JPH04330279A - Polypeptide - Google Patents

Abstract

PURPOSE:To obtain a polypeptide having a base sequence or aminoacylase I activity necessary for the genetic diagnosis or treatment of pulmonary small cell carcinoma and nephrocarcinoma by clarifying the gene structure and amino acid sequence of aminoacylase I. CONSTITUTION:A polypeptide having the amino acid sequence expressed by the sequence number 1 or 2 of the sequence table and exhibiting porcine aminoacylase I or human aminoacylase I activity or a base sequence coding the polypeptide and expressed by the sequence number 6 or 7 of the sequence table. A probe and primer can be prepared based on the DNA sequence and an antibody can be prepared based on the amino acid sequence.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a polypeptide having the amino acid sequence of aminoacylase I and its DNA sequence.

0002

[Prior art] Aminoacylase I [N-acylam
ino acid aminohydrolase,
(EC3.5.1.14.)] is an enzyme that hydrolyzes N-acylamino acids into organic acids and amino acids. It is widely present in animal tissues, molds, and bacteria, and in mammals, it has particularly high activity in the kidneys. The existence of this enzyme has been known for over a century, and its enzymatic properties are well known. For example, the substrate specificity of this enzyme is high, and only L-amino acids are used as substrates. Therefore, it is used on a laboratory or industrial scale to optically resolve L-amino acids from chemically synthesized racemic N-acylamino acids [New York City, John Wiley & Sons, Inc., 1961, Green. Stein J. M.
(Greenstein, J.M.) and Winitz, M., Chemistry of the Amin.
o Acids), Vol. 2, pp. 1753-1816]. Structurally, the molecular weight is 46,000 ± 2,000 Da (sodium lauryl sulfate-polyacrylamide electrophoresis method), 98,000 ± 5,000 Da (sedimentation equilibrium method),
It is presumed that it exists in the form of a dimer consisting of two identical subunits [Heese, D., Loffler, H.; G. (Loeffler, H.G.
) and Rohm K. H. (Roehm, K.H.), Biological Chemistry Hopf-Seiler (
Biol. Chem. Hoppe-Seyler
), Volume 369, Pages 559-566 (1988)]
. By the way, in small cell lung cancer and kidney cancer, human
Chromosomal deletions are frequently found in the p14-p23 region of the short arm of the chromosome. It is presumed that this enzyme is encoded in this region, and it has been confirmed that this enzyme activity is actually lost in certain small lung cell carcinoma cell lines [Miller Y. et al. E. (Miller,
Y. E. ), Kao B. (Kao, B.) and Gazder A. (Gazder, A.), American
Journal of Human Genetics (Amer.J.Hum.Genet.), No. 4
Volume 1, page A32 (1987)]. As described above, aminoacylase I is an extremely effective means for optical resolution of racemized amino acids. Furthermore, the DNA sequence encoding human aminoacylase I is considered to be useful as a marker for small cell lung cancer or renal cancer in the genetic diagnosis or treatment of the same cancer.

0003

[Problem to be Solved by the Invention] Aminoacylase I is widely distributed in animal tissues, and has been particularly purified as an enzyme from pig kidney [Henseling J. et al. ( Henselin
g, J. ) and Rohm K. H. , Biochimica e biophysica acta (Biochim.
Biophys. Acta), Volume 959, No. 3
70-377 (1988)] and is also commercially available. However, its genetic structure and amino acid sequence are still unknown. Furthermore, an industrially advantageous method for producing aminoacylase I is not disclosed. The purpose of the present invention is to clarify the gene structure and amino acid sequence of aminoacylase I, and to provide a nucleotide sequence necessary for genetic diagnosis or treatment of small cell lung cancer or renal cancer, and a polypeptide having aminoacylase I activity. There is a particular thing.

0004

[Means for Solving the Problems] To summarize the present invention, the first invention of the present invention is characterized by having an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. The present invention relates to a polypeptide having aminoacylase I activity. Furthermore, the second invention of the present invention relates to a base sequence encoding the polypeptide of the first invention.

The present invention will be explained in detail below. A known method can be used to clone the cDNA encoding aminoacylase I. For example, RN containing poly(A) is extracted from the kidney where aminoacylase I is particularly abundant.
A is extracted and purified using a cellulose carrier bound with oligo(dT). Using this as a template, reverse transcriptase is used to synthesize cDNA, using the Okayama-Berg method or the Gubler-Hoffman method (Gubl.
er-Hoffmann method), connect it to a plasmid or phage vector, introduce it into a host, and c
Create a DNA library. Such libraries are also commercially available, and can be purchased, for example, from Clonetech. In order to screen a cDNA clone encoding the desired aminoacylase I from a cDNA library, first, aminoacylase I
Determining the partial amino acid sequence of and deducing the synthetic D
NA probes must be created. To determine the partial amino acid sequence, first, purified aminoacylase I
The peptide is hydrolyzed by the action of a highly specific protein hydrolase, and the peptide is separated and purified using reversed-phase HPLC. It is effective to determine this by performing amino acid sequence analysis using the Edman degradation method. There are two methods for designing synthetic DNA probes from this partial amino acid sequence. One method is to synthesize all possible combinations of sequences. The other method is to synthesize and use long DNA using frequently used codons that have been investigated so far. In addition, if the base sequence of a cDNA encoding a certain type of aminoacylase I has been determined, a cDNA encoding an aminoacylase I of another origin may be used.
In order to screen NA clones, the cDNA of aminoacylase I whose sequence has already been revealed is used.
It is effective to use the A fragment as a probe. D.N.
The A probe may be chemically synthesized or purified by cutting it out with a restriction enzyme from a vector containing cDNA. To screen a library with a DNA probe, first the library is amplified on a plate, the grown colonies or plaques are transferred to a nitrocellulose or nylon filter, and the DNA is denatured by denaturation.
Fix A to the filter. This filter is incubated in a solution containing a DNA probe previously labeled with 32P, etc., and the DNA on the filter and probe D
A hybrid is formed with NA (hereinafter, this operation is referred to as hybridization). The incubation temperature is set based on the Tm (melting temperature) of the probe used. After hybridization, nonspecific adsorption is washed away and clones that have hybridized to the probe are identified by autoradiography. This operation is repeated to isolate clones for the next analysis. If the recombinant is Escherichia coli, culture a small amount in a test tube, extract the plasmid using a conventional method, perform a cleavage reaction with restriction enzymes, and perform agarose or acrylamide gel electrophoresis to extract the cloned insert fragment. Examine the generation of. Furthermore, the electrophoresis pattern is transferred to a nitrocellulose or nylon membrane, and hybridization is performed using the method described above to examine whether the inserted fragment forms a hybrid with the DNA probe. Finally, the base sequence of the inserted fragment is determined by a known method. When the recombinant is a phage, the clones are analyzed using basically the same steps. A previously cultured host E. coli is infected with the cloned phage, and phage DNA is prepared from the lysate. For specific preparation methods for phage DNA, see, for example, Section 100 of Sekibiochemistry Experiment Course 1 "Gene Research Methods II".
Page (Tokyo Kagaku Dojin Publishing). Phage D
The inserted fragment is confirmed by cutting the NA with a restriction enzyme and subjected to gel electrophoresis, and further, hybridization with the probe DNA is examined. Finally, the clone is confirmed by determining the base sequence. By comparing the determined base sequence with the amino acid sequence of the peptide fragment of aminoacylase I, C-terminal analysis, amino acid composition analysis, molecular weight, etc., the gene structure and amino acid sequence can be determined. In addition, the structural gene of the obtained cDNA is connected to an expression vector so that it can be expressed in a suitable host cell, for example, yeast, and by introducing it into the host cell and culturing it, a polypeptide having aminoacylase activity can be obtained. can be produced. Expression can be confirmed by measuring the activity using a conventional aminoacylase I activity assay method. For example, 2-acetamidoacrylic acid, L-alanine dehydrogenase, β-nicotinamide adenine dinucleotide disodium salt (NADH
), for example, a recombinant yeast cell extract is added to an aminoacylase I activity measurement system. At this time, 2-acetamidoacrylic acid is hydrolyzed by aminoacylase I and then deaminated to produce pyruvic acid and ammonia. Aminoacylase I activity can be measured by spectrophotometrically measuring the decrease in NADH consumed when L-alanine dehydrogenase synthesizes alanine. Conventional chromatographic techniques are used to purify aminoacylase I polypeptides from cultures of transformants. That is, for example, cultured bacterial cells are disrupted to obtain a supernatant. This can be fractionated by salting out using ammonium sulfate, and then the desired polypeptide can be obtained by chromatography such as hydrophobic, ion exchange, and gel filtration. Based on the above, the present invention has revealed the base sequence and primary structure of aminoacylase I, and provides a base sequence for genetic diagnosis of small cell lung cancer or kidney cancer, as well as a method for producing aminoacylase I using genetic engineering. It became possible.

[0006]

[Examples] Next, examples of the present invention will be shown, but these are not intended to limit the present invention.

Example 1 Porcine aminoacylase IcD
NA cloning (1-1) Screening from cDNA library <Identification and isolation of positive clones> Pig kidney poly(A
) RNA was obtained from Clonetech (USA) (
Code number 6683). From 2 μg of this poly(A) RNA, cDNA synthesis kit manufactured by Amersham (Code number R) was used.
P.N. 1256), cDNA was synthesized using an oligo(dT) primer. Next, cDNA was extracted in a cell-free system using Amersham's cDNA cloning system λgt10 (code number PRN.1257).
was integrated into the EcoRI site of λgt10 and packaged into lambda phage to prepare a cDNA library. However, the EcoRI linker represented by SEQ ID NO: 3 in the sequence listing was manufactured by Takara Shuzo Co., Ltd., and GIGAPACK GOLD manufactured by Stratagene (USA) was used for packaging. The cDNA library was used as a host strain in C600.
Using the hfl strain, approximately 16,000 plaques were formed on each of eight 14 cm x 10 cm square petri dishes. That is, C6 in L medium containing 4 mg/ml maltose.
0.1 ml of phage solution was mixed with 0.2 ml of a culture solution obtained by culturing 00hfl overnight at 37°C, and the mixture was incubated at 37°C for 15 minutes. To this, add agarose to soft agar (L medium to a final concentration of 0.6%), autoclave it,
(maintained at 0°C) was added, spread on an L-plate, and after solidification was kept at 37°C for about 10 hours to form phage plaques (hereinafter, this operation is abbreviated as plating). Next, two hybridization filters were prepared from this plate. That is, a nylon membrane made by Amersham [
product name Hybond-N] for 30 seconds, and then mixed with 0.5M NaOH, 1.5M N
5 minutes on filter paper soaked in a solution of aCl (denaturing), 0.5
M Tris-HCl buffer (pH
7.0), after treatment for 5 minutes (neutralization) on a filter paper soaked in a solution of 1.5M NaCl,
7.53 g of sodium citrate and 8.82 g of sodium citrate dissolved in 1 liter of water] and dried on filter paper (hereinafter this treatment will be abbreviated as filter treatment). The second filter treatment was performed with a contact time of 2 minutes between the plate and the nylon membrane. The filter was irradiated with a UV lamp for 5 minutes to immobilize the DNA. As a hybridization probe, pig kidney aminoacylase I
A mixture of 17 base synthetic DNAs (represented by SEQ ID NO: 4 in the sequence listing) deduced from the partial amino acid sequence of Sigma Corporation (Sigma) was used. The partial amino acid sequence was determined using a gas-phase peptide sequencer after the purified pig kidney aminoacylase I was digested with protein hydrolase Achromobacter protease I and fractionated by HPLC. SEQ ID NO: 5 in the sequence listing is the amino acid sequence. This synthetic DNA50
Mega label kit made by Takara Shuzo Co., Ltd. (code number 607)
0) and labeled with 32P using 8.6 x 108 cpm
Probe DNA with a specific activity of /μg was obtained. Using the entire amount of this probe and the filter prepared above, 6×SSC,
1% SDS 100μg/ml herring sperm DNA, 5x
Hybridization was carried out overnight at 45° C. in approximately 100 ml of a solution containing Denhardt's (containing bovine serum albumin, polyvinylpyrrolidone, and Ficoll at a concentration of 0.1% each). The filters were then washed for 10 minutes in 6x SSC at room temperature. After further washing twice for 10 minutes in 2×SSC at 40°C,
The filter was transferred onto filter paper to remove excess water. This was attached to Whatman 3MM filter paper, and an intensifying screen was applied to perform autoradiography at -70°C overnight. As a result, a total of 51 positive signals were obtained. Plaques at positions corresponding to these signals were placed on the agar at 0.00%.
2 ml of SM solution [NaCl 5.8 g, MgSO4
・7H2O 2g, 1M Tris-HCl buffer (
50 ml of 2% gelatin (pH 7.5) was dissolved in water to make a total volume of 1 liter] and suspended. Ten phages were appropriately selected from among these phage solutions and subjected to the following operations. These phage solutions were diluted and plated (approximately 300 plaques/φ9 cm round petri dish), and the same operation as above was performed (hereinafter abbreviated as secondary screening). As a result, a single plaque could be isolated for all clones. These clones were λpkA
They were named mA 1-10. (1-2) Base sequence analysis <Preparation of λDNA> The cloned phage was cultured in liquid using E. coli strain L87 as a host bacterium (Gene Research Methods II, p. 100, Tokyo Kagaku Dojin Publishing). This allows approximately 40 μg of λ to be prepared from 40 ml of culture solution.
I got the DNA. <Identification and extraction and purification of inserted fragment> The above prepared DNA 20
μg was incubated at 37°C for 1.5 hours with 120 units of EcoRI in 100 μl of 1× EcoRI buffer (composition listed in the Takara Shuzo Genetic Engineering Reagent Catalog), and then RNase A was added to a final concentration of 50 μg/ml for 10 minutes.
It was kept warm for minutes. Take out 5 μl from this reaction solution, 1.
The size of the inserted fragment was determined by electrophoresis on 0% agarose gel. As a result, it was found that a fragment with a size of 0.6 to 1.4 kb was inserted. Among these, λpkAmA 10 having the longest insert of 1.4 kb was selected and subjected to the following operation. The entire amount of the remaining reaction solution that had been previously digested with EcoRI was electrophoresed on a 1.0% agarose gel. After electrophoresis, the gel was stained with a 1 μg/ml ethidium bromide solution for 10 minutes, and then a portion containing the desired inserted fragment was cut out from the gel under ultraviolet irradiation. This was extracted and purified using Takara Shuzo Co., Ltd.'s EASYTRAP kit to obtain about 200 ng of a 1.4 kb DNA fragment. <Identification of restriction enzyme site of insert fragment and base sequence determination>λ
50 ng of insert of pkAmA 10 and M13mp18
(Takara Shuzo) RF DNA digested with EcoRI 50
ng ligation kit (Takara Shuzo code number 6)
Ligation was performed using 021). E. coli JM109 was transformed using a portion of this ligation reaction solution, and X-Gal (5-bromo-4-chloro-3-
0.2 ml of culture solution obtained by culturing JM109 overnight in L medium on soft agar containing indolyl-β-D-galactoside) and IPTG (isopropyl-β-D-thiogalactoside).
and spread it on an L-plate. This plate was incubated at 37° C. overnight and the white plaques formed were selected to obtain recombinants in which the fragment had been inserted. The obtained white plaques were cultured in liquid using Escherichia coli JM109 as the host bacteria, and RF DNA was prepared by the alkaline lysis method [Reference: 1982, published by Cold Spring Harbor Laboratory, T. Maniastis (T. Mani)
Molecular Cloning, A Laboratory Manual (MolecularC) et al.
loning, A Laboratory Ma
nual), page 368]. A part of this RF DNA was digested with EcoRI, and it was confirmed by 1% agarose gel electrophoresis that a 1.4 kb fragment had been inserted. Next, we examined the orientation of the inserted fragment and the presence of restriction enzyme sites. A portion of the obtained RF DNA was digested with several types of restriction enzymes and analyzed by 1% agarose gel electrophoresis. As a result, the restriction enzymes KpnI, SacI, StuI, P
It was found that one or two sites such as stI, Hind III, Hinc II, etc. were present. Also, M1
It was also found that clones were obtained in which a 1.4 kb fragment was inserted in the opposite direction to the vector DNA of 3mp18. MpkAmA for one direction
10-2, the other one MpkAmA 10-5
It was named. Next, in order to determine the entire nucleotide sequence of the 1.4 kb insert, MpkAmA 1
0-2, MpkAmA 10-5, D of various lengths
A derivative lacking NA was produced. First, MpkAmA
10-2 with KpnI, StuI and SmaI, PstI
, Hind III, MpkAmA 10-5 as Hi
ndIII, PstI, SacI, and KpnI, respectively, and cut at the restriction enzyme site within the multi-cloning site and the site within the inserted fragment to delete DNA of various sizes. Next, these vector DNAs were subjected to self-ligation and then introduced into E. coli JM109 to obtain a transformant. These derivative clones and MpkA
Single strand DNA (ssDNA) was prepared by culturing mA 10-2 and MpkAmA 10-5, and DNA sequencing was performed by the dideoxy method. As a result, the nucleotide sequence of the inserted fragment of λpkAmA 10 was determined. The results are shown in Figure 1. That is, FIG. 1 is a diagram showing a restriction enzyme map of porcine aminoacylase I cDNA, as well as portions where the base sequence has been determined and their orientations. This cD
From the results of the NA base sequence analysis, the entire base sequence and amino acid sequence of the porcine aminoacylase I constituent gene of SEQ ID NO: 6 in the sequence listing was determined. That is, SEQ ID NO: 6 is cDNA
The nucleotide sequence of one example of aminoacylase I and the corresponding amino acid sequence obtained from nucleotide sequence analysis are shown.

Example 2 Human aminoacylase IcD
NA Cloning (2-1) Screening from cDNA Library Human liver poly(A) RNA was obtained from Clontech. A human liver-λgt10-cDNA library was prepared from 2 μg of this poly(A) RNA in the same manner as in Example 1, and a filter was prepared. As a probe DNA for hybridization, λpkAmA 1
NcoI-StuI fragment (approximately 530 bp) in the 0 insert
) was used. That is, porcine aminoacylase IcDN
MpkAmA 10-2 DNA containing A was converted into Nco
After digestion with I and StuI, agarose gel electrophoresis was performed. After electrophoresis, the gel was stained with a 1 μg/ml ethidium bromide solution for 10 minutes, and then a portion containing the desired DNA fragment was excised from the gel under ultraviolet irradiation. This was extracted and purified using an Easy Trap kit. The obtained fragments were used with a random primer DNA labeling kit (Takara Shuzo Co., Ltd.).
It was labeled with 32P using code number 6045) and used for hybridization. Hybridization was performed under the same conditions as in Example 1. However, the temperature of hybridization was 65°C.
The final washing was carried out twice for 30 minutes in 0.2×SSC at 55°C. As a result, three positive signals were obtained. These plaques were subjected to secondary screening and isolated. This clone was named λhAmA1-3. (2-2) Base sequence analysis Prepare λDNA for the above three clones, and
After digestion, the size of the inserted DNA fragment was examined. Result 1
．． It was found that a DNA fragment of 1 to 1.4 kb was inserted. λhA with the longest insert of 1.4 kb
Excise the insert fragment from mA3 with EcoRI and insert it into M13m.
It was incorporated into p18. The obtained clones in both directions were named MhAmA3-2 and MhAmA3-3, respectively. Next, in order to determine the entire base sequence of the 1.4 kb inserted fragment, MhAmA3-2, MhAmA3
-3, derivatives with various sizes of DNA deleted were prepared. First, each RFDNA was transformed into Hinc II, Pst
Each DNA was digested with I and cut at the restriction enzyme site within the multi-cloning site and the site within the inserted fragment to delete DNA of various sizes. Next, these vector DN
After self-ligation of A, E. coli JM109
to obtain transformants. Also, Takara (TaKaR)
a) Delation kit for kilosequencing (Takara Shuzo)
Using this method, the inserted fragments of MhAmA3-2 and MhAmA3-3 were decomposed from the side of the annealing position of the sequencing primer to produce derivatives in which DNA of various sizes were deleted. MhAmA3-2 and MhAmA3-3 are cultured with appropriate ones of these derivatives.
sDNA was prepared and DNA sequencing was performed using the dideoxy method. As a result, the base sequence of the inserted fragment of λhAmA3 was determined. The results are shown in FIG. That is, FIG. 2 is a diagram showing the restriction enzyme map of human aminoacylase I cDNA, the portions where the base sequence was determined, and their orientations. From the results of this cDNA base sequence analysis, the entire base sequence and amino acid sequence of the human aminoacylase I constituent gene was determined. The results are shown in SEQ ID NO: 7 in the sequence listing. That is, SEQ ID NO: 7 shows the base sequence of an example of aminoacylase I obtained by cDNA base sequence analysis and the corresponding amino acid sequence.

Example 3 Construction of a plasmid expressing aminoacylase I polypeptide λpkAmA 10 or λhAmA
The part encoding aminoacylase I in the 3-insert fragment was cut out and pJM, an E. coli-yeast shuttle vector containing a yeast alcohol dehydrogenase promoter, was extracted.
124 [Reference: The Embo Journal (The
EMBO Journal), Volume 8, No. 2067
(1989)] (named pYAmA101 and pYhAmA 101, respectively)
was built. (3-1) Construction of porcine aminoacylase I-like polypeptide expression plasmid pYAmA101
MpkAmA 10-2RF DNA incorporated into 18,
It was digested with restriction enzymes NcoI and Eco47III, and the released DNA fragment of approximately 1230 bp was separated and purified. And a DNA blunting kit (Blunti)
ng Kit) (Takara Shuzo code number 6025) to blunt the NcoI protruding end. This was inserted into plasmid pJM124 which was digested with BamHI to remove the BamHI insert fragment and made into blunt ends. The obtained plasmid was named pYAmA101. That is, plasmid pYAmA101 encodes a polypeptide of 407 amino acid residues of porcine aminoacylase I downstream of the yeast alcohol dehydrogenase promoter. This plasmid pYAmA101 was transferred to yeast BJ
2168 [MATa, ura3-52, leu2, tr
p1, prc-407, prb1-1122, pep4
-3], or DKD-5D-H [MATa, leu
2-3, leu2-112, trp1, his3] using an alkali metal treatment method [Reference: Journal of Bacte.
riology) Volume 153, Page 163 (1983
)]. The yeast BJ2168 introduced with pYAmA 101 was transformed into Saccharomyces c
erevisiae BJ2168/pYAmA 10
1 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology [FERM P-12
002)]. (3-2) Construction of human aminoacylase I-like polypeptide expression plasmid pYhAmA 101 λhAmA3
Mh in which the EcoRI insert of
AmA3-3 RF DNA was digested with restriction enzymes SacI, E
After digestion with coRI, a DNA fragment of about 1360 bp released was separated and purified. And DNA blunting
The protruding ends were blunted using a kit (Takara Shuzo code number 6025). This is plasmid pJM124
was digested with BamHI to remove the BamHI insert fragment,
It was incorporated into a blunt-ended version. The resulting plasmid was named pYhAmA101. That is, plasmid p
YhAmA101 is a promoter of human aminoacylase I downstream of the yeast alcohol dehydrogenase promoter.
It encodes a polypeptide of 8 amino acid residues. This plasmid pYhAmA101 was transferred to yeast BJ2168 [MAT
a, ura3-5, leu2, trp1, prc-40
7, prb1-1122, pep4-3], or D
KD-5D-H [MATa, leu2-3, leu2-
112, trp1, his3] using an alkali metal treatment method [
Literature: Journal of Bacteriology, No. 1
53, p. 163 (1983)]. Yeast BJ2168 introduced with pYhAmA101 was transformed into S
accharomyces cerevisiae BJ
It was designated as 2168/pYhAmA101 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology.
No. 3 (FERM P-12003)].

Example 4 Expression of aminoacylase I polypeptide in yeast Saccharomyces c obtained in Example 3
erevisiae BJ2168/pYAmA 10
1 (FERM P-12002) or Sacc
haromyces cerevisiae BJ21
68/pYhAmA101 (FERM P-1200
3) with 20 μg/ml L-tryptophan, 30 μg
5 ml SD medium [0.67% Bacto yeast Nitrogen base w/o
Amino acid (Difco), 2% dextrose] was inoculated and cultured at 30°C for two nights to collect bacteria. Discard the supernatant, wash the bacterial cells with 2 ml of water, centrifuge, and add 400 μl of 0.2 M sodium phosphate buffer (
pH 7.2). An equal volume of glass beads (diameter 0.35 to 0.5 mm) was added to this, and the cells were crushed by stirring vigorously while cooling on ice. Glass beads and cell debris were removed by centrifugation, and the supernatant was collected and used as a yeast extract. The aminoacylase I activity of the extract thus obtained was measured by the acetamidoacrylic acid method [Reference: Analytical Biochemistry (Analytic)
al Biochemistry), Volume 165, Page 142 (1987)]. This will be explained in detail below. First, reaction buffer (40mM phosphoric acid, 40mM acetic acid, 40mM
(containing boric acid, 0.08mM cobalt chloride, 20mM ammonium sulfate, and adjusting the pH to 7.6 with sodium hydroxide) was prepared. Next, a substrate solution (1 mmol of 2-acetamidoacrylic acid dissolved in 1.2 ml of 1N sodium hydroxide aqueous solution and 0.8 ml of reaction buffer added), NADH solution (3. NADH dissolved to 2mM), 30U/ml
An L-alanine dehydrogenase solution was prepared. 700 μl of reaction buffer, 100 μl of substrate solution, 50 μl of NADH solution, 50 μl of L-alanine dehydrogenase solution
After mixing and preheating at 37°C for about 5 minutes, 100 μl of the above extract was added and kept at 37°C for 30 minutes. At this time, aminoacylase I activity was measured by measuring the decrease in absorption at a wavelength of 340 nm by NADH using a spectrophotometer. The enzyme activity was quantified by comparing it with that measured in the same manner using pig kidney aminoacylase I (Sigma) whose enzyme titer was known. As a result of aminoacylase I activity measurement, 100 μl of each of the previously prepared extracts had approximately 1 unit of aminoacylase I activity. In addition,
Yeast BJ2168, which does not carry the plasmid, was treated in the same manner, but almost no aminoacylase activity was observed (0.03 units/100 μl or less of extract).

[0011]

EFFECTS OF THE INVENTION Based on the above results, the present invention has revealed the amino acid sequences of porcine and human aminoacylase I and their DNA sequences, and provided a genetic engineering method for producing aminoacylase I polypeptides. In addition, by providing a target for investigating the presence or expression of aminoacylase I in living organisms, it is now possible to create probes and primers based on this DNA sequence, and to create antibodies based on the amino acid sequence. became possible.

[Sequence list] Sequence number: 1 Sequence length: 407 Sequence type: Number of amino acid chains: Single chain Topology: Linear sequence type: Peptide sequence characteristics: 1-407 Porcine aminoacylase I sequence: Met Ala Ser Lys Gly
Arg Glu Gly Glu His Pro S
er Val Thr Leu 1
5
10
15 Phe Arg Gln Tyr L
eu Arg Ile Arg Thr Val Gl
n Pro Glu Pro Asp
20
25
30 Tyr Gly Ala Al
a Val Ala Phe Leu Glu Glu
Arg Ala Arg Gln Leu
35
40
45 Gly Leu Gly
Cys Gln Lys Val Glu Val
Val Pro Gly His Val Val
50
55
60 Thr Val
Leu Thr Trp Pro Gly Thr A
sn Pro Thr Leu Ser Ser Il
e 65
70
75 Leu L
eu Asn Ser His Thr Asp Va
l Val Pro Val Phe Lys Glu
His
80 85
90Tr
p Ser His Asp Pro Phe Glu
Gly Phe Lys Asp Ala Asp
Gly Tyr
95 10
0 105
Ile Tyr Gly Arg Gly Ala
Gln Asp Met Lys Cys Val S
er Ile Gln
110
115 120
Tyr Leu Glu Ala Val A
rg Arg Leu Lys Val Glu Gl
y His His Phe
125
130 1
35 Pro Arg Thr Ile Hi
s Met Thr Phe Val Pro Asp
Glu Glu Val Gly
140
145
150 Gly His Gln Gly
Met Glu Leu Phe Val Lys
Arg Pro Glu Phe Gln
155
160
165 Ala Leu Arg
Ala Gly Phe Ala Leu Asp G
lu Gly Leu Ala Ser Pro
170
175
180 Thr Asp A
la Phe Thr Val Phe Tyr Se
r Glu Arg Ser Pro Trp Trp
185
190
195 Leu Ar
g Val Thr Ser Thr Gly Lys
Pro Gly His Gly Ser Arg
Phe 2
00 205
210 Ile
Glu Asp Thr Ala Ala Glu
Lys Leu His Lys Val Ile A
sn Ser
215 220
225
Ile Leu Ala Phe Arg Glu L
ys Glu Lys Gln Arg Leu Gl
n Ser Asn
230
235 240
Gln Leu Lys Pro Gly Al
a Val Thr Ser Val Asn Leu
Thr Met Leu
245
250 25
5 Glu Gly Gly Val Ala
Tyr Asn Val Val Pro Ala
Thr Met Ser Ala
260
265
270 Cys Phe Asp Phe
Arg Val Ala Pro Asp Val A
sp Leu Lys Ala Phe
275
280
285 Glu Glu Gln L
eu Gln Ser Trp Cys Gln Al
a Ala Gly Glu Gly Val
290
295
300 Thr Phe Gl
u Phe Val Gln Lys Trp Met
Glu Thr Gln Val Thr Ser
305
310
315 Thr Asp
Asp Ser Asp Pro Trp Trp
Ala Ala Phe Ser Gly Val P
he 32
0 325
330 Lys
Asp Met Lys Leu Ala Leu G
lu Leu Glu Ile Cys Pro Al
a Ser
335 340
345 T
hr Asp Ala Arg Tyr Ile Ar
g Ala Ala Gly Val Pro Ala
Leu Gly
350 3
55 360
Phe Ser Pro Met Asn His
Thr Pro Val Leu Leu His
Asp His Asp
365
370 375
Glu Arg Leu His Glu
Ala Val Phe Leu Arg Gly V
al Asp Ile Tyr
380
385
390 Thr Gln Leu Leu S
Er Ala Ser Val Pr
o Ala Leu Pro Ser
395
400
405 Glu Ser SEQ ID NO: 2 Sequence length: 408 Sequence type: Number of amino acid chains: Single chain Topology: Linear sequence type: Peptide sequence characteristics: 1-408 Human aminoacylase I sequence: Met Thr Ser Lys Gly
Pro Glu Glu Glu His Pro S
er Val Thr Leu 1
5
10
15 Phe Arg Gln Tyr L
eu Arg Ile Arg Thr Val Gl
n Pro Lys Pro Asp
20
25
30 Tyr Gly Ala Al
a Val Ala Phe Phe Glu Glu
Thr Ala Arg Gln Leu
35
40
45 Gly Leu Gly
Cys Gln Lys Val Glu Val
Ala Pro Gly Tyr Val Val
50
55
60 Thr Val
Leu Thr Trp Pro Gly Thr A
sn Pro Thr Leu Ser Ser Il
e 65
70
75 Leu L
eu Asn Ser His Thr Asp Va
l Val Pro Val Phe Lys Glu
His
80 85
90Tr
p Ser His Asp Pro Phe Glu
Ala Phe Lys Asp Ser Glu
Gly Tyr
95 10
0 105
Ile Tyr Ala Arg Gly Ala
Gln Asp Met Lys Cys Val S
er Ile Gln
110
115 120
Tyr Leu Glu Ala Val A
rg Arg Leu Lys Val Glu Gl
y His Arg Phe
125
130 1
35 Pro Arg Thr Ile Hi
s Met Thr Phe Val Pro Asp
Glu Glu Val Gly
140
145
150 Gly His Gln Gly
Met Glu Leu Phe Val Gln
Arg Pro Glu Phe His
155
160
165 Ala Leu Arg
Ala Gly Phe Ala Leu Asp G
lu Gly Ile Ala Asn Pro
170
175
180 Thr Asp A
la Phe Thr Val Phe Tyr Se
r Glu Arg Ser Pro Trp Trp
185
190
195 Val Ar
g Val Thr Ser Thr Gly Arg
Pro Gly His Ala Ser Arg
Phe 2
00 205
210 Met
Glu Asp Thr Ala Ala Glu
Lys Leu His Lys Val Val A
sn Ser
215 220
225
Ile Leu Ala Phe Arg Glu L
ys Glu Trp Gln Arg Leu Gl
n Ser Asn
230
235 240
Pro His Leu Lys Glu Gl
y Ser Val Thr Ser Val Asn
Leu Thr Lys
245
250 25
5 Leu Glu Gly Gly Val
Ala Tyr Asn Val Ile Pro
Ala Thr Met Ser
260
265
270 Ala Ser Phe Asp
Phe Arg Val Ala Pro Asp V
al Asp Phe Lys Ala
275
280
285 Phe Glu Glu G
ln Leu Gln Ser Trp Cys Gl
n Ala Ala Gly Glu Gly
290
295
300 Val Thr Le
u Glu Phe Ala Gln Lys Trp
Met His Pro Gln Val Thr
305
310
315 Pro Thr
Asp Asp Ser Asn Pro Trp
Trp Ala Ala Phe Ser Arg V
al 32
0 325
330 Cys
Lys Asp Met Asn Leu Thr L
eu Glu Pro Glu Ile Met Pr
o Ala
335 340
345 A
la Thr Asp Asn Arg Tyr Il
e Arg Ala Val Gly Val Pro
Ala Leu
350 3
55 360
Gly Phe Ser Pro Met Asn.
Arg Thr Pro Val Leu Leu
His Asp His
365
370 375
Asp Glu Arg Leu His
Glu Ala Val Phe Leu Arg G
ly Val Asp Ile
380
385
390 Tyr Thr Arg Leu L
eu Pro Ala Leu Ala Ser Va
l Pro Ala Leu Pro
395
400
405 Ser Asp Ser Sequence number: 3 Sequence length: 10 Sequence type: Number of nucleic acid strands: Double-stranded topology: Linear sequence type: Other nucleic acids (synthetic DNA) Sequence characteristics: 1-10 linker Sequence: CCGAATTCGG 10 Sequence number: 4 Sequence length: 17 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acids (synthetic DNA) Hypothetical sequence: NO Antisense :NO Sequence characteristics: 1-17 probe sequence: TGGATGGARA CNCARGT 17 Sequence number: 5 Sequence length: 6 Sequence type: Number of amino acid chains: Single chain Topology: Linear sequence type: Peptide fragment type: Intermediate fragment (porcine aminoacylase I) Sequence number: 6 Sequence length: 1221 Sequence type: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Anti Sense: NO Sequence characteristics: 1-1221 CDS (porcine aminoacylase I) Sequence: ATG GCC AGC AAG GGT CG
C GAG GGC GAG CAC CCA TCC
GTG ACG CTC 45 Me
t Ala Ser Lys Gly Arg Glu
Gly Glu His Pro Ser Val
Thr Leu 1
5
10 15
TTC CGT CAG TAC CTG CG
C ATC CGC ACC GTC CAG CCT
GAG CCC GAC 90 Ph
e Arg Gln Tyr Leu Arg Ile
Arg Thr Val Gln Pro Glu
Pro Asp
20
25 30
TAC GGG GCT GCT GTG GC
C TTC CTT GAG GAG AGA GCC
CGC CAG CTG 135 Ty
r Gly Ala Ala Val Ala Phe
Leu Glu Glu Arg Ala Arg
Gln Leu
35
40 45
GGC CTG GGC TGT CAG AA
A GTG GAG GTG GTA CCT GGC
CAT GTG GTG 180 Gl
y Leu Gly Cys Gln Lys Val
Glu Val Val Pro Gly His
Val Val
50
55 60
ACC GTG CTG ACC TGG CC
G GGC ACC AAC CCC ACA CTC
TCC TCC ATC 225 Th
r Val Leu Thr Trp Pro Gly
Thr Asn Pro Thr Leu Ser
Ser Ile
65
70 75
TTG CTC AAC TCC CAC AC
A GAT GTG GTG CCT GTC TTC
AAG GAG CAT 270 Le
u Leu Asn Ser His Thr Asp
Val Val Pro Val Phe Lys
Glu His
80
85 90
TGG AGT CAT GAC CCC TT
T GAG GGC TTC AAG GAT GCA
GAT GGC TAC 315 Tr
p Ser His Asp Pro Phe Glu
Gly Phe Lys Asp Ala Asp
Gly Tyr
95
100 105
ATC TAT GGC AGG GGC GC
C CAG GAC ATG AAG TGC GTC
AGC ATC CAG 360 Il
e Tyr Gly Arg Gly Ala Gln
Asp Met Lys Cys Val Ser
Ile Gln
110
115 120
TAC CTG GAG GCT GTG AGG
AGG CTG AAG GTT GAG GGC
CAC CAT TTC 405 Tyr
Leu Glu Ala Val Arg Arg
Leu Lys Val Glu Gly His H
is Phe
125
130 135
CCC AGA ACC ATC CAC ATG
ACC TTT GTG CCA GAT GAG G
AG GTT GGA 450 Pro
Arg Thr Ile His Met Thr P
he Val Pro Asp Glu Glu Va
l Gly
140 1
45 150
GGT CAC CAA GGC ATG GAG C
TC TTT GTG AAG CGG CCC GA
G TTC CAG 495 Gly H
is Gln Gly Met Glu Leu Ph
e Val Lys Arg Pro Glu Phe
Gln
155 16
0 165 G
CC CTG AGG GCT GGC TTT GC
C CTG GAT GAA GGC CTA GCC
AGC CCC 540 Ala Le
u Arg Ala Gly Phe Ala Leu
Asp Glu Gly Leu Ala Ser
Pro
170 175
180AC
T GAC GCC TTC ACT GTC TTT
TAC AGT GAG CGG AGC CCC
TGG TGG 585 Thr Asp
Ala Phe Thr Val Phe Tyr
Ser Glu Arg Ser Pro Trp T
rp
185 190
195 CTAs
AGG GTC ACG AGC ACT GGG
AAG CCA GGG CAT GGC TCG C
GC TTC 630 Leu Arg
Val Thr Ser Thr Gly Lys P
ro Gly His Gly Ser Arg Ph
e
200 205
210 ATT
GAG GAC ACA GCA GCA GAG A
AG CTG CAC AAG GTC ATC AA
C TCT 675 Ile Glu A
sp Thr Ala Ala Glu Lys Le
u His Lys Val Ile Asn Ser
2
15 220
225 ATC C
TG GCT TTT CGG GAG AAG GA
G AAG CAG AGG CTG CAG TCA
AAC 720 Ile Leu Al
a Phe Arg Glu Lys Glu Lys
Gln Arg Leu Gln Ser Asn
23
0 235
240 CAG CT
G AAG CCG GGG GCT GTG ACC
TCC GTG AAC CTG ACT ATG
CTA 765 Gln Leu Lys
Pro Gly Ala Val Thr Ser
Val Asn Leu Thr Met Leu
245
250
255 GAG GGT
GGC GTG GCC TAT AAC GTC
GTT CCT GCC ACC ATG AGT G
CC 810 Glu Gly Gly
Val Ala Tyr Asn Val Val P
ro Ala Thr Met Ser Ala
260
265
270 TGC TTT
GAC TTC CGC GTA GCA CCG G
AT GTG GAC CTG AAG GCT TT
C 855 Cys Phe Asp P
he Arg Val Ala Pro Asp Va
l Asp Leu Lys Ala Phe
275
280
285 GAG GAG C
AG CTG CAG AGT TGG TGC CA
G GCA GCT GGC GAG GGG GTC
900 Glu Glu Gln Le
u Gln Ser Trp Cys Gln Ala
Ala Gly Glu Gly Val
290
295
300 ACC TTT G
AG TTT GTT CAG AAG TGG AT
G GAG ACG CAA GTG ACA TCT
945 Thr Phe Glu Ph
e Val Gln Lys Trp Met Glu
Thr Gln Val Thr Ser
305
310
315 ACT GAT GA
C TCA GAC CCC TGG TGG GCA
GCA TTT AGT GGG GTC TTC
990 Thr Asp Asp Ser
Asp Pro Trp Trp Ala Ala
Phe Ser Gly Val Phe
320
325
330 AAG GAT ATG
AAG CTT GCC CTA GAG CTA
GAG ATC TGC CCT GCT TCC
1035 Lys Asp Met Lys
Leu Ala Leu Glu Leu Glu I
le Cys Pro Ala Ser
335
340
345 ACT GAC GCC
CGC TAT ATT CGT GCG GCG G
GG GTT CCG GCT CTG GGC
1080 Thr Asp Ala Arg T
yr Ile Arg Ala Ala Gly Va
l Pro Ala Leu Gly
350
355
360 TTC TCA CCC A
TG AAC CAC ACA CCG GTG CT
G CTC CAT GAC CAT GAT
1125 Phe Ser Pro Met As
n His Thr Pro Val Leu Leu
His Asp His Asp
365
370
375 GAG CGG CTG CA
T GAG GCC GTG TTT CTC CGT
GGG GTT GAC ATA TAC 1
170 Glu Arg Leu His Glu
Ala Val Phe Leu Arg Gly
Val Asp Ile Tyr
380
385
390 ACT CAG CTG CTG
TCT GCC TTG GCC AGC GTG
CCC GCA CTG CCC AGT 12
15 Thr Gln Leu Leu Ser
Ala Leu Ala Ser Val Pro A
la Leu Pro Ser
395
400
405 GAA AGC

122
1 Glu Ser

Sequence number: 7 Sequence length: 1224 Sequence type: Number of nucleic acid strands: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Hypothetical sequence: NO Antisense: NO Sequence characteristics: 1- 1224 CDS (human aminoacylase I) sequence: ATG ACC AGC AAG GGT CC
C GAG GAG GAG CAC CCA TCG
GTG ACG CTC 45 Me
t Thr Ser Lys Gly Pro Glu
Glu Glu His Pro Ser Val
Thr Leu 1
5 10
15 TTC
CGC CAG TAC CTG CGT ATC
CGC ACT GTC CAG CCC AAG C
CT GAC 90 Phe Arg
Gln Tyr Leu Arg Ile Arg T
hr Val Gln Pro Lys Pro As
p 20
25
30 TAT GGA G
CT GCT GTG GCT TTC TTT GA
G GAG ACA GCC CGC CAG CTG
135 Tyr Gly Ala Al
a Val Ala Phe Phe Glu Glu
Thr Ala Arg Gln Leu
35
40
45 GGC CTG GGC TGT
CAG AAA GTA GAG GTG GCA
CCT GGC TAT GTG GTG 1
80 Gly Leu Gly Cys Gln
Lys Val Glu Val Ala Pro G
ly Tyr Val Val
50
55 6
0 ACC GTG TTG ACC TGG C
CA GGC ACC AAC CCT ACA CT
C TCC TCC ATC 225 T
hr Val Leu Thr Trp Pro Gl
y Thr Asn Pro Thr Leu Ser
Ser Ile
65 70
75 TT
G CTC AAC TCC CAC ACG GAT
GTG GTG CCT GTC TTC AAG
GAA CAT 270 Leu Leu
Asn Ser His Thr Asp Val
Val Pro Val Phe Lys Glu H
is 80
85
90 TGG AGT
CAC GAC CCC TTT GAG GCC T
TC AAG GAT TCT GAG GGC TA
C 315 Trp Ser His A
sp Pro Phe Glu Ala Phe Ly
s Asp Ser Glu Gly Tyr
95
100
105 ATC TAT GCC AG
G GGT GCC CAG GAC ATG AAG
TGC GTC AGC ATC CAG
360 Ile Tyr Ala Arg Gly
Ala Gln Asp Met Lys Cys
Val Ser Ile Gln
110
115 1
20 TAC CTG GAA GCT GTG
AGG AGG CTG AAG GTG GAG G
GC CAC CGG TTC 405
Tyr Leu Glu Ala Val Arg A
rg Leu Lys Val Glu Gly Hi
s Arg Phe
125 13
0 135 C
CC AGA ACC ATC CAC ATG AC
C TTT GTG CCT GAT GAG GAG
GTT GGG 450 Pro Ar
g Thr Ile His Met Thr Phe
Val Pro Asp Glu Glu Val
Gly 140
145
150 GGT CAC
CAA GGC ATG GAG CTG TTC
GTG CAG CGG CCT GAG TTC C
AC 495 Gly His Gln
Gly Met Glu Leu Phe Val G
ln Arg Pro Glu Phe His
155
160
165 GCC CTG AGG G
CA GGC TTT GCC CTG GAT GA
G GGC ATA GCC AAT CCC
540 Ala Leu Arg Ala Gl
y Phe Ala Leu Asp Glu Gly
Ile Ala Asn Pro
170
175
180 ACT GAT GCC TTC ACT
GTC TTT TAT AGT GAG CGG
AGT CCC TGG TGG 585
Thr Asp Ala Phe Thr Val
Phe Tyr Ser Glu Arg Ser P
ro Trp Trp
185 1
90 195
GTG CGG GTT ACC AGC ACT G
GG AGG CCA GGC CAT GCC TC
A CGC TTC 630 Val A
rg Val Thr Ser Thr Gly Ar
g Pro Gly His Ala Ser Arg
Phe 20
0 205
210 ATG GA
G GAC ACA GCA GCA GAG AAG
CTG CAC AAG GTT GTA AAC
TCC 675 Met Glu Asp
Thr Ala Ala Glu Lys Leu
His Lys Val Val Asn Ser
215
220
225 ATC CTG GCA
TTC CGG GAG AAG GAA TGG C
AG AGG CTG CAG TCA AAC
720 Ile Leu Ala Phe A
rg Glu Lys Glu Trp Gln Ar
g Leu Gln Ser Asn
230
235
240 CCC CAC CTG AAA GA
G GGG TCC GTG ACC TCC GTG
AAC CTG ACT AAG 765
Pro His Leu Lys Glu Gly
Ser Val Thr Ser Val Asn
Leu Thr Lys
245
250 255
CTA GAG GGT GGC GTG GCC
TAT AAC GTG ATA CCT GCC A
CC ATG AGC 810 Leu
Glu Gly Gly Val Ala Tyr A
sn Val Ile Pro Ala Thr Me
t Ser 2
60 265
270 GCC A
GC TTT GAC TTC CGT GTG GC
A CCG GAT GTG GAC TTC AAG
GCT 855 Ala Ser Ph
e Asp Phe Arg Val Ala Pro
Asp Val Asp Phe Lys Ala
275
280
285 TTT GAG GAG
CAG CTG CAG AGC TGG TGC
CAG GCA GCT GGC GAG GGG
900 Phe Glu Glu Gln
Leu Gln Ser Trp Cys Gln A
la Ala Gly Glu Gly
290
295
300 GTC ACC CTA GAG T
TT GCT CAG AAG TGG ATG CA
C CCC CAA GTG ACA 945
Val Thr Leu Glu Phe Al
a Gln Lys Trp Met His Pro
Gln Val Thr
305
310 315
CCT ACT GAT GAC TCA AAC
CCT TGG TGG GCA GCT TTT
AGC CGG GTC 990 Pro
Thr Asp Asp Ser Asn Pro
Trp Trp Ala Ala Phe Ser A
rg Val
320 325
330 TGC
AAG GAT ATG AAC CTC ACT C
TG GAG CCT GAG ATC ATG CC
T GCT 1035 Cys Lys A
sp Met Asn Leu Thr Leu Gl
u Pro Glu Ile Met Pro Ala
335
340
345 GCC ACT GA
C AAC CGC TAT ATC CGC GCG
GTG GGG GTC CCA GCT CTA
1080 Ala Thr Asp Asn
Arg Tyr Ile Arg Ala Val
Gly Val Pro Ala Leu
350
355
360 GGC TTC TCA CCC
ATG AAC CGC ACA CCT GTG C
TG CTG CAC GAC CAC 112
5 Gly Phe Ser Pro Met A
sn Arg Thr Pro Val Leu Le
u His Asp His
365
370 375
GAT GAA CGG CTG CAT GA
G GCT GTG TTC CTC CGT GGG
GTG GAC ATA 1170 As
p Glu Arg Leu His Glu Ala
Val Phe Leu Arg Gly Val
Asp Ile
380 385
390 TAT
ACA CGC CTG CTG CCT GCC
CTT GCC AGT GTG CCT GCC C
TG CCC 1215 Tyr Thr
Arg Leu Leu Pro Ala Leu A
la Ser Val Pro Ala Leu Pr
o 395
400
405 AGT GAC A
G.C.

1224 Ser Asp Ser

[Brief explanation of the drawing]

FIG. 1 is a diagram showing a restriction enzyme map of the cDNA of porcine aminoacylase I, and the portion and orientation where DNA sequencing was performed.

FIG. 2 is a diagram showing a restriction enzyme map of the cDNA of human aminoacylase I, and the portion and orientation where DNA sequencing was performed.

Claims (2)

[Claims] 1. A polypeptide having aminoacylase I activity, characterized by having the amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2 in the sequence listing. 2. A base sequence encoding the polypeptide according to claim 1.