Name: Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation (Video) | JoVE Journal
Uploaded: 2019-01-16T13:30:00.000+00:00
Duration: 19 min 23 s
Description: Learn a fluorescent protease assay platform using recombinant fusion proteins and magnetic beads for high-throughput screening. This method enables real-time detection of proteolytic activity with HIV-1 protease.

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This work was supported in part by the GINOP-2.3.2-15-2016-00044 &#34;PHARMPROT teaming&#34; project and, also, financed by the Higher Education Institutional Excellence Programme of the Ministry of Human Capacities in Hungary, within the framework of the Biotechnology thematic program of the University of Debrecen. The authors are grateful for the members of the Laboratory of Retroviral Biochemistry for their scientific help during the assay development and also for their patience during filming the assay (especially to Norbert Kassay, Krisztina Jo&#243;n&#233; Mat&#250;z, and Vanda Toldi, who are appearing in the background of the video). The authors would also like to say special thanks to Gedeon Richter Plc., especially to Dr. Zolt&#225;n Urb&#225;nyi for allowing Be&#225;ta Boz&#243;ki&#39;s work in the Department of Biochemistry and Molecular Biology as a guest researcher. The authors would also like to extend their gratitude to Gy&#246;rgy Zsad&#225;nyi, Bal&#225;zs T&#337;gyi, Bal&#225;zs P&#246;st&#233;nyi, and Zolt&#225;n Kir&#225;ly from the Multimedia and E-learning Technical Center of the University of Debrecen for the professional assistance in audio and video production.

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The authors have nothing to disclose.

Due to the intensive industrial and academic investigations of proteolytic enzymes and the constant demand for expeditious and affordable HTS-compatible protease assay platforms accordingly, we have developed a magnetic-bead-based fluorescent protease assay. The assay is based on the use of recombinant fusion proteins which can be novel alternatives to the widely utilized synthetic peptide substrates.
In the developed assay format, the fusion protein substrates are immobilized to the surfaces of Ni-chelate-coated magnetic agarose beads. The substrate attachment is provided by the N-terminal His6 affinity tag of the fusion protein, which is directly fused to an MBP tag in order to facilitate the folding and enhance the water solubility of the substrate13. The MBP is followed by cleavage sites of TEV PR and a protease of interest. The former may serve as a control cleavage site in the assay, while the latter can be processed by the protease to be investigated. The cleavage site is interchangeable; a short dsDNA sequence coding for the cleavage site of interest can be inserted into the flexible &#39;cloning cassette&#39; of the expression plasmid by ligation. The recombinant fusion proteins contain a highly stable, monomeric fluorescent protein tag at the C-terminal, which enables the endpoint detection of the enzyme-liberated, fluorescent C-terminal cleavage products released upon proteolytic cleavage (Figure 1A). The purified fluorescent intact substrates solved in different buffers are also used for calibration to assess the molar concentrations of substrates and cleavage products. In addition, after fluorimetry, the assay components can be analyzed by SDS-PAGE, as well. Both native (nondenatured) and denatured fluorescent proteins can be visualized in the gel, immediately after the electrophoresis or after subsequent in-gel renaturation, respectively. This additional procedure-in combination with a conventional Coomassie Brilliant Blue staining-may be used efficiently for the verification of the assay results (Figure 1B).
The assay procedure consists of simple, easy-to-execute steps in a low-volume format that may be fully adapted to a high-throughput automatic environment. However, independently from performing the assay either manually or with an automation system, the following parts of the assay are considered to be crucial and need special attention while performing the procedure. i) Homogeneity of the magnetic bead solution. A homogeneous magnetic bead solution must be used throughout the assay, both in purification and washing steps. Particularly, the reliability of protease assays strongly depends on properly aliquoting the substrate-attached magnetic bead (SAMB) stock solutions. In order to increase the effectiveness of the suspension and dispersion, it is recommended to set the bead concentration between 2% and 10% (v/v). During sample preparation, the use of buffers supplemented with nonionic detergent (such as Triton X-100 or Tween 20) up to 2% may also decrease the adherence of the magnetic beads to plastic surfaces. The adherence of the beads to the walls of sample vials can be avoided if the bead suspensions are applied carefully to the bottoms of the vials instead of onto the walls of sample tubes. The homogeneity of the magnetic beads during the enzymatic reaction is also critical and can be ensured by continuously shaking the samples at 600 rpm during incubation. Beads are properly dispersed in rounded or flat-bottomed plastic wares, while the use of V-bottom vials is not recommended. A suboptimal result caused by improper bead homogenization is represented in Figure 4B. ii) Termination of reaction samples.&#160;Another advantage of the method is that the enzymatic reaction can be terminated without the use of heat denaturation treatment or any potentially interfering chemical agents15. The termination can be carried out simply by separating the magnetic beads from the reaction mixture, using a conventional magnetic particle concentrator. While the removed reaction buffer contains the active enzyme and the generated C-terminal fluorescent cleavage products, the uncleaved substrates remain attached to the beads. Due to the presence of the active enzyme in the reaction buffer, the separation procedure needs to be performed carefully for reliable endpoint detection. Before placing the sample vials into the concentrator, it is recommended to apply a short spin centrifugation. After placing the tubes into the concentrator, provide at least 15 s for the beads to be collected. Slight movement of the separator back-and-forth may facilitate the collection of the beads. Please consider that, during a manually performed separation, the termination usually takes more time than the initiation of the reactions. Therefore, an approximately 2 min registered delay is recommended between the initiations if the same incubation time needs to be applied to all samples.
The principle of the described proteolytic assay is relatively simple; however, the versatility of the system is guaranteed by the flexible and stable substrate structure. The individual optimization of the assay may be limited only by the compatibility of the affinity beads with the applied conditions, reagents, and additives. In agreement with the manufacturer&#39;s protocol, we also found that the affinity binding of substrates to the Ni-NTA bead surfaces substantially weakens at pH &#8804; 6.515. Therefore, it is recommended to apply substrate blank samples parallel to the reaction samples, and the rate of spontaneous substrate dissociation needs to be considered during the evaluation of results.
In those cases, where magnetic-bead-based assays cannot be performed due to the use of bead-incompatible components or a low pH, in-solution digestion of the purified recombinant substrates can be also applied. In these cases, the reaction mixtures can be analyzed by electrophoresis, and the proteins can be visualized in the gel based on the described protocol. To investigate proteolytic activity, in-solution digestion and in-gel detection of the proteins may also be alternative tools of fluorimetry. A novelty of the designed substrate system is the application of an in-gel renaturation step after denaturing SDS-PAGE. While native (nondenatured) fluorescent proteins retain their fluorescence during electrophoresis, the fluorescent property is abolished upon denaturation (Figure 7B). However, the fluorescence of denatured proteins can be partially recovered by the removal of SDS from the gel. Thus, a separation of reaction components using denaturing conditions makes not only the fluorescence-based but the molecular-weight-based identification possible. Another advantage of the fluorescent in-gel detection compared to the analysis of a Coomassie-stained gel is that the (native or renatured) fluorescent proteins can be easily identified in the gel based on their fluorescence (see Figure 7). This may be important if cleavage reactions are performed in samples containing nonfluorescent contaminants or proteins highly resembling the molecular weights of each other.
Protease assays using similarly designed substrates have already been published previously8,9,10, and although the cleavage site of interest in those cases was also located between an affinity tag and a fluorescent protein, the assay system presented here not only repeats the described ideas but combines the different advantages of the previous platforms and also completes them with further improvements: i) the utilization of an MBP fusion partner, ii) the presence of a TEV PR control cleavage site, iii) the use of newly engineered monomeric FPs, and iv) the application of a unique substrate calibration procedure. The assay itself was particularly designed to be useful for enzyme specificity and kinetic studies in a safe, time- and cost-efficient manner, without the need for expensive instrumentation. The method is aimed to be a suitable and affordable tool for both industrial and academic research purposes. Due to the flexibility of the &#39;cloning cassette&#39; of the expression plasmid, the system may be suitable for the fast and inexpensive generation of recombinant substrate libraries. The herein described assay is a feasible tool for the implementation of substrate specificity, enzyme mutagenesis, and inhibition studies and, also, provide an alternative tool to perform enzyme kinetics. The assay platform (from bacterial cell disruption to the determination of the kinetic parameters) can be adapted to an HTS- and automation-based environment and, potentially, may be applied in industrial protease inhibitor screening and/or antiviral drug development. In addition, the adaptation of the assay for competitive proteolysis is also in the future scope of our laboratory. In such a competitive assay, two different substrates-each containing a different cleavage site fused to a different C-terminal fluorescent tag-are intended to be used simultaneously in the same cleavage reaction to investigate the preference of the studied enzyme for the given target sequences. Furthermore, the use of a 96-well plate-adapted assay form (Figure 8) is also being optimized for mutation screening by using a series of substrates with modified cleavage site sequences in case of cysteine proteases.

Proteolytic enzymes belong to the most intensively investigated enzyme groups due to their importance in metabolic pathways and in industrial applications, as well. Their key role in viral diseases, the regulation of blood clotting, cancer, and cardiovascular and neurodegenerative diseases makes proteases prominent targets in the field of drug discovery. Therefore, the detailed characterization of substrate specificity and inhibitor profiling of the protease (PR) of interest is pivotal and is preferably performed by rapid, cost-efficient, and robust biochemical assays1,2,3.
Nowadays, the vast majority of in vitro protease assays applied in the field of drug discovery for compound profiling are homogeneous, fluorescent peptide-based, and high-throughput screening (HTS)-compatible platforms4. Moreover, labeled peptides are not only suitable for library screening, but they also offer great tools for the determination of enzyme kinetic parameters on the selected substrates. In other cases, where labeling of the substrate is not possible, separation-based assays may provide a possible solution to assess the kinetic properties of proteolytic reactions3.
Generally, in vitro protease assays are based on the use of two types of substrate: short peptides or whole proteins. In those cases, where the cleavage of short peptide sequences reflect the cleavage properties sufficiently, the following standard approaches are applicable: (i) examining standard protein substrates such as oxidized insulin B-chain, (ii) testing commercially available substrates of other proteases, (iii) screening synthetic and fluorescently labeled peptide libraries created by combinatorial chemistry, or (iv) using genetic methods, for instance, biological display technologies5,6. Besides the conventional classification, other novel platforms are also available for substrate generation (e.g., the formation of proteome-derived peptide libraries7 or special subtypes of genetic methods, like the recombinant fusion protein-based substrates8,9,10,11,12).
All of the above-mentioned substrate types and assays have their own advantages and limitations, and the development of assay formats combining and/or improving the advantages of the known platforms is still in demand. Here we describe a protocol for a separation-based fluorescent protease assay, which utilizes recombinant substrates. These fusion proteins consist of His6 and MBP tags fused to a control cleavage site of TEV PR, which is followed by the substrate sequence of interest that is directly connected to a C-terminal fluorescent protein (FP) (Figure 1A). The cloning of a DNA sequence coding for a cleavage site of interest into the 'cloning cassette' can be performed by a single ligation reaction into the expression plasmid, which has been linearized previously by restriction endonucleases.
<img alt="HIV-1 protease assay flowchart showing cleavage and fluorimetric analysis using PAGE and magnetic beads." class="xfigimg" src="/files/ftp_upload/58824/58824fig1.jpg" data-altupdatedat="1788250015000" data-alt="Figure 1"> 
Figure 1: Principle of the fluorescent protease assay. (A) The schematic representation of a fluorescent substrate and its cleavage by human immunodeficiency virus type 1 (HIV-1) protease is shown. The arrow indicates the cleavage position within the matrix/capsid cleavage site sequence of HIV-1 protease (VSQNY*PIVQ). (B) The fluorescent substrates can be used to analyze enzyme reactions by the Ni-NTA magnetic-bead-based assay and by polyacrylamide gel electrophoresis, as well, as is shown in the workflow diagram. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Although proteolytic assays using similar recombinant protein substrates-containing an affinity tag, a proteolytic cleavage site, and a fluorescent protein-have already been described8,9,10, the system presented here intends to integrate and improve on the advantages of these methods. An important difference is that the fusion protein substrates in this assay platform are equipped with MBP to enhance protein solubility13 and contain a control cleavage site for TEV PRs. Furthermore, the substrates contain new generation fluorescent proteins, which are highly stable and have a monomeric form to prevent substrate aggregation. Besides the previously published application of mTurquoise2- and mApple-fused forms14, here we also show results given by the use of a recombinant substrate containing a monomeric enhanced yellow fluorescent protein (mEYFP) fluorescent tag. Hereby we demonstrate the compatibility of the system with other fluorescent proteins and represent some general types of results that can be acquired by the protease assay.
The recombinant fusion proteins are expressed in E. coli BL21(DE3) cells and are used as substrates for the assay in a nickel-nitrilotriacetic acid (Ni-NTA)-coated magnetic-agarose-bead-attached form. The C-terminal cleavage products are liberated from the bead surface into the supernatant upon cleavage by the protease of interest. After the separation of the supernatant (containing the enzyme and the cleavage products) from the magnetic beads, the fluorescence can be measured to determine the cleavage properties of the enzyme. In contrast to the previously described methods, in the system presented here, the amounts of substrate and C-terminal cleavage products are uniquely quantified based on a detailed substrate calibration procedure. The assay system can be supported by an SDS-PAGE analysis of the assay samples; a subsequent fluorescent in-gel visualization may be applied immediately after the electrophoresis or after the in-gel renaturation of the nondenatured and denatured fluorescent components, respectively14.
The flexibility and structure of the 'cloning cassette' allow a time- and cost-efficient insertion of a wide variety of sequences into the construct and, thus, promotes the generation of substrate libraries. Since all assay steps are automation- and HTS-compatible, the system can be especially attractive for, for instance, protease specificity measurements and mutagenesis studies, or it may also be effectively utilized for industrial protease inhibitor screening and/or antiviral drug development, as well.
Enzyme kinetic parameters (kcat, Km) can be determined by the developed separation-based assay; therefore, it may be suitable to perform individual enzyme kinetic measurements, such as the time-course, substrate-dependent, and inhibition studies. This proves that the recombinant fusion protein substrates provide good alternatives for the frequently utilized synthetic oligopeptide substrates, and due to their high similarity to the polyprotein substrates, they represent the naturally occurring enzyme-substrate interactions more accurately.

<table><tbody><tr><td>10K Amicon tubes</td><td>Merck-Millipore</td><td>UFC501096</td><td></td></tr><tr><td>2-Mercaptoethanol (&amp;beta;-ME)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>M6250</td><td></td></tr><tr><td>40% Acrylamide/Bis solution 37.5:1</td><td>Bio-Rad</td><td>1610148</td><td></td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>Merck</td><td>100063</td><td></td></tr><tr><td>Agarose</td><td>SERVA</td><td>11404.04</td><td></td></tr><tr><td>Alpha Imager HP gel documentation system</td><td>ProteinSimple</td><td></td><td></td></tr><tr><td>Ammonium persulfate (APS)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>A3678</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>A9518</td><td></td></tr><tr><td>Beckman Coulter Allegra X-22 centrifuge</td><td>Beckman Coulter</td><td>392185</td><td></td></tr><tr><td>Black half-area plates</td><td>Greiner bio-One</td><td>675086</td><td></td></tr><tr><td>Bromophenol blue</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>B0126</td><td></td></tr><tr><td>CutSmart buffer (10x)</td><td>New England Biolabs</td><td>B7204S</td><td>For plasmid linearization (step 1.1.1)</td></tr><tr><td>Dark Reader transilluminator</td><td>Clare Chemical Research</td><td>DR-45M</td><td></td></tr><tr><td>DNase I&amp;nbsp;</td><td>New England Biolabs</td><td>M0303L</td><td></td></tr><tr><td>Dynamag-2&amp;nbsp;magnetic particle&amp;nbsp;concentrator</td><td>Thermo Fischer Scientific</td><td>12321D</td><td></td></tr><tr><td>Escherichia coli BL21(DE3) competent cells&amp;nbsp;</td><td>Thermo Fischer Scientific (Invitrogen)</td><td>C600003</td><td></td></tr><tr><td>Ethanol</td><td>Merc-Millipore</td><td>100983</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid&amp;nbsp; (EDTA)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>798681</td><td></td></tr><tr><td>Gel Loading Dye, Purple (6X)</td><td>New England Biolabs</td><td>B7024S</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>356350</td><td></td></tr><tr><td>Glycine</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>G7126</td><td></td></tr><tr><td>High-Speed Plasmid Mini Kit</td><td>GeneAid</td><td>PD300</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>56750</td><td></td></tr><tr><td>Isopropyl &amp;beta;-D-1-thiogalactopyranoside (IPTG)</td><td>Thermo Fischer Scientific (Invitrogen)</td><td>AM9464</td><td></td></tr><tr><td>Jouan CR 412 centrifuge</td><td>Jouan</td><td>CR412</td><td></td></tr><tr><td>Labinco LD-76 Rotator&amp;nbsp;</td><td>Labinco</td><td>7600</td><td></td></tr><tr><td>Luria-Bertani (LB) broth</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>L3022</td><td></td></tr><tr><td>Lysozyme&amp;nbsp;</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>L6876</td><td></td></tr><tr><td>Magnesium chloride</td><td>Scharlau</td><td>MA0036</td><td></td></tr><tr><td>MERCK eurolab ultrasonic bath</td><td>MERCK</td><td>USR54H</td><td></td></tr><tr><td>Millifuge Eppendorf spin centrifuge</td><td>Millipore</td><td>CT10</td><td></td></tr><tr><td>Mini-PROTEAN 3 Electrophoresis Cell</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>N,N,N&amp;prime;,N&amp;prime;-Tetramethylethylenediamine (TEMED)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>T9281</td><td></td></tr><tr><td>NanoDrop 2000</td><td>Thermo Fischer Scientific</td><td></td><td></td></tr><tr><td>NheI-HF restriction endonuclease</td><td>New England Biolabs</td><td>R3131L</td><td></td></tr><tr><td>Nickel(II) sulfate (NiSO4)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>656895</td><td></td></tr><tr><td>Ni-NTA magnetic agarose beads&amp;nbsp;</td><td>Qiagen</td><td>36113</td><td></td></tr><tr><td>Orbital shaker</td><td>Biosan</td><td>OS-20</td><td></td></tr><tr><td>PacI restriction endonuclease</td><td>New England Biolabs</td><td>R0547L</td><td></td></tr><tr><td>PageBlue Protein Staining Solution&amp;nbsp;</td><td>Thermo Fischer Scientific</td><td>24620</td><td></td></tr><tr><td>Phenylmethanesulfonyl-fluoride (PMSF)</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>P7626</td><td></td></tr><tr><td>Protein Lobind Micro-centrifuge tubes&amp;nbsp;</td><td>Eppendorf</td><td>22431102</td><td></td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>QIAGEN</td><td>28704</td><td></td></tr><tr><td>Snijders Press-to-Mix shaker&amp;nbsp;</td><td>Gemini</td><td>34524</td><td></td></tr><tr><td>Sodium-acetate trihydrate</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>S7670</td><td></td></tr><tr><td>Sodium-chloride</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>S9888</td><td></td></tr><tr><td>Sodium-hydroxide&amp;nbsp;</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>S5881</td><td></td></tr><tr><td>SYBR Green I Nucleic Acid Gel Stain</td><td>Thermo Fischer Scientific</td><td>S7563</td><td></td></tr><tr><td>T4 DNA ligase</td><td>Promega</td><td>M180A</td><td></td></tr><tr><td>Thermo shaker</td><td>Biosan</td><td>TS-100</td><td>with SC-24 accessory block</td></tr><tr><td>Tris</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>T1503</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich (St Louis, MO, USA)&amp;nbsp;</td><td>P2287</td><td></td></tr><tr><td>WALLAC VICTOR2 1420 multilabel counter</td><td>Wallac Oy, Turku, Finland</td><td></td><td></td></tr></tbody></table>

use of recombinant fusion proteins in a fluorescent protease assay platform and their in-gel renaturation

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.

Figure 1A shows the schematic structure of a representative fluorescent recombinant protein substrate which can be processed by HIV-1 PR at its specific cleavage site sequence. Figure 1B represents the substrate production and their possible applications in protease assays, including Ni-NTA magnetic-bead-based assay and/or PAGE.
To obtain reliable data by fluorimetry, a calibration procedure is required, in order to determine the quantities of fluorescent substrates and cleavage products. For this, the fluorescence intensity values of the different substrates in the different buffer conditions need to be measured and need to be correlated to their concentrations in the assayed concentration range (Figure 3). The slope values of the calibration curves can be applied to determine the amounts of substrates and cleavage products in the assay samples. The slopes of the calibration curves are independent of the cleavage site sequences inserted into the substrates (Table 11) and can potentially be used for a series of substrates fused to the same type of fluorescent protein. Zoom-in graphs are shown for all linear regressions, to enlarge the lower concentration ranges as well (Figure 3). It is important to note that the calibration needs to be performed carefully because a proper distribution of data points is required for a reliable calibration. For this reason, twofold serial dilution is applied to prepare the samples for calibration, because the R2 value indicates a good correlation between the concentration of fluorescent protein and fluorescence only if a sufficient number of data points have been used to cover the entire concentration range. Furthermore, experimental errors can highly affect the accuracy of the calibration; thus, a graphical evaluation of the regression lines may be also necessary.
A variety of enzymatic measurements can be performed by the protease assay, including an examination of the effect of the substrate concentration on reaction velocity (Figure 4A). By nonlinear regression, the data can be used to determine enzyme kinetic parameters (e.g., vmax and Km). An insufficient bead suspension and dispersion and an improper reaction termination may cause suboptimal results (Figure 4B), which are not suitable for calculating reliable enzyme kinetic values.
A dependence of the product formation on time can be determined by the assay (Figure 5A) (e.g., during the optimization of the cleavage reaction parameters). Enzyme activity in the presence of an inhibitor can also be investigated (Figure 5B) for the determination of the active enzyme concentration and inhibitory constant. Using the same methodology, effects of other inhibitors can also be screened by the assay.
The protease assay is useful when investigating the effects of pH on enzyme activity, as well. Figure 6A represents the dependence of enzyme activity on pH by the example of TEV PR, which has a wide optimal pH range (pH 6-9). If the pH dependence of enzyme activity is studied (or enzymes having an acidic pH optimum need to be measured), it is necessary to consider that the affinity binding of recombinant substrates to the beads may be restricted at slightly acidic pH. An elevated dissociation of the substrates from the beads (Figure 6B) may cause a distortion of the assay results. In order to consider the spontaneous substrate dissociation from the beads, the values measured for reaction samples need to be corrected by those of B samples.
Figure 7 shows that the nondenatured fluorescent proteins can be differentiated in the gel based on their colors, using blue light transillumination (Figure 7A). If the determination of the molecular weights of substrates/cleavage fragments is necessary, denaturing conditions can also be used for sample preparation, because fluorescent proteins can be partially renatured in the gel, and can be detected by UV illumination (Figure 7B) or by Coomassie staining (Figure 7C). If the R samples are analyzed, only the C-terminal cleavage products are visible (Figure 7C), while the N-terminal cleavage fragments and the uncleaved substrates remain attached to the beads. Occasionally, proteins may be partially denatured despite using nondenaturing conditions (Figure 7C), and while the nondenatured proteins are more abundant, denatured forms are also detectable in the sample. This phenomenon does not influence the detection of proteolytic cleavage but needs to be considered in the case of quantitative densitometry of nondenatured samples.
Although the detailed description is shown only for a 2 mL-tube-based assay, the assay can be adapted for a 96-well plate-based system (Figure 8), which has already been tested successfully in our laboratory (not shown). The 96-well plate-adapted format is fully compatible with the fluorimetric and electrophoretic analyses, as well, and the obtained data can also be evaluated based on the methods described in this paper.
<img alt="Enzyme kinetics analysis, linear plot, RFU vs. concentration, four protein assay results, cleavage vs. elution buffer." class="xfigimg" src="/files/ftp_upload/58824/58824fig3.jpg" data-altupdatedat="1788250015000" data-alt="Figure 3"> 
Figure 3: Calibration curves. Representative substrate calibration curves are demonstrated with the example of two recombinant substrates fused to different C-terminal fluorescent tags: (A and B) His6-MBP-VSQNY*PIVQ-mTurquoise2 and (C and D) His6-MBP-VSQNY*PIVQ-mEYFP. Zoom-in figures are also shown to represent the linear regression of data points in the 0-0.005 mM substrate concentration range. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Enzyme kinetic graphs for His-tagged fusion proteins, depicting reaction velocity vs substrate concentration." class="xfigimg" src="/files/ftp_upload/58824/58824fig4.jpg" data-altupdatedat="1788250015000" data-alt="Figure 4"> 
Figure 4: Determination of enzyme kinetic parameters. Substrate-dependent kinetic measurements were performed by HIV-1 PR (at a final active concentration of 41.2 nM). The initial velocity values were plotted against the substrate concentration and a Michaelis-Menten nonlinear regression analysis was performed. The error bars represent SD (n = 2). (A) A representative optimal result is shown with the example of His6-MBP-VSQNY*PIVQ-mApple fusion protein substrate. (B) A representative suboptimal result is also shown for the His6-MBP-KARVL*AEAM-mTurquoise2 substrate, where the setting of proper substrate concentrations was problematic due to an insufficient homogenization of the SAMB stock solution, while relatively high errors were caused by improper reaction termination. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Kinetic analysis graphs of His-tagged protein showing concentration vs. time and rate vs. log amp." class="xfigimg" src="/files/ftp_upload/58824/58824fig5.jpg" data-altupdatedat="1788250015000" data-alt="Figure 5"> 
Figure 5: Time-course and inhibitory study. (A)His6-MBP-VSQNY*PIVQ-mEYFP recombinant fusion protein substrate (at a final concentration of 0.00326 mM) was cleaved by HIV-1 PR (at a final active concentration of 41.2 nM), and the release of fluorescent PIVQ-mEYFP proteolytic fragments was measured to perform a time-course analysis. The measurements were carried out at five different time points. The error bars represent SD (n = 2). (B) His6-MBP-VSQNY*PIVQ-mEYFP was used as substrate (at 0.0015 mM) to determine the inhibitory effect of amprenavir on the activity of HIV-1 PR (at a total concentration of 163.8 nM). By plotting the data, the half maximal inhibitory concentration (IC50) could be assessed and the active enzyme concentration (a final active concentration of 41.2 nM) of the applied HIV-1 PR could also be calculated based on the inhibition curve. The error bars represent SD (n = 3). <a href="https://www.jove.com/files/ftp_upload/58824/58824fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Enzyme kinetics graph, pH effect on reaction rate and substrate dissociation, data analysis results." class="xfigimg" src="/files/ftp_upload/58824/58824fig6.jpg" data-altupdatedat="1788250015000" data-alt="Figure 6"> 
Figure 6: Studying dependence of enzyme activity and spontaneous substrate dissociation on pH. (A) The His6-MBP-VSQNY*PIVQ-mTurquoise2 substrate (in 0.033 mM) was used to measure the enzyme activity of TEV PR (at a final total concentration of 91.42 nM) in cleavage buffer set to a different pH, between the range of 6.5-8.5. The error bars represent SD (n = 2). The plotted data has been published previously14. (B) Based on the relative fluorescent intensity values of the substrate blank samples, the spontaneous dissociation of the His6-MBP-VSQNY*PIVQ-mTurquoise2 substrate (0.033 mM) from the magnetic beads was studied by using cleavage buffer with a different pH, between 6.0-8.5. The plotted data has been published previously14. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="HIV-1 PR denaturation analysis; transillumination, UV, Coomassie methods; gel electrophoresis results." class="xfigimg" src="/files/ftp_upload/58824/58824fig7.jpg" data-altupdatedat="1788250015000" data-alt="Figure 7"> 
Figure 7: Detecting proteins in the gel by different methods. (A) Uncleaved and HIV-1 PR-digested fusion protein substrates after nondenaturing sample preparation were visualized by blue light transillumination after SDS-PAGE. The cleavage reaction was performed by in-solution digestion. (B) Immediately after the PAGE, only nondenatured proteins could be detected in the gel by UV illumination, while after the removal of SDS, the previously denatured fluorescent proteins became partially renatured and detectable. The samples were prepared from the supernatants of the Ni-NTA magnetic-bead-based assay. (C) Coomassie staining can also be used for protein detection, after the in-gel renaturation. The SDS-present in the gel-may cause the partial denaturation of the native protein, but in native samples, the nondenatured forms are more abundant. The samples were prepared from the supernatants of the Ni-NTA magnetic-bead-based assay. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Bead-based assay method diagram and gel electrophoresis results for cleavage efficiency analysis." class="xfigimg" src="/files/ftp_upload/58824/58824fig8.jpg" data-altupdatedat="1788250015000" data-alt="Figure 8"> 
Figure 8: 96-well plate-based adaptation of the assay platform. (A) The assay can be performed not only in 2 mL tubes but in the wells of a 96-well plate, as well. Here we show the schematic representation for the application of the assay to study the specificity of a fictitious protease by using a series of fluorescent substrates, which may contain wild-type (wt) or mutated (mut-1 to mut-4) cleavage site sequences. For handling the magnetic beads, a 96-well compatible magnetic particle concentrator (MPC) is to be used in the experiments. All the indicated volumes are related to a single well. To compare the cleavage efficiency of the different substrates, substrate conversion can be assessed from the percentage of substrate-blank-corrected RFU values of the reaction samples, considering the substrate-blank-corrected RFU values of the related substrate control samples as 100. (B) After fluorimetry, the separated supernatants of the assay samples can also be analyzed by PAGE, and the fluorescent protein components can be analyzed directly or after in-gel renaturation in case of nondenaturing and denaturing sample preparation, respectively. The three different assay sample types are also illustrated in each figure: C = substrate control, B = substrate blank, and R = reaction. Substrate control samples are in elution buffer, while the substrate blank and the reaction samples are in cleavage buffer. <a href="https://www.jove.com/files/ftp_upload/58824/58824fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Fluorescent protein</td>
			<td>CV% of slopes (%)</td>
		</tr>
		<tr>
			<td>Elution</td>
			<td rowspan="2">mTurquoise2</td>
			<td>6.04</td>
		</tr>
		<tr>
			<td>Cleavage</td>
			<td>9.11</td>
		</tr>
		<tr>
			<td>Elution</td>
			<td rowspan="2">mApple</td>
			<td>10.92</td>
		</tr>
		<tr>
			<td>Cleavage</td>
			<td>12.68</td>
		</tr>
	</tbody>
</table>
Table 11: Coefficient of variance (CV%) values of the slopes of the substrate calibration curves. To test whether the fluorescence of the recombinant protein substrates is dependent on the inserted cleavage site, calibrations were performed by series of mApple- and mTurquoise2-fused substrates (six variants for each, containing different cleavage site sequences of HIV-1 protease), both in elution and cleavage buffers. We found that CV% values of the slopes are under 15%in all cases, which implies that a single substrate calibration can be utilized for the evaluation of the different measurements performed by substrate variants containing the same fluorescent tag.

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, ...

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