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Mix-and-Read Assays For Regulated Ubiquitination & Degradation of Proteins Using PathHunter® Technology

Mix-and-Read Assays For Regulated Ubiquitination & Degradation of Proteins Using PathHunter® Technology
Version:
0213

File Name/Number:
Ubiquitin

Year:
2013

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Ubiquitin-mediated degradation of signaling proteins is a primary means of signal regulation in a wide variety of cellular processes including inflammatory signaling, DNA damage responses, developmental pathways, and the cell cycle. However, observation of these events with the throughput necessary for modern drug discovery has been challenging. Numerous reporter proteins exist that can be used as fusion partners, however their size and stable tertiary structure impact the half-life of the targeted protein, limiting their utility. We reasoned that a smaller tag, lacking tertiary structure could be used as an inert fusion partner that could preserve the natural turnover of the protein target. To this end we employed the DiscoveRx® enzyme complementation system. In this system the fusion partner is a peptide that is less than 5kD making the system minimally invasive. The levels of the protein fusion can be quantified with a single reagent addition making this system amenable to high-throughput screening. Here we demonstrate the utility of the system across a wide range of targets and directly compare performance with luciferase fusions.