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Unintended inhibition of protein function using GFP nanobodies in human cells

View ORCID ProfileCansu Küey, View ORCID ProfileGabrielle Larocque, View ORCID ProfileNicholas I. Clarke, View ORCID ProfileStephen J. Royle
doi: https://doi.org/10.1101/590984
Cansu Küey
1Centre for Mechanochemical Cell Biology, Warwick Medical School, Gibbet Hill Road, Coventry, CV4 7AL, UK
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Gabrielle Larocque
1Centre for Mechanochemical Cell Biology, Warwick Medical School, Gibbet Hill Road, Coventry, CV4 7AL, UK
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Nicholas I. Clarke
1Centre for Mechanochemical Cell Biology, Warwick Medical School, Gibbet Hill Road, Coventry, CV4 7AL, UK
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Stephen J. Royle
1Centre for Mechanochemical Cell Biology, Warwick Medical School, Gibbet Hill Road, Coventry, CV4 7AL, UK
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  • For correspondence: s.j.royle@warwick.ac.uk
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Abstract

Tagging a protein-of-interest with GFP using genome editing is a popular approach to study protein function in cell and developmental biology. To avoid re-engineering cell lines or organisms in order to introduce additional tags, functionalized nanobodies that bind GFP can be used to extend the functionality of the GFP tag. We developed functionalized nanobodies, which we termed “dongles”, that could add, for example, an FKBP tag to a GFP-tagged protein-of-interest; enabling knocksideways experiments in GFP knock-in cell lines. The power of knocksideways is that it allows investigators to rapidly switch the protein from an active to an inactive state. We show that dongles allow for effective knocksideways of GFP-tagged proteins in genome-edited human cells. However, we discovered that nanobody binding to dynamin-2-GFP caused inhibition of dynamin function prior to knocksideways. While this limitation might be specific to the protein studied, it was significant enough to convince us not to pursue development of dongle technology further.

Footnotes

  • - Images in figures where transferrin uptake was quantified are now shown using the same minimum and maximum values per channel, for all images in the figure. - A missing box was added to Figure 1

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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Posted May 30, 2019.
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Unintended inhibition of protein function using GFP nanobodies in human cells
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle
bioRxiv 590984; doi: https://doi.org/10.1101/590984
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Unintended inhibition of protein function using GFP nanobodies in human cells
Cansu Küey, Gabrielle Larocque, Nicholas I. Clarke, Stephen J. Royle
bioRxiv 590984; doi: https://doi.org/10.1101/590984

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