JPH099975A - Human ubiquitin-conjugating enzyme gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new enzyme gene coding for a human ubiquitin- conjugating enzyme having a specific amino acid sequence, and to be used for e.g. producing enzymes useful for diagnosing various diseases such as cancer and metastasis and screening the therapeutic agents and preventives therefor. SOLUTION: This new gene contains a base sequence coding for an amino acid sequence of the formula, being useful for e.g. manifesting and detecting the human ubiquitin-conjugating enzyme protein valuable for diagnosing various diseases involving human ubiquitin-conjugating enzyme, morbid state elucidation of these diseases, e.g. diagnosis cancer and metastasis, and for screening or evaluation of the therapeutic agents and preventives for cancer and metastasis. This gene is obtained by the following process: a reverse transcriptase is made to act on a human fetus brain poly(A)+RNA to synthesize cDNA, which is, in turn, amplified by PCR process using a pair of primers consisting of a fragment of this gene followed by conducting a cloning.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human ubiquitin-conjugating enzyme gene, and more particularly to a novel gene expressing a human ubiquitin-conjugating enzyme.

[0002]

2. Description of the Related Art The ubiquitin system is essential for proper cellular processes and is preserved from yeast to humans. The system uses ubiquitin-activating enzyme (ub
iquitin-activating enzyme (UBA to E1), ubiquitin conjugating enzyme (UBC to E2), ubiquitin protein ligase (UBR to E)
3) and 26S proteasome particles. Ubiquitin is activated and transferred from UBA to several UBCs. UBC transfers ubiquitin to a target protein with or without UBR involvement. These ubiquitinated target proteins cause many cellular responses such as proteolysis, protein modification, protein transport, DNA repair, cell cycle control, replication control, stress response and immune response.

[0003] In the above system, UBC is an important component and is considered to have different substrate specificities, and is divided into a dozen or more subgroups which mediate the above-mentioned different cell functions. The members of different subgroups are similar to each other, especially near the active site cysteine, but the N-terminal region is conserved within the group, but not between the groups and has substrate specificity. Are believed to be involved in [Finly et al., Annu. Rev. Cell
Biol., 7, 25-26 (1991); Jentsch et al., Biochemica
et Biophysica Acta, 1089, 127-139 (1991); Herrshk
o et al., Anni. Rev. Biochem., 61, 761-807 (1992); J
entsch, Annu.Rev.Genet., 26, 179-207 (1992); Hochst
rasser, Curr.Opin.Cell Biol., 4, 1024-1031 (199
2)].

To date, 12 genes have been cloned from Saccharomyces cerevisiae (S. cerevisiae, including the first cloned UBC gene; RAD6),
Each gene has been assigned to various cellular functions. Considering the more complex functions in humans, it is possible that more UBC genes exist, but only a few human UBC genes have been cloned [Schneider et al.,
EMBO J., 9, 1431-1435 (1990); Koken et al., Proc.N
atl.Acad.Sci., USA, 88, 8865-8869 (1991); Liu et a
l., J. Biol. Chem., 267, 15829-15835 (1992); Plon et
al., Proc.Natl.Acad.Sci., USA, 90, 10484-10488 (1
993); Kaiser et al., J. Biol. Chem., 269, 8792-8802.
(1994)].

Recently, several new UBCs have been reported to play important roles in specific cell control processes such as cyclin-dependent cell cycle control and p53 processing [Hershko et al., J. Biol. Chem., 269,
4940-4946 (1994); Blumenfeld et al., J. Biol. Che
m., 269, 9574-9581 (1994); Ciechanover et al., JB
iol. Chem., 269 9582-9589 (1994)]. Furthermore, when the UBC9 gene, which is said to regulate the cell cycle from G2 phase to M phase, is lethal in yeast, it becomes lethal [Wolfgang Seufert, et.
al., Nature, 373, 78-81 (1995)]. Therefore, if it is possible to distinguish between normal cells and cancer cells, cancer cell growth can be negatively suppressed by suppressing the action of this enzyme in cancer cells in which cell growth is active.

[0006]

If a new human UBC gene other than those described above can be provided, the expression level in each cell and its structure and function can be analyzed. It is considered that it becomes possible to elucidate the pathological condition, diagnosis, treatment, etc. of the disease, and the present invention provides such a new UB.
The purpose is to provide the C gene.

[0007]

According to the present invention, there is provided a novel UBC gene comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.

Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in this specification are referred to as IUPA.
The provisions of C and IUB, “Guidelines for preparing specifications including base sequences or amino acid sequences” (Edited by the Patent Office), and symbols conventionally used in the field shall be followed.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION As a specific example of the gene of the present invention, "GEN-104C" will be shown in Examples described later.
02 ”and“ GEN-104C0205 ”, respectively, and those deduced from the DNA sequences possessed by the clones can be mentioned.
As shown in 2. This is 1 shown in SEQ ID NO: 1
474 nucleotides (nucleic acid) encoding 58 amino acids
It has an open reading frame of and its molecular weight is calculated to be 18006 Daltons. Therefore, the gene of the present invention may be hereinafter referred to as “human UBC (18 kda)”.

The human UBC (18 kda) of the present invention will be described in detail below.

That is, as described above, it has been conventionally known that the UBC family is involved in the functions of many cells and that many human UBCs have homology with already cloned yeast UBCs. , Recently several new UBC species have been shown to have cyclin-dependent cell cycle regulation,
Alternatively, it has been reported to play an important role such as p53 degradation [Hershko, A., et al., J. Biol. Chem., 269, 494.
0-4946 (1994); Blumenfeld, N., et al., J. Biol. Che
m., 269, 9574-9581 (1994); Ciechanover, A., et al.,
J. Biol. Chem., 269, 9582-9589 (1994)]. UB of others
Cysteine, which is the active site of C, is the human UBC of the present invention.
At (18 kda), the sequence corresponding to Cys-93 and blackening this cysteine was well conserved. Further, like almost all UBCs, the amino acids at the 1st and 2nd positions of the human UBC (18kda) of the present invention were methionine and serine. The human U
The 8th amino acid of BC (18 kda) is arginine, and the basic amino acid residue found around this position in other proteins was presumed to be important for the interaction with UBA [Sullivan, Ml. , et al., J. Biol.Ch
em., 266, 23878-23885 (1991)]. In addition, some proline residues (69-P
XXPP-73) was well preserved [Ellison, K.
S., et al., J. Biol. Chem., 266, 24116-24120 (199
1)].

From the above results, the human UBC of the present invention (18
It was strongly suggested that kda) belongs to the UBC family. In addition, this human UBC (18 kda)
Was suspected to be involved in the distant relationship to the mammalian UBC2. Because, some limited homology to the mammalian UBC2 gene was revealed by the identification of the gene of the present invention. And there was no homology with other UBC genes. Furthermore, the human UBC (18 kda) of the present invention is UB
It shares some of the specific conserved residues with the C2 protein, is similar in size to the UBC2 protein, and shows some higher homology with the UBC2 protein than other subgroups. . The predicted open reading frame of human UBC (18 kda) of the present invention was 158 amino acids long and was composed of only one UBC region (Class I). The open reading frame shows that human UBC (18 kda) is a specific UB for substrate recognition.
May request R protein help. Its water affinity is similar to those of all UBC2s. Furthermore, except for the polyacidic column, it was also in agreement with the water affinity pattern of RAD6.

However, the human UBC of the present invention (18 k
da) is not UBC2 but all UBC2s that are considered to be classified into a novel subgroup of the UBC family.
The protein was tightly retained not only in the segment around the active site cysteine, but also in the N-terminal region. They appear to be associated with UBC2-specific functions such as DNA repair and mutagenesis. In comparison, human UBC
(18 kda) showed clearly low homology with UBC2 in the N-terminal region. It may exhibit different substrate specificities and differentiated functions. However, human UBC
It remains unclear whether (18 kda) is involved in DNA repair, but this gene must play an important role in basic cell function.

According to previous studies, S. cerevisiae, Sc.
pombe and D. melanogaster carry a single RAD6 homolog, whereas mouse and human species carry a highly conserved RAD6 homolog even at the two nucleic acid levels. And they can functionally supplement the inactive RAD6 gene. The duplication of this gene is considered to have occurred in the Jura stage (Jurassi era), and their duplicated UBC2 gene is RAD6.
(Koken, MH, et al., Proc.Natl.Acad.Sci., USA., 8
8, 8865-8869 (1991)], it is highly preserved. The human UBC (18 kda) gene is a mammalian UB
Given some homology with the C2 gene, it may be derived from the same ancestral gene as the mammalian UBC2 gene. And it may have changed to playing different roles. Human UBC (18kda)
It is unclear whether the homologous gene of was found in other species.

Also, as shown below, Northern blot analysis revealed that a major 1.35 kb of this gene and two other copies of different size (copy) were found in all normal human tissues studied. showed that.

The gene of the present invention is represented by a single-stranded DNA sequence, for example, as shown in SEQ ID NO: 2, and the present invention includes a DNA sequence complementary to the single-stranded DNA sequence or both of them. Including components that also include. The sequence of the gene of the present invention shown in SEQ ID NO: 2 is an example of one combination of codons representing each amino acid residue encoded thereby, and the gene of the present invention is not limited to this and It is, of course, possible to have a DNA base sequence that is selected by combining arbitrary codons. Selection of the codon can be carried out according to a conventional method, for example, the frequency of codon usage of the host to be used can be considered [Nucl. Acids Res.,
9, 43-74 (1981)].

Further, in the gene of the present invention, a part of the amino acid sequence shown above is replaced with an amino acid or amino acid sequence,
A DNA sequence which is modified by deletion, addition, etc. and has the same function and encodes the same compound is also included. Production, modification (mutation) and the like of these polypeptides may occur naturally, and the natural gene (the gene of the present invention) may be modified, for example, by post-translational modification or by a genetic engineering technique, for example, cytospecific mutagenesis [ Methods in
Enzymology, 154, p350, 367-382 (1987); ibid. 100, p46.
8 (1983) ； Nucleic Acids Research, 12, p9441 (198
4); sequel biochemistry experiment course 1 “Gene Research Method II”, edited by the Japanese Biochemical Society, p105 (1986)]
Chemical synthesis means such as the phosphoric acid triester method and the phosphoric acid amidite method [J. Am. Chem. Soc., 89, p4801 (1967);
p3350 (1969); Science, 150, p178 (1968); Tetrahed
ron Lett., 22, p1859 (1981); ibid. 24, p245 (1983)], and can be obtained by synthesizing a DNA mutated or a combination thereof.

The gene of the present invention can be easily and stably used for the above human ubiquitin conjugating enzyme (UBC) by utilizing this, that is, by incorporating the gene into a microbial vector and culturing a transformed microorganism. Can be expressed.

The human ubiquitin-conjugating enzyme obtained by using the gene of the present invention can also be used to prepare a specific antibody. The component used here as an antigen may be a protein produced in large quantities according to the above-mentioned genetic engineering method, the obtained antibody may be either a polyclonal antibody or a monoclonal antibody, and the antibody is a human ubiquitin conjugating enzyme. Purification, measurement,
It can be advantageously used for identification and the like.

The gene of the present invention can be easily produced by a general genetic engineering technique based on the sequence information of the gene of the present invention disclosed by the present invention [Molecu
larCloning 2nd Ed, Cold Spring Harbor Laboratory P
ress (1989); Second Biochemistry Laboratory "Gene Research Methods I, I"
I, III ", edited by the Japanese Biochemical Society (1986), etc.].

This is desired by using a suitable probe or antibody specific to the gene of the present invention from, for example, a human cDNA library (prepared according to a conventional method from an appropriate source cell in which the human ubiquitin conjugating enzyme gene is expressed). It can be carried out by selecting a clone [Proc. Natl. Aca
d. Sci. USA, 78, 6613 (1981); Science, 222, 778
(1983) etc.].

In the above-mentioned method, examples of the source cells include various cells expressing the human ubiquitin-conjugating enzyme gene, cultured cells derived from tissues or tissues, and the like. Separation of total RNA, separation and purification of mRNA from the cells. , CDNA conversion (synthesis), cloning thereof, and the like can be carried out by conventional methods. Further, cDNA libraries are commercially available, and in the present invention, those cDNA libraries, for example, various cDNA libraries commercially available from Clontech Lab. Inc. can be used.

Screening of the gene of the present invention from a cDNA library can be carried out according to the above-mentioned usual method. Examples of the screening method include c
By immunological screening using a human ubiquitin-conjugating enzyme-specific antibody against the protein produced by DNA,
Method for selecting corresponding cDNA clone, desired DN
Examples thereof include plaque hybridization, colony hybridization and the like using a probe that selectively binds to the A sequence. As the probe used here, a DNA sequence or the like chemically synthesized based on information on the DNA sequence of the gene of the present invention is generally used. Of course, the gene of the present invention or a fragment thereof which has already been obtained is also used. Can be used as a probe.

When obtaining the gene of the present invention, P
DN by CR method [Science, 230, 1350-1354 (1985)]
The A / RNA amplification method can be preferably used. Especially when it is difficult to obtain full-length cDNA from a library, the race method [RACE: Rapid amplification of cDNA ends;
Experimental Medicine, 12 (6), 35-38 (1994)], especially 5'-race (RACE) method [Frohman, MA, et al., Proc. Natl. Ac.
Ad.Sci., USA., 8, 8998-9002 (1988)] is preferable. Primers to be used when employing such a PCR method can be appropriately set based on the sequence information of the gene of the present invention which has already been revealed by the present invention, and can be synthesized according to a conventional method.

The amplified DNA / RNA fragment can be isolated and purified according to the conventional method described above, for example, gel electrophoresis.

The gene of the present invention or various DNs obtained above
The determination of the base sequence of the A fragment and the like can be performed according to a conventional method,
For example, the dideoxy method [Proc. Natl. Acad. Sci. USA, 7
4, 5463-5467 (1977)] and the Maxam-Gilbert method (Met
hod in Enzymology, 65, 499 (1980)] and the like. Such determination of the nucleotide sequence can be easily performed even using a commercially available sequence kit or the like.

According to the use of the gene of the present invention, a usual gene recombination technique [eg, Science, 224, p1431 (1984);
Biochem. Biophys. Res. Comm., 130, p692 (1985);
Proc. Natl. Acad. Sci. USA, 80, p5990 (1983) and the references cited above], a recombinant human ubiquitin-conjugating enzyme can be obtained. More specifically, the production of the human ubiquitin-conjugating enzyme is carried out by preparing a recombinant DNA capable of expressing the gene of the present invention in a host cell, introducing the recombinant DNA into the host cell, transforming, and culturing the transformant. It is done by

As the host cell, either a eukaryote or a prokaryote can be used. The eukaryotic cells include cells such as vertebrates and yeast, and the vertebrate cells include, for example, COS cells [Cel cells]
l, 23, 175-182 (1981)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains [Pr
oc. Natl. Acad. Sci. USA, 77, 4216-4220 (1980)] and the like are often used, but are not limited to these.

As a vertebrate expression vector, a promoter usually located upstream of the gene to be expressed,
Those having an RNA splice site, a polyadenylation site, a transcription termination sequence and the like can be used, which may further have an origin of replication if necessary. As an example of the expression vector, for example, pSV2dhfr [Mol. Cell. Biol., 1, 854 (198) carrying an SV40 early promoter is used.
1)] etc. can be illustrated. As eukaryotic microorganisms, yeasts are generally used, and among them, yeasts belonging to the genus Saccharomyces can be advantageously used. Examples of the expression vector for eukaryotic microorganisms such as yeast include pAM82 [Proc. Natl. Acad.
Sci. USA, 80, 1-5 (1983)] can be used.

As a vector for expressing the gene of the present invention, a prokaryotic gene fusion vector can be preferably used. Specific examples of the vector include, for example, a GST domain having a molecular weight of 26000, as shown in Examples described later. Examples thereof include pGEX-2TK and pGEX-4T-2 (derived from S. japonicum).

Escherichia coli and Bacillus subtilis are commonly used as prokaryotic hosts. In the present invention, a plasmid vector that can be replicated in the host bacterium is used, and a promoter and SD (Shine and And) are provided upstream of the gene so that the gene of the present invention can be expressed in the vector. It is preferable to use an expression plasmid having a Dalgarno) nucleotide sequence and a start codon (eg, ATG) necessary for initiation of protein synthesis. As Escherichia coli (Escherichia coli) as the above-mentioned host,
li) K12 strain and the like are often used, and pBR322 and its improved vector are often used as the vector, but not limited to these and various known strains and vectors can also be used. As the promoter, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, PL / PR promoter and the like can be used.

Various general methods can be adopted as a method for introducing the desired recombinant DNA thus obtained into a host cell and a method for transforming it. The resulting transformant can be cultured by a conventional method, and the desired human ubiquitin-conjugating enzyme encoded by the gene of the present invention is produced and expressed by the culture. As the medium used for the culturing, various media commonly used can be appropriately selected and used according to the adopted host cells, and the culturing can also be carried out under conditions suitable for the growth of the host cells.

As described above, the target recombinant human ubiquitin-conjugating enzyme is expressed, produced, accumulated or secreted in the transformant cells, extracellularly or on the cell membrane.

Recombinant human ubiquitin conjugating enzyme can be subjected to various separation operations utilizing its physical properties, chemical properties, etc. [“Biochemical Data Book II”, 1175-1259, if desired.
Page, 1st edition, 1st print, June 23, 1980, Tokyo Kagaku Dojin; Biochemistry, 25 (25), 8274-8277 (1986); Eu
r. J. Biochem., 163, 313-321 (1987), etc.]. Specific examples of the method include ordinary reconstitution treatment, treatment with a protein precipitating agent (salting out method),
Centrifugation, osmotic shock method, ultrasonic disruption, ultrafiltration,
Examples include various liquid chromatography such as molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HPLC), dialysis method, combinations of the above, and the like. An example of the method is affinity chromatography using a column to which ubiquitin is bound.

Further, based on the sequence information of the gene of the present invention clarified by the present invention, expression of the gene of the present invention in various human tissues can be obtained by utilizing, for example, part or all of the nucleotide sequence of the gene. Can be detected. This can be performed according to a conventional method, for example, RT-PCR (Reverse transcribed-Polymerase chain).
reaction) (Kawasaki, ES, et al., Amplificatio
n of RNA.In PCR Protocol, A Guide to methods and
applications, Academic Press, Inc., SanDiego, 21-2
7 (1991)] and Northern blotting analysis [Molecular Cloning, Cold Spring Har.
bor Laboratory, (1989)], etc.

When the PCR method is adopted, the primer used is not limited as long as it is unique to the gene of the present invention capable of specifically amplifying only the gene of the present invention, and is based on the genetic information of the present invention. The array can be set appropriately. Usually this is 20-
It may have a partial sequence of about 30 nucleotides. A preferred example thereof is as shown in Examples below.

Therefore, the present invention relates to such human UBC (1
It also provides primers and / or probes useful for detection unique to the 8 kda) gene.

[0038]

INDUSTRIAL APPLICABILITY According to the present invention, a novel human ubiquitin-conjugating enzyme gene is provided, and by using the gene, the expression of the gene in various tissues can be detected and the human ubiquitin-conjugating enzyme can be genetically produced. It becomes possible, and with these,
As described above, it is possible to analyze the functions of the human ubiquitin system or ubiquitin-conjugating enzyme, which is diverse and deeply involved in basic activities in cells, and to diagnose various diseases in which it is involved, for example, cancer, Screening and evaluation of therapeutic and preventive agents for cancer and cancer metastasis can also be performed.

[0039]

EXAMPLES Examples will be given below to explain the present invention in more detail.

[0040]

Example 1 (1) Cloning and DNA Sequencing Sequence analysis of a cDNA clone arbitrarily selected from a human fetal brain cDNA library (Otsuka GEN Research Institute, Otsuka Pharmaceutical Co., Ltd.) revealed homology with the mammalian UBC2 gene. We found a highly active clone of 890 bp (excluding poly A) and used it as GEN-104C0.
Named 2. As a result of sequence analysis, it was confirmed that the clone lacked the 5'portion of the gene of the present invention.

The above sequence analysis was carried out by the dideoxynucleotide chain termination method from a double-stranded template (automatic cycle and automatic reading sequence kit, Pharmacia).
According to A. L. F. It analyzed using the DNA sequencer (Pharmacia). Also, the homology is UWGC.
G's FASTA program [Pearson, et al., Proc.Na
tl.Acad.Sci., USA., 85, 2444-2448 (1988), based on database (GenBank, EMBL] search.

(2) 5'-Race A cDNA clone containing the 5'portion of the gene of the present invention was isolated by the 5'race method using a commercially available kit (5'-AmpliFINDERTMRACE Kit, Clontech) as follows. .

Gene-specific primer P used here
1. The same nested primer P2 containing the XhoI endonuclease cleavage site and the anchor primer were synthesized by a conventional method, and their sequences are shown in Table 1 below.

[0044]

[Table 1]

That is, 0.1 μg of human fetal brain poly (A) + RNA was treated with reverse transcriptase (Superscript ™ II RNase HR).
Reverse Transcriptase, Life Technologies) was used for reverse transcription using a random hexamer to obtain cDNA, which was amplified by the first PCR using P1 primer and anchor primer.

Then, 1 μl of 50 μl of the first PCR reaction was amplified by the second PCR using the nested P2 primer and the anchor primer. The second PCR product was then analyzed by 1.5% agarose gel electrophoresis. A single strand having a length of about 500 nucleobases was detected, and this product was used as a vector (pT7Blue (R) TV).
vectorBlue (R) T-vector, Novagen)
, And a plurality of clones showing an insertion of an appropriate size were selected.

Five of the 5'race clones obtained from two independent PCR reactions were sequenced and had the same sequence but different length (500-600 bases).
Was confirmed.

The clone named GEN-104C0205, which is the longest one, was selected from the above five clones, and the above-mentioned GEN-104C02 and GEN-104C were selected.
Both strands of 0205 have primer extension and deletion (primer
The complete coding sequence and the 5'side and 3'side sequences of the gene of the present invention were determined by the complete sequencing by the extention and deletion methods).

The above results are shown in FIG.

The figure shows the partial clone GEN-104C0.
2 and GEN-104C0205 are listed as the full length of the human UBC (18 kda) gene of the present invention.
EN-GEN-104C02 is 260 of the gene of the present invention.
~ 1148 bases covered, GEN-104C0
205 covers the same 1 to 521 bases. Also,
In the figure, coding regions are indicated by open boxes, non-coding regions are indicated by lines, and sequencing strategies are indicated by arrows.

(3) Gene UBC of the present invention (18 kda)
First, the coding region of human UBC (18 kda)
Based on the sequence information obtained above, 2
Two primers (C1 primer and C2 primer)
It was created. The sequences of homonuclear primers are shown in Table 1 above.

The C1 primer used here was BamH.
The C2 primer was the antisense primer containing the I site and the EcoRI site.

Using the above primers, human fetal brain m
PCR was performed using cDNA derived from RNA to amplify the cDNA, and a product with a size of about 500 bases was obtained. The amplified cDNA is ethanol-precipitated, the cDNA is cleaved with restriction enzymes BamHI and EcoRI, the cleaved product is extracted with phenol, and ethanol-precipitated again to produce a prokaryotic gene fusion vector p.
It was inserted into the BamHI-EcoRI site of the GEX-2TK vector (Pharmacia) and subcloned. The fusion vector comprises BamHI and Ec
Has an oRI cleavage site, and has the desired gene product and molecular weight of 26
Is an expression vector expressing a fusion protein with 000 GST domain (derived from S. japonicum).

An expression inducer IPTG (Isopropyl-β-D-thio-Galactopyranoside) was added to the pGEX-2TK vector.
Takara Shuzo (TAKARA), positive clones were selected, and one of them was named 104C02-2TK-15,
The clone was sequenced to confirm that the inserted position was correct and the sequence was identical to the sequence obtained in the above cloning. This clone is human UBC
It was used for purification of the (18 kda) recombinant protein.

(4) Gene UBC of the present invention (18 kda)
Expression of E. coli containing pGEX-2TK-15 was cultured overnight, diluted to a concentration of 1/10 and cultured in Amp (+) LB medium for 2 hours. At this point, add IPTG to a final concentration of 1
μM and added.

The expression-inducing medium was further cultured for 5 hours and then centrifuged for washing. The centrifuged product is PBS
After washing with (-), the washed pellet was resuspended in buffer and sonicated on ice for 15 minutes. The cell lysate was pelleted by centrifugation, the supernatant was removed and applied to a glutathione-Sepharose column for affinity purification, and the column was washed with 20-column-volume PBS (-). The above washing column was treated with thrombin, and gel filtration was applied to a Sephacreel-200 column (manufactured by Pharmacia) to collect the product obtained by separating the target protein from the fusion protein, and collecting the fractions.

The fraction containing the recombinant protein of the human UBC (18 kda) gene was concentrated and its homogeneity was confirmed. At this stage, the molecular weight of this product is 22 kd
a (by SDS-PAGE). The molecular weight calculated was 19 kda after the cleavage of thrombin and the addition of 5 amino acids at the N-terminal.

Thus, from the final 2 liters of culture solution, 1.
7 mg of recombinant protein was obtained.

(5) Activity measurement of human UBC (18 kda) as human ubiquitin-conjugating enzyme Human UBC (18 kda as human ubiquitin-conjugating enzyme
The activity measurement of da) was determined by whether human UBC (18 kda) can obtain a ubiquitin moiety by thiol bond. The recombinant human UBC (18 kda) contained a target portion of fetal bovine heart protein kinase in its N-terminus.

We have labeled and purified it with gamma-32p-ATP.

The ubiquitin-conjugating enzyme assay is carried out by the method of Trier [Treier et al., Cell, vol.78, pp787-798.
(1994)] and Haas' method [Haas et al., J. Biol. Che.
m., vol.263, 13258-13267 (1988)].

That is, 50 μl of the reaction mixture was incubated at 37 ° C. for 2 hours.
Incubated for minutes. The reaction mixture is 70%
Rabbit reticulocyte lysate (Dromega, Catalog No. L4151, 1 x UBC buffer [50 mM
Tris HCl (pH 7.5), 5 mM ATP, 5 m
MgCl2 and 0.2 mM DTT]), 1 unit of yeast inorganic pyrophosphate, 20 μM ubiquitin, 400 nM of 32P-104C02. For control, any one component of the 70% rabbit reticulocyte lysate, ubiquitin and 32P-104C02 was removed from the reaction mixture.

Further, the above reaction mixture was mixed with 2 × SDS-PAGE loading buffer containing an equal amount of β-mercaptoethanol and boiled for 5 minutes. As a target, 2 x SDS without β-mercaptoethanol in the reaction mixture
-Mixed with PAGE sample buffer and incubated for 30 minutes on ice.

The obtained results are shown in FIG.

In the figure, the leftmost end is lane 1, and lanes 2, 3, 4, ...
Lane 2 showing all components / 2ME (+)
Indicates all components / 2ME (−), lane 3 indicates 32P-104C02 (−) / 2ME (−), and lane 4 indicates ubiquitin (−) / 2ME (−). In addition, in the figure, the upper band shows 32P-104C02 (hUBC (18
kda) -ubiquitin, 31.0 kda), and the lower band is 32P-104C02 itself (hUBC (18
kda) and 21.5 kda) are shown.

From the figure, the following can be seen. That is, lane 2
Shows that 32P-104C02 was linked by a ubiquitin moiety, and lane 1 is probably a thiol ester bond, as separated by boiling at 2ME. Lane 5 also shows that the component (probably UBA) was required for ubiquitin binding. Considering the results from lane 4, it can be seen that the rabbit reticulocyte lysate also contained sufficient ubiquitin.

The thus obtained gene UBC (1 of the present invention
The nucleic acid sequence of 8 kda) is as shown in SEQ ID NO: 2, and the amino acid sequence of the human ubiquitin conjugating enzyme encoded thereby is as shown in SEQ ID NO: 1.

It should be noted that, in SEQ ID NO: 2, 118 to 12
The 0th ATG encodes the starting methionine,
The stop codon (TAA) is located at positions 592-594. In addition, the polyadenylation signal is 1133 to 113.
The poly A sequence located at position 8 and immediately after the 3'end is not shown. Furthermore, 2 used for 5'race
The two primers are 502-524 and 53, respectively.
It is located in the 7th to 560th positions.

The gene UBC (18 kda) of the present invention has a total length of 1148 bases and the 5'non-coding region is relatively GC.
It is rich and the combination of CGC is 5 between 38 and 52 bases.
Repeated times, the optimal polyadenylation signal is
It was followed by 16 bases of poly (A) start. The open reading frame of the gene consisted of 474 bases encoding 158 amino acids, and the calculated molecular weight was 18,006 daltons. Of particular interest, the estimated pI was 8.86, higher than those of other UBCs known to date.

The human UBC (18 kda) gene showed some homology at the nucleic acid level with the mammalian UBC2 gene. The human UBC (18 kda) gene is the yeast UBC
There was no homology with the 2 gene and the RAD6 gene. The human UBC (18 kda) gene coding sequence includes human E2 (Mw = gene (human HHR6B) and human HH
It showed 56% and 59% identity with the R6A gene, respectively. Both of them are characterized by the yeast RAD6 [Schneider, e
t al., EMBO J., 9, 1431-1435 (1990); Koken, et al.,
Proc.Natl.Acad.Sci., USA., 88, 8865-8869 (199
1)) Regarding the 5'non-coding region and the 3'non-coding region, there was no homology with those of the two human UBC genes described above.

Amino acid level human UBC (18 kd
a) For proteins, UBC2 from yeast to mammals
It showed some high homology with the gene. Therefore, all UBC
The proteins were particularly similar in the region containing the active site cystein. Human UBC (18 kda) protein also showed some homology with other UBC proteins (multiple aligment). For more precise study of conserved regions, multiple alignments of human U
Performed on BC (18 kda), human E2 (Mw = 17000) and some yeast UBC proteins. As a result, it was suggested that some of the strictly conserved residues among all UBC proteins are involved in the interaction with ubiquitin or UBA. The somewhat higher homology between the UBC2 protein and human UBC (18 kda) is due to these two UBC2 proteins and human UBC.
Shown as a result of several specifically conserved residues at (18 kda). It remains unclear whether these residues are important for UBC2-specific function. The deduced protein had four cysteine residues containing the expected active site cysteine (Cys-93). Cys-93 appeared to be the active site cysteine involved in accepting activated ubiquitin molecules from UBA. The tightly conserved residues around Cys-93 may be involved in the interaction of ubiquitin with UBA as described above. Human UBC
The hydropathy of (18 kda) for water is
Cys-93 followed by a highly acidic peptide (98-
Except for EEDKD-102), it was very similar to those of other UBC2 proteins.

Human UB in some adult human tissues
Expression of C (18 kda) mRNA was detected by G
Analyzed by Northern blotting using EN-104C02 (including 3'non-coding region). 3.3 kb and 1.35 k in all eight tissues tested
Two different sized transcripts from b were exposed for a short time (4 h
r). The 1.35 kb transcript was the most abundant type and was in size consistent with the cDNA clone obtained in the present invention. After long-term exposure (24h
r), a weak 6.4 kb band also appeared.

(6) Northern Blot Analysis Human UBC (18kda) mRN in normal human tissues
The expression of A was evaluated by Northern blot probed with GEN-104C02 (including non-coding region).

Northern Blot analysis is informative, M
Human Multiple Tissue Nothern blot;
Clontech).

That is, the clone GEN-104C02 was
T3 promoter in the vector (pBluescript) and-
First PCR with 40 promoter sequence primers
Followed by a second PCR with primers for the T3 and T7 promoter sequences.

The second PCR product was purified and [ ³² P] -d
CTP (random primed DNA labeling kit,
A probe was prepared by labeling with Boehringer Mannheim.

The blot was prehybridized for 3 hours and then
18 hours at 42 ° C., 50% formamide / 5 × SSC /
10 x Denhardt's solution / 2% SDS solution (100 μg /
Bridizing was performed in a solution containing 1 ml of denatured salmon sperm DNA. 2 x SSC / 0.05% SDS at room temperature for 1 hour, then 0.1 x SSC / 0.1% SDS at 50 ° C.
Washed down for 1 hour. Filters were exposed at -80 ° C for 4 or 24 hours.

The results are shown in FIG. Each lane in the figure is approximately 2
It contains μg of poly (A) + RNA and is derived from the following tissues respectively. Lane 1: Heart Lane 2: Brain Lane 3: Placenta Lane 4: Lung Lane 5: Liver Lane 6: Skeletal muscle Lane 7: Kidney Lane 8: Pancreas

The lower panel is the result of a control using a beta actin (β-Actin) probe for adjusting the amount of RNA under load.

3.3 kb in 8 tissues tested
And two different size transcripts of 1.35 kb were detected. It was universally expressed in all tissues. The 1.35 kb transcript is the major species and was consistent in size with the cDNA clone obtained in the present invention. In addition, in long exposure (24 hours), a faint band of about 6.4 kb was confirmed.

(7) Chromosome mapping Chromosome mapping was performed on human UBC (18 kda).
Using two cosmid clones corresponding to chromosomal DNA, direct R-banding fluorescein in situ hybridization [FI] was performed on 100 representative R band (pro) metaphase plates.
SH, Takahashi et al., Hum. Genet., 86, 14-16 (199
0); Takahashi et al., Him.Genet., 88, 119-121 (199
1)].

As a result, 39% showed complete twin spots in both homologues, 51% showed incomplete signal and / or twin spots in one or both homologues,
The other (10%) showed no spots. The signal was localized to the p13.3 band of chromosome 16 and this gene was therefore assigned to the 16p13.3 band.

[0083]

[Sequence list]

SEQ ID NO: 1 Sequence length: 158 amino acids + stop codon Sequence type: amino acid Topology: linear Sequence type: protein Sequence: Met Ser Gly Ile Ala Leu Ser Arg Leu Ala Gln Glu Arg Lys Ala Trp 1 5 10 15 Arg Lys Asp His Pro Phe Gly Phe Val Ala Val Pro Thr Lys Asn Pro 20 25 30 Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cys Ala Ile Pro Gly Lys 35 40 45 Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Lys Leu Arg Met Leu Phe 50 55 60 Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro 65 70 75 80 Leu Phe His Pro Asn Val Tyr Pro Ser Gly Thr Val Cys Leu Ser Ile 85 90 95 Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Ile Thr Ile Lys Gln Ile 100 105 110 Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pro Asn Ile Gln Asp Pro 115 120 125 Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gln Asn Arg Val Glu Tyr 130 135 140 Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Phe Ala Pro Ser * 145 150 155

SEQ ID NO: 2 Sequence length: 1148 Sequence type: Nucleic acid Number of chains: single Topology: linear Sequence type: DNA (cDNA) Sequence features: Characteristic symbols: CDS Location: 118. .594 Method of characterizing: E sequence: AGCGGGCTCC GGAGGGAAGT CCCGAGACAA AGGGAAACGC CGCCGCCGCC GCCCCGCTCG 60 GTCCTCCACC TGTCCGCTAC GCTCGCCAGG GCTGCGGCCG CCCGAGGGAC TTTGAAC 117 ATG TCG GCA GLA GAG AAG CAG ACA GAGAGAG CAG AGA CAG ACA CAG ACA CGA Leu Ala Gln Glu Arg Lys Ala Trp 1 5 10 15 AGG AAA GAC CAC CCA TTT GGT TTC GTG GCT GTC CCA ACA AAA AAT CCC 213 Arg Lys Asp His Pro Phe Gly Phe Val Ala Val Pro Thr Lys Asn Pro 20 25 30 GAT GGC ACG ATG AAC CTC ATG AAC TGG GAG TGC GCC ATT CCA GGA AAG 261 Asp Gly Thr Met Asn Leu Met Asn Trp Glu Cys Ala Ile Pro Gly Lys 35 40 45 AAA GGG ACT CCG TGG GAA GGA GGC TTG TTT AAA CTA CGG ATG CTT TTC 309 Lys Gly Thr Pro Trp Glu Gly Gly Leu Phe Lys Leu Arg Met Leu Phe 50 55 60 AAA GAT GAT TAT CCA TCT TCG CCA CCA AAA TGT AAA TTC GAA CCA CCA 357 Lys Asp Asp Tyr Pro Ser Ser Pro Pro Lys Cys Lys Phe Glu Pro Pro 65 70 75 80 TTA TTT CAC CCG AAT GTG TAC CCT TCG GGG ACA GTG TGC CTG TCC ATC 405 Leu Phe His Pro Asn Val Tyr Pro Ser Gly Thr Val Cys Leu Ser Ile 85 90 95 TTA GAG GAG GAC AAG GAC TGG AGG CCA GCC ATC ACA ATC AAA CAG ATC 453 Leu Glu Glu Asp Lys Asp Trp Arg Pro Ala Ile Thr Ile Lys Gln Ile 100 105 110 CTA TTA GGA ATA CAG GAA CTT CTA AAT GAA CCA AAT ATC CAA GAC CCA 501 Leu Leu Gly Ile Gln Glu Leu Leu Asn Glu Pro Asn Ile Gln Asp Pro 115 120 125 GCT CAA GCA GAG GCC TAC ACG ATT TAC TGC CAA AAC AGA GTG GAG TAC 549 Ala Gln Ala Glu Ala Tyr Thr Ile Tyr Cys Gln Asn Arg Val Glu Tyr 130 135 140 GAG AAA AGG GTC CGA GCA CAA GCC AAG AAG TTT GCG CCC TCA TAA 594 Glu Lys Arg Val Arg Ala Gln Ala Lys Lys Phe Ala Pro Ser * 145 150 155 GCAGCGACCT TGTGGCATCG TCAAAAGGAA GGGATTGGTT TGGCAAGAAC TTGTTTACAA 654 CATTTTTGCA AATCTAAAGT TGCTCCATAC AATGACTAGT CACCTGGGGG GGTTGGGCGG 714 GCGCCATCTT CCATTGCCGCCGCG TGTG CGGTCTCGAT TCGCTGAATT GCCCGTTTCC 774 ATACAGGGTC TCTTCCTTCG GTCTTTTGTA TTTTTGATTG TTATGTAAAA CTCGCTTTTA 834 TTTTAATATT GATGTCAGTA TTTCAACTGC TGTAAAATTA TAAACTTTTA TACTTGGGTA 894 AGTCCCCCAG GGGCGAGTTC CTCGCTCTGG GATGCAGGCA TGCTTCTCAC CGTGCAGAGC 954 TGCACTTGGC CTCAGCTGGC TGTATGGAAA TGCACCCTCC CTCCTGCCGC TCCTCTCTAG 1014 AACCTTCTAG AACCTGGGCT GTGCTGCTTT TGAGCCTCAG ACCCCAGGTC AGCATCTCGG 1074 TTCTGCGCCA CTTCCTTTGT GTTTATATGG CGTTTTGTCT GTGTTGCTGT TTAGAGTAAA 1134 TAAACTGTTT ATAT 1148

[Brief description of drawings]

FIG. 1 Partial clones GEN-104C02 and GEN
-104C0205 was replaced with human UBC (18
kda) and the total length is shown.

FIG. 2 is a drawing showing the results of Northern blot analysis of the human UBC (18 kda) gene of the present invention.

FIG. 3 Human UBC (18 kd according to Example 1- (5)
It is drawing which shows the activity measurement result of a).

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location (C12N 9/88 C12R 1:19)

Claims (1)

[Claims] 1. A human ubiquitin conjugating enzyme gene comprising a base sequence encoding the amino acid sequence represented by SEQ ID NO: 1.