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

Abstract

1An effective method to modulate the stability of proteins is essential to biological 2 research. Herein, we describe a new technology that allows conditional stabilization of 3 proteins based on masking of a degron tag by a specific intracellular protein cue. A 4 target protein is fused to a degron tag and an affinity sensor domain. When the sensor 5 detects its target protein, the degron is effectively concealed and the target protein is 6 rescued. By introducing nanobodies as the sensor, we allow for virtually any endogenous 7 protein to be targeted. In a model system using yeast cytosine deaminase, we 8 demonstrate low cell death background yet maintain the ability to elicit strong activation 9 and prodrug-mediated cell killing using GFP as the rescue protein. The flexibility in 1 0 choosing different masking targets provides a straightforward method to generalize the 1 1 strategy for conditional protein rescue in a wide range of biological contexts, including 1 2 oncoprotein detection.