JP3040280B2 - Human 8-oxodeoxyguanosine triphosphate degrading enzyme, and cDNA and cDNA clones encoding this enzyme - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a human 8-oxodeoxyguanosine triphosphate degrading enzyme (hereinafter sometimes referred to as 8-oxodGTPase) which suppresses mutation and carcinogenesis in human cells, and an 8-oxodGTPase. It is related to cDNA encoding More specifically, the present invention relates to a clone of the above cDNA,
The present invention relates to a transformant having an NA clone and 8-oxodGTPase produced from this transformant.

[0002] The 8-oxod GTPase and cDNA of the present invention are useful for, for example, diagnosis and treatment of cancer, or development of new anticancer agents.

[0003]

2. Description of the Related Art Recent advances in genetic engineering technologies and the results of molecular biological research using them have been remarkable, and active research has been conducted at the genetic level on human carcinogenic mechanisms. A new dimension is being developed in the diagnosis and treatment of cancer. Gene-level knowledge of carcinogenic mechanisms has traditionally included cell chromosome D
Activation of an oncogene or inactivation of a tumor suppressor gene by a point mutation of NA is known. Such point mutations in DNA are caused by various factors, one of which is a transversion mutation due to oxidation of guanine, a DNA component.

That is, a DNA molecule is composed of four types of bases, adenine (A), thymine (T), guanine (G) and cytosine (C), as is well known. G and C are paired to form a double-stranded DN
When guanine (G) is oxidized by, for example, oxygen in the air or metabolism in a living body, this oxidized guanine (8-oxoguanine) becomes adenine (A) in addition to cytosine (C). A). As a result, the original GC pairing is mutated to a completely different TA pairing through DNA division and replication.

[0005] Moreover, such oxidation of guanine is caused by D
It has also been shown to occur at a much higher frequency at the nucleotide level than at NA. That is, deoxyguanosine triphosphate (dGTP), which is a guanine precursor for DNA synthesis, is relatively easily oxidized in cells, and
Oxodeoxyguanosine triphosphate (8-oxodGT
P). And this 8-oxodGTP is dG
When used in DNA synthesis instead of TP, the same base mismatch occurs as described above, resulting in a point mutation in the gene DNA. In the case of mammals, such mutations in gene DNA may lead to canceration of cells.

On the other hand, 8-oxo GT is present in the cells of an organism.
There is also an enzyme that degrades P, and this enzyme is 8-oxodG
It is thought that the degradation of gene DNA is suppressed by decomposing TP to 8-oxodGMP. For example, in the case of E. coli, such MutT proteins have been known to have enzymatic activity, also the inventors of the present invention, E. coli strains deficient for the gene (mut T) encoding MutT proteins naturally suddenly Have a very high probability of mutation (Maki & Sekiguc
hi: Nature, 355 , 273-275, 1992 ) .

Further, the inventors of the present invention have proposed a human tissue.
Also have the same enzymatic activity as MutT protein of Escherichia coli.
Look for the presence of the enzyme (8-oxodGTPase).
This enzyme has been successfully separated and purified (Mo, Ma
ki & Sekiguchi: Proc. Natl. Acad. Sci. U.S.A.,8
911021-11025, 1992). like this
In addition, an enzyme that degrades 8-oxodGTP in human tissues
Element, and G.G.
Mutation by C → TA substitution is effectively prevented
This enzyme can be used industrially
New forms of cancer therapy and anti-cancer
It is considered to be extremely useful for the development of agents.

The present invention has been made in view of the circumstances described above. The inventors of the present invention have succeeded in industrializing human 8-oxodGTPase which has been successfully separated and purified by clarifying its molecular structure. It is intended to be used for the use of. Further, the present invention provides the human 8-o
cDNA encoding xodGTPase and this cDNA
It is also an object to provide a genetic engineering material that allows for easy manipulation of NA and mass production of enzymes.

[0009]

Means for Solving the Problems The present invention solves the above-mentioned problems by providing a protein extracted and purified from cultured human cells, comprising a peptide having at least the amino acid sequence of SEQ ID NOS: 1 to 5. Human 8-oxodG
Provides TPase. Further, the present invention provides the human 8-
cDNA encoding oxodGTPase,
Also provided are NA clones, cDNA clone recipient cells transformed by the cDNA clones, and 8-oxoD GTPase produced by the recipient cells.

Hereinafter, the present invention will be described in detail.
The human 8-oxodGTPase of the present invention comprises, for example, M
o et al. (Mo, Maki & Sekiguchi: Proc. Natl. Acad.
Sci. USA, 89 , 11021-11025, 199
According to 2), it can be extracted and purified from cultured human cells. There is no particular limitation on human cells, and they may be directly isolated from human tissues, or may be known human cell lines. However, 8-oxod GTP
When considering the content of a protein having an ase activity,
It is preferable to use a cell line derived from a cancer cell.

[0011] The molecular structure (amino acid sequence, molecular weight, etc.) of the extracted and purified 8-oxoD GTPase can be analyzed by using a known method and apparatus. In the present invention, as shown in Example 1, a partial sequence of an enzyme was identified by separating a peptide from a purified protein and analyzing its amino acid sequence.

The cDNA of the present invention can be synthesized by allowing reverse transcriptase to act on mRNA extracted from human cells. CD of interest from pool of synthesized cDNAs
When isolating NA, for example, an antibody screening method may be performed using an antibody against the above-mentioned peptide as a probe, or an oligonucleotide synthesized based on the peptide sequence, as exemplified in Examples 2 and 3.
Perform PCR using primers and obtain the target cDNA
May be isolated and amplified.

The cDNA fragment obtained as described above is subcloned into a known vector to obtain d
It is possible to determine the salt sequence by the ideoxy method. The nucleotide sequence of the cDNA of the present invention thus determined is as shown in SEQ ID NO: 6 in the sequence listing. Further, by searching the protein translation region from the nucleotide sequence of the cDNA thus determined, the entire amino acid sequence and molecular weight of 8-oxodGTPase can be estimated. The entire amino acid sequence of 8-oxodGTPase thus estimated is as shown in SEQ ID NO: 6, and has a molecular weight of about 17,900 daltons.

The cDNA clone of the present invention comprises the above-mentioned cD
It can be prepared by incorporating the NA fragment into a known vector. Also, as exemplified in Example 4, 8-
Only the cDNA portion corresponding to the translation region of oxodGTPase may be inserted into the vector. The type of the vector to be used is not particularly limited, and can be appropriately selected in consideration of the purpose of use of the cDNA clone, compatibility with the cDNA fragment to be inserted, and the like. The same applies to the recipient cells, and any host-vector system can be used. For example, when Escherichia coli is used as a recipient cell, a plasmid or phage can be used as a vector, and when an animal cell is used as a recipient cell, a viral vector can be used.

Furthermore, by cloning the cDNA of the present invention as described above, and culturing the recipient cells into which the clone has been introduced, human 8-oxodGTPase can be easily and mass-produced. Host in this case
As a vector system, it is preferable to use an Escherichia coli-plasmid vector system in view of handling, clone copy number, protein expression level, and the like. In addition, Escherichia coli is a protein having its own 8-oxo GTPase activity (M
It is particularly preferable to use a mutant ( mut T gene mutant) which does not express utT protein).

As described above, extraction and purification are performed directly from human cells.
Or human 8-ox of the present invention expressed from cDNA
Since odGTPase effectively prevents cell mutation by purifying nucleotide pools in cells, it can be used for the development of new anticancer and anticancer agents. Moreover, this human 8-oxodGTPa
By preparing an antibody against se, it is possible to estimate the risk of carcinogenesis and to diagnose the progress or prognosis of cancer. Furthermore, by expressing the cDNA of the present invention in a living body, it becomes possible to suppress cancer caused by point mutation of the gene DNA.

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.

[0018]

EXAMPLES Example 1 (Purification of Human 8-oxodGTPase and Determination of Amino Acid Partial Sequence) Method of Mo et al. (Mo, Maki & Sekiguchi: Pr
oc. Natl. Acad. Sci. USA, 89 , 11021-11.
025, 1992) to improve human 8-oxodGT
Pase was extracted and purified as follows.

A crude extract was prepared from 1 × 10 ¹⁰ Jurkat cells, a human T-cell leukemia cell line, and proteins were precipitated with 0.33 g / ml ammonium sulfate.
(25 mM Tris · HCl, pH 7.5; 0.1 m
M EDTA; 2 mM DTT; 15% glycerin) and dialyzed against the same buffer to obtain a protein standard. This protein sample was added to a DEAE BiO Gel A column (bed volume: 200 ml, manufactured by Bio Rad), fractionated, and fractionated.
After washing with 30 ml of buffer A, 0-0.5 M NaC
The protein was eluted from the column with 625 ml of buffer A containing 1 linear gradient.

Next, the fraction having enzyme activity (32 m
1.5 × 10 ⁶ enzyme units per liter)
This was dialyzed twice against Buffer A, added to a HiTrap Heparin column (15 ml bed volume, manufactured by Pharmacia), and a flow-through fraction (containing 1.4 × 10 ⁶ enzyme units in 48 ml) was collected. Was added to a Q-Sepharose HP packed column (bed volume: 20 ml, manufactured by Pharmacia). After washing the column with 75 ml of buffer A, 0-0.5
75 ml of buffer A containing a linear gradient of M NaCl
Eluted the protein. The fraction containing 8-oxdGTPase activity (3 ml containing 8.2 × 10 ⁵ enzyme units) was dialyzed against 1000 ml of buffer A,
MonoQHR5 / 5 (1 ml bed volume, Pharmacia
And eluted with 30 ml of buffer A containing a linear 0-0.4 M NaCl gradient.

The activity of 8-oxodGTPase is as follows:
The degree of hydrolysis from oxodGTP to 8-oxodGMP was measured as an index. That is, 20 mM Tr
is · HCl (pH 8.0), 4 mM MgCl ₂ , 4
0 mM NaCl, 20 μM 8-oxodGTP, 80
The eluted enzyme fraction was added to a reaction solution composed of μm / ml bovine serum albumin and 10% glycerin, so that the total amount was 1%.
2.5 μl, reacted at 30 ° C. for 20 minutes,
After adding 1 l of 50 mM EDTA to stop the reaction, a part of the reaction solution was analyzed by thin layer chromatography.

Of the fractions eluted from the above MonoQ column, 33 and 34 fractions having 8-oxoD GTPase activity were mixed (containing 1.6 × 10 ⁵ units of activity), and the mixture was mixed with 15% SDS-polyacrylamide gel. After electrophoresis in gel, the gel was stained with 0.3M ZnCl ₂ and
The band containing the 8 kd protein was cut out of the gel,
0.25M TrisHCl (pH 9.0): 0.25
Washed three times with M EDTA solution. Next, an elution buffer (20 mM Tris base: 150 mM glyceri)
n: 0.01% SDS) and the Amicon Cen.
Electrophoresis was performed at 250 V for 2 hours using a tiluter to elute the protein. FIG. 1 is an electrophoresis pattern diagram.

The protein eluted in this manner is
con10, concentrate with 20% trichloroacetic acid,
The SDS was removed with acetone, and the purified protein (5.0 × 1) was removed.
0 ⁴ including 8-oxodGTPase activity units) was obtained (see FIG. 2). Next, a plurality of types of peptides were separated from the purified protein, and their amino acid sequences were determined.

That is, the purified protein is reduced, S-alkylated, and digested with lysyl endopeptidase.
ocosorb 5-ODS-H column (2.1 × 100mm)
The peptides were separated by reversed-phase high-performance liquid chromatography using HPLC. Then 8 containing 0.06% trifluoroacetic acid
-80% acetonitrile linear concentration gradient (flow rate 0.2 m
The peptide was eluted in l). Peptides were monitored by absorbance at 210 nm.

Thus, five types of peptides were isolated from the purified protein. The amino acid sequence of each of these peptides was determined using a gas-phase sequencer 473A (manufactured by Applied Biosystems) and a dedicated reagent and program. The amino acid sequence of each peptide is as shown in SEQ ID NOs: 1 to 5 in the sequence listing. Example 2 (Synthesis of cDNA fragment) A cDNA fragment was synthesized by the following two steps. (1) Oligonucleotide primers were synthesized based on the amino acid sequence (SEQ ID NOS: 1-5) of the peptide separated from the purified 8-oxodGTPase protein obtained in Example 1.

Next, cDNA was synthesized by allowing reverse transcriptase to act on 10 μg of mRNA (poly (A) ⁺ RNA) extracted from Jurkat cells, and using this cDNA as a template, a PC was prepared using the above synthetic oligonucleotide primers.
By the R method, a cDNA portion consisting of 59 base pairs corresponding to a partial amino acid of 8-oxoGTPase was amplified.

The cDNA fragment thus amplified was incorporated into a plasmid vector Bluescript KS ^+, and the nucleotide sequence of the cDNA region was determined according to a conventional method. (2) The 5'-side and 3'-side from the center of the 59 base sequence of the cDNA synthesized and determined in the above (1), and the known cDN
A Library (Hayakawa, Koike & Sekiguchi: J. Mo
l. Bio., 213 , 729-747, 1990) to prepare synthetic oligonucleotide primers in the regions corresponding to the 3 'and 5' sides of the vector sequence.

Using these two sets of primers,
PCR was performed using the DNA sample of the NA library as a template, and the cDNA was divided into 5 'and 3' sides and amplified. FIG. 3 shows c in the above steps (1) and (2).
FIG. 2 is a schematic diagram showing the relationship between DNA, vector / plasmid, and primers for PCR. That is, the black portion indicates the cDNA, and the white portion indicates the region near the cleavage of the vector plasmid. Also, SRα and A
n indicates the SRα promoter on the vector plasmid and the polyA addition signal from the SV40 virus, respectively. Furthermore, the positions of the primers used in the PCR in step (1) and their 5 ′ → 3 ′ directions are indicated by ○ · ● and arrows, and the primers in step (2) are also indicated by Δ, ▲ and □
■ and each arrow. Example 3 (Determination of base sequence of cDNA) The two sets of cDNA fragments amplified in Example 2 were respectively
It was incorporated into script KS ⁺ and the nucleotide sequence was determined according to a conventional method. The results are as shown in SEQ ID NO: 2 in the sequence listing.

Next, a protein translation region was searched based on the nucleotide sequence of the cDNA. As a result, as shown in SEQ ID NO: 6, the amino acid sequence of human 8-oxodGT
Pase consists of 156 amino acid residues and its molecular weight was estimated to be about 17,900 daltons. This molecular weight almost coincided with the molecular weight of 18 kd of the Jurkat cell-derived protein estimated by electrophoresis in Example 1.

Further, the amino acid sequence deduced from the nucleotide sequence of the cDNA contained almost completely five peptides (SEQ ID NOS: 1 to 5) separated from the purified protein in Example 1. That is, the peptide sequence of SEQ ID NO: 1 is
It corresponds to the sequence at positions 133 to 138 in the amino acid sequence of SEQ ID NO: 6, and similarly, SEQ ID NO: 2 to 39-63,
SEQ ID NO: 3 is 139-156, SEQ ID NO: 4 is 115-
130 and SEQ ID NO: 5 correspond to 67-89, respectively.

Further, this cDNA has 5'-end and 3'-end.
Each end has an untranslated region. On the 3 'side, a poly (A) addition signal sequence (AATAAA) downstream of the translation termination codon (TAG) and a subsequent poly
(A) Sequence is present. Example 4 (Creation of cDNA clone) cDN determined in Example 3
Based on the nucleotide sequence of A and its translation region, human 8-ox
The cDNA encoding odGTPase is replaced with a series of D
It was recovered as an NA fragment.

That is, the cDNA whose nucleotide sequence is shown in SEQ ID NO: 6 has a recognition sequence for the restriction enzyme NcoI (CCATGG: 55-) at the position containing the translation initiation codon (ATG).
Utilizing the NcoI recognition sequence, an oligonucleotide primer starting from this NcoI recognition sequence was synthesized. On the 3 'side, a translation stop codon (T
AG) to the BamHI recognition sequence (GATC: 527-5)
Oligonucleotide primers starting at base number 24) were synthesized.

Next, mRNA extracted from Jurkat cells
A reverse transcriptase is allowed to act on to prepare a cDNA preparation, and PCR is performed using the above primers as a template,
A DNA fragment was synthesized and amplified. Thus, human 8
C containing the entire coding region of -oxodGTPase
DNA was recovered as a NcoI / BamHI DNA fragment.

[0034] The recovered DNA fragment was ligated to the plasmid vector pTrc99A and the NcoI /
Each was integrated into the BamHI site to create cDNA clones, which were named plasmids p24T and p24N. Among them, p24T was replaced with SU derived from E. coli K12 strain.
The transformed strain SURE / p24T was prepared by introducing the strain into the RE strain and deposited on June 7, 1993 with the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Accession No. FERM P-13).
676). Example 5 (Expression of cDNA in E. coli)
xodGTPase cDNA was expressed, and (1) the enzyme activity and (2) the suppression effect of the mutation occurrence rate were examined. (1) Measurement of enzyme activity cDNA clone pramid p24 prepared in Example 4
N was introduced into Escherichia coli MK602, and transformed strain MK602 was introduced.
602 / p24N. As a control, plasmid vector pTV119N was also used for MK.
MK602 / pTV119N was introduced into strain 602. Escherichia coli MK602 is a mutant strain of the mut T gene, and Escherichia coli itself has a defect in 8-oxo GTPase activity.

These transformed strains MK602 / p24
N and MK602 / pTV119N, respectively, in LB
1 mM isopropylthiogalactoside (IPT
G) After shaking culture at 37 ° C. for 12 hours under induction, the cells were collected by centrifugation. Next, a lysis buffer containing 0.5 mM phenylmethylsulfonyl fluoride (25 mM
Tris · HCl, pH 7.6; 2 mM DTT;
The harvested cells were suspended in 0.1 mM EDTA (15% glycerin), sonicated, and incubated at 4 ° C. for 15 minutes at 15,000.
The mixture was centrifuged at xg to prepare a crude extract. These crude extracts were subjected to 8-oxodG by the method of Mo et al.
TPase activity was measured.

Next, 800 μl of a crude extract (protein content: 1.5 mg / ml) prepared from each transformant was used.
To a DEAE Bio Gel A column (bed volume 4 ml, Bio R
After washing with 15 ml of lysis buffer, fractionation was carried out with a concentration gradient of 0-0.4 M NaCl in the same buffer, and 2-oxo GTPase activity was measured for 2 μl of each fraction.

The results are as shown in FIG. 4 and FIG. 5, and the crude extract (FIG. 4) and the fraction by column (FIG. 5)
In any of the above, the expression product of Escherichia coli harboring the cDNA clone p24N has high 8-oxodGTPas
e activity was observed. Also, as shown in FIG.
In the AE column fraction, a low activity peak other than the high enzyme activity peak in MK602 / p24N was observed.
/ PTV119N, it is presumed that these are the activities of nucleoside triphosphate degrading enzymes having broad substrate specificity. (2) Suppressive effect of spontaneous mutation The cDNA clone with low copy number, p24T, prepared in Example 4 was introduced into Escherichia coli MK602 strain to prepare transformant MK602 / p24T.

The transformed bacterium MK602 / p24T was
The cells were cultured in a medium containing the antibiotic rifampicillin, and the inhibitory effect of 8-oxodGTPase on the mutation appearance rate was observed using the frequency of emergence of resistant bacteria against rifampicillin as an index. That is, the transformed bacterium MK602 / p24T containing a cDNA clone was cultured at 37 ° C. for 24 hours in the presence of 1 mM IPTG, appropriately diluted, and seeded on an LB agar medium having a rifampicillin concentration of 100 μg / ml, and incubated at 37 ° C. overnight. After culturing, the number of resistant bacteria was counted.

As a control, a normal mut
E. coli wild-type strain having T gene (MK601), mut
T gene mutant (MK602) and MK602 (MK602) having the plasmid vector pTrc99A
/ PTrc99A) was similarly cultured, and the number of rifampicillin-resistant bacteria was observed. The results are as shown in Table 1, and the cDNA of human 8-oxodGTPase was used.
The occurrence rate of the mutant strain (rifampicillin-resistant strain) of Escherichia coli MK602 / p24T harboring E. coli was higher than that of the wild-type strain MK601, but was clearly lower than that of the MK602 / pTrc99A and MK602 strains having the plasmid vector pTrc99A.

[0040]

[Table 1]

From the results of (1) and (2) above, the cDNA clone of the present invention was reliably expressed in Escherichia coli, and the expressed human 8-oxoDTPase had high enzymatic activity. It was proved that it had an excellent inhibitory effect. In particular, the inhibitory effect on spontaneous mutation is due to the action of human 8-oxodGTPase to purify the intracellular nucleotide pool.
It is suggested that -oxodGTPase greatly contributes to suppression of human spontaneous carcinogenesis.

[0042]

As described in detail above, the present invention provides 8-oxo which suppresses human cell mutation and carcinogenesis.
dGTPase and cDNA and cDNA clones encoding this enzyme are provided. These open new avenues for the diagnosis and treatment of cancer, the development of effective anticancer drugs, and the like.

[0043]

[Sequence list]

SEQ ID NO: 1 Sequence length: 6 Sequence type: amino acid Topology: Linear Sequence type: Peptide SEQ ID NO: 2 Sequence length: 25 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Val Gln Glu Gly Glu Thr Ile Glu Asp Gly Ala Arg Arg Glu Leu 1 5 10 15 Gln Glu Glu Ser Gly Leu Thr Val Xaa Ala 2025 (Xaa represents Asp or Trp in the above sequence) SEQ ID NO: 3 Sequence length: 18 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Phe Gln Gly Gln Asp Thr Ile Leu Asp Tyr Thr Leu Arg Glu Val 1 5 10 15 Asp Thr Val SEQ ID NO: 4 Sequence length: 16 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Xaa Met Trp Pro Asp Asp Ser Tyr Trp Phe Xaa Xaa Leu Xaa Gln 1510 15 Lys (In the above sequence, the first Xaa is Ser or A
sp, the eleventh Xaa represents Pro or Gln, and the twelfth Xaa represents Ile, Leu or Ala.
Is shown. The Xaa at the 14th position is unknown.) SEQ ID NO: 5 Sequence length: 23 Sequence type: Amino acid Topology: Linear Sequence type: Peptide Sequence: Val Gly Gln Ile Val Phe Glu Phe Val Gly Glu Pro Glu Leu Met 1 5 10 15 Asp Val His Val Phe Xaa Thr Asp 20 SEQ ID NO: 6 Sequence length: 676 Sequence type: Nucleic acid Topology: Linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens cell Line: Jurkat cell line Sequence characteristics Symbol indicating characteristics: polyA signal Location: 652. . 657 Method for determining feature: S Symbol representing feature: polyA site Location: 671 Method for determining feature: P Symbol representing feature: 3′UTR Location: 525. . 676 Method for determining feature: S Symbol representing feature: 5 'UTR Location: 1. . 56 Method for determining feature: S Symbol representing feature: mat peptide Location of occurrence: 57. . 524 Method for determining characteristics: S sequence: CTGCAG 6 GGGGGGGGGG GGGCGCGGCG GGGCGAGCGG CGGTGCAGAA CCCAGGGACC 56 ATG GGC GCC TCC AGG CTC TAT ACC CTG GTG CTG GTC CTG CAG CCT 101 Met Gly Ala Ser Arg Leu Tyr Thr Leu Val Leu Val Leu Val 5 10 15 CAG CGA GTT CTC CTG GGC ATG AAA AAG CGA GGC TTC GGG GCC GGC 146 Gln Arg Val Leu Leu Gly Met Lys Lys Arg Gly Phe Gly Ala Gly 20 25 30 CGG TGG AAT GGC TTT GGG GGC AAA GTG CAA GAA GGA GAG ACC ATC 191 Arg Trp Asn Gly Phe Gly Gly Lys Val Gln Glu Gly Glu Thr Ile 35 40 45 GAG GAT GGG GCT AGG AGG GAG CTG CAG GAG GAG AGC GGT CTG ACA 236 Glu Asp Gly Ala Arq Arq Glu Leu Gln Glu Glu Ser Gly Leu Thr 50 55 60 GTG GAC GCC CTG CAC AAG GTG GGC CAG ATC GTG TTT GAG TTC GTG 281 Val Asp Ala Leu His Lys Val Gly Gln Ile Val Phe Glu Phe Val 65 70 75 GGC GAG CCT GAG CTC ATG GAC GTG CAT GTC TTC TGC ACA GAC AGC 326 Gly Glu Pro Glu Leu Met Asp Val His Val Phe Cys Thr Asp Ser 80 85 90 ATC CAG GGG ACC CCC GTG GAG AGC GAC GAA ATG CGC CCA TGC TGG 371 Ile Gln Gly Thr Pro Val Glu Ser Asp Glu Met Arg Pro Cys Trp 95 100 105 TTC CAG CTG GAT CAG ATC CCC TTC AAG GAC ATG TGG CCC GAC GAC 416 Phe Gln Leu Asp Gln Ile Pro Phe Lys Asp Met Trp Pro Asp Asp 110 115 120 AGC TAC TGG TTT CCA CTC CTG CTT CAG AAG AAG AAA TTC CAC GGG 461 Ser Tyr Trp Phe Pro Leu Leu Leu Gln Lys Lys Lys Phe His Gly 125 130 135 TAC TTC AAG TTC CAG GGT CAG GAC ACC ATC CTG GAC TAC ACA CTC 506 Tyr Phe Lys Phe Gln Gly Gln Asp Thr Ile Leu Asp Tyr Thr Leu 140 145 150 CGC GAG GTG GAC ACG GTC TAG CGGGAGCCCA GGGCAGCCCC TGGGCAGGAG 557 Arg Glu Val Asp Thr Val ... GGTGAAAAA 676

[Brief description of the drawings]

FIG. 1 is an electrophoretogram of a purified protein sample extracted from Jurkat cells.

FIG. 2 shows the measurement results of 8-oxo GTPase activity in each of the fractions A, B, and C collected from the gel shown in FIG.

FIG. 3 shows cDN in the cDNA synthesis step of the present invention.
FIG. 2 is a schematic view illustrating the relationship between A, a vector, and a primer.

FIG. 4 shows 8-oxodGTPase of a crude extract in transformed E. coli harboring a cDNA clone of the present invention.
It is an activity measurement result.

FIG. 5 shows the enzyme activity when the crude extract of FIG. 4 was subjected to column analysis.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ identification symbol FI (C12N 9/12 C12R 1:19) (58) Investigated field (Int.Cl. ⁷ , DB name) C12N 15/00-15 / 90 C12N 1/00-1/38 C12N 9/00-9/99 BIOSIS (DIALOG) GenBank / EMBL / DDBJ (GENETYX) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A human 8-amino acid having the nucleotide sequence of SEQ ID NO.
Encodes oxodeoxyguanosine triphosphate degrading enzyme
CDNA. 2. The cDNA of claim 1 is integrated into a vector.
CDNA clone. 3. An E. coli SURE / p24T (FERM).
P-13676).
The cDNA clone of claim 2. 4. The cDNA clone according to claim 2 or 3,
Transformed cDNA clone recipient cells. 5. The mutT gene mutant Escherichia coli.
4 cDNA clone recipient cells. 6. The recipient cell of claim 5, wherein
A protein to be produced, the amino acid represented by SEQ ID NO: 6
Recombinant human 8-oxodeoxyguanosine having the sequence
Triphosphate degrading enzyme.