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1. Designing and Preparing the Peptides
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Resuspend the peptide according to the manufacturer&#39;s recommendations by gently pipetting it up and down. For example, resuspend peptides in 70% ethanol to the final concentration of 1 mM.</li>
	<li>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.</li>
	<li>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 &#181;L). Store aliquots at -20&#176; C.</li>
</ol>
2. Preparing the Fluorescence Plate Reader
<ol>
	<li>Turn on the plate reader and wait until the self-test is finished.</li>
	<li>Open the operating software on the attached computer and make sure it is connected to the plate reader.</li>
	<li>Open the temperature setting and set it to 30 &#176;C (or the required temperature for optimal performance for the protease).</li>
	<li>Click on Control | Instrument Setup to set up the experiment.</li>
	<li>Choose Kinetic.</li>
	<li>Select Fluorescence.</li>
	<li>Enter Excitation and Emission wavelengths: 330 nm and 390 nm respectively.</li>
	<li>Unselect Auto Cut-off.</li>
	<li>Select Medium, normal sensitivity.</li>
	<li>Choose a runtime of 1 h for the assay and select one measurement every 60 s.</li>
	<li>Set 5 s mixing before the first measurement and 3 seconds before each measurement</li>
	<li>Select wells to read and start the assay.</li>
</ol>
3. Preparing the Assay
<ol>
	<li>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.</li>
	<li>Chill the buffer on ice.</li>
	<li>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 &#34;leakage&#34; from adjacent wells.</li>
	<li>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 &#181;L. For trypsin, use 0.5 &#181;L of 160 nM TPCK trypsin.</li>
	<li>Per sample prepare 3 technical replicates per assay in a total volume of 100 &#181;L 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.</li>
	<li>Pipette the appropriate amount of assay buffer (94.5 &#181;L as described under Step 3.1) into each well.</li>
	<li>Add the protease to each well. For both, furin and TPCK trypsin, use 0.5 &#181;L per sample. To 6 wells (3 wells for a blank control and 3 wells for a peptide control), add 0.5 &#181;L buffer instead of the respective protease.</li>
	<li>Add the peptide to a final concentration of 50 &#181;M in each well except the blank control. In this case, pipette 5 &#181;L peptide was prepared as described under Step 1.3. Add 5 &#181;L 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.</li>
	<li>Insert the plate into the fluorescence plate reader and click start.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Peptides</td>
			<td>Biomatik</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Furin</td>
			<td>NEB</td>
			<td>P8077S</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, TPCK-treated</td>
			<td>Sigma-Aldrich</td>
			<td>4352157-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-040-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax Gemini XPS</td>
			<td>Molecular Devices</td>
			<td>XPS</td>
			<td></td>
		</tr>
		<tr>
			<td>SoftMax Pro 6.5.1&#160;</td>
			<td>Molecular Devices</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate (solid black polystyrene with a flat bottom and non-treated)</td>
			<td>Costar</td>
			<td>3915</td>
			<td></td>
		</tr>
		<tr>
			<td>Light damping tubes</td>
			<td>Watson Lab</td>
			<td>131-915BL or 131-915BR</td>
			<td></td>
		</tr>
	</tbody>
</table>

a fluorogenic peptide cleavage assay to screen the proteolytic activity of proteases

Source: Jaimes, J. A., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> 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.

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

1. Designing and Preparing the Peptides

	Acquire the sequence of the fusion protein of interest from a public database such as NCBI ...

Enveloped viruses with transmembrane spike proteins contain a proteolytic cleavage site.
Membrane-bound proteases on host cells cleave the spike proteins, exposing fusion peptides that facilitate virus entry.
To begin, take wild-type peptides with the canonical cleavage site and variant peptides, each bearing a unique mutation at the site.
The fluorogenic peptides contain a fluorophore and a quencher. Due to proximity, the quencher absorbs the fluorescence of the fluorophore.
Take a multi-well plate on ice, containing assay buffer and host proteases.
Add the wild-type and variant peptides into separate wells. Incubate at an appropriate temperature for optimum protease activity.
The protease cleaves the peptides, separating the quencher from the fluorophore and enabling its fluorescence emission.
Measure the change in the fluorescence signal to determine the rate of proteolytic cleavage.
A higher rate for the wild-type sequence indicates higher proteolytic activity, while a lower rate for the variants indicates altered cleavage efficiency.&#160;&#160;

a-fluorogenic-peptide-cleavage-assay-to-screen-proteolytic-activity

Universidad San Sebastian

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Immunology

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Specialized Assays

69 Views. 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...

Video: A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases - Concept

Detection of Protease Activity by Fluorescent Peptide Zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

JoVE Journal

Cancer Research

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, consequently, cardiovascular disease. Although PCSK9 proteolysis is required for its full hypercholesterolemic effect, the evaluation of its proteolytic function is challenging: PCSK9 is only known to cleave itself, undergoes only a single turnover, and after proteolysis, retains its substrate in its active site as an auto-inhibitor. The methods presented here describe an assay which overcomes these challenges. The assay focuses on intermolecular proteolysis in a cell-based context and links successful cleavage to the secreted luciferase activity, which can be easily read out in the conditioned medium. Via sequential steps of mutagenesis, transient transfection, and a luciferase readout, the assay can probe PCSK9 proteolysis under conditions of either genetic or molecular perturbation in a high-throughput manner. This system is well suited for both the biochemical evaluation of clinically discovered missense single-nucleotide polymorphisms (SNPs), as well as for the screening of small-molecule inhibitors of PCSK9 proteolysis.

This protocol presents a method to evaluate the proteolytic activity of an intrinsically low-activity, single turnover protease in a cellular context. Specifically, this method is applied to evaluate the proteolytic activity of PCSK9, a key driver of lipid metabolism whose proteolytic activity is required for its ultimate hypercholesterolemic function.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a single-turnover protease which regulates serum low-density lipoprotein (LDL) levels and, ...

Biochemistry

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

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

Transcript

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

Detection of Protease Activity by Fluorescent Peptide Zymography

A High-Throughput Luciferase Assay to Evaluate Proteolysis of the Single-Turnover Protease PCSK9

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation