KR20040087358A - Screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lamda phage - Google Patents

Abstract

PURPOSE: A screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage is provided, thereby easily screening ubiquitin and ubiquitin-like protein specific proteases and simultaneously measuring activity of enzymes, so that the enzymes are useful for development of drugs for treating brain neuronal diseases. CONSTITUTION: The screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lambda phage comprises the steps of: (1) cloning Ub, UbI and cI genes, preparing a mutant of cI gene, preparing an expression plasmid of Ub-cI* or UbI-cI* fusion protein, and preparing transformed E. coli using the expression plasmid; (2) introducing cDNA library into the transformed E. coli using recombinant lambda-phage; (3) selecting E. coli expressing desired protein by heat shock at 42 deg. C for 2 hours; and analyzing enzyme activity of the expressed protein, wherein the Ub-cI* or UbI-cI* fusion protein is produced by fusing ubiquitin-like protein Nedd8 or SUMO-1 with cI protein mutant(cI*); and the fusion protein expression plasmid is pACYC184.

Description

Screening method for ubiquitin and ubiquitin-like protein specific proteases using cI gene of lamda phage}

The present invention relates to a novel method for selecting ubiquitin (Ub) and ubiquitin-like protein (Ubl) degrading enzymes. In other words, the present invention relates to a novel method for selecting an enzyme gene having a function of degrading the C-terminus of ubiquitin or ubiquitin-like protein from a λ-phage cDNA library using a λ-phage cI gene that regulates the life history of Escherichia coli. .

Ubiquitin or ubiquitin-like proteases selected by this method have been reported to be associated with proteins fused with ubiquitin or ubiquitin-like proteins, neurodegenerative diseases such as Alzheimer's or Huntington's disease, Turner syndrome, X-chromosomes and It may be used to identify the causes of diseases related to ovarian cancer and to develop therapeutics.

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

Ubiquitin-like proteins such as Nedd8, SUMO-1, and ISG15 have been identified, which have about 30% similarity with the amino acid sequence of ubiquitin, which are reported to play an important role in transport through nuclear membranes, transcriptional regulation, and carcinogenesis. (Yeh et al., (2000) Gene 248, 1, Review); Muller et al., (2001) Nat Rev Mol Cell Biol. 2, 202, Review).

Ubiquitin and ubiquitin-like proteins are made of peptides consisting of 4-6 amino acids at the C-terminus or precursors fused with certain ribosomal proteins. 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.

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

Protein molecules bound to ubiquitin-polymers in a branch form are degraded in cells by an ATP-dependent protease complex called proteasome (Hershko A & Ciechanover A. (1998) Annu Rev Biochem. 67, 425, Review). . Proteins that are cleaved after ubiquitin binding are cyclins that regulate the cell cycle, transcription factors such as Jun / Fos and NF-kappaB that regulate gene expression, and EGF and PDGF that regulate cell growth and differentiation. Growth factor and p53 known as tumor suppressor protein. (Hu et al., (2002) J Biol. Chem. 277, 16528; Amir et al., (2002) J Biol Chem. 277, 23253; Baron V & Schwartz M., (2000) J Biol Chem. 275, 39318; Scheffner et al., (1995) Nature 373, 81).

Ubiquitin-like proteins are similar to ubiquitin in a variety of proteins, including PML (leukemia-associated protein), RanGAP1 (Ran-GTPase activating protein), p53 (tumor inhibitor), and IkappaBalpha (NF-kappaB inhibitor). Combined with. However, the binding of proteins with ubiquitin-like proteins does not cause the degradation of proteins like the binding of ubiquitin-polymers (Tanaka et al., (1998) Mol. Cells. 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).

In order for ubiquitin or ubiquitin-like proteins to be active, the C-terminus of the molecule made in the precursor form must be cleaved, so the activity of the protease that cuts the C-terminus of the ubiquitin or ubiquitin-like protein is determined by It is essential to produce monomers that enable them to perform several functions in vivo (Hershko et al., (1998), 67, 425, Review). In addition, the binding and dissociation process of the ubiquitin or ubiquitin-like protein of the main regulatory protein will play an essential role in the reversible regulation of the physiological function of the cell, similar to the process of phosphorylation / dephosphorylation of the protein.

The ubiquitin produced in the precursor form is formed into ubiquitin monomers by cleaving α-peptide bonds at the linkages. This process is a ubiquitin-specific degrading enzyme (DUB), an enzyme that cleaves peptide bonds after ubiquitin. Is catalyzed by. Such DUB enzymes include two groups of cysteine proteases, UBP-protease (ubiquitin-specific processing protease) and ubiquitin carboxy-terminal hydrolase (Tobias & Varshavsky, (1991) J. Biol. Chem. 266, 12021; Pickart & Rose, (1985) J. Biol. Chem. 260, 7903).

The SUMO-specific protease that cleaves peptide bonds after the G-G residues at the C-terminus of the precursor molecule of SUMO, one of the ubiquitin-like proteins, is a SUMO-protease. Looking at the SUMO-protease known to date, yeast Ulp1 and Ulp2, mammalian SUSP1, SENP1, SMT3IP1, and SMT3IP2 / Axam, and cuts the isopeptide bond between SUMO1 and binding protein to remove SUMO-1 (Gong et al., (2000) J. Biol. Chem. 275, 3355; Nishida et al., (2000) Eur. J. Biochem. 267, 6423; Kadoya et al., (2002) Mol Cell Biol. 22, 3803).

One of the common properties of ubiquitin or ubiquitin-like proteolytic enzymes is ubiquitin or ubiquitin-like, regardless of the type of amino acid at the N-terminus of the protein linked to the ubiquitin or ubiquitin-like protein (the first amino acid is proline). It is possible to cleave the peptide bond between the protein and the binding protein. Therefore, the same method of gene selection can find both ubiquitin and ubiquitin-like proteases.

It has been reported that ubiquitin and ubiquitin-like proteins and their 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 It has a sequence very similar to the sequence of a conserved cysteine / histidine site known to be characteristic of ubiquitin-like proteases (Sakamoto K M. (2002) Mol Genet Metab. 77, 44). These results strongly suggest that these diseases can be caused by overexpression or lack of ubiquitin or ubiquitin-like protease. Therefore, finding ubiquitin or ubiquitin-like proteolytic enzymes that specifically act on proteins associated with the ubiquitin or ubiquitin-like protein associated with the disease, isolating their genes, and conducting studies on three-dimensional structure and function It is essential for the cause of the disease and the development of treatments.

What has been widely used as a method of screening for genes using E. coli is through cDNA library gene expression using E. coli and λ-phage, a parasitic bacteriophage. This method puts the c-DNA library into the λ-phage and then provides E. coli as a host to obtain a large amount of phage, but eventually to find a DNA-hybridization reaction using radiolabeled DNA regardless of the function of the protein. Phage with cDNA were selected (US Pat. No. 5,128,256.).

As another method, there was a method for enabling protein expression of a desired gene, but in order to select phage expressing a desired protein, there was a difficulty in securing an antibody to the protein in advance (US Pat. No. 5,128,256.).

That is, the above two methods are limited to screening genes as part of DNA or selecting corresponding genes from a large amount of proteins, and thus functional screening of the proteins of the selected genes to obtain a protein having activity in the cell at one time. Was not possible.

In order to improve this point, in the present invention, using the life history of λ-phage, the activity of the protein expressed by the desired gene is linked to the survival of Escherichia coli, whereby the E. coli expressing the unwanted gene dies, Only E. coli expressing survived to be selected.

The life history of E. coli due to λ-phage infection can be largely divided into lysogenic cycle and lysogenic cycle. In the case of lysogenic cycle, λ-phage breaks E. coli, causing the host E. coli to die. The phage gene is inserted into the host gene and multiplies with E. coli.

At this time, the cI gene is a transcriptional repression gene and interferes with the transcription of various genes induced by the lytic cycle, thereby inducing the lysogenic cycle. In order to perform this function, the cI gene must bind to DNA in the form of a single-dimer, which is unable to bind DNA when expressed in the form of a fusion of another protein to the N-terminus of cI. It can't be done. Thus, using these characteristics of the cI gene, it is possible to control the life history of E. coli due to λ-phage infection. That is, when fusion of ubiquitin or C-terminus of ubiquitin-like protein is expressed at the N-terminus of cI protein, Escherichia coli is induced by lytic cycle and dies because cI gene cannot bind DNA. At this time, when the C-terminus of the ubiquitin or ubiquitin-like protein hydrolyzes the cI fusion protein by the respective enzyme, E. coli can survive in the lysing cycle.

At this time, Escherichia coli protein called RecA is involved in controlling the life history of Escherichia coli by the cI gene. RecA is an E. coli recombinase involved in DNA modification and has been reported to cleave between arginine 112 and glycine 113 of the cI protein. When the cI protein is cut by RecA, the part forming single dimer and the part binding to DNA are separated from each other, and as a result, the cI gene loses its original role in maintaining the lysogenic cycle. This RecA is expressed when E. coli is exposed to harsh environments (eg heat shock), which can control when E. coli escapes E. coli. That is, in the present invention, even when lytic phenomena are induced by heat shock, in order to ensure that E. coli expressing the desired gene does not die, a mutation is introduced at a site known to be specifically recognized and cut by RecA to perform the same function. CI mutants (hereinafter cI ^* ) were made that do not result in cleavage by RecA (Sices, HJ & Kristie ™ (1998) Proc. Natl. Acad. Sci. 95, 2828).

Therefore, the inventors made a plasmid expressing a fusion protein of the N-terminus and ubiquitin or ubiquitin-like protein C-terminus of the cI protein by utilizing the functional characteristics of the cI gene on the life history of Escherichia coli, and then λ-phage Infection with E. coli causes the expression of the fusion protein, thereby forming a condition in which E. coli is induced into the lytic cycle and dies. The present invention was then completed by selecting pBlueScript clones containing ubiquitin or ubiquitin-like protease cDNA from the surviving strains when infecting the λ-ZAP II cDNA library with the transformed strain and verifying the activity of the enzyme.

It is an object of the present invention to provide a new method for the simple and efficient screening of protease that cuts the C-terminus of ubiquitin or ubiquitin-like protein.

First degree. It is a schematic diagram explaining the life history of phage and gene selection method using cI gene.

2nd degree. It is a structural diagram of a plasmid expressing the fusion protein of the ubiquitin or ubiquitin-like protein and lambda-phage cI protein formed according to Example 3.

3rd degree. In Example 5, the gene selected by this invention, that is, an enzyme that hydrolyzes the ubiquitin C-terminus and an enzyme that hydrolyzes the C-terminus of ubiquitin-like proteins (Nedd8 and SUMO1), inhibits phage plaque formation. It is a photograph of the agar culture dish of the Luria-Bertani medium confirmed that.

4th. In Example 5, it is a photograph of agar culture dish of Luria-Bertani medium confirming that UBP95, the ubiquitin degrading enzyme, inhibits plaque formation by phage.

5th. In Example 5, the introduction of the newly selected ubiquitin lyase gene into human cell line h293 to confirm that the selected gene has activity as a protease followed by a decrease in the fusion protein of cI and ubiquitin in E. coli cells. It is the photograph confirmed using the antibody (a). In the same way, the ubiquitin-like proteins (Nedd8 and SUMO1) of the C-terminal enzyme activity is confirmed photo (b & c).

The present invention relates to methods for screening proteolytic enzymes that cleave the C-terminus of ubiquitin or ubiquitin-like proteins and useful plasmids used therein.

The present invention provides a plasmid expressing a fusion protein in which the N-terminus of the cI protein is bound to each C-terminus of a ubiquitin or ubiquitin-like protein.

The present invention provides recombinant E. coli transformed with a plasmid expressing a fusion protein in which each C-terminus of the ubiquitin or ubiquitin-like protein is bound to the N-terminus of the cI protein.

The present invention performed the gene search in the following configuration.

1.Preparation of plasmids expressing Ub-cI ^* or Ubl-cI ^* fusion proteins and transformed Escherichia coli (cI ^* is a mutant of cI)

1.1. Cloning of Ub, Ubl and cI Genes

1.2. mutant (cI ^* ) production of the cI gene

1.3. Cloning of Ub-cI ^* or Ubl-cI ^* Fusion Plasmids and E. Coli Transformation

2. Introduction of cDNA Library Using Genetically Engineered λ-phage

3. Selection of E. coli strains expressing the desired protein by thermal shock (42 ° C, 2 hours)

4. Confirmation of Enzyme Activity of Expressed Protein

Through the following examples will be described the present invention in detail.

These examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1 Cloning of Ub or Ubl and λ-phage cI Gene

Ubiquitin and ubiquitin-like proteins were cloned from cDNA obtained by performing Reverse Transcription-Polymerase Chain Reaction (RT-PCR). As a template, RNA was isolated from the blood of a healthy adult. Each sequence is listed in Genbank as follows.

Ubiquitin: 28278206, ubiquitin B [Homo sapiens].

Nedd8: Genbank No. 461287, ubiquitin-like protein [Homo sapiens].

Sumo-1: Genbank No. 1762972, Human ubiquitin-related protein SUMO-1 mRNA, complete cds

ISG15: Genbank No. 27362956, Homo sapiens ubiquitin-like protein ISG15 mRNA, complete cds

In the case of the cI gene, DNA extracted from wild-type lambda-phage was cloned by performing a polymerase chain reaction as a template.

cI gene is listed in Genbank as follows.

cI: Genbank No. 3915849, Repressor protein CI

The primers used for reverse transcription-polymerase chain reaction and polymerase chain reaction are as follows.

Ubiquitin Forward: 5'-GAC TAG TAT GCA GAT CTT CGT GAA GAC TCT-3 '

Ubiquitin Reverse: 5'-GAG GAT CCA TCC CAC CTC TGA GAC GGA GTA-3 '

Nedd8 Forward: 5'-GAC TAG TAT GCT AAT TAA AGT GAA GAC GCT G-3 '

Nedd8 Reverse: 5'-GAG GAT CCT CCT CCT CTC AGA GCC AAC ACC-3 '

Sumo-1 Forward: 5'-GAC TAG TAT GTC TGA CCA GGA GGC AAA AC-3 '

Sumo-1 Reverse: 5'-GAG GAT CCA CCC CCC GTT TGT TCC TGA TAA-3 '

ISG-15 Forward: 5'-GAC TAG TAT GGG CTG GGA CCT GAC GGT G-3 '

ISG-15 Reverse: 5'-GAG GAT TCG CCT CCC CGC AGG CGC AGA T-3 '

cI Forward: 5'-GAG GAT CCA TGA GCA CAA AAA AGA ATA CAT TA-3 '

cI reverse: 5'-GAG AGC TCT CAG CCA AAC GTC TCT TCA GGC-3 '

Using the above primer and template, the reverse transcriptase used for reverse transcriptase-polymerase chain reaction was M-MLV (Promega, USA), and the polymerase used for reverse transcriptase-polymerase chain reaction and polymerase chain reaction was pfu Turbo (Stratagene). , USA).

In the case of ubiquitin and ubiquitin-like proteins, reverse transcription was performed using RNA isolated purely from healthy adult blood as a template, according to the protocol provided by Promega. That is, dNTP was reacted with M-MLV buffer at 37 ° C. for 1 hour. Thereafter, the mixture was placed at 90 DEG C for 10 minutes to inactivate reverse transcriptase-polymerase and then used as a template for polymerase chain reaction. In the polymerase chain reaction, the reaction mixture was mixed according to the instructions of Stratagene, followed by reaction at 94 ° C. for 3 minutes, followed by 30 cycles of reaction at 94 ° C. 30 seconds, 52 ° C. 30 seconds, and 72 ° C. for 1 minute. The polymerization was carried out at 72 ° C. for 5 minutes.

The polymerase chain reaction of cI was also mixed with the reactants as described above, followed by DNA denaturation at 94 ° C. for 3 minutes, followed by 30 times of 94 ° C. 30 seconds, 52 ° C. 30 seconds, and 72 ° C. 1 minute 30 seconds. The polymerization was carried out at 72 ° C. for 5 minutes.

The ubiquitin, ubiquitin-like protein or cDNA of cI obtained from the above reverse transcriptase-polymerase chain reaction and polymerase chain reaction were isolated using a Quia-QUICK spin column (Quiagen, Germany).

Purely isolated ubiquitin or ubiquitin-like protein and cI cDNA were cloned into pGEM T-easy vector (Promega, USA), respectively. The amount of pGEM T-easy vector used was 50 ng, and each DNA fragment was inserted into the pGEM T-easy vector at a ratio of 4 times the number of molecules. The DNA ligase was treated with 5 units of T4 DNA ligase (promega). . As described above, the vectors cloned with Ub-cI ^* and Ubl-cI ^* were transformed into E. coli, and then purified in large quantities to be used for subsequent experiments.

Example 2 Formation of cI Protein Mutants

To make a cI mutant protein that is not degraded by the protease RecA, glycine 112 between amino acids 110 and 113 (Q-A-G-M) was changed to arginine. For this purpose, site-directed mutagenesis using polymerase chain reaction was performed using the following primers. Using pfu Turbo as a polymerase, the annealing temperature was set to 60 ° C, and the other matters were carried out in the same manner as in the use of the positional mutagenesis kit (Stratagene). Thus obtained cI mutant gene was confirmed that the mutant was correctly generated through sequencing.

cI mutagenesis primers:

5'-CCCTGTTTTTTCTCATGTTCAGGCAcggATGTTCTCACCTGAGC-3 '

5'-GCTCAGGTGAGAACATccgTGCCTGAACATGAGAAAAAACAGGG-3 '

Lowercase letters indicate parts that are substituted with mutant amino acids.

Example 3 Formation of Expression Plasmids and Escherichia Coli Transgenic Strains Expressing Ub-cI ^* and Ubl-cI ^* Fusion Proteins (cI ^* is a cI Protein Mutant)

Ubiquitin and ubiquitin-like proteins in the pGEM T easy vector (Promega, USA) were cloned into the Spe1-BamH1 position of the pACYC184 vector, and then the cI mutant gene was cloned into the BamH1-Sac1 position. The pACYC184 vector contains a site that is resistant to the antibiotic chloramphenicol (FIG. 2).

In the present invention, the use of pACYC184 as a fusion protein expression plasmid of ubiquitin or ubiquitin-like protein and cI protein mutant is to solve the compatibility problem due to replication origin. That is, in order to use a replication source different from the ColE1 replication source of pBlueScript, which is a cytoplasmic plasmid form of the E. coli strain of the λ-ZAP II vector (Stratagene, USA) containing the cDNA library.

The prepared pACYC184-Ub-cI ^* or pACYC184-UBL-cI ^* vector was infected with XL-1 Blue Escherichia coli to form each transformed E. coli strain.

Example 4 Gene Selection

The transformed XL-1 Blue Escherichia coli (pACYC184-Ub-cI ^* or pACYC184-Ubl-cI ^* ) obtained in Example 3 was converted to tetracycline (12.5 μg / ml), chloramphenicol (34 μg / ml) magnesium sulfate (10 mM ), And cultured with Luria-Bertani culture containing maltose (0.2%) at 37 ° C. until OD ₆₀₀ reached 0.6. The λ ZAP II library was infected with a MOI index of 0.1 and incubated at 37 ° C. for 15 minutes, followed by 0.1 mM isopropyl-thio-beta-D-galactopyranoside (isopropyl-thio-β-D-galactopyranoside, Was applied to Luria-Bertani agar medium in a 100 mm dish containing IPTG). After 8 hours of incubation at 37 ° C., the cells were kept at 42 ° C. for 2 hours, and then cultured at 37 ° C. until colonies were seen. In this way, 145 colonies were selected from a total of 100 dishes, of which 81 were ubiquitin degrading enzymes and the rest were ubiquitin-like proteinases.

Example 5 Validation of Gene Selection Process

Clones obtained in Example 4 were transformed to pACYC184-Ub-cI ^* or pACYC184-UBL-cI ^* E. coli strains to confirm the formation of plaque compared to when wild-type phage infection (Fig. 3, A).

That is, plaques were generated when ubiquitin-C terminal hydrolase was added to pACYC184-Ub-cI ^* coli strain and UBP 95 was added to pACYC184-SUMO-cI ^* coli strain. On the other hand, when the ubiquitin degrading enzyme clone obtained in Example 4 was transformed into pACYC184-Ub-cI ^* Escherichia coli (FIG. 3, E & F), Nedd8 was cut from Nedd8-cI ^* fusion protein, one of the ubiquitin-like proteins. Surviving clones (FIG. 3, G & H) and SUMO were cut from SUMO-cI ^* fusion proteins to identify surviving clones, respectively (FIG. 3, I).

In addition, UBP-95 gene, which is already known to specifically recognize ubiquitin and is known as an enzyme that cuts its C-terminus, was also confirmed that plaque was not formed (FIG. 4).

Example 6 Activity Verification of Selected Enzymes

The activity of the protein expressed by the gene obtained from the clone obtained through the gene selection method developed as the present invention as ubiquitin degrading enzyme or ubiquitin-like protease was measured as follows.

Each ubiquitin-protease, SUMO-protease and Nedd8-protease clones identified in Example 5 were cloned into protein expression plasmids (pCDNA3.1), and then cloned into NIH3T3 cells with HA-Ub, Myc-SUMO, or HA-Nedd8. Infected. Extracts from infected NIH3T3 cells were used to develop proteins by SDS-polyacrylamide gel electrophoresis, followed by Western blotting using antibodies against HA or Myc, which are tagged with ubiquitin, SUMO or Nedd8 (FIG. 5). .

As a result, when overexpressing only HA-Ub, Myc-SUMO, or HA-Nedd8, a band of fusion proteins bound to ubiquitin, SUMO or Nedd8, respectively, was identified (Fig. 5A-lane 2; 5B-lane 1; 5C- Lane 1). Protein bands bound to such ubiquitin, SUMO or Nedd8 are each ubiquitin-protease (FIG. 5A, lanes 3 & 4), SUMO-protease (FIG. 5B, lanes 2 & 3) and Nedd8-protease (FIG. 5C, lane 2 When expressed together with & 3), it was found to be remarkably reduced, and it was confirmed that each of the clones obtained in Example 5 had enzymatic activity.

Conventional gene selection method has been widely used as a method for selecting a desired gene using E. coli, cDNA library gene expression using λ-phage to select a gene corresponding to a specific probe or to express a gene for a large amount of protein It is limited to screening, and also functional screening is impossible for the protein of the selected gene to obtain a protein having activity in the cell at one time.

The present invention relates to a novel method for screening ubiquitin and ubiquitin-like proteolytic enzymes, wherein the CI gene of ubiquitin or ubiquitin-like protein is derived from a λ-phage cDNA library by using the λ-phage cI gene that controls the life history of Escherichia coli. It has a merit different from the conventional gene selection method in that a functional gene having an enzymatic activity that breaks down a terminal can be selected. In addition, this screening method does not require a specific DNA probe or antibody as compared to the existing method, and the characteristic that can be easily measured by the expression of the gene at the same time can be said that the great advantage.

Ubiquitin and Ubiquitin-Like Proteins and Their Degrading Enzymes Continue to report direct or indirect association with neurodegenerative diseases such as Alzheimer's disease or Huntington's disease, and various human diseases, such as Turner syndrome and ovarian cancer associated with X-chromosomes. It is becoming. 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 It has a sequence very similar to the nucleotide sequence of a conserved cysteine / histidine site, which is known as a ubiquitin-like protease. These results strongly suggest that these diseases can be caused by overexpression or lack of ubiquitin or ubiquitin-like protease.

Therefore, through this screening method, ubiquitin and ubiquitin-like proteins and their degrading enzymes are identified, their respective genes are separated, and the three-dimensional structure and function are traced to contribute to the cause of various diseases and to develop therapeutics. It is suggested that

Claims (13)

Fusion protein combining cI protein mutant (cI *) with ubiquitin protein. Fusion protein combining cI protein mutant (cI *) with ubiquitin-like protein (Nedd8) Fusion protein combining cI protein mutant (cI *) with ubiquitin-like protein (SUMO-1) The mutant fusion protein of claim 1, wherein one or more amino acids are substituted. The plasmid according to claim 1-4, which is capable of expressing a fusion protein, pACYC184 The expression plasmid pACYC184-Ub-cI * according to claim 5. Expression plasmid pACYC184-SUMO1-cI * according to claim 5 The expression plasmid pACYC184-Nedd8-cI * according to claim 5 The transformant of claim 5, wherein the host cell is transformed with the expression plasmid. 10. The transformant of claim 9, wherein the bacterial XL-1 strain was transformed with the expression plasmid pACYC184-Ub-cI *. 10. The transformant of claim 9, wherein the bacterial XL-1 strain was transformed with the expression plasmid pACYC184-Nedd8-cI *. 10. The transformant of claim 9, wherein the bacterial XL-1 strain was transformed with the expression plasmid pACYC184-SUMO1-cI *. All methods for obtaining an E. coli strain expressing a fusion protein in Example 3 and expressing a protein that hydrolyzes the fusion protein