Towards a survival-based cellular assay for rapid selection of protease
inhibitors
Abstract
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: ENLYFQG 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.