title: Development of FRET assay into quantitative and high-throughput screening technology platforms for protein-protein interactions.
abstract: Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research and is a very powerful tool in elucidating protein interactions in many cellular processes. Ubiquitination and SUMOylation are multi-step cascade reactions, involving multiple enzymes and protein-protein interactions. Here we report the development of dissociation constant (K (d)) determination for protein-protein interaction and cell-based high-throughput screening (HTS) assay in SUMOylation cascade using FRET technology. These developments are based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused with SUMO1 and Ubc9, respectively. The developments in theoretical and experimental procedures for protein interaction K (d) determination and cell-based HTS provide novel tools in affinity measurement and protein interaction inhibitor screening. The K (d) determined by FRET between SUMO1 and Ubc9 is compatible with those determined with other traditional approaches, such as isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR). The FRET-based HTS is pioneer in cell-based HTS. Both K (d) determination and cell-based HTS, carried out in 384-well plate format, provide powerful tools for large-scale and high-throughput applications.
authors.affiliation: Department of Bioengineering, Bourns College of Engineering, University of California at Riverside, 900 University Avenue, Riverside, CA 92521, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.forename: Vipul
authors.initials: V
authors.lastname: Madahar
authors.forename: Jiayu
authors.initials: J
authors.lastname: Liao
mesh_terms: D057005:Bioengineering; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D025941:Protein Interaction Mapping / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism; D058207:Sumoylation; D020349:Surface Plasmon Resonance; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9