MP046:Liu:An in vitro High Sensitive FRET-based High-throughput Screening Assay for SENP Inhibitors in SUMOylation Pathway
Yan Liu 1, Jiayu Liao 12*
1Department of Bioenginnering and 2 Institute for Integrative Genome Biology, Bourns College of Engineeing, University of California-Riverside, 900 University Avenue, Riverside, CA, 92521
Ubiquitin or ubiquitin-like proteins (ULPs), such as SUMO (small ubiquitin-related modifier), play important roles in diverse cellular processes, including regulation of cell cycle, transcriptional regulation, cell survival and death, DNA damage response, and stress response. SUMO is processed into mature form from its precursor, pre-SUMO, by its specific protease- Sentrin/SUMO-specific proteases (or SENPs). Conjugated SUMO is then removed from the substrate by SENPs to re-start the SUMOylation cascade.
Here we report the development of a high sensitive in vitro FRET-based high-throughput screening (HTS) assay of SENP1. This assay is based on steady state and high efficiency of fluorescent energy transfer between CyPet and YPet fused to pre-SUMO1. We optimized the assay and validate the screening platform by non-specific SENP inhibitors (NEM). Our FRET-based high-throughput screening assay provides a powerful tool for large-scale and high-throughput applications. The robust and reproducible FRET-based HTS assay can be expanded to other protease inhibitor discoveries.
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