KR102121863B1 - Method for Analyzing Proteinase Activity using luciferase - Google Patents

Abstract

The present invention relates to a method for measuring the activity of a protease using a luciferase. When the protease has activity, the substrate peptide is decomposed, and the luciferase is dissociated to decrease the bioluminescence signal, so that the protease is Activity can be easily measured.

Description

Method for Analyzing Protease Activity Using Luciferase{Method for Analyzing Proteinase Activity using luciferase}

The present invention relates to a method for measuring the activity of a protease using luciferase.

Cells secrete enzymes into extracellular fluids, primarily through mediated endoplasmic reticulum and Golgi. Extracellular matrix metalloproteinase (MMP) is a protease secreted from cancer cells and stromal cells and is an important enzyme for cell migration, metastasis and tissue remodeling. Quantitative evaluation of MMP activity in multiple tissues or cells provides important clues to distinguish normal and abnormal development and provides the basis for developing new disease treatment strategies. So far, zymographic techniques have been used for a long time as a standard method for analyzing MMP activity, which easily measures the activity of different MMPs depending on the degradation and molecular weight of the preferred substrate. However, zymographic technology takes a long time in the process of protein unfolding and refolding, and SDS (sodium dodecyl sulfate) dissociates TIMPs (tissue inhibitor of metalloproteinase) from MMP during electrophoresis. There is a drawback in how many MMPs exist in complex form with TIMPs. For rapid and direct measurement of MMP activity, colorimetry, electrochemical measurement or highly sensitive fluorescence measurement using fluorescent dyes, proteins and nanomaterials has been developed. However, when used in real samples, colorimetric or electrochemical measurements can interfere with the measurement signal, and fluorescence signals can be disturbed by absorption or scattering of light in biological samples. To avoid this problem, fluorophores and nanomaterials exhibiting long wavelength emission have been used for in vivo imaging, but complicated synthesis process, expensive cost and low quantum yield Due to this, it is still a hindrance to actual use.

Bioluminescence (BL) measurements using luciferases over the past few decades have allowed sensitive and selective detection of targets of most cell and tissue types due to the lack of extensive emission range and the need for incidental excitation. Was used.

Bioluminescence based detection is mainly used for intracellular sensing, such as a bacterial sensor, gene reporter or bioluminescence resonance energy transfer (BRET) system for protein-protein interaction. However, despite the wide range of applications of luciferace, in order to extend bioluminescence measurement in a real sample to a large-capacity analysis method, it must be possible to fix the luciferace to a surface.

In order to solve the above problems, the present inventors produced a conjugate in which luciferase, a protease substrate and biotin were fused, and immobilized the conjugate on the surface through biotin-streptavidin binding to enhance the activity of protease. The present invention was completed by confirming that it can be measured.

1.Jeftinija, S., J Cell Mol Med., 2006, 10, 273-2791 2.Page-McCaw, A. et.al., Nat Rev Mol Cell Bio., 2007, 8, 221-233 3. Snoek-van Beurden, P.A.M. et.al., Biotechniques., 2005, 38, 73-83 4. Kim, G.B. et.al., Biosens Bioelectron., 2013, 41, 833-839 5. Zhang, C.Q. et.al., Biochip J., 2016, 10, 198-207 6. Fernandez, A. et.al., Chem Soc Rev., 2016, 45, 1182-1196 7.Yang, J. et.al., Bba-Mol Cell Res., 2007, 1773, 400-407 8. Kim, Y.P. et.al., Anal Chem., 2008, 80, 4634-4641 9.Frangioni, J.V., Current opinionin chemical biology., 2003, 7, 626-634 10. Paley, M.A. et.al., MedChemComm., 2014, 5, 255-267 11.Kim, Y.P. et.al., Chem Commun., 2010, 46, 76-78 12. Dawson, P.E. et.al., Science., 1994, 266, 776-779

One object of the present invention is to provide a method for measuring the activity of a protease comprising the following steps:

⒜ fixing the fusion protein of luciferase and protease substrate peptide to the surface through a linker;

⒝ contacting the fixed fusion protein with luciferin; And

⒞ contacting the product of 결과 with a sample to be tested for protease activity.

Another object of the present invention is to provide a kit for measuring the activity of a protease, comprising a fusion protein of a luciferase and a protease substrate peptide and a linker that fixes the fusion protein to the surface.

In order to achieve the above object, an aspect of the present invention

⒜ fixing the fusion protein of luciferase and protease substrate peptide to the surface through a linker;

⒝ contacting the fixed fusion protein with luciferin; And

It provides a method for measuring the activity of protease, comprising the step of contacting a sample to be tested for protease activity with the product of ⒝.

In the present invention, when the protease is present in the sample and has activity, the substrate peptide of the protease is decomposed, and the luciferase dissociates from the surface. Since the dissociated luciferase is removed by washing, the bioluminescence signal is reduced, and the activity of the protease can be measured by confirming this signal change.

As used herein, the term'luciferase' refers to an oxidase that causes bioluminescence by oxidizing the substrate luciferin and refers to Photinus Pyralis , fireflies, Renilla reniformis , sea pansy, Gaussia Princeps ), Oplophorus gracilirostris (sea shrimp).

In one embodiment of the present invention, the luciferase of the mussel is Renilla luciferase from Renilla reniformis, Gaussia luciferase from Gaussian Princes, and Opus luciferase It can be selected from the group consisting of Optophorus luciferase and Firefly luciferase derived from Fortinus pyralis, but is not limited thereto.

As used herein, the term'proteinase' refers to a hydrolase that hydrolyzes a peptide bond of a protein to make an amino acid or peptide. It is contained in a lot of animal and plant tissues and cells, and is classified into exopeptidase and endopeptidase according to the decomposition pattern.

In one embodiment of the present invention, the protease may be an extracellular matrix metalloproteinase (MMP). The extracellular matrix metalloproteinase is a type of endopeptidase that breaks down the extracellular matrix and contains a metal atom at the active site. The extracellular matrix protease is MMP 1, MMP 2, MMP 3, MMP 7, MMP 8, MMP 9, MMP11, MMP 12, MMP 13, MMP 14, MMP15, MMP 16, MMP 17, MMP 24 and MMP It may be selected from the group consisting of 25, preferably MMP 7.

As used herein, the term'protease peptide of a protease' is a peptide recognized and cleaved by a protease, and may include any amino acid sequence recognized by the protease to be analyzed and cleaved. .

In one embodiment of the invention, the substrate peptide of the protease may include the sequence of SEQ ID NO: 6. The peptide sequence shown in SEQ ID NO: 6 (GGVPLSLTMGG) can be used as a substrate for an extracellular matrix protease, and has a k cat/ K _M value that is 10 times higher for MMP-7 compared to MMP-2 or MMP-9. It is particularly useful for measuring MMP-7 activity.

As used herein, the term'linker' is a medium for fixing a fusion protein to a surface, and one end of the linker is connected to a substrate peptide of a protease. The other end is connected by a material that specifically binds to a linker fixed to the surface by covalent bonding, hydrogen bonding, or the like, and as a result, the conjugate is fixed to the surface. One or two or more substances may be used as the linker, and may be selected from the group consisting of biotin, glutathione, histidine, lectine, or derivatives thereof, but is not limited thereto. no. For example, when biotin is used as a linker, streptavidin may be immobilized on the surface.

In one embodiment of the present invention, the fusion protein of the luciferase and the protease substrate peptide may be in the form of a conjugate interconnected with a linker. For example, a fusion protein and GyrA (intein) combination of a luciferase and a protease substrate peptide is first produced using a recombinant vector. Thereafter, as shown in FIG. 1 of the present invention, a conjugate of luciferase-proteinase-substrate peptide-linker can be prepared by performing a substitution reaction of GyrA and biotin.

The intein (intein) is a region in which the protein is cut in the process of protein splicing (folding) before folding (folding) in the form of a completed form. In some proteins, the precursor protein after translation is part of it by autocatalytic reaction. Is cut and the rest is recombined to become a mature protein. At this time, the region to be cut is called intein, and the region that remains as a mature protein is called extein.

In one embodiment of the present invention, the luciferin of the murine is luciferin, firefly luciferin, snail luciferin ( Latia neritoides luciferin), the bacterial luciferin ( Achromobacter fischerii luciferin) and coelenterazine (coelenterazine), preferably coelenterazine- h may be used.

In one embodiment of the present invention, the method detects a change in the bioluminescence signal of the luciferase after the step ⒞, and when the bioluminescence signal decreases, there is protease activity in the sample, and when the bioluminescence signal is maintained, the sample It may further include the step of determining that there is no enzymatic activity of the protease.

In the present invention, the'sample' means a composition to be analyzed by containing or presumed to contain a protease to be analyzed, and may be a biological sample. The biological sample may be tissue, cells, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems, but is not limited thereto.

Another aspect of the present invention provides a kit for measuring the activity of a protease, comprising a fusion protein of a luciferase and a protease substrate peptide and a linker that fixes the fusion protein to the surface.

The kit may further include a microplate in which luciferin and a fusion protein, which are substrates for bioluminescence of luciferase, are fixed, and the microplate may be attached with a linker to fix the fusion protein.

Using the method according to an example of the present invention, it is possible to easily measure the activity of a protease by measuring a change in the bioluminescence signal of luciferase.

A of FIG. 1 schematically shows a process of preparing a fusion protein ( R luc8-pep-Bio) linked with luciferase, a substrate peptide, and biotin, and FIG. 1B shows proteolysis using R luc8-pep-Bio A method for measuring the activity of an enzyme is schematically shown.
2A, R luc8-m7-GyrA was treated with 2-mercaptoethane-sulfonic acid (MESA) alone (lane 2), MESA and CK-biotin co-treatment (lane 3), and MESA and L-cysteine, respectively. SDS-PAGE was performed after treatment (lane 4), and lanes 5 and 6 were purified R luc8-m7-Bio and R luc8-m7-C (C is L-cysteine) to SDS-PAGE, respectively. The result of separation is shown.
B of FIG. 2 shows the binding thickness after loading R luc8-m7-Bio (biotinylated fusion protein) or R luc8-m7-C (non-biotinylated fusion protein) on the streptavidin coated microplate surface. binding thickness).
Figure 3 shows the results of reacting with various concentrations of MMP-7 by fixing R luc8-m7-Bio to a microplate (A), and measuring the bioluminescence signal after reaction (B) and displaying the bioluminescence signal in a graph. One result (C) is shown.
Figure 4 shows the results of measuring the activity of MMP-7 in various concentrations of mouse serum (A), cell culture medium (B) and cell tissue extract (C).

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are intended to illustrate one or more embodiments by way of example, and the scope of the present invention is not limited to these examples.

Experiment method

reagent

Activated extracellular matrix metalloproteinase-7 (hereinafter referred to as MMP-7) and extracellular matrix metalloproteinase-2 (hereinafter referred to as MMP-2) are Millipore Sigma ( MA, USA). Coelenterazine - h (coelenterazine- h) and is purchased from (AZ, USA) Nanolight Technologies, nickel (Ⅱ) - nitrilotriacetic acid (Ni (II) -nitrilotriacetic acid, Ni-NTA) resin and Nutri avidin-coated NeutrAvidin (NA)-coated white 96-well plates were purchased from Thermo Fisher Scientific Inc. (USA). L-arabinose, 2-mercaptoethane-sulfonic acid (MESA) and L-cysteine (L-Cys) were purchased from Sigma-Aldrich (USA). . In addition, cysteine-lysine-biotin (Cys-Lys-Biotin, CK-biotin) was synthesized from Peptron Inc. (Daejeon, Korea), and RIPA buffer (radioimmunoprecipitation assay buffer) was purchased from Cell Signaling Technology (MA, USA). .

R Luc8 -pep- GyrA Plasmid production

Renilla RenifomisRenilla reniformis; Sea pansy)-derived luciferase mutant 8RLuc8 gene and Mycobacterium genophy (Mycobacterium xenopi) To insert the substrate sequence of MMP-7 (GGVPLSLTMGG, SEQ ID NO: 6; hereinafter referred to as m7) between the GyrA gene encoding Mxe GyrA intein derived from pBAD-RThe m7pep-GyrA gene was constructed using the Luc8-GyrA plasmid. As a production method, continuous PCR (consecutive polymerase chain reaction) using the following five primers was used, and the primer sequence is as follows.

(SEQ ID NO: 1) 1st F-primer: 5′-ACA ATG GGT GGT GAA TTC TGC ATC ACG-3′

(SEQ ID NO: 2) 2nd F-primer: 5′-TA CCT CTG TCA CTG ACA ATG GGT GGT G-3′

(SEQ ID NO: 3) 3rd F-primer: 5′-C GAG GGA GGA GTA CCT CTG TCA CT-3′

(SEQ ID NO: 4) 4th F-primer: 5′-CCG CTC GAG GGA GGA GTA CCT-3′

(SEQ ID NO: 5) Common R-primer: 5′-ATGC GAA TTC ACC ACC GCT ACG CAG ACT TAC AAT ACC ACC CTG CTC GTT CTT CAG-3′

Subsequently, the PCR product was digested with a combination of XhoI/HindIII restriction enzyme, and ligated with the pBAD- R Luc8-GyrA plasmid digested with the same restriction enzyme to prepare a pBAD- R Luc8-m7-GyrA plasmid.

R Luc8 -m7-Bio and R luc8 -pep-C fusion protein production

To construct a fusion protein with His ₆ -tag at the C-terminus, the pBAD- R Luc8-m7-GyrA plasmid was transformed into E. coli TOP10 competent cells. The transformed cells were cultured at 37° C. for 16 hours while stirring at 200 rpm/min in 0.5 L of LB liquid medium (Luria-Bertani broth) containing ampicillin (100 μg/ml). Then, when the absorbance of the medium reached 0.7, 0.2% arabinose was added to induce protein expression, and further cultured at 37° C. for 5 hours. After incubation, E. coli cells are recovered by centrifugation and 50 mM phosphate-buffered saline (PBS) containing lysis buffer [10 mM imidazole, 300 mM sodium chloride (NaCl) and 1 mg/mL lysozyme) , pH 8.0] The cells were resuspended in 30 ml. E. coli cells were sonicated for 10 minutes with 10 second on and 10 second off cycles, and centrifuged at 5,200x g for 30 minutes at 4°C. Only the supernatant was recovered and filtered with a syringe filter (Minisart) of 0.45 μm pore size. The filtered soluble lysate was mixed with 1 ml of a 50% Ni-NTA agarose slurry (thermo Scientific) equilibrated with lysis buffer and reacted at 4° C. for 16 hours with stirring. After the reaction is finished, the E. coli lysate-Ni-NTA mixture is loaded into a 5 ml column (Thermo Scientific) whose bottom outlet is blocked with a cap, and the sample flowing out through the column after removing the cap of the bottom outlet ( flow-through sample). The column was washed 4 times with washing buffer (50 mM PBS containing 20 mM imidazole and 300 mM sodium chloride, pH 8.0), and elution buffer (50 mM PBS containing 500 mM imidazole and 300 mM sodium chloride, pH 8.0 ) To elute the protein from the column. The eluted protein fraction is further purified with a PD-10 desalting column (GE Healthcare), and the purified protein is concentrated with Microcon (YM-50; Millipore) and then in 100 mM PBS (pH 7.4). Resuspension. The protein concentration was calculated using an extinction coefficient of 91,010 M ^-1 cm ^-1 after measuring absorbance at 280 nm.

R luc8-m7-Bio (R luc8 m7--biotin) or a non-biotinylated form of R luc8-m7-C (R luc8-m7- cysteine) to make, by using amino-thiol derivative (aminothiol derivative) R Luc8 Splicing of -m7-GyrA was performed. Briefly, MESA (40 μl at 50 mM) and CK-biotin (20 μl at 1 mM) and purified R luc8-m7-GyrA-His ₆ fusion protein (40 μl at 50 M) were added to the reaction buffer (0.1 mM PBS). , pH 8.0). In addition, L-Cys (20 μl at 1 mM) was used instead of CK-biotin for R luc8-m7-C production. Each reaction mixture was reacted overnight at 4° C. in a dark place with light stirring. To purify the cleavage product, 200 μl of the reaction mixture and 100 μl of a 50% Ni-NTA agarose slurry were mixed with stirring at 4° C. for 1 hour. After centrifuging the agarose mixture, the supernatant protein was further purified with a microcone to remove the remaining MESA, CK-biotin and L-Cys. The desired product ( R luc8-m7-Bio or R luc8-m7-C) was confirmed by SDS-PAGE, the absorbance was measured at 280 nm, and the concentration was confirmed using an extinction coefficient of 63,495 M ^{- 1} cm ^-1 .

sample preparation

HT-1080 (human fibrosarcoma cells, MMP-7 positive) and HT-29 (human colon adenocarcinoma cells, MMP-7 negative) cells are T75 tissue culture flasks (SPL Life Science, Korea) and Cultured at 37°C using RPMI 1640 medium containing 10% FBS. After 2 days, the cell culture medium was replaced with serum-free medium and cultured for 24 hours, and the medium was collected in a 15 ml tube using a sterile pipette. The collected medium was centrifuged (6,800x g , 10 min) and then concentrated with centricon (Centron, Amicon Ultra-4 Centifugal Filters 10 K; Merck Millipore).

The mouse tissue extract was prepared by immersing the mouse tissue in PBS containing 1% Triton-X-100 and 1% (v/v) protease inhibitor, followed by sonication at 4°C for 5 seconds. After grinding, the water-soluble fraction was stored at -80°C for long-term storage.

The total protein concentration in the culture medium or mouse tissue extract was confirmed according to the manufacturer's instructions using the Bradford method.

Of fusion proteins Biotinylation Check whether

To confirm the biotinylation of the purified fusion protein, surface analysis was performed with biolayer interferometry (BLItz instrument, Fortebio, USA). Before binding measurements, streptavidin-coated optical probes (#18-5019; Fortebio, USA) were equilibrated for 10 min in loading buffer (PBS with 0.2 mg/mL BSA). Ordered. After measuring the initial baseline for 60 seconds, R luc8-m7-Bio (4 μl at 1 μM in PBS) was loaded on the streptavidin coated sensor for 180 seconds. Thereafter, washing was performed for 120 seconds while flowing PBS to the biosensor, and the same process was repeated with a negative control R luc8-m7-C (4 μl at 1 μM in PBS).

MMP Active measurement

For surface passivation and activation, each well of the NA coated plate was pretreated with TBS buffer (pH 7.4) containing 0.1% BSA and 0.05% Tween-20. R Luc8-m7-Bio (90 μl at 70 μg/mL, corresponding to 1.94 μM) and MMP enzyme (reaction buffer) dispersed in reaction buffer (20 mM Tris with 5 mM CaCl ₂ and 100 mM sodium chloride, pH 7.6) To each solution, 10 μl of the dispersed solution was mixed.

For measuring MMP activity in real biological samples, R Luc8-m7-Bio (80 μl at 70 μg/ml) dispersed in the reaction buffer and 20 μl of cell culture medium or mouse tissue lysate (protein concentration 50 μg/m) Ml). As a control, R Luc8-m7-Bio dispersed in the reaction buffer was mixed with 20 µl (50 µg/ml) of bovine serum albumin (BSA) under the same conditions.

100 μl of a solution of R Luc8-m7-Bio and MMP enzyme, R Luc8-m7-Bio and cell culture medium, or a mixture of R Luc8-m7-Bio and mouse tissue lysate was reacted for 2 hours at 37° C. To each well of the NA coated plate, 95 μl of the above reaction was added. The plate was left at room temperature for 1 hour to induce the reaction of biotin and streptavidin, and the surface was washed three times with washing buffer. 100 μl of PBS was added to each well of the plate, and coelenterazine- h (100 μl at 2 μg/ml in PBS) was added. The bioluminescence spectrum was measured at 400 to 650 nm with a multimode plate reader (Varioskan, Thermo Scientific, Inc., USA). Bioluminescence images were obtained using a chemiluminescence/bioluminescence image collection system (Fusion SL, Vilber Lourmat, Torcy, France).

Experiment result

R luc8 -pep-Bio fusion protein production

The intein protein GyrA used in the present invention is stable through a self-splicing process in the presence of synthetic cysteine-lysine-biotin (CK-biotin) and 2-malcaptoethane-sulfonic acid (MESA). Alpha-thioester is produced. As a result, due to the octane of the C- terminal fusion of the R luc8 it is ligated to the C- terminus of the CK- biotin R luc8. MESA was used as a nucleophilic compound to promote N-terminal cleavage of intein.

The R luc8-pep-GyrA recombinant vector (pep represents a peptide substrate) containing the peptide substrate of MMP was expressed in E. coli, and biotinylated R luc8-pep-Bio was produced through an intein-mediated ligation process. (Fig. 1 A). Purified R luc8-pep-Bio is used to monitor MMP activity, and after reacting with MMP in the actual sample, the peptide substrate is cleaved, leaving biotin on the NA coated microplate surface (Fig. 1B). . The bioluminescent signal can be generated at the surface of the microplate in the presence of the luciferase substrate coelenterazine- h , which is inversely proportional to the amount of MMP present in the sample.

R luc8 -m7-Bio of fusion protein Intein -medium Biotinylation Confirm

The R luc8-m7-GyrA protein was subjected to SDS-PAGE treated with MESA and/or biotin, cysteine under different conditions. As a result, as shown in FIG. 2A, R luc8-m7-GyrA protein (lane 1) was treated with MESA alone (lane 2), MESA and CK-biotin treatment (lane 3), and MESA and L-cysteine (lane When 4)) was treated together, it was confirmed that two splicing protein fragments (lanes 2 to 4) were formed compared to the original protein (lane 1). L-cysteine, which does not have a biotin moiety, was used as a negative control to compare the attachment of biotin through a trans-thioesterification process in intein and cysteine derivatives (L-cysteine). The results indicate that GyrA was cleaved by MESA in the R luc8-m7-GyrA protein and replaced with CK-biotin or L-cysteine (lanes 3 and 4). The product was purified using Ni-NTA agarose beads to remove His ₆ tagged GyrA protein from the reaction mixture in lanes 3 and 4. Thereafter, as a result of separating the purified R luc8-m7-Bio (from lane 3) and R luc8-m7-C (from lane 4) by SDS-PAGE, there was no significant difference in gel mobility of the two proteins (A in FIG. 2) Lanes 5 and 6). This is presumed to be because the molecular weights of the two proteins are almost similar.

For biolayer interferometry analysis, the binding thickness when biotinylated ( R luc8-m7-Bio) and non-biotinylated ( R luc8-m7-C) proteins were loaded onto the surface of a streptavidin coated microplate ( binding thickness) increased with saturation curve up to 4 minutes. This means that Rluc8-m7-bio is strongly bound to the surface of the microplate (solid line in B of FIG. 2). In contrast, no significant binding was seen when Rluc8-m7-C was loaded (dotted line in FIG. 2B). These experimental results indicate that the biotin group was successfully introduced into the C-terminus of R luc8-m7-GyrA through an intein-mediated splicing process to form R luc8-m7-Bio.

MMP -7 activity measurement

It was confirmed that R luc8-m7-Bio was able to measure the activity of MMP-7. MMP-7, also known as matrilysin, was selected as a model protease because it has broad specificity for various substrates of extracellular matrix including casein, gelatin, fibronectin and proteoglycan. In addition, MMP-7 is known to promote tumor invasion involving MMP-2 and MMP-9.

NA coated microplate After fixing R luc8-m7-Bio, it was reacted with active MMP-7 in various concentration ranges (0, 1, 10, 100 and 1000 ng/ml). As a result, the signal intensity of the bioluminescence image significantly decreased as the concentration of the active MMP-7 increased (A in FIG. 3), and this result was consistent with the signal change of the bioluminescence spectrum of R luc8-m7-Bio. (Fig. 3B). On the other hand, MMP-2 did not show a significant signal reduction in the same concentration range (FIG. 3C ). As a result of the experiment, it was found that the detection limit of MMP-7 activity was in the range of 1 to 10 ng/ml concentration, which is similar to or slightly increased from the previously reported results. The peptide substrate used (GGVPLSLTMG) is known to have a k cat/ K _M value that is 10 times higher for MMP-7 compared to MMP-2 or MMP-9. These results indicate that R luc8-m7-Bio can be used for quantitative measurement of MMP-7 activity with high specificity and high sensitivity.

In order to confirm that R luc8-m7-Bio acts on a real biological sample, a bioluminescence signal was confirmed after reacting mouse serum containing no active MMPs with R luc8-m7-Bio. As a result, although the bioluminescence signal was slightly changed according to the serum concentration, a significant decrease in the bioluminescence signal was not confirmed in mouse serum at various concentrations (A in FIG. 4 ). These results suggest that the luciferase probe, R luc8-m7-Bio, is stable for serum and that bioluminescence signal measurements on the microplate surface are useful for monitoring complex substances.

Culture medium (protein concentration 50) of HT-1080 cells (MMP-7 secretion) and HT-29 cells (MMP-7 non-secreting) to further investigate whether MMP-7 activity can be detected in biological samples by the above method ㎍ / ㎖) was carried out the same experiment. To measure relative MMP activity, BSA at a concentration equal to the protein concentration in the culture medium was used as a negative control.

As a result of the experiment, a significantly reduced bioluminescence signal (high MMP-7 activity) was observed in the culture medium of HT-1080 compared to the control group. In contrast, a slightly reduced signal (low MMP-7 activity) was observed in the culture medium of HT-29 (FIG. 4B ). In addition, as a result of experimenting with the same method of extracting mouse tissues including brain and testis (total protein concentration of 50 μg/ml), it was found that the activity of MMP-7 was relatively low compared to HT-1080 culture medium. There was (Fig. 4C).

Through the results thus far, it has been confirmed that the method using R luc8-m7-Bio can be usefully used to monitor and compare the activity of various proteases in biological samples in a high-efficiency and multiple manner. Bioluminescence based methods have many advantages over other colorimetric or fluorescence methods. Luciferase, a highly stable enzyme, can detect the activity of specific enzymes in complex samples, such as serum and tissue extracts, without autofluorescence or other background noise. In addition, biotinylated luciferase provides a surface immobilization method to quickly read bioluminescent signals. Unlike the conventional zymography method based on time-consuming, gel electrophoresis limited to several MMPs, the method of the present invention based on peptide substrates simply measures the activity of a wide range of proteases. Can be.

So far, the present invention has been focused on the preferred embodiments. Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered in terms of explanation, not limitation. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be interpreted as being included in the present invention.

<110> Industry-University Cooperation Foundation Hanyang University <120> Method for Analyzing Proteinase Activity using luciferase <130> P18U10C0591_P20180269KR <150> KR 10-2018-0026770 <151> 2018-03-07 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 1st F-primer <400> 1 acaatgggtg gtgaattctg catcacg 27 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 2nd F-primer <400> 2 tacctctgtc actgacaatg ggtggtg 27 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 3rd F-primer <400> 3 cgagggagga gtacctctgt cact 24 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 4th F-primer <400> 4 ccgctcgagg gaggagtacc t 21 <210> 5 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> common R-primer <400> 5 atgcgaattc accaccgcta cgcagactta caataccacc ctgctcgttc ttcag 55 <210> 6 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> substrate peptides of MMP-7 <400> 6 Gly Gly Val Pro Leu Ser Leu Thr Met Gly Gly 1 5 10

Claims (9)

⒜ fixing the fusion protein of the matrix peptide of extracellular matrix metalloproteinase (MMP) 7 and luciferase to the surface through a linker;
⒝ contacting the fixed fusion protein with luciferin; And
⒞ contacting the result of ⒝ with a sample to test the activity of the extracellular matrix metalloproteinase 7,
The substrate peptide comprises the peptide sequence of SEQ ID NO: 6, extracellular matrix metalloproteinase 7 activity measurement method.
The method of claim 1, wherein the method detects a change in the bioluminescence signal of the luciferase after the step ⒞, and when the bioluminescence signal decreases, there is activity of the extracellular matrix metalloproteinase 7 in the sample, and the bioluminescence signal is maintained. When the sample is further comprising the step of determining that there is no enzyme activity of the extracellular matrix metalloproteinase 7, the method for measuring the activity of the extracellular matrix metalloproteinase 7.
The group of claim 1, wherein the luciferase of the genus is composed of Renilla luciferase, Gaussia luciferase, Oplophorus luciferase and firefly luciferase. Is selected from, extracellular matrix metalloproteinase 7 activity measurement method.
delete According to claim 1, wherein the linker of ⒜ is selected from the group consisting of biotin (biotin), glutathione (glutathione), histidine (histidine) and lectin (lectin), activity measurement of extracellular matrix metalloproteinase 7 Way.
delete delete According to claim 1, The luciferin is selected from the group consisting of firefly luciferin (firefly luciferin), snail luciferin ( Latia neritoides luciferin), bacterial luciferin ( Achromobacter fischerii luciferin) and coelenterazine (coelenterazine). Method for measuring the activity of matrix metalloproteinase 7.
The substrate peptide of the extracellular matrix metalloproteinase 7 and the fusion protein of luciferase and a linker that fixes the fusion protein to the surface, wherein the substrate peptide comprises the peptide sequence of SEQ ID NO: 6, extracellular matrix Kit for measuring activity of metalloproteinase 7.

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