KR100807182B1 - - 2 Ubiquitin-like Protein Specific Protease UfSP2 and Process for Preparing the Same - Google Patents

Abstract

A novel protease is provided to show the specific protease activity on Ufm1, which acts on an important mechanism in a cell, thereby being widely utilized for investigating the important mechanism in the cell where the Ufm1 involves. A Ufm1-specific protease 2(UfSP2) includes an amino acid sequence of SEQ ID : NO. 1. A method for preparing the UfSP2 comprises the steps of: (a) culturing a transformant which is prepared by transforming E. coli BL21 using an expression plasmid of pMAL-c2X-UfSP2; and (b) obtaining the UfSP2 from the cultured fungus bodies.

Description

Ubiquitin-like Protein Specific Protease UfSP2 and Process for Preparing the Same}

1 is a genetic map of pMAL-c2X-UfSP2, which is an expression vector for UfSP2 expression.

Figure 2 is an electrophoresis picture showing the result of cutting the GST-Ufm1-HA with UfSP2 and UfSP2 C249S.

Figure 3 is an electrophoresis picture showing the result of cutting GST-Ufm1-HA, GST-Ub-HA, GST-SUMO-1-HA or GST-ISG15-HA with UfSP2.

The present invention relates to ubiquitin-like protein specific protease UfSP2 and a method for preparing the same. More specifically, the present invention provides a ubiquitin-like protein, Ufm1-specific protease, Ufm1-specific protease (Ufm1-specific protease 1, UfSP2), a gene encoding an electric UfSP2, a foot comprising an electric gene. A method for producing UfSP2 from current plasmids, transformants transformed with electroexpressing plasmids and electrical transformants.

Ubiquitin protein consisting of 76 amino acids in all eukaryotes is synthesized in the form of several ubiquitin in a cell in the cell or a fusion protein in which a specific ribosome protein is linked to the C-terminus of the ubiquitin by an α-peptide bond. (Lund et al., J. Biol. Chem., 260: 7609, 1985; Finley et al., Nature, 338: 401, 1989).

The ubiquitin protein, through ubiquitination that binds to other proteins in the cell, including abnormal protein degradation, cell cycle regulation, carcinogenic proteolysis signaling functions, transcription regulation, stress response, signaling pathways, It is known to be involved in various intracellular reactions, such as the presentation of antigen (Wilkinson KD, Annu. Rev. Nutr., 15: 161, 1995; Hershko and Ciechanover, Annu. Rev. Biochem., 61: 761, 1992 Jentsch, Annu. Rev. Genet., 26: 179, 1992; Glotzer et al., Nature, 349: 132, 1991; Bond and Sciesinger, Mol. Cell. Biol., 6: 4602, 1986). This ubiquitination is accomplished by the carboxyl terminal glycine of ubiquitin performing a peptide bond with the lysine group of the target protein, and additionally by performing a peptide bond with the lysine group of the ubiquitin whose C-terminus of ubiquitin is already bound to the target protein. This binding reaction is carried out by a group of complex enzymes consisting of a series of Ub-activating enzyme (E1), Ub-conjugating enzyme (E2) and Ub-ligaing enzyme (E3) enzymes (see Hochstrasser, M., Annu. Rev). Genet., 30: 405, 1996; Varshavsky, A., Trends Biochem.Sci., 22: 383, 1997; Hershko and Ciechanover, Annu. Rev. Biochem., 65: 801, 1998).

On the other hand, the synthesized fusion protein or ubiquitinated protein conjugate can release the ubiquitin monomer by the de-ubiquitinating enzyme (DUB), such a free ubiquitin monomer can be recycled intracellularly ( Piotrowski et al., J. Biol. Chem., 272: 23712, 1997; Swaminanthan and Hochstrasser, Mol. Biol. Chem., 10: 2583, 1999; Lam et al., Nature, 385: 737, 1997) .

The DUB not only plays a role in liberating ubiquitin, but is also known to be involved in various intracellular control processes such as fruit fly development, transcription inhibition, cytokine response, and growth regulation (Huang et al., Science 270). : 1828, 1995; Moazed and Johnson, Cell 86: 667, 1996; Naviglio et al., EMBO J., 17: 3241, 1998). This DUB is a ubiquitin that cleaves ubiquitin-specific protease (UBP) and ubiquitin-specific protease (UBP) and ubiquitin C-terminal amides and esters, which liberate ubiquitin from ubiquitin binding proteins linked by α-peptide or ε-isopeptide bonds. Ubiquitin C-terminal hydrolase (UCH), a C-terminal hydrolase (Tobias and Varshavsky, J. Biol. Chem., 266: 12021, 1991; Pickart and Rose, J. Biol. Chem., 260). : 7903, 1985). UBP and UCH have the common property of cleaving peptide bonds between ubiquitin and protein regardless of the amino acid sequence of the protein bound to ubiquitin.

Meanwhile, Ub-like proteins (Ubl) such as Nedd8, SUMO, ISG15, and Ufm1 have about 30% similarity with the amino acid sequence of ubiquitin (Kawakami et al., EMBO J. 20: 4003, 2001; Saitoh and Hinchey, J. Biol. Chem., 275: 6252, 2000; Potter et al., J. Biol. Chem., 274: 25061, 1999; Komatsu et al., EMBO J. , 23: 1977, 2004). These ubiquitin-like proteins are combined with various proteins such as promyelocytic leukemia protein (PML), ran-GTPase activating protein (RANGAP1), tumor suppressor (p53), and inhibitor of NF-kappaB (IKBα) in a similar manner to ubiquitin. It is known to form a conjugate and can be liberated from the conjugate through proteolytic enzymes specific for each Ubl, and although it does not cause degradation of the bound protein, it can modulate the intracellular activity of the bound protein. Tanaka et al., Mol. Cell, 8: 503, 1998; Hochstrasser, Nat. Cell Biol., 2: E153-E157, 2000; Melchior F., Annu. Rev. Cell. Dev. Biol , 16: 591, 2000; Yeh et al., Gene, 248: 1, 2000; Muller at al., Nat. Rev. Mol. Cell Biol., 2: 202, 2001; Komatsu et al., EMBO J. , 23: 1977, 2004).

Recently, a new Ubl, ubiquitin-fold modifier 1 (Ufm1), consisting of 83 amino acids present only in nematode and multicellular organisms, has been reported. Ufm1 consists of the C-terminal valine and glycine, 16 on ubiquitin and its amino acid sequence. Similarity was reported in% and very similar to ubiquitin and other ubiquitin-like proteins in the tertiary structure (Sasakawa et al., Biochem. Biophys. Res. Commun., 343: 21, 2006). However, since Ufm1 specific protease that can dissociate Ufm1 from the conjugate formed by the binding of Ufm1 and protein has not been identified yet, there is a difficulty in researching the intracellular activity of Ufm1. In particular, the need to identify Ufm1-specific proteases has emerged.

Thus, the present inventors made a thorough research to identify the Ufm1-specific protease, a novel protease consisting of 461 amino acids and the gene of 1386bp encoding the enzyme, the enzyme is Ufm1-specific protein It confirmed that it exhibits a decomposition activity, and this invention was completed.

After all, the first object of the present invention is to provide a Ufm1-specific protease that exhibits Ufm1-specific proteolytic activity.

It is a second object of the present invention to provide a gene encoding an electric enzyme.

It is a third object of the present invention to provide an expression vector containing the electric gene.

A fourth object of the present invention is to provide a transformant transformed with the expression vector.

It is a fifth object of the present invention to provide a method for preparing a Ufm1 specific protease, comprising the step of culturing an electric transformant and obtaining the electric enzyme therefrom.

The present inventors conducted various studies to identify the Ufm1-specific protease, and as a result, to confirm that there is a protease having Ufm1-specific proteolytic activity in the brain, liver and kidney of the rat, and to purify it It was.

That is, the whole protein obtained from the rat brain, liver and kidneys was sequentially applied to ammonium sulfate precipitation method, ion exchange chromatography and hydrophobic chromatography to obtain an active fraction. At this time, the active fraction was confirmed using the enzyme activity reaction using the substrate GST-Ufm1-HA protein.

Then, in order to purify the Ufm1-specific protease from the previously obtained active fraction, Flag-Ufm1-VME (vinylmethylester) capable of covalently binding to the cysteine residue present in the active site of UfSP was added to the active fraction. After mixing and reacting, immunoprecipitation with an anti-Flag antibody was performed to purify Ufm1-specific protease.

The purified protease was analyzed using MS / MS analysis and mass data analysis database (MASCOT), which is a proteomic analysis method, and it was confirmed that LOC70240 is a protein whose function is not known to date.

On the other hand, as a result of searching for a protein having homology with the protein, it was possible to search for AAH05503 protein having about 45% amino acid sequence homology. The AAH05503 protein is a protein whose function has not been confirmed so far, and is composed of 461 amino acids (SEQ ID NO: 1), expressed from a gene having a size of 1386 bp (SEQ ID NO: 2), and in terms of function, It was confirmed that no similarity was shown.

Accordingly, by constructing an expression vector capable of expressing the AAH05503 protein, the protein was expressed and the enzyme activity thereof was measured. As a result, the expressed protein exhibited Ufm1-specific protease activity. That is, the protein recognizes the C-terminal glycine of Ufm1, a ubiquitin-like protein, and can cleave the protein bound to the C-terminal of Ufm1, and the 249th amino acid plays an important role in this enzymatic activity. In addition to the ubiquitin-like proteins SUMO-1 and ISG15 other than Ufm1, as well as the above-described enzyme activity for ubiquitin, the protein was found to be a Ufm1-specific protease.

Thus, the inventors named the protein "UfSP2 (Ufm1-specific protease 2)".

Since the protease of the present invention exhibits proteolytic activity specific to Ufm1, a protein that acts on intracellular major mechanisms, it may be widely used for the study of the intracellular major mechanisms involving Ufm1.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Purification and Identification of Ufm1-specific Protease (UfSP)

Example 1-1 Construction of Substrate for Measuring Enzyme Activity of UfSP

First, to measure the enzyme activity of UfSP, glutathione-S-transferase (GST) is bound to the N-terminus of Ufm1, and hemagglutinin (HA, hemagglutinin) is attached to the C-terminus. Substrates that were bound GST-Ufm1-HA proteins were constructed.

Specifically, a fragment obtained by binding a gene encoding hemagglutinin (HA) to the 3′-end of the Ufm1 gene was obtained. A pGEX4T vector containing gene encoding GST (Amersham Pharmacia, USA) was cut with restriction enzyme BamH1, and the obtained fragment was inserted to construct an expression vector pGEX4T-Ufm1-HA.

E. coli strain BL21DE3 (Novagen, USA) was transformed with pGEX4T-Ufm1-HA, which was previously constructed, and the transformed strain was cultured at 37 ° C. in LB medium, which is a medium for E. coli culture. While culturing, the absorbance (OD ₆₀₀ ) of the culture was measured, and isopropyl-thio-beta-D-galactopyranoside (isopropyl-thio-β- was added so that the final concentration of IPTG was 1 mM at the time when the absorbance became 0.4. D-galactopyranoside (IPTG) was added and incubated at 37 ° C. for 4 hours. After the incubation was completed, the cells were centrifuged at 6,000 rpm for 10 minutes to obtain the cells, which were suspended in PBS, and then subjected to an ultrasonic grinder to disrupt the cells. The lysate was centrifuged at 43,000 rpm for 2 hours to obtain a supernatant, which was subjected to a glutathione Sepharose column (Amersham Pharmacia, USA) to construct the substrate GST-Ufm1-HA protein.

Using the prepared substrate, the method for measuring the enzymatic activity of UfSP was as follows: 30 μl of reaction buffer (100 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 5% (v / v) glycerol, pH 7.8) 1 μg of the substrate (GST-Ufm1-HA) and 10 μl of the sample were mixed, reacted at 37 ° C. for 2 hours, and then the reactant was subjected to 12% (w / v) SDS-PAGE to obtain C- By confirming the presence of hemagglutinin at the terminal, the enzyme activity of UfSP that specifically cleaves the C-terminus of Ufm1 was measured.

Example 1-2 : Partial Purification of UfSP Using Column Chromatography

Brain, liver and kidney extracted from the mice were homogenized in lysis buffer (25 mM Tris, 1 mM β-mercapto ethanol, 1 mM EDTA, 10% glycerol, pH 8.5) and centrifuged at 13,000 rpm for 30 minutes to obtain a supernatant. An ammonium sulfate precipitation method using 30-60% (w / v) ammonium sulfate was applied to the supernatant to obtain a precipitate fraction including UfSP. The obtained precipitate fractions were dissolved in buffer (25 mM Tris, 1 mM β-mercaptoethanol, 1 mM EDTA, 10% glycerol, 50 mM NaCl, pH 8.5), which were then subjected to Q-Sepharose (Q-Sepharose, Amersham Pharmacia, USA) column chromatography. It was applied to graphi (mobile phase: concentration gradient buffer in which 50-300 mM NaCl was dissolved) to obtain an active fraction containing UfSP. The obtained active fractions were dialyzed in a dialysis solution containing 1M ammonium sulfate (25 mM Tris, 1 mM β-mercaptoethanol, 1 mM EDTA, 10% glycerol, 50 mM NaCl, pH 8.5), and then phenyl sepharose (phenyl Sepharose, Amersham Pharmacia). , USA) Column chromatography (mobile phase: concentration gradient dialysis solution containing 1-0M ammonium sulfate) to obtain an active fraction containing UfSP eluted by a dialysis solution containing 0.5M ammonium sulfate.

Example 1-3 Purification of UfSP Using Flag-Ufm1-VME (vinylmethylester)

Example 1-3-1 Construction of Flag-Ufm1-VME

In order to purify UfSP from the active fraction obtained in Example 1-2, Flag-Ufm1-VME, which is capable of covalently binding to the cysteine residue present in the active site of UfSP, was constructed (see Gan-Erdene et. al. , J. Biol. Chem., 278: 28892-28900, 2003).

Specifically, the genes encoding amino acids 1 to 82 of Ufm1 using the restriction enzymes Nde I and Sap I are expressed in the intein protein gene of the pTYB1 (New England Biolabs) vector containing the intein protein gene. Inserted at the 5'-end. Subsequently, a pTYB1-Flag-Ufm1-intein vector was constructed by inserting a DNA fragment having the following nucleotide sequence encoding the FLAG (Sigma) protein sequence at the NdeI cleavage site present at the 5 'position of Ufm1.

sense: 5'-tatgatcgactacaaagacgatgacgataaaca-3 '(SEQ ID NO: 3)

antisense: 5'-tatgtttatcgtcatcgtctttgtagtcgatca-3 '(SEQ ID NO: 4)

E. coli BL21DE3 was transformed with the constructed vector, and the transformed strain was incubated in LB medium at 37 ° C. until OD ₆₀₀ = 0.4, and the culture solution was maintained at 15 ° C., so that the final concentration was 0.3 mM. IPTG was added to the culture and incubated for 16 hours. After the incubation was completed, the cells were centrifuged at 6,000 rpm for 10 minutes to obtain cells, which were suspended in lysate buffer solution (20 mM Tris-HCl, 150 mM NaCl, pH 8.0), and the cells contained in the suspension were ultrasonically pulverized. , Was centrifuged at 43,000 rpm for 2 hours to obtain a supernatant.

The obtained supernatant and chitin resin (chitin resin, New England Biolabs, USA) were mixed at 4 ° C. for 2 hours, and filled in a column, followed by primary buffer solution (20 mM HEPES, 50 mM CH ₃ COONa, pH 6.5). ), Followed by washing buffer solution containing 50 mM MESNa (sodium 2-mercaptoethanesulfonate). Then, a secondary buffer (20 mM HEPES, 50 mM CH ₃ COONa, pH 7.0) was added to obtain Flag-Ufm1-MESNA eluted from the column.

1 ml of Flag-Ufm1-MESNa obtained previously, 0.1 ml of 2M glycine vinylmethylester tosylate, 0.1 ml of 2M N-hydroxysuccinimide, 0.04 ml of 5M Tris pH 7.0 and 0.76 ml of tertiary distilled water was mixed and reacted at 37 ° C. for 4 hours, and then the reaction was applied to S-Sepharose (S-Sepharose, Amersham Pharmacia, USA) column chromatography to purify Flag-Ufm1-VME.

Example 1-3-2 Purification of UfSP Using Flag-Ufm1-VME

The protein contained in the active fraction obtained in Example 1-2 was mixed with Flag-Ufm1-VME purified in Example 1-3-1 at 1: 1 (w / w), and 12 hours at 37 ° C. During the reaction, Flag-Ufm1-VME was covalently bound to the cysteine residue present in the active site of UfSP through the VME group. Anti-Flag antibody was added to the reaction and immunoprecipitation was performed to purify UfSP bound with Flag-Ufm1-VME.

Example 1-4 Identification of UfSP by Proteomics Method

The UfSP purified in Example 1-3-2 was applied to 12% (w / v) SDS-PAGE, and the results were used for the proteomic analysis method of MS / MS analysis and mass data analysis database (MASCOT, http As a result of the analysis using the method, the purified UfSP was confirmed to be LOC70240, a protein whose function is not known until now.

In addition, as a result of searching for a protein homologous to the protein LOC70240 through blast search, it was possible to search for AAH05503 protein having an amino acid sequence homology of about 45%. The AAH05503 protein is a protein whose function has not been confirmed so far, and is composed of 461 amino acids (SEQ ID NO: 1), expressed from a gene having a size of 1386 bp (SEQ ID NO: 2), and in terms of function, It was confirmed that no similarity was shown.

Example 2 Cloning and Nucleotide Sequence and Amino Acid Sequence of UfSP Gene

Using a cDNA library of mouse brain tissue as a template, PCR was performed using primers having the following nucleotide sequence to obtain a gene encoding AAH05503 protein:

Forward: 5'-ctggatccatggatatactcttcagaataag-3 '(SEQ ID NO: 5)

Reverse: 5'-gagtcgacttaaagagcattaggtcgttg-3 '(SEQ ID NO: 6)

The obtained gene was inserted into pGEM-T Easy vector (Promega Co. Ltd., USA) and applied to an automated DNA sequence analyzer (373A, Applied Biosystems, USA) to determine the DNA sequence of the AAH05503 gene ( See SEQ ID NO: 2). The gene of SEQ ID NO: 2 was found to express a protein with a molecular weight of about 52 kDa, consisting of base pairs of 1386 bp, consisting of 461 amino acids (see SEQ ID NO: 1).

Thus, the inventors named the protein expressed from the electric gene "UfSP2 (Ufm1-specific protease 2)".

Example 3 Construction of UfSP2 Expression Vector and Protein Expression

Example 3-1 Construction of a UfSP2 Expression Vector

In order to express UfSP2 from the UfSP2 gene obtained in Example 2, the gene was cloned into the bacterial expression vector pMALc2X (New England Biolabs, USA) to construct a vector for UfSP2 expression.

That is, the UFSP2 gene cloned into the pGEM-T Easy vector constructed in Example 2 was cloned into the pMALc2X vector using the restriction enzymes BamHI and SalI to construct pMAL-c2X-UfSP2 as a protein expression vector (see FIG. One). 1 is a genetic map of pMAL-c2X-UfSP2, which is an expression vector for UfSP2 expression.

On the other hand, using pMAL-c2X-UfSP2 as a template, using a primer having the following nucleotide sequence, PCR using a site-specific mutation kit (QuickChange ^™ Site-Directed Mutagenesis kit, Stratagene, USA), UfSP2 PMAL-c2X-UfSP2 C249S, a vector for mutant UfSP2 expression, was constructed by substituting serine for the 249 th cysteine residue of:

Forward: 5'-gacaatggttggggcagtgcttataggtccc-3 '(SEQ ID NO: 7)

Reverse: 5'-gggacctataagcactgccccaaccattgtc-3 '(SEQ ID NO: 8)

Since each of the constructed expression vectors has a gene of maltose binding protein (MBP) derived from pMALc2X, the C-terminus of MBP is coupled to the N-terminus of UfSP2 (MBP-UfSP2). It was predicted that the protein would be expressed.

Example 3-2 Expression of UfSP2

PMAL-c2X-UfSP2 and pMAL-c2X-UfSP2 C249S, which were previously constructed, were respectively introduced into Escherichia coli BL21 to prepare respective transformants, which were incubated at 600 nm while incubated in LB medium for Escherichia coli culture (including 100 μg / ml ampicillin). Absorbance (OD ₆₀₀ ) was measured at. When the measured absorbance was 0.6, IPTG was added to the electric culture so as to reach a final concentration of 1 mM, and then cultured for 3 hours.

After the incubation was completed, each culture solution was centrifuged at 5,000 rpm for 5 minutes to obtain cultured cells, and dissolved in lysis buffer (20 mM Tris-HCl, 5 mM β-mercaptoethanol, 200 mM NaCl, 1 mM EDTA, pH 7.8). After suspension, the cells were pulverized by sonication, and the electric cell grinding was centrifuged at 15,000 × g for 1 hour to obtain each cell extract as a supernatant. In order to purify MBP-UfSP2 and MBP-UfSP2 C249S, respectively, from each obtained cell extract, each cell extract was applied to an Amylose (Amylose, New England Biolabs, USA) column, eluted with 10 mM maltose solution, Each active fraction comprising -UfSP2 and MBP-UfSP2 C249S was obtained and electrophoresed to identify each protein.

Then, in order to separate MBP from MBP-UfSP2 and MBP-UfSP2 C249S, Factor Xa (Sigma Chem. Co., USA), a cleavage enzyme capable of cleaving between MBP and UfSP2, was used for MBP-UfSP2 and MBP-UfSP2 C249S. After mixing to the active fraction in a ratio of 1mg per 100mg, and reacted for 1 hour at 37 ℃, the reaction was dialyzed with a dialysis solution containing 20mM NaCl (20mM Tris-HCl, pH 8.0), which was then DEAE-sepa UfSP2 and UfSP2 C249S were purified by application to Rose (DEAE-Sepharose, Amersham Pharmacia, USA) column chromatography (mobile phase: concentration gradient dialysis solution containing 25-500 mM NaCl).

Example 4 Determination of Enzyme Activity of UfSP2 and UfSP2 C249S

Example 4-1 Determination of Enzyme Activity of UfSP2 and UfSP2 C249S Against Ufm1

To determine if UfSP2 and UfSP2 C249S have protease activity against GST-Ufm1-HA, reaction buffer (100 mM Tris-HCl, 1 mM DTT, 1 mM EDTA, 5% (v / v) glycerol, pH 7.8) 40 1 μl, UfSP2 1, 2, 3, 5 μg purified in Example 3-2 or 5 μg of UfSP2 C249S and 1 μg of GST-Ufm1-HA were mixed and reacted at 37 ° C. for 1 hour, followed by Example 1 Electrophoresis was carried out in the same manner to confirm the cleavage of the HA bonded to the C-terminus of Ufm1. In this case, Ufm1 having GST coupled to the N-terminus was used as a control (see FIG. 2).

Figure 2 is an electrophoresis picture showing the results of cleavage of GST-Ufm1-HA with UfSP2 and UfSP2 C249S, lane 1 represents the control, lane 2 represents GST-Ufm1-HA, lane 3 is 1 μg of GST- The reaction result of Ufm1-HA and 1 μg of UfSP2 is shown, and the lane 4 shows the reaction result of 1 μg of GST-Ufm1-HA and 2 μg of UfSP2. Lane 5 shows 1 μg of GST-Ufm1-HA and 3 μg. Shows the reaction result of UfSP2, lane 6 shows the reaction result of 1 μg GST-Ufm1-HA and 5 μg UfSP2, and lane 7 shows the reaction result of 1 μg GST-Ufm1-HA and 5 μg UfSP2 C249S. Indicates.

As shown in FIG. 2, UfSP2 was able to cleave HA bound to GST-Ufm1-HA (lanes 3 to 6), but UfSP2 C249S was not able to cleave HA bound to GST-Ufm1-HA. (Lane 7). From the results of FIG. 2, it can be seen that the 249th amino acid of UfSP2 plays an important role in the enzymatic activity of UfSP2, and it can be seen that UfSP2 recognizes the C-terminal glycine of Ufm1.

Example 4-2 Measurement of Enzyme Activity of UfSP2 on Ubiquitin and Ubiquitin-Like Proteins

First, in addition to the GST-Ufm1-HA protein constructed in Example 1-1, in order to construct a substrate protein including ubiquitin (Ub) and ubiquitin-like proteins SUMO-1 and ISG15, Ub1 instead of the gene of Ufm1 GST-Ub-HA, GST-SUMO-1-HA and GST-ISG15-HA, respectively, by using the same method as in Example 1-1, except that the gene of SUMO-1 or ISG15 was used. Prepared.

Then, Example 4 was used, except that UfSP2 was used as the enzyme and GST-Ufm1-HA, GST-Ub-HA, GST-SUMO-1-HA or GST-ISG15-HA were used as substrates. In the same manner as in 1, the enzyme activity of UfSP2 on each substrate was measured (see FIG. 3). Figure 3 is an electrophoresis picture showing the result of cutting GST-Ufm1-HA, GST-Ub-HA, GST-SUMO-1-HA or GST-ISG15-HA with UfSP2. As shown in FIG. 3, UfSP2 cleaved HA bound to GST-Ufm1-HA, but not HA bound to GST-Ub-HA, GST-SUMO-1-HA and GST-ISG15-HA. Could.

From the above results, it was confirmed that UfSP2 of the present invention is a Ufm1 specific protease that specifically cleaves the C-terminus of Ufm1 and does not exhibit enzymatic activity with respect to ubiquitin or other ubiquitin-like proteins.

As described and demonstrated in detail above, the present invention relates to a Ufm1-specific protease Ufm1-specific protease (Ufm1-specific protease 2, UfSP2), a ubiquitin-like protein, a gene encoding an electric enzyme, and an electric gene. It provides an expression plasmid comprising, a transformant transformed with the electric expression plasmid and an electric enzyme from the electric transformant. Since the protease of the present invention exhibits proteolytic activity specific to Ufm1, which is a protein acting on intracellular major mechanisms, it will be widely used in studies for the identification of major intracellular mechanisms involving Ufm1.

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Claims (7)

Ufm1-specific protease 2 (UfSP2) having the amino acid sequence of SEQ ID NO: 1. A gene having the nucleotide sequence of SEQ ID NO: 2 and encoding the Ufm1-specific protease of claim 1. An expression plasmid comprising the gene of claim 2. The method of claim 3, wherein pMAL-c2X-UfSP2 Expression plasmid. A transformant transformed with the expression plasmid of claim 3. The method of claim 5, E. coli BL21 was transformed with pMAL-c2X-UfSP2 Transformants. A method for preparing UfSP2, comprising culturing the transformant of claim 5 and obtaining Ufm1-specific protease 2 (UfSP2) from the cultured cells.

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