Construction and application of protein degrader cell screening based on HaloTag
Objective The cell screening model for stable expression of HaloTag protein was constructed for the activity screening of proteolysis targeting chimeras(PROTAC)degraders and the development of E3 ubiquitin ligase ligands.Methods Lentivirus technology was used to construct HEK-293T cell lines stably expressing HA-Halo and Flag-Halo-NLS proteins,and Western Blotting was used to detect the levels of HA-Halo and Flag-Halo-NLS proteins targeted by HaloTag degrader VHL-HP3.Results Cell models of HEK-293T stably expressing HA-Halo and Flag-Halo-NLS proteins were successfully constructed;compound VHL-HP3 significantly degraded the levels of HA-Halo and Flag-Halo-NLS proteins and both showed significant degradation activity at 0.3 μmol/L.Conclusion Stably expressing HaloTag protein cell lines could be used as model cells for screening the activity of PROTAC degraders targeting HaloTag protein,providing a high-throughput screening method for the development of E3 ubiquitin ligase ligands of marine drug origin.
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