KR20110021522A - Sumo-ubc9 fusion protein enhnacing sumo modification of target proteins containing a sumo-interacting motif(sim) - Google Patents

Abstract

PURPOSE: A novel SUMO conjugation enzyme and a method for screening a SUMO-targeting protein using the same are provided to prepare sumoylated protein and to analyze function. CONSTITUTION: A SUMO conjugation enzyme has an amino acid sequence of sequence number 2. A gene encoding the SUMO conjugation enzyme has a base sequence of sequence number 1. An expression vector producing the SUMO conjugation enzyme contains the gene encoding the SUMO conjugation enzyme. A composition for enhancing interaction with a SUMO target protein contains the SUMO conjugation enzyme or gene thereof as an active ingredient. The SUMO target protein contains SIM(SUMO-interacting motif).

Description

 SUMO-Ubc9 fusion protein enhnacing SUMO modification of target proteins containing a SUMO-interacting motif (SIM) to increase the SOMMO-Mc9 fusion protein of proteins containing SMO-Uc9 fusion protein (SIM)

The present invention provides a novel SUMO conjugation enzyme (small ubiquitin-like modifier conjugation enzyme), a gene encoding the same, a composition for increasing interaction with a hair growth target protein including the same, a composition for identifying a hair growth target protein, and a combination thereof. It relates to a screening method of the used hair growth target protein.

Small ubiquitin-like modifiers (SUMOs) are covalently modified to various proteins to alter their location in cells, their stability, and their ability to interact with other proteins and nucleic acids. In particular, hydration of proteins affects the function of various proteins associated with transcription, migration of macromolecules, nucleic acid structure maintenance, nucleic acid DNA metabolism, cell signaling, and neurodegenerative disorders.

Hydrocyclization of the protein is mediated by the E1, E2, and E3 enzymes. Specifically, in the hair growth pathway, the hair growth precursor protein is hydrolyzed by C terminal double-glycine motifs present at the C terminus, and the hydrolyzed hair growth precursor protein is heteromeric E1 hair activating enzyme (SAE1 / SAE2). and thioester (thioester) after the activation to form a bond (ATP and Mg ₂ + - dependent reaction), moves back to form a thioester bond with the conjugated enzyme E2 animal hair (e. g., Ubc9). The hairs are then assisted by E3 hair ligases or migrate directly from the E2 hairs conjugation enzyme (eg, Ubc9) to the lysine residues of the substrate protein.

In ubiquitination, the family of ubiquitin E3 hydrophilases provides substrate specificity, but only a few mammalian E3 hydrophilic enzymes (eg, PIAS family members, RanBP2, and Pc2) have been reported for hydrophilization. In culturing, Ubc9 can bind directly to the modified lysine residues of the target protein via a conserved motif, ψKXE / D (ψ, a bulky hydrophobic residue), so that Ubc9 as well as E3 ligase Modulate substrate specificity for hydrocyclization.

Hydrogenation of proteins is reversibly regulated by a group of sentrin / SUMO-specific proteases with three well-conserved catalytic triads (His, Asp, and Cys).

In mammals, three hair protein, hair hair-1, hair hair-2 and hair hair-3 have been identified and hair hair-1 has about 45% sequence homology with hair hair-2 and hair hair 3, hair hair-2 and hair hair-3. The liver has about 95% amino acid sequence homology.

Since hair growth occurs at low levels, there are many limitations in identifying new targets of hair and understanding its function by protein hair growth. Recently, several studies have reported that Ubc9 activity is regulated by direct interaction with or induction of hair. The hairs have been found to bind directly to Ubc9 and this non-covalent interaction promotes the formation of SUMO chains.

The inventors have therefore developed a novel hair conjugation enzyme and compared to intact Ubc9, the novel protein of the present invention significantly increases the conjugation activity of target proteins including SIMs such as Sp100 and human cytomegalovirus IE2. And the present invention was completed. In addition, analysis using the IE2 SIM mutant revealed that the novel proteins of the present disclosure increase the hydration of proteins comprising SIM. According to the results of the pull-down assay through cell extracts as described in the specific examples of the present invention, the novel proteins of the present invention bind to more diverse cellular proteins than Ubc9 and have a stronger affinity to interact with SIM-containing proteins. As a result, it is expected to be useful for identifying a hair growth target protein, producing a SUMO-modified protein, and regulating the hair growth pathway of cells.

An object of the present invention is a novel SUMO conjugation enzyme (SEMO conjugation enzyme; small ubiquitin-like modifier conjugation enzyme) comprising the amino acid sequence of SEQ ID NO: 2, genes encoding the amino acid sequence of SEQ ID NO: 2, and To provide an expression plasmid that produces a conjugated enzyme.

An object of the present invention is to provide a composition for increasing interaction with a SUMO-interacting motif (SIM) containing protein comprising the amino acid sequence of SEQ ID NO: 2 or a conjugate conjugation enzyme or a gene encoding the same as an active ingredient. .

It is an object of the present invention to provide a composition for identifying a hair growth target protein comprising a hair growth conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same, and a method for screening the hair growth target protein using the same.

In one aspect, the present invention provides a novel SUMO conjugation enzyme (small ubiquitin-like modifier conjugation enzyme) comprising the amino acid sequence of SEQ ID NO: 2.

The hair conjugation enzyme fuses the N-terminus of Ubc9 with the hair conjugation residue (K14) disrupted to the C-terminus of the sumo-1 precursor protein with six carboxyl terminal amino acids including the double-glycine residue. SUMO-Ubc9 fusion protein (FIG. 1).

In another aspect, the present invention provides a gene encoding an amino acid sequence of SEQ ID NO. Preferably, the base sequence of the gene may be SEQ ID NO: 1, but is not limited thereto.

In another aspect, the present invention provides an expression vector containing the gene to produce a hair conjugation enzyme.

According to a specific embodiment of the present invention, plasmids such as pDEST17 (Invitrogen), pDEST53 (Invitrogen), etc. were used, but only if the plasmid was able to express a conjugation conjugate enzyme containing the gene and having the amino acid sequence of SEQ ID NO: 2 It doesn't work.

In another aspect, the present invention provides a composition for increasing interaction with a hair growth target protein comprising a hair growth conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient.

The hair growth target protein is a substrate protein which is conjugated and activated by the hair growth conjugation enzyme, and preferably includes both the C-terminal carboxyl group of the hair growth protein and a protein which forms an isopeptide bond. The binding of the hair protein to the ε-amino group of the lysine residue of the hair hair target protein is called SUMOylation. The hair growth target protein is preferably a SIM containing hair growth target protein comprising a SUMO-interacting motif (SIM) region.

According to a specific experimental example of the present invention, as a result of analyzing the binding pattern to the hair growth target protein, it was confirmed that the novel hair growth conjugation enzyme of the present invention had a significantly increased conjugate activity compared to Ubc9 (FIG. 2B, FIG. 2). 3C left). In addition, the novel hydrocapital conjugation enzyme of the present application binds to a wider variety of proteins than Ubc9 (Figure 4A left) and has a significantly higher binding affinity with substrate proteins (Figure 4A right).

As such, increased interaction with the hairy target protein includes, for example, increased conjugation activity or affinity to the hairy target protein or increased diversity of the interacting protein.

The composition of the present invention enables mass production of hydration proteins, and studies the correlation of various cancers, leukemias, diabetes mellitus, dementia, etc. related to hydration of proteins, and is useful as a means for research and development of the therapeutic agents and treatment methods thereof. Can be used.

In addition, it can be used to prevent and / or treat various cancers, leukemias, diabetes mellitus, dementia and the like which are related to protein hydration by increasing the interaction with the vesicle target protein.

In another aspect, the present invention provides a composition for identifying a hair growth target protein comprising a hair growth conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same as an active ingredient.

According to a specific experimental example of the present invention, as a result of analyzing the binding pattern to the hair growth target protein, the novel hair growth conjugation enzyme of the present invention has a binding affinity to the hair growth target protein and conjugates and activates the hair growth conjugation enzyme of the present invention. It was confirmed that the protein is recognized.

The composition of the present invention may further include a stabilizer and a carrier commonly used in biologics, auxiliary active ingredients, and the like, in addition to the hydrophilic conjugation enzyme, which is an active ingredient of the present application, and a buffer solution that stably maintains the structure or physiological activity of the protein. Alternatively, the reaction solution may be included.

In another aspect, the present invention provides a method for screening a target protein using a hair dye conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2 or a gene encoding the same.

Preferably, the method comprises the steps of: 1) treating the matrix protein with a hydrophobic conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2; 2) identifying SUMOylated proteins in the substrate protein; 3) analyzing the results obtained in step 2 may include the step of determining the sieved protein as a sieving target protein in the matrix protein.

In the first step, the substrate protein is hydrolyzed by treating a hydrophilic conjugation enzyme comprising the amino acid sequence of SEQ ID NO: 2. The matrix protein may be a protein mixture isolated from E. coli, yeast, cell line, or the like, and may also be one kind of protein purified by mass expression.

The second step is to identify the hydrocyclized protein in the matrix protein, but the hydrophilized protein can be identified by various methods known in the art, but according to the specific experimental example of the present invention, the Coomassie blue staining method and the immunoblotting method are used. Can be used.

In step 3, the result obtained in step 2 is analyzed to determine the hydrolyzed protein as the target hair protein.

The hair growth target protein is preferably a protein comprising a SUMO-interacting motif (SIM).

According to a specific experimental example of the present invention, by analyzing the hydration pattern of the SIM-containing substrate protein and SIM-deleted substrate protein using the novel hair-conjugation enzyme of the present application, only SIM-containing substrate protein was efficiently modified, It was confirmed that the conjugation (FIG. 3B), the SIM deletion substrate protein showed similar conjugation activity to Ubc9, while the SIM containing substrate protein showed significantly higher conjugation activity than Ubc9.

As described above, the novel proteins of the present disclosure can bind to more diverse cellular proteins than Ubc9 and can interact with SIM-containing proteins with a stronger affinity, thereby identifying the SUMO-modified protein It is expected to be used for the production of proteins as well as for regulating the hair marrow pathway of cells.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example  1. Experiment Method and Preparation

1-1. Cell culture and transient DNA  Transfection ( Cell culture and transient DNA transfection )

293T and human foreskin fibroblast (HF) cells were cultured and [H. R. Lee, DJ Kim, JM Lee, CY Choi, BY Ahn, GS Hayward, and JH Ahn, Ability of the human cytomegalovirus IE1 protein to modulate sumoylation of PML correlates with its functional activities in transcriptional regulation and infectivity in cultured fibroblast cells, J Virol 78 (2004) 6527-6542.

1-2. Plasmid ( Plasmids )

Plasmids expressing GST-SAE2 / SAE1 and His-Ubc9 are described by JH Ahn, Y. Xu, WJ Jang, MJ Matunis, and GS Hayward, Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9, J Virol 75 (2001) 3859-3872. PET-17b (Novagen) -based plasmid, which expresses T7-SUMO-1 _GG , was obtained from Chaon Hoon (Chonnam National University, Republic of Korea). Using Gateway technology (Invitrogen), plasmids expressing flag-SUMO-1 and HA-IE2 were generated in the pSG5 background [S. Green, I. Issemann, and E. Sheer, A versatile in vivo and in vitro eukaryotic expression vector for protein engineering, Nucleic Acids Res 16 (1988) 369.], generating expression plasmids for myc-IE2 on the pCS3-MT background It was. Plasmids for GST-IE1, GST-PML VI, GST-IE2 (135-289), and GST-Sp100A were also generated in pGEX-derived background using gateway technology. GST-RanGAP1 (NΔ419) expression plasmids are described by DA Sampson, M. Wang, and MJ Matunis, The small ubiquitin-like modifier-1 (SUMO-1) consensus sequence mediates Ubc9 binding and is essential for SUMO-1 modification, J Biol Chem 276 (2001) 21664-21669.

To generate plasmids expressing IE2 SIM-mutant, IVIS residues between 200 and 203 were replaced with A residues using the Stratagene QuickChange site-directed mutagenesis protocol. Ubc9 K14R mutants were also generated using the same mutagenesis protocol. His-tagged SUMO-1-Ubc9 (K14R) fusion protein was substituted for pDEST17 (substituted by UMO9 after SUMO-1ΔGG (i.e., truncated sumo-1 with 6 carboxyl terminal amino acids containing double-glycine residues deleted). Invitrogen) in the background. In addition, plasmids for the GFP-Ubc9 and GFP-SUMO-Ubc9 fusion constructs were generated in the pDEST53 (Invitrogen) background by replacing Ubc9 or SUMO-Ubc9 after GFP using gateway technology.

1-3. In beat Confiscation  analysis( In in vitro SUMOylation assays )

Recombinant GST fusion protein was expressed in E. coli and purified on Glutathione-Agarose 4B (Peptron) according to the manufacturer's instructions. Histidine-tagged proteins were also expressed in E. coli and purified on Ni-NTA beads (Invitrogen). T7-SUMO-1GG was expressed in E. coli and subsequently purified by DEAE-Sephacel columns (Bio-Rad) and Sephacryl S-200 gel filtration chromatography (Amersham). Typical hydration reactions include 70 nM GST-SAE2 / SAE1, 0.1-3 μM His-Ubc9 or His-SUMO-Ubc9, and 9 μM SUMO-1 _GG [50 mM Tris-HCl (pH 7.5), 10 mM MgCl2 in buffer. , 1 mM DTT, and 5 mM ATP] in 30 μl. The reaction mixture was allowed to hatch for 1 hour at 37 ° C. The reaction was terminated using SDS sample buffer containing β-mercaptoethanol, and the reaction product was separated by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis (PAGE).

1-4. Antibodies ( Antibodies )

Single Anti-His (H-3) and anti-GFP (B-2) mice conjugated with Horseradish peroxidase (HRP), anti-Daxx rabbit PAb (M-112), and anti-GST MAb (B-14) Clonal antibody (MAb) was purchased from Santa Cruz. Anti-HA rat MAb (3F10) and anti-myc mouse MAb (9E10) conjugated with HRP were purchased from Roche. Anti-flag mouse MAb M2 was obtained from Sigma. Rabbit anti-PML PAb is described in J. H. Ahn, Y. Xu, WJ Jang, MJ Matunis, and GS Hayward, Evaluation of interactions of human cytomegalovirus immediate-early IE2 regulatory protein with small ubiquitin-like modifiers and their conjugation enzyme Ubc9, J Virol 75 (2001) 3859- 3872., anti-IE1 PAbs were made in rabbits using purified IE1 protein, and anti-Sp100 rabbit PAbs were obtained from Paul D. Ling (Baylor College of Medicine, Houston, TX, USA). It was supplied.

1-5. Immunoblot  analysis( Immunoblot analysis ).

For hydrocephalization analysis in transfected cells, cells were washed with PBS containing 5 mM NEM, and samples were prepared by boiling the cells in SDS loading buffer. Purified cell extracts were then isolated using SDS-PAGE according to immunoblotting standard procedures.

1-6. Pull-down analysis ( Pull - down Assays)

HF cells (2 x 107) were lysed with cold PBS, 1% NP-40, 5% glycerol, and 1% complete protease inhibitor cocktail (Sigma). Cell extracts were pre-washed with Ni-NTA beads and incubated for 6 hours at 4 ° C. with 100 μg of His-Ubc9 or His-SUMO-Ubc9 fusion proteins immobilized on Ni-NTA agarose beads. Beads were washed with 20 mM Tris-HCl (pH 8.0) containing 100 mM NaH 2 PO 4 and 25 mM imidazole. Binding proteins were diluted in SDS loading buffer at 100 ° C. and analyzed by Coomassie Blue staining or immunoblotting analysis.

Experimental Example  One. wild - type Ubc9  see SIM Remarkably high on substrates containing Conjuge Having eating activity SUMO - Ubc9  Fusion protein

SUMO-fused Ubc9 (K14R) fusion constructs were made to determine whether to increase the N-terminal hairlo fusion protein conjugating activity of Ubc9 compared to native Ubc9 (FIG. 1A). In this fusion protein, the original hair conjugation region (K14) of Ubc9 was completely destroyed. Since the hairs fused at the N-terminus of Ubc9 lack a double glycine residue, these hairs molecules are not processed by the hair protease. Both Ubc9 and SUMO-Ubc9 proteins were histidine tagged at the N-terminus and expressed in E. coli and partially purified for the utility of in vitro assays (FIG. 1B). We first compared the conjugation activity of Ubc9 and SUMO-Ubc9 using a SIMM-deficient hair marrow substrate such as RanGAP1 (NΔ419), IE1, and PML VI. In in vitro hydrotreatment analysis using these substrates, the conjugation activity of wild-type Ubc9 and SUMO-Ubc9 fusion proteins was similar (FIG. 2A). Then we tested two different hair marrow substrates such as Sp100A and HCMV's IE2 (hair including SIM). In vitro hydrocyclization analysis showed that SUMO-Ubc9 fusion protein had significantly more conjugating activity in both Sp100A (2.5-fold) and IE2 (9-fold) than wild-type Ubc9 (FIG. 2B).

Experimental Example  2. SIM Happening in a dependent manner SUMO - Ubc9  By fusion protein Increased IE2 Confiscation

In order to examine whether the increased conjugating activity of the SUMO-Ubc9 fusion protein requires SIM in the target protein, an IE2 SIM mutant was used. IE2 SIM is one of several proposed matching sequence of (h, hydrophobic;; a, acidic X, any amino acid) ( for example, h - h -XSXS / T- a - a - a) and is similar to SIM (IVISDSEEE from 200 to 208). We replace IE2 (135-289) (including the hydrophilic region (K175 and K180) and SIM) and its SIM mutant version, and IE2 (mSIM) (4 consecutive IVIS residues between 200-203 at A residues) To be included) (FIG. 3A).

In in vitro hydrotreatment analysis, the SUMO-Ubc9 construct efficiently modified wild-type IE2 (approximately 40% of the total IE2) but the SIM mutant did not (Figure 3B, top). The degree of culturing of IE2 SIM mutants by the Ubc9 or SUMO-Ubc9 construct did not increase (FIG. 3B, bottom). Compared to wild-type Ubc9, the result shows that in order to increase the conjugation activity of SUMO-Ubc9 for IE2, SIM must be present in IE2. We also compared the conjugation activity of wild-type Ubc9 and SUMO-Ubc9 in co-transfected cells. The results showed that the conjugation activity for IE2 of the SUMO-Ubc9 fusion protein was much higher than that of wild-type Ubc9 in transfected cells (FIG. 3C, left). In addition, the increased activity of the SUMO-Ubc9 construct was not detected in the IE2 SIM mutant (FIG. 3C, right). These results show that increased hydrophilization of IE2 by the SUMO-Ubc9 fusion protein occurs in a SIM-dependent manner.

Experimental Example  3. Ubc9 see SIM More efficiently interact with inclusion substrates SUMO - Ubc9  Fusion protein

To assess whether the SUMO-Ubc9 fusion protein is useful for identifying new hair marrow substrates, the binding patterns of wild-type Ubc9 and SUMO-Ubc9 fusion proteins to cellular proteins were compared using pull-down assay methods. As a result, it was found that SUMO-Ubc9 binds to more diverse cellular proteins than Ubc9 (FIG. 4A, left).

In addition, the SUMO-Ubc9 fusion protein showed significantly higher binding affinity for Sp100 proteins and Daxx, known as two SIM-containing substrates (FIG. 4A, right two). These data suggest that SUMO-Ubc9 may be useful for identifying new substrates. In addition, these results demonstrate that SUMO-Ubc9 fusion proteins have much higher conjugation activity to SIM-containing hair growth targets (Sp100 and IE2) than wild-type Ubc9, whereas SIM deleted hair growth targets (RanGAP1 (NΔ419) , PML VI, and IE1), their activities were similar. Importantly, increased hydrophilization of IE2 by the SUMO-Ubc9 fusion protein requires intact SIM in IE2. In addition, the results using the pull-down assay method demonstrated that the SUMO-Ubc9 fusion protein has increased binding activity to the SIM-containing substrate.

The ability to identify new hair growth targets and to understand the function of protein hair growth is usually limited to low levels of hair growth. In this regard, the designed SUMO-Ubc9 fusion protein will be useful for identification, production, and characterization of SUMO-modified proteins in vivo as well as in vivo. The results also show that the hydromycinization of Ubc9 plays a role in regulating the hydropathic pathway. SIMs have been found in the hair growth targets as well as in the E3 hair ligation enzymes such as PIAS proteins and RanBP2 and in SAE2 of E1. Therefore, in addition to target identification, hydrocyclization of Ubc9 also recognizes E1 and E3 enzymes. In this regard, the SUMO-Ubc9 fusion protein will also be useful for regulating the hair marrow pathway of cells.

1 SUMO - Ubc9  The structure of the fusion protein is shown.

(A) The structures of the Ubc9 and SUMO-Ubc9 fusion proteins are shown.

In the SUMO-Ubc9 fusion protein, the hairs with the C-terminal 6 amino acids deleted were fused to the N-terminus of Ubc9 (K14R) via a two amino acid (Lys and Leu) linker. The treatment area common to the hair growth precursor (after the underlined double glycine residues) is indicated by the arrow.

(B) The amounts (0.5, 1, and 1.5 μg) of His-Ubc9 and His-SUMO-Ubc9 constructs used in the in vitro assays were compared according to Coomassie Blue staining.

 2 is Ubc9  And SUMO - Ubc9  fusion Homogeneous  Invitro conspiracy to various substrates Conjugation  The activity is compared.

(A) GST-RanGAP1 (NΔ419) while increasing the amount of His-Ubc9 or His-SUMO-Ubc9 (for RanGAP1 (NΔ419); 0.1, 0.2, 0.3 μM / PML VI or IE1; 1, 2, 3 μM) In vitro hydrotreatment was performed using GST-PML VI, or GST-IE1. The reaction product was subjected to immunoblotting with SDS-PAGE (for RanGAP1 (NΔ419); 12% / for PML VI or IE1; 8%) and anti-GST (for RanGAP1), anti-PML, or anti-IE1 Ab. Analyzed. The amount of unmodified and SUMO-modified proteins was counted by counting using Scion Image software (Scion Corporation) and described the percent protein modified based on the total amount of protein.

(B) It was performed using GST-Sp100A or GST-IE2 (135-289) in a manner similar to the invitro hydrolysis analysis method. Reaction products were analyzed by immunoblotting using SDS-PAGE [8% for Sp100A and 12% for IE2 (135-289)] and anti-GST [for IE2 (135-289)] or anti-Sp100 Abs. The amount of hydrocyclized protein was quantified as described above.

3 is SUMO - Ubc9 fusion construct of IE2 Conspiracy SIM Indicates dependence.

(A) an IE2 construct comprising an intact or mutant SIM and hydrophilic regions (K174 and K180).

(B) In vitro hematuration reaction was performed with GST-IE2 (135-289) or GST-IE2 (135-289 / mSIM with increasing amounts of His-Ubc9 or His-SUMO-Ubc9) (1, 2, or 3 μM). ) Was performed. This reaction product was analyzed using immunoblotting with SDS-PAGE (12%) and anti-GST Ab. The amount of sieved protein was quantified as in FIG. 2A.

(C) HF cells were coatfected with plasmid. After 48 hours, whole cell extracts were prepared and separated using SDS-PAGE (8%). Immunoblotting was performed using anti-myc Ab and anti-HA Ab to detect unmodified or SUMO-modified IE2 (full-length) (Top panels). Expression levels of wild-type Ubc9 and SUMO-Ubc9 fusion constructs were determined by immunoblotting with anti-GFP Ab (bottom panels). The% modified protein is described based on the total amount of protein.

4 is by the pull-down analysis method Ubc9  And SUMO - Ubc9 Binding capacity to cellular proteins.

(A) Pre-washed cell lysates prepared from HF cells were incubated with His-Ubc9 or His-SUMO-Ubc9 fusion proteins. Some of His-Ubc9 or His-SUMO-Ubc9 bound proteins were purified with Ni-NTA resin, isolated by SDS-PAGE, coomassie blue staining (left) and anti-Sp100 or anti-Daxx Abs. By immunoblotting (two on the right).

(B) Coomassie blue staining showed 5% of the fixed His-Ubc9 and His-SUMO-Ubc9 fusion proteins used in the binding reaction compared to the control.

<110> Sungkyunkwan University Foundation for Corporate Collaboration <120> SUMO-Ubc9 fusion protein enhnacing SUMO modification of target          proteins containing a SUMO-interacting motif (SIM) <130> PA090293KR <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 765 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of SUMO-Ubc9 fusion <400> 1 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 atcatactcc aaaagaactg 240 ggaatggagg aagaagatgt gattgaagtt tatcaggaac aaacgaagct ttcggggatc 300 gccctcagca gactcgccca ggagaggaga gcatggagga aagaccaccc atttggtttc 360 gtggctgtcc caacaaaaaa tcccgatggc acgatgaacc tcatgaactg ggagtgcgcc 420 attccaggaa agaaagggac tccgtgggaa ggaggcttgt ttaaactacg gatgcttttc 480 aaagatgatt atccatcttc gccaccaaaa tgtaaattcg aaccaccatt atttcacccg 540 aatgtgtacc cttcggggac agtgtgcctg tccatcttag aggaggacaa ggactggagg 600 ccagccatca caatcaaaca gatcctatta ggaatacagg aacttctaaa tgaaccaaat 660 atccaagacc cagctcaagc agaggcctac acgatttact gccaaaacag agtggagtac 720 gagaaaaggg tccgagcaca agccaagaag tttgcgccct cataa 765 <210> 2 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> aminoacid sequence of SUMO-Ubc9 fusion <400> 2 Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys   1 5 10 15 Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser              20 25 30 Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys          35 40 45 Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe      50 55 60 Leu Phe Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro Lys Glu Leu  65 70 75 80 Gly Met Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu Gln Thr Lys                  85 90 95 Leu Ser Gly Ile Ala Leu Ser Arg Leu Ala Gln Glu Arg Arg Ala Trp             100 105 110 Arg Lys Asp His Pro Phe Gly Phe Val Ala Val Pro Thr Lys Asn Pro         115 120 125 Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cys Ala Ile Pro Gly Lys     130 135 140 Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Lys Leu Arg Met Leu Phe 145 150 155 160 Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro                 165 170 175 Leu Phe His Pro Asn Val Tyr Pro Ser Gly Thr Val Cys Leu Ser Ile             180 185 190 Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Ile Thr Ile Lys Gln Ile         195 200 205 Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pro Asn Ile Gln Asp Pro     210 215 220 Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gln Asn Arg Val Glu Tyr 225 230 235 240 Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Phe Ala Pro Ser                 245 250  

Claims (10)

SUMO conjugation enzyme (small ubiquitin-like modifier conjugation enzyme) having an amino acid sequence of SEQ ID NO: 2. A gene encoding the hydrophilic conjugation enzyme of claim 1. The gene according to claim 2, wherein the nucleotide sequence of the gene is SEQ ID NO: 1. An expression vector comprising the gene of claim 2 or 3, wherein said expression vector produces a hair conjugation enzyme. Claim 1, comprising a conjugation enzyme of claim 1 or the gene of claim 2 as an active ingredient, a composition for increasing the interaction with the hair target protein. 6. The composition of claim 5, wherein said hydrocephalus target protein comprises a SUMO-interacting motif (SIM). A composition for identifying a hydrophilic target protein comprising the conjugate enzyme of claim 1 or the gene of claim 2 as an active ingredient. 1) treating the substrate protein with the enzyme of claim 1; 2) identifying SUMOylated proteins in the substrate protein; And 3) analyzing the results obtained in step 2 to determine the hydrolyzed protein as the hairy target protein in the matrix protein. 10. The method of claim 8, wherein the step 2 identification of the hydrolyzed protein is Coomassie Blue staining (Coomassie Blue staining) or immunoblotting method (Immunoblot analysis) characterized in that the method for screening the target hair. 10. The method of claim 8 or 9, wherein said hair growth target protein comprises a SUMO-interacting motif (SIM).

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