Towards a survival-based cellular assay for rapid selection of protease inhibitors

We are describing a method tailored for in-cell selection of protease inhibitors. In this method, a target protease is co-expressed with a selective substrate, the product of which kills the host cells. Therefore, it can be applied to identify potential inhibitors, based on cell host survival, when inhibition of the target protease occurs. The method would be suitable for selection of inhibitors from intracellularly generated inhibitor libraries, like SICLOPPS, for instance. TEV protease was chosen for this proof-of-concept. The genetically encoded selective substrate is a single polypeptide chain, composed of three parts: (1) ccdB protein that can cause host cell death when it accumulates inside cell, (2) a protease cleavage sequence, that can be changed according to the target protease: ENLYFQG sequence for TEV substrate ( - predicted cleavage site) and (3) the ssrA sequence (AANDENYALAA), which drives the polypeptide to degradation by the ClpX/ClpP complex inside host E. coli cells. Conditions for controlled expression of this substrate, in which its steady-state concentration did not provoke significant death of host cells, were established. Co-expression of active TEV protease and this selective substrate (ccdB-ENLYFQG-ssrA) caused the death of a significant host cell population, while control assays with an inactive mutant TEV Asp81Asn did not. Details of the methodology used are given, providing the basis for the application of similar systems for other proteases of interest.