Cited by 510Ubiquitin and proteasome pathwaysAdvanced Fluorescence Microscopy TechniquesRNA Research and Splicing

Fluorescent fusion protein knockout mediated by anti-GFP nanobody

Caussinus, Emmanuel, Kanca, Oguz, Affolter, Markus·2012·Nature Structural & Molecular Biology

Abstract

The use of genetic mutations to study protein functions in vivo is a central paradigm of modern biology. Recent advances in reverse genetics such as RNA interference and morpholinos are widely used to further apply this paradigm. Nevertheless, such systems act upstream of the proteic level, and protein depletion depends on the turnover rate of the existing target proteins. Here we present deGradFP, a genetically encoded method for direct and fast depletion of target green fluorescent protein (GFP) fusions in any eukaryotic genetic system. This method is universal because it relies on an evolutionarily highly conserved eukaryotic function, the ubiquitin pathway. It is traceable, because the GFP tag can be used to monitor the protein knockout. In many cases, it is a ready-to-use solution, as GFP protein-trap stock collections are being generated in Drosophila melanogaster and in Danio rerio.

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Crossref · 10.1038/nsmb.2180

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  • deGradFP, a fusion of an anti-GFP nanobody with the F-box domain from Drosophila melanogaster Slmb, efficiently degrades GFP-tagged proteins in D. melanogaster.

    “One such approach, named deGradFP, utilized a fusion of an anti-GFP nanobody with the F-box domain from the Drosophila melanogaster Slmb protein, which is a substrate receptor component of the SCF E3 ligase complex, and demonstrated efficient degradation of GFP-tagged proteins in D. melanogaster (Fig. 3b) [50, 51].”

    From Advances in targeted degradation of endogenous proteins (2019) · cites this paper as [51]

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Authors
Caussinus, E., Kanca, O., & Affolter, M.
Affiliations
University of Basel (CH)
Corresponding author
  • Caussinus, Emmanuel
  • Affolter, Markus
Venue
Nature Structural & Molecular Biology
Published
2011-12-11
Volume
19
Issue
1
Pages
117-121
ISSN
  • 1545-9985 (ISSN-L)
  • 1545-9993
OpenAlex
W1995701587
Cited by
510
References
39
Citations by year
2026: 27 · 2025: 46 · 2024: 44 · 2023: 34 · 2022: 50 · 2021: 40 · 2020: 45 · 2019: 47 · 2018: 35 · 2017: 40 · 2016: 31 · 2015: 25 · 2014: 24 · 2013: 17 · 2012: 5
FWCI
5.98
Type
journal article
Language
en
Open access
closed
Locations
  • Nature Structural & Molecular Biology (publishedVersion)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Ubiquitin and proteasome pathways
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
Keywords
anti-GFP nanobody, nanobodies, F-box domain, ubiquitin pathway, protein degradation, reverse genetics, Drosophila melanogaster, zebrafish
MeSH terms
Amino Acid Sequence, Animals, Antibodies, Base Sequence, Cytoskeletal Proteins, Drosophila melanogaster, Embryo, Nonmammalian, HeLa Cells, Histones, Humans, Luminescent Proteins, Models, Biological, Molecular Sequence Data, Recombinant Fusion Proteins, Wings, Animal, Blotting, Western, Homeodomain Proteins, Microscopy, Confocal, Drosophila Proteins, Animals, Genetically Modified, Green Fluorescent Proteins, POU Domain Factors, Gene Knockout Techniques, Proteolysis

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