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

This method can help answer key questions in the biochemistry field, such as the proteolytic modification of proteins, as well as the characterization of inhibitors and proteases and peptidases. The main advantage of this technique is that it allows a rapid screening of the proteolytic activity of proteases on peptides, representing the cleavage side of the given protein. The use here is to study proteases that activate viral fusion.

The first step in peptide design is to acquire the sequence of the fusion protein of interest from a public database such as NCBI or the virus pathogen database. Choose the protease recognition site proceeding the fusion peptide and include two to three amino acids upstream and downstream of this sequence. When ordering the peptide, modify it with the fluorescence resonance energy transfer or FRET pair, Mca at the end terminus and Dnp at the c terminus.

During the assay, Mca is excited and emits light energy that is quenched by Dnp as long as the pair is in close vicinity to each other. However, if cleavage occurs, Dnp will not be able to quench the emission which can then be read by the fluorescence plate reader. Resuspend the peptide according to the manufacturers recommendations by gently pipetting up and down.

For example, resuspend in 70%ethanol to a final concentration of one millimolar. If the peptide does not resuspend very well by pipetting, place the tube containing the peptide and the solvent in a sonication bath until it is fully resuspended. Dispense 100 microliter aliquots of the peptide into light dampening or resistant tubes to protect the peptide from bleaching.

Store the aliquots at minus 20 degrees Celsius. Begin this procedure by turning on the plate reader and waiting until the self test is finished. Next, open the operating software on the attached computer and make sure it is connected with the plate reader.

Open the temperature setting and set it to the required temperature for optimal performance for the protease, which is 30 degrees Celsius, in this case. To set up the experiment, click on control instrument set-up, choose kinetic, and then select fluorescence. Enter an excitation wavelength of 330 nanometres and an emission wavelength of 390 nanometres.

Unselect the auto cut-off and select medium/normal sensitivity. Choose a run time of one hour for the assay and select one measurement every 60 seconds. Set five seconds of mixing before the first measurement, and three seconds before each measurement.

Lastly, select the wells to read. Prepare the appropriate assay buffers for the proteases as described in the text protocol and chill the buffers on ice. Place a solid black polystyrene non-treated flat bottom 96-well plate on ice with a thin metal plate underneath to support cooling and stability.

It is essential to use a black plate as the assay plate, to prevent fluorescent leakage from adjacent wells. Per peptide, prepare three technical replicates per assay, and a total volume of 100 microliters per sample. Pipette the appropriate amount of assay buffer into each well of the assay plate.

Add 0.5 microliters of protease to each well. To six wells, add 0.5 microliters of buffer instead of the respective protease. Three of these will be blind controls and the other three will be peptide controls.

Add five microliters of the peptide to a final concentration of 50 micromolar to each well, except the blind controls. To each of the three blind control wells, add five microliters of buffer instead of peptide. Insert the plate into the flourescents plate reader and click start.

Prior to starting the data analysis, save the experiment file. Click on export, and export the file as a txt. Import the txt file into a spreadsheet.

Start the analysis by creating a graph, plotting the relative fluorescent units on the y-axis against the time on the x-axis for each technical replicate per sample. Select the data range where the graph is in a linear range, and the closest to the start of the fluorescence increase. Plot the selected data on a second graph and add a linear trend line.

In the trend line options, select display equation on the chart. The equation will show V max, which corresponds to the slope of the trend line. Calculate the average V max for each sample from the three technical replicates.

After repeating the experiment twice more, to obtain three biological replicates, calculate the standard deviation based on the data from the three independent biological replicates. A furin cleavage assay of human MERS coronavirus, EMC/2012 S2 prime site, and camel-derived double-mutant strain HKU205, along with single mutant variants of EMC/2012 revealed that the EMC/2012 peptide was efficiently cleaved, while almost no cleavage of the S2 prime peptide of HKU205 occurred. Cleavage of the EMC/2012 A-S mutated S2 prime peptide was strongly reduced, and there was almost no cleavage of the EMC/2012 S to I mutated S2 prime peptide.

Another furin cleavage assay showed only minimal cleavage of the camel-derived Morocco 213 S2 prime site, compared to the human MERS coronavirus, EMC/2012 S2 prime site. Furin cleavage was observed for the prototypical S1/S2 peptide, FECV I and 4 S1/S2, but not in the mutated S1/S2 peptide, FIPV I Black S1/S2. Similarly, furin was able to cleave the prototypical S2 prime peptide, FECV II 1683 S2 prime, but not the mutated S2 prime peptides, FECV I and 4 S2 prime, FIPV I Black S2 prime, and FIPV II 1146 S2 prime.

Following this procedure, western blot analysis or amino-fluorescence assays, using the full length protein of interest can be performed to verify protein cleavage, or in this case, if cleavage results in a fusogenic version of the viral fusion protein.