# Towards a Survival-Based Cellular Assay for the Selection of Protease Inhibitors in Escherichia coli

## TL;DR

This paper introduces a new method to find protease inhibitors by using cell survival in Escherichia coli.

## Contribution

A novel survival-based cellular assay is developed for selecting protease inhibitors in E. coli.

## Key findings

- Co-expression of active TEV protease and a selective substrate caused significant host cell death.
- In contrast, inactive TEV mutant did not cause cell death.
- The method can be adapted for other proteases by changing the cleavage sequence.

## Abstract

We describe a method tailored to the in-cell selection of protease inhibitors. In this method, a target protease is co-expressed with a selective substrate, the product of which kills host cells. Therefore, the method can be applied to identify potential inhibitors based on cell host survival when inhibition of the target protease occurs. The TEV protease was chosen for this proof-of-concept experiment. The genetically encoded selective substrate is a single polypeptide chain composed of three parts: (1) a ccdB protein, which can cause host cell death when it accumulates inside the cell; (2) a protease cleavage sequence that can be changed according to the target protease, in this case the TEV substrate ENLYFQ↓G (↓-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. In our experiment, co-expression of the 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.