Journal
DEVELOPMENTAL CELL
Volume 35, Issue 4, Pages 513-525Publisher
CELL PRESS
DOI: 10.1016/j.devcel.2015.10.016
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Funding
- ACRF
- National Health and Medical Research Council of Australia [APP1045092, APP1037320, APP1058565, APP1041929]
- Australian Research Council (ARC) Centre of Excellence in Convergent Bio-Nano Science and Technology
- ARC [DP120103930]
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Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines.
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