JP2001128680A - Human deubiquitinase - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a human deubiquitinase and a material for producing the enzyme by the gene manipulation. SOLUTION: A human deubiquitinase having a specific amino acid sequence, an expression vector encoding the enzyme, an expression vector carrying the polynucleotide, a transformant by the expression vector capable of producing the enzyme, and an antibody against the enzyme are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The invention of this application relates to a novel human deubiquitinase. More specifically, the invention of this application relates to a deubiquitinating enzyme useful for elucidating cell functions and for developing therapeutic methods and therapeutics for human diseases such as canceration of cells, and genetic engineering for producing the enzyme. It is about materials.

[0002]

2. Description of the Related Art Ubiquitin is a protein having a molecular weight of 7,500, which is ubiquitously present in eukaryotic cells. In recent years, it has been revealed that ubiquitin binds to proteins (short-lived proteins) such as oncogene products and growth factor receptors in multiple chains (polyubiquitination), which is recognized and degraded by the proteasome. Has been attracting attention as a part of the sex protein degradation mechanism.

On the other hand, as an enzyme that cleaves ubiquitin from a ubiquitin-protein complex or polyubiquitin, a deubiquitinase (or a ubiquitin-specific protease [Ubps] or a ubiquitin C-terminal hydrolase) is known. Ubps forms a large protein family (Wilkinson, Annu. Rev. Nutr. 1
5: 161-189, 1995), certain Ubps produce monomeric ubiquitin by cleaving polyubiquitin precursors or by recycling ubiquitin from partially degraded proteins (Tobias and Varshavsky, J. Biol.
Chem. 266: 12021-12028, 1991; Baker et al., J. Bio.
l. Chem. 267: 23364-23375, 1992; Papaand Hochstras
ser, Naure 366: 313-319, 1993).

[0004] Recent studies have revealed the following other essential cell functions of Ubps in higher eukaryotes. Drosophila Fat-facets protein
(FAF) regulates developing ocular cells (Huang et a
l., Science 270: 1828-1831, 1995). In addition, Drosophila D-Ubp-64E (Henchoz et al., Mol. Cell. Biol.
16: 5717-5725, 1996) and yeast UBP3 (Moazed and Jo
hnson, Cell 86: 667-677, 1996) affects silencing of gene expression. Aplysia Ap-uch is required for long-term memory accumulation (Hegde et al., Cell
89: 115-126, 1997). Furthermore, murine DUB-1 (Zhu et a
l., Proc. Natl. Acad. Sci. USA 93: 3275-3279, 199
6) and DUB-2 (Zhu et al., J. Biol. Chem. 272: 51-
57, 1997) describes cytokine-induced U that affects cell proliferation.
bps.

[0005]

As described above, it is known that deubiquitinase plays an important role in various essential functions of higher eukaryotic cells. Therefore, the novel human deubiquitinase is extremely useful, for example, for elucidating the mechanism of abnormal cell proliferation in humans, or for developing effective therapies and anticancer agents for treating cancer.

Further, the present invention is useful for elucidating information transmission and processing mechanisms in brain and nerve cells, and for treating degenerative diseases and developing preventive drugs. The invention of this application has been made in view of the circumstances described above, and has as its object to provide a novel human deubiquitinase.

Another object of the present invention is to provide various genetic engineering materials for producing the human deubiquitinase.

[0008]

Means for Solving the Problems The inventors of the present application have:
The RS447 gene was isolated as a novel tandem repetitive DNA sequence from the human chromosome 4p15 region (Kogi, et al., Genom).
ics 42: 278-283, 1997), classifying this RS447 as a megasatellite repeat based on a relatively large 4.7 kb basic unit, tandem structure, and polymorphism (Gondo et al.
al., Genomics 54: 39-49, 1998).

[0009] Further, the inventors have found that the megasatellite unit of RS447 contains an open reading frame (ORF) of 1590 bp, and that this 0RF encodes a deubiquitinase.

This application provides the following inventions (1) to (7) based on the above findings by the inventors. (1) A human deubiquitinase having the amino acid sequence of SEQ ID NO: 2. (2) A polynucleotide encoding the human deubiquitinase of the invention (1) and having at least the 242-1834 base sequence of SEQ ID NO: 1. (3) An expression vector carrying the polynucleotide of the invention (2). (4) A transformant using the expression vector of the invention (4), which is capable of producing the enzyme of the invention (1). (5) An antibody against the human deubiquitinase of the invention (1). (6) The promoter sequence of the human deubiquitinase gene. (7) A transcription initiation promoter for anti-human deubiquitinase mRNA.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The enzyme of the invention (1) (hereinafter referred to as RS4)
47-bUb), a method of isolating from human organs, cell lines, etc., a method of preparing a peptide by chemical synthesis based on the amino acid sequence provided by this application, or a polynucleotide of the invention (2) Can be obtained by a method of producing with recombinant DNA technology, etc., but a method of obtaining with recombinant DNA technology is preferably used. For example, RS447-bUb can be expressed in vitro by preparing RNA from a vector having the polynucleotide of the invention (2) by in vitro transcription and performing in vitro translation using the RNA as a template. Further, by recombining the translation region into an appropriate expression vector by a known method, the polynucleotide is encoded in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, mammalian cells, and plant cells. RS447-bUb can be expressed in large amounts.

When RS447-bUb of the invention (1) is produced by expressing a polynucleotide by in vitro translation, for example, the polynucleotide of the invention (2) is recombined into a vector having an RNA polymerase promoter [invention ( 3)], when added to an in vitro translation system such as a rabbit reticulocyte lysate or a wheat germ extract containing an RNA polymerase corresponding to the promoter, the RS447-b of the invention (1) can be obtained.
Ubs can be produced in vitro. Examples of the RNA polymerase promoter include T7, T3, SP6 and the like. Vectors containing these RNA polymerase promoters include pKA1, pCDM8, p
T3 / T718, pT7 / 319, pBluescript II
And the like.

When RS447-bUb of the invention (1) is produced by expressing a polynucleotide in a microorganism such as Escherichia coli, an origin, a promoter capable of replicating in the microorganism, a promoter,
An expression vector [Invention (3)] was prepared by recombining the polynucleotide of the invention (2) into an expression vector having a ribosome binding site, a DNA cloning site, a terminator, and the like. A host cell was transformed with the expression vector. Thereafter, if the obtained transformant [invention (4)] is cultured, RS447-bUb encoded by this polynucleotide can be mass-produced in a microorganism. At this time, an RS447-bUb fragment containing an arbitrary region can be obtained by adding and expressing a start codon and a stop codon before and after an arbitrary translation region. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving this fusion protein with an appropriate protease, only the protein portion encoded by the cDNA can be obtained. Examples of expression vectors for Escherichia coli include pUC, pBluescript I
I, pET expression system, pGEX expression system and the like can be exemplified.

When the RS447-bUb of the invention (1) is produced by expressing a polynucleotide in a eukaryotic cell, the polynucleotide of the invention (2) is added with a promoter, splicing region and poly (A). Recombinant into an expression vector for eukaryotic cells having a site or the like [Invention (3)], if introduced into eukaryotic cells [Invention (4)], the RS447-bUb of the invention (1) can be used in eukaryotic cells Can be produced. As expression vectors, pKA1, pCDM8, pSVK3, pMSG, p
Examples include SVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYES2. Also, p
IND / V5-His, pFLAG-CMV-2, pE
If GFP-N1, pEGFP-C1 or the like is used as an expression vector, it can be expressed as a fusion protein to which various tags such as His tag, FLAG tag, and GFP are added. As eukaryotic cells, monkey kidney cells COS
7, mammalian cultured cells such as Chinese hamster ovary cells CHO, budding yeast, fission yeast, silkworm cells, Xenopus egg cells and the like are generally used, provided that they can express RS447-bUb of the invention (1), It can be any eukaryotic cell. In order to introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a liposome method, and a DEAE dextran method can be used.

After the RS447-bUb of the invention (1) is expressed in prokaryotic cells or eukaryotic cells, the desired enzyme can be isolated and purified from the culture by a combination of known separation procedures. . For example, treatment with a denaturant such as urea or a surfactant, ultrasonic treatment, enzymatic digestion, salting out or solvent precipitation,
Dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAG
E, isoelectric focusing, ion exchange chromatography,
Examples include hydrophobic chromatography, affinity chromatography, and reverse phase chromatography.

The RS447-bUb of the invention (1) includes a peptide fragment (5 or more amino acid residues) consisting of any partial amino acid sequence of the amino acid sequence of SEQ ID NO: 2. These peptide fragments can be used as antigens for producing antibodies. Further, RS447-bUb of the invention (1)
After being translated, it undergoes various modifications in the cell. Therefore, these modified enzymes are also RS447 of the invention (1).
Included in -bUb range. Examples of such post-translational modifications include N-terminal methionine elimination, N-terminal acetylation, sugar chain addition, limited degradation by intracellular protease, myristoylation, isoprenylation, phosphorylation and the like.

The polynucleotide of the invention (2) can be obtained by a method based on chemical synthesis or a method for screening a human genomic library. To clone a polynucleotide of interest from a genomic library, an oligonucleotide is synthesized based on the base sequence of any part of SEQ ID NO: 1 provided by this application, and this is used as a probe by a known method. Screening by colony or plaque hybridization may be performed. Further, an oligonucleotide that hybridizes to both ends of the target polynucleotide is synthesized, and using this as a primer, the PCR of the invention (2) is performed by a PCR method using genomic DNA isolated from human cells as a template. Nucleotides can also be prepared.

In general, polymorphism due to individual differences is frequently observed in human genes. Accordingly, 242-1834 of SEQ ID NO: 1
Polynucleotides in which one or more nucleotides have been added, deleted and / or substituted by other nucleotides in the base sequence are also included in the scope of the present invention.

Similarly, an RS447-bUb having one or more amino acids added, deleted and / or substituted with another amino acid resulting from these changes also has an RS447-bUb having the amino acid sequence of SEQ ID NO: 2. As long as it has the activity of, it is included in the scope of the present invention.

Further, the polynucleotide of the invention (2) includes a DNA fragment (10 bp or more) consisting of any partial nucleotide sequence of the 242-1834 nucleotide sequence of SEQ ID NO: 1. In addition, DN consisting of a sense strand and an antisense strand
The A fragment is also included in this range.

The antibody of the invention (5) can be obtained from serum after immunization of an animal using the enzyme of the invention (1) as an antigen. As the antigen, a peptide chemically synthesized based on the amino acid sequence of SEQ ID NO: 2 or RS447-bUb expressed in eukaryotic cells or prokaryotic cells can be used. Alternatively, it can be produced by introducing the above eukaryotic cell expression vector into an animal muscle or skin by injection or gene gun, and then collecting serum (for example, described in JP-A-7-313187). Method). As animals, mice, rats, rabbits, goats, chickens and the like are used. If B cells collected from the spleen of the immunized animal are fused with myeloma to produce a hybridoma, the monoclonal antibody against RS447-bUb of the invention (1) can be produced.

The invention (6) is a promoter of a gene encoding human deubiquitinase, which is a DNA sequence containing a CAAT box at position -179 of the RS447 gene, as described later.

Further, the invention (7) is a transcription initiation promoter of the RS447 ORF that transcribes human deubiquitinase mRNA, and as described below, the position of SEQ ID NO: 1
It is a DNA sequence having a nucleotide sequence of 153 to 241.

[0024]

EXAMPLES Hereinafter, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to the following examples. 1. Materials and Methods 1-1. Construction of Expression Vector The ORF (1590 bp) of RS447 was amplified by PCR, and pGE
X-2TK (Pharmacia) BamHI-EcoRI site or pET2
8b (Novagen) inserted into the NdeI-BamHI site, and
PGEX-2TK / RS447-dU expressing -RS447-dUb protein
b, and pET28b / RS447-dUb expressing His ₆ -RS447-dUb
Was built.

A mutant plasmid pGEX-2TK / expressing a mutant RS447-dUb in which Cys at position 89 of SEQ ID NO: 2 has been replaced with Ser.
RS447-dUb (S89) and pET28b / RS447-dUb (S89) were constructed. These plasmids contain mutagenic primers:
5'-ATG GGA AAT ACT AGT TAG GTG AAC GCT TCC TTG-3 '
(Sense) 5'-GTT CAC GTA ACT AGT ATT TCC CAT ATT C
It was constructed using a rapid conversion site-directed mutagenesis kit (Stratagene) using TG GAG-3 '(antisense).

The human ubiquitin gene monomer and green fluorescent protein were amplified by PCR from human genomic DNA or pEGFP-1 plasmid (Clontech), respectively. These PCR products were ligated to the BamHI-SalI sites of pET28b, His ₆ - and T7 peptide tag ubiquitin -GFP
A fusion protein was produced. This expression unit is RS447-dU
b Inserted into a compatible plasmid vector along with the expression vector. Similarly, the NcoI-SalI insert carrying the His _6- and T7 peptide-labeled ubiquitin-GFP gene was replaced with p15A
HindIII-Sa of plasmid pLysE (Novagen) containing origin of replication
Connected to the lI site. This plasmid (pLysE / T7 / Ub-GF
P) expresses T7 / Ub-GFP protein by Tc promoter in pLysE. PGEX-2TK / RS447-dUb containing RS447-dUb
BamHI-EcoRI insert of ligated into BamHI-EcoRI site of pcDNA3.1 / HisB, His ₆ - and Xpress peptide labeled RS447
-dUb was expressed. AflII- obtained from this plasmid
The XbaI insert was added to the pTracerCMV vector AflII-
Introduced to XbaI site, pTracerCMV / Xpress-RS447-dUb
Was built. This construct is Xpress labeled RS44
7-dUb is expressed in mammalian cells. 1-2. Zoo Blot Analysis 2 μg of genomic DNA (Clontech) from various animal species
It was digested with EcoRI, electrophoresed on a 1% agarose gel, and transferred to a Hybond-N ⁺ nylon membrane (Amersham) under alkaline conditions. The described ³² P-labeled probe was prepared by a random priming method (Feinberg and Vogelstein, Anal. Biochem. 13
2: 6-13, 1983), hybridization is carried out at 65 ° C. for 18 hours by a known method (Nguyen et al., Am. J.
Hum. Genet. 30: 601-611, 1988). The final wash was performed at 50 ° C. with 2 × SSC, 0.1% SDS. 1-3. Northern blot analysis Human multiplex poly (A) ⁺ RNA tissue Northern (MTM ^™ ) blot (Clontech) was analyzed using ³² P-labeled antisense RS447 ORF.
Hybridized with a DNA probe (242-1831 position). A single-stranded antisense DNA probe was prepared by a known method (Espelund et al., Nucleic. Acids. Res. 15: 2327-23).
41, 1990).
Performed in QuikHyb solution (Stratagene) at 65 ° C. for 18 hours. Hybridization filters were washed at 65 ° C. with 2 × SSC and 0.1% SDS. 1-4.5 'RACE cRACE (circular or concatemeric first-strand
cDNA-mediated RACE method (Maruyama et al., Nucleic A
cids Res. 23: 3796-3797, 1995) was used to perform 5'RACE. The cDNA is a 5'-phosphorylated primer complementary to positions 417-431 of RS447 and AMV reverse transcriptase XL (Takar
a) was used to synthesize from human liver poly (A) ⁺ RNA (Clontech). After digesting the template mRNA with RNaseH, single-stranded c
DNA was circularized or concatamerized with T4 RNA ligase. Using a fixed amount (aliquot) as a template, primer shift PCR (nef) with pfu polymerase (Stratagene)
Sted PCR) was used to amplify the region across the concatemer junction. The first and second PCR amplifications were performed at positions 205-2
Complementary RS447 primers were used for 24 and 268-287 and 175-194 and 387-406, respectively. PCR product is pCR
-Blunt vector (Invitrogen)
Sequencing was performed using the qDyeDeoxy Terminator Cycle Sequencing Kit (ABI) and ABI373A sequencer. 1-5. 3′RACE 3′-RACE is prepared by a known method (Frohman et al., Proc. Nat.
l. Acad. Sci USA, 85: 8998-9002, 1988). Human liver poly (A) ⁺ RNA (Clontech) was converted to oligo (d
T) -Adapter primer and AMV reverse transcriptase XL (Takar
Reverse transcription was performed using a). Aliquots were PCR amplified using LA Taq polymerase (Takara) with adapter primers and primers specific to positions 396-413 of RS447. The specificity of the amplified 2.3 kb was confirmed by Southern blot analysis using the RS447 ORF sequence as a probe. P
The CR product was isolated from agarose gel, blunt-ended with pfu polymerase (Invitrogen), and cloned into pCR-Blunt vector (Invitrogen). RS447 ORF
Clones containing the insert were colony hybridized (Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Lab
oratory, Cold Spring Harbor, NY. 1989), probed with the RS447 ORF fragment and sequenced as described above. 1-6. GFP Fluorescent Reporter Assay PstI-EcoRI (3079-4746) and EcoRI-NdeI of RS447
The fragment (1-2054) was ligated and cloned into the PstI-NdeI site of pUC19. Using this plasmid as a template, RS
The upstream sequence of the 447 ORF was PCR amplified. HindIII labeled sense primer and BamHI labeled antisense primer,
And RS using ExTaq DNA polymerase (Takara)
Fragments of various lengths upstream of 447 were amplified. These amplified fragments were converted to an enhanced green fluorescent protein (EGFP) vector without a promoter [pEGFP-1 (Clontech)].
At the HindIII-BamHI site. CAAT box
(5'-CCCAAGCTTGTCTGGCTTATCGCGCACCTGATG-3 ').
It was designed to constitute a reporter plasmid with ox. All inserts of these reporter plasmids were confirmed by DNA sequencing.

COS7 cells were prepared using a Lipofectamine reagent (GibcoBR) in a 6-well plate according to the manufacturer's protocol.
L). G cells for 15 hours
1.5 μg of FP-reporter plasmid, pcDNA3.1 / His B / Lac
0.5 μg of Z (Invitrogen) and Lipofectamine reagent
Incubated in OptiMEM (GibcoBRL) containing 6 μl. After transfection, cells were incubated for 6 hours in DMEM with 10% fetal calf serum. Transfer cells from each well to 150 μl Reporter Lysis Buffer (Promega)
The mixture was thawed at 0 ° C., centrifuged, and 100 μl of each supernatant was placed in a well of a Cytoplate 96 plate (PerSeptive Biosystems). G
FP fluorescence using CytoFluor II fluorescence plate reader (PerSeptiv
e Biosystems) with an excitation wavelength of 485 ± 20 nm and 530 ±
Measured at an emission wavelength of 25 nm. To normalize transfection efficiency, LacZ activity of each cell extract was measured using a β-galactosidase enzyme assay system (Promega). 1-7. Transient and stabilized transfection of CRS447 A9 cells (2 × 10 ⁵ cells / 6-well dish) were cultured in DMEM containing 10% fetal calf serum (GibcoBRL) for 24 hours. OptiMEM (G
ibcoBRL), the cells were washed with 6 μl of Lipofectamine (GibcoBRL) and 2 μg of CRS447 in OptiME
Incubated in 1 ml of M. CRS447 is a cosmid from pWE15 containing an RS6 copy (Kogi et al., Genomi
cs 42: 278-283, 1997). After 15 hours, the transfection medium was removed and the cells were further incubated with OptiMEM for 8 hours. After washing the cells twice with PBS, the cells were washed with RIPA buffer (50 mM TrisHCl (pH 8), 0.15 M NaC
l, 1% IGEPAL (Sigma) 200 μl, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM P
MSF and 1x protease inhibitor cocktail] 200
Dissolved in μl at 4 ° C. for 30 minutes. Protease inhibitor cocktails include leupeptin, chymotrypsin, pepstatin A, and antipain each at 10 mg / ml DMSO
Prepared as a 1000 × stock solution containing the solution. The cell extract is collected in an Eppendorf tube, vortexed, and
Centrifuged at xg for 5 minutes. The protein in the supernatant fraction is S
Separated by DS-PAGE (Laemmli, Nature 277: 680-685,
1970), anti-RS447-d using ECL detection set (Amersham)
Analyzed by Western blotting with Ub antibody.

CRS447 was also digested with ScaI to a linear form and stable transformation was performed as described above. After removing the transfection medium,
Cells were incubated in DMEM containing 10% fetal calf serum. Twenty-four hours later, cells were trypsinized, split into different dilutions and selected in media containing 0.75 mg / ml G418 (GibcoBRL). Drug-resistant colonies were isolated, subcultured in the presence of G418, and subjected to Western blotting as described above. 1-8. Deubiquitination assay The pLysE / T7 / Ub-GFP plasmid and one of the following plasmids [pET28b, pET28b / RS447-dUb, pET28bRS447-dUb (S
89), pGEX-2TK, pGEX-2TK / dUb, and pGEX-2TK / dUb
(S89)] was added to E. coli JM109 for the pGEX-2TK strain,
The T28b strain was co-transformed into E. coli BL21 (DE3). These transformants were 34 for the pGEX-2TK line.
The strain was cultured at 37 ° C. in liquid L-broth containing μg / ml chloramphenicol and 100 μg / ml ampicillin, or the 50 μg / ml kanamycin for the pET28b strain. 600nm
After reaching an optical density of 0.6, 0.1 mM IPTG was added and RS447-
dUb protein was induced. One hour later, collect the cells and
Dissolve in sample buffer (Laemmli, Nature 227: 68
0-685, 1970), sonicated briefly. These E.col
The protein extract from the transformant was subjected to SDS gel electrophoresis (Laemmli, Nature 227: 680-685, 1970), and the anti-GFP
Antibody (Clontech), Anti-T7 Antibody (Novagen), and Anti-R
The analysis was performed by Western blotting using the S447-dUb antibody. The signal was detected by an ECL western blotting detection set (Amersham). 1-9. Preparation of Anti-RS447-dUb Antibody and Detection of RS447-dUb in Mammalian Cells The middle part of the RS447 ORF, the HindIII-BglII fragment at positions 441-846 of the 4.7-kb RS447 repeat unit, was isolated from E. coli.
His ₆ - expressed as a tagged protein. The expressed protein was Ni-NTA agarose (Qiagen) under denaturing conditions of 6M urea.
Bound. After elution with 0.5M imidazole, the protein was separated using a Model 491 Prep Cell (Bio-Rad).
Purified by S-PAGE. The antiserum was raised by injecting the purified protein into Japanese white rabbits. Anti-His ₆ labeled antibodies in serum were removed by fractionation through affigel-10 (Bio-Rad) with His ₆ labeled proteins pre-bound. Anti-RS447-dUb antibody was electrophoresed and PVDF
Purified by affinity for the purified antigen transferred to the membrane (Smith and Fisher, J. Cell Biol. 99: 20-28, 198
Four). pTracerCMV / Xpress-RS447-dUb was purified by COS using Lipofectamine reagent (BRL) according to the manufacturer's protocol.
7 cells were transfected. Collect transfectants, wash with PBS, and add SDS sample buffer (Laemml
i, Nature 227: 680-685, 1970) and lightly sonicated. Immunoblotting was performed as described above using affinity-purified antibodies, and signals were detected with an ECL western blotting set (Amersham). 2. Result 2-1. The RS447 megasatellite ORF is a 4.7 kb RS447 unit conserved in various mammals.
One large ORF of 1590 bp was expected (FIGS. 1A and 1B). Zo probed with complete 4.7 kb RS447 DNA
o Blots show that there are several homologous fragments in various mammals (Gondo et al., Genomics 54: 39-49, 199).
8). To determine if these sequences are homologous to the RS447 ORF itself, an ORF specific probe as well as 3.1 kb DNA from the non-coding region of RS447.
The fragments were reprobed separately. ORF probe is monkey,
Homologous sequences were detected in rabbit, dog, bovine, and Chinese hamster genomic DNA (FIG. 2A), but blots with non-coding probes revealed only human and monkey bands (FIG. 2B).

Further, using genomic DNA from various animal species, RS447OR from positions 913 to 1195 (282 bp) was used.
A portion of F was PCR amplified and the target sequence was successfully amplified from human, monkey, rabbit, and dog genomic DNA. Sequencing analysis of these amplified DNA fragments confirmed high sequence homology to each other. Monkey, rabbit, and dog amplified sequences showed 95%, 74%, and 75% homology, respectively, as compared to the human sequence (data not shown).

From the above results, it was confirmed that the ORF region of RS447 was conserved in various mammalian species. 2-2. RS447 ORF is a transcribed sequence The conservation of RS447 ORF in mammals suggests that RS447 behaves as an intrinsic gene in mammalian cells.
To demonstrate its expression, Northern blot hybridization of poly (A) ⁺ RNA from multiple human tissues was performed. The RS447 ORF probe detected a 2.4 kb transcript in heart, brain, liver, and skeletal muscle (FIG. 3).

Further, in order to determine the transcription initiation site of the RS447 ORF, the cD
Rapid amplification of NA 5 'end (5'RACE) was performed (Maruyama e
t al., Nucleic Acids Res. 23: 3796-3797, 1995). RS447 of human liver poly (A) ⁺ RNA
Reverse transcribed with a 47-specific antisense primer, amplified and cloned into a plasmid vector. Of the 10 clones sequenced, all sequences showed that the transcription initiation site was at position 153 of RS447 (FIG. 1).
B).

The 3 'end of the RS447 mRNA is also labeled with a 3'RACE
(Frohman et al., Proc. Natl. Acad.
Sci USA, 85: 8998-9002, 1988). Human liver poly (A) ⁺ RN
RS447 mRNA of A was reverse transcribed with an oligo (dT) adapter primer. Using this 3 'adapter primer and a 5' gene specific primer located at positions 396 to 413 of RS447, a 2.3 kb cDNA fragment from RS447 was amplified and cloned into a plasmid vector.
Sequencing analysis of 5 individual clones was RS447
The transcription termination site for mRNA is at position 2608 of RS447.
And 2628. All clones contained a poly (A) terminus in addition to the two poly (A) added sequences found at positions 2562 and 2603 (AATAAA). Although some base pair changes were seen, nonetheless, all 5'RACE and 3'RACE products were RS447
The sequence was derived, and no splice sequence was found.

From the above results, it was confirmed that the size of the RS447 transcript was 2.47 kb. This was in good agreement with the band size detected by Northern blot analysis using the RS447 ORF probe (FIG.
3). 2-3. RS447 ORF Transcription is Driven by Promoter Sequence in RS447 To determine whether there is a promoter for the RS447 ORF, the promoter activity of the DNA fragment subcloned from the RS447 sequence was examined. At the beginning,
The large fragment immediately upstream of the RS447 ORF (-566 to -1
Until +1 is the A of the ATG start codon and negative integers are in the 3 'direction) into a promoterless green fluorescent protein (GFP) vector. Next, this was transfected into COS7 cells, and the promoter activity was confirmed by GFP fluorescence. In addition, this RS447 ORF upstream sequence (−156
6 to -1) was truncated, and the promoter activity of the obtained fragment was quantified (FIG. 4). The minimal region from -179 to -1 was found to have promoter activity, but-
Activity was disrupted in clones containing deletions from 95 to -1. These data indicate that the region from -179 to -96 contains sequences responsible for minimal promoter activity. CAAT box (GATTGGCT) in this area
Was found, but no TATA box. Changing the two base pairs in this CAAT box (from CAAT to CAGA)
Loss of promoter activity was confirmed.

From these results, it can be seen that position -179 of RS447
The CAAT box was confirmed to be essential for the transcription of the RS447 ORF. 2-4. RS447 ORF encodes a novel human deubiquitinase RS447 ORF (1590 bp) encodes a putative polypeptide of 530 amino acids (FIG. 1B). The nucleotide sequence around the start codon (GACATGG) is the minus of the Kozak consensus sequence.
Guanines at positions 3 and +4 are shown. A BLAST database search for the complete polypeptide sequence revealed strong homology to several members of the deubiquitinase family. These enzymes (ubiquitin-specific proteases (UBPs) or ubiquitin C-terminal hydrolases) cleave ubiquitin from ubiquitin-protein complexes. The conserved Cys and His regions, which are thought to form the active center of the deubiquitinase and have already been identified within this enzyme family, are also RS447OR
It was found in the F protein (FIG. 5A). Among deubiquitinase, RS447 polypeptide is similar in many parts of the total amino acids to murine DUB-1 and DUB-2, two highly similar (88% same) cytokine-induced deubiquitinase. Showed sex. That is, RS447 ORF
Showed 47% identity to DUB-1 and 48% identity to DUB-2. Not only is the similarity limited to the Cys and His regions, but the conserved amino acid sequences also vary from positions 1-55, 374-440, and 468-46.
Except for 9, it is widely seen. These variable regions are likely to contribute to the functional differences seen between these enzymes. 2-5. The protein encoded by RS447 (RS447-d
Ub) acts as a deubiquitinase. To examine whether RS447 ORF encodes an active deubiquitinase, we expressed RS447-dUb and analyzed its deubiquitination activity. The RS447 ORF was subcloned into the bacterial expression vectors pET28b and pGEX-2TK to express (His) ₆ -or GST-tagged RS447-dUb. The resulting expression vectors pET28b-RS447-dUb and pGEX-2TK-R
S447-dUb is T7 / Ub-GFP in which T7 peptide-labeled ubiquitin is fused to the N-terminal of green fluorescent protein (GFP)
Was transformed into E. coli together with a compatible plasmid expressing. After RS447-dUb induction, Western blot was performed with anti-GFP, anti-T7 peptide, and anti-RS447-dUb antibody. Anti-GFP and anti-T7 peptide antibodies are pET28b
Or in the cells transformed with pGEX-2TK control vector
A 40 kDa T7 / Ub-GFP fusion protein was detected (FIGS. 6A and 6B, His and GST). In cells expressing RS447-dUb, an anti-GFP antibody was identified in a 27 kDa band and its molecular weight corresponded to GFP (FIG. 6A, His-dUb and GST-dU).
b). The anti-T7 peptide antibody did not recognize this 27 kDa protein (FIG. 6B, His-dUb and GST-dUb). These results indicate that expression of RS447-dUb in E. coli cooperates with deubiquitination of the T7 / Ub-GFP protein.

To demonstrate the deubiquitination activity associated with RS447-dUb, the putative catalytic Cys-89 residue of RS447-dUb was mutated to serine. The Cys-89 residue in the Cys box was conserved in all deubiquitinase and was shown to be part of the catalytically active site (Papa and Hochstrasse).
r, 1993; Huang et al., 1995; Hegde et al., 1997; Z
hu et al., 1996, 1997). The mutant RS447-dUb (S89) was expressed to the same extent as the wild-type RS447-dUb (FIG. 6C).
S447-dUb (S89) did not deubiquitinate the T7 / Ub-GFP protein [FIGS. 6A and 6B, His-dUb (S89) and
GST-dUb (S89)]. These results indicate that RS447-dUb is an active deubiquitinase and the Cys-89 residue of RS447-dUb is essential for its enzymatic activity. 2-6. Expression of RS447 ORF causes deubiquitinase production Antibodies to RS447-dUb were prepared and tested whether the RS447 ORF was translated into deubiquitinase in human cells. Western blot analysis of the affinity purified anti-RS447-dUb antibody revealed that the electrophoretic mobility was RS447-dUb.
60 kD in human cells, consistent with the predicted molecular weight of dUb
a The RS447-dUb protein (FIG. 7, lymphoblasts) was identified. In addition, in transfected COS7 cells expressing the Xpress peptide fusion RS447-dUb protein, anti-Xpres
Both the s antibody and the anti-RS447-dUb antibody have the same molecular weight as the predicted molecular weight of the Xpress epitope-tagged RS447-dUb 63
kDa was detected (FIG. 7, COS7 / Xp.dUb). These results indicate that RS447 ORF is an active transcription unit capable of producing a 63 kDa deubiquitinase. 2-7. Transfection of a cosmid clone containing 6 tandem copies of RS447 expresses RS447-dUb In each of the above studies, it was confirmed that the RS447 gene encodes a novel functionally expressed deubiquitinase . Since RS447 is normally tandemly repeated on the human chromosome, the expression of CRS447, a cosmid clone containing six tandem copies of RS4477 was also examined (Kogi et al., 1997). That is, to determine whether a tandem copy of RS447 can express RS447-dUb, CRS447 was transfected into mouse A9 cells, and the expression of RS447-dUb was examined. Anti-RS447-d
Western blot analysis of Ub antibodies revealed that RS447- in A9 cells transiently transfected with CRS447.
dUb was detected (FIG. 8A). Also, a stable cell line containing CRS447 was isolated. Expression of RS447-dUb was also confirmed in cell extracts prepared from these stable cell lines (FIG. 8B).

From the above results, the tandemly repeated RS
It was confirmed that the 447 gene was able to produce RS447-dUb protein. In addition, expression of the RS447 gene was also examined by Northern blotting. With the antisense RS447 ORF probe, these cell lines A and
2.4 and 4 kb transcripts were detected in poly (A) ⁺ RNA isolated from D (FIG. 9A). Poly (A) ⁺ R from clone A
Perform 5 'RACE on NA and start transcription at RS447
At position 153. Therefore, RS447
Is expected to use two different transcription termination sites. 3'RACE was performed to confirm this possibility.
Positions 2310 to 2331 to amplify the downstream region of RS447
RS447 specific primers were used. The PCR reaction amplified two different fragments, 0.3 and 1.7 kb in size, respectively. Sequencing analysis of these DNA fragments indicated that the transcription termination site for RS447 mRNA was at positions 2628 and 3974 of RS447, respectively, and that there was no splicing sequence.
All clones contained the poly (A) terminus in addition to the three poly (A) added sequences (AATAAA) found at positions 2562, 2603 and 3951.

From the above results, expression of two different sizes of the RS447 transcript was confirmed. This difference was derived from the length of the 3 'untranslated sequence. 2-8. Expression of high molecular weight antisense transcript of RS447 The sense transcript of RS447 ORF was detected with an antisense RS447 ORF probe (FIG. 9A). Sense RS447O
The same filter was retested with the RF probe. A prominent high molecular weight smear band was detected in poly (A) ⁺ RNA from clone D with a sense probe (FIG. 9B). The same smear signal was detected in clone D after prolonged exposure of X-ray film (data not shown). In contrast, the 2.4 and 4 kb R
The amount of S447 sense transcript was not very different between clones A and B (FIG. 9A). Southern blots probing RS447 show that the integrated copy number of RS447 in clone III is higher than in clone I (FIG. 9).
C).

From the above results, the amount of the antisense transcript of RS447 is proportional to the integrated copy number of RS447, but the amount of the sense transcript of RS447 in clone III is expected from the integrated copy number of the cell. Less than

In addition, Northern blots of poly (A) ⁺ RNA from multiple human tissues were retested with the sense RS447 probe. Then, a remarkable high molecular weight RS447 antisense transcript was detected in the brain (FIG. 10).

From the above results, it was confirmed that the high-molecular-weight antisense transcript of RS447 was actually expressed in at least several human brain and nerve tissues.

[0041]

As described in detail above, the invention of the present application provides a novel human deubiquitinase and a genetic engineering material for producing the enzyme in a large amount and uniformly.

[0042]

[Sequence List] SEQUENCE LISTING <110> Japan Science and Technology Corporation <120> Human deubiquitinase <130> NP99303-YS <140> <141> <160> 2 <170> PatentIn Ver 2.0 <210> 1 <211> 4746 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (242) .. (1834) <400> 1 gaattcccag cgatgccagg ggacacaccc tgtgactcct tcctgaattg agtgctgata 60 tttgattggc ttatcgcca cctgatgagt gggtggggtg ttcgcggttg gtgggggtga 120 cttacagaag ggctgatgcg gccagagagc tcgtcatttg aagactctct cggaagggat 180 agcgtctttc tgcaacctgc ggtcccagca gacaaacctt gtgatcctcg ttccagtcga 240 c atg gag gac gac tca ctc tac ttg gga ggt gag tgg cag ttc aac cac 289 Met Glu Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn His 1 5 10 15 ttt cca aaa ctc aca tct tct cgg ccc gat gca gct ttt gct gaa atc 337 Phe Pro Lys Leu Thr Ser Ser Arg Pro Asp Ala Ala Pla Ahe Glu Ile 20 25 30 cag cgg act tct ctc cct gag aag tca cca ctc tca tgt gag acc cgt 385 Gln Arg Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser Cys Glu Thr Ar g 35 40 45 gtc gac ctc tgt gac gat ttg gct cct gtg gca aga cag ctt gct ccc 433 Val Asp Leu Cys Asp Asp Leu Ala Pro Val Ala Arg Gln Leu Ala Pro 50 55 60 agg gag aag ctt cct ctg agt agc agg aga cct gct gcg gtg ggg gct 481 Arg Glu Lys Leu Pro Leu Ser Ser Arg Arg Pro Ala Ala Val Gly Ala 65 70 75 80 ggg ctc cag aat atg gga aat acc tgc tac gtg aac gct tcc ttg gag 529 Gly Leu Gln Asn Met Gly Asn Thr Cys Tyr Val Asn Ala Ser Leu Glu 85 90 95 tgg ctg aca tac aca ccg ccc ctt gcc aac tac atg ctg tcc cgg gag 577 Trp Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu 100 105 110 cac tct caa acg tgt cat cgt cac aag ggc tgc atg ctc tgt act atg 625 His Ser Gln Thr Cys His Arg His Lys Gly Cys Met Leu Cys Thr Met 115 120 125 caa gct cac atc aca cgg gcc ctc cac aat cct ggg cac gtc atc cag 673 Gln Ala His Ile Thr Arg Ala Leu His Asn Pro Gly His Val Ile Gln 130 135 140 ccc tca cag gca ttg gct gct ggc ttc cat aga ggc aag cag gaa gat 721 Pro Ser Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln Glu Asp 145 150 155 160 gcc cat gaa ttt ctc atg ttc act gtg gat gcc atg aaa aag gca tgc 769 Ala His Glu Phe Leu Met Phe Thr Val Asp Ala Met Lys Lys Ala Cys 165 170 175 ctt ccc ggg cac aag cag gta gat cat cac tct aag gac acc acc ctc 817 Leu Pro Gly His Lys Gln Val Asp His His Ser Lys Asp Thr Thr Leu 180 185 190 atc cac caa ata ttt gga ggc tac tgg aga tct caa atc aag tgt ctc 865 Ile His Gln Ile Phe Gly Gly Tyr Trp Arg Ser Gln Ile Lys Cys Leu 195 200 205 cac tgc cac ggc att tca gac act ttt gac cct tac ctg gac atc gcc 913 His Cys His Gly Ile Ser Asp Thr Phe Asp Pro Tyr Leu Asp Ile Ala 210 215 220 ctg gat atc cag gca gct cag agt gtc cag caa gct ttg gaa cag ttg 961 Leu Asp Ile Gln Ala Ala Gln Ser Val Gln Gln Ala Leu Glu Gln Leu 225 230 235 240 gtg aag ccc gaa gaa ctc aat gga gag aat ggt tat gtt 1009 Val Lys Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Val 245 250 255 tgt ctc cag agg gcg ccg gcc tcc aag acg tta act tta cac acc tct 1057 Cys Leu Gln Arg Ala Pro Ala Ser Lys Thr Leu T hr Leu His Thr Ser 260 265 270 gcc aag gtc ctc atc ctt gta ttg aag aga ttc tcc gat gtc aca ggc 1105 Ala Lys Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp Val Thr Gly 275 280 285 aac aag att gcc aag aat gtg caa tat cct gag tgc ctt gac atg cag 1153 Asn Lys Ile Ala Lys Asn Val Gln Tyr Pro Glu Cys Leu Asp Met Gln 290 295 300 cca tac atg tct cag cag aac aca gga cct ctt gtc tat gtc ctc tat 1201 Ser Gln Gln Asn Thr Gly Pro Leu Val Tyr Val Leu Tyr 305 310 315 320 gct gtg ctg gtc cac gct ggg tgg agt tgt cac aac gga cat tac ttc 1249 Ala Val Leu Val His Ala Gly Trp Ser Cys His Asn Gly His Tyr Phe 325 330 335 tct tat gtc aaa gct caa gaa ggc cag tgg tat aaa atg gat gat gcc 1297 Ser Tyr Val Lys Ala Gln Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala 340 345 350 gag gtc acc gcc tct agc atc act tctgt agt caa cag gcc tac 1345 Glu Val Thr Ala Ser Ser Ile Thr Ser Val Leu Ser Gln Gln Ala Tyr 355 360 365 gtc ctc ttt tac atc cag aag agt gaa tgg gaa aga cac agt gag agt 1393 Val Leu Phe Tyr Ile Gln Ly s Ser Glu Trp Glu Arg His Ser Glu Ser 370 375 380 gtg tca aga ggc agg gaa cca aga gcc ctt ggc gca gaa gac aca gac 1441 Val Ser Arg Gly Arg Glu Pro Arg Ala Leu Gly Ala Glu Asp Thr Asp 385 390 395 400 agg cga gca acg caa gga gag ctc aag aga gac cac ccc tgc ctc cag 1489 Arg Arg Ala Thr Gln Gly Glu Leu Lys Arg Asp His Pro Cys Leu Gln 405 410 415 gcc ccc gag ttg gac gag cac ttg gtg gaga gaga gaa agc 1537 Ala Pro Glu Leu Asp Glu His Leu Val Glu Arg Ala Thr Gln Glu Ser 420 425 430 acc tta gac cac tgg aaa ttc ctt caa gag caa aac aaa acg aag cct 1585 Thr Leu Asp His Trp Lys Phe Leu Gln Glu Gln Asn Lys Thr Lys Pro 435 440 445 gag ttc aac gtc aga aaa gtc gaa ggt acc ctg cct ccc gac gta ctt 1633 Glu Phe Asn Val Arg Lys Val Glu Gly Thr Leu Pro Pro Asp Val Leu 450 455 460 gtg att cat caa tca aaa tac aag tgt ggg atg aag aac cat cat cct 1681 Val Ile His Gln Ser Lys Tyr Lys Cys Gly Met Lys Asn His His Pro 465 470 475 480 gaa cag caa agc tcc ctg cta aac ctc tct tcg acg acc ccg aca cat 1729 Gl u Gln Gln Ser Ser Leu Leu Asn Leu Ser Ser Thr Thr Thr Pro Thr His 485 490 495 cag gag tcc atg aac act ggc aca ctc gct tcc ctg cga ggg agg gcc 1777 Gln Glu Ser Met Asn Thr Gly Thr Leu Ala Ser Leu Arg Gly Arg Ala 500 505 510 agg aga tcc aaa ggg aag aac aaa cac agc aag agg gct ctg ctt gtg 1825 Arg Arg Ser Lys Gly Lys Asn Lys His Ser Lys Arg Ala Leu Leu Val 515 520 525 tgc cag tga tctcagtgac gaccag gaccagacacg Gln 530 cacacgcaca cacacagaca cacacataac tacacccaga agcgcgcacg taaacacaca 1934 cacacccaca caaacacgaa caccgtcaat cctacataaa ctaatgagga gcccaagttt 1994 ctgtctctac aacagggaca actggatagt gatggctaca tctcaggatg agcccgcata 2054 tgggaaacat caagttttgg ggtcgtgagt cttccgaacc tctggaggga ctgtctgagt 2114 gtttgtgttc atgataggcg acattcagtg tgtatttctg aatatgacct accgacgtgt 2174 aggtttgcgt gtgaggtaat tgcaggggac tcggtttcgt attttctctt ggggtgtgtt 2234 tcattcgtca gttgttggtc ggcatgagaa ggtgaaaggt ggctcatgtg ggacatccgt 2294 ggatcattct cgccaccttg aatagtggaa gctggaatgc atttggaaga gaagaacggt 2354 gctcttcttt cttccccggg ctcgccgttt ttacactggt tcctgaatgg acctcaggcg 2414 ccctgggact tgtgctcttg ctggaaccca cataacgccg gaagcggaca gaccgacttg 2474 cctgtttcac ggtgcccgct tcccatgagt cgaaacggaa aattttccca cgggcatgta 2534 agtcatctgg aagtaagctg tattgataat aaaggaaagc aaacacagga gtgtgtgtat 2594 tcaactgaaa taaattcaga aagccctgaa atcaatctca ctgggtgtgt ttaaaaatgg 2654 catttgggga atttctgggt catttgtcca gctgcgaaag ctgcatctct gaagcacagt 2714 ccctgtcccg cagtgagact tatttatccg acgtggtgtt tccgtggaaa tgattgtggg 2774 aaatggcccc ttccttttct ctatttgctg attagacttc atggtccctt tctcgtcagg 2834 tacagtgatc aaagttgacc aaccccagag gaaagctgcc cagggcacaa ctcagggctc 2894 cgtagaacca cagaatcttg ggcgcaaccc tgctcaagca cccaaatgtg catacgaaca 2954 gggtctccgt gtgacgtgtg tgaaaactac agtgtgatga gcatgactgg cagacagctt 3014 atcgattggg ctcccctcaa aatcggttat gagcattcaa gcacaccgat gcccaggtcc 3074 cggctgcagg aataagaccc tccagggtct tgtgtgaagc ctcggcatct gcattgctca 3134 tgcttctggg gatcattctc ctgaaaatgg tggctccttt ctccctgtgg agcatctttc 3194 t aagcagtgc tcttttcttc ccccaggaca ctttacatcc ggcacaggaa gccttctgat 3254 ggagcacacc tggcccatga aaagacaagg gaaagaaacg ggggcaaagg tcacagtcct 3314 ctcatcccat catcctcctt aaaatcatcc taatttcatg ggccctgaag ccagggctgt 3374 ttctttacac ctagaggcct tggcgccggg cctcaattcc gccctgttcc ttaccgtcta 3434 agacatgttg ggaaaatccc tagagccagg atcttcattc ccgctaagcc agacagccgg 3494 aagacacacc caaattctgt ccctcttact tcagggaaca tgtccacttt cggcagcatt 3554 acaattttgg caccaaatgt gctaactgca attccaccat acaatgcgta actggaaatg 3614 gaggcaacat ctccgatcct gaacgatcga tgcgagaatc caggatatgc acggcttatt 3674 ttggcctttt cccactgaaa caagggccag tattaaaaat ggcacgctat cctctgtttc 3734 actccctgct tttaaacgtc tccgatgttt ctccctgaga cagcgcctca cttccgtcag 3794 ccgggctttt ctacggtata attttccttg tttgcttttg tccaaattag aactttttat 3854 ttcatctcta ggaaacgttg atccattatc acatacgtat ggaaatatta tcacacatgc 3914 tgtgagatac gttgttttta ttttcatcaa ttccttaata aacaaaaggt tatagctggg 3974 ataccttctg agttctcaag ttttttgttt cgtgttttct taaactgccg tcgcacgtcc 4034 gaaacc gctc actatgcagt gtcatgaccg tctctctttt ctggcaaaca taaatttggg 4094 gattgtcatc aattagtctc tcggggattg catgatttcc ccaaaggctt tcacagtcta 4154 ctttgtgcac tgagtatctc tacaaacttc agtgcatgtt tctaccattt gatgctttct 4214 tatttggcaa tctagcttcc acaagagcat ttcatgcaaa gacttgtctt gttctccact 4274 ggcaggtaat ttcactcgga cagagaatca ataggctcaa cgtggaaagg ttatcgctgg 4334 aaggtctgtt tgattccacg gatctctcct ttctcactag ggaagaaaat acgctgtgct 4394 aaatactata cttcattgac tattctcagg tcagaaagcg cactttcgac ttcttgtcct 4454 tccgtcgctg agaggatgat ggcagctgcc aaaagtacct acttggaggt tcatcccagc 4514 acaaacacac acacacacac gcccccccac acacacacac aaacacactc acacacacac 4574 acgcacacgg tttcctaggt aaagatttct tccctgccat tgctttacct aaaataaggc 4634 aactgtgagg ccgctgtccc aacccggtta cactcctatt atatgtgcct atcatcctga 4694 ggagtaattt gattcaggtg ttctggaagt catgctgtgg gctgtgtctg tt 4746 <210> 2 <211> 530 <212> PRT <213> Homo sapiens <400> 2 Met Glu Asp Asp Ser Leu Tyr Leu Gly Gly Glu Trp Gln Phe Asn His 1 5 10 15 Phe Pro Lys Leu Thr Ser Ser Arg P ro Asp Ala Ala Phe Ala Glu Ile 20 25 30 Gln Arg Thr Ser Leu Pro Glu Lys Ser Pro Leu Ser Cys Glu Thr Arg 35 40 45 Val Asp Leu Cys Asp Asp Leu Ala Pro Val Ala Arg Gln Leu Ala Pro 50 55 60 Arg Glu Lys Leu Pro Leu Ser Ser Arg Arg Pro Ala Ala Val Gly Ala 65 70 75 80 Gly Leu Gln Asn Met Gly Asn Thr Cys Tyr Val Asn Ala Ser Leu Glu 85 90 95 Trp Leu Thr Tyr Thr Pro Pro Leu Ala Asn Tyr Met Leu Ser Arg Glu 100 105 110 His Ser Gln Thr Cys His Arg His Lys Gly Cys Met Leu Cys Thr Met 115 120 125 Gln Ala His Ile Thr Arg Ala Leu His Asn Pro Gly His Val Ile Gln 130 135 140 Pro Ser Gln Ala Leu Ala Ala Gly Phe His Arg Gly Lys Gln Glu Asp 145 150 155 160 Ala His Glu Phe Leu Met Phe Thr Val Asp Ala Met Lys Lys Ala Cys 165 170 175 Leu Pro Gly His Lys Gln Val Asp His His Ser Lys Asp Thr Thr Leu 180 185 190 Ile His Gln Ile Phe Gly Gly Tyr Trp Arg Ser Gln Ile Lys Cys Leu 195 200 205 His Cys His Gly Ile Ser Asp Thr Phe Asp Pro Tyr Leu Asp Ile Ala 210 215 220 Leu Asp Ile Gln Ala Ala Gln Ser Val Gln Gln Ala Leu Glu Gln Leu 225 230 235 240 Val Lys Pro Glu Glu Leu Asn Gly Glu Asn Ala Tyr His Cys Gly Val 245 250 255 Cys Leu Gln Arg Ala Pro Ala Ser Lys Thr Leu Thr Leu His Thr Ser 260 265 270 Ala Lys Val Leu Ile Leu Val Leu Lys Arg Phe Ser Asp Val Thr Gly 275 280 285 Asn Lys Ile Ala Lys Asn Val Gln Tyr Pro Glu Cys Leu Asp Met Gln 290 295 300 Pro Tyr Met Ser Gln Gln Asn Thr Gly Pro Leu Val Tyr Val Leu Tyr 305 310 315 320 Ala Val Leu Val His Ala Gly Trp Ser Cys His Asn Gly His Tyr Phe 325 330 335 Ser Tyr Val Lys Ala Gln Glu Gly Gln Trp Tyr Lys Met Asp Asp Ala 340 345 345 Glu Val Thr Ala Ser Ser Ile Thr Ser Val Leu Ser Gln Gln Ala Tyr 355 360 365 Val Leu Phe Tyr Ile Gln Lys Ser Glu Trp Glu Arg His Ser Glu Ser 370 375 380 Val Ser Arg Gly Arg Glu Pro Arg Ala Leu Gly Ala Glu Asp Thrasp 385 390 395 395 400 Arg Arg Ala Thr Gln Gly Glu Leu Lys Arg Asp His Pro Cys Leu Gln 405 410 415 Ala Pro Glu Leu Asp Glu His Leu Val Glu Arg Ala Thr Gln Glu Ser 420 425 430 Thr Leu Asp His Trp Lys Phe Leu Gln Glu Gln Asn LysThr Lys Pro 435 440 445 Glu Phe Asn Val Arg Lys Val Glu Gly Thr Leu Pro Pro Asp Val Leu 450 455 460 Val Ile His Gln Ser Lys Tyr Lys Cys Gly Met Lys Asn His His Pro 465 470 475 480 480 Glu Gln Gln Ser Ser Leu Leu Asn Leu Ser Ser Thr Thr Pro Thr His 485 490 495 Gln Glu Ser Met Asn Thr Gly Thr Leu Ala Ser Leu Arg Gly Arg Ala 500 505 510 Arg Arg Ser Lys Gly Lys Asn Lys His Ser Lys Arg Ala Leu Leu Val 515 520 525 Cys Gln 530

[Brief description of the drawings]

FIG. 1 is a test result showing that the presence of the 1590 bp ORF is within the 4.7-kb RS447 repeat unit. A: 4.7-kb
The location of the estimated ORF in RS447. Arrows and arrow heads indicate estimated ORFs. The solid bar is RS447
Of the 4.7-kb EcoRI fragment. The size of the largest ORF is indicated by the arrow. B: Nucleotide and deduced amino acid sequence of RS447 ORF.

FIG. 2 shows test results confirming the conservation of the RS447 ORF sequence in mammalian species. 2 μg of EcoRI digested genomic DNA from each animal species was electrophoresed, blotted onto a nylon membrane and hybridized with a ³² P-labeled probe. A: 153
A 1.6 kb EcoRI-Kpnl fragment of RS447 consisting of 9 bp ORF was used as a probe. B: 3.1 kb KpnI-E of RSS447
The coRI fragment was used as a non-coding region probe. C: The positions of these probes are illustrated in RS447.

FIG. 3 shows test results confirming the expression of RS447 in human tissues. A: Multiple poly (A) ⁺ RNA tissue northern blots of human were analyzed with ³² P-labeled antisense RS447 ORF.
It hybridized with the DNA probe (positions 242-1183). Lane 1; heart 2; brain 3; placenta 4, lung 5;
Liver, 6; muscle, 7; kidney, and 8; pancreas.

FIG. 4 shows the test results confirming that the RS447 repeating unit exhibits promoter activity. The deletion construct containing the RS447 ORF upstream region was ligated to the non-promoter green fluorescent protein (GFP) gene. Promoter activity was quantified by measuring GFP fluorescence.

FIG. 5 is an amino acid sequence homolog of RS447-dUb and several deubiquitinase enzymes. A: Sequence of the Cys and His regions of RS447-dUb, B: Deubiquitinase family member; yeast UBP1 and Doa4, Drosophila FA
F, human Unp and Tre2, and mouse DUB-1 and DUB-2
Are some arrays. Identical amino acid sequences are shown in shaded boxes.

FIG. 6 shows the test results of testing the deubiquitination activity of RS447-dUb. T7 / Ub-GFP, the fusion proteins of ubiquitin and green fluorescent protein labeled T7 His ₆
Labeled peptide (lane 1), His ₆ -RS447-dUb (lane 1)
2), His ₆ -RS447-dUb (S89) (lane 3), GST (lane
4), GST-RS447-dUb (lane 5), and GST-RS447-dUb
(S89) (lane 6). Western blots show anti-GFP (panel A), anti-T7 label (panel B), and anti-R
The test was performed with each antibody of S447-dUb (panel C). As shown in each panel, the anti-GFP antibody detected T7 / Yb-GFP and deubiquitinated GFP (panel A), and the anti-T7 labeled antibody detected T7 / Ub-GFP.
(Panel B), and the anti-RS447-dUb antibody is His ₆ -R
S447-dUb and GST-RS447-dUb were detected (Panel C).

FIG. 7 shows the results of detection of RS447-dUb in human cells.
Epstein-Barr virus transformation costs and lymphoblasts (lane 1), COS7 cells (lane 2), and Xpress-RS4
Protein extract from Cos7 cells expressing 47-dUb (lane 3) was subjected to SDS gel electrophoresis, and anti-RS447-dUb antibody (panel
Immunoblotting was performed with A) and anti-Xpress antibody (panel B).

FIG. 8. Cosmid containing 6 copies of RS447 (CRSS44
7 shows the results of testing the expression of RS447-dUb in A9 cells transfected in 7). CRSS447 was transfected into mouse A9 cells. Cell extracts from transient (A) and stable (B) transformants were subjected to SDS gel electrophoresis,
Immunoblot with anti-RS447-dUb antibody.

FIG. 9. RS447 in CRS447 stable cell lines A and D.
-The results of testing the expression of dUb sense and antisense transcripts. Poly (A) ⁺ RNA (clone A and D) from CRS447 stable transformants was hybridized with a ³² P-labeled antisense (A) or sense (B) RS447 ORF DNA probe. Southern blot hybridization tested with RS447 (C).

FIG. 10. Antisense RS447OR in human tissue
It is the result of having examined the expression of F. Multiple human poly (A) ⁺ RNA tissue northern blots were prepared using ³² P-labeled RS447O
Hybridized with an RF DNA probe (positions 242-1831). Lane 1, heart 2, brain 3, placenta 4, lung
5; liver, 6; muscle, 7; kidney, and 8; pancreas.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 5/10 C12N 15/00 ZNAA 9/48 5/00 A F term (Reference) 4B024 AA01 BA11 BA53 CA03 CA07 CA12 DA02 EA04 FA02 GA13 HA01 4B050 CC04 CC05 DD07 LL01 4B065 AA90X AA91X AA93Y AB01 AC14 BA02 CA31 CA44 4H045 AA10 BA10 BA41 CA40 DA89 EA28 FA72 FA74 HA06

Claims (7)

[Claims] 1. A human deubiquitinase having the amino acid sequence of SEQ ID NO: 2. 2. A polynucleotide encoding the human deubiquitinase of claim 1 and having at least the 242-1834 base sequence of SEQ ID NO: 1. 3. An expression vector carrying the polynucleotide of claim 2. 4. A transformant which is a transformant of the expression vector according to claim 3, which is capable of producing the enzyme according to claim 1. 5. An antibody against the human deubiquitinase of claim 1. 6. A promoter sequence of a human deubiquitinase gene. 7. An anti-human deubiquitinase mRNA
Transcription initiation promoter.