Fusion protein substrate for protease activity measurement

Abstract

The purpose of the present invention is to provide a novel protease activity measurement method that can be used in a high-throughput manner and enables activity measurement based on the full-length amino acid sequence of a protease.　Provided is a fusion protein substrate for protease activity measurement, said fusion protein substrate containing, in order from the N-terminal side, (i) a signal sequence, a reporter region, a protease target region and a membrane-binding region, or (ii) a signal sequence, a protease target region, a reporter region and a membrane-binding region.

Description

Fusion protein substrates for measuring protease activity

[0001] The present invention relates to a fusion protein substrate for measuring protease activity, a kit for measuring protease activity, and a method for measuring protease activity.

[0002] The ISG15 protein contains two ubiquitin-like domains and is used for post-translational modification (ISG15 modification) by being added to target proteins in the cell in the same way as ubiquitin.

[0003] ISG15 protein expression is induced by interferon stimulation, viral infection, etc., and ISG15 modification is known to play an important role in innate immunity, such as antiviral immunity. Studies using ISG15-deficient mice have shown that ISG15 modification is important for antiviral activity against influenza virus, herpesvirus, and togavirus (Non-Patent Document 1). It has also been reported that ISG15 modification contributes to resistance to intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV) (Non-Patent Document 2).

[0004] On the other hand, some viral proteins are known to undergo ISG15 deconjugation to evade host immunity based on ISG15 modification. Deconjugation is a reaction that cleaves the ISG15 protein portion from ISG15-modified proteins. ISG15 deconjugating enzymes are a type of protease, and examples include the Nsp3 protein of coronaviruses and the L protein of Crimean-Congo hemorrhagic fever virus. Since inhibiting the ISG15 deconjugating activity of viral proteins is expected to inhibit viral replication while simultaneously activating the host immune system, ISG15 deconjugating enzymes are considered ideal targets for drug discovery.

[0005] Western blotting using antibodies such as anti-ISG15 antibodies has been used to detect ISG15 modification and de-ISG15 modification in cells. While this method has the advantage of being able to directly detect ISG15 modification and de-ISG15 modification in cells, it has the drawback of being inapplicable to drug discovery screening due to its low throughput.

[0006] Furthermore, a common method for evaluating protease activity in vitro involves reacting purified recombinant proteins with fluorescently labeled substrate peptides. However, this method requires the purification of active recombinant proteins, which necessitates the use of partial fragments in the case of proteases with large molecular weights or proteases containing transmembrane domains, and the proteins used as substrates are limited to short peptides. Therefore, this method is limited in the range of applicable proteases and substrate proteins, and the detected activity does not adequately reflect the original protease activity.

[0007] Therefore, there is a need for a new method for measuring protease activity that allows high throughput and enables activity measurement based on the full-length amino acid sequence of the protease.

[0008] Lenschow DJ, et al., PROc Natl Acad Sci USA, 2007, 104: 1371-1376.Ritchie KJ, et al., Nature Medicine, 2004, 10: 1374-1378.

[0009] An object of the present invention is to provide a new method for measuring protease activity, which allows for high throughput and enables activity measurement based on the full-length amino acid sequence of a protease.

[0010] To solve the above-mentioned problems, the present inventors have created a new fusion protein substrate for measuring protease activity, which contains a signal sequence, a reporter region, a protease target region, and a transmembrane domain. When this fusion protein substrate is introduced into cells together with a viral protease and expressed on the cell membrane, fragments cleaved by the protease activity are released extracellularly. Protease activity can be measured by detecting the reporter activity of the released fragments in the cell supernatant. The present inventors applied this method to the Nsp3 protein, a protease of the coronavirus SARS-CoV2, and successfully detected its ISG15 deactivation activity. Subsequently, the present inventors found that applying this method to proteases derived from feline coronavirus (FeCoV), human T-cell leukemia virus type 1 (HTLV-1), and African swine fever virus enabled highly sensitive detection of protease activity in general, leading to the completion of the present invention. Based on the above-mentioned research results, the present invention provides the following:

[0011] (1) A fusion protein substrate for measuring protease activity, comprising, from the N-terminus, (i) a signal sequence, a reporter region, a protease target region, and a membrane-binding region, or (ii) a signal sequence, a protease target region, a reporter region, and a membrane-binding region. (2) The fusion protein substrate according to (1), wherein the protease is a viral protease or a deubiquitinating enzyme. (3) The fusion protein substrate according to (2), wherein the viral protease is derived from Coronaviridae, Retroviridae, Asfarviridae, Flaviviridae, Caliciviridae, Picornaviridae, Poxviridae, Herpesviridae, Adenoviridae, Togaviridae, or Matonaviridae. (4) The fusion protein substrate according to (3), wherein the target region of the viral protease derived from Coronaviridae comprises the amino acid sequence set forth in SEQ ID NO: 4, 12, or 13. (5) The fusion protein substrate according to (3), wherein the target region of a viral protease derived from the Retroviridae family comprises any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 21 to 27 and 62 to 70. (6) The fusion protein substrate according to (3), wherein the target region of a viral protease derived from the Asfarviridae family comprises any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 41 to 46. (7) The fusion protein substrate according to (3), wherein the target region of a viral protease derived from the Flaviviridae family comprises any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 73 to 77, 80 to 84, and 86 to 90. (8) The fusion protein substrate according to (3), wherein the target region of a viral protease derived from the Caliciviridae family comprises any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 104 to 108 and 110 to 113. (9) The fusion protein substrate according to (3), wherein the target region of a viral protease derived from the Picornaviridae family comprises any of the amino acid sequences selected from the group consisting of SEQ ID NOs: 93 to 102. (10) The fusion protein substrate according to (2), wherein the target region of the deubiquitinase comprises a ubiquitin protein, a SUMO protein, or an ISG15 protein.(11) The fusion protein substrate according to (10), wherein the ubiquitin protein consists of the amino acid sequence set forth in SEQ ID NO: 33 or 59, an amino acid sequence having 90% or more amino acid identity thereto, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added. (12) The fusion protein substrate according to (10), wherein the SUMO protein consists of the amino acid sequence set forth in SEQ ID NO: 47, 56, 48, 57, 49, 58, or 60, an amino acid sequence having 90% or more amino acid identity thereto, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added. (13) The fusion protein substrate according to (10), wherein the ISG15 protein consists of the amino acid sequence set forth in SEQ ID NO: 4 or 55, an amino acid sequence having 90% or more amino acid identity thereto, or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added. (14) A nucleic acid encoding the fusion protein substrate according to any of (1) to (13). (15) A gene expression vector comprising, in an expressible state, the nucleic acid according to (14). (16) A host cell comprising the gene expression vector according to (15). (17) The host cell according to (16), further comprising, in an expressible state, a gene expression vector comprising, in an expressible state, a base sequence encoding a protease that cleaves the protease target region. (18) A kit for measuring protease activity, comprising one or more selected from the group consisting of the fusion protein substrate according to any one of (1) to (13), the nucleic acid according to (14), the gene expression vector according to (15), and the host cells according to (16) and (17). (19) A method for measuring protease activity, comprising: a reaction step of reacting a host cell expressing a fusion protein substrate for measuring protease activity in a solution with a test protease, wherein the fusion protein substrate comprises, in order from the N-terminus, a signal sequence, a reporter region, a protease target region, and a membrane-binding region; a recovery step of recovering a supernatant from the solution after the reaction; and a measurement step of measuring reporter activity based on the reporter region in the supernatant.(20) A method for measuring protease activity, comprising: a reaction step of reacting a host cell expressing a fusion protein substrate for measuring protease activity in solution with a test protease, wherein the fusion protein substrate comprises, in order from the N-terminus, a signal sequence, a protease target region, a reporter region, and a membrane-binding region; and a measurement step of measuring reporter activity based on the reporter region in the host cell after the reaction. (21) The method according to (19) or (20), in which the test protease cleaves the fusion protein substrate. (22) The method according to any one of (19) to (21), in which the viability of the host cell is measured after the measurement step. This specification includes the disclosure of Japanese Patent Application No. 2021-095348, from which the present application claims priority.

[0012] The fusion protein substrate of the present invention provides a novel method for measuring protease activity that allows high throughput and enables activity measurement based on the full-length amino acid sequence of the protease.

[0013] Figure showing the alignment of the amino acid sequence of ISG15 protein. Figure showing the structure of the ssISG sensor expression plasmid and ssISG sensor. Figure 2A shows the structure of the ssISG sensor expression plasmid. Figure 2B shows the structure of the ssISG sensor. Figure showing the structure of the coronavirus polyprotein PP1ab. The PP1ab protein is cleaved by the protease activity of the Nsp3 protein and Nsp5 protein to produce 15 types of non-structural proteins (Nsp1 to Nsp16). The gray triangle indicates the cleavage site of the Nsp3 protein, and the black triangle indicates the cleavage site of the Nsp5 protein. Figure showing the structure of the ssNsp3 expression plasmid and ssNsp3 protein. Figure 4A shows the structure of the ssNsp3 expression plasmid. Figure 4B shows the structure of the ssNsp3 protein. PL2 proindicates a papain-like protease domain. Figures 5A and 5B show an exemplary principle and results of a method for evaluating ISG15 deconjugation activity. Figure 5B shows an example of the principle of the method for evaluating ISG15 deconjugation activity. Figure 5B shows the results of detecting luciferase activity in the culture supernatant of cells expressing the ssNsp3(WT) protein or the ssNsp3(CHAA) protein together with the ssISG sensor. Figure 5C shows the results of detecting luciferase activity in the culture supernatant of cells expressing the ssNsp3(WT) protein with a signal sequence at the N-terminus or the Nsp3(-ss) protein without a signal sequence together with the ssISG sensor. The presence or absence of a signal sequence (ss) at the N-terminus of the NSP protein is indicated by + / -. The results are the average of six experiments, and the error bars indicate the standard deviation. Figure 5C shows the structure of the ssISG sensor and the results of evaluating the ISG15 deconjugation activity of each ssISG sensor. Figure 6A shows the structures of the ssISG sensor (GG), ssISG sensor (AA), ssISG sensor (ΔD1), and ssISG sensor (ΔD1+2). Figure 6B shows the results of luciferase activity detection in the culture supernatant of cells expressing each ssISG sensor together with the ssNsp3(WT) protein or ssNsp3(CHAA) protein. The results represent the average of six experiments, and the error bars indicate the standard deviation. This figure shows the structures of the coronavirus polyprotein PP1ab and the ssPRO-TM sensor. The PP1ab protein is cleaved by the protease activity of the Nsp3 and Nsp5 proteins to produce 15 nonstructural proteins (Nsp1 to Nsp16). The gray triangle indicates the cleavage site of the Nsp3 protein, and the black triangle indicates the cleavage site of the Nsp5 protein. The sequences of the cleavage sites between Nsp4 / Nsp5 and between Nsp5 / Nsp6 are shown on the right side of the figure. The structure of the PRO-TM sensor is shown below the figure. 1 shows the results of detecting luciferase activity in the culture supernatant of cells expressing the ssNsp5(WT) protein or ssNsp5(C144A) protein together with the ssNsp4 / 5-TM sensor or ssNsp5 / 6-TM sensor.Results are shown as averages from n=6 experiments, with error bars indicating standard deviation. Figure 11 shows the structures of the HTLV-1 polyprotein GAG-PRO-POL and the ssPRO-TM sensor. The GAG-PRO-POL protein is cleaved by the protease activity of the PRO protein to produce individual proteins, such as GAG and PRO proteins. The seven sequences cleaved by the PRO protein are shown in the center of the figure. Results are shown for luciferase activity detected in the culture supernatant of cells expressing ssGAG-PRO (WT or D32A) protein, along with the ssPRO-TM sensor. Results are shown as averages from n=4 experiments, with error bars indicating standard deviation. Figure 11 shows the results of evaluating the inhibitory activity of the protease inhibitor GC376. Figure 11A shows the structure of the protease inhibitor GC376. Figure 11B shows the results of evaluating the protease activity of the SARS-CoV2 Nsp3 and Nsp5 proteins and the HTLV-1 PRO protein under different GC376 concentrations. Figure 11C shows the results of measuring cell viability after culture using a WST8 assay. Figure 11D shows the structures of African swine fever virus polyproteins pp220 and pp60. The pp220 protein is cleaved by the S273R protease to generate five proteins. Similarly, the pp60 protein generates three proteins. The black triangle indicates the cleavage site of the S273R protease. Figure 11E shows the structure of an ssPRO-TM sensor containing the target sequence of the S273R protease. The six cleaved sequences are shown at the top of the figure. Figure 11F shows the results of detecting luciferase activity in the culture supernatant of cells expressing the ssPRO-TM sensor and the pS273R (WT or C232A) protein. The results show the average of four experiments, and the error bars indicate the standard deviation. Figure 11F shows the structure of each sensor and the results of evaluating its protease activity. Figure 15A shows the structures of the hSUMO-1 sensor, hSUMO-2 sensor, hISG15 sensor, and 2xUb sensor. FIG. 15B shows the results of detecting luciferase activity in the culture supernatant of cells expressing each sensor alone or each sensor together with the pS273R (WT or C232A) protein.The results are the average of n=6 experiments, and the error bars indicate the standard deviation.

[0014] 1. Fusion Protein Substrate for Measuring Protease Activity 1-1. Overview A first aspect of the present invention is a fusion protein substrate for measuring protease activity. The fusion protein substrate of the present invention comprises an N-terminal signal sequence and a C-terminal membrane-binding region, with a reporter region and a protease target region between them. When the fusion protein substrate of this aspect is cleaved by a protease, a fragment containing the reporter region is detected.

[0015] 1-2. Definitions The following terms frequently used in this specification are defined below.

[0016] As used herein, "ISG15 (Interferon-Stimulated Gene 15kDa) protein" refers to a protein containing ubiquitin-like domain 1 and ubiquitin-like domain 2 (Figure 1). Similar to ubiquitin, ISG15 protein functions in post-translational modification by being attached to other proteins.

[0017] As used herein, the term "ISG15 protein" includes ISG15 proteins derived from any species. Specifically, the term "ISG15 protein" includes human ISG15 proteins (e.g., the mature human ISG15 protein consisting of the amino acid sequence set forth in SEQ ID NO: 4 or the precursor human ISG15 protein consisting of the amino acid sequence set forth in SEQ ID NO: 34) and their orthologs. Examples of orthologs of human ISG15 proteins include the precursor porcine ISG15 protein consisting of the amino acid sequence set forth in SEQ ID NO: 35, the feline ISG15 protein consisting of the amino acid sequence set forth in SEQ ID NO: 36, and the bovine ISG15 protein consisting of the amino acid sequence set forth in SEQ ID NO: 37 (Figure 1). Porcine and human ISG15 proteins are produced as precursor proteins, and then the C-terminal amino acid sequence (8 amino acids in humans) from the "LRLRGG" sequence is deleted to form the mature ISG15 protein. In contrast, feline and bovine ISG15 proteins are produced as mature forms with the C-terminal sequence "LRLRGG" (Figure 1).

[0018] As used herein, the term "ISG15 gene" refers to a gene encoding an ISG15 protein. Specific examples of ISG15 genes include those encoding mature or precursor ISG15 proteins consisting of the amino acid sequence shown in SEQ ID NO: 4 or 34, such as the human ISG15 gene consisting of the nucleotide sequence shown in SEQ ID NO: 38 or 39.

[0019] As used herein, "ISG15 conjugation" refers to a protein modification in which an ISG15 protein is added to a target protein. In this modification, the ISG15 protein is covalently attached, primarily to lysine residues, of the target protein. ISG15 conjugation is a post-translational modification that, like ubiquitination, is catalyzed by three types of enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3). The enzymes that catalyze the ISG15 conjugation of proteins are referred to as "ISG15 conjugation enzymes."

[0020] As used herein, "ISG15 depletion" refers to a reaction that cleaves the bond between an ISG15 protein and its bound target protein. In this process, the amide bond is typically cleaved by hydrolysis or elimination at the C-terminal end of the "LRLRGG" sequence located at the C-terminus of the mature ISG15 protein. The ISG15 depletion process is included in protease reactions.

[0021] As used herein, the term "ISG15 deconjugating enzyme" refers to an enzyme that catalyzes the ISG15 deconjugation modification. ISG15 deconjugating enzymes are a type of protease. Examples of ISG15 deconjugating enzymes include virus-derived enzymes and host-derived enzymes. Examples of virus-derived ISG15 deconjugating enzymes include the Nsp3 protein of coronaviruses and the L protein of Crimean-Congo hemorrhagic fever virus. Examples of host-derived ISG15 deconjugating enzymes include the USP18 (ubiquitin-specific peptidase 18) protein. USP18 is also known as a deubiquitinase.

[0022] "Protease" is a general term for enzymes that catalyze the cleavage or degradation of peptides or proteins, and is also called peptidase or proteinase. Generally, proteases cleave substrates by hydrolysis or elimination of amide bonds. Proteases include cysteine ​​proteases, serine proteases, threonine proteases, aspartic acid proteases, metalloproteases, glutamic acid proteases, and asparagine peptide lyases. Examples of serine proteases include plasmin, thrombin, subtilisin, chymotrypsin, trypsin, elastase, kallikrein, granzyme, chymase, FSAP protein, and V8 protein. Examples of cysteine ​​proteases include papain, cathepsin B, cathepsin H, cathepsin L, and calpain, as well as papain-like proteases such as the Nsp3 protein described below and 3C-like proteases such as the Nsp5 protein. Examples of aspartic acid proteases include pepsin and renin. Examples of metalloproteases include collagenase, gelatinase, thermolysin, ACE protein, and ADAMTS protein. Further examples of proteases include deubiquitinating enzymes, ISG15 deconjugating enzymes, signal peptidases, secretases (e.g., α-secretase, β-secretase, and γ-secretase), cathepsins, meprin A, and meprin B. Proteases are classified into exopeptidases, which cleave a single amino acid at the N- or C-terminus of a peptide or protein, and endopeptidases, which cleave internal peptide bonds. In this specification, preferred proteases are endopeptidases. The protease may be a full-length protease or an active fragment thereof.

[0023] As used herein, the term "viral protease" encompasses proteases derived from any virus. It is known that viral proteases may be involved in cleaving polyproteins during viral replication. Specific examples of viruses from which viral proteases are derived include those described below. Examples of viral proteases include NSP2 protein, NSP3 protein, NSP5 protein, L protease, 3C protease, NS6 protease, NS2B-NS3 protease, and S273R protease (pS273R). Specific examples of viral proteases in single-stranded positive-strand RNA viruses include the NSP2 protein of the Nidoviridae order (e.g., Arteriviridae); the NSP3 and NSP5 proteins of the Coronaviridae family; the L protease and 3C protease of the Picornaviridae order (e.g., foot-and-mouth disease virus); the NS6 protease of the Caliciviridae family (e.g., norovirus); the NS2B-NS3 protease of the Flaviviridae family (e.g., Japanese encephalitis virus, Zika virus, dengue virus, hepatitis C virus, and swine fever virus); and proteases of the Retroviridae family (e.g., human immunodeficiency virus (HIV), human T-cell leukemia virus type 1 (HTLV-1), and bovine leukemia virus (BLV)).

[0024] As used herein, the term "deubiquitinating enzyme" refers to an enzyme that catalyzes a deubiquitination reaction. 95 types of deubiquitinating enzymes are known in humans, and those classified as cysteine ​​proteases or metalloproteases are known. Examples of deubiquitinating enzymes include the USP superfamily, OTU superfamily, MJD superfamily, UCH superfamily, and SUMO desulfonating enzymes. Specific examples of deubiquitinating enzymes belonging to the USP superfamily include USP1, USP2, USP3, USP4, USP5, USP6, USP7, USP8, USP9X, USP9Y, USP10, USP11, USP12, USP13, USP14, USP15, USP16, USP17, USP17L2, USP17L3, USP17L4, and USP17L5, in addition to the aforementioned USP18 protein, which can also catalyze ISG15 deubiquitination. , USP17L7, USP17L8, USP19, USP20, USP21, USP22, USP23, USP24, USP25, USP26, USP27X, USP28, USP29, USP30, USP31, USP32, USP33, USP34, USP35, USP36, USP37, USP38, USP39, USP40, USP41, USP42, USP43, USP44, USP45, and USP46 proteins. Specific examples of deubiquitinating enzymes belonging to the OTU superfamily include the OTUB1 and OTUB2 proteins. Specific examples of deubiquitinating enzymes belonging to the MJD superfamily include the ATXN3 and ATXN3L proteins. Specific examples of enzymes belonging to the UCH superfamily include the BAP1, UCHL1, UCHL3, and UCHL5 proteins. Specific examples of de-SUMOylating enzymes include the SENP1 and SENP2 proteins. In deubiquitination by deubiquitinating enzymes, the amide bond is cleaved at the C-terminal end of the "LRLRGG" sequence, which is normally located at the C-terminus of ubiquitin, by hydrolysis or elimination, similar to the above-mentioned ISG15 deconjugation modification.

[0025] As used herein, the term "fusion protein substrate" refers to a fusion protein that can function as a substrate for a protease. The fusion protein substrate contains at least a sequence that can be a target for protease cleavage, and can contain a reporter region for detecting fragments generated after cleavage by the protease, a functional module such as a localization signal (e.g., a transmembrane domain), etc.

[0026] As used herein, the term "virus" is not limited and may refer to either a DNA virus or an RNA virus. Examples of DNA viruses include Asfarviridae, Poxviridae (e.g., smallpox virus), Herpesviridae (e.g., herpes simplex virus, EB virus, and varicella-zoster virus), and Adenoviridae. Examples of RNA viruses include Retroviridae (e.g., human T-cell leukemia virus type 1 and human immunodeficiency virus (e.g., HIV-1 and HIV-2)), Togaviridae, Coronaviridae (e.g., SARS-CoV and SARS-CoV2), Flaviviridae (e.g., dengue virus, yellow fever virus, Japanese encephalitis virus, hepatitis C virus, and swine fever virus), Caliciviridae (e.g., norovirus, feline calicivirus), Filoviridae (e.g., Ebola virus), Matonaviridae (e.g., rubella virus), and Picornaviridae (e.g., poliovirus, foot-and-mouth disease virus).

[0027] Coronaviruses are relatively large RNA viruses that belong to the Coronaviridae family and have a genome of approximately 30 kb of positive-strand RNA. Viruses in the Coronaviridae family are broadly divided into the Lethovirinae and Orthocoronavirus subfamily. The Orthocoronavirus subfamily includes the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus genera. Examples of Alphacoronavirus include human coronavirus NL63, human coronavirus 229E, feline coronavirus, and porcine epidemic diarrhea virus. Examples of Betacoronavirus include MERS-CoV (Middle East respiratory syndrome coronavirus), SARS-CoV (SARS coronavirus), and SARS-CoV2 (also known as the novel coronavirus (nCoV) or Wuhan seafood market pneumonia virus). Examples of Gammacoronavirus include avian infectious bronchitis virus.

[0028] The coronavirus genome contains the approximately 20 kb ORF1ab gene at the 5' end, which encodes 15 nonstructural proteins (Nsp1 to Nsp16 proteins) (Fig. 3). The ORF1ab gene produces a large polyprotein, pp1ab, consisting of approximately 7,000 amino acid residues, including the Nsp1 to Nsp16 proteins. The pp1ab protein is then cleaved into 15 independent proteins (Nsp1 to Nsp16 proteins). The cleavage at 14 sites between the Nsp1 to Nsp16 proteins is mediated by the Nsp3 protein (PL3), a papain-like protease. pro ) and the Nsp5 protein, a 3C-like protease (also called 3CL proThe three cleavages of Nsp1 / Nsp2, Nsp2 / Nsp3, and Nsp3 / Nsp4 are catalyzed by the Nsp3 protein, while the remaining 11 cleavages are catalyzed by the Nsp5 protein. Because the Nsp5 protein catalyzes 11 cleavages, it is also called the "main protease." The Nsp3, Nsp4, and Nsp6 proteins contain transmembrane domains and are involved in the formation of double membrane vesicles (DMVs), which are important for coronavirus replication. In addition, the Nsp3 protein, which is involved in the three cleavages of the pp1ab protein, has ISG15 deconjugation activity and is known to be involved in evading host immunity through ISG15 modification.

[0029] Within the Coronaviridae family, the structure of the Nsp3 protein can vary depending on the virus species. The Nsp3 proteins of viruses belonging to the Alphacoronavirus and Betacoronavirus clade A contain two papain-like protease domains, PL1 and PL2. pro and PL2 pro On the other hand, the Nsp3 proteins of viruses classified into clades B, C, and D of the Betacoronavirus genus, including SARS-CoV2, and the Deltacoronavirus genus, contain PL1 pro Excluding PL2 pro Only contains (Figure 3).

[0030] As used herein, the term "retrovirus" is not limited to viruses belonging to the Retroviridae family, and examples include human T-cell leukemia virus type 1 (HTLV-1) and human immunodeficiency virus (HIV).

[0031] As used herein, "Asfarvirus" refers to any virus belonging to the Asfarviridae family. The only known virus belonging to the Asfarviridae family is African swine fever virus (ASFV). African swine fever virus transmits African swine fever, a highly fatal infectious disease that infects pigs and wild boars and is characterized by fever and hemorrhagic lesions throughout the body. African swine fever virus can be transmitted by ticks or through animal-to-animal contact. African swine fever virus has a genome consisting of linear double-stranded DNA of approximately 170 kb to 190 kb. The polyprotein pp220 (also known as CP2475L) encoded by the African swine fever virus genome is cleaved by the S273R protease to produce five distinct proteins (p5, p34, p14, p37, and p150). The polyprotein pp60 (also called CP530R) encoded by the viral genome is cleaved by the S273R protease to generate three independent proteins (p15, p35, and p8).

[0032] A "linker peptide" is a peptide that can be inserted between the fused moieties in the fusion protein substrate of the present invention so that each fused moiety can perform its intended function. The length of the linker peptide is not limited, but examples include a length of 3 to 100 amino acids, preferably 5 to 50 amino acids. Peptides containing many amino acids with relatively small side chains, such as serine and glycine, are often used.

[0033] A "tag peptide" is a peptide that can be used to label proteins. It is typically a short peptide consisting of a dozen to several tens of amino acids and is used for protein detection or purification. Typically, a nucleotide sequence encoding the tag peptide is linked to the 5' or 3' end of a gene encoding the protein to be labeled, and the protein is expressed as a fusion protein with the tag peptide, thereby achieving labeling. Various types of tag peptides have been developed in the field, and any tag peptide may be used. The tag peptide may be an epitope tag. Specific examples of tag peptides include FLAG, HA, His, DAP, PA, GST, myc, MBP tags, and streptavidin.

[0034] As used herein, the term "active fragment" refers to a polypeptide fragment that contains a partial region of a protein and retains at least 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, or an equivalent amount of the activity of the full-length protein. The amino acid length of the active fragment is not particularly limited as long as it retains the activity of the protein.

[0035] As used herein, the term "plurality" refers to an integer of 2 or more, for example, an integer of 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.

[0036] As used herein, "amino acid identity (amino acid sequence identity)" refers to the percentage (%) of matching amino acid residues in the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when the sequences are aligned by inserting appropriate gaps into one or both sequences as needed to maximize the number of identical amino acid residues. "Nucleotide identity (nucleotide sequence identity)" can be determined in a similar manner.

[0037] As used herein, "amino acid substitution" refers to substitutions among the 20 types of amino acids that constitute natural proteins. Amino acid substitutions are preferably within conservative amino acid groups that have similar properties, such as charge, side chain, polarity, and aromaticity. Examples of such substitutions include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, PRO), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp).

[0038] 1-3. Configuration The fusion protein substrate of the present invention comprises an N-terminal signal sequence and a C-terminal membrane-binding region, with a reporter region and a protease target region between them. The reporter region can be located either N-terminal or C-terminal to the protease target region.

[0039] In one embodiment, the fusion protein substrate of the present invention comprises, in order from the N-terminus, a signal sequence, a reporter region, a protease target region, and a membrane-binding region. This arrangement is preferred when the size of the protease target region is relatively small, for example, when the protease target region is 1000 amino acids or less, 900 amino acids or less, 800 amino acids or less, 700 amino acids or less, 600 amino acids or less, 500 amino acids or less, 400 amino acids or less, 300 amino acids or less, 200 amino acids or less, 150 amino acids or less, 100 amino acids or less, 50 amino acids or less, 40 amino acids or less, 30 amino acids or less, 20 amino acids or less, or 10 amino acids or less, and / or 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, 50 kDa or less, 40 kDa or less, 30 kDa or less, 20 kDa or less, 15 kDa or less, 10 kDa or less, 5 kDa or less, 4 kDa or less, 3 kDa or less, 2 kDa or less, or 1 kDa or less.

[0040] In another embodiment, the fusion protein substrate of the present invention comprises, in order from the N-terminus, a signal sequence, a protease target region, a reporter region, and a membrane-binding region. This arrangement is preferred when the size of the protease target region is relatively large, for example, when the protease target region is 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 100 or more, 150 or more, 200 or more, 300 or more, 400 or more, 500 or more, 600 or more, 700 or more, 800 or more, 900 or more, or 1000 or more amino acids, and / or 1 kDa or more, 2 kDa or more, 3 kDa or more, 4 kDa or more, 5 kDa or more, 10 kDa or more, 15 kDa or more, 20 kDa or more, 30 kDa or more, 40 kDa or more, 50 kDa or more, 60 kDa or more, 70 kDa or more, 80 kDa or more, 90 kDa or more, or 100 kDa or more. In such cases, the reporter activity of the reporter region may be suppressed or inhibited by the presence of the protease target region, and thus the reporter activity may be increased by its release after cleavage. By detecting the change in reporter activity, protease activity can be assessed.

[0041] As used herein, the term "signal sequence" refers to an extracellular localization signal required for the extracellular secretion of a protein biosynthesized by gene expression, and is also called a signal peptide. A signal sequence may contain a region composed of hydrophobic amino acids. After translation, a signal sequence is cleaved and removed by a signal peptidase before being extracellularly translocated. Signal sequences are present at the N-terminus of many secretory proteins and membrane proteins, and are, for example, 15 to 30 amino acids long. A signal sequence may be derived from any biological species, either human or non-human, such as insect cells or viruses. Examples of human-derived signal sequences include the immunoglobulin κ light chain signal sequence (SEQ ID NO: 1) and variants thereof having one or more amino acid insertions, deletions, substitutions, and / or additions.

[0042] As used herein, the term "reporter region" refers to a region that can be used as a label when detecting a fragment generated by protease cleavage from the fusion protein substrate of the present invention (when the reporter region is located on the N-terminal side of the protease target region, this refers to a fragment released extracellularly; when the reporter region is located on the C-terminal side of the protease target region, this refers to a fragment that remains fixed to the membrane). Typically, this refers to a polypeptide that can determine the presence or absence of a target fragment or a polypeptide that can measure the amount of the fragment based on the reporter activity contained therein. Examples of reporter regions include fluorescent proteins, luminescent proteins, and enzyme proteins.

[0043] As used herein, the term "reporter activity" refers to an activity contained in a reporter region that enables detection of the reporter region. Examples include the fluorescent activity of a fluorescent protein, the luminescent activity of a luminescent protein, and the enzymatic activity of an enzyme protein.

[0044] A "fluorescent protein" refers to a protein that emits fluorescence of a specific wavelength when irradiated with excitation light of a specific wavelength. It may be either natural or non-natural. Furthermore, there are no particular limitations on the excitation wavelength or fluorescence wavelength. Specific examples include CFP, BFP, RFP, mCherry, DsRed (including derivatives such as 3xP3-DsRed), YFP, PE, PerCP, APC, GFP (including derivatives such as EGFP and 3xP3-EGFP), and the like.

[0045] The term "luminescent protein" refers to a substrate protein that can emit light without requiring excitation light or an enzyme that catalyzes the luminescence of the substrate protein. Examples include luciferin or aequorin as the substrate protein and luciferase (e.g., Renilla luciferase, Gaussia luciferase, firefly luciferase, and bacterial luciferase) as the enzyme.

[0046] A "chromoprotein" is a protein, usually an enzyme, involved in the biosynthesis of a pigment or capable of chemical detection by a pigment when a substrate is provided. Specific examples of such enzymes include horseradish peroxidase (HRP), alkaline phosphatase (AP), β-galactosidase (LacZ), β-glucuronidase (GUS), and melanin-based pigment synthesis proteins.

[0047] In addition to the above examples, a tag peptide or epitope sequence that can be detected by an antibody or the like can also be used as the reporter region.

[0048] As used herein, the term "protease target region" refers to a region that can be a cleavage target for a protease. Examples of protease target regions include regions that can be targeted by any of the proteases described above. The protease target region may be, for example, a target region for a viral protease, a target region for a deubiquitinating enzyme, or a target region for an ISG15 deconjugating enzyme.

[0049] Specific examples of target regions of viral proteases include, but are not limited to, the target sequence of a coronavirus Nsp3 protein (e.g., the amino acid sequence of the mature or precursor form of human ISG15 protein shown in SEQ ID NO: 4 or 34), the target sequence of a coronavirus Nsp5 protein (e.g., SEQ ID NO: 12 or 13), the target sequence of a PRO protein that is a human T-cell leukemia virus type 1 (HTLV-1) protease (e.g., SEQ ID NOs: 21 to 27), the target sequence of a pS273R protein that is an African swine fever virus (ASFV) protease (e.g., the pp220 p5 / p34 cleavage site, pp220 p34 / p14 cleavage site, pp220 p14 / p37 cleavage site, pp220 p37 / p150 cleavage site, pp60 p15 / p35 cleavage site, and pp60 p35 / p8 cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 41 to 46), and the target sequence of a human immunodeficiency virus 1 (Human Immunodeficiency Virus ... target sequences of the HIV-1 (HIV-1) protease (SEQ ID NO: 61) (e.g., HIV MA / CA cleavage site, HIV CA / p2 cleavage site, HIV p2 / NC cleavage site, HIV NC / p1 cleavage site, HIV p1 / p6 cleavage site, HIV TF / PR cleavage site, HIV PR / RT cleavage site, HIV RT / RH cleavage site, and HIV RH / IN cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 62 to 70); target sequences of the HCV NS2-3 protein (SEQ ID NO: 71) or HCV NS3 protein (SEQ ID NO: 72), which are proteases of Hepatitis C virus (HCV) (e.g., HCV NS2 / NS3 cleavage site, HCV NS3 / NS4A cleavage site, HCV NS4A / NS4B cleavage site, HCV NS4B / NS5A cleavage site, and HCV NS5A / RdRp cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 73 to 77); The target sequences of the CSFV Npro protein (SEQ ID NO: 78) or CSFV NS3 protein (SEQ ID NO: 79), which are proteases of the CSFV virus (CSFV) (for example, the CSFV Npro / C cleavage site, the CSFV NS3 / NS4A cleavage site, and the CSFV NS4A cleavage site, which are the amino acid sequences shown in SEQ ID NOs: 80 to 84, respectively)a target sequence of the JEV NS3 protein (SEQ ID NO: 85), which is a protease of Japanese encephalitis virus (JEV) (for example, a JEV C / pr cleavage site, a JEV NS2B / NS3 cleavage site, a JEV NS3 / NS4A cleavage site, a JEV NS4A / NS4B cleavage site, and a JEV NS4B / NS5 cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 86 to 90); a target sequence of the FMDV Npro protein (SEQ ID NO: 91) or FMDV 3Cpro protein (SEQ ID NO: 92), which is a protease of Foot-and-mouth disease virus (FMDV) (for example, an FMDV L / VP4 cleavage site, an FMDV VP3 / VP1 cleavage site, and an FMDV VP4 cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 93 to 102, respectively); VP1 / 2A cleavage site, FMDV 2B / 2C cleavage site, FMDV 2C / 3A cleavage site, FMDV 3A / 3B cleavage site, FMDV 3B1 / 3B2 cleavage site, FMDV 3B2 / 3B3 cleavage site, FMDV 3B / 3C cleavage site, and FMDV 3C / 3D cleavage site), target sequences of the NoV NS6 protein (SEQ ID NO: 103) which is a protease of Norovirus (NoV) (e.g., NoV NS2 / NS3 cleavage site, NoV NS3 / NS4 cleavage site, NoV NS4 / NS5 cleavage site, NoV NS5 / NS6 cleavage site, and NoV NS6 / NS7 cleavage site consisting of the amino acid sequences shown in SEQ ID NOs: 104 to 108, respectively), FCV protease Examples of target sequences include the Pro protein (SEQ ID NO: 109) (for example, the FCV p32 / p39 cleavage site, the FCV p39 / p30 cleavage site, the FCV p30 / p13 cleavage site, and the FCV p13 / Pro cleavage site, each consisting of the amino acid sequences shown in SEQ ID NOs: 110 to 113).

[0050] Specific examples of target regions for deubiquitinases include ubiquitin proteins, SUMO proteins, and ISG15 proteins. The biological species from which ubiquitin proteins, SUMO proteins, and ISG15 proteins are derived are not particularly limited. Examples of ubiquitin proteins include human ubiquitin protein (SEQ ID NO: 33), mouse ubiquitin protein (SEQ ID NO: 33), and yeast ubiquitin protein (SEQ ID NO: 59). Note that ubiquitin proteins can serve as target sequences for USP18 proteins. SUMO proteins are not limited as long as they are SUMO family proteins, and examples include SUMO-1 proteins (e.g., human SUMO-1 protein (SEQ ID NO: 47), mouse SUMO-1 protein (SEQ ID NO: 56)), SUMO-2 proteins (e.g., human SUMO-2 protein (SEQ ID NO: 48), mouse SUMO-2 protein (SEQ ID NO: 57)), SUMO-3 proteins (e.g., human SUMO-3 protein (SEQ ID NO: 49), mouse SUMO-3 protein (SEQ ID NO: 58)), and yeast SMT3 protein (SEQ ID NO: 60). Examples of ISG15 proteins include the mature human ISG15 protein (SEQ ID NO: 4) and mouse ISG15 protein (SEQ ID NO: 55). The ubiquitin protein, SUMO protein, and ISG15 protein may have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid identity to any of the amino acid sequences shown above (e.g., human ubiquitin protein (SEQ ID NO: 33), human SUMO-1 protein (SEQ ID NO: 47), human SUMO-2 protein (SEQ ID NO: 48), human SUMO-3 protein (SEQ ID NO: 49), or human ISG15 protein (SEQ ID NO: 4)), or may have one or more amino acid deletions, substitutions, or additions in any of the above amino acid sequences.

[0051] Specific examples of target regions for ISG15 deconjugation enzymes include the target sequences of the coronavirus Nsp3 protein and USP18 protein mentioned above.

[0052] The protease target region is not necessarily limited to a region that is actually cleaved by a protease, but may also be a region that has the potential to be a cleavage target, such as a candidate sequence.

[0053] As used herein, the term "membrane-binding domain" refers to a domain that stably integrates or attaches the fusion protein substrate of the present invention to a membrane. Examples of membrane-binding domains include membrane proteins or fragments thereof, as described below, and artificial transmembrane domains that primarily contain hydrophobic amino acid residues.

[0054] As used herein, the term "membrane protein" includes integral membrane proteins, peripheral membrane proteins, and lipid-anchored proteins.

[0055] An "integral membrane protein" is a protein that can be at least partially embedded in a membrane, and includes integral monotopic proteins and transmembrane proteins. An integral monotopic protein is a membrane protein that does not completely span the membrane but protrudes from only one side of the membrane. A transmembrane protein is a membrane protein that completely spans the membrane. Transmembrane proteins are divided into single-pass transmembrane proteins that have one transmembrane domain and multi-pass transmembrane proteins that have two or more transmembrane domains. A specific example of a single-pass transmembrane protein is an immunoglobulin heavy chain. A specific example of a multi-pass transmembrane protein is a choline transporter, a histamine H1 receptor, and a G protein-coupled receptor.

[0056] A "peripheral protein" is a protein that is not itself embedded in the membrane, but is anchored to the membrane by association with lipids or integral membrane proteins.

[0057] A "lipid-anchored protein" is a protein that is anchored to a membrane by lipids added through lipid modification. Specific examples include glycosylphosphatidylinositol (GPI)-conjugated proteins, prenylated proteins, cholesterol-conjugated proteins, and fatty acid-acylated (e.g., S-palmitoylated and N-myristoylated) proteins.

[0058] Membrane protein fragments include portions of any of the above membrane proteins that can be anchored to a membrane. For example, fragments containing one or more transmembrane domains of transmembrane proteins include the transmembrane domains of CD3, CD4, CD8, CD28, and IL-2 receptor.

[0059] In one embodiment, the membrane binding region may be derived from the same species and / or from the same protein as the signal sequence.

[0060] The fusion protein substrate of the present invention may contain a linker peptide, a tag peptide, and the like, as needed, in addition to the above-mentioned signal sequence, reporter region, protease target region, and membrane-binding region.

[0061] 1-4. Effects When the fusion protein substrate of the present invention contains, in order from the N-terminus, a signal sequence, a reporter region, a protease target region, and a membrane-binding region, cleavage of the protease target region results in the release of a fragment containing the reporter region outside the cell, and reporter activity can be detected in the cell supernatant.

[0062] When the fusion protein substrate of the present invention comprises, in order from the N-terminus, a signal sequence, a protease target region, a reporter region, and a membrane-binding region, a change in reporter activity resulting from the release of suppression or inhibition by cleavage of the protease target region can be detected in cells.

[0063] 2. Nucleic Acids Encoding Fusion Protein Substrates 2-1. Overview A second aspect of the present invention is a nucleic acid encoding a fusion protein substrate.

[0064] 2-2. Configuration The "nucleic acid encoding a fusion protein substrate" may be any nucleic acid encoding any of the fusion protein substrates described in the first aspect. The base sequence of such a nucleic acid is not limited. For example, a codon-optimized base sequence or a base sequence with an initiation codon (ATG) added to the 5' end is also exemplified. Specific examples of nucleic acids encoding a fusion protein substrate include nucleic acids consisting of the base sequences shown in SEQ ID NOs: 8, 15, 17, 29, and 32.

[0065] 3. Gene Expression Vector Comprising a Nucleic Acid Encoding a Fusion Protein Substrate 3-1. Overview A third aspect of the present invention is a gene expression vector (hereinafter referred to as a "fusion protein substrate expression vector") comprising, in an expressible state, the nucleic acid encoding the fusion protein substrate described in the second aspect. The gene expression vector of this aspect can be introduced into cells such as mammalian cells to express the induced protein substrate.

[0066] 3-2. Configuration The gene expression vector of this embodiment comprises the nucleic acid of embodiment 2 and a promoter, and is capable of expressing a fusion protein substrate in cells. In addition to the nucleic acid and promoter components, the gene expression vector may also contain other components such as a marker gene, an intron, an enhancer, a terminator, a replication origin, and / or a polyA signal, as necessary.

[0067] As used herein, the term "gene expression vector" refers to a vector that contains a gene or a gene fragment (hereinafter referred to as "gene, etc.") in an expressible state and includes an expression unit that can control the expression of the gene, etc. The gene expression vector may be a plasmid vector or a viral vector.

[0068] As used herein, "in an expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter. Known vectors include plasmid vectors and viral vectors, and any of these vectors can be used. Generally, a plasmid vector that is easy to manipulate for genetic recombination is sufficient.

[0069] The plasmid vector may be a commercially available expression vector for mammalian cells, such as the pSI vector from PROmega, or a shuttle vector that can replicate between mammalian cells and bacteria such as E. coli.

[0070] Viral vectors that can be used include retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Replication-deficient viral vectors that do not autonomously replicate in infected cells may also be used.

[0071] As used herein, a "promoter" refers to a gene expression regulatory region that can control the expression of a gene, etc., located downstream (3'-end side) in a cell into which a gene expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location where the gene, etc., under their expression control is expressed. A ubiquitous promoter is a promoter that controls the expression of a target gene, etc. (a target gene, etc.) in all cells, i.e., the entire host organism. A site-specific promoter is a promoter that controls the expression of a target gene, etc., only in specific cells or tissues.

[0072] Furthermore, promoters are classified into constitutively active promoters, expression-inducible promoters, and stage-specific active promoters based on the timing of expression. Constitutively active promoters can constitutively express a target gene, etc. in a cell. Expression-inducible promoters can induce the expression of a target gene, etc. in a cell at any stage. Stage-specific active promoters can induce the expression of a target gene, etc. in a cell only at a specific stage of development. All of these promoters can be considered overexpression promoters because they can result in excessive expression of a target gene in a host cell.

[0073] In the gene expression vector of this embodiment, the promoter is a promoter that can induce the expression of a nucleic acid encoding a fusion protein substrate in cells. Since the target cells into which the gene expression vector of the present invention is introduced are preferably mammalian cells, such as human-derived cells, examples of promoters that can express downstream genes in these cells include the CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, EF1α promoter, and Ub promoter.

[0074] As used herein, a "marker gene" refers to a gene encoding a marker protein, also known as a selectable marker or reporter protein. A "marker protein" refers to a peptide whose activity can be used to determine the presence or absence of expression of a marker gene. Detection of activity may involve direct detection of the activity of the marker protein itself, or indirect detection via a metabolite, such as a dye, generated by the activity of the marker protein. Detection may be biological (including detection via binding of peptides or nucleic acids, such as antibodies or aptamers), chemical detection (including enzyme reaction detection), physical detection (including behavioral analysis detection), or sensory detection by the detector (including detection via vision, touch, smell, hearing, and taste).

[0075] The type of labeled protein encoded by the marker gene is not particularly limited, as long as its activity can be detected by methods known in the art. Labeled proteins that are less invasive to transformants during detection are preferred. Examples include tag peptides, drug-resistance proteins, chromoproteins, fluorescent proteins, and luminescent proteins.

[0076] "Drug resistance proteins" are proteins, many of which are enzymes, that confer resistance to drugs such as antibiotics added to culture media. Examples include β-lactamase, which confers resistance to ampicillin, aminoglycoside 3'-phosphotransferase, which confers resistance to kanamycin, tetracycline efflux transporter, which confers resistance to tetracycline, and CAT (chloramphenicol acetyltransferase), which confers resistance to chloramphenicol.

[0077] As used herein, the term "enhancer" is not particularly limited as long as it can enhance the expression efficiency of a gene or a fragment thereof in a vector.

[0078] As used herein, a "terminator" refers to a sequence that can terminate transcription of a gene or the like expressed by the activity of the promoter. The type of terminator is not particularly limited. Preferably, the terminator is derived from the same organism as the promoter. Particularly preferred is a terminator that is paired with the promoter on the genome in a single gene expression control system.

[0079] 4. Host Cell Comprising a Gene Expression Vector 4-1. Overview A fourth aspect of the present invention is a host cell. The host cell of this aspect comprises the fusion protein substrate expression vector described in the third aspect.

[0080] The host cell of this embodiment contains a fusion protein substrate expression vector as an essential component and a protease expression vector as an optional component. The fusion protein substrate expression vector as an essential component is similar to that described in the third embodiment, so the host cell and optional components will be described below.

[0081] The type of host cell is not limited herein. The host cell may be any cell capable of expressing the fusion protein substrate on its membrane and capable of being cleaved by a protease; therefore, it is not limited to mammalian cells. The host cell may be either a prokaryotic or eukaryotic cell. Examples of prokaryotic cells include bacterial cells such as Escherichia coli cells. Examples of eukaryotic cells include fungal cells (e.g., yeast cells), algae cells, plant cells, protozoan cells, insect cells, nematode cells, fish cells, avian cells (e.g., chicken cells), and mammalian cells (e.g., mouse cells, chimpanzee cells, and human cells). Mammalian cells are preferred. Specific examples of mammalian cells include, but are not limited to, CHO cells, COS cells, Vero cells, HEK293 cells, HeLa cells, and NIH3T3 cells. The host cell may be either an adherent cell or a suspension cell, with adherent cells being more preferred.

[0082] A "protease expression vector" is a gene expression vector that contains a nucleic acid encoding a protease, with a signal sequence added to the N-terminus as needed, and a promoter, and is capable of expressing a protease in cells. In addition to the nucleic acid and promoter as components, the protease expression vector may also contain components such as a marker gene (selection marker), an enhancer, an intron, a terminator, a replication origin, and / or a polyA signal as needed. The protease expression vector may be a plasmid vector or a viral vector.

[0083] The protease expressed by the protease expression vector is a protease that cleaves or has the potential to cleave the protease target region contained in the fusion protein substrate that is co-transfected into the host cell.

[0084] 5. Protease Activity Measurement Kit 5-1. Overview A fifth aspect of the present invention is a protease activity measurement kit. The protease activity measurement kit of this aspect includes one or more selected from the group consisting of the substrate of the first aspect, the nucleic acid of the second aspect, the expression vector of the third aspect, and the host cell of the fourth aspect. The protease activity measurement kit of this aspect allows protease activity to be evaluated by quantifying it.

[0085] 5-2. Configuration The protease activity measurement kit of this embodiment comprises one or more selected from the group consisting of the fusion protein substrate of the first embodiment, the nucleic acid encoding the fusion protein substrate of the second embodiment, the fusion protein substrate expression vector of the third embodiment, and a host cell comprising the fusion protein substrate expression vector of the fourth embodiment. The configurations of the substrate, nucleic acid, gene expression vector, and host cell are as described in the first to fourth embodiments. Therefore, detailed explanations will be omitted here.

[0086] In addition to the substrate, nucleic acid, gene expression vector, and / or host cell, the protease activity measurement kit of this embodiment may also include a cell culture medium, a gene introduction reagent, an agent such as an antibiotic to be added to the medium, etc., a luminescent substrate, a chromogenic substrate, or an antibody for detecting reporter activity, or instructions for use.

[0087] 6. Method for Measuring Protease Activity 6-1. Overview The sixth aspect of the present invention is a method for measuring protease activity. According to the measurement method of the present invention, protease activity can be measured using the full-length amino acid sequence of a protease. Because the measurement method of the present invention does not require destruction of host cells to measure protease activity, the viability of host cells can also be measured after measuring protease activity. When the reaction step in the measurement method of the present invention is carried out in the presence of a protease inhibitor, protease inhibitory activity can also be evaluated.

[0088] The specific configuration of each step of the method for measuring protease activity in this embodiment differs depending on whether the reporter region is located on the N-terminal side or the C-terminal side of the protease target region in the fusion protein substrate used for measuring protease activity. Therefore, the two cases will be explained below.

[0089] 6-2-1. When the reporter region is located on the N-terminal side of the protease target region When the reporter region is located on the N-terminal side of the protease target region in the fusion protein substrate, the fusion protein substrate comprises, from the N-terminus, a signal sequence, a reporter region, a protease target region, and a membrane-binding region. In this case, the measurement method of this embodiment includes a reaction step, a recovery step, and a measurement step as essential steps, and a vector introduction step as an optional step. Each step is described in detail below.

[0090] (1) Vector introduction step The "vector introduction step" is a step of introducing the fusion protein substrate expression vector described in the third aspect and / or the protease expression vector described above into a host cell. The specific configurations of the fusion protein substrate expression vector and the protease expression vector are as described in the third and fourth aspects. There are no particular limitations on the method for introducing each vector into a host cell.

[0091] When each vector is a plasmid vector, a gene transfer method (transformation method) known in the art, such as that described in Green & Sambrook, 2012, Molecular Cloning: A Laboratory Manual Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, may be used. Examples of such methods include lipofection, electroporation, microinjection, calcium phosphate, DEAE-Dextran, and particle bombardment.

[0092] When each vector is a viral vector, methods for infecting cells with the virus are known in the art. A functional substance that improves viral infection efficiency, such as fibronectin or a fibronectin fragment (e.g., RetroNectin (registered trademark) or Vecofusin-1 (registered trademark), which are fibronectin fragments having a heparin-binding site), may be used for the introduction of the viral vector.

[0093] (2) Reaction step: The "reaction step" is a step in which a host cell expressing a fusion protein substrate for measuring protease activity is reacted with a test protease in a solution. The purpose of this step is to react the test protease with the fusion protein substrate on the membrane of the host cell.

[0094] The host cells reacted with the test protease in this step are as described in the fourth embodiment. For example, host cells into which a fusion protein substrate expression vector and / or a protease expression vector have been introduced in the vector introduction step, which is the optional step described above, may be used.

[0095] The test protease reacted with the host cell in this step is not limited. For example, the test protease may be one that cleaves the fusion protein substrate expressed in the host cell, one that may cleave the fusion protein substrate, or one that is unknown whether it cleaves the fusion protein substrate or not. Furthermore, the test protease may be a full-length amino acid sequence or an active fragment that has protease activity.

[0096] The method for providing the test protease in solution is not limited.

[0097] In one embodiment, the test protease can be co-expressed in a host cell that expresses a fusion protein substrate. This method can be used whether the test protease is a membrane protein or a secreted protein, but is particularly preferred when the test protease is a membrane protein.

[0098] In another embodiment, the test protease may be expressed in a host cell different from the host cell expressing the fusion protein substrate. In this case, the method for providing the test protease in solution can be easily determined by one skilled in the art depending on the type of test protease, such as a membrane protein, a secreted protein, or other proteins (e.g., cytoplasmic proteins). For example, the test protease may be provided in solution as host cells expressing the test protease, their culture supernatant, their cell lysates, or proteins isolated or purified from any of these.

[0099] In a further embodiment, the protease under test is a recombinant protein and can be added in solution.

[0100] The type of solution used in this reaction is not limited as long as it allows the fusion protein substrate and protease to react with each other. The solution may be, for example, a medium capable of supporting the host cells as living cells.

[0101] The medium may be any medium commonly used in cell culture and known in the art. The medium may be a basal medium, serum-free medium, low-serum medium, or serum-supplemented medium, but typically a basal medium, such as a standard cell culture medium. The term "standard cell culture medium" refers to a versatile basal medium primarily used for culturing various types of mammalian cells. Specific examples include Eagle Minimum Essential Medium (MEM), Dulbecco's Modified Eagle Medium (DMEM), Ham's Nutrient Mixture F10 (Ham's F10), Ham's Nutrient Mixture F12 (Ham's F12), M199, High Performance Medium 199, Roswell Park Memorial Institute-1640 (RPMI-1640), and Dulbecco's Modified Eagle Medium / Ham's Nutrient Mixture F12 (DMEM / F12). The mixing ratio of DMEM / F12 is not particularly limited. Preferably, DMEM and F12 are mixed at a weight concentration ratio of the components ranging from 6:4 to 4:6. Specific compositions of standard cell culture media are known in the art and may be prepared based on the compositions described in appropriate literature (e.g., Kaech S. and Banker G., 2006, Nat. PROtoc., 1(5): 2406-2415). Alternatively, commercially available media from manufacturers such as Thermo Fisher Scientifics and Wako Pure Chemical Industries may be used.

[0102] The reaction conditions for this step can be culture conditions known in the art that are appropriate for the type and origin of the host cell. Typically, the reaction can be carried out at 5% CO and 37°C.

[0103] The reaction time for this step varies depending on the type of host cell and protease, but to achieve the above-mentioned objective of this step, it is usually 1 to 72 hours, preferably 24 to 48 hours.

[0104] In one embodiment, the reaction in this step can be carried out in the presence of a test substance that is a candidate substance for a protease inhibitor. In this case, the measurement method of the present invention can be used to measure the protease inhibitory activity of a protease inhibitor. The type of test substance is not particularly limited. The test substance can be any substance, specifically, natural molecules (e.g., amino acids, peptides, oligopeptides, polypeptides, proteins, nucleic acids, lipids, carbohydrates (e.g., sugars), steroids, glycopeptides, glycoproteins, proteoglycans, etc.), synthetic analogs or derivatives of natural molecules (e.g., peptidomimetics, nucleic acid molecules (e.g., aptamers, antisense nucleic acids, double-stranded RNA (RNAi)), etc.), and non-natural molecules such as small molecular weight compounds (e.g., small inorganic compounds and small organic compounds), as well as mixtures thereof.

[0105] The test substance may be a single substance tested independently, or a mixture of several candidate test substances (e.g., a library, etc.) Examples of libraries containing multiple test substances include synthetic compound libraries and peptide libraries (e.g., combinatorial libraries).

[0106] The effect and efficacy of a test substance can also be examined under several conditions. Such conditions include the time or duration of treatment with the test substance, the amount (large or small), and the number of times. For example, multiple doses can be established by preparing a dilution series of the test substance.

[0107] (3) Recovery step: The "recovery step" is a step of recovering a supernatant from the solution after the reaction. The purpose of this step is to obtain a supernatant that may contain fragments cleaved from the fusion protein substrate and released extracellularly in the above-mentioned reaction step, and to separate the supernatant from host cells containing uncleaved fusion protein substrate.

[0108] The recovery method in this step is not limited as long as it can separate the supernatant containing the fragments containing the reporter region released extracellularly in the reaction step from the host cells. For example, the supernatant containing the fragments containing the reporter region can be obtained by centrifugation, filtration, sedimentation, decantation, pipetting, or a combination thereof. All of these methods can basically be performed according to conventional methods in the field. When the host cells are adherent cells, the supernatant can also be easily recovered using a pipette.

[0109] (4) Measurement step: The "measurement step" is a step of measuring reporter activity based on the reporter region in the supernatant. In this step, the reporter activity contained in the supernatant is detected and quantified, thereby enabling quantitative analysis of protease activity.

[0110] The measurement method in this step varies depending on the type of reporter activity contained in the reporter region, but can be easily determined by one skilled in the art. When the reporter region is derived from a fluorescent protein, the fluorescent activity contained in the supernatant can be measured based on the excitation wavelength and fluorescence wavelength corresponding to the type of fluorescent protein. When the reporter region is derived from a luminescent protein, the luminescent activity in the supernatant can be measured by adding a luminescent substrate of the luminescent protein (e.g., luciferin). When the reporter region is based on an enzyme such as HRP, the enzyme activity in the supernatant can be measured by adding a chromogenic substrate (e.g., HRP substrates such as TMB, 4-CN, DAB, AP substrates such as BCIP / NBT, and LacZ substrates such as X-Gal).

[0111] In one embodiment, the viability of the host cells can be measured after the measuring step. Methods for measuring the viability of host cells are known. For example, the viability of the cells can be measured by detecting the uptake of a dye such as neutral red, trypan blue, or ALAMAR blue into the cells.

[0112] Furthermore, when the above-described reaction step is carried out in the presence of a test substance, the protease activity measured in this step provides protease inhibitory activity, and the test substance can be identified as a protease inhibitor based on the results. For example, the test substance can be identified as a protease inhibitor when the measured protease activity is 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 20% or less, 10% or less, 5% or less, 1% or less, or 0.1% or less compared to the value in the absence of treatment with the test substance.

[0113] 6-2-2. When the reporter region is located on the C-terminal side of the protease target region When the reporter region is located on the C-terminal side of the protease target region in the fusion protein substrate, the fusion protein substrate comprises, from the N-terminus, a signal sequence, a protease target region, a reporter region, and a membrane-binding region. In this case, the measurement method of this embodiment includes a reaction step and a measurement step as essential steps, and a vector introduction step as an optional step. Here, the vector introduction step and reaction step are as described in "(1) Vector introduction step" and "(2) Reaction step" in "6-2-1. When the reporter region is located on the N-terminal side of the protease target region" above, and only the configuration of the measurement step will be described below.

[0114] The "measurement step" is a step of measuring reporter activity based on the reporter region in the host cell after the reaction step. In this step, a change in reporter activity in the host cell due to the release of suppression or inhibition caused by cleavage in the protease target region is detected, and therefore the obtained reporter activity is preferably compared with a reference reporter activity, such as a control that has not reacted with the test protease.

[0115] The reporter activity measurement method in this step can be the same as that for measuring the reporter activity in the supernatant in "6-2-1. When the reporter region is located on the N-terminal side of the protease target region" above, but applied directly to host cells. In the reporter activity measurement method in this step, the supernatant after the reaction step may or may not be removed.

[0116] In the method for measuring reporter activity in this step, the reporter activity measured when the test protease is not reacted with host cells expressing the fusion protein substrate can be used as a negative control. By comparing the reporter activity with that of the negative control, the protease activity of the test protease can be evaluated.

[0117] According to the measurement method of the present invention, protease activity can be evaluated by detecting reporter activity based on the reporter region in the supernatant or in the host cells. In addition, by performing the reaction step in the presence of a test substance, the inhibitory effect on protease activity can also be evaluated.

[0118] The advantage of the measurement method of the present invention is that protease activity can be measured in living cells without destroying the host cells. Based on this advantage, the method of the present invention can also measure cell viability after the measurement step. For example, when a protease inhibitory effect is confirmed in the presence of a test substance in drug screening, it is necessary to distinguish between direct inhibitory effects on the protease and other indirect effects (e.g., inhibitory effects on cell viability). In this respect, the method of the present invention can provide a method with extremely high throughput.

[0119] Example 1: Development of a method for evaluating ISG15 deconjugation activity (Objective) To develop a method for evaluating ISG15 deconjugation activity and to detect the ISG15 deconjugation activity of the Nsp3 protein, a protease of SARS-CoV2.

[0120] (Methods and Results) (1) Preparation of ssISG sensor expression plasmid We prepared ssISG sensor as a fusion protein to serve as a substrate for detecting ISG15 deconjugation activity. ssISG sensor is a fusion protein composed of the following (a) to (f) linked in order from the N-terminus:

[0121] (a) signal sequence of human immunoglobulin κ light chain (SEQ ID NO: 1); (b) secreted luciferase (ssGluc) derived from Gaussia princeps (SEQ ID NO: 2); (c) flexible linker (GGGGS; SEQ ID NO: 3); (d) mature human ISG15 protein (SEQ ID NO: 4); (e) flexible linker (3×GGGGS; SEQ ID NO: 5); (f) transmembrane domain derived from immunoglobulin heavy chain (SEQ ID NO: 6).

[0122] The structure of the ssISG sensor is shown in Figure 2B. The full-length amino acid sequence of the ssISG sensor is shown in SEQ ID NO: 7. The ssISG sensor gene (SEQ ID NO: 8), which encodes this ssISG sensor and whose base sequence was codon-optimized based on codon usage in human cells, was inserted into the mammalian expression vector pCI (Promega). The structure of the ssISG sensor expression plasmid thus constructed is shown in Figure 2A.

[0123] (2) Example of the Principle of the Method for Evaluating ISG15 Deconjugation Activity The principle of the method for evaluating ISG15 deconjugation activity will be described below. The ssISG sensor consisting of (a) to (f) above is translocated to a membrane, and the signal sequence (a) above is cleaved and removed, resulting in an ISG sensor consisting of (b) to (f) above. In this specification, the protein consisting of (b) to (f) after cleavage of the signal sequence is referred to as the "ISG sensor" to distinguish it from the "ssISG sensor" before cleavage of the signal sequence.

[0124] When the ISG sensor is cleaved between (d) and (e) above (the C-terminal position of the "LRLRGG" sequence) by the ISG15 deconjugation activity, a fragment consisting of (b) to (d) above containing luciferase activity is released outside the cell. This released fragment is detected in the cell supernatant based on its luciferase activity. On the other hand, if the ISG sensor is not cleaved between (d) and (e) above, the ISG sensor remains on the cell membrane surface, and luciferase activity is not detected in the cell supernatant.

[0125] The principle of the method for evaluating ISG15 deconjugation activity using the ssISG sensor and the ssNSP3 protein described below is illustrated in Figure 5A. The ISG sensor can be cleaved by the ISG15 deconjugation activity, for example, in the endoplasmic reticulum, in secretory vesicles, and / or on the plasma membrane.

[0126] (3) Preparation of wild-type or inactive ssNsp3 expression plasmids To express the SARS-CoV2 Nsp3 protein as a target for measuring ISG15 deconjugation activity, we prepared an ssNsp3(WT) expression plasmid that expresses an ssNsp3 protein in which a signal sequence has been added to the N-terminus of the wild-type SARS-CoV2 Nsp3 protein (referred to herein as "ssNsp3(WT) protein").

[0127] The ssNsp3(WT) protein contains the immunoglobulin kappa light chain signal sequence (SEQ ID NO: 1) at the N-terminus and wild-type SARS-CoV2 (nCoV) Nsp3 (SEQ ID NO: 9) at the C-terminus, and its full-length amino acid sequence is shown in SEQ ID NO: 10 (Figure 4B).

[0128] The ssNsp3(WT) gene (SEQ ID NO: 11), which encodes the ssNsp3(WT) protein and whose nucleotide sequence was codon-optimized based on codon usage in human cells, was inserted into the mammalian expression vector pCI (Promega). The structure of the ssNsp3(WT) expression plasmid thus constructed is shown in Figure 4A.

[0129] Furthermore, as a negative control for ssNsp3(WT), we prepared an ssNsp3(CHAA) expression plasmid containing the ssNsp3(CHAA) gene encoding the ssNsp3 protein in which both cysteine ​​at position 111 and histidine at position 272, which are thought to be the active sites of the Nsp3 protein, have been replaced with alanine (referred to herein as "ssNsp3(C111A / H272A) protein" or "ssNsp3(CHAA) protein," etc.).

[0130] (4) Evaluation of ISG15 deconjugation activity The ssNsp3(WT) expression plasmid or the ssNsp3(CHAA) expression plasmid was introduced into HeLa cells together with the ssISG sensor expression plasmid.

[0131] Specifically, 1 x 10 HeLa cells 6 A total of 10 μg of DNA was transiently introduced into each cell by electroporation using a gene introduction device "Nepa21" (Nepagene Co., Ltd.). The ratio of the introduced DNA was ssISG sensor expression plasmid: ssNsp3 (WT or CHAA) expression plasmid = 1:9.

[0132] Cells were grown at 4 × 10 in DMEM supplemented with 10% FBS. 5 The cells were suspended in 0.1 mL / mL of IgG and cultured in a 96-well plate at 0.1 mL / well for 24 hours. The culture supernatant was then separated from the cells and the luciferase activity in the culture supernatant was measured.

[0133] As a result, high levels of luciferase activity were observed in the culture supernatant of cells co-transfected with the ssNsp3(WT) expression plasmid (Fig. 5B, "WT"), showing a signal / background ratio of approximately 45.3-fold higher than that of the negative control transfected with the empty vector (Fig. 5B, "-"). On the other hand, no luciferase activity was detected in the culture supernatant of cells co-transfected with the ssNsp3(CHAA) expression plasmid above that observed with the empty vector (Fig. 5B, "CHAA").

[0134] (5) Investigation of the necessity of a signal sequence To investigate the necessity of a signal sequence in the ssNsp3 protein, we constructed an Nsp3(-ss) expression plasmid expressing the wild-type Nsp3 protein derived from SARS-CoV2 without an N-terminal signal sequence (hereafter referred to as the "Nsp3(-ss) protein"). Using the same method as described in (4) above, the ssNsp3(WT) expression plasmid or the Nsp3(-ss) expression plasmid was introduced into HeLa cells together with the ssISG sensor expression plasmid, and luciferase activity in the culture supernatant was measured.

[0135] The results are shown in Figure 5C. High levels of luciferase activity were observed in the culture supernatant of cells cotransfected with the ssNsp3(WT) expression plasmid, but no increase in luciferase activity was observed above background levels in cells cotransfected with the Nsp3(-ss) expression plasmid. The Nsp3 protein lacking the signal sequence likely does not enter secretory vesicles and does not efficiently associate with the ISG sensor.

[0136] Example 2: Investigation of the region required for ISG15 deconjugation in the ISG15 protein (Objective) A modified ssISG sensor was prepared by modifying the ssISG sensor constructed in Example 1. The region required for ISG15 deconjugation in the ssISG sensor was investigated by evaluating the ISG15 deconjugation activity of the ssNsp3 protein on the modified ssISG sensor.

[0137] (Methods and Results) (1) Construction of modified ssISG sensor Based on the ssISG sensor constructed in Example 1 (hereinafter referred to as "ssISG sensor (GG)"), the following modified ssISG sensors were fabricated: ssISG sensor (AA), ssISG sensor (ΔD1), and ssISG sensor (ΔD1+2).

[0138] The "ssISG sensor (AA)" is a mutant of the "ssISG sensor (GG)" in which both glycines in the "LRLRGG" sequence adjacent to the cleavage site of the ISG15 deconjugation modification were replaced with alanines (Fig. 6A, "2) AA").

[0139] "ssISG sensor (ΔD1)" is a mutant of ssISG sensor (GG) lacking ubiquitin-like domain 1 (the region from positions 7 to 71 in SEQ ID NO: 4) (Fig. 6A, "3) ΔD1").

[0140] "ssISG sensor (ΔD1+2)" is a mutant of ssISG sensor (GG) in which both ubiquitin-like domains 1 and 2 (the region from positions 7 to 148 in SEQ ID NO: 4) are deleted, leaving only 15 amino acids containing the "LRLRGG" sequence (Figure 6A, "4) ΔD1+2").

[0141] In order to express each of the above modified ssISG sensors, modified ssISG sensor expression plasmids containing the modified ssISG sensor genes were prepared.

[0142] (2) Examination of ISG15 deconjugation activity of the modified ssISG sensor Using a method similar to that described in (4) of Example 1, the ssISG sensor expression plasmid or the modified ssISG sensor expression plasmid was introduced into HeLa cells together with the ssNsp3(WT) expression plasmid or the ssNsp3(CHAA) expression plasmid, and the ISG15 deconjugation activity was evaluated.

[0143] The results are shown in Figure 6B. As a result, high levels of luciferase activity were detected in the culture supernatant of cells co-expressing ISG sensor (GG) and ssNsp3 (WT) proteins. In contrast, in ssISG sensor (AA), in which the "LRLRGG" sequence was replaced with the "LRLRAA" sequence, luciferase activity was barely detectable, and de-ISG15 activity was almost completely abolished. Furthermore, in ssISG sensor (ΔD1), in which ubiquitin domain 1 was deleted, luciferase activity was significantly reduced, and de-ISG15 activity was attenuated. In ssISG sensor (ΔD1+2), in which both ubiquitin-like domains 1 and 2 were deleted, luciferase activity was barely detectable, and no de-ISG15 activity was observed.

[0144] These results demonstrate that the ISG15 deconjugating enzyme not only recognizes the short amino acid sequence "LRLRGG," but also recognizes the structure of the ISG15 protein, which contains two ubiquitin-like domains, and performs the deconjugation modification. Therefore, conventional in vitro evaluation methods using short peptide substrates are insufficient for evaluating the activity of proteases with ISG15 deconjugation activity or for screening compounds that inhibit its function, and are therefore unable to accurately detect ISG15 deconjugation activity. In contrast, the method for evaluating ISG15 deconjugation activity based on the ssISG sensor of the present invention uses a fusion protein containing the mature full-length ISG15 protein as a substrate, and is a significantly superior evaluation method compared to conventional methods.

[0145] Example 3: Development of a method for evaluating FeCoV protease activity (Objective) A method for evaluating the protease activity of the Nsp5 protein, which is a protease of feline coronavirus (FeCoV), was developed.

[0146] (Methods and Results) (1) Preparation of ssPRO-TM sensor expression plasmid The ssPRO-TM sensor was prepared as a fusion protein that serves as a substrate for detecting protease activity. The ssPRO-TM sensor is a fusion protein in which the portion of the above-mentioned ssISG sensor corresponding to the mature human ISG15 protein (d) is replaced with the cleavage target sequence of the Nsp5 protein (3CLpro) derived from feline coronavirus (FeCoV). Specifically, it is a fusion protein composed of the following (a) to (f) linked in order from the N-terminus (Figure 7, bottom).

[0147] (a) signal sequence of human immunoglobulin κ light chain (SEQ ID NO: 1) (b) secreted luciferase (ssGluc) derived from Gaussia princeps (SEQ ID NO: 2) (c) flexible linker (GGGGS; SEQ ID NO: 3) (d) Nsp5 cleavage target sequence (specifically, either (d1) or (d2) described below) (e) flexible linker (3×GGGGS; SEQ ID NO: 5) (f) transmembrane domain derived from immunoglobulin heavy chain (SEQ ID NO: 6)

[0148] Here, either (d1) or (d2) below was used as the Nsp5 cleavage target sequence in (d) above: (d1) an amino acid sequence consisting of 20 amino acids around the boundary between Nsp4 and Nsp5 in the FeCoV polyprotein PP1ab (shown as "Nsp4 / Nsp5 cleavage site" in Figure 7, with the cleavage site indicated by "|"; SEQ ID NO: 12); (d2) an amino acid sequence consisting of 20 amino acids around the boundary between Nsp5 and Nsp6 in the FeCoV polyprotein PP1ab (shown as "Nsp5 / Nsp6 cleavage site" in Figure 7, with the cleavage site indicated by "|"; SEQ ID NO: 13).

[0149] When the Nsp5 cleavage target sequence in (d) above consists of the amino acid sequence described in (d1) or (d2) above, the ssPRO-TM sensor is referred to as an "ssNsp4 / 5-TM sensor" or an "ssNsp5 / 6-TM sensor," respectively. The full-length amino acid sequence of the ssNsp4 / 5-TM sensor is shown in SEQ ID NO: 14, and the nucleotide sequence of the ssNsp4 / 5-TM sensor gene encoding the ssNsp4 / 5-TM sensor is shown in SEQ ID NO: 15. The full-length amino acid sequence of the ssNsp5 / 6-TM sensor is shown in SEQ ID NO: 16, and the nucleotide sequence of the ssNsp5 / 6-TM sensor gene encoding the ssNsp6 / 7-TM sensor is shown in SEQ ID NO: 17.

[0150] (2) Example of the Principle of the Protease Activity Evaluation Method The principle of the protease activity evaluation method will be described below as an example. After the signal sequence is cleaved and removed, the ssPRO-TM sensor (ssNsp4 / 5-TM sensor or ssNsp5 / 6-TM sensor) consisting of the above (a) to (f) becomes the PRO-TM sensor (Nsp4 / 5-TM sensor or Nsp5 / 6-TM sensor) consisting of the above (b) to (f). When the PRO-TM sensor is cleaved at the center position of the above (d) by protease activity, a fragment containing the above (b) to (c) and containing luciferase activity is released outside the cell, and luciferase activity based on this released fragment is detected in the cell supernatant.

[0151] (3) Preparation of wild-type or inactive ssNsp5 expression plasmids To express the Nsp5 protein derived from feline coronavirus (FeCoV) as a target for measuring protease activity, an ssNsp5(WT) expression plasmid was prepared that expresses an ssNsp5 protein in which a signal sequence was added to the N-terminus of the wild-type Nsp5 protein (hereinafter referred to as "ssNsp5(WT) protein").

[0152] The ssNsp5(WT) protein contains an immunoglobulin κ light chain signal sequence (SEQ ID NO: 1) at the N-terminus and wild-type FeCoV Nsp5 (SEQ ID NO: 18) at the C-terminus, and its full-length amino acid sequence is shown in SEQ ID NO: 19. Furthermore, to express the ssNsp5(WT) protein, an ssNsp5(WT) expression plasmid containing the ssNsp5(WT) gene (SEQ ID NO: 20) was constructed.

[0153] Furthermore, as a negative control for the ssNsp5(WT) protein, we prepared an ssNsp5(C144A) expression plasmid containing the ssNsp5(C144A) gene encoding an inactive Nsp5 protein (referred to herein as "ssNsp5(C144A) protein" or similar), in which cysteine ​​at position 144, which is assumed to be the active site of the Nsp5 protein, has been replaced with alanine.

[0154] (4) Evaluation of protease activity The ssNsp4 / 5-TM sensor expression plasmid or ssNsp5 / 6 sensor expression plasmid was co-transfected with the ssNsp5(WT) expression plasmid or ssNsp5(C144A) expression plasmid into HeLa cells and cultured for 24 hours. The culture supernatant was then separated from the cells and the luciferase activity in the culture supernatant was measured.

[0155] The results are shown in Figure 8. Higher levels of luciferase activity were observed in the culture supernatant of cells cotransfected with the ssNsp5(WT) expression plasmid compared with cells transfected with the empty vector. On the other hand, in cells cotransfected with the ssNsp5(C144A) expression plasmid, luciferase activity in the culture supernatant was reduced to the same level as in cells transfected with the empty vector.

[0156] These results demonstrate that the PRO-TM sensor can measure the activity of viral proteases within cells.

[0157] Example 4: Development of a method for evaluating HTLV-1 protease activity (Objective) A method for evaluating the activity of protease derived from human T-cell leukemia virus type 1 (HTLV-1) was developed.

[0158] (Methods and Results) (1) Preparation of ssPRO-TM sensor expression plasmid The ssPRO-TM sensor was prepared as a fusion protein that serves as a substrate for detecting the protease activity of the PRO protein, which is the HTLV-1 protease. The ssPRO-TM sensor in this example is a fusion protein in which the site corresponding to (d) in the ssPRO-TM sensor of Example 3 is replaced with the cleavage target sequence of the HTLV-1 PRO protein. Specifically, it is a fusion protein in which the following (a) to (f) are linked in order from the N-terminus (Figure 9, bottom):

[0159] (a) signal sequence of human immunoglobulin κ light chain (SEQ ID NO: 1) (b) secreted luciferase (ssGluc) derived from Gaussia princeps (SEQ ID NO: 2) (c) flexible linker (GGGGS; SEQ ID NO: 3) (d) PRO cleavage target sequence (specifically, any of (d1) to (d7) described below) (e) flexible linker (3×GGGGS; SEQ ID NO: 5) (f) transmembrane domain derived from immunoglobulin heavy chain (SEQ ID NO: 6)

[0160] Here, any of the following (d1) to (d7) was used as the target sequence for PRO cleavage in (d) above. (d1) An amino acid sequence consisting of 20 amino acids before and after the boundary between MA and CA in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "MA / CA cleavage site" in Figure 9, with the cleavage position indicated by "|"; SEQ ID NO: 21). (d2) An amino acid sequence consisting of 20 amino acids before and after the boundary between CA and NC in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "CA / NC cleavage site" in Figure 9, with the cleavage position indicated by "|"; SEQ ID NO: 22). (d3) An amino acid sequence consisting of 20 amino acids before and after the boundary between TF1 and PR in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "TF1 / PR cleavage site" in Figure 9, with the cleavage position indicated by "|"; SEQ ID NO: 23). (d4) An amino acid sequence consisting of 20 amino acids before and after the boundary between PR and p1 in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "PR / p1 cleavage site" in Figure 9). (d5) an amino acid sequence consisting of 20 amino acids before and after the boundary between p1 and RT in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "p1 / RT cleavage site" in Figure 9, and the cleavage position is shown by "|"; SEQ ID NO: 25); (d6) an amino acid sequence consisting of 20 amino acids before and after the boundary between RT and RH in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "RT / RH cleavage site" in Figure 9, and the cleavage position is shown by "|"; SEQ ID NO: 26); (d7) an amino acid sequence consisting of 20 amino acids before and after the boundary between RH and IN in the polyprotein GAG-PRO-POL of HTLV-1 (shown as "RH / IN cleavage site" in Figure 9, and the cleavage position is shown by "|"; SEQ ID NO: 27);

[0161] When the PRO cleavage target sequence in (d) above consists of the amino acid sequence described in (d1) above, the ssPRO-TM sensor will be referred to as an ssMA / CA-TM sensor, etc., and the same will be used for (d2) to (d7). The full-length amino acid sequence of the ssPRO-TM sensor corresponding to (d1) above is shown in SEQ ID NO: 28. The ssPRO-TM sensors corresponding to (d2) to (d7) above have the same amino acid sequence as (d1) except for the PRO cleavage target sequence. The nucleotide sequence of the ssPRO-TM sensor gene corresponding to (d1) above is shown in SEQ ID NO: 29. The nucleotide sequence of the ssPRO-TM sensor gene corresponding to (d2) to (d7) above is the same as (d1) except for the PRO cleavage target sequence.

[0162] (2) Example of the Principle of the Protease Activity Evaluation Method The principle of the protease activity evaluation method will be described below as an example. After the signal sequence is cleaved and removed, an ssPRO-TM sensor (e.g., ssMA / CA-TM sensor) consisting of the above (a) to (f) becomes a PRO-TM sensor (e.g., MA / CA-TM sensor) consisting of the above (b) to (f). When the PRO-TM sensor is cleaved by protease activity at the center position of the above (d), a fragment containing the above (b) to (c) and containing luciferase activity is released outside the cell, and the luciferase activity based on this released fragment is detected in the cell supernatant.

[0163] (3) Preparation of wild-type or inactive ssGAG-PRO expression plasmids In order to express the HTLV-1-derived GAG-PRO protein as a target for measuring protease activity, an ssGAG-PRO(WT) expression plasmid was prepared that expresses an ssGAG-PRO protein in which a signal sequence was added to the N-terminus of the wild-type GAG-PRO protein (hereinafter referred to as "ssGAG-PRO(WT) protein" or similar).

[0164] The ssGAG-PRO(WT) protein contains an immunoglobulin κ light chain signal sequence (SEQ ID NO: 1) at the N-terminus and the wild-type GAG-PRO protein derived from HTLV-1 (SEQ ID NO: 30) at the C-terminus, and its full-length amino acid sequence is shown in SEQ ID NO: 31. Furthermore, to express the ssGAG-PRO(WT) protein, a wild-type ssGAG-PRO(WT) expression plasmid containing the ssGAG-PRO(WT) gene (SEQ ID NO: 32) was constructed.

[0165] Furthermore, as a negative control for the ssGAG-PRO(WT) protein, an ssGAG-PRO(D32A) expression plasmid was prepared containing the ssGAG-PRO(D32A) gene encoding an ssGAG-PRO protein in which aspartic acid at position 32, presumed to be at the active site of the PRO protein, was substituted with alanine (herein referred to as "ssGAG-PRO(D32A) protein" etc.).

[0166] (4) Evaluation of protease activity One of the seven ssPRO-TM sensor expression plasmids was co-transfected with an ssGAG-PRO (WT or D32A) expression plasmid into HeLa cells and cultured for 24 hours. The culture supernatant was then separated from the cells and the luciferase activity in the culture supernatant was measured.

[0167] As a result, in all cases where seven types of ssPRO-TM sensor expression plasmids were transfected, higher levels of luciferase activity were observed in the culture supernatant of cells co-transfected with the ssGAG-PRO(WT) expression plasmid compared to cells transfected with the empty vector (Figure 10, "WT" vs. "-"). On the other hand, in cells transfected with the ssGAG-PRO(D32A) expression plasmid, luciferase activity in the culture supernatant was reduced to the same level as in cells transfected with the empty vector (Figure 10, "D32A" vs. "-").

[0168] Therefore, it was demonstrated that the PRO-TM sensor can be widely applied to measure the activity of viral proteases.

[0169] Example 5: Evaluation of the protease inhibitory activity of GC376 (Fig. 11A), a protease inhibitor against the FeCoV Nsp5 protein, was evaluated for its inhibitory activity against the SARS-CoV2 Nsp3 and Nsp5 proteins and the HTLV-1 PRO protein. GC376 is a known 3C-like protease inhibitor that inhibits the replication of porcine epidemic diarrhea virus (Porcine Epidemic Diarrhea Virus) (Ye G., et al., Viruses, 2020, 12(2):240).

[0170] (Methods and Results) HeLa cells were transfected with the ssISG sensor expression plasmid and ssNsp3(WT) expression plasmid prepared in Example 1; the ssNsp4 / 5-TM sensor expression plasmid and ssNsp5(WT) expression plasmid prepared in Example 3; or the ssPR / p1-TM sensor expression plasmid and ssGAG-PRO(WT) expression plasmid prepared in Example 4, and cultured for 24 hours in the presence of GC376 (Carbosynth, BG167367) at the concentrations shown in the figure. Luciferase activity in the culture supernatant was then measured. Cells transfected with ssGluc containing the human immunoglobulin κ light chain signal sequence were used as a negative control.

[0171] The results are shown in Figure 11B. GC376 inhibited the protease activity of the SARS-CoV2 Nsp5 protein in a concentration-dependent manner, while it did not affect the activity of the SARS-CoV2 Nsp3 protein or the HTLV-1 PRO protein.

[0172] Cell viability was also measured using the WST8 assay. An equal volume of 4% WST8 solution (Viable Cell Count Reagent SF (Nacalai Tesque, 07553-44)) was added to the cell culture medium, and the cells were cultured at 37°C for 2 hours in the presence of 5% CO2. The absorbance (450 nm and 650 nm) was then measured. No inhibitory effect on cell viability was observed under any of the conditions (Figure 11C).

[0173] These results indicate that GC376 does not affect the secretion or activity of ssGluc, does not inhibit cell viability, and therefore specifically inhibits the protease activity of the Nsp5 protein derived from SARS-CoV2. These results also demonstrate that the method of the present invention is useful for discovering protease inhibitors.

[0174] Example 6: Development of a method for evaluating the activity of protease derived from African swine fever virus (ASFV) (Objective) A method for evaluating the activity of protease pS273R derived from African swine fever virus (ASFV) was developed.

[0175] (Methods and Results) (1) Preparation of ssPRO-TM sensor expression plasmid The ssPRO-TM sensor was prepared as a fusion protein that serves as a substrate for detecting the protease activity of the ASFV protease, S273R protease (also referred to as "pS273R protein"). The ssPRO-TM sensor in this example is a fusion protein in which the site corresponding to (d) in the ssPRO-TM sensor of Example 3 is replaced with the cleavage target sequence of the ASFV pS273R protein. Specifically, it is a fusion protein in which the following (a) to (f) are linked in order from the N-terminus (lower panel in Figure 13):

[0176] (a) signal sequence of human immunoglobulin κ light chain (SEQ ID NO: 1) (b) secreted luciferase (ssGluc) derived from Gaussia princeps (SEQ ID NO: 2) (c) flexible linker (GGGGS; SEQ ID NO: 3) (d) pS273R cleavage target sequence (specifically, any of (d1) to (d6) described below) (e) flexible linker (3×GGGGS; SEQ ID NO: 5) (f) transmembrane domain derived from immunoglobulin heavy chain (SEQ ID NO: 6)

[0177] Here, any of the following (d1) to (d6) was used as the pS273R cleavage target sequence in (d) above, which corresponds to the pS273R cleavage sites in the ASFV polyproteins pp220 and pp60 shown in Figure 12. (d1) An amino acid sequence consisting of 23 amino acids before and after the boundary between p5 and p34 in the ASFV polyprotein pp220 (shown in Figure 13 as "pp220 p5 / p34 cleavage site", with the cleavage site indicated by "|"; SEQ ID NO: 41). (d2) An amino acid sequence consisting of 23 amino acids before and after the boundary between p34 and p14 in the ASFV polyprotein pp220 (shown in Figure 13 as "pp220 p34 / p14 cleavage site", with the cleavage site indicated by "|"; SEQ ID NO: 42). (d3) An amino acid sequence consisting of 23 amino acids before and after the boundary between p14 and p37 in the ASFV polyprotein pp220 (shown in Figure 13 as "pp220 p14 / p37 cleavage site", with the cleavage site indicated by "|"; SEQ ID NO: 43). (d4) an amino acid sequence of 23 amino acids before and after the boundary between p37 and p150 in the ASFV polyprotein pp220 (illustrated as "pp220 p37 / p150 cleavage site" in Figure 13, with the cleavage position indicated by "|"; SEQ ID NO: 44); (d5) an amino acid sequence of 23 amino acids before and after the boundary between p15 and p35 in the ASFV polyprotein pp60 (illustrated as "pp60 p15 / p35 cleavage site" in Figure 13, with the cleavage position indicated by "|"; SEQ ID NO: 45); (d6) an amino acid sequence of 23 amino acids before and after the boundary between p35 and p8 in the ASFV polyprotein pp60 (illustrated as "pp60 p35 / p8 cleavage site" in Figure 13, with the cleavage position indicated by "|"; SEQ ID NO: 46);

[0178] (2) Preparation of wild-type or inactive pS273R expression plasmids To express the ASFV-derived pS273R protein as a target for measuring protease activity, a pS273R(WT) expression plasmid was prepared, which expresses the pS273R(WT) protein, which has a signal sequence added to the N-terminus of the wild-type pS273R protein consisting of the amino acid sequence shown in SEQ ID NO: 53.

[0179] Furthermore, as a negative control for the pS273R(WT) protein, we constructed a pS273R(C232A) expression plasmid containing the pS273R(C232A) gene encoding the pS273R(C232A) protein in which the cysteine ​​residue at position 232, which is assumed to be the active site of the pS273R protein, was replaced with an alanine residue.

[0180] (3) Evaluation of protease activity One of the six ssPRO-TM sensor expression plasmids was co-transfected with the pS273R (WT or C232A) expression plasmid into HeLa cells and cultured for 24 hours. The culture supernatant was then separated from the cells and the luciferase activity in the culture supernatant was measured.

[0181] As a result, in all cases where six types of ssPRO-TM sensor expression plasmids were transfected, higher levels of luciferase activity were observed in the culture supernatant of cells co-transfected with the pS273R(WT) expression plasmid compared to cells transfected with the empty vector (Fig. 14, "pS273R WT" vs. "-"). On the other hand, in cells transfected with the pS273R(C232A) expression plasmid, the luciferase activity in the culture supernatant was lower than that of cells transfected with the empty vector (Fig. 14, "pS273R C232A" vs. "-").

[0182] Therefore, it was demonstrated that the ssPRO-TM sensor can detect the activity of viral protease derived from ASFV.

[0183] Example 7: Evaluation of cleavage activity against SUMO protein, ISG15 protein, and ubiquitin protein (Objective) The protease activity of the pS273R protein is evaluated using SUMO protein, ISG15 protein, and ubiquitin protein as cleavage targets.

[0184] (Methods and Results) (1) Construction of hSUMO-1 sensor, hSUMO-2 sensor, hISG15 sensor, and 2xUb sensor Various sensors (hSUMO-1 sensor, hSUMO-2 sensor, hISG15 sensor, and 2xUb sensor) shown in Figure 15A were constructed. Each sensor is a fusion protein consisting of the following (a) to (f) linked in order from the N-terminus (Figure 15A): (a) signal sequence of human immunoglobulin κ light chain (SEQ ID NO: 1) (b) secreted luciferase (ssGluc) derived from Gaussia princeps (SEQ ID NO: 2) (c) flexible linker (GGGGS; SEQ ID NO: 3) (d) cleavage target sequence (specifically, one of (d1) to (d4) described below) (e) flexible linker (3xGGGGS; SEQ ID NO: 5) (f) transmembrane domain derived from immunoglobulin heavy chain (SEQ ID NO: 6)

[0185] Here, any of the following (d1) to (d4) was used as the cleavage target sequence in (d) above. The sensors containing (d1) to (d4) are referred to as the "hSUMO-1 sensor," "hSUMO-2 sensor," "hISG15 sensor," and "2xUb sensor," respectively. Expression plasmids expressing each sensor were constructed: (d1) human SUMO-1 (hSUMO-1) protein (SEQ ID NO: 47); (d2) human SUMO-2 (hSUMO-2) protein (SEQ ID NO: 48); (d3) mature human ISG15 protein (SEQ ID NO: 4); and (d4) 2xUb sequence (SEQ ID NO: 52). (2) Evaluation of protease activity of S273R protease against various sensors. Using the same method as in (4) of Example 1, various sensor expression plasmids were introduced into HeLa cells together with the pS273R (WT or C232A) expression plasmid, and protease activity was evaluated.

[0186] The results are shown in Figure 15B. Higher levels of luciferase activity were observed in the culture supernatants of cells co-expressing various sensors with the pS273R(WT) protein than in cells transfected with the empty vector (Figure 15B, "pS273R WT" vs. "-"). On the other hand, luciferase activity in the culture supernatants of cells transfected with the pS273R(C232A) expression plasmid was lower than that of cells transfected with the empty vector (Figure 15B, "pS273R C232A" vs. "-"). These results demonstrate that the S273R protease can cleave SUMO proteins, ISG proteins, and ubiquitin proteins. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

Claims

1. A fusion protein substrate for measuring protease activity, which, in order from the N-terminal side, (i) a signal sequence, a reporter region, a protease target region, and a membrane-binding region, or (ii) a signal sequence, a protease target region, a reporter region, and a membrane-binding region The fusion protein substrate comprising the same.

2. The fusion protein substrate according to claim 1, wherein the protease is a viral protease or a deubiquitinating enzyme.

3. The fusion protein substrate according to claim 2, wherein the viral protease is derived from the family Coronaviridae, Retroviridae, Asfarviridae, Flaviviridae, Caliciviridae, Picornaviridae, Poxviridae, Herpesviridae, Adenoviridae, Togaviridae, or Matonaviridae.

4. The fusion protein substrate according to claim 3, wherein the target region of the viral protease derived from the family Coronaviridae comprises the amino acid sequence shown in SEQ ID NO: 4, 12, or 13.

5. The fusion protein substrate according to claim 3, wherein the target region of the viral protease derived from the family Retroviridae comprises any amino acid sequence selected from the group consisting of SEQ ID NOs: 21-27 and 62-70.

6. The fusion protein substrate according to claim 3, wherein the target region of the viral protease derived from the family Asfarviridae comprises any amino acid sequence selected from the group consisting of SEQ ID NOs: 41-46.

7. The fusion protein substrate according to claim 2, wherein the target region of the deubiquitinating enzyme comprises a ubiquitin protein, a SUMO protein, or an ISG15 protein.

8. A nucleic acid encoding the fusion protein substrate according to claim 1.

9. A gene expression vector containing the nucleic acid according to claim 8 in an expressible state.

10. A host cell containing the gene expression vector according to claim 9.

11. The host cell according to claim 10, further comprising a gene expression vector containing a nucleotide sequence encoding a protease that cleaves the protease target region in an expressible state.

12. A protease activity measurement kit comprising one or more selected from the group consisting of the fusion protein substrate according to any one of claims 1-7, the nucleic acid according to claim 8, the gene expression vector according to claim 9, and the host cells according to claims 10 and 11.

13. A method for measuring protease activity, comprising: a reaction step of reacting a host cell expressing a fusion protein substrate for measuring protease activity in a solution with a protease to be tested, wherein the fusion protein substrate contains, in order from the N-terminal side, a signal sequence, a reporter region, a protease target region, and a membrane-binding region; a recovery step of recovering the supernatant from the solution after the reaction; and a measurement step of measuring the reporter activity based on the reporter region in the supernatant. The method as described above.

14. A method for measuring protease activity, comprising: a reaction step of reacting a host cell expressing a fusion protein substrate for measuring protease activity in a solution with a protease to be tested, wherein the fusion protein substrate contains, in order from the N-terminal side, a signal sequence, a protease target region, a reporter region, and a membrane-binding region; and a measurement step of measuring the reporter activity based on the reporter region in the host cell after the reaction. The method as described above.

15. The method according to claim 14, wherein the viability of the host cell is measured after the measurement step.