KR100194124B1 - Method for measuring protease activity isolated from Turnip Mosaic Virus C5 - Google Patents

Abstract

The present invention relates to a method for measuring the activity of a protease isolated from Mosaic virus C5 (Turnip Mosaic Virus C5, hereinafter abbreviated as TuMV-C5), which is a kind of potyvirus.

Specifically, the present invention is a method of measuring the activity of protease at the C-terminus of NIa protein, which plays an important role in viral proliferation by being involved in the maturation process of polyprotein expressed in TuMV-C5, wherein the amino acid to which the protease can act The present invention relates to a method for determining the activity of a protease simply, quickly and accurately by identifying a sequence, synthesizing the peptide, and using the peptide as a substrate.

Description

Method for measuring protease activity isolated from Turnib Mosaic Virus C5

Figure 1 shows the heptapeptide sequences of cleavage sites recognized by the NIa protease of TuMV.

*: New internal self-cutting site of NIa protease.

2 shows the degradation activity of the NIa 27kDa protease against the ex vivo translational product of pTNC labeled with [ ³⁵ S] methionine.

Lanes 1,2,3 are E. coli XL1-Blue induced cell eluate containing pGEX-KG, pGPOPR, pMPOPR, lane 4 is GST fusion protein, lane 5 is protease purified by mono S chromatography, lane 6 The KSI fusion protein purified by silver mono S chromatography, lane 7, is the result of putting together 2 μg of thrombin.

3 analyzes the degradation activity of NIa protease against the synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His-Gln-Leu-Asn-Glu. a, b and c are chromatograms when the reaction mixture of the peptide substrate and the NIa protease were incubated for 0, 20 and 40 minutes, respectively, and the peaks at the retention times of 8.5 minutes and 12.5 minutes correspond to the product and the substrate, respectively.

4 shows the results of enzyme activity measured with proresamine based on the synthetic nonapeptide, acetyl-Glu-Pro-Thr-Val-Tyr-His-Glu-Thr-Leu-amide. 1 is distilled water, 2 is NIa protease only, 3, 5, 6 and 7 are reacted with substrate for 0, 30, 60, 90 minutes, and 4 is NH ₂ -Thr-Leu-amide as a control. .

5 shows the results of continuous activity measurements confirmed by fluorescence using single-Ala-Thr-Val-Tyr-His-Gln-Thr-Leu-p-nitroanilide as a substrate.

6 shows the results of enzyme activity assayed with TNBS using nonapeptide as a substrate. 1, 2 is when no substrate is added, 3, 4 is when the substrate is added with the enzyme and 5,6 is when NH ₂ -Thr-Leu-amide of the substrate control.

FIG. 7 shows a Lineweaver-Burke plot to determine the dynamic constants of NIa proteases for the peptide substrate, Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-Asn-Glu. The X- and Y-axis units are mM ⁻¹ and M ⁻¹ · mg · min, respectively, and each point represents the mean value from three independent measurements.

[Field of use of the invention]

The present invention relates to a method for measuring the activity of a protease isolated from a Turnip Mosaic Virus C5 (Turnip Mosaic Virus C5, hereinafter abbreviated as TuMV-C5) which is a kind of potyvirus.

Specifically, the present invention relates to a method for measuring the activity of proteases at the C-terminus of NIa proteins, which play an important role in viral proliferation by participating in the polyprotein maturation process expressed in TuMV-C5. The present invention relates to a method for determining the activity of a protease simply, quickly and accurately by identifying a sequence, synthesizing the peptide, and using the peptide as a substrate.

[Background of invention]

Fortiviruses are plant viruses and are a broad and severely damaging viral species that infects almost all major crops, such as cabbage, potatoes, peppers, tobacco, tomatoes, and flowers. For example, the high morbidity rate caused by viruses is high even in highlands, which are known to have relatively low morbidity caused by bugs and bacteria, and the damage caused by viruses has recently spread. Virus infections in crops are very serious. The productivity of the crop is reduced by 10% to 50%, the product rate is lowered, and also after the product becomes a serious damage to quality control. In spite of the enormous damage of crops caused by viral diseases, pesticides as antivirals have not been developed until recently.

[Prior technology]

The pesticides developed so far have been mostly control of visible bee and host cells such as herbicides, insecticides and fungicides, and bacteria and fungi with different life mechanisms. This is because the viruses share the mechanism of life phenomena with the host cells, which makes it difficult to isolate and identify them, as well as to make pesticides that target only the virus without damaging the host. Indeed, the development of antiviral pesticides is at a very early stage, with an example of an antiviral plant developed and patented by Monsanto. (US910224, Jan. 94). The method involves injecting a plant from a viral genomic gene of potato into a plant to produce a transgenic antiviral potato variety. Since this is a plant transformation rather than the development of pesticides in a strict sense, this method requires that one crop transforms dozens of useful varieties and keeps the genetics stable while keeping the genes stable. In addition, since it shows resistance only to specific viruses, many viral genes have to be injected and replicated in plants.

On the other hand, when looking at the development of antiviral drugs as a medicine, a method of inhibiting the action of enzymes essential for the survival of the virus is being used.

Since viruses have a very simple gene structure, few proteins can be targeted in the development of antiviral agents. Proteases are useful for developing drugs with high selectivity as antiviral targets. Proteases generally have very high selectivity and specificity due to the nature of the enzyme and thus have a specific structure. In particular, viral proteases have different properties from the proteases in the host and are also essential enzymes for the growth of the virus. Moreover, it acts at an early stage of viral replication, making it a good target enzyme for the development of antiviral control agents.

Therefore, researches on protease have been conducted for the development of antiviral agents, and recently, some antiviral agents have been developed as medicines using proteases. For example, protease inhibitors developed in human immunodeficiency virus (HIV), a virus that causes AIDS (acquired immunodeficiency syndrome), have a pronounced antiviral effect. In addition, recently developed HIV-1 protease inhibitors show very potent inhibitory activity and specificity even at low concentrations below nM.

The inventors of the present invention have devised a method for developing an antiviral agent by developing a protease-inhibiting substance essential for the survival of the virus, and have attempted to develop a pesticide for controlling a virus that causes enormous damage to crops. In particular, we have developed a pesticide against the ternib mosaic virus, a type of fortivirus that causes the most damage among plant viruses.

The present inventors isolate and purify the protease which plays an essential role in the maturation of polyprotein, which is a major step in the proliferation of TuMV-C5 virus, in order to develop a maternal virus control agent according to the invention filed as the same as the present invention It was revealed, the manufacturing method and the characteristics thereof were revealed. The present inventors seek to provide a novel method for measuring the proteolytic activity of the protease.

[Summary of invention]

An object of the present invention is to provide a method for measuring the protease activity of the NIa protein at the C-terminus of TuMV-C5.

Provided is an amino acid sequence recognized by a TuMV-C5 protease, a method of synthesizing the peptide and measuring the activity of the protease with a substrate.

Synthetic peptides that can be used as substrates in the protease activity measurement method of the present invention include amino acid sequences of KEVVHQA, PTVYHQT, EAVNHQS, EPVTHEA, TAVYAQT, ACVYHQA, VPVDHES, QASQPSG.

The present invention also provides a method for measuring the activity of a protease based on the in vitro detoxification product of TuMV-C5.

Accordingly, the present invention provides a method for measuring the activity of protease using the product of cloning and expressing genes of NIb and CP proteins as a substrate.

The TuMV-C5 protease activity measuring method of the present invention can be used to measure the inhibitory ability of protease activity inhibitors.

In order to measure the protease activity inhibitory ability, it is measured using a protease, a synthetic peptide, and an inhibitor. As a protease that can be used in the method for measuring the protease inhibitory ability of the present invention, proteases of 27 kDa and 25 kDa may be used.

Detailed description of the invention

Hereinafter, the present invention will be described in detail.

Turnip mosaic virus (TuMV) is a species of fortivirus and belongs to the superfamily of picornaviruses, which are (+) RNA strand viruses. The genome of fortiviruses is about 10 kb in length, and the 3'-end is polyadenylated, and the 5'-end is covalently linked to a protein (VPg) produced by the virus (Ward and Shukla, 1991; Dougherty and Carrington, 1988). The RNA of the Tunnip mosaic virus is translated into a polyprotein that is processed into at least nine mature proteins by three viral proteases (see Figure 1). Among the proteins of the virus, the N-terminal protein (P1) and helper component-protease (HC-Pro) autocatalytically cut only their respective C-terminals, and the nuclear inclusion NIa protease (Nuclear Inclusion a Protease) Involved in cutting the rest of the polyprotein (Carrington et al., 1989; Verchot et al., 1991; Carrington and Dougherty, 1987).

The NIa of the fortivirus consists of a protein with two functional sites: the VPg region at the N-terminus of 22 kDa and the C-terminal protease region at 27 kDa, and the C-terminal protease region has a sequence similar to the serine protease family similar to trypsin. Dougherty and Park, 1991; Murphy et al., 1990; Gorbalenya et al., 1989.

The NIa 27kDa protease self-cleaves at the C-terminus to produce 25kDa protein, which in turn undergoes secondary self-cleavage to produce 24kDa protein. This cleavage occurs in a cis manner, and the 25 kDa protein obtained by self-cutting also retains proteolytic activity.

As a method for measuring the activity of NIa 27kDa protease, there is a method for measuring the specific proteolytic activity in TuMV-C5 using an in vitro translation product or a synthetic peptide as a substrate, respectively.

1) When using an in vitro detoxification product as a substrate

The RNA of the Turnip Mosaic Virus is translated into polyprotein, which is converted into 9 mature proteins of P1, HC-Pro, P3, 6K ₁ , CI, 6K ₂ , NIa, NIb, and CP by proteases. Dual N-terminal protein (P1) and helper component-protease (HC-Pro) auto-catalytically cut their respective C-terminus, while NIa protease cuts the rest of the site, namely P3-6K ₁ , 6K ₁ -CI, CI -6K ₂ 6K ₂ ,-involved in cutting NIa, NIa- NIb and NIb-CP sites.

Therefore, in order to measure the activity of the NIa protease, a protein expressed by cloning the P3-6K ₁ , 6K ₁ -CI, CI-6K ₂ , 6K ₂ -NIa, NIa-NIb, and / or NIb-CP sites can be used as a substrate. (See Figure 1).

In vitro detoxification products comprising NIb-CP sites are obtained from plasmid pTNC using rabbit reticulocyte lysate. It is a molecular weight 65 kDa protein that includes the C-terminal half of the NIb protein and the entire capsid protein (CP). The 65 kDa protein used as substrate was cut into 32 kDa and 33 kda proteins by enzymatic activity such as GST fusion protease, maltose binding protein fusion protease or purified protease, which is consistent with the expected size from cleavage between NIb and the outer protein. However, this substrate is not cut by intracellular proteases or thrombin. In addition, the three mutated proteases have no degradation activity against the substrate, but the 25-kDa protease and the deletion mutant protease (ProΔ23aa) with the C-terminus cut off can degrade the substrate (see also Figure 2). .

2) In case of using synthetic peptide as substrate

Looking at the amino acid sequence of the site where the NIa protease cuts, namely P3-6K ₁ , 6K ₁ -CI, CI-6K ₂ , 6K ₂ -NIa, NIa-NIb, and NIb-CP sites, the P3-6K ₁ site is KEVVHQA, 6K ₁ -CI site is PTVYHQT, CI-6K ₂ site is EAVNHQS, 6K ₂ -NIa site is EPVTHEA, NIa-NIb site is TAVYAQT, VPg-protease site is VPVDHES and NIb-CP site is ACVYHQA in NIa to be.

Looking at these sequences, homology can be found, where the third amino acid is V, the fifth amino acid is H or A, and the sixth is Q or E. In addition, the NIa protease not only cuts between proteins in the polyprotein, but also self-cleaves at the C-terminus, resulting in a protein from 27kDa to 25kDa. The amino acid sequence of this self-cleaving site is QASQPSG, which is a completely different sequence from the interprotein cleavage sequence of the polyprotein. Thus, it can be seen that the NIa protease specifically recognizes at least the eight sequences.

The present inventors have identified the amino acid sequence of the recognition site of the NIa protease, and made a synthetic peptide according to the amino acid sequence of the recognition site and used it as a substrate for protease activity measurement. Synthetic peptides can be prepared based on these eight sequences and used as substrates for measuring protease activity. First, Glu-Pro-Thr- containing a heptapeptide cleavage sequence between 6K ₁ and CI proteins present in the polyprotein of TuMV. Undecapeptide of Val-Tyr-His-Gln-Thr-Leu-Asn-Glu and nonapeptide of acetyl-Glu-Pro-Thr-Val-Tyr -His-Gln-Thr-Leu-amide as substrate use.

The NIa protease cleaves between Gln and Thr in the peptide substrate. For undecapeptides, the enzyme produces Glu-Pro-Thr-Val-Tyr-His-Gln and Thr-Leu-Asn-Glu, so HPLC analysis is performed. Absorbance at 230nm was measured to identify peptides with Tyr residues. As shown in FIG. 3, with the incubation time, the peak of the product increases at the retention time of 8.5 minutes, and the peak of the substrate decreases at the retention time of 12.5 minutes. By collecting the fractions at the peaks of the substrate and the product and analyzing their molecular mass by mass spectrometry, it can be seen that it contains the peptide of the mass expected at each peak.

In addition, floresamine (fluorescamine) can be used to measure the change in fluorescence produced by substrate peptide cleavage to determine the activity of the enzyme (see FIG. 4).

In addition, changes in fluorescence caused by cleavage of the substrate were observed using single-Ala-Thr-Val-Tyr-His-Gln-Thr-Leu-p-nitroanilide as the substrate. Since p-nitroanilide acts as an energy receptor to eliminate fluorescence, it is possible to measure protease activity continuously (see Figure 5).

In addition, the cleavage of the substrate by the enzyme activity is also observed as the color change of the reaction mixture using 2,4,6-trinitro-sulfonic acid. NH ₂ -Thr-Leu-amide was used as a control, the color change of the reaction mixture can be confirmed by measuring the absorbance (see Figure 6).

As described above, in order to measure the proteolytic activity of the NIa protease of TuMV-C5, first, a 27 kDa, 25 kDa, or the like may be used as the NIa protease. The 27 kDa protease self-cleaves to break down into an active 25 kDa and an inactive 24 kDa protease. As the substrate, any of the peptides including the recognition sequence of the protease can be used.

3) Determination of kinetic constant of protease

The properties of the previously purified NIa protease are investigated through analysis of the Lineweaver-Burk plot (see Figure 7). Kinetic constants are determined by an in vitro identification system using purified NIa 27kDa protease and undecapeptide as substrate. The K _m and V _max values can be determined to be 1.15 ± 0.16mM and 0.74 ± 0.091 M / mg / min, respectively, by regression analysis of the data from three independent measurements. These K _m and V _max values are 17 and 2 times higher, respectively, than those determined using peptides containing cleavage sites between the NIb and CP proteins as substrates for the NIa protease of TEV (Parl et al., 1995). K _cat and K _cat / K _m are calculated as 0.33 ± 0.041 sec ^-1 and 290 ^-1 M ^-1 sec ^-1 , respectively, using a molecular weight of 27kDa for the protease. This data suggests that cleavage between NIb and CP throwback is more efficient than cleavage between 6K ₁ and CI proteins.

Hereinafter, the present invention will be described in more detail with reference to Examples.

The following examples are merely illustrative of the present invention, and the content of the present invention is not limited by the examples.

[Proteolytic Activity of NIa Protease]

The proteolytic activity of the NIa protease was confirmed using a substrate having a cleavage site of the NIa protease. As substrate, an in vitro detoxification product of NIb and CP protein containing a cleavage site for protease and an undecapeptide or nonapeptide comprising a cleavage site of 6K ₁ protein and columnar inclusion protein (CI) were used. .

Example 1

Measurement of Proteolytic Activity Based on in Vitro Detoxification Products of NIb and CP Proteins

(1-1) Preparation of NIb and CP Proteins

In order to use the ex vivo translation products of NIb and CP proteins including cleavage sites for proteases, pTNCs were prepared by cloning the genes of NIb and CP proteins. First, two clones, pVT4 and pVT5, were obtained from the cDNA library. pVT4 contained 3'-terminal nucleotides at 8144 of the TuMV-C5 genome in pBluescript SK (-), and pVT5 contained 6950 to 8504 nucleotides in pUC19. The location of the nucleotides was determined based on the sequence of the TuMV Canadian type genome.

pVT4 was digested with HindIII and DNA fragments to be inserted from 8428 to 3'-end were isolated by electroelution. pVT5 was digested with HindIII to isolate the DNA containing both the vector and the DNA fragments from 6950 to 8427 by electroelution. Both isolated DNA fragments were joined to prepare a plasmid pVT45 comprising 6950 to 3'-terminal nucleotides in pUC19. The pVT45 was digested with Sac I and Pst I to obtain a DNA fragment of about 2 kb that contained half the 3'-end of the gene encoding nuclear inclusion protein b (NIb) and the entire gene of the outer protein (CP). This DNA fragment was linked to the plasmid pTM1 containing the T7 promoter and the 5'-terminal nontranslated site of Encephalomyocarditis Virus to prepare pTNC.

The NIa protease activity of TuMV in the crude cell eluate or purified solution was confirmed using a polyprotein having a cleavage site of the NIa protease as a substrate. The plasmid pTNC gene, which includes the 3'-terminal half of the NIb gene and the entire CP gene, was digested with SalI and linearized and used for in vitro transcription using T7 RNA polymerase (New Egland Biolab.). In vitro, RNA transcripts were read in rabbit blood cell eluate, and proteins synthesized ex vivo were labeled with [ ³⁵ S] methionine as recommended by Promega.

(1-2) Measurement of proteolytic activity

NIa protease activity was measured in identification buffer [20 mM HEPES (pH 7.4), 10 mM magnesium chloride, 0,5 mM DTT, 10 mM calcium chloride] using the ex vivo translation product as a substrate. The reaction product with the substrate was analyzed by 10% SDS-PAGE and the gel was dried and exposed to X-ray film (Agfasa).

Example 2

[Measurement of Proteolytic Activity Based on Synthetic Undecapeptide]

(2-1) Activity measurement by HPLC analysis

To determine the degree of catalytic activity of the NIa protease, a synthetic undecapeptide, Glu-Pro-Thr-Val-Tyr-His, containing a cleavage site of 6K ₁ protein and columnar inclusion protein (CI) -Gln-Leu-Asn-Glu or nonapeptide, acetyl-Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-amide was used as substrate. A typical assay method was used, in which 0.58 mM or 0.2 mM peptide substrate and crude cell eluate or 1.5 μg purified protease were added to 24 μl of identification buffer [20 mM HEPES (pH 7.4), 10 mM magnesium chloride, 0.5 mM DTT, 10 mM potassium chloride] was used. After the reaction mixture was reacted at 25 ° C., 300 μl of 10% acetic acid was added to stop the reaction when the formation of the product did not exceed 15%. The resulting 150 μl mixture was mixed with acetonitrile concentration gradient (8% to 18%) with 0.05% trifluoroacetic acid (w / v) on a Vidac C ₁₈ column (Vydac C ₁₈ , 4.6 mm ID × 25 cm). 10 minutes) and HPLC (Hewlett Packard HP1090) using an elution rate of 1 ml / min. Peaks from the product and the substrate were identified by measuring the adsorption at 230 nm and the degree of cleavage was calculated by summing the area of the peaks (see Figure 3).

(2-2) Activity measurement using fluorescamine

Synthetic nonapeptide, acetyl-Glu-Pro-Thr-Val-Tyr-His-Gln-Thr-Leu-, comprising a cleavage site between the 6K ₁ protein of the TuMV polyprotein and the columnar inclusion protein as a substrate for activity measurement Amides were used. As a reaction mixture for measuring activity, 24 μl of confirmation buffer (20 mM HEPES, pH 7.4, 10 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT) containing 0.2 mM peptide substrate and 1.5 μg of purified protease was used. It was. 76 µl of 2% SDS was added to the reaction mixture to stop the reaction for each hour. Then, 0.01% floresamine dissolved in 100 µl of acetone was added thereto, followed by reaction at room temperature for 20 minutes. Proresamine attacked the primary amine groups of peptides produced by cleavage of substrate peptides to produce floresamine binding compounds that were excited at 386 nm and maximally released at 466 nm. The resulting fluorescence was measured by excitation at 386 nm and emission at 466 nm. As a result, the longer the reaction between the substrate and the protease enzyme, the more fluorescence emitted (see FIG. 4).

(2-3) Continuous activity measurement method using fluorescent substrate

In order to measure proteolytic activity continuously, a continuous activity measurement method based on fluorescence was developed. Dansyl-Ala-Thr-Val-Tyr-His-Gln-Thr-Leu-p-nitroanilide was used as a substrate for continuous activity measurement. At this time, the monosil group was prepared as an energy acceptor that eliminates the fluorescence of p-nitroanilide. As a reaction mixture for measuring activity, 24 μl of confirmation buffer (20 mM HEPES, pH 7.4, 10 mM MgCl ₂ , 10 mM KCl, 0.5 mM DTT) containing 0.1 mM peptide substrate and 1.5 μl of purified protease was used. The total volume was 80 μl. The progress of the reaction was measured by observing the fluorescence change in excitation at 328 nm and emission at 422 nm caused by cleavage of the substrate. As a result, the change in fluorescence was clearly distinguished by the reaction of the enzyme and the substrate (see Fig. 5).

(2-4) Activity measurement method using 2,4,6-trinitrobenzene-sulfonic acid (TNBS)

As a substrate for measuring activity, a synthetic nonapeptide, acetyl-Glu-Pro-Thr-Val-Tyr-His-Gln- Thr-Leu-amide, containing a cleavage site between 6K ₁ protein and columnar inclusion protein, was used. NH ₂ -THr-Leu-amide was used as a control. Reaction mixtures for measuring activity include 24 μl of confirmation buffer (20 mM sodium borate, pH 8.3, 10 mM MgCl ₂ , 10 mM) containing 0.5 mM peptide substrate, 1.5 µg peptide substrate and 1.5 µg purified protease. KCl ₂ , 0.5 mM DTT) was used. 100 µl of 0.25 M sodium borate (pH 10.5) was added to the reaction mixture to stop the reaction.0.2 M TNBS was prepared in 0.25 M sodium borate solution, and then 12 µl was added to the reaction mixture. The reaction was carried out at room temperature for 30 minutes. 500 μl of ₃ mM Na ₂ SO ₃ and 0.2 M KH ₂ PO ₄ solution were added to stabilize color and the progress of peptide decomposition was measured by measuring absorbance at 405 nm (see FIG. 6).

Example 3

[Determining Dynamic Constants of NIa Protease]

In order to understand the catalytic mechanism and enzymatic properties of NIa protease, K _m and K _max values for undecapeptide substrates synthesized with purified protease were measured. Weaver line obtained by the measurement condition check the activity of the purified protease in the range of 1.75mM substrate concentration in 0.029mM described above - the buck drawings analyzed to determine the K _m and K _max value. It was 62.5 mg / l in the final concentration mixture of purified protease for each substrate concentration. Protease concentrations were determined using the Bradford Identification Kit (Pierce, Inc.) (see Figure 7).

As described above, the system for confirming the NIa protease activity of TuMV-C5 using a synthetic peptide as a substrate is very effective and very effective, and thus can be very useful for the development of protease inhibitors in relation to virus eradication in plants.

The tunip mosaic virus of the present invention is a kind of fortivirus. In terms of evolution, the similarity of genes to the various viral species belonging to this group is very high, and the sequence of the recognition site of the protease is very similar. Ultimately, it is possible to develop a wide range of antiviral agents with a fairly broad spectrum. It can be applied not only to TuMV, but also to antiviral agents that inhibit various virus proliferations, including TEV, which is a kind of the same fortivirus and has a significant effect on tobacco farming.

Therefore, the method for measuring the NIa protease activity of TuMV-C5 of the present invention is simple, accurate and rapid, and can be used for the development of new antiviral pesticides because many samples can be measured at one time.

Claims (9)

Method for measuring proteolytic activity using a peptide comprising the amino acid sequence of () () V () HQ (), which is a region recognized by NIa protease of TuMV-C5 expressed by cloning genes of NIb and CP proteins . The method of claim 1, wherein the peptide substrate comprises a KEVVHQA amino acid sequence. The method of claim 1, wherein the peptide substrate comprises a PTVYHQT amino acid sequence. The method of claim 1, wherein the peptide substrate comprises an EAVNHQS amino acid sequence. The method of claim 1, wherein the peptide substrate comprises an EPVTHEA amino acid sequence. The method of claim 1, wherein the peptide substrate comprises a TAVYAQT amino acid sequence. The method of claim 1, wherein the peptide substrate comprises an ACVYHQA amino acid sequence. The method of claim 1, wherein the peptide substrate comprises a VPVDHES amino acid sequence. The method of claim 1, wherein the peptide substrate comprises a QASQPSG amino acid sequence.