Artificial protein sensor for protease activity and inhibition

Abstract

The present invention discloses a reliable protease activity biosensor for determining the cleavage of a protease recognition site that can be viewed by FRET, fluorescence change or by SDS-PAGE electrophoresis. This biosensor consists of a globular domain, a flexible connector with a variable protease recognition / cleavage area and a second globular domain. The proposed invention can be useful for detecting proteases and assessing the activity thereof, as well as finding novel protease inhibitors or monitoring the efficiency of therapeutic agents in patients undergoing treatment. The proposed biosensor is an engineered protein from the rational engineering of proteins which makes this molecule unique and not available in nature.

Description

[0001] ARTIFICIAL PROTEIN SENSOR OF ACTIVITY AND INHIBITION OF PROTEIN HANDLES

[0002] TECHNICAL FIELD

[0003] The present invention is in the field of biotechnology, specifically, it relates to a protein biosensor designed for the detection and analysis of protease activity. Furthermore, the invention encompasses compounds that incorporate the aforementioned biosensor, either independently or in association with other molecules, so that they can be used as reagents or as part of a method for detecting functional proteases or novel inhibitors of these enzymes. This invention has practical applications in several areas, such as organic biochemistry, pharmacy, virology, medicine, and enzymology, as it can be used to confirm the presence of proteases, analyze their activity, and determine the inhibitory capacity of a compound, whether known or unknown.

[0004] BACKGROUND OF THE INVENTION

[0005] Proteases are enzymes responsible for breaking peptide bonds in proteins and have a variety of functions in different biosystems. Their basic function is related to the destruction of proteins for cell turnover or defense, but these molecules also have a specialized role as molecular scissors or specific catalysts for the production of new protein products. This activity allows proteases to control cellular behavior, survival, or death in all organisms; they are capable of influencing DNA replication, transcription, cell proliferation, differentiation, neurogenesis, immunity, senescence, necrosis, apoptosis, among others. Evidence of this is that alterations in proteolytic activity are linked to pathologies such as cancer, neurodegeneration, and immune and inflammatory diseases.In the specific case of microbial and viral proteases, they are involved in replication or constitute virulence factors. Therefore, methods for measuring protease activity are useful for studying diseases, finding new protease inhibitor drugs and evaluating their therapeutic efficacy (López-Otín C, Bond JS et al. 2008). Current published assays include peptide substrates that present a donor fluorophore and a quencher acceptor molecule at each end that, through Frequence Resonance Energy Transmission (FRET), generate a fluorescent signal change that indicates the presence of an active protease. These substrates have a cleavage site that, when cut by the protease, separates the donor fluorophore from the quencher, generating a fluorescent signal that can be measured by fluorometry.These substrates are short peptides that are chemically generated where amino acids and chromophores are added by solid-phase synthesis. This generates a production limitation and an added economic cost. Examples of this are the works of Ma et al. (2020), Sacco et al. (2020), Fu et al. (2020), Chamakuri et al. (2021 ), Drazic et al. (2021 ), Coelho et al. (2020), Jin et al. (2020), Steuten et al. (2021 ) and Rut et al. (2021 ).

[0006] The search for related patents similarly shows kits or methods for detecting active proteases using peptide substrates with fluorescent signal (CN111896504A, US10962529B1, WO2010000591 A2,

[0007] W09000399A1 ) or Surface Enhanced Raman Scattering (SERS, EP2019587A2 ). Unlike all these predecessors, the invention in question does not have a peptidic nature by chemical synthesis, but is constituted by an engineered protein (Figure 1 ) that can be efficiently produced in an Escherichia coli (E. coli) bacterial system, eliminating the production and cost limitations of the aforementioned peptide sensors. Additionally, it presents a design with a single cysteine ​​in each domain, one at the end of the other, which allows the specific, reactive thiol addition of a fluorophore in the carboxyl and amino terminal domains, eliminating the dependence on modification by synthesis. Finally, peptide substrates are not easily visualized by gels for protein visualization due to their size, which limits the use of these sensors with basic visualization methods in laboratories worldwide.

[0008] The patents most similar to our invention are

[0009] US7109293B2 System for detecting protease and W02005054433A2 A NOVEL "CLEAVE-N-READ" SYSTEM FOR PROTEASE ACTIVITY ASSAY AND METHODS OF USE THEREOF, which develop chimeric substrates composed of one or more protease cleavage sites with a fluorescent protein module for in vivo assays.

[0010] Unlike these, the present invention is not a chimeric protein, but rather an engineered one, which involves modifying a single protein to obtain a sensor, rather than adding multiple proteins. This is reflected in the size of 25 kDa vs. their counterparts of 70 and 90 kDa respectively, which implies greater production efficiency. In turn, these chimeric sensors use fluorescent proteins, which, while allowing in vivo function, limit the visualization of these sensors in protein gels. The denaturating nature of these gels eliminates the fluorescent function of these sensors, limiting visualization to insensitive dyes such as bromophenol blue and losing the high sensitivity provided by fluorescence.

[0011] BRIEF DESCRIPTION OF THE INVENTION

[0012] The present invention is an engineered biosensor that provides accurate and versatile detection of protease activity. This biosensor, expressed in E. coli, overcomes the production and cost limitations associated with synthetic peptide substrates. The biosensor is composed of three parts: a globular domain, a flexible linker with a variable amino acid region that generates the protease activity, and a second globular domain, all of which are encoded in a plasmid expression vector.

[0013] The invention offers two variants of the biosensor adapted to various experimental needs. The original form, a non-fluorescent biosensor, provides a direct and efficient method for visualizing protease activity by SDS-PAGE with simple protein staining. The second variant, a fluorescent version of the biosensor, incorporates a pair of fluorophores at the N- and C-termini of the protein, allowing detection by Fluorescence Resonance Energy Transmission (FRET). This version allows the acquisition of detectable fluorescent signals in the presence of protease activity, both in gels and in fluorescence readers, increasing the versatility of the biosensor.

[0014] This biosensor represents a more efficient and adaptable method for studying protease activity, with important applications in biomedical research and therapeutic efficacy evaluation. Thus, the invention provides alternatives that adapt to a variety of protease activity detection and analysis needs.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] FIGURE 1. Schematic representation of the expression vector of the protein substrate of the invention and of the Biosensor. The expression vector carries a bacterial, viral, plant or mammalian promoter, followed by a tandem DNA sequence encoding a protein with: 1) an amino-terminal globular domain with a single cysteine, 2) a flexible linker having a variable region of a protease recognition / cleavage site, 3) a second carboxyl-terminal globular domain with a single cysteine. The biosensor produced by this vector is functional in a fluorescent or non-fluorescent form.

[0017] FIGURE 2. Schematic representation of an example of a biosensor construction for the detection and evaluation of protease activity. Generation of the PEPROT-S biosensor, a substrate for the detection of SARS-CoV-2 Mpro protease activity. The use of the pET-24(+) plasmid with an expression construct based on the E. coli IF3 protein (IF3ec) is shown, which comprises the coding sequences for: 1) the N-terminal globular domain of IF3ec, 2) the flexible linker with the protease recognition / cleavage region, 3) the C-terminal globular domain of IF3ec engineered to contain a single, surface-exposed cysteine.

[0018] FIGURE 3. Modification of the biosensor for the detection of protease activity with a fluorescent signal. To create this fluorescent version, a pair of fluorophores is added to the protein biosensor, one acceptor and one quenched. These fluorophores are located in the N- and C-terminal domains of the protein, allowing the detection of fluorescence signals through the fluorescence resonance energy transfer (FRET) technique in a fluorescence reader. The biosensor in its fluorescent version maintains the same recognition and cleavage capacity by the protease, and its detection can be performed either directly by fluorescence or indirectly with Coomassie blue staining in an SDS-PAGE gel. In this way, the biosensor can be used in two forms: the original (non-fluorescent) and the modified (fluorescent), expanding the possibilities for detection and analysis of protease activity.(A) Schematic of the double-labeling process: First, the globular domain is labeled at the C-terminal of the cysteine ​​with a maleimide fluorophore (acceptor fluorophore). After denaturation of the biosensor, the cysteine ​​hidden in the N-terminal domain is exposed, allowing its labeling with a maleimide fluorophore (donor fluorophore). (B) In the specific case of the fluorescent version PEPROT-S (fluo), double labeling is performed, incorporating two fluorophores, one acceptor (Atto-540Q) and the other donor (Atto-488), in the N- and C-terminal domains of the protein that makes up the biosensor. Final product, PEPROT-SF biosensor, ready for use in the detection and fluorescent analysis of protease activity.

[0019] FIGURE 4. Schematic diagram of the principle of the assay using the biosensor of the present invention. The figure shows: (A) Biosensor in the absence of protease, where the acceptor fluorophore and the quencher fluorophore are close together and no fluorescent signal is observed by FRET in a fluorescence reader. The biosensor can be observed in an SDS-PAGE gel directly as a single fluorescent band or with Comassie blue staining as a band of characteristic size (22 KDa); (B) Biosensor in the presence of protease, where the acceptor fluorophore and the quencher fluorophore are far apart and fluorescent signal is observed by FRET in a fluorescence reader. Both remnants of the biosensor can be observed in an SDS-PAGE gel directly as two nearby fluorescent bands or with Comassie blue staining as two bands smaller than the complete biosensor (<12 KDa).

[0020] FIGURE 5. Demonstration of the operation of the biosensor of the present invention.

[0021] (A) Both forms of the PEPROT-S biosensor, the fluorescent (PEPROT-SF) and the non-fluorescent form, are visualized on a Coomassie blue-stained SDS-PAGE gel as bands of characteristic size. When protease (Mpro +) cleaves both forms of the biosensor, the intensity decreases and the remaining domains (CTD and NTD) are visualized on the SDS-PAGE gel as two bands smaller than the original biosensor (indicated by arrows). (B) Only the fluorescent form of the PEPROT-SF biosensor can be directly visualized on an SDS-PAGE gel using a transilluminator. As in (A), when protease (Mpro +) cleaves the biosensor, the remaining domains (CTD and NTD) are visualized as two bands smaller than 12 KDa (indicated by arrows). (C) Use of the PEPROT-SF biosensor in a fluorescence reader. In the presence of protease (Mpro), the acceptor and quencher fluorophores at the ends of the biosensor separate, resulting in a fluorescent signal observable by FRET (squares).In contrast, in the absence of protease or in the presence of a protease inactivated with an inhibitor (N3), the acceptor and quencher fluorophores remain close, resulting in an absence of fluorescent signal by FRET (circles and triangles).

[0022] FIGURE 6. Confirmation of protease recognition specificity (Mpro). Assays were performed with a variant of the biosensor lacking the recognition or cleavage region (called NoRECON), in both fluorescent and non-fluorescent forms. These did not exhibit cleavage signals in SDS-PAGE gels or FRET readings, even in the presence of the protease, as evidenced by the absence of any remaining domains smaller than 12 KDa. The arrows indicate the absence of biosensor cleavage.

[0023] FIGURE 7. Evaluation of the effect of protease concentration on biosensor activity. In Figures (A) and (B), the biosensor is visualized on an SDS-PAGE gel, either with Coomassie blue staining or by direct fluorescent visualization, as a band of characteristic size, indicated by an arrow as PEPROT-S. When Mpro protease is present, the remnant domains of the biosensor become evident, visualized on the SDS-PAGE gel as two nearby fluorescent bands or as two bands smaller than 12 KDa with Coomassie blue staining. As the concentration of Mpro protease is reduced from 0.1 to 0 pM, a gradual decrease in the biosensor activity is observed, directly proportional to the increase in protease concentration, and the gradual appearance of the two remnant domains.Figure (C) demonstrates that the FRET fluorescent signal, measured in a fluorescence reader, is inversely related to the concentration of Mpro protease during the first 20 minutes of reaction.

[0024] FIGURE 8. Evaluation of the effect of inhibitor concentration on biosensor activity in the presence of protease. In Figures (A) and (B), the biosensor is visualized on an SDS-PAGE gel, either with Coomassie blue staining or by direct fluorescent visualization, as a band of characteristic size, indicated by an arrow as PEPROT-S. When the protease Mpro is present, the decrease in biosensor activity and the appearance of the remnant domains become evident, visualized on the SDS-PAGE gel as two nearby fluorescent bands or as two smaller bands with Coomassie blue staining. With the introduction of the protease inhibitor N3 at concentrations ranging from 0.1 to 100 pM, no protease activity is observed, evidenced by the absence of the remnant domains. Only when the concentration of the inhibitor N3 is less than or equal to 0.01 pM, the remnant domains can be visualized.Figure (C) demonstrates that the FRET fluorescent signal, measured in a fluorescence reader, is detected only when the concentration of the N3 inhibitor is less than or equal to 0.01 pM during the first 20 minutes of reaction.

[0025] DESCRIPTION OF THE INVENTION

[0026] The present invention is a protein biosensor based on engineered recombinant proteins, designed to detect and evaluate protease activity. Unlike chemically synthesized peptide-based sensors, this biosensor can be produced economically and efficiently through recombinant expression in a bacterial system such as Escherichia coli (Figure 1).

[0027] The biosensor design involves an expression plasmid vector that includes a promoter (bacterial, viral, plant, or mammalian) followed by a tandem DNA sequence encoding a three-dimensional protein. This protein consists of three key elements: 1) a globular domain with a single cysteine ​​at the amino terminus, 2) a flexible linker with a variable amino acid region that serves as a protease recognition / cleavage site, and 3) a second, carboxyl-terminal, globular domain containing a single cysteine. The biosensor produced from this vector can operate in two formats: fluorescent and non-fluorescent.

[0028] The fluorescent version of the biosensor (Figure 3) incorporates a pair of fluorophores, one acceptor and one quencher, located at the N- and C-terminal domains of the protein. This configuration allows the detection of protease activity by fluorescence resonance energy transfer (FRET). The principle of the FRET assay (Figure 4) in a fluorescence reader is based on the proximity of the fluorophores: in the absence of protease, the acceptor fluorophore and the quencher fluorophore are close, resulting in a low fluorescent signal. However, in the presence of protease, the recognition / cleavage site in the flexible region is cleaved, which separates the fluorophores and increases the fluorescent signal detected by FRET. This change can be measured and quantified in real time or at the end of the assay using a fluorescence reader.

[0029] In an SDS-PAGE gel, the two biosensor fragments resulting from protease cleavage can be observed as two nearby bands of smaller size compared to the entire sensor. Therefore, protease activity can be observed by the disappearance of the entire protein and the appearance of the two lower molecular weight domains. In the fluorescent version of the biosensor, these bands can be visualized directly in the gel using the fluorescence of the fluorophores. In the non-fluorescent version of the biosensor, these bands can be visualized after staining the gel with Coomassie blue. (Figure 4)

[0030] The principle of double-labeling the biosensor protein with fluorophores is based on the unique properties of cysteines in the protein. Cysteine ​​is one of the few amino acids that can form covalent bonds with other molecules, thus allowing the direct coupling of fluorophores to the protein. For this purpose, the biosensor of the present invention features a single cysteine ​​in each of the protein domains (N- and C-terminal). These cysteines can be found naturally in the N- and C-terminal positions of the biosensor's base protein, or the protein can be engineered to incorporate them, as long as one of them is exposed and the other hidden. This design allows for a sequential labeling process with the two different fluorophores. First, the exposed cysteine ​​is labeled under conditions that do not allow the reaction of the hidden cysteine.The physicochemical environment is then modified to expose the second cysteine ​​and allow its labeling. This sequential process ensures that each fluorophore couples to the corresponding cysteine, enabling the formation of a functional FRET system with the fluorophores located at opposite ends of the protein (Figure 3).

[0031] An example of the application of this technology is the PEPROT-S biosensor, specifically designed to detect the activity of the SARS-CoV-2 Mpro protease. As shown in Figure 2, the development of this biosensor uses the pET-24(+) plasmid with an expression construct based on the E. coli IF3 protein (IF3ec). The construct includes the coding sequences for the N-terminal globular domain of IF3ec, a flexible linker with the recognition / cleavage sequence TSAVLQSGFRK, and the C-terminal globular domain of IF3ec. The fluorescent version of PEPROT-S, PEPROT-SF, can also be produced, allowing the detection of Mpro protease activity by the FRET technique in a fluorescence reader (Figure 3).

[0032] To obtain PEPROT-SF, PEPROT-S is modified with a double-labeling strategy based on Chulluncuy et al., 2016; Nakamoto et al. 2021 . In the first step, the exposed cysteine ​​at the C-terminus is labeled with Atto-540Q maleimide under optimal conditions. Intensive dialysis and controlled incubation ensure efficient labeling, followed by purification of the Biosensor-CTD540Q product using a HiTrap SP HP column. In the second step, the biosensor is denatured to expose the buried cysteine ​​at the N-terminus, which is then labeled with Atto-488 maleimide. The final product, PEPROT-SF, is purified and evaluated for purity and labeling efficiency.

[0033] The biosensor was validated through a series of experiments designed to measure its efficiency, specificity, and response to different experimental conditions. Figures 5 and 6 illustrate the biosensor's performance and selectivity, respectively. The biosensor demonstrated a clear response to protease activity, indicating its functionality. Additionally, it showed high selectivity, responding exclusively to the cleavage site of interest, demonstrating its specificity.

[0034] A crucial aspect of this invention is its ability to measure and adapt to different experimental variables, which was analyzed in depth. The biosensor not only detects the presence of the protease, but its signal varies proportionally with the protease concentration (Figure 7), providing a quantitative measure of protease activity. Furthermore, the biosensor can detect and quantify the presence of protease inhibitors. As the concentration of an inhibitor increased, a corresponding decrease in the biosensor signal was observed (Figure 8). This feature is essential for studies seeking to evaluate the efficacy of protease inhibitors. This series of experiments confirms that the biosensor can be used accurately and efficiently to detect and quantify protease activity in a variety of experimental settings, thus demonstrating its versatility and reliability.

[0035] Sequences

[0036] This section details a series of amino acid sequences in a bioengineered protein designed to detect the activity of various proteases. The engineered sequence, SEQ ID No. 1, corresponds to a modified biosensor protein that has been optimized to effectively detect protease activity in a variety of experimental settings.

[0037] In particular, the amino acid sequence in the biosensor's flexible linker, starting at amino acid 88, can be modified to accommodate different proteases. At this site, designated (Xaa)n, different amino acid sequences corresponding to the recognition sites of various proteases can be inserted. This makes our biosensor highly adaptable and capable of detecting a variety of proteases from different species and associated with diverse pathological conditions.

[0038] Examples of such protease-specific recognition sites and their corresponding DNA sequences, which can be inserted into position 88 of the biosensor protein, are presented in Table 1 . These include, for example, the Chikungunya virus nsP2 protease, the Zika virus NS2B-NS3 protease, and the SARS-CoV-2 primary protease, among others. In addition, Table 1 also contains the specific recognition sequence of the Mpro protease used in the PEPROT protein example in this invention.

[0039] This ability to swap recognition sites allows the biosensor to be used to study a wide range of proteases and evaluate the efficacy of protease inhibitors in a variety of contexts, from basic research to clinical applications.

[0040] In addition to the amino acid sequences, the DNA sequence encoding the bioengineered protein designed for the detection of protease activity, designated SEQ ID No. 2, is also provided herein. This DNA sequence includes regions encoding the amino acid sequences corresponding to the specific protease recognition sites.

[0041] It is important to note that, as with amino acid sequences, the DNA sequence can be modified to accommodate different proteases. In particular, a segment of the DNA sequence designated (N)n can be modified to encode any protease-cleaving peptide sequence. This means that the DNA sequence can be tailored to produce biosensor proteins capable of detecting different proteases.

[0042] This ability to customize the DNA sequence of the biosensor protein enhances its versatility and expands its potential for the detection of a variety of proteases of interest in various scientific and clinical applications. To complete this section, the sequences corresponding to the specific example of the PEPROT biosensor are provided. The sequences include the protein sequence, identified with SEQ ID No. 3, and the DNA sequence, with SEQ ID No. 4. These sequences are fundamental to the design and operation of the PEPROT biosensor, and are applicable to both its fluorescent and non-fluorescent versions. With this, it is expected that this information will facilitate the understanding and eventual replication or adaptation of this biosensor for a variety of applications in the field of protease activity detection SEQ ID No. 1

[0043] MKGGKRVQTARPNRINGEIRAQEVRLTGLEGEQLGIVSLREALEKAEEAGVDL

[0044] VEISPNAEPPVCRIMDYGKFLYEKSKSSKEQKKK(Xaa)nQKVIQVKEIKFRPGT

[0045] DEGDYQVKLRSLIRFLEEGDKAKITLRFRGREMAHQQIGMEVLNRVKDDLQEL

[0046] AWESFPTKICGRQMIMVLAPKKKQ

[0047] SEQ ID N°2

[0048] AtgAAAGGCGGAAAACGAGTTCAAACGGCGCGCCCTAACCGTATCAATGGC

[0049] GAAATTCGCGCCCAGGAAGTTCGCTTAACAGGTCTGGAAGGCGAGCAGCT

[0050] TGGTATTGTGAGTCTGAGAGAAGCTCTGGAGAAAGCAGAAGAAGCCGGAG

[0051] TAGACTTAGTCGAGATCAGCCCTAACGCCGAGCCGCCGGTTTGTCGTATA

[0052] ATGGATTACGGCAAATTCCTCTATGAAAAGAGCAAGTCTTCTAAGGAACAG

[0053] AAGAAAAAG(N)nCAAAAAGTTATCCAGGTTAAGGAAATTAAATTCCGTCCTG

[0054] GTCAGATGAAGGCGACTATCAGGTAAAACTCCGCAGCCTGATTCGCTTTC

[0055] TCGAAGAGGGTGATAAAGCCAAAATCACGCTGCGTTTCCGCGGTCGTGAG

[0056] ATGGCGCACCAGCAAATCGGTATGGAAGTGCTTAATCGCGTGAAAGACGA

[0057] TTTGCAAGAACTGGCAGTGGTCGAATCCTTCCCAACGAAGATCTGTGGCC

[0058] GCCAGATGATCATGGTGCTCGCTCCTAAAAAAAAAAAAAAA

[0059] SEQ ID No. 3

[0060] MKGGKRVQTARPNRINGEIRAQEVRLTGLEGEQLGIVSLREALEKAEEAGVDL

[0061] VEISPNAEPPVCRIMDYGKFLYEKSKSSKEQKKKTSAVLQSGFRKQKVIQVKEI

[0062] KFRPGTDEGDYQVKLRSLIRFLEEGDKAKITLRFRGREMAHQQIGMEVLNRVK

[0063] DDLQELAWESFPTKICGRQMIMVLAPKKKQ

[0064] SEQ ID No. 4

[0065] AtgAAAGGCGAAAACGAGTTCAAACGGCGCGCCCTAACCGTATCAATGGC

[0066] GAAATTCGCGCCCAGGAAGTTCGCTTAACAGGTCTGGAAGGCGAGCAGCT

[0067] TGGTATTGTGAGTCTGAGAGAAGCTCTGGAGAAAGCAGAAAAGCCGGAG

[0068] TAGACTTAGTCGAGATCAGCCCTAACGCCGAGCCGCCGGTTTGTCGTATA

[0069] ATGGATTACGGCAAATTCCTCTATGAAAAGAGCAAGTCTTCTAAGGAACAG

[0070] AAGAAAAAGaccagcgccgtgctgcagagcggctttcgcaaaCAAAAAGTTATCCAGGTT

[0071] AAGGAAATTAAATTCCGTCCTGGTACAGATGAAGGCGACTATCAGGTAAAAA

[0072] CTCCGCAGCCTGATTCGCTTTTCTCGAAGAGGGTGATAAAGCCAAAATCAC GCTGCGTTTCCGCGGTCGTGAGATGGCGCACCAGCAAATCGGTATGGAA

[0073] GTGCTTAATCGCGTGAAAGACGATTTGCAAGAACTGGCAGTGGTCGAATC

[0074] CTTCCCAACGAAGATCTGTGGCCGCCAGATGATCATGGTGCTCGCTCCTA

[0075] AGAAGAAACAGtaa

[0076] TABLE 1: Examples of specific protease recognition and cleavage sites PREFERRED EMBODIMENT OF THE INVENTION

[0077] The following is a detailed example of the preferred embodiment of the invention, encompassing all the steps necessary for the proper functioning of the biosensor, specifically the PEPROT-S and PEPROT-SF biosensors. This example is purely illustrative and in no way limits the scope of the invention.

[0078] PEPROT-S

[0079] OBTAINING PEPROT-SF

[0080] The implementation of the fluorescent version of the biosensor involves a technique known as double labeling, which allows the incorporation of two fluorophores, one acceptor and one quencher, at the N- and C-termini of the protein that makes up the biosensor. In this sense, the biosensor features unique cysteines that facilitate the binding of various fluorophore pairs. Conceptually, this allows the biosensor to be adapted to diverse protease activity detection and analysis needs.

[0081] In the specific case of PEPROT-S (fluo), based on the IF3ec protein mutated in the C-terminal domain to allow the introduction of an exposed cysteine, double labeling follows the following protocol:

[0082] The biosensor is subjected to extensive dialysis in labeling buffer (50 mM Hepes pH: 7.1, 100 mM NH4Cl, 10% glycerol, 0.5 mM TCEP) to remove traces of 2-mercaptoethanol, since this reducing agent strongly inhibits the coupling of maleimide dyes to cysteines.

[0083] First, the C-terminus of the engineered cysteine ​​is labeled, as it is exposed and reacts efficiently with maleimide derivatives. A molar excess of Atto-540Q maleimide is incubated on the biosensor in the labeling buffer for 20 min. The reaction is stopped with the addition of 6 mM 2-mercaptoethanol.

[0084] The C-terminally modified biosensor is purified from excess fluorophore using a HiTrap SP HP column. After washing with 100 mM NH4Cl buffer, a one-step elution is performed using 3 mL of 1 M NH4Cl buffer. The CTD540Q biosensor is then dialyzed against labeling buffer containing 2 M urea. Denaturation of the biosensor exposes the cysteine ​​normally buried in the N-terminal domain. The denatured protein is incubated with a molar excess of Atto-488 maleimide for 1 h at room temperature with gentle shaking.

[0085] The biosensor modified in both domains (PEPROT-SF) is purified from excess fluorophore using the HiTrap SP HP column. The eluted proteins are dialyzed against storage buffer (Hepes pH: 7.1, 100 mM NH4Cl, 10% Glycerol, 6 mM 2-mercaptoethanol) and stored in small aliquots at -80 °C. The purity and efficiency of the labeling are evaluated by 15% SDS-PAGE, where fluorescence is observed under a UV transilluminator and total protein by Coomassie blue staining.

[0086] Thus, the fluorescent version of the PEPROT-S biosensor is achieved, which maintains the same protease recognition and cleavage capabilities as its non-fluorescent version, but adds the ability to detect via fluorescence. Similarly, other fluorescent variants containing a single fluorophore in any of their domains are obtained. These PEPROT-SF variants maintain the ability to be observed by SDS-PAGE.

[0087] PROTEASE ACTIVITY DETECTION ASSAY CONDITIONS:

[0088] The test conditions cover the composition of the solution (10 mM phosphate buffer (pH: 7), 100 mM NaCl, 1 mM TCEP, 5% Glycerol) in which the cleavage reaction of the invention (0.5 pM PEPROT-SF) by 0.2 pM MPro, the SARS-Cov2 Mpro protease, will be carried out. PROCEDURE:

[0089] 1 ) Block the required wells of a 96-well Nunc plate with 100 pL of blocking solution (100 mM Carbonate Buffer (pH:8.6, 0.5% BSA) for 1 hour at 37 °C.

[0090] 2) Prepare the reaction mix according to the number of wells using the following conditions: a. Total volume (TV): Total volume per well, 60 pL x Number of wells. b. Reaction mix volume (VM): [Total volume per well- Volume of elements (VE, inhibitors, sensor, M-pro, DMSO, Buffer)] x Number of wells. c. Add 0.1 mg / mL of BSA (VB) d. Mix the required volume of M-Pro cutting reaction buffer (VM-VB) and VB

[0091] 3) Dispense the reaction mixture, M-pro, inhibitors into a multi-well plate and finally add the PEPROT-SF sensor or variants to each corresponding well.

[0092] 4) Read the plate in the fluorescence reader.

[0093] 5) Place the volume of each well in 1.5 mL capacity centrifuge tubes.

[0094] 6) Centrifuge at 10,000 rpm for 5 minutes.

[0095] 7) Place the reaction volume in new centrifuge tubes.

[0096] 8) Add Laemmli buffer (LaB) (60 pL 2X or 12 pL 6X)

[0097] 9) Load 20 pL onto 15% SDS-PAGE gels.

[0098] 10) Run at 100V for 5 min and then at 180V for 55 min.

[0099] 11) Observe the gels in a blue light transilluminator.

[0100] If an inhibitory activity test or inhibition curve is required, incubate the M-Pro and the inhibitors to be tested at room temperature before adding the substrate (sensor) between steps 2 and 3. PLATE READING CONDITIONS:

[0101] In conclusion, this invention provides an invaluable and low-cost tool for the detection and evaluation of protease activity. Although a specific embodiment has been described, it is understood that the invention may be implemented in various ways, without departing from the scope of the invention.

Claims

CLAIM 1. A protease activity biosensor, comprising a protein with two domains, each with a single cysteine, linked by an engineered peptide bond composed of a flexible and interchangeable connector containing a specific recognition site for a protease, and wherein the resulting protein can be efficiently produced.

2. The biosensor according to claim 1, wherein the N and C terminal domains, with single cysteine, are labeled with fluorophores compatible with the FRET technique, enabling the creation of a fluorescent version of the biosensor.

3. The biosensor according to claims 1 and 2, which allows the observation of protease activity through three independent methods: monitoring of donor emission by FRET, monitoring of acceptor emission by FRET, and SDS-PAGE electrophoresis.

4. The biosensor according to claims 1, 2 and 3, wherein the protease activity can be detected in an SDS-PAGE gel, observing the separation of the globular domains of the biosensor by the action of the protease and being reflected as two fluorescent bands if the fluorescent version of the biosensor is used.

5. The biosensor according to claims 1 to 4, which allows the detection of protease activity by SDS-PAGE electrophoresis, not only through the visualization of fluorescent bands corresponding to the globular domains, but also through a standard protein staining, such as Coomassie blue, allowing the observation of the cleavage in the peptide bond by the action of the protease.

6. The biosensor according to claims 1 to 5, wherein the FRET signals can be measured in vitro using equilibrium, steady-state and single-molecule techniques.

7. Use of the biosensor according to any of claims 1 to 5 for measuring protease activity in a variety of experimental environments.

8. Use of the biosensor according to any of claims 1 to 5 for evaluating the efficacy of protease inhibitors, including, but not limited to, small molecules and drug candidates.

9. Use of the biosensor according to any of claims 1 to 5 to study the mechanism of action of proteases and their inhibitors.

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