Ultrasensitive Scaffold-Dependent Protease Sensors with Large Dynamic Range

The rational construction of synthetic protein switches with predefined input–output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor-engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein–protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of α-thrombin and prostate specific antigen with a dynamic range covering 5 orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers, and actuators