Directed evolution of the 3C protease from Coxsackievirus using a novel fluorescence-assisted intracellular method.

This reporter consists of an aggregation-prone peptide fused to a fluorescent protein via a linker that contains the corresponding substrate sequence. Cleavage of the substrate rescues the fluorescent protein from aggregation, resulting in increased fluorescence that correlates to proteolytic activity, which can be monitored using flow cytometry. In one round of flow-cytometric cell sorting, we isolated an efficiently cleaved TEV substrate from a 1:100,000 background of non-cleavable sequences, with around 6000- fold enrichment. We then engineered the 3C protease from Coxsackievirus B3 (CVB3 3Cpro) towards improved proteolytic activity on the substrate LEVLFQ↓GP. We isolated highly proteolytic active variants from a randomly mutated CVB3 3Cpro library with up to 4-fold increase in activity. The method enables simultaneous measurement of proteolytic activity and protease expression levels and can therefore be applied for protease substrate profiling, as well as directed evolution of proteases. PMID: 30521472 [PubMed - as supplied by publisher]
Source: Biological Chemistry - Category: Chemistry Tags: Biol Chem Source Type: research