(79f) Conditional Protein Rescue (CPR) By Binding-Induced Protective Shielding

The rational regulation of protein concentration remains an elusive problem in synthetic biology, and no solutions currently exist that allow the intracellular concentration of a protein of interest (POI) to be regulated by the cellular environment. To that end, we propose a conditional protein rescue (CPR) system: a protein modulator capable of translating information concerning endogenous cellular proteins to desirable changes in the concentration of a POI. The POI is fused to a destabilizing domain (DD) and a small sensor domain, a product which—by virtue of the DD—is inherently unstable via proteasomal degradation. In the absence of the sensor’s target, the protein remains unstable and exists at low intracellular concentrations. When the sensor’s target is present, the interaction with the sensor sterically masks the DD and rescues the POI from degradation.

As an initial proof-of-concept, we used YFP as our POI and the short peptide SpyTag as the sensor. When SpyTag interacts with its target, SpyCatcher, a covalent bond is formed, providing maximum bond strength and thus optimal conditions for CPR to occur. YFP levels remained low in the absence of SpyCatcher, and upon co-expression with SpyCatcher, YFP levels rose rapidly indicating CPR functions as predicted. Furthermore, adding an ubiquitin-like (UbL) domain to the N-terminus of YFP enhanced the degradation kinetics and reduced background to undetectable levels while still allowing for rescue in the presence of SpyCatcher. Cancer is often marked by dramatic changes in a cell’s proteome. If this information could be accurately translated into a therapeutic output, then that technology opens the door to a next generation of “smart therapeutics”. To demonstrate this conceptually, we changed the sensor in CPR to a camel single-domain antibody fragment (nanobody), a small, monomeric protein with high specificity towards its antigen. As an output POI, we chose yeast cytosine deaminase (yCD), a prodrug converting enzyme capable of converting the innocuous prodrug 5-fluorocytosine (5-FC) to the cytotoxic drug 5-fluorouracil (5-FU). GFP served as a visually traceable cancer-protein surrogate and the target of a well-characterized nanobody. The nanobody was not large enough to effect protein degradation, and no cytotoxicity was observed in the absence of GFP, even when 5-FC was administered. However, when GFP was co-expressed, cell death was observed on levels similar to when 5-FU was administered directly. This demonstrates that CPR can be used to control a wide variety of proteins in different applications.