Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction.
A new baculovirus-based fluorescence resonance energy transfer (Bv-FRET) assay for measuring multimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone receptor (GnRH-R) was capable of forming oligomeric complexes in the plasma membrane under normal physiological conditions. The mouse gonadotropin-releasing hormone receptor GnRH-R was used to evaluate the efficiency and potential applications of this assay. Two chimeric constructs of GnRH-R were made, one with green fluorescent protein as a donor fluorophore and the other with enhanced yellow fluorescent protein as an acceptor fluorophore. These chimeric constructs were coexpressed in an insect cell line (BTI Tn5 B1-4) using recombinant baculoviruses. Energy transfer occurred from the excited donor to the acceptor when they were in close proximity. The association of GnRH-R was demonstrated through FRET and the fluorescence observed using a Leica TSC-SPII confocal microscope. FRET was enhanced by the addition of a GnRH agonist but not by an antagonist. The Bv-FRET assay constitutes a highly efficient, reliable and convenient method for measuring protein-protein interaction as the baculovirus expression system is superior to other transfection-based methods. Additionally, the same insect cell line can be used routinely for expressing any recombinant proteins of interest, allowing various combinations of molecules to be tested in a rapid fashion for protein-protein interactions. The assay is a valuable tool not only for the screening of new molecules that interact with known bait molecules, but also for confirming interactions between other known molecules.
Cheung
Timothy C
TC
Developmental Biology Research Group, Research School of Biological Sciences, The Australian National University, Canberra, Australia. tcheung@liai.org
Hearn
John P
JP
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D007313:Insecta; D008164:Luminescent Proteins / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011966:Receptors, LHRH / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D008164:Luminescent Proteins; D011966:Receptors, LHRH; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins