Conditional Protein Rescue (CPR) by Binding-Induced Protective Shielding

An effective method to modulate the stability of proteins is essential to biological research. Herein, we describe a new technology that allows conditional stabilization of proteins based on masking of a degron tag by a specific intracellular protein cue. A target protein is fused to a degron tag and an affinity sensor domain. When the sensor detects its target protein, the degron is effectively concealed and the target protein is rescued. By introducing nanobodies as the sensor, we allow for virtually any endogenous protein to be targeted. In a model system using yeast cytosine deaminase, we demonstrate low cell death background yet maintain the ability to elicit strong activation and prodrug-mediated cell killing using GFP as the rescue protein. The flexibility in choosing different masking targets provides a straightforward method to generalize the strategy for conditional protein rescue in a wide range of biological contexts, including oncoprotein detection.