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.