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Unintended perturbation of protein function using GFP nanobodies in human cells

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Küey, Cansu, Larocque, Gabrielle, Clarke, Nicholas I. and Royle, Stephen J. (2019) Unintended perturbation of protein function using GFP nanobodies in human cells. Journal of Cell Science, 132 (21). jcs234955. doi:10.1242/jcs.234955

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Official URL: http://dx.doi.org/10.1242/jcs.234955

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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. The function of GFP-tagged tumor protein D54 (TPD54, also known as TPD52L2) in anterograde traffic was also perturbed by dongles. While these issues potentially limit the application of dongles, we discuss strategies for their deployment as cell biological tools.

Item Type: Journal Article
Subjects: Q Science > QH Natural history
Q Science > QP Physiology
Divisions: Faculty of Medicine > Warwick Medical School > Biomedical Sciences > Cell & Developmental Biology
Faculty of Medicine > Warwick Medical School > Biomedical Sciences
Faculty of Medicine > Warwick Medical School
SWORD Depositor: Library Publications Router
Library of Congress Subject Headings (LCSH): Green fluorescent protein, Cell membranes , Endocytosis, Cytology
Journal or Publication Title: Journal of Cell Science
Publisher: The Company of Biologists
ISSN: 1477-9137
Official Date: 10 October 2019
Dates:
DateEvent
10 October 2019Published
6 November 2019Available
3 October 2019Accepted
Volume: 132
Number: 21
Article Number: jcs234955
DOI: 10.1242/jcs.234955
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
MR/P018947/1[MRC] Medical Research Councilhttp://dx.doi.org/10.13039/501100000265
UNSPECIFIEDUniversity of Warwickhttp://dx.doi.org/10.13039/501100000741
UNSPECIFIED[EPSRC] Engineering and Physical Sciences Research Councilhttp://dx.doi.org/10.13039/501100000266
EP/L016494/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
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