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


RESULTS
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.


CONCLUSIONS
This technique should greatly speed the discovery of functional fusion proteins, genetically encodable sensors, and optimized fluorescence resonance energy transfer pairs.
Sheridan
Douglas L
DL
Department of Cellular and Molecular Physiology, Yale University Medical School, 330 Cedar St, New Haven, CT 06520, USA. Douglas.Sheridan@yale.edu
Berlot
Catherine H
CH
Robert
Antoine
A
Inglis
Fiona M
FM
Jakobsdottir
Klara B
KB
Howe
James R
JR
Hughes
Thomas E
TE
D002460:Cell Line; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics*; D016254:Mutagenesis, Insertional / Q000379:methods*; D017510:Protein Folding; D021122:Protein Subunits / Q000235:genetics; D018091:Receptors, AMPA / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D014162:Transfection
D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D008164:Luminescent Proteins; D021122:Protein Subunits; D018091:Receptors, AMPA; D011993:Recombinant Fusion Proteins