Home › Biochemistry › A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Biochemistry JoVE (Open Access) Citable · DOI

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

DOI: 10.3791/58892-v
What you'll learn
  • ✓ Design and prepare fluorogenic peptides for protease cleavage assays
  • ✓ Set up and configure fluorescence plate reader for real-time kinetic measurements
  • ✓ Analyze protease activity data and interpret cleavage efficiency results
  • ✓ Adapt assay for viral fusion proteins and custom amino acid motifs
Protocol

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Difficulty
intermediate
Total time
~3–4 hours per assay run (including peptide synthesis and plate reader setup)
Biosafety
BSL-2

Steps

1
Design and prepare fluorogenic peptides

Design peptide substrates representing viral fusion protein cleavage sites or target amino acid motifs. Synthesize or order peptides with N-terminal fluorophore and C-terminal quencher labels.

▶ 00:31
2
Configure fluorescence plate reader settings

Set up the fluorescence plate reader with appropriate wavelengths for peptide fluorophore excitation and emission, establish kinetic read parameters, and validate instrument calibration.

▶ 02:06
3
Prepare assay mixture and reaction wells

Combine protease, fluorogenic peptide substrate, and assay buffer in microplate wells. Establish positive and negative controls for protease activity validation.

▶ 03:11
4
Analyze cleavage kinetics and quantify activity

Extract fluorescence time-course data, calculate reaction rates and cleavage efficiency, and generate dose–response or mutation comparison curves.

▶ 04:30
5
Interpret mutation effects on protease specificity

Compare cleavage efficiency across wild-type and mutant spike protein sequences to determine how amino acid substitutions alter furin and other protease recognition.

▶ 05:37
💬 Comments coming soon