title: An in vitro Förster resonance energy transfer-based high-throughput screening assay for inhibitors of protein-protein interactions in SUMOylation pathway.
abstract: Förster resonance energy transfer (FRET) is a powerful tool in biological research and has been widely used in the study of biomolecular interactions. SUMOylation is an important post-translational modification that is involved in many key biological processes. As a multi-step cascade reaction, SUMOylation involves multiple enzymes and protein-protein interactions. Here, we report the development of an in vitro FRET-based high-throughput screening (HTS) assay in SUMOylation. This assay is based on steady state and high efficiency of the fluorescent energy transfer between CyPet and YPet fused to SUMO1 and Ubc9, respectively. We optimized the assay and performed a small-scale pilot study to validate the screening platform. Carried out in 384-well plate format, our FRET-based HTS provides a powerful tool for large-scale and high-throughput applications.
authors.affiliation: Department of Bioengineering, University of California, Riverside, California 92521, USA.
authors.forename: Yang
authors.initials: Y
authors.lastname: Song
authors.forename: Jiayu
authors.initials: J
authors.lastname: Liao
mesh_terms: D000465:Algorithms; D004247:DNA / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression / Q000187:drug effects; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D053858:Metabolic Networks and Pathways / Q000187:drug effects; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015203:Reproducibility of Results; D025842:SUMO-1 Protein / Q000378:metabolism; D059629:Signal-To-Noise Ratio; D058207:Sumoylation / Q000187:drug effects*; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D014508:Urea / Q000494:pharmacology; D000098843:Ubiquitin-Conjugating Enzyme UBC9
chemical_list: D011993:Recombinant Fusion Proteins; D025842:SUMO-1 Protein; C495501:SUMO1 protein, human; D014508:Urea; D004247:DNA; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9