KR20070034362A - Method for Measuring Activity of Specific Degrading Enzyme of Specific Substrate Protein Using Ecotin-Fusion Protein - Google Patents

Abstract

The present invention relates to a method for measuring the activity of a specific degrading enzyme of a particular substrate protein.

The present invention (1) the fusion to the carboxyl terminus of the polypeptide comprising a site cleaved by a specific degrading enzyme of a specific matrix protein and the amino acid at any one selected from the group consisting of GST, GFP, MBP Fusion to obtain a fusion protein; (2) performing a substrate-enzyme reaction with the specific substrate proteinase using the fusion protein as a substrate; And (3) heating the reaction product of the substrate-enzyme reaction to separate the supernatant by centrifugation, and measuring the inhibitory activity of trypsin activity of the supernatant. Provided are methods for measuring enzyme activity.

In the case of using the method for measuring the activity of specific enzymes of specific substrate proteins of the present invention, the activity of specific enzymes of specific substrate proteins can be measured quickly and simply and efficiently on a fluorescence meter through the degree of inhibition of trypsin activity.

Ubiquitin, ubiquitin-like protein, protease, ecotin, trypsin

Description

Method for Assaying the Activity of Substrate Specific Proteases Utilizing Ecotin Fusion Protein}

1 is a view showing the structure of a plasmid expressing the GST, ubiquitin, ecotin fusion protein produced through the process of Example 1.

2 is a schematic diagram of the activity measurement method performed in Example 3.

Figure 3 is an electrophoresis picture of the intermediate reaction of the fusion protein of the present invention and various amounts of ubiquitin-specific degrading enzyme 69 in the reaction process carried out in Example 3.

4 is a diagram showing the results of inhibition of trypsin activity obtained in Example 3. FIG.

The present invention relates to a method for measuring the activity of a specific degrading enzyme of a particular substrate protein. More specifically, the present invention relates to a method for measuring the activity of a specific degrading enzyme of a specific substrate protein, which is capable of quantitative activity and is quick, simple and efficient.

Ubiquitin is a small protein consisting of 76 amino acids and is present in all eukaryotic cells. In general, ubiquitin plays a key role in various diseases as well as cell growth and differentiation (Sakamoto Km, 2002, Mol Genet Metab. 77, 44; Hammond AL, 2002, Prog Mol Subcell Biol. 29,61).

In addition, neuronal precursor cell expressed (NEDD8), a small ubiquitin-like variant (SUMO), which is a developmentally inhibited neuronal progenitor gene (NEDD8), which has about 30% similarity with the amino acid sequence of ubiquitin. like modifier), interferon stimulated gene 15, and ubiquitin fold modifier 1 (UFM 1) have been identified. These include ubiquitin-like proteins such as transport through the nuclear membrane, transcriptional regulation, and carcinogenesis. Has been reported to play a very important role (Yeh et al, 2000, Gene, 248, 1, review; Muller et al, 2001, Nat Rev Mol Cell Biol, 2, 202, review; Komatsu M et al, 2004, EMBO J, 23, 1997).

Ubiquitin or ubiquitin-like proteins are attached to various proteins in cells by a series of enzyme groups called E1, E2, and E3, respectively, and another ubiquitin or ubiquitin-like protein molecule is attached to a ubiquitin or ubiquitin-like protein molecule once attached to the protein. Each can be attached continuously by isopeptide bonds in a branched form.

Protein molecules bound to branched ubiquitin polymers are degraded in cells by ATP-dependent protease complexes called proteasomes (Hershko A, Ceinchanover A, 1998, Annu Rev Biochem, 67, 425, review). Proteins that are cleaved after ubiquitin binding are cyclins that regulate cell cycles, transcription factors such as Jun / Fos and NF-kappaB that regulate gene expression, and EGF and PDGF that regulate cell growth and differentiation. P53 known as a factor and tumor suppressor protein (Hu et al, 2002, J Biol Chem, 277, 16528; Amir et al, 2002, J Biol Chem, 277, 23, 253; Baron V, Schwartz M, 2000, J Biol Chem, 275, 39318; Scheffiner et al, 1995, Nature, 373, 81).

Ubiquitin-like proteins work in a very similar way to ubiquitin, with various proteins such as PML (leukemia-associated protein), RanGAP1 (Ran-GTPase activating protein), p53 (tumor inhibitor), and IkappaBalpha (NF-kappaB inhibitor). To combine. However, the binding of the ubiquitin-like protein to the target protein does not cause degradation of the protein as the binding of the ubiquitin polymer, but rather regulates the function of the target protein (Tanaka et al, 1998, Mol Cell, 8, 503; Hochstrasser M , 2000, Nat Cell Biol, 2, E153-E157; Melchior F, 2000, Annu Rev Cell Dev Biol, 16, 591; Yeh et al, Gene, 2000, 248, 1; Muller et al, 2001, Nat Rev Mol Cell Biol, 2, 202).

These ubiquitin or ubiquitin-like proteins (UFM1, SUMO, NEDD8, ISG15) are made of precursors in the form of fused peptides or specific ribosomal proteins consisting of 4-6 amino acids at the carboxy terminus. In particular, ubiquitin is not a monomer, but is also made in the form of a ubiquitin polymer in which several ubiquitin molecules are connected in series.

Therefore, in order for ubiquitin or ubiquitin-like proteins to be active, the carboxy terminus of a protein made in precursor form must be cleaved, and the activity of proteolytic enzymes that cleave the carboxy terminus of ubiquitin or ubiquitin-like proteins produces monomers of ubiquitin or ubiquitin-like proteins. It is essential to be able to perform various functions in vivo (Hershko et al, 1998, 67, 425, review).

The protease performs a dissociation process between a major regulatory protein and ubiquitin or ubiquitin-like protein to perform a function of reversibly regulating the physiological function of the cell like the process of phosphorylation / dephosphorylation of the protein.

Deubiquitinating enzyme (DUB), a ubiquitin-specific degrading enzyme that performs this function, is a group of ubiquitin-specific processing protease (UBP) and ubiquitin carboxy terminus belonging to Cystein protease. Ubiquitin C-terminal hydrolase (UCH) (Tobias; Varshavsky, 1991, J Biol Chem 266, 12021; Pickart, Rose, 1985, J Biol Chem, 260, 7903).

In the case of SUMO, one of the ubiquitin-like proteins, the specific enzymes are yeast ubiquitin-like protein specific protease 1 (Ulp1) and ubiquitin-like protein-specific protease 2 (Ulp2, ubiquitin). -like protein specific protease 2), mammalian SUMO-specific protease 1 (SUSP1), centrin-specific protease 1 (SENP1, sentrin-specific protease 1), sterol methyltransferase 3 ( SMT3, sterol methyltransferase 3) specific isopeptidase 1 (SMT3IP1, SMT3-specific isopeptidase 1), and sterol methyltransferase 3 (SMT3) specific isopeptase2 (SMT3IP2, SMT3-specific isopeptidase 2) Similar to the DUB enzyme, it has been reported to cleave the isopeptide bond between SUMO and the binding protein to remove SUMO (Gong et al, 2000, J Biol Chem, 275, 3355; Nishida et al, 2000, Eur J Biochem, 267, 6423; Kado ya et al, 2002, Mol Cell Biol, 22, 3803).

It is reported that ubiquitin or ubiquitin-like proteins and their specific degrading enzymes are directly or indirectly related to various human diseases. For example, in neurodegenerative diseases such as Alzheimer's disease or Huntington's disease, the accumulation of ubiquitin polymer-bound proteins is a symptom, and genes associated with abnormal corneal development, Turner syndrome, and ovarian cancer associated with X-chromosomes are ubiquitin or ubiquitin. It has a sequence very similar to that of the conserved Cystein / Histidin site, which is known to be characteristic of similar proteases (Sakamoto KM, 2002, Mol Genet Metab, 77, 44). Such evidence strongly suggests that such diseases can be caused by overexpression or lack of ubiquitin or ubiquitin-like protease.

In the past, the method of measuring the activity of proteolytic enzymes is to express a protein including a site recognized by a specific protease from a ubiquitin or ubiquitin-like protein and simultaneously decompose the protein to act as cis or trans. After expressing the enzyme, there is a method of determining the activity by observing the difference of the pattern developed on the SDS-polyacrylamide gel.

This activity measurement method has provided good information for the study of proteolytic enzymes, but it took a lot of time and effort for electrophoresis for it, and in some cases, each result was obtained by using an antibody for each protein. It had a drawback to perform lotting and the like.

In addition, Ub-PEST was made by fusing a peptide having a sequence of MHISPPEPESEEEEEHYC followed by ubiquitin to make Ub-PEST, and then a substrate labeling radioactive substance ¹²⁵ I using a tyrosine group in the PEST sequence using iodine-bead There is a way to use it. When the radiolabeled Ub-PEST ¹²⁵ I is reacted with DUB enzyme, the ¹²⁵ I-labeled PEST peptide is released from ubiquitin. Treatment of this reaction with trichloro acetic acid (TCA), which is a strong acid, causes the ubiquitin moiety to be denatured and precipitated, and the PEST peptide portion released by the DUB enzyme is dissolved in the supernatant. The radioactivity of this supernatant can be measured using a radiometer to determine the activity of the DUB enzyme (Lee et al, 1998, Biol. Proced. Online 1, 92).

However, the method has the disadvantage of having a risk of using a radioactive substance harmful to the human body and the hassle of having to perform the experiment only where the radioactive test equipment and facilities are equipped.

N-benzyloxycarbonyl-leucine- using a peptide having the same sequence as the four amino acids of the C terminus of ubiquitin and the fluorescent substance 7-amido-4-methylcoumarin (AMC) Arginine-glycine-glycine-7-amido-4-methylcoumarin (Z-LRGG-AMC, N-benzyloxycarbonyl-Leu-Arg-Gly-Gly-7-amido-4-methylcoumarin) or at the carboxy terminus of ubiquitin There is a method for measuring activity using Ub-AMC bound to the fluorescent material AMC. After reacting the produced Z-LRGG-AMC or Ub-AMC with ubiquitin-specific protease, the amount of fluorescence emitted from the reactant is measured by a fluorometer. AMC is released when it is bound to an amino acid and when it is not bound. Since the wavelength is different, the activity of the ubiquitin specific degrading enzyme can be measured by changing the fluorescence at a specific wavelength.

This method has a disadvantage in that the procedure for performing experiments has to be complicated, such as the process of labeling expensive fluorescent substances on once purified ubiquitin or ubiquitin-like protein and purifying only the desired protein from the reactants again (Stein et. al, 1995, Biochemistry, 34, 12616; Dang et al, 1998, Biochemistry, 37, 1868).

As mentioned above, conventional methods for measuring the activity of specific degrading enzymes for ubiquitin or ubiquitin-like proteins have disadvantages such as risk, economy, and inconvenience. On the other hand, recently reported studies of ubiquitin or ubiquitin-like proteins or their specific degrading enzymes have been linked to various life phenomena and diseases, suggesting the need for the development of more efficient, rapid and economic enzyme assays.

In order to solve the above problems, an object of the present invention is to provide a method for measuring activity of a specific degrading enzyme of a specific substrate protein, which is capable of quantitative activity measurement and is quick, simple and efficient.

In particular, it is an object to provide a new method for measuring the activity of specific degrading enzymes of ubiquitin or ubiquitin-like proteins.

In order to achieve the above object, the present invention

(1) Fusion by fusion of ecotin to the carboxy terminus of a polypeptide comprising a site cleaved by a specific degradation enzyme of a specific matrix protein and fusion of any one protein selected from the group consisting of GST, GFP, and MBP to an amino terminus Obtaining a protein;

(2) performing a substrate-enzyme reaction with the specific substrate proteinase using the fusion protein as a substrate; And

(3) heating the reaction product of the substrate-enzyme reaction, separating the supernatant by centrifugation, and measuring the inhibitory activity of trypsin activity of the supernatant, wherein the specific degrading enzyme of the specific substrate protein is included. It provides a method for measuring the activity of.

The method for measuring activity of a specific degrading enzyme of a specific substrate protein of the present invention may have a molecular weight of 20,000 to 50,000 Daltons of any one protein selected from the group consisting of GST, GFP, and MBP.

In the method for measuring the activity of a specific degrading enzyme of a specific substrate protein of the present invention, the specific substrate protein may be ubiquitin or ubiquitin-like protein.

The method for measuring the activity of a specific degrading enzyme of a specific matrix protein of the present invention may be any one selected from the group consisting of UFM1, SUMO, NEDD8 and ISG15.

In addition, the present invention is a fusion to the carboxyl terminus of the polypeptide comprising a site cleaved by a specific degradation enzyme of a specific substrate protein and the fusion of any one protein selected from the group consisting of GST, GFP, MBP to the amino terminus The obtained fusion protein is provided.

The fusion protein of the present invention may have a molecular weight of 20,000 to 50,000 Daltons of any one protein selected from the group consisting of GST, GFP, and MBP.

In the fusion protein of the present invention, the specific matrix protein may be ubiquitin or ubiquitin-like protein.

The fusion protein of the present invention may be any one selected from the group consisting of UFM1, SUMO, NEDD8 and ISG15.

The present invention also provides a vector expressing the aforementioned fusion protein.

The present invention also provides a transformant, characterized in that transformed by the above-described vector.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for measuring the activity of a specific degrading enzyme of a specific substrate protein, which is ubiquitin (Ubiquitin) or neural precursor cell expressed in developmentally inhibited development (NEDD8, neural precursor cell expressed, developmentally downregulated 8 ), Small ubiquitin-like modifiers (SUMO), interferon stimulated gene 15 (ISG15, interferon stimulated gene 15), and ubiquitin-like proteins 1 (UFM 1, ubiquitin fold modifier 1) Listen and explain.

The fusion protein used as a substrate of the enzyme-substrate reaction in the present invention is fused at the carboxy terminus of the cleavage site of the ubiquitin or ubiquitin-like protein to the ecotin protein, a trypsin activity inhibitory protein, at the amino terminus glutathione S-transferase (GST, glutathione S-transferase (GFP), green fluorescent protein (GFP), maltose-binding protein (MBP), and the like.

In the present invention, by using the fusion protein as a substrate, an ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) by performing an enzyme-substrate reaction with a ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) degrading enzyme The activity of the enzyme is measured.

The fusion protein of the present invention will contain an ecotin protein that acts as an inhibitor of trypsin at the carboxy terminus of the substrate protein of ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15) specific degradation enzymes. Depending on the type of ubiquitin or ubiquitin-like protein, it is called Ub-Ecotin, SUMO-Ecotin, ISG15-Ecotin or UFM1-Ecotin.

The fusion protein of the present invention comprises an amino acid sequence corresponding to a cleavage site of a substrate protein of GST, GFP, MBP and the like, ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15) specific degradation enzymes Polypeptides and genes or oligonucleotides encoding ecotin proteins can be prepared by binding and expressing them using genetic recombination techniques.

By using the fusion protein of the present invention as a substrate, the enzyme-substrate reaction with protease, in particular, ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) specific degradation enzyme, and using the resulting product By measuring the degree of inhibition against trypsin, the activity of ubiquitin or ubiquitin-like proteins (UFM1, SUMO, NEDD8, ISG15, etc.) specific degrading enzymes can be measured.

That is, by adding proteolytic enzyme to the substrate of the fusion protein of the present invention, the ecotin, which was fused to the carboxy terminus of the cleavage site of the substrate, was separated from the original substrate, and the reaction was heated at 100 ° C. for 5 minutes, and then 13,000 rpm. After centrifugation for 10 minutes at, the portion where the GST, GFP or MBP and the ubiquitin or ubiquitin-like protein are fused at the amino site of the cleavage site of the substrate protein of the present invention is precipitated by denaturation such as GST, GFP or MBP, Only the heat-stable ecotin proteins remain in the supernatant. When the inhibitory activity of trypsin of the ecotin protein in the supernatant is measured, ubiquitin or ubiquitin-like proteins (UFM1, SUMO, NEDD8, ISG15, etc.) The degree of activity of specific degradation enzymes can be measured.

GST, GFP, or MBP in the amino region of the cleavage site of the substrate protein of the present invention is only an example, and is a protein that is less stable to heat than the ecotin protein, so that it is denatured by heat even at a temperature at which no degeneration of the ecotin protein occurs. If this happens, when heated at the temperature range, the ecotin protein does not precipitate, but is easily denatured and precipitated, and at the same time, if the protein is applicable to protein purification, the protein is fused to the amino region of the cleavage site of the substrate protein of the present invention. Can be used.

In addition, in the protease-enzyme-substrate reaction using the fusion protein of the present invention as a substrate, the inhibitory activity of the inhibitor or the inhibitor candidate can be quickly and easily measured by adding an activity regulation candidate. This method may be useful for primary screening of many candidates to develop an activity modulator of ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) specific degradation enzymes. That is, the method for measuring the activity of the ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) specific degradation enzymes of the present invention can be applied to find a substance that modulates the activity of these enzymes. This can be measured by comparing the substrate-enzyme reaction with the activity of the enzyme in the absence of the substance and the activity of the protease in the presence of the inhibitor.

In the above, the method for measuring the activity of proteolytic enzymes has been described based on the method for measuring the activity of specific enzymes such as ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.). Only, it can be widely applied to measure the activity of the protease having the activity of cleaving a specific sequence site of the substrate, such as ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) specific enzyme. That is, in the fusion protein of the present invention, the protease in the substrate of the protease instead of the polypeptide containing the site cleaved by the specific enzyme for ubiquitin or ubiquitin-like protein (UFM1, SUMO, NEDD8, ISG15, etc.) By constructing a fusion protein using a polypeptide comprising a site cleaved by the, and using the fusion protein as a substrate to apply the method of the present invention in the process of performing a substrate-enzyme reaction with the protease The activity of the enzyme can be measured.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the following examples do not limit the scope of the present invention.

The following reference describes the conventional manipulations required for the expression of recombinant proteins, and the examples describe the expression of the fusion proteins and the activity of the protease of the present invention.

<Reference Example 1> DNA digestion and fragment recovery using restriction enzyme

Restriction enzymes and buffers used were purchased from Takara and the reaction volume was usually 20ul to 30ul in a sterile 1.5ml eppendorf tube and reacted for 2 to 3 hours at a reaction temperature of 37 ° C. The composition of the 10-fold buffer used for restriction enzyme reaction is as follows.

1) 10-fold TAKARA Buffer K: 200mM Tris-HCl, 100mM MgCl ₂ , 10mM Dithio Traceol, 1M KCl, pH 8.5

2) 10-fold TAKRA buffer M: 100 mM Tris-HCl, 100 mM MgCl ₂ , 10 mM Dithiothreitol, 500 mM NaCl, pH 7.5

DNA fragments were recovered by staining the electrophoretic agarose gel with ethidium bromide to cut the desired fragments and then isolated using JBI's Gel Extract kit.

Reference Example 2 Liggatin Reaction

For conjugation reactions, the conjugation enzyme was usually ligase (Gibco-BRL) and a 10-fold conjugation buffer (660 uM Tris-HCl, pH7.5, 50 uM MgCl2, 10 mM dithioerythritol, 10 mM ATP). 10 unit ligase was used. The reaction conditions were usually at least 5 hours at 16 ° C.

Reference Example 3 E. coli Transformation

E. coli XL1blue was used as E. coli host cell. Host cells were inoculated in 500 ml of liquid medium (1% bacterium-trypton, 5% yeast extract, 1% NaCl) and shaken at 37 ° C. until absorbance at 600 nm was 0,5 to 1.0. Then, the cells were harvested by centrifugation and washed twice with 500 ml of cold distilled water. 4 ml of cold 10% glycerol solution was added to uniformly disperse the cells. 50ul of the cell suspension obtained above was taken, 2ul of the conjugation reaction mixture was added and allowed to stand at 0 ° C for 2 minutes. After applying an electric shock of 2.5kV using a Bio-rad electroporator (electroporator) of 1ml LB solution (Luria-Bertani Medium) was added and incubated at 37 ℃ for 1 hour. After the incubation was completed, the culture was applied to a solid medium containing 50ug / ml of empicillin and applied, and incubated overnight at 37 ℃ to form colonies.

Reference Example 4 Synthesis of Oligonucleotide (Primary for Polymerase Chain Reaction)

Oligonucleotide synthesis service from Bioneer was used.

Reference Example 5 Polymerase Chain Reaction (PCR)

100ng, 5ul of 10-fold buffer solution (10mM Tris-HCl, pH8.8, 1.5mM MgCl ₂ , 50mM KCl, 0.1% Triton X-100), 2ul of dNTP mixed solution (dATP, dGTP, dCTP, dTTP) 2.5mM), 50ng of primer each, 0.5ul of taq polymerase was added and distilled water was added to make the total volume 50ul. Circulation program using a temperature circulator (GeneAmp PCR system 2700, Applied Biosystems) is 95 ℃, 30 seconds; 55 ° C., 30 seconds; The cycle was repeated 30 times at 72 ° C. for 30 seconds. After the reaction, the reaction product was subjected to electrophoresis using an agarose gel, stained with ethidium bromide, the desired sections were cut, and then separated using a JBI gel extract kit. The DNA was used in an appropriate amount for the next experiment.

Example 1 Preparation of Plasmid pQE30-GST-Ub-Ecotin Expressing Recombinant Protein

(Step 1) Preparation of Plasmid with Ecotin

Primers Ecotin-F (KpnI) of SEQ ID NO: 1 and primers Ecotin-R (HindIII) of SEQ ID NO: 2 were synthesized to make plasmids with ecotin. Using the primers, only the ecotin was obtained from the plasmid pBS-Ecotin containing the entire ecotin gene through polymerase chain reaction. The obtained ecotin gene was digested with Takara buffer M with restriction enzymes Kpn I and Hind III and isolated on agarose gel. Vector pQE30 (Qiagen) to be used in the conjugation reaction was digested with restriction enzymes KpnI and HindIII in Takara buffer M and isolated on agarose gel. Ten units of ligase were added to 10 μl of the obtained ecotin gene and 4 μl of the cleaved vector pQE30, and distillation was added so that the total volume was 20 μl, followed by reaction at 16 ° C. for 12 hours. Subsequently, E. coli XL1blue roll transformation was performed to find the desired recombinant plasmid and named pQE30-Ecotin.

(Step 2) Preparation of plasmid containing ubiquitin-ecotin fusion gene

Primers Ub-F (BamHI) of SEQ ID NO: 3 and primers Ub-R (Kpn I) of SEQ ID NO: 4 were synthesized to fuse ecotin and ubiquitin. Using the primer, only ubiquitin was obtained from polymerase chain reaction from pET-Ub containing the entire ubiquitin gene. The obtained ubiquitin gene was digested in Takara buffer K with restriction enzymes BamHI and KpnI and then separated on agarose gel. The vector pQE30-Ecotin to be used in the conjugation reaction was digested with restriction enzymes BamH1I and KpnI in Takara buffer K and isolated on agarose gel. Ten units of ligase were added to 10 μl of the ubiquitin gene obtained above and 4 μl of the cleaved vector pQE-Ecotin, and distilled water was added to a total volume of 20 μl, followed by reaction at 16 ° C. for 12 hours. Subsequently, E. coli XL1blue E. coli was transformed to find the desired recombinant plasmid and named pQE30-Ub-Ecotin.

(Step 3) Preparation of the plasmid expressing the GST-ubiquitin-ecotin fusion protein

Primer GST-F (BamHI) of SEQ ID NO: 5 and primers GST-R (BamHI) of SEQ ID NO: 6 were synthesized to insert the GST gene into pQE30-Ub-Ecotin. Using the primers, only the GST portion of the plasmid pGEX5T-1 (Pharmacia) containing the GST gene was obtained through a polymerase chain reaction. The obtained GST gene was digested with restriction enzyme BamHI in Takara buffer K and isolated on agarose gel. The vector pQE30-Ub-Ecotin to be used for the conjugation reaction was digested with Takara buffer K with restriction enzyme BamHI and treated on Takara dephosphoryase CIAP and isolated on agarose gel. Ten units of ligase were added to 10 μl of the GST gene obtained above and 4 μl of the cut vector pQE30-Ub-Ecotin, and distilled water was added so that the total volume was 20 μl, followed by reaction at 16 ° C. for 12 hours. Subsequently, E. coli XL1blue was transformed to find the desired recombinant plasmid and named pQE30-GST-Ub-Ecotin.

Example 2 Expression and Purification of his-GST-Ub-Ecotin Substrate Protein

(Step 1) Induction test of GST-ubiquitin-ecotin fusion protein

A plasmid capable of expressing a fusion protein (pQE30-GST-Ub-Ecotin) was transformed into Escherichia coli M15 [pREP4] and incubated in a solid medium at 37 ° C. for one day. Colonies formed the next day were inoculated in 3 ml LB liquid medium (Luria-Bertani Medium) containing 60 μg / ml of empicillin and kanamycin and incubated at 37 ℃ for about 3 hours. IPTG (Isopropylthiogalactoside) was added thereto to have a final concentration of 1 μM and further incubated at 37 ° C. for 3 hours. After incubation, 1 ml of the culture medium was taken to obtain only cells through a centrifuge. The cells were mixed well by adding 100 μl of 1 × sample loading buffer. After the mixture obtained above was boiled for 5 minutes, cooled to room temperature, 10 μl was taken to perform 12% SDS-PAGE, and the expression of the fusion protein was observed through Coomassie brilliant blue staining.

(Step 2) Expression and Purification of Water-Soluble Fusion Proteins

The E. coli cells transformed in step 1 were incubated overnight at 37 ° C. in LB medium containing 50 μl / ml of empicillin and kanamycin, and then 10 ml of the culture solution was added to 1 liter of LB medium and the culture was continued. When shaken at 37 ℃ culture was added IPTG (Isopropylthiogalactoside) to the final concentration of 0.4mM when the absorbance of the bacteria is about 0,4 at the wavelength of 650nm. About 4 hours after the addition of IPTG, the culture was stopped and bacterial cell precipitates were collected by centrifugation at 13,000 rpm for 10 minutes using a centrifuge (Sorvall RC5C plus, SS-34 rotor). Cells prior to purification

Was frozen at −20 ° C. to facilitate cell destruction. Suspended by adding 100 ml of buffer solution (25 mM Tris, pH 8.0, 1 mM EDTA, 0.2 N NaCl) to the recombinant E. coli cell precipitate obtained in 1 liter culture, and then 50% with an ultrasonic grinder (Sonics; Vibra Cell, USA) in an ice bath. Cells were destroyed by sonication for about 3 minutes at an output of 50% and an efficiency cycle of 50%. After standing in an ice bath for 2 minutes, the above crushing process was repeated twice to completely crush the cells. The cell homogenate thus obtained was centrifuged at 13,000 rpm for 30 minutes using a high-speed centrifuge (Sorvall RC5C plus, SS-34 rotor) to obtain a supernatant in which soluble protein was dissolved.

On the other hand, a glutathione Sepharose 4B column was prepared to separate the GST-fused protein. The column was washed with buffer volume 2 (0.1 M Tris-HCl, pH8.5, 0.5 M NaCl) in about 5 times the volume of the bed. Subsequently, the resultant was washed with buffer volume 3 (0.1 M sodium acetate, pH 7.5, 0.5 M NaCl) to about 5 times the volume of the bed. Repeat this process three times to wash the column thoroughly and finally wash it with 10 bed volumes of phosphate buffer (0.14M NaCl, 27mM KCl, 1.5mM KH ₂ PO ₄ , 8.1mM Na ₂ HPO ₄ , pH7.4) The final preparation is completed.

This smoothed glutathione Sepharose 4B column (Pharmacia Biotech AB, Sweden) was passed the supernatant of the soluble protein obtained above at a rate of 2 ml per minute. Subsequently, at least 30 bed volumes of phosphate buffer were run through the column to remove unadsorbed protein. Glutathione elution buffer (50 mM Tris-HCl, pH7.5, 10 mM reduced glutathione) was then added to elute the proteins adsorbed on the resin at a rate of 2 ml per minute to collect the adsorbed proteins. The collected protein was subjected to 10% SDS-polyacrylamide gel electrophoresis to confirm the approximate molecular weight and purity of the purified protein. The purity was over 95% and the approximate molecular weight was 52,000 Daltons. This is shown in FIG. 3. Column C of FIG. 3 shows purified his-GST-Ub-Ecotin, and column 2 shows purified his-GST-Ub (G76A), which transforms the 76th amino acid glycine of ubiquitin into alanine. ) -Ecotin is shown.

This protein sample was obtained using only glutathione-free protein using a dilution membrane. The method used was according to the manufacturer's instructions, the buffer used was 50mM Tris-HCl, pH7.5. In addition, since the protein was expressed in the pQE30 plasmid, six histidine amino acids were bound to the amino terminus of GST.

Example 3 Method for Measuring Protease Activity Using Protease and His-GST-Ub-Ecotin Substrate Protein

In order to establish a method for measuring the activity of protease, first, the ubiquitin-specific degrading enzyme UBP69 of rat, which is the prior patent application of the present inventors (Application No. 10-2003-0021548), was purified. The degradation activity of this purified protease was confirmed using his-GST-Ub-Ecotin substrate protein purified in step 2 of Example 2. His-GST-Ub-Ecotin substrate protein, as shown in Figure 1, GST protein is attached to the amino terminus of ubiquitin, and the carboxy terminus is attached to the ecotin protein, when the substrate is specifically degraded by protease, the carboxy The terminal ecotin is pulled away from the original substrate protein.

100 mM Tris, 1 mM 1,4-dithio-DL-threitol (DTT, 1,4-dithio-DL-threitol), 1 mM ethylenediaminetetraacetic acid (EDTA), 5% glycerol to determine the appropriate activity In a buffer of pH 7.8 containing 1 μg of his-GST-Ub-Ecotin as a substrate and an appropriate amount of ubiquitin-specific degrading enzyme UBP69 was reacted at 37 ° C. for 1 hour. The reaction occurring at this time can be confirmed through the SDS-PAGE results in FIG. In order to terminate the reaction of the previous reaction, 150ul of 0.1% bovine serum albumin (BSA) was added and heated at 100 ° C for 10 minutes. The sample was centrifuged at 13,000 rpm for 4 minutes at high speed, and only the supernatant in which heat-soluble soluble ecotin was dissolved was taken. 10 μl of the supernatant was taken, 2 ng of trypsin and 50 μl of buffer solution (200 mM Tris, 40 mM calcium chloride, pH 8.0) were mixed to a total volume of 90 μl with distilled water and incubated at room temperature for 10 minutes. N-benzoyl-arginine-7-amido-4-methylcoumarin (Bz-R-AMC, N-benzoyl-Arg-7-amido-4-methylcoumarin), a fluorescent substrate for measuring trypsin activity, was 10ul of 1mM concentration was added so that the total volume was 100ul. This sample was measured by fluorescence of 7-amido-4-methylcoumarin (AMC) using a fluorimeter (FLUOSTAR optima) (active wavelength: 355 nm, emission wavelength). 460 nm)

4 shows this result. As the amount of UBP69, a ubiquitin-specific degrading enzyme, increases, ecotin is released from the matrix protein, which further inhibits the activity of trypsin.

In the above, the present invention has been described with reference to Examples, but the present invention is not limited thereto.

That is, it will be understood by those skilled in the art that the technical configuration of the present invention may be implemented in other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the embodiments described above are to be understood as illustrative in all respects and not as restrictive.

As described above, in the case of using the method for measuring the activity of a specific degrading enzyme of a specific matrix protein of the present invention, the specific degrading enzyme of a specific matrix protein can be rapidly and simply and efficiently used on a fluorescence meter through the degree of inhibition of trypsin activity. Activity can be measured.

In addition, the present invention can be usefully used for the primary screening of candidate substances for regulating the activity of specific degradation enzymes of specific substrate proteins.

In addition, in the protease-enzyme-substrate reaction using the fusion protein of the present invention as a substrate, the addition of an activity regulator can measure the regulatory activity quickly and simply and efficiently.

<110> seoul national university industry foundation <120> Method for Assaying the Activity of Substrate Specific Proteases          Utilizing Ecotin Fusion Protein <160> 15 <170> KopatentIn 1.71 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Ecotin-F (Kpn1): a primer for PCR <400> 1 agacttcgtg gtggtaccgc agaaagcgtc cag 33 <210> 2 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Ecotin-R (Hind 3): a primer for PCR <400> 2 cagctaatta agcttttagc gaactaccgc gtt 33 <210> 3 <211> 30 <212> DNA <213> Artificial Sequence <220> Ub-F (BamH1): a primer for PCR <400> 3 gggcccctgg gatccatgca gattttcgtg 30 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence <220> Ub-R (Kpn1): a primer for PCR <400> 4 ctggacgctt tctgcggtac caccacgaag tct 33 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> GST-F (BamH1): a primer for PCR <400> 5 catcacggat ccatgtcccc tatactaggt 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> GST-R (BamH1): a primer for PCR <400> 6 cacgaaaatc tgcatggatc ccaggggccc 30 <210> 7 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> Sequence of Human Ubiquitin <400> 7 atgcagattt tcgtgaaaac ccttacgggg aagaccatca ccctcgaagt tgaaccctcg 60 gatacgatag aaaatgtaaa ggccaagatc caggataagg aaggaatacc tcctgatcag 120 cagagactga tctttgctgg caagcagctg gaagatggac gtactttgtc tgactacaat 180 attcaaaagg agtctactct tcatcttgtg ttgagacttc gtggtggt 228 <210> 8 <211> 249 <212> DNA <213> Artificial Sequence <220> <223> sequence of Human UFM1 (ubiquitin fold modifier 1) <400> 8 atgtcgaagg tttcctttaa gatcacgctg acgtcggacc cacggctgcc gtacaaagta 60 ctcagtgttc ctgaaagtac acctttcaca gcagtcttaa agtttgcagc agaagaattt 120 aaagttcctg ctgcaacaag tgcaattatt accaatgatg gaataggaat aaatcctgca 180 cagactgctg gaaatgtttt tctaaaacat ggttcagaac tgcggattat tcctagagat 240 cgtgttgga 249 <210> 9 <211> 291 <212> DNA <213> Artificial Sequence <220> <223> sequence of Human SUMO (small ubiquitin-like modifier) <400> 9 atgtctgacc aggaggcaaa accttcaact gaggacttgg gggataagaa ggaaggtgaa 60 tatattaaac tcaaagtcat tggacaggat agcagtgaga ttcacttcaa agtgaaaatg 120 acaacacatc tcaagaaact caaagaatca tactgtcaaa gacagggtgt tccaatgaat 180 tcactcaggt ttctctttga gggtcagaga attgctgata ataatactcc aaaagaactg 240 ggaatggagg aagaagatgt gattgaagtt tatcaggaac aaacgggggg t 291 <210> 10 <211> 228 <212> DNA <213> Artificial Sequence <220> <223> sequence of Human NEDD8 (neural precursor cell expressed,          developmentally downregulated 8) <400> 10 atgctaatta aagtgaagac gctgaccgga aaggagattg agattgacat tgaacctaca 60 gacaaggtgg agcgaatcaa ggagcgtgtg gaggagaaag agggaatccc cccacaacag 120 cagaggctca tctacagtgg caagcagatg aatgatgaga agacagcagc tgattacaag 180 attttaggtg gttcagtcct tcacctggtg ttggctctga gaggagga 228 <210> 11 <211> 471 <212> DNA <213> Artificial Sequence <220> <223> sequence of Human ISG15 (interferon stimulated gene 15) <400> 11 atgggctggg acctgacggt gaagatgctg gcgggcaacg aattccaggt gtccctgagc 60 agctccatgt cggtgtcaga gctgaaggcg cagatcaccc agaagattgg cgtgcacgcc 120 ttccagcagc gtctggctgt ccacccgagc ggtgtggcgc tgcaggacag ggtccccctt 180 gccagccagg gcctgggccc tggcagcacg gtcctgctgg tggtggacaa atgcgacgaa 240 cctctgagca tcctggtgag gaataacaag ggccgcagca gcacctacga ggtccggctg 300 acgcagaccg tggcccacct gaagcagcaa gtgagcgggc tggagggtgt gcaggacgac 360 ctgttctggc tgaccttcga ggggaagccc ctggaggacc agctcccgct gggggagtac 420 ggcctcaagc ccctgagcac cgtgttcatg aatctgcgcc tgcggggagg c 471 <210> 12 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> sequence of Ecotin <400> 12 gcagaaagcg tccagccact ggaaaaaatc gcgccttatc cacaagctga aaaagggatg 60 aagcgtcagg tgattcagtt aaccccgcaa gaagatgaat ctaccctgaa agtagaactg 120 ttaatcggtc agacgctgga agtcgattgc aatttgcatc gtctcggcgg gaagctggaa 180 aacaaaacgc tggaaggctg gggctatgat tattatgtct ttgataaagt cagttccccg 240 gtttcaacga tgatggcctg cccggatggc aagaaagaga agaaatttgt caccgcgtat 300 ctgggcgatg ctggaatgct gcgttacaac agcaagctgc cgatcgtggt gtatacgcca 360 gacaatgtag atgtgaagta ccgcgtctgg aaggcggaag agaaaattga caacgcggta 420 gttcgctaa 429 <210> 13 <211> 693 <212> DNA <213> Artificial Sequence <220> <223> sequence of GST (glutathione S-transferase) <400> 13 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 ctggaagttc tgttccaggg gcccctggga tcc 693 <210> 14 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> sequence of MBP (maltose-binding protein) <400> 14 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggg 1149 <210> 15 <211> 729 <212> DNA <213> Artificial Sequence <220> <223> sequence of GFP (green fluorescent protein) <400> 15 atgggtaaag gagaagaact tttcactgga gttgtcccaa ttcttgttga attagatggt 60 gatgttaatg ggcacaaatt ttctgtcagt ggagagggtg aaggtgatgc aacatacgga 120 aaacttaccc ttaaatttat ttgcactact ggaaaactac ctgttccatg gccaacactt 180 gtcactactt tctcttatgg tgttcaatgc ttttcaagat acccagatca tatgaaacgg 240 catgactttt tcaagagtgc catgcccgaa ggttatgtac aggaaagaac tatatttttc 300 aaagatgacg ggaactacaa gacacgtgct gaagtcaagt ttgaaggtga tacccttgtt 360 aatagaatcg agttaaaagg tattgatttt aaagaagatg gaaacattct tggacacaaa 420 ttggaataca actataactc acacaatgta tacatcatgg cagacaaaca aaagaatgga 480 accaaagtta acttcaaaat tagacacaac attgaagatg gaagcgttca actagcagac 540 cattatcaac aaaatactcc aattggcgat ggccctgtcc ttttaccaga caaccattac 600 ctgtccacac aatctgccct ttcgaaagat cccaacgaaa agagagacca catggtcctt 660 cttgagtttg taacagctgc tgggattaca catggcatgg atgaactata caagtccgga 720 tctagataa 729

Claims (10)

(1) Fusion by fusion of ecotin to the carboxy terminus of a polypeptide comprising a site cleaved by a specific degradation enzyme of a specific matrix protein and fusion of any one protein selected from the group consisting of GST, GFP, and MBP to an amino terminus Obtaining a protein; (2) performing a substrate-enzyme reaction with the specific substrate proteinase using the fusion protein as a substrate; And (3) heating the reaction product of the substrate-enzyme reaction and centrifuging to separate the supernatant, and measuring the trypsin activity inhibition of the supernatant; Method for measuring the activity of a specific degrading enzyme of a specific substrate protein comprising a. The method of claim 1, Method for measuring the activity of a specific degrading enzyme of a specific substrate protein, characterized in that the molecular weight of any one selected from the group consisting of GST, GFP, MBP is 20,000 to 50,000 Daltons. The method of claim 1, The specific substrate protein is a ubiquitin or ubiquitin-like protein, characterized in that the activity of the specific degradation enzyme of the specific substrate protein, characterized in that. The method of claim 3, The ubiquitin-like protein is any one selected from the group consisting of UFM1, SUMO, NEDD8 and ISG15. A fusion protein obtained by fusing an ecotin at a carboxy terminus of a polypeptide comprising a site cleaved by a specific cleavage enzyme of a specific matrix protein and at least one protein selected from the group consisting of GST, GFP, and MBP at an amino terminus. The method of claim 5, The fusion protein, characterized in that the molecular weight of any one selected from the group consisting of GST, GFP, MBP is 20,000 to 50,000 Daltons. The method of claim 5, The specific substrate protein is fusion protein, characterized in that ubiquitin or ubiquitin-like protein. The method of claim 7, wherein The ubiquitin-like protein is a fusion protein, characterized in that any one selected from the group consisting of UFM1, SUMO, NEDD8 and ISG15.  The vector which expresses the fusion protein of any one of Claims 5-8. A transformant transformed with the vector described in claim 9.

Images