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Dev Cell. 2015 Nov 23;35(4):513-25. doi: 10.1016/j.devcel.2015.10.016. Epub 2015 Nov 12.

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

Author information

1
Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia.
2
Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia.
3
Institute for Molecular Bioscience, University of Queensland, QLD 4072, Australia; Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia. Electronic address: r.parton@imb.uq.edu.au.

Abstract

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.

PMID:
26585296
DOI:
10.1016/j.devcel.2015.10.016
[Indexed for MEDLINE]
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