An In Vitro Forster Resonance Energy Transfer-Based High-Throughput Screening Assay for Inhibitors of Protein-Protein Interactions in SUMOylation Pathway

Forster 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 proteinprotein 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 SUMO 1 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.