A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases
Transcrição
Enveloped viruses with transmembrane spike proteins contain a proteolytic cleavage site.
Membrane-bound proteases on host cells cleave the spike proteins, exposing fusion peptides that facilitate virus entry.
To begin, take wild-type peptides with the canonical cleavage site and variant peptides, each bearing a unique mutation at the site.
The fluorogenic peptides contain a fluorophore and a quencher. Due to proximity, the quencher absorbs the fluorescence of the fluorophore.
Take a multi-well plate on ice, containing assay buffer and host proteases.
Add the wild-type and variant peptides into separate wells. Incubate at an appropriate temperature for optimum protease activity.
The protease cleaves the peptides, separating the quencher from the fluorophore and enabling its fluorescence emission.
Measure the change in the fluorescence signal to determine the rate of proteolytic cleavage.
A higher rate for the wild-type sequence indicates higher proteolytic activity, while a lower rate for the variants indicates altered cleavage efficiency.
