Optimizing in Vitro Assay Conditions for Metalloprotease Falcilysin

SMSU Event Center BC

Malaria is a parasitic disease causing nearly 500,000 deaths each year, with the most severe form of disease caused by Plasmodium falciparum. Our group focuses on falcilysin (FLN), an essential metalloprotease expressed by P. falciparum during host cell infection. In this study, we set out to optimize in vitro assay conditions for recombinant FLN activity to facilitate inhibitor screenings against this enzyme. In this series of experiments, various parameters such as enzyme, substrate, and detergent concentrations were optimized alongside pH conditions. This assay uses a FRET substrate containing a peptide linker between EDANS and Dabcyl chromophores. This results in fluorescence upon proteolytic cleavage, and substrate turn over can be monitored by detecting emission at 490 nm. Multiple concentrations of enzyme (0.015-0.12 µg/mL) and of substrate (0.85-27.2 µM) were tested in a 96-well format to identify conditions producing a robust signal-to-noise ratio (S/N) over a reasonable time frame. We determined that a concentration of 0.06-0.12 µg/mL protease, 3.4 µM substrate and 0.01%-0.03% triton x-100 gave a linear signal with an S/N of 5:1 for at least 10 minutes. This assay was further characterized to identify potential time and position dependent artefacts.