JPH09262A - Human ubiquitin-conjugating enzyme E2 and cDNA encoding the same - Google Patents

Abstract

(57) [Summary] [Object] To provide ubiquitin-conjugating enzyme E2, which has an important role in intracellular proteolytic system, and human cDNA encoding the enzyme. [Structure] Human c encoding ubiquitin-conjugating enzyme E2
It consists of cloning of DNA, expression of the protein encoded by this human cDNA by E. coli, and measurement of the ubiquitin binding activity of the expression product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel cDNA derived from mRNA expressed in human cells, and a ubiquitin-conjugating enzyme E2 encoded by the cDNA. The human cDNA of the present invention can be used as a gene diagnostic probe or a gene source for gene therapy. Further, it can be used as a gene source for mass-producing the protein encoded by the cDNA. The protein of the present invention is one of the ubiquitin-conjugating enzymes E2 and can be used as a reagent for ubiquitin system research.

[0002]

2. Description of the Related Art The ubiquitin system is one of intracellular proteolytic systems and is a ubiquitin activating enzyme E.
1 (E1), ubiquitin-conjugating enzyme E2 (E2), and ubiquitin-discriminating enzyme E3 (E3) [Hershko, A. et al. and Ciechano
ver. A. , Annu. Rev .. Biochem.
61: 761-807 (1992)]. Ubiquitin (U
b) is E1 via C-terminal glycine residue in the presence of ATP
Form a thioester bond with the cysteine residue of. Then, when E1-Ub reacts with E2, the activated Ub becomes E1.
To E2 and E2-Ub is formed. Further E
2-Ub ubiquitinates the target protein in the presence of E3. The target protein labeled with ubiquitin is finally degraded by the proteasome in an energy-dependent manner.

Recently, it has become clear that abnormalities in the ubiquitin system are associated with many diseases [May.
er, R.R. J. et al. , Biochim. Bi
ophys. Acta 1089: 141-157 (1
991)]. For example, Drosophila bendles
A malformation called s has been reported to be caused by a single amino acid mutation in E2 [Muralidhar,
M. G. FIG. and Thomas, J. et al. B. , Neur
on 11: 253-266 (1993)]. It is considered that E2 determines the specificity of the target protein for ubiquitination, and 10 or more kinds of E2 have been isolated in yeast. Moreover, it is known that these abnormalities cause abnormalities in cell division and cell proliferation. Although more types of E2 exist in humans and are predicted to be associated with various diseases, the number of E2s isolated so far is small, and more isolation and functional elucidation of E2s are desired. ing.

[0004]

An object of the present invention is to provide a human cDNA encoding a novel ubiquitin conjugating enzyme E2, and a protein encoded by this human cDNA.

[0005]

[Means for Solving the Problems] As a result of earnest studies, the present inventors have found that human c that encodes a novel ubiquitin-conjugating enzyme E2.
The DNA was cloned to complete the present invention. That is,
The present invention provides a protein containing the amino acid sequence represented by SEQ ID NO: 1, which is a novel ubiquitin-conjugating enzyme E2. The present invention also provides a cDNA encoding the above protein, the cDNA including the nucleotide sequence represented by SEQ ID NO: 1.

The human cDNA of the present invention is c derived from human cells.
It can be cloned from a DNA library.
cDNA is synthesized using poly (A) ⁺ RNA extracted from human cells as a template. The human cells may be cells that have been extracted from the human body by surgery or the like, or cultured cells. In the examples, poly (A) ⁺ RNA isolated from human epidermoid carcinoma cell line KB was used. cDNA is Okayama-Berg
Method [Okayama, H .; and Berg, P.M. ,
Mol. Cell. Biol. 2: 161-170
(1982)], Gubler-Hoffman method [G
ubler, U .; and Hoffman, J .; Ge
ne 25: 263-269 (1983)] and the like. However, in order to efficiently obtain a full-length clone, a method using a vector primer as described in the Examples is desirable. The identification of cDNA is
It is performed by determining the entire base sequence by sequencing, searching for a known protein having a sequence similar to the amino acid sequence predicted from the base sequence, expressing the protein by in vitro translation, expressing in E. coli, and measuring the activity of the expression product. The activity was measured by adding the expression product to a rabbit reticulocyte lysate containing labeled ubiquitin (containing rabbit E1),
It is performed by confirming that a thioester conjugate of the expression product and ubiquitin is formed by DS-polyacrylamide gel electrophoresis.

The cDNA of the present invention is characterized by containing the base sequence represented by SEQ ID NO: 1. For example, the sequence represented by SEQ ID NO: 2 has a base sequence of 1203 bp, It had an open reading frame of 459 bp. This open reading frame encodes a protein consisting of 152 amino acid residues. This protein is a Drosophila ubiquitin-conjugating enzyme E2 [Muralidha at the amino acid sequence level.
r, M. G. FIG. and Thomas, J. et al. B. , Ne
uron 11: 253-266 (1993)] and 8
It has a high similarity of 0.1%.

By screening a human cDNA library prepared from the cell line used in the present invention using an oligonucleotide probe synthesized based on the nucleotide sequence of the cDNA shown in SEQ ID NO: 1 or SEQ ID NO: 2. A clone identical to the cDNA of the present invention can be easily obtained.

In general, human genes are frequently found to have polymorphisms due to individual differences. Therefore, SEQ ID NO: 1 or SEQ ID NO: 2
In the present invention, a cDNA in which one or more nucleotides are added, deleted and / or replaced with another nucleotide is also included in the scope of the present invention.

Similarly, the resulting 1
Alternatively, a protein in which a plurality of amino acids are added, deleted and / or substituted with other amino acids also falls within the scope of the present invention.

The cDNA of the present invention also includes a cDNA fragment (10 bp or more) containing any partial base sequence of the base sequence represented by SEQ ID NO: 1 or 2. In addition, a DNA fragment consisting of a sense strand and an antisense strand also falls into this category. These DNA fragments can be used as a probe for gene diagnosis.

The protein of the present invention can be expressed in vitro by preparing RNA from the vector having the cDNA of the present invention by in vitro transcription and performing in vitro translation using this as a template. When the translation region is recombined into an appropriate expression vector by a known method, E. coli,
A large amount of the encoded protein can be expressed in Bacillus subtilis, yeast, animal cells and the like. Alternatively, a peptide can be prepared by chemical synthesis based on the amino acid sequence of the present invention.

The protein of the present invention includes a fusion protein with any other protein as long as it has a ubiquitin conjugating enzyme E2 activity. For example, a fusion protein with the maltose-binding protein mentioned in the examples can be exemplified.

[0014]

EXAMPLES The present invention will now be described in detail with reference to examples, but the present invention is not limited to these examples. D
The basic operation and enzymatic reaction for recombination of NA are
Reference ["Molecular Cloning. AL
“Aboratory Manual”, Cold Sp
ring Harbor Laboratory, 19
89]. Unless otherwise specified, the restriction enzymes and various modifying enzymes used were those manufactured by Takara Shuzo. The buffer composition of each enzyme reaction and the reaction conditions were in accordance with the attached instructions.
cDNA synthesis is described in the literature [Kato, S. et al. el al. ,
Gene 150: 243-250 (1994)].

Preparation of poly (A) ⁺ RNA A human epidermoid carcinoma cell line KB (ATCC CRL 17) was cultured in MEM medium containing 10% fetal calf serum at 37 ° C. in a 5% CO ₂ stream to give 1 g of cells. Got This is 5.5
After being dissolved in 16 ml of M guanidinium thiocyanate solution, it was dissolved in the literature [Okayama, H .; et al. , "M
methods in Enzymology "Vo
l. 164, Academic Press, 198
7] to prepare mRNA. 20 mM Tris-HCl buffer (pH 7.6), 0.5 M NaCl, 1 mM
It was applied to an oligo dT cellulose column washed with EDTA to obtain 300 μg of poly (A) ⁺ RNA according to the above-mentioned literature.

Preparation of cDNA library 10 μg of the above poly (A) ⁺ RNA was dissolved in 100 mM Tris-HCl buffer (pH 8), and 1 unit of RNase-free bacterial-derived alkaline phosphatase was added,
The reaction was carried out at 37 ° C for 1 hour. After extracting the reaction solution with phenol,
Ethanol precipitation was performed, and the pellet was 50 mM sodium acetate (pH 6), 1 mM EDTA, 0.1% 2-
Mercaptoethanol, 0.01% Triton X-
It was dissolved in 100 solution. To this, 1 unit of tobacco-derived acid pyrophosphatase (manufactured by Epicenter Technologies) was added and reacted at 37 ° C. for 1 hour in a total volume of 100 μl. After the reaction solution was extracted with phenol, ethanol precipitation was performed and the pellet was dissolved in water.

Decapped poly (A) ⁺ RNA,
DNA-RNA chimera oligonucleotide (5'-dG
-DG-dG-dG-dA-dA-dT-dT-dC-
dG-dA-G-G-A-3 ') 3 nmol 50 mM
Tris-HCl buffer (pH 7.5), 0.5 mM ATP,
5 mM MgCl2, 10 mM 2-mercaptoethanol, dissolved in 25% polyethylene glycol aqueous solution,
50 units of T4 RNA ligase was added, and the mixture was reacted at 20 ° C. for 12 hours in a total volume of 30 μl. After the reaction solution was extracted with phenol, ethanol precipitation was performed and the pellet was dissolved in water.

After digesting pKA1 (JP-A-4-117292) with KpnI, about 60 dT tails were added with a terminal transferase. This was digested with EcoRV to remove the dT tail on one side and used as a vector primer.

⁶ μg of the chimeric oligo-capped poly (A) ⁺ RNA prepared above was added to 1.2 μm of the vector primer.
50mM Tris-HCl buffer (p
H8.3), 75 mM KCl, 3 mM MgCl ₂ ,
10 mM dithiothreitol, 1.25 mM dNTP
It was dissolved in a (dATP + dCTP + dGTP + dTTP) solution, 200 units of reverse transcriptase (GIBCO-BRL) was added, and reacted at 42 ° C. for 1 hour in a total volume of 20 μl.
After extracting the reaction solution with phenol, perform ethanol precipitation,
The pellet was added with 50 mM Tris-HCl buffer (pH 7.5),
100 mM NaCl, 10 mM MgCl ₂ , 1 mM
Dissolved in dithiothreitol solution. EcoRI
100 units were added, and the whole was reacted at 37 ° C. for 1 hour with a total volume of 20 μl. After the reaction solution was extracted with phenol, ethanol precipitation was performed, and the pellet was mixed with 20 mM Tris-HCl buffer (pH
7.5), 100 mM KCl, 4 mM MgCl ₂ ,
It was dissolved in 10 mM (NH ₄ ) ₂ SO ₄ , 50 μg / ml bovine serum albumin solution. E. coli DNA ligase 6
0 unit was added and reacted at 16 ° C. for 16 hours. 2 μl of 2 mM dNTP, E. coli DNA polymerase I4 in the reaction solution
Unit, 0.1 unit of E. coli RNaseH was added, and
The reaction was conducted at 1 ° C for 1 hour and then at 22 ° C for 1 hour.

Escherichia coli DH1 using the cDNA synthesis reaction solution
2S (GIBCO-BRL) was transformed. Transformation was performed by the electroporation method. A part of the transformant was plated on 2 x YT agar medium containing 100 µg / ml ampicillin and cultured overnight at 37 ° C. Pick up any colonies generated on agar, 100 μg /
3 ml by inoculating 2 ml of 2xYT medium containing ml ampicillin
After culturing at 7 ° C for 2 hours, helper phage MK13KO7
(Pharmacia) was further infected and further cultured overnight at 37 ° C. The culture solution is centrifuged to separate the cells into a supernatant and a supernatant, and double-stranded plasmid DNA is separated from the cells by an alkaline lysis method.
Single-stranded phage DNA was isolated from the supernatant by a conventional method. The double-stranded plasmid DNA was double-digested with EcoRI and NotI and then subjected to 0.8% agarose gel electrophoresis to determine the size of the cDNA insert. On the other hand, single-stranded phage DNA undergoes a sequence reaction using a fluorescent dye-labeled M13 universal primer and Taq polymerase (a kit manufactured by Applied Biosystems), and then subjected to a fluorescent DNA sequencer (Applied Biosystems) to prepare 5 cDNAs. 'End about 400b
The base sequence of p was determined. The sequence data was filed as a homo protein cDNA bank database.

CDNA Cloning The nucleotide sequence of a clone arbitrarily selected from the above cDNA library was determined, the obtained nucleotide sequence was converted into an amino acid sequence of 3 frames, and a protein database was searched with these sequences. Analysis software is GE
NETYX-MAC (manufactured by Software Development Co.) was used. As a result, the protein encoded by clone HP00686 was identified as Drosophila ubiquitin-binding protein E.
2 was found to have similarity at the amino acid sequence level. The structure of this clone is shown in FIG. When the entire nucleotide sequence of the cDNA insert was determined, it had a structure consisting of a 63 bp 5'untranslated region, a 459 bp open reading frame, a 681 bp 3'untranslated region, and a 80 bp poly (A) tail ( SEQ ID NO: 2). The open reading frame encodes a protein consisting of 152 amino acid residues, and when a protein database was searched using this sequence, a high similarity of 80.1% with the amino acid sequence of Drosophila ubiquitin-binding protein E2 was found over the entire region. Had. Table 1 shows a comparison of the amino acid sequences of the protein HP686 (HP) of the present invention and the Drosophila ubiquitin-binding protein E2 (E2). * Indicates the same amino acid residue as the protein of the present invention ,. Represents an amino acid residue similar to that of the protein of the present invention.

Table 1 _________________________________________________________________ HP MAGLPRRIIKETQRLLAEPVPGIKAEPDESNARYFHVVIAGPQDSPFEGGTFKLELFLPE * .. ************ .. ******. * ***. ******* ... **. *******. ********* E2 MSSLPRRIIKETQRLMQEPVPGINAIPDENNARYFHVIVTGPNDSPFEGGVFKLELFLPE HP EYPMAAPKVRFMTKIYHPNVDKLGRICLDILKDKWSPALQIRTVLLSIQALLSAPNPDDP. ***. *. ***********. *. ******** . *************. **************** E2 DYPMSAPKVRFITKIYHPNIDRLGRICLDVLKDKWSPALQIRTILLSIQALLSAPNPDDP HP LANDVAEQWKTNEAQAIETARAWTRLYAMNNI ******* **. ***. * *. **. **. ** ... E2 LANDVAELWKVNEAEAIRNAREWTQKYAVED _________________________________________________________________________________

The sequence of the obtained cDNA is used to generate a nucleotide sequence database GenBank / EMBL / DDB.
As a result of searching for J, cD partially matching the cDNA of the present invention represented by SEQ ID NO: 2 in the EST database
It was found that the partial sequence of NA (registration number T96902 etc.) was registered. However, even if the partial sequences match, there is no guarantee that this fragment and the full-length cDNA of the present invention are derived from the same mRNA. Moreover, the amino acid sequence and function of the protein which may be encoded cannot be known only from this sequence.

[0024] Using the vector containing the cDNA of protein synthesis present invention by in vitro translation was carried out in vitro translation by T _N T rabbit reticulocyte lysate kit (Promega). At this time, [ ³⁵ S] methionine was added, and the expression product was labeled with a radioisotope.
Both reactions were performed according to the protocol attached to the kit. After subjecting the expression product to SDS-polyacrylamide gel electrophoresis, autoradiography is performed,
The molecular weight of the translation product was determined. As a result, the cDNA of the present invention
A produced a translation product with a molecular weight of approximately 18 kDa. This value was in agreement with the predicted molecular weight 17137 of the protein predicted from the nucleotide sequence represented by SEQ ID NO: 2, and it was shown that this cDNA surely encodes the protein represented by SEQ ID NO: 2.

Expression by Escherichia coli Two oligonucleotide primers represented by the following formula were synthesized by an automatic DNA synthesizer (Applied Biosystems) according to the attached protocol. PR1 5'-GCGGTCTCGATGGCCGGGCTGCCCCGCA-3 'PR2 5'-GCGTCGACTTAAATATTATTCATGGCAT-3' Plasmid pHP00686 1 ng and primer PR
PCR using 100 pmole of each of 1 and PR2
The cDNA translation region was amplified with a kit (Takara Shuzo). After phenol extraction and ethanol precipitation, 20 units of B
After digestion with saI (New England Biolabs), Klenow treatment was performed, followed by digestion with SalI,
The reaction product was subjected to 1.5% agarose gel electrophoresis, and a DNA fragment of about 500 bp was excised from the gel and purified.

Then 1 μg of pMAL ^™ -c2 (New England Biolabs) was added to 20 units of XmnI.
(New England Biolabs Co., Ltd.) and SalI, and then subjected to 0.6% agarose gel electrophoresis,
A 6.7 kbp DNA fragment was excised from the gel. After ligating the vector fragment and the cDNA fragment with a ligation kit, Escherichia coli JM109 was transformed. Plasmid pMALHP686 was prepared from the transformant, and the target recombinant was confirmed by the restriction enzyme cleavage map. The structure of the obtained plasmid is shown in FIG.

10 ml of an overnight culture of pMALHP686 / JM109 was added to 100 μg / ml ampicillin-containing rich medium (containing 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 2 g of glucose per liter) 50
The suspension was suspended in 0 ml and shake-cultured at 37 ° C., and isopropylthiogalactoside was added to 1 mM when A600 reached about 0.5. After further culturing at 37 ° C for 3 hours, the cells were collected by centrifugation, and the cells were collected into a column buffer (10 mM Tris-HCl, pH 7.4, 200 mM NaC).
1, 1 mM EDTA) in 25 ml. This solution was sonicated and then centrifuged, and the supernatant was added to a bed volume of 3.5 ml.
Amylose column (New England Biolabs). After washing the column with 8 times the column volume of the column buffer, 20 ml of the column buffer containing 10 mM maltose was used to bind maltose-binding protein / HP686.
The fusion protein was eluted to obtain 14.4 mg of fusion protein. When the fusion protein was subjected to SDS-polyacrylamide gel electrophoresis, a single band was observed at a position of about 59 kDa. This molecular weight value is consistent with the expected molecular weight of the maltose binding protein / E2 fusion protein.

Maltose binding protein / E2 fusion protein 1
1 μg of Factor Xa was added to 200 μl of a column buffer containing 00 μg, and reacted at 4 ° C. for 12 hours.
The reaction mixture was applied to an amylose column and the fractions that passed through were collected to obtain 48 μg of E2 protein. When subjected to SDS-polyacrylamide gel electrophoresis, a band was observed at a position of about 17 kDa corresponding to the E2 protein of the present invention.

Ubiquitin-binding activity measurement cDNA library of human lymphoma cell line U937
(Described in JP-A-4-117292)
The nucleotide sequence of the selected clone was determined and
The polyubiquitin cDNA represented was obtained. This cDNA
16 μg of plasmid containing_NT rabbit reticulocytes
Lysate 30 μl, buffer (included in kit) 2.4 μl,
1.2 μl of amino acid mixture (without methionine),
[³⁵S] methionine (Amersham) 2.4 μl
(0.37 MBq / μl), T7 RNA polymerase
1.2μl, total volume 60μ including RNasin 1.2μl
The reaction was carried out at 23 ° C. for 90 minutes in 1 l of the reaction solution. further,
To 7 μl of this reaction solution, 3 μg of the fusion protein and a buffer solution (200
mM Tris-HCl buffer, pH 7.5, 40 mM MgC
l _Two4 mM ATP, 0.4 mM dithiothreitol
2) Add 3 μl to make a total volume of 12 μl and at 23 ° C for 20 minutes
Reacted. SDS sampling in 5 μl of the obtained reaction solution
Buffer (50 mM Tris-HCl buffer, pH 6.8,
2% SDS solution, 0.1% bromophenol blue, 1
5% of 0% glycerol) was added to SD under non-reducing conditions.
Subjected to S-polyacrylamide gel electrophoresis. Auto
As a result of radiography, a position of about 67 kDa
Band was recognized. This molecular weight is
It corresponds to the sum of the molecular weights of chitin. In addition, 100m in the reaction solution
SDS sample containing M2-mercaptoethanol
Buffer and heat treatment at 95 ° C for 3 minutes, reducing conditions
This band disappears when subjected to electrophoresis under
And, due to the complex containing thioester bond
It has been shown. In addition, by factor Xa treatment, malt
The same results were obtained with the source-binding protein part removed.
A test showed that E2 and ubiquitin formed a thioester bond.
A combined band of 24 kDa was observed. that's all
From the results of E. coli obtained by expressing in E. coli,
It was shown to have chitin binding activity.

[0030]

INDUSTRIAL APPLICABILITY The present invention provides a human cDNA encoding a novel ubiquitin-conjugating enzyme E2, a protein encoded by this human cDNA, and a fusion protein containing this protein. By using the cDNA of the present invention, the recombinant protein can be expressed in a large amount. The recombinant protein can be used as a research reagent.

[0031]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 456 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence ATG GCC GGG CTG CCC CGC AGG ATC ATC AAG GAA ACC CAG CGT TTG CTG 48 Met Ala Gly Leu Pro Arg Arg Ile Ile Lys Glu Thr Gln Arg Leu Leu 1 5 10 15 GCA GAA CCA GTT CCT GGC ATC AAA GCC GAA CCA GAT GAG AGC AAC GCC 96 Ala Glu Pro Val Pro Gly Ile Lys Ala Glu Pro Asp Glu Ser Asn Ala 20 25 30 CGT TAT TTT CAT GTG GTC ATT GCT GGC CCT CAG GAT TCC CCC TTT GAG 144 Arg Tyr Phe His Val Val Ile Ala Gly Pro Gln Asp Ser Pro Phe Glu 35 40 45 GGA GGG ACT TTT AAA CTT GAA CTA TTC CTT CCA GAA GAA TAC CCA ATG 192 Gly Gly Thr Phe Lys Leu Glu Leu Phe Leu Pro Glu Glu Tyr Pro Met 50 55 60 GCA GCC CCT AAA GTA CGT TTC ATG ACC AAA ATT TAT CAT CCT AAT GTA 240 Ala Ala Pro Lys Val Arg Phe Met Thr Lys Ile Tyr His Pro Asn Val 65 70 75 80 GAC AAG TTG GGA AGA ATA TGT TTA GAT ATT TTG AAA GAT AAG TGG TCC 288 Asp Lys Leu Gly Arg Ile Cys Leu Asp Ile Leu Lys Asp Lys Trp Ser 85 90 95 CCA GCA CTG CAG ATC CGC ACA GTT CTG CTA TCG ATC CAG GCC TTG TTA 336 Pro Ala Leu Gln Ile Arg Thr Val Leu Leu Ser Ile Gln Ala Leu Leu 100 105 110 AGT GCT CCC AAT CCA GAT GAT CCA TTA GCA AAT GAT GTA GCG GAG CAG 384 Ser Ala Pro Asn Pro Asp Asp Pro Leu Ala Asn Asp Val Ala Glu Gln 115 120 125 TGG AAG ACC AAC GAA GCC CAA GCC ATA GAA ACA GCT AGA GCA TGG ACT 432 Trp Lys Thr Asn Glu Ala Gln Ala Ile Glu Thr Ala Arg Ala Trp Thr 130 135 140 AGG CTA TAT GCC ATG AAT AAT ATT 456 Arg Leu Tyr Ala Met Asn Asn Ile 145 150

SEQ ID NO: 2 Sequence length: 1203 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: Organism name: Homo = sapiens Cell type: Class Epidermal cancer Cell line: KB Clone name: HP00686 Sequence features: Characteristic symbols: CDS Location: 64. . 522 Sequences ACTCGTGCGT GAGGCGAGAG GAGCCGGAGA CGAGACCAGA GGCCGAACTC GGGTTCTGAC 60 AAG ATG GCC GGG CTG CCC CGC AGG ATC ATC AAG GAA ACC CAG CGT TTG 108 Met Ala Gly Leu CLA GLA GCA Lega CLA GCA LGA CCT GGC ATC AAA GCC GAA CCA GAT GAG AGC AAC 156 Leu Ala Glu Pro Val Pro Gly Ile Lys Ala Glu Pro Asp Glu Ser Asn 20 25 30 GCC CGT TAT TTT CAT GTG GTC ATT GCT GGC CCT CAG GAT TCC CCC TTT 204 Ala Arg Tyr Phe His Val Val Ile Ala Gly Pro Gln Asp Ser Pro Phe 35 40 45 GAG GGA GGG ACT TTT AAA CTT GAA CTA TTC CTT CCA GAA GAA TAC CCA 252 Glu Gly Gly Thr Phe Lys Leu Glu Leu Phe Leu Pro Glu Glu Tyr Pro 50 55 60 ATG GCA GCC CCT AAA GTA CGT TTC ATG ACC AAA ATT TAT CAT CCT AAT 300 Met Ala Ala Pro Lys Val Arg Phe Met Thr Lys Ile Tyr His Pro Asn 65 70 75 GTA GAC AAG TTG GGA AGA ATA TGT TTA GAT ATT TTG AAA GAT AAG TGG 348 Val Asp Lys Leu Gly Arg Ile Cys Leu Asp Ile Leu Lys Asp Lys Trp 80 85 90 95 TCC CCA GCA CTG CAG ATC CGC ACA GTT CTG CTA TCG ATC CAG GCC TTG 396 Ser Pro Ala Leu Gln Ile Arg Thr Val Leu Leu Ser Ile Gln Ala Leu 100 105 110 TTA AGT GCT CCC AAT CCA GAT GAT CCA TTA GCA AAT GAT GTA GCG GAG 444 Leu Ser Ala Pro Asn Pro Asp Asp Pro Leu Ala Asn Asp Val Ala Glu 115 120 125 CAG TGG AAG ACC AAC GAA GCC CAA GCC ATA GAA ACA GCT AGA GCA TGG 492 Gln Trp Lys Thr Asn Glu Ala Gln Ala Ile Glu Thr Ala Arg Ala Trp 130 135 140 ACT AGG CTA TAT GCC ATG AAT AAT ATT TAAATTGATA CGATCATCAA G 540 Thr Arg Leu Tyr Ala Met Asn Asn Ile 145 150 TGTGCATCAC TTCTCCTGTT CTGCCAAGAC TTCCTCCTCT TTGTTTGCAT TTAATGGACA 600 CAGTCTTAGA AACATTACAG AATAAAAAAG CCCAGACATC TTCAGTCCTT TGGTGATTAA 660 ATGCACATTA GCAAATCTAT GTCTTGTCCT GATTCACTGT CATAAAGCAT GAGCAGAGGC 720 TAGAAGTATC ATCTGGATTG TTGTGAAACG TTTAAAAGCA GTGGCCCCTC CCTGCTTTTA 780 TTCATTTCCC CCATCCTGGT TTAAGTATAA AGCACTGTGA ATGAAGGTAG TTGTCAGGTT 840 AGCTGCAGGG GTGTGGGTGT TTTTATTTTA TTTTATTTTA TTTTATTTTT GAGGGGGGAG 900 GTAGTTTAAT TTTATGGGCT CCTTTCCCCC TTTTTTGGTG ATCTAATTGC ATTGGTTAAA 960 AGCAGCTAAC CAGGTCTTTA GAATATGCTC TAGC CAAGTC TAACTTTATT TAGACGCTGT 1020 AGATGGACAA GCTTGATTGT TGGAACCAAA ATGGGAACAT TAAACAAACA TCACAGCCCT 1080 CACTAATAAC ATTGCTGTCA AGTGTAGATT CCCCCCTTCA AAAAAAGCTT GTGACCATTT 1140 TGTATGTCTG GTCTGGAAAC TTCTGTTTTATTAATATATATA

SEQ ID NO: 3 Sequence length: 2192 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: cDNA to mRNA Origin: Organism name: Homo = sapiens Cell type: Lymphoma Cell line: U937 Clone name: HP00184 Sequence characteristics: Characteristic symbol: CDS Location: 69. . 2126 Method of characterizing: E-sequence TAGTTCCGTC GCAGCCGGGA TTTGGGTCGC GGTTCTTGTT TGTGGATCGC TGTGATCGTC 60 ACTTGACA ATG CAG ATC TTC GTG AAG ACT CTG ACT GGT AAG ACC ATC ACC 110 Met Gln Ile Phe Val Lys 5 Thr Leu Thr Thr Iu Thr GAG GTT GAG CCC AGT GAC ACC ATC GAG AAT GTC AAG GCA AAG ATC 158 Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile 15 20 25 30 CAA GAT AAG GAA GGC ATC CCT CCT GAC CAG CAG AGG CTG ATC TTT GCT 206 Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala 35 40 45 GGA AAA CAG CTG GAA GAT GGG CGC ACC CTG TCT GAC TAC AAC ATC CAG 254 Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln 50 55 60 AAA GAG TCC ACC CTG CAC CTG GTG CTC CGT CTC AGA GGT GGG ATG CAA 302 Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln 65 70 75 ATC TTC GTG AAG ACA CTC ACT GGC AAG ACC ATC ACC CTT GAG GTG GAG 350 Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu 80 85 90 CCC AGT GAC ACC ATC GAG AAC GTC AAA G CA AAG ATC CAG GAC AAG GAA 398 Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu 95 100 105 110 GGC ATT CCT CCT GAC CAG CAG AGG TTG ATC TTT GCC GGA AAG CAG CTG 446 Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu 115 120 125 GAA GAT GGG CGC ACC CTG TCT GAC TAC AAC ATC CAG AAA GAG TCT ACC 494 Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr 130 135 140 CTG CAC CTG GTG CTC CGT CTC AGA GGT GGG ATG CAG ATC TTC GTG AAG 542 Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Val Lys 145 150 155 ACC CTG ACT GGT AAG ACC ATC ACC CTC GAG GTG GAG CCC AGT GAC ACC 590 Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr 160 165 170 ATC GAG AAT GTC AAG GCA AAG ATC CAA GAT AAG GAA GGC ATT CCT CCT 638 Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro 175 180 185 190 GAT CAG CAG AGG TTG ATC TTT GCC GGA AAA CAG CTG GAA GAT GGT CGT 686 Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg 195 200 205 ACC CTG TCT GAC TAC AA C ATC CAG AAA GAG TCC ACC TTG CAC CTG GTA 734 Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val 210 215 220 CTC CGT CTC AGA GGT GGG ATG CAA ATC TTC GTG AAG ACA CTC ACT GGC 782 Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly 225 230 235 AAG ACC ATC ACC CTT GAG GTC GAG CCC AGT GAC ACT ATC GAG AAC GTC 830 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 240 245 250 AAA GCA AAG ATC CAA GAC AAG GAA GGC ATT CCT CCT GAC CAG CAG AGG 878 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg 255 260 265 270 TTG ATC TTT GCC GGA AAG CAG CTG GAA GAT GGG CGC ACC CTG TCT GAC 926 Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 275 280 285 TAC TAC AAC ATC CAG AAA GAG TCT ACC CTG CAC CTG GTG CTC CGT CTC AGA 974 Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg 290 295 300 GGT GGG ATG CAG ATC TTC GTG AAG ACC CTG ACT GGT AAG ACC ATC ACC 1022 Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr 305 310 315 CTC GAAGTG GAG CCG AGT GAC ACC ATT GAG AAT GTC AAG GCA AAG ATC 1070 Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile 320 325 330 CAA GAC AAG GAA GGC ATC CCT CCT GAC CAG CAG AGG TTG ATC TTT GCC 1118 Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala 335 340 345 350 GGA AAA CAG CTG GAA GAT GGT CGT ACC CTG TCT GAC TAC AAC ATC CAG 1166 Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln 355 360 365 AAA GAG TCC ACC TTG CAC CTG GTG CTC CGT CTC AGA GGT GGG ATG CAG 1214 Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln 370 375 380 ATC TTC GTG AAG ACC CTG ACT GGT AAG ACC ATC ACT CTC GAG GTG GAG 1262 Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu 385 390 395 CCG AGT GAC ACC ATT GAG AAT GTC AAG GCA AAG ATC CAA GAC AAG GAA 1310 Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu 400 405 410 GGC ATC CCT CCT GAT CAG CAG AGG TTG ATC TTT GCT GGG AAA CAG CTG 1358 Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu 4 15 420 425 430 GAA GAT GGA CGC ACC CTG TCT GAC TAC AAC ATC CAG AAA GAG TCC ACC 1406 Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr 435 440 445 CTG CAC CTG GTG CTC CGT CTT AGA GGT GGG ATG CAG ATC TTC GTG AAG 1454 Leu His Leu Val Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Val Lys 450 455 460 ACC CTG ACT GGT AAG ACC ATC ACT CTC GAA GTG GAG CCG AGT GAC ACC 1502 Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr 465 470 475 ATT GAG AAT GTC AAG GCA AAG ATC CAA GAC AAG GAA GGC ATC CCT CCT 1550 Ile Glu Asn Val Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro 480 485 490 GAC CAG CAG AGG TTG ATC TTT GCT GGG AAA CAG CTG GAA GAT GGA CGC 1598 Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg 495 500 505 510 ACC CTG TCT GAC TAC AAC ATC CAG AAA GAG TCC ACC CTG CAC CTG GTG 1646 Thr Leu Ser Asp Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val 515 520 525 CTC CGT CTT AGA GGT GGG ATG CAG ATC TTC GTG AAG ACC CTG ACT GGT 1694 Leu Arg Leu Arg Gly Gly Met Gln Ile Phe Va l Lys Thr Leu Thr Gly 530 535 540 AAG ACC ATC ACT CTC GAA GTG GAG CCG AGT GAC ACC ATT GAG AAT GTC 1742 Lys Thr Ile Thr Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val 545 555 555 AAG GCA AAG ATC CAA GAC AAG GAA GGC ATC CCT CCT GAC CAG CAG AGG 1790 Lys Ala Lys Ile Gln Asp Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg 560 565 570 TTG ATC TTT GCT GGG AAA CAG CTG GAA GAT GGA CGC ACC CTG TCT GAC 1838 Leu Ile Phe Ala Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp 575 580 585 590 TAC AAC ATC CAG AAA GAG TCC ACC CTG CAC CTG GTG CTC CGT CTC AGA 1886 Tyr Asn Ile Gln Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg 595 600 605 GGT GGG ATG CAG ATC TTC GTG AAG ACC CTG ACT GGT AAG ACC ATC ACC 1934 Gly Gly Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr 610 615 620 CTC GAG GTG GAG CCC AGT GAC ACC ATC GAG AAT GTC AAG GCA AAG ATC 1982 Leu Glu Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys Ala Lys Ile 625 630 635 CAA GAT AAG GAA GGC ATC CCT CCT GAT CAG CAG AGG TTG ATC TTT GCT 2030 Gln Asp Lys Glu Gly Il e Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala 640 645 650 GGG AAA CAG CTG GAA GAT GGA CGC ACC CTG TCT GAC TAC AAC ATC CAG 2078 Gly Lys Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr Asn Ile Gln 655 660 665 670 AAA GAG TCC ACT CTG CAC TTG GTC CTG CGC TTG AGG GGG GGT GTC 2123 Lys Glu Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Val 675 680 685 TAAGTTTCCC CTTTTAAGGT TTCAACAAAT TTCATTGCAC TTTCCTTTCA ATAACATTTTC 2

[Brief description of drawings]

FIG. 1 is a diagram showing the structure of clone HP00686.

FIG. 2 Fusion protein expression vector pMALH for E. coli
It is a figure which shows the structure of P686.

Continuation of front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12Q 1/68 9453-4B C12Q 1/68 A // (C12N 15/09 ZNA C12R 1:91) (C12N 9 / 64 C12R 1:19) (C12P 21/02 C12R 1:19)

Claims (3)

[Claims] 1. A protein comprising the amino acid sequence represented by SEQ ID NO: 1. 2. c containing the nucleotide sequence represented by SEQ ID NO: 1
DNA. 3. The cDNA according to claim 2, which consists of the base sequence represented by SEQ ID NO: 2.