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

Author(s)

Oyadomari, William Y. ; Carvalho, Elizangela A. ; Machado, Gabriel E. ; Machado, Ana Julia O. ; Santos, Gabriel S. ; Marcondes, Marcelo ; Oliveira, Vitor

• Document type: Journal article
• Source: BIOTECH; v. 14, n. 1, p. 10-pg., 2025-03-07.

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 down arrow G (down arrow-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. (AU)

• FAPESP's process: 23/07904-4 - Study of molecular and cellular mechanisms of mental disorders: clinical studies and animal models
• Grantee: William Yoshio Agliardi Oyadomari
• Support Opportunities: Scholarships in Brazil - Post-Doctoral
• FAPESP's process: 20/09678-3 - Selection of inhibitors for the NS2/NS3 proteases from Dengue and Zika viruses by cellular high-throughput assays coupled with cyclic peptide libraries
• Grantee: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Support Opportunities: Regular Research Grants
• FAPESP's process: 21/11936-3 - Center for Translational Science and Biopharmaceutical Development
• Grantee: Benedito Barraviera
• Support Opportunities: Research Grants - Science Centers for Development
• FAPESP's process: 23/09167-7 - Engineering antibodies and bacteria for tumor diagnosis and therapeutics.
• Grantee: Vitor Marcelo Silveira Bueno Brandão de Oliveira
• Support Opportunities: Regular Research Grants