KR20090030089A - Fusion protein for detecting caspase-3 activity and method for detecting caspase-3 activity using the same - Google Patents

Abstract

A fusion protein for detecting caspase-3 activity is provided to minimize interference in interaction between the caspase-3 and the peptide stroma, to improve sensitivity and selectivity and to be useful for the treatment of the apoptosis-related diseases. A fusion protein for detecting caspase-3 activity comprises a protein-L-fluorescence protein for the solid support bond. L is the linker including DEVD amino acid sequence. The for the solid support bond is glutathinoe-S transferase(GST), Maltose Binding Protein(MBP) or 6xHistidine. The fluorescence protein is EGFP(enhanced green fluorescent protein), ECFP(enhanced cyanfluorescent protein), EBFP(enhanced blue fluorescent protein), EYFP(enhanced yellow fluorescent protein) or RFP(red fluorescent protein).

Description

Fusion Protein for Detecting Caspase-3 Activity and Method for Detecting Caspase-3 Activity Using the Same}

The present invention provides a fusion protein for detecting the activity of caspase-3, which provides improved sensitivity and selection in measuring caspase activity, a protein chip having the fusion protein immobilized on a substrate, and the caspase-3 using the same. It relates to an activity detection method.

Apoptosis is the action of genetically conserved cells that play an important role in the pathogenesis and treatment of many diseases (Ashkenazi, A. & Dixit, VM, Science, 281: 1305, 1998; Elmore, S., Toxieol . Pathol ., 35: 495, 2007; Nagata, S., Cell , 88: 355, 1997). Cells in which apoptosis occurs include unique morphological and biochemical changes, such as membrane remodeling, nuclear condensation, DNA fragmentation, and activation of intracellular cysteine degrading enzyme families called caspases. (Earnshaw, WC et al ., Annu . Rev. Biochem., 68: 383, 1999; Hengartner, MO, Nature , 407: 770, 2000; Thornberry, NA & Lazebnik, Y., Science , 281: 1312, 1998). Caspases involved in apoptosis can be distinguished according to their substrate specificity (Thornberry, NA et. al ., J. Biol. Chern ., 272: 17907, 1997), in particular, primary effector caspase, including caspase-3 or caspase-7, has a preference for substrates comprising the four amino acid recognition sequence DEVD. It is found frequently in the caspase matrix of the cytoplasm or nucleus (Thornberry, NA et. al ., J. Biol . Chern ., 272: 17907, 1997; Zhivotovsky, B., Essays Biochem ., 39:25, 2003). Because caspase-3 is degraded by nucleosomal fragmentation of chromosomes and promotes the activity of other effector caspases, this protease is known to play a pivotal role in the practice of apoptosis (Boatright, KM & Salvesen). , GS, Curro. Opin. Cell Biol ., 15 : 725, 2003; Grutter, MG, Struet . Biol ., 10 : 649, 2000). Activated caspase-3 also contains poly- (ADP-ribose) polymerase (PARP), inhibitor of caspase-activated deoxyribonuclease (CAD), and cell death. death) to cause alpha-cuts a plurality of the cell substrate, such as fabric gave (alpha-fodrin) (Counis, MF & Torriglia, A., Biochem cell Biol ., 78: 405, 2000; Janicke, RU et al ., J. Biol . Chem ., 273: 15540, 1998; Stroh, C. & Schulze-Osthoff, K., Cell Death . Differ ., 5: 997, 1998).

Recent studies indicate that FRET-based assays can be used to detect the degradation activity of caspase-3 (E1phick, LM et al ., Anal . Bioehem., 349: 148, 2006; Jones, J. et al ., J. Biomol . Screen , 5: 307, 2000; Luo, KQ et al . , Biochem . Biophys . Res . Commun ., 283: 1054, 2001; Wu, Y. et al. , Cancer Lett ., 235: 239, 2006). FRET occurs when two different fluoroproteins (donor and acceptor) are linked to each other and the transfer of energy takes place from the donor to the donor. When the caspase-3 activity is measured in a spectrum-based method, the linker is a protease. When separated by, no FRET is observed. FRET-based methods are very useful for understanding other intracellular processes, but it is very difficult to obtain quantitative results because FRET measurements can be hindered by overlapping exitation intensities between donor and recipient wavelengths (Nguyen, AW). & Daugherty, PS, Nat Biotechnol, 23:..... 355, 2005; Tsien, RY, Annu Rev Biochem, 67: 509, 1998).

The most popular experimental method for monitoring the activation of caspase-3 is a fluorometric assay using an artificial fluorogenic substrate (DEVD-AFC) for caspase-3 (Stennicke , HR & Salvesen, GS, J. Bioi . Chern ., 272: 25719, 1997). However, although fluorescence-based assays are sensitive and suitable for measuring small amounts of enzymatic activity, the biological activity of peptide substrates can be influenced by artificial labels, thereby caspase-3 and peptide substrates. There was a problem that interference could occur during interaction (Su, J. et. al ., Anal . Chern ., 78: 4945, 2006). In addition, Korean Patent Publication No. 2002-8122 also has the same problem as the invention to measure the amc fluorescence emitted by mixing the DEVD- amc substrate to measure the caspase activity present in the organelle membrane.

On the other hand, Korean Patent Publication No. 2003-76734 concerning a recombinant fluorescent protein prepared by inserting a caspase cognitive sequence into a variant insertion fluorescent protein (Peridot), and a cationic quencher is cut out by the caspase to fluoresce the fluorescence Korean Patent Application Publication No. 2006-113882, which is a method of analyzing enzyme activity by inhibiting it, has been published, but its utilization and sensitivity are not high in measuring caspase activity. This is because the quench-based measurement method is not an indirect monitoring method, but an indirect measurement method in which the quencher cut by the caspase measures a decrease in fluorescence generated by quenching the fluorescent agent again. There is a need for a method for measuring the activity of caspase-3, which provides improved sensitivity and selection.

The present inventors minimize the problem of interference due to the interaction between caspase-3 and the peptide substrate due to fluorometric assay using a conventional artificial fluorogenic substrate (DEVD-AFC), Efforts have been made to develop a fusion protein for detecting the activity of caspase-3 and a detection method using the same, which are simple, economical and quantitative measurements. As a result, a solid support binding protein, a fluorescent protein, a protein for binding the solid support and a fluorescent protein A fusion protein for detecting the activity of caspase-3 comprising a linker located between and comprising a DEVD amino acid sequence was prepared, and then the caspase-3 vs. It was confirmed that the activity can be detected economically and quantitatively without interference of peptide substrate interaction, and the present invention To complete.

In conclusion, the main object of the present invention is to provide a fusion protein for detecting the activity of caspase-3 and a protein chip immobilized on a substrate, which provides improved sensitivity and selection in measuring caspase activity. have.

Another object of the present invention is to provide a protein chip in which the fusion protein and the fusion protein are immobilized on a substrate, and a method for detecting the activity of caspase-3 using the protein chip.

In order to achieve the above object, the present invention provides a fusion protein for detecting the activity of caspase-3 consisting of a protein-L-fluorescent protein for solid support binding:

Wherein L is a linker comprising the DEVD amino acid sequence of SEQ ID NO: 1.

SEQ ID NO 1: Asp Glu Val Asp (DEVD)

In the present invention, the solid support binding protein is selected from the group consisting of glutathione-S transferase (GST), maltose binding protein (MBP) and hexahistidine (6 × Histidine) It may be characterized by. As the solid support, beads, membranes, slides, plates, test tubes, streptavidin-coated spheres, polymer microspheres, silica microspheres, organic nanoparticles, inorganic nanoparticles, and the like can be used. It may be characterized by including a molecular material that binds to the support binding protein. In other words, when GST is used as a solid support binding protein, glutathione is used, amylose is used when MBP is used, and nickel is coated or spotted on the surface of the solid support when hexahistidine is used. To be combined.

In the present invention, the fluorescent protein is a group consisting of enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP). May be selected from, but is not limited to.

In the present invention, the linker may be characterized by consisting of 6 to 9 amino acids. In this case, preferably, the linker comprises a linking peptide consisting of two glycines (glycine) at the N and C ends of the DEVD amino acid sequence of SEQ ID NO: 1.

The present invention also provides a protein chip for detecting the activity of caspase-3, wherein the fusion protein is immobilized on a substrate.

In the present invention, the substrate may be characterized in that the metal, preferably the metal uses gold (Au).

In the present invention, when GST is used as the protein for binding the solid support of the fusion protein to the metal substrate, the fusion protein can be immobilized on the substrate by binding the GST to glutathione on the surface of the glutathione metal substrate. Meanwhile, the protein chip according to the present invention can fix the fusion protein on the substrate by binding the GST to glutathione on the surface of the glutathione glass substrate. Here, the "glutathione metal substrate" refers to a metal chip in which glutathione is coated or spotted on the surface of the metal chip, and the "glutathione glass substrate" refers to a glass chip in which glutathione is coated or spotted on the surface of the glass chip.

In the present invention, in the case of using maltose binding protein (MBP) as a protein for solid support binding of the fusion protein fixed to the protein chip, amylose may be used instead of glutathione, and solid support binding When hexahistidine (6 × Histidine) is used as the soluble protein, nickel can be used to prepare protein chips for detecting the activity of caspase-3.

The present invention also provides a method for detecting caspase-3 activity, wherein the fusion protein is used.

Caspase-3 activity detection method using a fusion protein according to the present invention comprises the steps of (a) fixing the fusion protein to a solid support; (b) treating caspase-3 with the fusion protein immobilized on the solid support; And (c) analyzing the fluorescence signal of the fluorescent protein of the caspase-3 treated fusion protein to detect the activity of the caspase-3.

The method for detecting the activity of caspase-3 using the fusion protein also includes: (a) treating caspase-3 with the fusion protein; And (b) performing immunoblotting on the caspase-3 treated fusion protein using an antibody against the protein for binding to the solid support of the fusion protein to detect the activity of the treated caspase-3. It may be characterized in that it comprises a.

The present invention also provides a method for detecting caspase-3 activity using a protein chip in which the fusion protein for detecting the activity of caspase-3 is immobilized on a substrate.

When the substrate of the protein chip according to the present invention is a metal, the caspase-3 activity detection method according to the present invention comprises: (a) introducing and reacting caspase-3 on the surface of the protein chip; And (b) analyzing the surface plasmon resonance (SPR) image of the reacted protein chip to detect the activity of the introduced caspase-3. At this time, it is preferable to use gold (Au) as the metal.

Specifically, the SPR image analysis of step (b) is performed by analyzing the SPR image of the caspase-3 introduced metal chip and the SPR image of the caspase-3 unintroduced metal chip (glutathione metal chip to which the fusion protein is fixed). In comparison, when there is a change in the SPR image, it can be determined that caspase-3 is active, and at this time, the activity of caspase-3 can be quantified to the extent of the change of the SPR image.

Caspase-3 activity detection method further comprises the steps of (a) introducing and reacting caspase-3 on the surface of the protein chip according to the present invention; And (c) analyzing the fluorescence image of the reacted protein chip to detect the activity of the introduced caspase-3.

The fusion protein for detecting the activity of caspase-3 according to the present invention may be prepared by the following steps. That is, (a) amplifying a gene encoding a fluorescent protein using a primer comprising a nucleotide sequence corresponding to the DEVD amino acid sequence; (b) introducing the amplified product into a plasmid containing a protein support for solid support binding to generate a fusion gene; And (c) transforming the plasmid containing the fused gene into a microorganism, and then expressing the fused gene.

Preferably, the primer is a 5 'primer using a base sequence consisting of the sequence of SEQ ID NO: 2, 3' primer can be used a base sequence consisting of the sequence of SEQ ID NO: 3.

SEQ ID NO: 5'-CGGGATCCGGAGGGGATGAGGTGGATGGGGGCATGGTGAGCAAGGGCGAG

SEQ ID NO: 5'-CGGAATTCCTTGTACAGCTCGTCCATG

Caspase-3, on the other hand, is known to play an important key role in the process of apoptosis, and the regulation of caspase-3 enables therapeutic strategies targeting the apoptosis pathway. 1 illustrates a fusion protein for detecting caspase-3 activity according to the present invention. As shown in FIG. 1, activated caspase-3 has a cleavage activity against a DEVD amino acid sequence, that is, immediately after the DEVD sequence. Cleavage of the peptide followed by the Asp sequence.

Fluorescent proteins constituting the fusion protein of the present invention not only allow the measurement of fluorescence changed by caspase activity, but also enable an analytical approach to measure the reduced molecular weight of the truncated fluorescent protein.

As described in detail above, the present invention provides a fusion protein for detecting the activity of caspase-3, a protein chip to which the fusion protein is immobilized, and a caspa using the same. A third activity detection method is provided.

The method for detecting the activity of caspase-3 using the fusion protein according to the above is a simple, economical, label-free system, thereby minimizing the problem of interference of caspase-3 versus peptide substrate interaction.

In conclusion, the present invention is useful for effectively establishing a treatment strategy for various apoptosis-related diseases targeting the pathway of apoptosis by providing a more sensitive and selective method of detecting caspase-3 activity.

Through the following examples will be described the present invention in more detail. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

< Example  1> Caspase -3 ( caspase -3) for activity detection Fusion protein  Produce

1. Strains, Vectors, and Enzymes

E. coli Strain DH5α was used as a host for subcloning and E. coli BL21 (DE3) (Novagen, W1) was used as a host for gene expression. E. coli in the experiment Strains were incubated in 37 ℃ LB medium, ampicillin was treated to the strain containing the plasmid at a concentration of 50mg / l. PBluescript SK + (Stratagene, CA) was used as a vector for subcloning and amplification of the full-length gene, and pGEX 4T-1 (Amersham Pharmacia Biotech, CA) was used as a vector for expression. And modifiying enzymes were purchased from Boehringer-Mannheim (Germany) and used according to the supplier's recommendations. QIAEX gel extraction kit (QIAEX gel extraction kit, Qiagen, Germany) was used to prepare the vector DNA.

2. Gene Cloning, Expression and Isolation of Recombinant Fusion Proteins

In order to prepare glutathione-S transferase (GST) -L-EGFP (L is a linker including the DEVD amino acid sequence), a fusion protein for detecting caspase-3 activity, GST: DEVD Cloning of: EGFP fusion gene was performed. Wherein the GST: DEVD: EGFP fusion gene corresponds to a linker (L) comprising (i) a GST gene, (ii) an EGFP gene and (iii) the DEVD amino acid sequence, and as a base sequence connecting the GST and EGFP genes. Means a fusion gene.

To clone the GST: DEVD: EGFP fusion gene, the full-length gene encoding the EGFP was amplified by polymerase chain reaction (PCR) using a 5 'primer of SEQ ID NO: 2 and a 3' primer of SEQ ID NO: 3, The 5 and 3 ends were designed to include Bam HI and Eco RI restriction enzyme cleavage sites.

SEQ ID NO: 5'-CGGGATCCGGAGGGGATGAGGTGGATGGGGGCATGGTGAGCAAGGGCGAG

SEQ ID NO: 5'-CGGAATTCCTTGTACAGCTCGTCCATG

The PCR product was isolated using a DNA purification kit (DNA purification kit, Qiagen), and then cleaved at the Bam HI and Eco RI restriction enzyme sites. The resulting DNA fragments were then introduced into the pGEX 4T-1 plasmid containing the GST gene, resulting in in-frame fusion of pGST: DEVD: EGFP. At this time, the GST: DEVD: EGFP fusion gene was confirmed by DNA sequencing. For expression of recombinant fusion proteins, the pGST: DEVD: EGFP was replaced with Escherichia. coli BL21 (DE3). Transformed cells were induced by 1 mM IPTG (isopropyl β-D-thiogalactopyranoside), incubated for an additional 3 hours, and then centrifuged at 6,000 × g for 10 minutes at 4 ° C. Thereafter, 10 ml of crude cell lysate was loaded on a GST miniexcellose affinity column (Biorogen Co., Korea), and 10 ml of balanced buffer (PBS, pH) to separate the fusion protein GST: DEVD: EGFP from the obtained product. 7.4) and washed three times. Here, 'GST: DEVD: EGFP' refers to a fusion protein of EGFP, which is a linker (L) -fluorescent protein comprising a GST protein-DEVD amino acid sequence, and is referred to as 'GST: DEVD: EGFP' hereinafter for convenience. On the other hand, GST: EGFP, a fusion protein of GST and EGFP as a negative control group, was produced using a PCR technique, and was also separated by the same method as above.

As a result, as shown in Figure 2, the isolated GST: EGFP fusion protein and GST: DEVD: EGFP fusion protein according to the present invention was found to correspond to approximately 51kDa and 52kDa on SDS-PAGE, each of the fusion proteins The molecular weight of was consistent with that expected. The linker of the GST: DEVD: EGFP fusion protein prepared using the primers of SEQ ID NOS: 2 and 3 includes DEVD, the sequence for caspase-3 cleavage, and the linker at the N- or C- terminus of DEVD (FIG. 1). Linker-1 and Linker-2) are each composed of two glycines.

The separated fusion protein GST: EFGP with GST: DEVD: EGFP is a caspase-3 and then decomposing activity characteristic in For in vitro monitoring, immunoblotting analysis, glutathione-agarose bead analysis, SPR image protein chip technology and fluorescence based detection methods were analyzed as in the following examples.

< Example  2> Caspase -3 ( caspase -3) for activity detection Fusion proteins  Preparation of Used Protein Chips

Bare gold chip (K-MAC, Korea) was converted to concentrated 'Pirnaha' solution (70% (v / v) H ₂ SO ₄ , 30% (v / v) H ₂ O ₂ ) Washed and rinsed thoroughly with distilled water and ethanol. Gold chips were then immersed in ethanol containing 10 mM MUA (11-mercapto-1-undecanoic acid) for 16 hours. A self-assembled monolayer (SAM) of MUA self-assembled on a gold chip was prepared with 0.1 M NHS (N-hydroxy-succinimide) and 0.4 M EDC (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride) in distilled water. The mixture was incubated for 10 minutes and activated with hydroxylsuccinimidyl ester. After surface activity, PBS buffer (pH 7.4) containing 1 mg / ml of amino-dextran was treated on the gold surface and allowed to react for 1 hour. The hydrophilic dextran surface was then activated for 2 hours using 0.1 mg / ml BMPS (N- (β-maleimidopropyloxy) succinimide ester) in PBS buffer (pH 7.4). The chips were then incubated for 2 hours in PBS buffer (pH 7.4) containing 1 mg / ml L-glutathione (L-glutathione (reduced form)) to modify the activated gold surface, and the chips were washed with distilled water. . The resulting glutathione gold chip was preserved in 4 ° C. PBS buffer (pH 7.4). MUA and NHS were purchased from Sigma-Aldrich, EDC and BMPS were purchased from Pierce (IL, USA) and amino-dextran (MW 500,000) was purchased from Molecular Probes (OR, USA).

Thereafter, the protein chip of the present invention was prepared by immobilizing GST: DEVD: EGFP reporter, a fusion protein for detecting the activity of caspase-3 prepared in Example 1, on the surface of glutathionylated gold chip. .

< Example  3> Caspase -3 ( caspase -3) for measuring activity SDS - PAGE  And Immunoblotting  analysis( SDS - PAGE and immunoblotting assay )

E. coli transformed with pGST: DEVD: EGFP obtained in Example 1 The culture medium of BL21 (DE3) was suspended in 50mM Tris-HCL buffer (pH 8.0), cell disrupted by ultrasound, and centrifuged at 10,000 g for 10 minutes to separate the soluble and insoluble fragments. The total cell lysate, ie soluble and insoluble lysates, was redissolved in a 12% SDS gel and then transferred to the nitrocellulose membrane. Gels were stained with Coomassiae Brilliant Blue R250, and protein quantitation was determined by using a densitometer (densitometer, Bio-Rad Model GS-700, CA) and scanning at 300 dpi gray scale pixel density with a scanner (HP ScanJet 6100C, CA). The intensity of the band of gels stained with was determined by reading, and protein concentration was determined by Bradford method based on bovine serum albumin. Immunoblotting assays were performed using antibodies to Glutathion S-transferase (GST) as described above (Porter, AG & Janicke, RU, Cell). Death . Differ., 6:99, 1999).

Esherichia coli Active caspase-3 expressed in BL21 (DE3) was isolated as described in Stennicke and Salvesen (Nguyen, AW & Daugherty, PS, Nat . Biotechnol ., 23 : 355, 2005). In order to confirm the cleavage efficiency of the GST: DEVD: EGFP fusion protein by the reaction of the separated active caspartate-3, the synthetic fluorescent substrate (DEVD) used for the fluorescence analysis of the isolated GST: EGFP and GST: DEVD: EGFP fusion proteins Incubation at a concentration of caspase-3 known to obtain complete cleavage cleavage of -AFC).

As a result, as shown in Fig. 3, the relative caspase-3 activity was found to be approximately proportional to the concentration, and the increase of caspase-3 activity was the concentration at which caspase-3 degrading activity was expected to be saturated. It lasted up to 300 U / ml.

Thus GST: DEVD: decomposing activity of caspase-3 using the EGFP fusion proteins in 300 U / ml of caspase-3 was treated for in vitro monitoring. Immunoblotting analysis was performed using anti-GST antibodies on GST: EGFP and GST: DEVD: EGFP fusion proteins treated with caspase-3. As a result, as shown in FIG. 4, most of the fragments of GST: DEVD: EGFP were completely cut when compared to the fragments of GST: EGFP. This means that the composition of GST: DEVD: EGFP can be used as a reporter for the detection of caspase-3 degradation activity.

< Example  4> Caspase -3 ( caspase -3) for measuring activity Glutathione - Agarose Bead  analysis( Glutathine - agarose bead assay )

The GST: DEVD: EGFP fusion protein prepared in Example 1 was immobilized on glutathione-acarrose to detect caspase-3 activity in the bead regime. Beads combined with GST: DEVD: EGFP became green fluorescence and were easily recognized, which was used for caspase-3 dependent degradation activity monitoring. In culture with activated caspase-3, the EGFP moiety is isolated from GST: DEVD: EGFP immobilized on glutathione-acarrose beads (FIG. 5).

10 μl glutathione-agarose beads combined with GST: EGFP or GST: DEVD: EGFP were decomposed at 20 ° C. in 20 mM PBS (phosphate buffered saline) buffer (pH 7.4) with 300 (U / ml) caspase-3. After incubation for 1 hour, the cells were washed three times with the same buffer. After this wash, EGFP was isolated from the fusion protein in 10 μl glutathione-agarose beads combined with GST: DEVD: EGFP. As shown in FIG. 6, the EGFP fluorescence signal disappeared completely when GST: DEVD: EGFP was incubated with caspase-3 for 1 hour, but it was correlated with the treatment of activated caspase-3 for GST: EGFP. Green fluorescence is shown. In other words, it can be seen that the method can also be applied to monitor the activity of caspase-3.

< Example  5> Caspase -3 ( caspase -3) for measuring activity SPR Imaging  Use of the system

For SPR imaging analysis to detect caspase-3 activity, a two-dimensional surface plasmon resonance (2D-SPR (surface plasmon resonance) imaging system was used. As a light source, a 150-W quartz tungsten-halogen lamp (Schott, Germany) was used, and light was obtained using a liquid light guide (Oriel Instruments, USA). goniometer arm (Physik Instrumente, Germany).

The light aimed by the lens was passed through a narrow interference filter (750 nm, Δλ = 1 nm; Oriel Instruments) and a polarizer (Newport, USA) that converts it into a particularly monochromatic, linearly polarized light beam, the diameter of which is Was approximately 2 cm. The gold-slotted gold sensor chip is optically coupled with a prism coupler (Korea Electro-optics, Korea) using an index matching oil (n _D = 1.517) and placed in the center of the goniometer. It was electrically controlled by a DC Servo motor controller (Physik Instrumente). The images reflected from the gold chip were taken with a ½ inch CCD (charge coupled device) camera (Sony, Japan), while contrast images were monitored with a personal computer. The combination of lenses was placed in front of the bare CCD chip to obtain a clear image. The image was then digitally stored on a personal computer using a B / W frame grabber (National Instrument, USA).

The glutathione gold chip used to analyze caspase-3 dependent degradation activity using the SPR imaging system was prepared in Example 2, and GST was used to monitor the degradation activity of caspase-3 using the SPR imaging system. Active caspase-3 was treated on the surface of the gold chip to which the: DEVD: EGFP fusion protein was immobilized. In theory, disappearance of EGFP results in a corresponding decrease in molecular weight of the GST: DEVD: EGFP reporter, resulting in a change in the degree of SPR image signal (FIG. 7).

As a result of measuring the SPR image, it was observed that the SPR image on the gold surface was brighter in GST: EGFP, and the relatively visual image in GST: DEVD: EGFP was substantially blurred, which is a fusion protein immobilized on the surface of the chip. Indicates that EGFP has been separated from GST: DEVD: EGFP. That is, it showed that active caspase-3 cleaved behind Asp (D) of the DEVD sequence on a protein chip to which the GST: DEVD: EGFP fusion protein was immobilized (FIG. 8). This was consistent with the fluorescence peptide analysis of FIG. 3. The SPR image was recorded with an angle of incidence slightly smaller than the SPR angle of the chip surface corresponding to only PBS or BSA with the largest difference in intensity among the SPR images. For this reason the brighter areas in the SPR image indicate the presence of the original GST: DEVD: EGFP protein on the gold chip surface (FIG. 8).

Referring to Figure 8, the fusion protein GST: DEVD: EGFP according to the present invention showed that the intensity of the SPR signal is relatively weakened when the active caspase-3 treatment, which is due to the degradation activity of caspase-3 The separation of EGFP (approximately 25 kDa) from the fusion protein GST: DEVD: EGFP means that only GST: DEVD (27 kDa) remains on the gold chip. However, bright SPR images of GST: EGFP on the gold surface were observed to be independent of caspase-3 treatment. PBS or BSA was used as the negative control and did not show any visual image.

< Example  6> Caspase -3 ( caspase -3) Fluorescence based for activity measurement Monitoring

In order to visualize caspase-3 cleavage activity for the GST: DEVD: EGFP fusion protein on the chip, glass chips immobilized with GST: DEVD: EGFP were prepared through the same procedure as the one adopted for the SPR analysis on the gold chip. After immobilizing the GST: DEVD: EGFP fusion protein on a glass chip coated with glutathione, 300 U / ml of active caspase-3 was treated on a glass chip immobilized with the fusion protein GST: DEVD: EGFP for fluorescence-based analysis. It was. Fluorescence images were measured using a GenPix 4200A 488mm laser and microarray scanner (Axon Inc., USA).

Isolation of EGFP protein appears to decrease green fluorescence, and can easily measure the degradation activity of caspase-3 using a fluorescence reader. Therefore, in order to visualize EGFP, the degradation activity of caspase-3 on the fusion protein GST: DEVD: EGFP on glass chips was measured with a fluorescence microscope equipped with a green filter. As a result, as shown in Figure 9, when incubated with active caspase-3, no green fluorescence was detected on the surface of the glass chip to which the fusion protein GST: DEVD: EGFP was fixed, which is a fusion protein GST: DEVD: Degradation occurred behind the Asp residues of the DEVD site of EGFP, indicating that the EGFP protein was isolated from the fusion protein GST: DEVD: EGFP immobilized on a glass chip.

Meanwhile, FIG. 10 is a quantitative result of analyzing the caspase-3 activity by fluorescence-based detection, and is a line plot profile picture showing fluorescence intensity where a line passes in order to quantitatively indicate fluorescence intensity. In other words, the method can be used to quantitatively measure the activity of caspase-3.

As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, such a specific description is only a preferred embodiment, which is not limited by the scope of the present invention Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

1 is a diagram illustrating a fusion protein for detecting caspase-3 activity according to the present invention having a DEVD sequence which is a caspase-3 specific cleavage site.

Figure 2 is a photograph showing the size of the control group GST: EGFP fusion protein and GST: DEVD: EGFP fusion protein according to the present invention on the SDS-PAGE.

3 is a graph showing the relative caspase-3 activity according to the concentration of caspase-3.

4 is a photograph showing the results of immunoblotting using GST: EGFP fusion protein as a control and GST: DEVD: EGFP fusion protein according to the present invention.

FIG. 5 is a schematic view of caspase-3 treatment in GST: DEVD: EGFP immobilized on glutathione-akarose beads.

6 is a photograph showing the results of glutathione-agarose beads analysis for the control GST: EGFP fusion protein and GST: DEVD: EGFP fusion protein according to the present invention.

7 is a schematic for SPR image analysis based on caspase-3 activity.

8 is a photograph showing the results of SPR image analysis for the GST: DEVD: EGFP fusion protein according to the present invention.

9 and 10 are photographs (GenePix 4200A 488nm laser) showing the results of analysis of caspase-3 activity by fluorescence-based detection.

10 is a line profile picture quantitatively showing the results of analysis of caspase-3 activity by fluorescence-based detection.

<110> Technology Licensing Center Dong-A University <120> PHARMACEUTICAL COMPOSITION FOR PREVENTING OR TREATING SEPSIS          CONTAINING WKTMVm OR ITS DERIVATIVES <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 acagcctgta ctttcgac 18 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctggaagtta gagcccgttc 20 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 gtcaagatca acagaagaac c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gggctctctc aagactataa gg 22 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atggatgatg atatggccgc g 21 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tctccatgtc gtcccagttc 20  

Claims (15)

Fusion protein for detecting caspase-3 activity consisting of protein-L-fluorescent protein for solid support binding: Wherein L is a linker comprising a DEVD amino acid sequence. The method of claim 1, wherein the solid support binding protein in the group consisting of glutathione-S transferase (GST), maltose binding protein (Maltose Binding Protein (MBP) and hexahistidine (6 × Histidine) Fusion protein, characterized in that selected. The method of claim 1, wherein the fluorescent protein is composed of enhanced green fluorescent protein (EGFP), enhanced cyan fluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP), enhanced yellow fluorescent protein (EYFP) and red fluorescent protein (RFP). Fusion protein, characterized in that selected from the group. According to claim 1, wherein the linker is a fusion protein, characterized in that consisting of 6 to 9 amino acids. The protein chip for detecting caspase-3 activity, wherein the fusion protein of any one of claims 1 to 4 is immobilized on a substrate. 6. The protein chip of claim 5, wherein the substrate is a metal. The protein chip according to claim 6, wherein the protein for binding to the solid support of the fusion protein is GST, and the GST of the fusion protein is fixed by binding to glutathione on the surface of the glutathione metal substrate. 8. The protein chip of claim 7, wherein the metal is gold (Au). The protein chip according to claim 5, wherein the protein for binding the solid support of the fusion protein is GST, and the GST is fixed by binding to glutathione on the surface of the glutathione glass substrate. A method for detecting the activity of caspase-3, comprising using the fusion protein of claim 1. The method for detecting activity of caspase-3 according to claim 10, comprising the following steps: (a) immobilizing the fusion protein of claim 1 on a solid support; (b) treating caspase-3 with the fusion protein immobilized on the solid support; And (c) detecting the activity of the caspase-3 by analyzing the fluorescent signal of the fluorescent protein of the caspase-3 treated fusion protein. The method for detecting activity of caspase-3 according to claim 10, comprising the following steps: (a) treating caspase-3 with the fusion protein of claim 1; And (b) detecting the activity of the treated caspase-3 by performing immunoblotting on the caspase-3 treated fusion protein using an antibody against the protein for binding to the solid support of the fusion protein. A method for detecting activity of caspase-3, comprising using the protein chip of claim 5. Caspase-3 activity detection method comprising the following steps: (a) introducing and reacting caspase-3 on the surface of the protein chip of claim 6; And (b) detecting the activity of the introduced caspase-3 by analyzing a surface plasmon resonance (SPR) image of the reacted protein chip. Caspase-3 activity detection method comprising the following steps: (a) introducing and reacting caspase-3 on the surface of the protein chip of claim 5; And (c) detecting the activity of the introduced caspase-3 by analyzing fluorescence images of the reacted protein chips.

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