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA.
3
National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA.
4
1] National Center for Microscopy and Imaging Research, University of California at San Diego, La Jolla, California, USA. [2] Department of Neurosciences, University of California at San Diego, La Jolla, California, USA.

Abstract

APEX is an engineered peroxidase that functions as an electron microscopy tag and a promiscuous labeling enzyme for live-cell proteomics. Because limited sensitivity precludes applications requiring low APEX expression, we used yeast-display evolution to improve its catalytic efficiency. APEX2 is far more active in cells, enabling the use of electron microscopy to resolve the submitochondrial localization of calcium uptake regulatory protein MICU1. APEX2 also permits superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins.

PMID:
25419960
PMCID:
PMC4296904
DOI:
10.1038/nmeth.3179
[Indexed for MEDLINE]
Free PMC Article

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