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

Proteases are one of the most investigated groups of biomolecular supports in academic and industrial research. Their powerful role in several physiological and pathological processes makes them a prominent target in the field of drug discovery as well. Regarding their intensive research, there is a great and continuous demand for the development of time-and cost-saving high-throughput screening-compatible fluorescence protease assay platforms.

Here, we would like to introduce a protocol for the application of a separation-based high-throughput screening-compatible fluorescence assay platform using both mTurquoise2-and mApple-fused recombinant fusion protease substrates. Additionally, we would like to demonstrate a method for the in-gel renaturation of the substrates and the cleavage fragments of the assay samples after denaturing SDS-PAGE as well. All the experiments are carried out on the example of HIV-1 virus protease.

The expression plasmid carries the coding sequence of a hexa histadine affinity tag and a maltose binding fusion protein followed by a control cleavage site of tobacco etch virus protease, a cloning cassette, and a C-terminal fluorescent protein. Linearize the expression plasmid by PacI and NheI restriction endonucleases. Perform the annealing of the Escherichia coli codon optimized forward and reverse oligonucleotide primers that code for the substrate sequence of interest and flank by PacI and NheI cohesive ends.

Perform the insertion of the annealed primers into the linearized expression plasmid by ligation. For protein expression, transform BL21(DE3)competent cells by the ligation mixture. The fluorescent proteins will be in the same open reading frame with the N-terminal fusion tags only after a successful ligation.

A few days after transformation, the colonies containing the expression plasmid coding the inserted cleavage site of interest will show visible fluorescence with or even without using a Dark Reader blue transilluminator. Add 10 microliters glycerol stock of the BL21(DE3)cells carrying the expression plasmid coding for the cleavage site sequence of interest to five milliliters LB medium containing 100 micrograms per milliliter ampicillin in a 50 milliliter centrifuge tube. Incubate the suspension at 37 degrees for 15 hours while constantly shaking.

Transfer five milliliters bacterial culture to 50 milliliters fresh LB medium containing 100 micrograms per milliliter ampicillin. Grow the cells at 37 degrees to an absorbance of 0.6 to 0.8 at 600 nanometer wavelengths. At this point, induce protein expression by the addition of IPTG.

Hereafter, incubate the culture for three hours at 37 degrees while continuously shaking. After the incubation, transfer 25 milliliters of the culture into clean 50 milliliter centrifuge tubes, and harvest the cells by centrifugation. Discard the supernatant and store the bacterial cell pellets at minus 70 degrees for at least an hour.

Cell pellets containing the successfully expressed fluorescent proteins show clearly a fluorescence with or even without using a Dark Reader blue transilluminator. Place the frozen cell pellet on ice and let it thaw for 15 minutes. Add two milliliters lysis buffer to the pellet and suspend the cells.

Hereafter, add protease inhibitor, free the suspension by lyzozyme and DNase, suspend the cells, and incubate the suspension on ice for 15 minutes. Transfer two times one milliliter suspensions into 1.5 milliliter microcentrifuge tubes and sonicate the suspensions for three minutes in rounds of 10 seconds of sonication and five seconds break. Centrifuge the tubes at 10, 000 g for 20 minutes at room temperature.

Cleared bacterial cell lysates containing the fluorescent substrate of interest show visible fluorescence with or even without using a Dark Reader blue transilluminator. The procedure of the developed protease assay starts with the preparation of the assay samples. Firstly, the recombinant substrates are incubated with the affinity magnetic beads, and the substrate-attached magnetic beads, abbreviated as SAMBs, are formed.

SAMBs are washed several times, and finally the SAMB stock solution is prepared by the aliquoting of which assay samples are created. The reactions are initialized by the addition of the protease solution. Upon cleavage by the protease, the proteolytic fragments are released into the supernatant.

The reaction is terminated by the separation of the magnetic beads from the reaction buffer that contains the fluorescent cleavage products and the enzyme. The supernatants are applied to the wells of a microtiter plate and the fluorescence is determined by fluorimetry. Calibration curves are generated using purified fluorescent substrates solved in each assay buffers.

Concentration of the C-terminal fluorescent cleavage products and also that of the applied substrate in the assay samples are determined based on the slope of the calibration curves. Place the tube containing new or recycled nickel-NPA magnetic agarose beads in a magnetic particle concentrator. Beads may stick to the wall and/or into the lid of the microcentrifuge tube.

Therefore, turn the concentrator upside-down in every direction to make sure that all of the beads are collected. Remove the supernatant and discard it. Add 1.8 milliliters lysis buffer to the beads and remove the closed tubes from the concentrator.

Suspend the beads in the tube by shaking and/or turning the tube upside-down until beads are completely homogeneous. Place the tubes back in the concentrator and turn it upside down in every direction to make sure that all of the beads are collected. Open the tube and discard the supernatant.

Add cleared bacteria-sterile lysate containing the substrate of interest and remove the closed tube from the concentrator. Turn the tubes upside-down until the beads are completely homogeneous and rotate the tubes by rotator, slowly, at room temperature for 30 minutes. SAMBs show visible fluorescence with or even without using a Dark Reader blue transilluminator.

Wash the SAMBs three times by 1.8 milliliter 1%Tween 20, washing buffer, and cleavage buffer. Add cleavage buffer to the washed SAMBs to create a SAMB stock solution. If using two-milliliter protein-low-binding microcentrifuge tubes, the volume of the applied cleavage buffer is up to 1.9 milliliters.

After the addition of the buffer, do not shake or turn the tube upside-down. Close the tube and remove it from the concentrator. Prepare two-milliliter protein-low-binding microcentrifuge tubes for the assay samples.

Suspend the SAMB stock solution until it is completely homogeneous and measure the amount of substrate to be analyzed in the reactions by immediately transferring the SAMB stock solution into the sample vials. The volume of the SAMB stock solution to be transferred is recommended to be 25 to 300 microliters, but it is to be set according to the individual experimental design. Place the sample tubes containing the aliquoted SAMB suspensions into the concentrator.

Carefully remove the supernatant from the SAMBs and discard it. Remove the tubes from the concentrator and carefully add the reaction buffer to the SAMBs. The volume of the added reaction buffer is to be calculated according to the individual experimental design, but it is recommended to set the final volume of the reaction mixture between 50 and 150 microliters if two-milliliter tubes are used.

Close the lids of the tubes. Now, the samples are ready for being initialized. Add the enzyme solution to the reaction samples.

Stir up the beads carefully by gently moving the tubes and place the tubes immediately into the already-shaking thermoshaker. In the case of substrate blank samples and substrate control samples, add cleavage buffer and elution buffer respectively. Thirty seconds prior to the end of incubation, take the sample out from the thermoshaker and spin it promptly.

Place the tube onto the concentrator and let the tube stand for 15 seconds. The collection of the SAMBs may be facilitated by slightly moving the concentrator back and forth. Open the lid and transfer the supernatant carefully into a plate or a new tube.

Do not touch the concentrated beads by the tip of the pipette. The collected supernatant of the substrate control samples and the reaction samples with a high degree of cleavage may show visible fluorescence with or even without using a Dark Reader blue transilluminator. Transfer two times 30 microliters of the separated sample supernatants into a black half-area microplate.

Measure the fluorescence by fluorimeter using the appropriate filters according to the applied fluorescent protein. For the purification of the fluorescent substrates, prepare the same solution as described previously, and discard the supernatant. Add 400 to 600 microliters elution buffer to the SAMBs and rotate the tubes by rotator slowly at room temperature for five minutes.

Transfer the eluate into new protein-low-binding microcentrifuge tubes. It shows clearly visible fluorescence with or without using a Dark Reader blue transilluminator. After purification, buffer exchange, and the measurement of the protein content, prepare a twofold serial dilution in at least eight steps, both from the elution-buffer-and the cleavage-buffer-solved substrate solutions, using elution or cleavage buffer for dilution respectively.

Transfer 30 microliters of each dilution point into a black half-area microplate. Measure the fluorescence by fluorimeter using the same settings that were applied in the measurement of the assay sample supernatants. As an example for data processing, the substrate-dependent kinetic measurements of HIV-1 protease was performed on an mTurquoise2-fused form of the recombinant substrate coding the cleavage site between the matrix and the capsid proteins in the natural gapic protein precursor.

Plot the corrected relative fluorescence intensity values against the molar concentration of the purified substrates, solve either the cleavage or elution buffer, and perform linear regression. Calculate the molar concentration of the C-terminal cleavage products in the reaction samples using the slope of the cleavage-buffer-based calibration curve. Also calculate the applied substrate concentration in the reaction samples based on the molar concentration of the eluted substrate in the substrate control samples using the slope of the elution-buffer-based calibration curve.

Initial velocity values were calculated from the amount of the C-terminal cleavage fragments and then plotted against the applied substrate concentration. Also kinetic parameters were determined by Michaelis-Menten non-linear regression analysis. After performing the nickel-NTA magnetic bead-based assay, the assay supernatants can be analyzed by polyacrylamide gel electrophoresis as well.

However, beside the analysis of the assay samples, it is also possible to analyze the purified fluorescent substrates and/or the cleavage fragments after in-solution digestion. For in-solution digestion, aliquote the purified fluorescent substrates to be digested dissolved in cleavage buffer into 1.5 milliliter microcentrifuge tubes, and add the enzyme solution to the samples. Incubate the samples according to the experimental design and terminate the reactions by the procedure of the sample preparation for PAGE.

For nondenatured sample preparation, mix the samples to be analyzed with nondenaturing sample loading buffer containing no reducing agents. In the case of denatured sample preparation, mix the samples to be analyzed with denaturing sample loading buffer and expose the samples to heat treatment at 95 degrees for 10 minutes. Apply the non-denatured and the denatured samples into the wells of the SDS-polyacrylamide gel and perform electrophoresis.

Remove the gel cassette from the running module. Non-denatured samples are already visible in the gel even by naked eye or with the Dark Reader blue transilluminator. In order to detect the fluorescent components of the denatured samples on the transilluminator, perform the in-gel renaturation by washing the SDS out from the gel.

Add 100 milliliters distilled water to the gel and rinse it at least for 30 minutes. Visualize the fluorescent proteins by placing the unstained gel onto to the Dark Reader blue transilluminator. The recombinant fluorescent fusion proteins were engineered to be used as substrates in proteolytic assays.

Upon cleavage by the protease at the specific cleavage site, the C-terminal fluorescent cleavage fragment is released and its fluorescence can be detected. Their use in nickel-NTA magnetic bead-based assay and the fluorescent detection by PAGE analysis have been optimized using HIV-1 protease. However, the developed assay platform can be suitable for other proteases as well.

Calibration curves of the purified substrates solved in cleavage or elution buffer are illustrated on the examples of both mTurquoise2-and mEYFP-fused form of the recombinant substrate coding the cleavage side between the matrix and the capsid proteins of the natural HIV anechoic protein. The nickel-NTA magnetic bead-based assay is a useful tool for examining the effect of substrate concentration on reaction velocity and thereby also makes the determination of the enzyme kinetic parameters such as Vmax and Km possible. Diagram A shows an optimal result obtained after a successfully-performed analysis of substrate-dependent activity on an mApple-fused substrate.

In contrast, diagram B presents a sub-optimal result where the insufficient homogenization of the SAMB stock solution causes problems in setting up the proper substrate concentrations while improper termination of the reaction samples results in relatively high errors. The assay also provides a suitable tool for performing time-course and inhibitory studies as well. Diagram A demonstrates the time-dependence of the proteolytic cleavage of HIV-1 protease on an mEYFP-fused substrate, while diagram B represents the inhibitory effect of amprenavir on the cleavage reaction.

From the data obtained by the inhibitory study, both the active enzyme concentration and the inhibitory constant can be determined. The assay can also be optimized to study the dependence of enzyme activity on pH, as it is represented by diagram A on the example of tobacco etch virus protease. Diagram B indicates a limitation of the assay by pH and shows that neutral or slightly alkalic pH is fully compatible with the measurements, while acidic pH facilitates spontaneous dissociation of the substrates from the surface of the magnetic beads.

The present figure shows the purified intact fluorescent substrates, the fluorescent C-terminal cleavage fragments, generated by in-solution digestion by HIV-1 protease. The fluorescent components can be detected in the SDS-containing polyacrylamide gel by blue light transillumination right after the electrophoresis if non-denaturing conditions were applied during sample preparation. The different fluorescent proteins can be differentiated based on their color.

After the magnetic bead-based assay, the non-denatured fluorescent proteins in the sample supernatants can be detected in the gel by UV illumination as well. In contrast, if denaturing conditions are applied during sample preparation, the denatured proteins cannot be detected in the gel immediately after the SDS PAGE. However, the denatured fluorescent proteins can be partially renatured by the removal of the SDS from the gel and become detectable.

Therefore, the SDS PAGE is followed by the in-gel renaturation procedure. The renaturation ability of the fluorescent proteins makes their identification possible not only based on their fluorescence but also based on their molecular weight. Here we demonstrated the protocol for the use of our recently-developed magnetic bead-based fluorescent protease assay platform.

We have demonstrated the use of this system on the example of enzyme kinetic studies on HIV-1 protease. We used mTurquoise-and mApple-fused forms of the recombinant assay substrate, which contained the cleavage site sequence of the protease. Besides the fluorimetric analysis, the assay samples were subsequently analyzed by SDS PAGE as well.

We have demonstrated that the denatured fluorescent proteins can be partially renatured in the gel after SDS PAGE which provides both fluorescent and molecular-weight based identification of the substrate and the proteolytic fragments.