Protein-level silencing technologies hold promise both as a research tool for exploring protein function and as therapeutics for accelerating the turnover of disease-related proteins. To this end, we have previously demonstrated the efficacy of “ubiquibodies”, engineered protein chimeras that combine the activity of E3 ubiquitin ligases with the target specificity of designer binding proteins (DBPs) to selectively and controllably steer target proteins to the ubiquitin proteasome pathway for degradation. Importantly, ubiquibodies are modular - simply swapping DBPs can generate a new ubiquibody with specificity for a different substrate protein. Unfortunately, pre-existing DBPs (scFvs, FN3s, DARPins, etc.) do not exist for many desired cellular proteins. To address this limitation, we have developed a universal ubiquibody based on a bacterial E3 ubiquitin ligase that specifically targets GFP or virtually any mammalian target protein fused to GFP. The resulting ubiquibody exhibits extraordinary silencing efficiency against a wide array of GFP fusion proteins that vary in size, subcellular localization, and method of expression (i.e., transient vs. stable). Because numerous fluorescent protein fusions already exist or can be rapidly created, our GFP-specific ubiquibody is poised to enable proteolytic silencing on a proteome-wide scale without the need for further engineering or optimization.

Proteolytic silencing of diverse mammalian protein targets using a functionally remodeled bacterial E3 ubiquitin ligase

255th National Meeting (2018)

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