A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Abstract

Source: Jaimes, J. A., et al. A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation. J. Vis. Exp. (2019).

This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

Protocol

1. Designing and Preparing the Peptides

1. Acquire the sequence of the fusion protein of interest from a public database such as NCBI (https://www.ncbi.nlm.nih.gov/) or the virus pathogen database (https://www.viprbrc.org). Choose the protease recognition site preceding the fusion peptide and include 2-3 amino acids upstream and downstream of this sequence.
2. When ordering the peptides, modify them with the FRET pair 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitrophenyl (DNP) at the C-terminus.
   NOTE: Several suppliers provide these modifications. Price and delivery time depend on the sup....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Peptides	Biomatik	N/A
Furin	NEB	P8077S
Trypsin, TPCK-treated	Sigma-Aldrich	4352157-1KT
HEPES	Sigma-Aldrich	H3375
CaCl2	Sigma-Aldrich	C1016
2-Mercaptoethanol	Sigma-Aldrich	M6250
Triton-X100	Sigma-Aldrich	X100
PBS	Corning	21-040-CV
SpectraMax Gemini XPS	Molecular Devices	XPS
SoftMax Pro 6.5.1	Molecular Devices	N/A
96-well plate (solid black polystyrene with a flat bottom and non-treated)	Costar	3915
Light damping tubes	Watson Lab	131-915BL or 131-915BR