Our collaboration with the Sapkota and Ganley Labs is now published in Cell Chemical Biology.<\/p>\n
Highlights<\/strong><\/p>\n
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Ligand (L)-inducible AdPROM consists of Halo conjugated to a target protein binder<\/li>\n
Target protein is degraded with HaloPROTAC in cells expressing L-AdPROM construct<\/li>\n
HaloPROTAC-mediated target protein degradation using L-AdPROM system is reversible<\/li>\n
Degradation using HaloPROTAC L-AdPROM impacts target protein biological functionRead the Article open access here.<\/li>\n<\/ul>\n
Title<\/strong>: Inducible Degradation of Target Proteins through a Tractable Affinity-Directed Protein Missile System<\/p>\n
Authors<\/strong>: Luke M Simpson, Thomas J Macartney, Alice Nardin, Luke J Fulcher, Sascha R\u00f6th, Andrea Testa, Chiara Maniaci, Alessio Ciulli, Ian G Ganley, Gopal P Sapkota<\/p>\n
Summary<\/strong>:<\/p>\n
The affinity-directed protein missile (AdPROM) system utilizes specific polypeptide binders of intracellular proteins of interest (POIs) conjugated to an E3 ubiquitin ligase moiety to enable targeted proteolysis of the POI. However, a chemically tuneable AdPROM system is more desirable. Here, we use Halo-tag\/VHL-recruiting proteolysis-targeting chimera (HaloPROTAC) technology to develop a ligand-inducible AdPROM (L-AdPROM) system. When we express an L-AdPROM construct consisting of an anti-GFP nanobody conjugated to the Halo-tag, we achieve robust degradation of GFP-tagged POIs only upon treatment of cells with the HaloPROTAC. For GFP-tagged POIs, ULK1, FAM83D, and SGK3 were knocked in with a GFP-tag using CRISPR\/Cas9. By substituting the anti-GFP nanobody for a monobody that binds H- and K-RAS, we achieve robust degradation of unmodified endogenous RAS proteins only in the presence of the HaloPROTAC. Through substitution of the polypeptide binder, the highly versatile L-AdPROM system is useful for the inducible degradation of potentially any intracellular POI.<\/p>\n
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