A widespread approach to study the function of a protein of interest is to tag it with GFP, which involves genome editing to integrate the GFP tag into the DNA. Frequently, a biological question requires further tags and thus re-engineering of the GFP cell line. In their Tools and Resources article, Stephen Royle and colleagues (Kuey et al., 2019) explore the possibility of exploiting the already existing GFP tag to introduce new tags without the need for re-engineering a cell line. To tackle this question, they developed nanobodies they termed ‘dongles’ that have an affinity for GFP through a GFP-binding protein enhancer. The authors demonstrate that these GFP-targeted dongles will bind to any Aequorea victoria GFP (avGFP) derivatives (for example mAzurite, EBFP2, sfGFP, mEmerald and EGFP) and by attaching FKBP domains to dongles, they were able to extend the function of GFP. FKBP domains heterodimerize with FRB domains in the presence of rapamycin, and by overexpressing the FRB-containing Mitotrap and the dongle, they were able to relocalise a GFP-tagged protein to mitochondria. Despite the promising results on the use of dongles being able to retrofit GFP-tagged cell lines for additional functionality, the authors importantly report that some dongles inhibit the function of the tagged protein. The authors warn that this unintended effect could produce potentially misleading results and thus further technical development will be required before nanobodies can be used broadly for assays.
A careful look at GFP nanobodies in cells
A careful look at GFP nanobodies in cells. J Cell Sci 1 November 2019; 132 (21): e2101. doi:
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