A new way to rapidly create functional, fluorescent fusion proteins: random insertion of GFP with an in vitrotransposition reaction

Douglas L Sheridan, Catherine H Berlot, Antoine Robert, Fiona M Inglis, Klara B Jakobsdottir, James R Howe, Thomas E Hughes

The jellyfish green fluorescent protein (GFP) can be inserted into the middle of another protein to produce a functional, fluorescent fusion protein. Finding permissive sites for insertion, however, can be difficult. Here we describe a transposon-based approach for rapidly creating libraries of GFP fusion proteins. We tested our approach on the glutamate receptor subunit, GluR1, and the G protein subunit, alphas. All of the in-frame GFP insertions produced a fluorescent protein, consistent with the idea that GFP will fold and form a fluorophore when inserted into virtually any domain of another protein. Some of the proteins retained their signaling function, and the random nature of the transposition process revealed permissive sites for insertion that would not have been predicted on the basis of structural or functional models of how that protein works. This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.