JPH11332579A - Tsc501 gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new TSC501 gene having a specific amino acid sequence, containing a base sequence encoding proteins having acetyl transferase activity, capable of expression in kidney and liver-specific manner, and useful as a medicament metabolism promoter or the like. SOLUTION: This new TSC501 gene is such one as to contain a base sequence encoding either a protein having an amino acid sequence of the formula or a protein having an amino acid sequence wherein one or more amino acids are deleted, substituted or added in the amino acid sequence of the formula and also having acetyl transferase activity, be capable of expression in kidney- and liver-specific manner, and related to medicament metabolism, therefore being useful as a medicament metabolism promoter or the like. This gene is obtained by screening a human normal kidney cDNA library constructed by conventional method using human normal kidney cDNA, using, as probe, a cDNA fragment consisting of a labeled partial sequence thereof.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a gene which is specifically expressed in the kidney and liver and is involved in drug metabolism, a novel protein encoded by the gene, and a specific antibody thereof.

[0002]

2. Description of the Related Art The activity of proteins synthesized in vivo, drugs and toxins taken into the body is regulated by enzymes that catalyze various modification reactions such as phosphorylation, methylation, and acetylation. It has been known.

[0003] Among the above enzymes, acetyltransferase is acetyl coenzyme A
It catalyzes an acetylation reaction that transfers an acetyl group from (acetyl CoA) to proteins, drugs, and poisons, and is active in metabolism of various drugs administered to the body, toxins taken into the body, and unnecessary proteins. And it is said to have mainly a drug metabolizing action. However, the detailed mechanism of drug metabolism has not been elucidated at present.

Elucidation of the physiological roles of enzymes involved in drug metabolism such as acetyltransferase and the information obtained thereby are important for elucidation of the drug metabolism mechanism.
Not only in the field of basic science research, but also in the field of medicine, it is desired from the aspect of new drug development.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide the above-mentioned information required in the art, and in particular, has homology to acetyltransferase and is involved in drug metabolism in vivo. An object of the present invention is to provide a novel protein and a gene encoding the same.

As a result of repeated search for genes derived from various human tissues, the present inventors have found that they are specifically expressed in kidney and liver, have a certain homology with various acetyltransferases, Successful isolation and identification of a new gene encoding an enzyme that regulates metabolism related to the present invention led to the completion of the present invention.

[0007]

That is, according to the present invention,
TSC501 gene containing a nucleotide sequence encoding any of the following proteins (a) and (b), in particular, SEQ ID NO:
The gene containing the nucleotide sequence represented by No. 2 and a gene such as a human gene are provided.

(A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1;
Alternatively, a protein comprising an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added, and having an acetyltransferase activity.

According to the present invention, there is provided a TSC5 comprising any one of the following polynucleotides (a) and (b):
The present invention provides the gene of the present invention, which is a human gene, especially a human gene.

(I) a polynucleotide comprising all or a part of the nucleotide sequence represented by SEQ ID NO: 3; (ii) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 3
A polynucleotide that hybridizes under stringent conditions to a polynucleotide.

Further, according to the present invention, there is provided the above gene which is a DNA fragment used as a specific probe or a specific primer for detecting the TSC501 gene.

In addition, according to the present invention, there is provided a recombinant TSC501 protein which is any one of the above-mentioned proteins (a) and (b) and an antibody capable of binding thereto.

Hereinafter, the abbreviations of amino acids, peptides, base sequences, nucleic acids and the like in the present specification are referred to as IUPAC.
-IUB regulations [IUPAC-IUB Communication on Biologi
calNomenclature, Eur.J. Biochem., 138 : 9 (198
4)], "Guidelines for preparation of specifications containing base sequences or amino acid sequences" (edited by the Patent Office) and conventional symbols in the relevant field.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION As a specific example of the gene of the present invention, a gene deduced from the DNA sequence of a PCR product named “TSC501a” shown in Examples described later can be mentioned. Is as shown in SEQ ID NO: 3.

The gene is represented by SEQ ID NO: 1.
Human cDNA (open length: about 1.0 kb) containing an open reading frame encoding a novel 7-amino acid sequence (estimated molecular weight: about 25 KDa), an enzyme that regulates drug metabolism and is specifically expressed in kidney and liver. And
The enzyme is thought to play an important role in drug metabolism.

A protein (hereinafter referred to as "TSC5 gene") which is an expression product of the gene of the present invention (hereinafter referred to as "TSC501 gene").
01 protein))
Is the FASTA program (Person WR, et al., Pr.
oc. Natl. Acad. Sci., USA., 85 , 2444-2448 (1988))
As a result of homology analysis using the GenBank / EMBL database, the (1) gentamicin
3'-acetyl transferase (gentamicin 3'-ac
etyltransferase (AAC (3) -I); W. Wohlleben, et al.,
Mol. Gen. Genet., (1989), 217: 202-208), (2) Ribosomal protein-alanine acetyltransferase (r
ibosomal-protein-alanine acetyltransferase (Rim
I); A. Yoshikawa, et al., Mol. Gen. Genet. (1987),
209: 481-488) and (3) streptothricin acetyltransfera
se (Sta T); S. Horinouchi, et al., J. Bacteriol.,
(1987), pp. 1929-1937) and 29% and 26%, respectively.
And 27% homology, and significant identity was confirmed with these and other various acetyltransferases.

Analysis of the above protein by computer analysis of the protein structure suggests that one transmembrane region is present.
C501 protein was found to be highly likely to be a cell membrane protein.

As a result of Northern analysis, the TSC501 of the present invention
The gene was found to be specifically expressed in normal kidney and liver. Thus, the above kidney and liver are organs that mainly perform drug metabolism, and the gene of the present invention was found to be a gene related to drug metabolism from the viewpoint of being specifically expressed in these organs. .

As described above, the provision of the TSC501 gene of the present invention and its gene product provide useful information particularly for elucidating the mechanism of drug metabolism and its regulation in living organisms. In particular, can be suitably used as a metabolic accelerator for various drugs to be administered to a living body.

The gene of the present invention is, specifically, SEQ ID NO:
Examples include a gene containing a nucleotide sequence encoding a protein consisting of the amino acid sequence represented by 1 or a gene containing a nucleotide sequence represented by SEQ ID NO: 2, but are not particularly limited thereto. It may be a gene having a certain modification therein, or a gene having a certain homology with the above nucleotide sequence.

That is, the gene of the present invention comprises a protein having an amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted or added, and having acetyltransferase activity. Encoding base sequences and genes containing them are also included. Here, the degree of “deletion, substitution or addition of amino acids” and their positions are determined by the modified protein,
There is no particular limitation as long as it has the same function as the protein consisting of the amino acid sequence represented by SEQ ID NO: 1.
The plurality includes two or more, usually several.

The modification (mutation) of these amino acid sequences
May naturally occur due to, for example, mutation or post-translational modification, and may be artificially performed based on a naturally-derived gene (for example, a specific example gene of the present invention). The gene of the present invention includes all modified genes having the above-mentioned properties, regardless of the cause and means of such modification / mutation.

As the above-mentioned artificial means, for example, cytospecific mutagenesis [Methods in Enzym
ology, 154 : 350, 367-382 (1987); id. 100 : 468 (198
3); Nucleic Acids Res., 12 : 9441 (1984); Lectures on Sequel Chemistry Experiment 1, Genetic Research Method II, edited by The Japanese Biochemical Society, p105
(1986)] and chemical synthesis means such as the phosphate triester method and the phosphate amidite method [J. Am. Che.
m. Soc., 89 : 4801 (1967); 91 : 3350 (1969); Scien
ce, 150 : 178 (1968); Tetrahedron Lett., 22 : 1859 (1
981); and 24 : 245 (1983)] and combinations thereof.

As one embodiment of the gene of the present invention, a gene comprising a polynucleotide containing all or a part of the nucleotide sequence shown by SEQ ID NO: 3 can be exemplified. The open reading frame included in this nucleotide sequence is an example of one combination of codons indicating each amino acid residue of the above amino acid sequence (SEQ ID NO: 1). The gene of the present invention is not limited to this example, and each amino acid residue It is of course possible to have a base sequence in which arbitrary codons are selected in combination. The selection of the codon can be carried out according to a conventional method, for example, by considering the codon usage frequency of the host to be used [Nc
leic Acids Res, 9:. 43 (1981) ].

The gene of the present invention is, for example, SEQ ID NO:
As shown in the specific example of No. 3, it is represented as a base sequence of single-stranded DNA. However, the present invention naturally includes a polynucleotide comprising a base sequence complementary to such a base sequence and a component containing both of them. Encompasses also cDNA
It is not limited to such DNA.

Further, as described above, the gene of the present invention is not limited to a polynucleotide comprising all or a part of the nucleotide sequence shown in SEQ ID NO: 3, but has a certain homology with the nucleotide sequence. It also includes those consisting of a base sequence. Such a gene hybridizes at least with a DNA consisting of the nucleotide sequence represented by SEQ ID NO: 3 under the following stringent conditions,
Those which are not desorbed even by washing under certain conditions are mentioned.

That is, the base sequence having a certain homology includes a DNA having the base sequence of SEQ ID NO: 3 and 6 ×
It hybridizes under the conditions of 65 ° C. overnight in SSC or under the conditions of overnight at 37 ° C. in 4 × SSC containing 50% formamide, and desorbs from the DNA even under the condition of washing in 2 × SSC at 65 ° C. for 30 minutes. Includes base sequences that do not separate. Here, SSC is a standard saline-citrate buffer (standard saline citrate; 1 × SSC = 0.15M NaCl,
0.015M sodium citrate).

The gene of the present invention can be easily produced and obtained by general genetic engineering techniques on the basis of sequence information on specific examples [Molecular Cloning
2d Ed, Cold Spring Harbor Lab. Press (1989); see Seismic Chemistry Laboratory Course “Gene Research Methods I, II, III”, edited by The Biochemical Society of Japan (1986), etc.).

Specifically, a cDNA library is prepared from an appropriate source in which the gene of the present invention is expressed according to a conventional method, and the desired library is prepared from the library using an appropriate probe or antibody specific to the gene of the present invention. By selecting a clone, the gene of the present invention can be obtained (for the above-mentioned method, for example, Proc. Natl. Acad. Sci., USA., 78 :
6613 (1981); Science, 222 : 778 (1983), etc.].

In the above, the origin of the cDNA is as follows:
Examples include various cells and tissues expressing the gene of the present invention, and cultured cells derived therefrom. Total RNA from these
, MRNA isolation and purification, cDNA acquisition and cloning thereof can all be carried out according to conventional methods.
Note that cDNA libraries are commercially available. In the present invention, commercially available cDNA libraries, for example, various cDNA libraries available from Clontech Lab. Inc. can also be used.

The method for screening the gene of the present invention from a cDNA library is not particularly limited, either, and any conventional method can be used. As a specific method, for example, c
A method for selecting a corresponding cDNA clone by immunoscreening using a specific antibody for a protein produced by DNA, a plaque hybridization method using a probe that selectively binds to a target DNA sequence,
Examples include a colony hybridization method and the like, and combinations thereof.

The probe used herein can be generally exemplified by DNA chemically synthesized based on the information on the nucleotide sequence of the gene of the present invention. Can also be used well.

Further, the screening for the gene of the present invention comprises:
A protein interaction cloning method using a TSC501 protein instead of the above specific antibody (protein interaction clon)
oning procedure) or a method using a sense primer and an antisense primer set based on the nucleotide sequence information of the gene of the present invention as a screening probe.

According to the present invention, a differential display method (Liand P.,
et al., Science, 257 , 967-971 (1992)), the mR between cells under different conditions or a plurality of different cell groups.
The expression of NA can be directly compared and examined.

When obtaining the gene of the present invention, DNA / RNA by PCR (Science, 230 : 1350 (1985)) is used.
An amplification method can also be suitably used. Particularly, when it is difficult to obtain a full-length cDNA from the library, the lace method (RACE: Rapid amplification of cDNA ends; Experimental Medicine, 12 (6): 35 (1994)), particularly 5′-race (5'-
RACE) method [Proc. Natl. Acad. Sci., USA., 8 : 899
8 (1988)] is preferred. 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 isolation and purification of the amplified DNA / RNA fragment can be carried out according to a conventional method as described above, for example, by gel electrophoresis.

The gene of the present invention or various DNs obtained above
The A fragment can be obtained by a conventional method, for example, the dideoxy method [Proc. Natl.
ad.Sci., USA., 74 : 5463 (1977)] or the Maxam-Gilbert method [Method in Enzymology, 65 : 499 (1980)], etc., and conveniently using a commercially available sequence kit or the like. The sequence can be determined.

According to the use of the gene of the present invention, its gene product (protein or enzyme) can be easily and stably produced in a large amount by using a general genetic engineering technique. Therefore, the present invention relates to the TSC50 of the present invention.
The present invention also provides a vector containing one gene (expression vector), a host cell transformed with the vector, and a method for producing the TSC501 protein by culturing the host cell.

The production of the protein is carried out by a conventional gene recombination technique [Science, 224 : 1431 (1984); Biochem. Biophys.
es. Comm., 130 : 692 (1985); Proc. Natl. Acad. Sc
i., USA., 80 : 5990 (1983) and the references cited above)
Can be implemented according to

As the host cell, any of prokaryote and eukaryote can be used. For example, prokaryote hosts widely include commonly used ones such as Escherichia coli and Bacillus subtilis. Escherichia coli, especially those contained in the Escherichia coli K12 strain can be exemplified. Eukaryotic host cells also include
Cells such as vertebrates and yeast are included, and the former includes, for example, COS cells [Cell, 23 : 175 (198
1)] and Chinese hamster ovary cells and their dihydrofolate reductase-deficient strains (Proc. Natl. Acad. Sc.
i., USA., 77 : 4216 (1980)]. As the latter, yeast cells of the genus Saccharomyces can be suitably used.
Of course, it is not limited to these.

When a prokaryotic cell is used as a host, a vector capable of replicating in the host cell is used, and a promoter and an SD (Shine & Sand) are arranged upstream of the gene so that the gene of the present invention can be expressed in the vector.・ Dargano)
An expression plasmid to which a base sequence and an initiation codon (for example, ATG) necessary for initiation of protein synthesis are added can be suitably used. As the above vector, generally, a plasmid derived from Escherichia coli, for example, pBR322, pBR325, pUC1
2, pUC13 and the like are often used, but not limited thereto, and various known vectors can be used. Commercially available products of the above-mentioned vector used in an expression system using Escherichia coli include, for example, pGEX-4T (Amersham Pharmac.
ia Biotech), pMAL-C2, pMAl-P2 (Ne
w England Biolabs), pET21, pET21 / la
cq (Invitrogen), pBAD / His (Invitrogen)
Company).

When the vertebrate cell is used as a host, the expression vector usually has a promoter, an RNA splice site, a polyadenylation site, a transcription termination sequence, etc., located upstream of the gene of the present invention to be expressed. Which may optionally have a replication origin. Specific examples of the expression vector include, for example, pS having the early promoter of SV40.
V2 dhfr [Mol. Cell. Biol., 1 : 854 (1981)]. In addition to the above, various known commercial vectors can be used. Commercially available products of such a vector used in an expression system using animal cells include, for example, pEGF
PN, pEGFP-C (Clontrech), pIND (I
nvitrogen), pcDNA3.1 / His (Invitroge
nF) and animal cells such as pFastBacH
T (GibciBRL), pAcGHLT (PharMing
en), pAc5 / V5-His, pMT / V5-Hi
s, pMT / Bip / V5-his (above Invitrogen
And the like for insect cells.

A specific example of an expression vector when a yeast cell is used as a host is, for example, pAM82 having a promoter for the acid phosphatase gene [Proc.
Natl. Acad. Sci., USA., 80 : 1 (1983)]. Commercially available expression vectors for yeast cells include, for example, pPI
CZ (Invitrogen), pPICZα (Invitrogen)
Etc. are included.

There is no particular limitation on the promoter. When Escherichia is used as a host, for example, tryptophan (trp) promoter, lpp promoter, lac promoter, recA promoter, PL / PR promoter and the like can be preferably used. When the host is a Bacillus genus,
SP01 promoter, SP02 promoter, penP promoter and the like are preferable. When yeast is used as a host, for example, a pH05 promoter, a PGK promoter, a GAP promoter, an ADH promoter and the like can be suitably used. When an animal cell is used as a host, preferred promoters include SV40-derived promoter, retrovirus promoter, metallothionein promoter, heat shock promoter, cytomegalovirus promoter, SRα promoter and the like.

Incidentally, as the expression vector of the gene of the present invention, an ordinary fusion protein expression vector can also be preferably used. Specific examples of the vector include glutathione-S-
Examples include pGEX (Promega) for expression as a fusion protein with transferase (GST).

The method for introducing and transforming a desired recombinant DNA (expression vector) into a host cell is not particularly limited, and various general methods can be employed. The resulting transformant can also be cultured according to a conventional method, and by the culture, the desired TSC50 encoded by the gene of the present invention is obtained.
One protein is expressed and produced, and accumulated or secreted inside, outside, or on the cell membrane of the transformant.

As the medium used for the above-mentioned culture, various media commonly used depending on the host cell used can be appropriately selected and used, and the culture can be carried out under conditions suitable for the growth of the host cell.

The thus obtained recombinant protein (TSC5
01 protein), for example, [ ^14C ] -acetyl-CoA and TSC5
01 protein in the presence of antibiotics (drugs) such as gentamicin and streptomycin to produce [ ¹⁴
C] -drug is diffused into a scintillator which is an organic layer, and the amount of the drug is measured by a liquid scintillation counter [Sambrook, J. Fritsch, E., et al.
F. & Maniatis, T., Molecular Cloning, A Laboratory
Mannual, Second Edition (Cold Spring Harbor, New Y
ork, Cold SpringHarbor Laboratory Press), 1989, 1
6.30-16.55 / 16.56-16.67; Ausubel, FM, et al., Curen
t Protocols in Molecular Biology (New York, John W
iley andSons, 1990, 9.0-9.5 / 9.6.1-9.6.9].

The culture of the above transformant is described in more detail
The method can be carried out, for example, as follows using a cultured cell containing a transformant obtained by incorporating the desired TSC501 gene into the above-described expression vector and a cell extract of the cultured cell. That is, first, the cells were washed with 10 ml of PBS, and then the cells were washed with a rubber postman.
After suspending in BS and centrifuging at 2000 rpm at 4 ° C. for 5 minutes, the precipitate (cells) is collected. Next, 100 µl of 0.25 M Tris-HCl (pH 7.
5) After suspending to 1 to 2 mg / ml,
After a freezing operation using liquid nitrogen (or by leaving it in a -80 ° C ultra-low temperature refrigerator for 5 minutes) and a thawing operation are repeated four times, the mixture is centrifuged at 15000 rpm at 4 ° C for 10 minutes, and the supernatant is collected.

The supernatant thus recovered can be stored at −80 ° C. if it is not immediately subjected to measurement. For example, a part of the supernatant is subjected to a bio-Rad protein assay or the like to remove the protein. Can also be quantified.

20 to 200 μg of the protein in the above supernatant was added to 0.
Make up to 25 μl using 25 M and squirrel-hydrochloric acid (pH 7.5) and add 50 μl of 0.1 μl to the protein solution.
0.25 M Tris-HCl (pH 7.5) containing Ci [ ¹⁴ C] chloramphenicol (Amersham, etc.) and 10 μl
10 mM acetyl-CoA (Wako Pure Chemical Industries, Sigma, etc.)
And incubate at 37 ° C. for 1 hour. To this, 300 μl of ethyl acetate is added, and the mixture is stirred using a vortex mixer, centrifuged at 15,000 rpm, 4 ° C. for 10 minutes, 250 μl of the supernatant is transferred to another 1.5 ml tube, and 250 μl of ethyl acetate is further added. Next, the mixture is stirred using a vortex mixer, centrifuged in the same manner as described above, and 250 μl of the supernatant is added to the 1.5 ml tube. A total of 500 μl of ethyl acetate was evaporated to dryness using a concentrating centrifuge, the extract was dissolved in 20 μl of ethyl acetate, and 0.5 μl of each was placed on a silica gel thin plate at intervals of 1 cm using a disposable micropipette or using a pipetman. To spot.

The chamber was saturated with chloroform-methanol (95: 5) in advance, separated for 20 to 30 minutes, dried several minutes later at room temperature, and subjected to autoradiography overnight to obtain an acetylated product. Separate counts for each non-acetylated form were analyzed separately with an image analyzer (Fu
ji: BSA2000) or liquid scintillation counter (Beckman, etc.)
The acetyltransferase activity of the TSC501 protein can be measured.

The recombinant protein (TSC501 protein)
If desired, it can be separated and purified according to various separation procedures utilizing its physical properties, chemical properties, etc. [“Biochemical Data Book II”, pp. 1175-1259, 1st edition, 1st printing, 1980 June 23, issued by Tokyo Chemical Co., Ltd .; Bioc
hemistry, 25 (25): 8274 (1986); Eur. J. Biochem., 1
63 : 313 (1987).

Examples of the method include, for example, ordinary reconstitution treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock method, sonication, ultrafiltration, molecular sieve chromatography ( Gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, high performance liquid chromatography (HP
Examples thereof include various liquid chromatography such as LC), dialysis, combinations thereof, and the like. A particularly preferable example of the above method is affinity chromatography using a column to which a specific antibody against the protein of the present invention is bound.

Thus, the present invention also provides a novel TSC501 protein itself obtained, for example, as described above. The protein has homology to acetyltransferase and is characterized by having an acetylation reaction promoting action (acetyltransferase activity) or a drug metabolizing action, and is useful in the pharmaceutical field as described above. .

The TSC501 protein can also be used as an immunizing antigen for preparing a specific antibody of the protein. The components used as antigens here are:
For example, it can be a protein or a fragment thereof produced in large amounts by the above-described genetic engineering technique, and by using these antigens, desired antisera (polyclonal antibodies) and monoclonal antibodies can be obtained.

The method of producing the antibody itself is well understood by those skilled in the art, and the present invention can follow these conventional methods [Seismochemistry Laboratory Course “Immunobiochemical Research Method”, The Japanese Biochemical Society] (1986)).

As the immunized animal used for obtaining the above antiserum, for example, normal animals such as rabbits, guinea pigs, rats, mice and chickens can be arbitrarily selected. Immunization methods using the above antigens and blood collection are also common. It can be implemented according to the law.

For obtaining a monoclonal antibody, fusion cells of plasma cells (immunocytes) and plasmacytoma cells of an animal immunized with the above immunizing antigen are prepared according to a conventional method, and a clone producing the desired antibody is selected therefrom. However, it can be carried out by culturing the clone. The immunized animal is generally selected in consideration of compatibility with a plasmacytoma cell used for cell fusion, and usually a mouse or rat can be advantageously used. Immunization can be carried out in the same manner as in the case of the above antiserum, and if necessary, an immunizing antigen can be used in combination with a usual adjuvant or the like.

The plasmacytoma cells used for the fusion are not particularly limited. For example, p3 (p3 / x63-Ag8) [Natu
re, 256 : 495-497 (1975)], p3-U1 [Current Top
icsin Microbiology and Immunology, 81 : 1-7 (197
8)], NS-1 [Eur. J. Immunol., 6 : 511-519 (197
6)], MPC-11 [Cell, 8 : 405-415 (1976)], S
Various myeloma cells such as P2 / 0 [Nature, 276 : 269-271 (1978)], rat R210 [Nature, 277 : 131-133 (1979)], etc. and cells derived therefrom are used. it can.

The fusion of the above immune cells and plasmacytoma cells can be carried out in the presence of a conventional fusion promoter such as polyethylene glycol (PEG) or Sendai virus (HVJ) according to a known method. Separation of the desired hybridoma can also be performed [Meth. In Enzym
ol., 73 : 3 (1981);

Further, the search for the antibody-producing strain of interest and the monocloning are also carried out by a conventional method. For example, the search for the antibody-producing strain is carried out by the ELISA method using the above-mentioned immunizing antigen [Me
th.in Enzymol., 70 : 419-439 (1980)), plaque method,
Spot method, agglutination reaction method, octeroney (Ouchterlon
It can be carried out according to various methods generally used for detecting antibodies, such as y) method and radioimmunoassay.

The desired antibody can be collected from the hybridoma obtained in this manner by culturing the hybridoma by a conventional method to obtain a culture supernatant thereof, or by administering the hybridoma to a mammal compatible with the hybridoma and growing the hybridoma. And the like. The former method is suitable for obtaining high-purity antibodies, and the latter method is suitable for mass production of antibodies. The antibody thus obtained can be further purified by ordinary means such as salting out, gel filtration, and affinity chromatography.

The antibody thus obtained is the TSC of the present invention.
It is characterized by having binding property to the 501 protein, which can be advantageously used for purification of the TSC501 protein, measurement or identification thereof by an immunological technique, and the like. The present invention also provides such a novel antibody.

Further, based on the sequence information of the gene of the present invention revealed by the present invention, for example, by utilizing a part or all of the nucleotide sequence of the gene, the gene of the present invention in an individual or various tissues can be used. Expression can be detected.

Such detection can be performed according to a conventional method, for example, RT-PCR [Reverse transcribed-Polyme
rase chain reaction; ES Kawasaki, et al., Amplif
ication of RNA.In PCR Protocol, A Guide to method
s and applications, AcademicPress, Inc., SanDiego,
21-27 (1991)) and Northern blot analysis (Molecular Cloning, Cold Spring Harbor Lab.
(1989)] and in situ RT-PCR [Nucl.
s., 21 : 3159-3166 (1993)], their measurement at the cell level such as in situ hybridization, and NASBA
Method (Nucleic acidsequence-based amplification, Natu
re, 350 : 91-92 (1991)] and other various methods.

In the case where the RT-PCR method is employed, the primers used are not particularly limited as long as they are specific to the gene of the present invention capable of specifically amplifying only the gene of the present invention. The sequence can be appropriately set based on the sequence. Usually, it can have a partial sequence of the order of 20 to 30 nucleotides.

As described above, the present invention relates to the TS according to the present invention.
It also provides a DNA fragment used as a specific primer and / or a specific probe for detecting the C501 gene.

[0069]

According to the present invention, there is provided a novel TSC5 having acetyltransferase activity or drug metabolism.
The present invention provides a protein 01, a gene encoding the same, and use thereof to provide a technique useful for elucidation of a drug metabolism mechanism in a living body, development of a new drug, and the like.

[0070]

The present invention will now be described in further detail with reference to examples.

[0071]

EXAMPLE 1 (1-1) [.gamma. ³³ P] exposed labeled with [.gamma. ³³ P] ATP to confirm the human gene expressed in expression method tissue-specific technique labeled with ATP The method was used. The procedure of the method is essentially as follows:
(Liang P., et al., Science, 257 , 967-971 (1992)).

That is, 13 human tissues (adult brain, fetal brain,
Lung, liver, stomach, pancreas, spleen, mammary gland, bladder, placenta, testicle,
Kidney and heart: 0.2 μg of poly A RNA isolated from each of the clones (Clontech) was added to 8 μl of diethylpyrocarbonate-treated water in 3 μl of 3′-anquard oligo dT primer G (T) 15MA. (M is a mixture of G, A and C) and heated at 65 ° C. for 5 minutes. To this solution was added 4 μl of 5 × first strand buffer (BRL) and 2 μl of 0.1 M DT.
T (BRL), 1 μl of 250 mM dNTPs (B
RL), 1 μl of ribonuclease inhibitor (40 units; TOYOBO) and 1 μl of Superscript II reverse transcriptase (200 units; BRL) were added. The final volume of each reaction was 20 μl. After culturing each solution at 37 ° C. for 1 hour, the solution was diluted by a factor of 2.5 by adding 30 μl of distilled water, and -20 until use.
Stored at ° C.

The cDNA was amplified by PCR in the presence of [γ- ³³ P] ATP-labeled (Amersham) 3′-uncarded primer. PC of this cDNA
R amplification was performed as follows. That is, each 20 μl
2 μl RT reaction mixture, 2 μl 10 × PCR buffer (Takara), 4 μl 2.5 m
M dNTPs, Ex Taq DNA polymerase of 0.25μl (5 units / ml: Takara Co., Ltd.), [γ- ³³ P]
ATP-labeled 25 pmol 3'-Uncard oligo-dT primer and 25 pmol 5'-primer (N
o. 20, a 10-mer deoxyoligonucleotide having an arbitrary sequence of 5′-GATCTGACAC-3 ′
Primer). The PCR was performed under the following conditions. That is, 95 ° C. for 3 minutes, 40 ° C. for 5 minutes and 72 ° C. for 5 minutes as one cycle, and then 95 cycles of 0.5 minute, 40 ° C. for 2 minutes and 72 ° C. for 1 minute for 40 cycles. Finally, the reaction was carried out at 72 ° C. for 5 minutes.

The PCR reaction samples were extracted with ethanol, resuspended in formamide sequencing dye, and reacted on a 6% acrylamide 7.5 M urea sequencing gel. The gel was dried without fixing and autoradiography was performed overnight.

(1-2) The sub cDNA of the amplified cDNA fragment
Cloning A mark containing radioactive ink was previously placed on a 3MM filter paper on which a dried gel had been placed, and a gel containing a band containing the cDNA of interest was cut out together with the autoradiogram to remove the gel together with the 3MM filter paper. 300μ
The mixture was stirred for 1 hour with 1 dH ₂ O. After removing the polyacrylamide gel and the filter paper,
μl of 10 mg / ml glycogen and 0.3 M NaO
Recollection by ethanol precipitation in the presence of Ac
Redissolved in μl dH ₂ O. 5 μl for reamplification
Of this solution was used. PCR conditions and primers were the same as for the first PCR. The re-amplified product of an appropriate size was recovered again as the first PCR product, and the PCR product was then purified from HUC of pUC118 vector (Takara).
Clone at the II site. The nucleic acid sequence is ABI37
7 Determined by an automatic sequencer (manufactured by Applied Biosystems).

According to the above method, fetal brain (F. brain), adult brain (A. brain), lung (Lung), liver (Liver), stomach (Sto
mRNA), using mRNA isolated from the pancreas, the testis, the prostate, the spleen, the spleen, the breast, the placenta, the placenta, the heart and the kidney. FIG. 1 shows the results of comparing different expression patterns.

From the figure, it can be seen that the P
CR amplification was performed, and one PCR product specifically expressed in kidney and liver was confirmed.

This P cloned and sequenced
The CR product was named TSC501a. This product is 1
It consisted of 16 nucleotides. FASTA program (Person WR, et al., Proc. Natl. Acad. Sci., U.
SA, 85 , 2444-2448 (1988)).
Comparison of the DNA sequence in the k / EMBL database with the nucleotide data revealed that the PCR product had no homology to any other known DNA sequence.

(1-3) Screening of cDNA A human normal kidney cDNA library was prepared using oligo (dT)-
Primed human normal kidney cDNA and Uni-ZAP ^™
It was constructed using XR (Stratagene). 1x1
0 the entire ^six clones were isolated by the above method,
The screening was performed using a cDNA fragment labeled with [α- ³² P] -dCTP. Positive clones were selected and their inserted cDNAs were cloned into pBluescript II SK
(-) Cut out in vivo.

As a result, about 50% of TSC501a
Zero plaques were identified. From this result, the amount of transcription between the total RNAs was calculated to be about 0.05%. T
The assembled cDNA sequence homologous to SC501 (TSC5
01) is 6 which encodes a protein having a 227 amino acid sequence.
It contained 960 nucleotides including an 81 nucleotide open reading frame.

The deduced amino acid sequence of the TSC501 protein has a signal motif at the N-terminal part (Leu ^55- Ala ⁷⁸ ) and a transmembrane domain at the C-terminal part (Gln ^198- Pro ^220). It has been revealed that the protein encoded by the gene of the present invention is a membrane protein.

In addition, comparisons with known proteins were made in public databases using the FASTA program. The result is shown in FIG.

FIG. 2 shows the amino acids of each protein represented by one letter and their sequences compared. In the figure, the same amino acids are highlighted.

As shown in the figure, the TSC501 protein was obtained from (1) gentamicin 3'-acetyltransferase (gen
tamicin 3'-acetyltransferase (AAC (3) -I); W. Wohlle
ben, et al., Mol. Gen. Genet., (1989), 217: 202-20
8, "AAC (3) -I" in the figure), (2) Ribosomal protein-alanine acetyltransferase (ribosom
al-protein-alanine acetyltransferase (Rim I); A. Y
oshikawa, et al., Mol. Gen. Genet. (1987), 209: 48
1-488, shown as “RimI” in the figure) and (3) streptothricin acetyltransferase
acetyltransferase (Sta T); S. Horinouchi, et al.,
J. Bacteriol., (1987), pp. 1929-1937.
T "), respectively, and were 29%, 26% and 27% identical, confirming significant identity. This means that T
This shows that the SC501 protein is a new member of the acetyltransferase family.

(2) Expression in Tissues To examine the expression profile of TSC501 in the tissues, Northern blot analysis using various human tissues was performed.

For Northern blot analysis, human MTN was used.
(Multiple-Tissue Northern) Blots I and II (Clontech) were used. The cDNA fragment consists of T3 and T
PC using a primer set of 7 promoter sequences
Labeled with [α- ³² P] -dCTP by R. The membrane containing the amplification product was pre-hybridized (conditions according to the product protocol), and then hybridization was performed according to the product protocol.

After hybridization, the washed membrane was exposed to autoradiography at -80 ° C. for 48 hours.

As human tissues, a heart (Heart) and a brain (Bra)
in), placenta (Placenta), lung (Lung), liver (Live
r), skeletal muscle (Sk. muscle), kidney (Kidney), pancreas (P
ancreas), spleen (Spleen), thymus (Thymus), prostate (Prostate), testis (Testis), ovary (Ovary), small intestine (Small int.) and colon (Colon). An approximately 1.0 kb transcript homologous to was specifically observed in kidney and liver. This result was consistent with the expression patterns seen in different expression methods (see FIG. 1).

[0089]

[Sequence List] (1) GENERAL INFORMATION: (i) APPLICANT: Ostuka Pharmaceutical Co., Ltd. (ii) TITLE OF INVENTION: TSC501 gene (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: PatentIn Release # 1.0, Version # 1.30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER : (B) FILING REFERENCE: B48JP (C) FILING DATE: 26-MAY-1998

(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 227 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi ) SEQUENCE DESCRIPTION: SEQ ID NO: 1: Met Ala Pro Cys His Ile Arg Lys Tyr Gln Glu Ser Asp Arg Gln Trp 1 5 10 15 Val Val Gly Leu Leu Ser Arg Gly Met Ala Glu His Ala Pro Ala Thr 20 25 30 Phe Arg Gln Leu Leu Lys Leu Pro Arg Thr Leu Ile Leu Leu Leu Gly 35 40 45 Gly Pro Leu Ala Leu Leu Leu Val Ser Gly Ser Trp Leu Leu Ala Leu 50 55 60 Val Phe Ser Ile Ser Leu Phe Pro Ala Leu Trp Phe Leu Ala Lys Lys 65 70 75 80 Pro Trp Thr Glu Tyr Val Asp Met Thr Leu Cys Thr Asp Met Ser Asp 85 90 95 Ile Thr Lys Ser Tyr Leu Ser Glu Arg Gly Ser Cys Phe Trp Val Ala 100 105 110 Glu Ser Glu Glu Lys Val Val Gly Met Val Gly Ala Leu Pro Val Asp 115 120 125 Asp Pro Thr Leu Arg Glu Lys Arg Leu Gln Leu Phe His Leu Phe Val 130 135 140 Asp Ser Glu His Arg Arg Gln Gly Ile Ala Lys Ala Leu Val Arg Thr 145 150 155 160 Val Leu Gln Phe Ala Arg Asp G ln Gly Tyr Ser Glu Val Ile Leu Asp 165 170 175 Thr Gly Thr Ile Gln Leu Ser Ala Met Ala Leu Tyr Gln Ser Met Gly 180 185 190 Phe Lys Lys Thr Gly Gln Ser Phe Phe Cys Val Trp Ala Arg Leu Val 195 200 205 Ala Leu His Thr Val His Phe Ile Tyr His Leu Pro Ser Ser Lys Val 210 215 220 Gly Ser Leu 225

(2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 684 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATGGCTCCTT GTCACATCCG CAAATACCAG GAGAGCGACC GCCAGTGGGT TGTGGGCTTG 60 CTCTCCCGGG GGATGGCCGA GCATGCCCCA GCCACCTTCC GGCAATTGCT GAAGCTGCCT 120 CGAACCCTCA TACTCTTACT TGGGGGGCCC CTCGCCCTAC TCCTGGTCTC TGGATCCTGG 180 CTTCTAGCCC TCGTGTTCAG CATCAGCCTC TTCCCTGCCC TGTGGTTCCT TGCCAAAAAA 240 CCCTGGACGG AGTATGTGGA CATGACATTG TGCACAGACA TGTCTGACAT TACCAAATCC 300 TACCTGAGTG AGCGTGGCTC CTGCTTCTGG GTGGCTGAGT CTGAAGAGAA GGTGGTGGGC 360 ATGGTAGGAG CTCTGCCTGT TGATGATCCC ACCTTGAGGG AGAAGCGGTT GCAGCTGTTT 420 CATCTCTTTG TGGACAGTGA GCACCGTCGT CAGGGGATAG CAAAAGCCCT GGTCAGGACT 480 GTCCTCCAGT TTGCCCGGGA CCAGGGCTAC AGTGAAGTTA TCCTGGACAC CGGCACCATC 540 CAGCTCTCTG CTATGGCCCT CTACCAGAGC ATGGGCTTCA AGAAGACGGG CCAGTCCTTC 600 TTCTGTGTGT GGGCCAGGCT AGTGGCTCTT CATACAGTTC ATTTC ATCTA CCACCTCCCT 660 TCTTCTAAGG TAGGGAGTCT G 681

(2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 960 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (cDNA) (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 169..849 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CTAGAATTCA GCGGCCGCTG AATTCTAGTC CTGGATGCCA GTGAGCGGCT GAGAGCTGAA 60 GCTCCCGAGA CACTCAGC TCTTGTGGTG ACAGTCTGAC GTAAAGGCGT GCAGGGAGGC 120 CTAGCTCTGT CTCCTGGACT TAGAGATTTC AGACACAGAA GTCTGTCC ATG GCT CCT 177 Met Ala Pro 1 TGT CAC ATC CGC AAA TAC CAG GAG AGC GAC CGC CAG TGG GTT GTG GGC GG Cys Hisg Grp Arg Gly 5 10 15 TTG CTC TCC CGG GGG ATG GCC GAG CAT GCC CCA GCC ACC TTC CGG CAA 273 Leu Leu Ser Arg Gly Met Ala Glu His Ala Pro Ala Thr Phe Arg Gln 20 25 30 35 TTG CTG AAG CTG CCT CGA ACC CTC ATA CTC TTA CTT GGG GGG CCC CTC 321 Leu Leu Lys Leu Pro Arg Thr Leu Ile Leu Leu Leu Gly Gly Pro Leu 40 45 50 GCC CTA CTC CTG GTC TCT GGA TCC TGG CTT CTA GCC CT C GTG TTC AGC 369 Ala Leu Leu Leu Val Ser Gly Ser Trp Leu Leu Ala Leu Val Phe Ser 55 60 65 ATC AGC CTC TTC CCT GCC CTG TGG TTC CTT GCC AAA AAA CCC TGG ACG 417 Ile Ser Leu Phe Pro Ala Leu Trp Phe Leu Ala Lys Lys Pro Trp Thr 70 75 80 GAG TAT GTG GAC ATG ACA TTG TGC ACA GAC ATG TCT GAC ATT ACC AAA 465 Glu Tyr Val Asp Met Thr Leu Cys Thr Asp Met Ser Asp Ile Thr Lys 85 90 95 TCC TAC CTG AGT GAG CGT GGC TCC TGC TTC TGG GTG GCT GAG TCT GAA 513 Ser Tyr Leu Ser Glu Arg Gly Ser Cys Phe Trp Val Ala Glu Ser Glu 100 105 110 115 GAG AAG GTG GTG GGC ATG GTA GGA GCT CTG CCT GTT GAT GAT CCC ACC 561 Glu Lys Val Val Gly Met Val Gly Ala Leu Pro Val Asp Asp Pro Thr 120 125 130 TTG AGG GAG AAG CGG TTG CAG CTG TTT CAT CTC TTT GTG GAC AGT GAG 609 Leu Arg Glu Lys Arg Leu Gln Leu Phe His Leu Phe Val Asp Ser Glu 135 140 145 CAC CGT CGT CAG GGG ATA GCA AAA GCC CTG GTC AGG ACT GTC CTC CAG 657 His Arg Arg Gln Gly Ile Ala Lys Ala Leu Val Arg Thr Val Leu Gln 150 155 160 TTT GCC CGG GAC CAG GGC TAC AGT GAA GTT ATC CTG GAC ACC GGC ACC 705 Phe Ala Arg Asp Gln Gly Tyr Ser Glu Val Ile Leu Asp Thr Gly Thr 165 170 175 ATC CAG CTC TCT GCT ATG GCC CTC TAC CAG AGC ATG GGC TTC AAG AAG 753 Ile Gln Leu Ser Ala Met Ala Leu Tyr Gln Ser Met Gly Phe Lys Lys 180 185 190 195 ACG GGC CAG TCC TTC TTC TGT GTG TGG GCC AGG CTA GTG GCT CTT CAT 801 Thr Gly Gln Ser Phe Phe Cys Val Trp Ala Arg Leu Val Ala Leu His 200 205 210 ACA GTT CAT TTC ATC TAC CAC CTC CCT TCT TCT AAG GTA GGG AGT CTG 849 Thr Val His Phe Ile Tyr His Leu Pro Ser Ser Lys Val Gly Ser Leu 215 220 225 TGA TCTCTTTCTG TGTGTATTGG TCAGAATAGA ATCCATTCAG CTGTAGCAGC 901

[Brief description of the drawings]

FIG. 1 is a photograph substituted for a drawing, showing the results of different mRNA expression in sub-cloning of the amplified cDNA fragment according to (1-2) of Example 1.

FIG. 2 is a diagram comparing the amino acid sequence homology between the expressed protein of the TSC501 gene of the present invention and a known protein.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12Q 1/68 C12P 21/08 // C12P 21/08 A61K 37/48 ADQ (C12N 15/09 ZNA C12R 1:91)

Claims (7)

[Claims] 1. A TSC501 gene comprising a nucleotide sequence encoding any of the following proteins (a) and (b): (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (b) a protein having the amino acid sequence represented by SEQ ID NO: 1
Or a protein comprising an amino acid sequence in which a plurality of amino acids are deleted, substituted or added, and having acetyltransferase activity 2. The TSC501 gene according to claim 1, wherein the nucleotide sequence is represented by SEQ ID NO: 2. 3. A TSC501 gene consisting of any one of the following polynucleotides (a) and (b): (i) a polynucleotide containing all or a part of the nucleotide sequence represented by SEQ ID NO: 3 (ii) a sequence DNA consisting of the base sequence represented by No. 3
Polynucleotides that hybridize under stringent conditions 4. The gene according to claim 1, which is a human gene. 5. The gene according to claim 3, which is a DNA fragment used as a specific probe or a specific primer for detecting the TSC501 gene. 6. A recombinant TSC5 of the following (a) and (b):
01 protein. (A) a protein consisting of the amino acid sequence represented by SEQ ID NO: 1; (b) a protein having the amino acid sequence represented by SEQ ID NO: 1
Or a protein comprising an amino acid sequence in which a plurality of amino acids are deleted, substituted or added, and having acetyltransferase activity [7] an antibody having a binding property to the TSC501 protein according to [6];