tailieunhanh - Báo cáo khoa học: 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 measuringmultimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone recep-tor (GnRH-R) was capable of forming oligomeric com-plexes 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. | Eur. J. Biochem. 270 4973-4981 2003 FEBS 2003 doi Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction Timothy C. Cheung and John P. Hearn Developmental Biology Research Group Research School of Biological Sciences The Australian National University Canberra Australia 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