JPH05276960A - Asparaginyl endopeptitase gene - Google Patents

Abstract

PURPOSE:To obtain a gene of asparaginylendopeptitase and its DNA sequence. CONSTITUTION:An asparaginylendopeptitase gene. Its DNA sequence includes e.g. a DNA sequence each expressed by sequence numbers 1-8 of sequence table and having 1323 sequence length or a DNA sequence hybridizable therewith. They are discovered or derived from a seed of Canavaliaensiformis. Thereby, it is made possible to industrially produce asparaginylendopeptitase.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a base sequence encoding asparaginyl endopeptidase and its amino acid sequence.

[0002]

The present inventors have found a protease (hereinafter referred to as asparaginyl endopeptidase) that selectively hydrolyzes only the C-terminal amide bond of an L-asparagine residue (JP-A-3-160997). ), And an enzyme clarified to have a strong transpeptidation activity (JP-A-4-23995). This enzyme is a protease found in the seeds of jack bean (Canavalia ensiformis), its substrate specificity is strict, and it is possible to selectively cleave only the C-terminal of asparagine residues, various synthetic peptides, It has been confirmed that a protein is used as a substrate [Ibid.
7, or Matsushita et al., Protein Nucleic Acid Enzyme, Vol. 36, p. 730 (1991)]. The activity of this enzyme was not decreased even when it was allowed to stand at 30 ° C for 20 minutes in the presence of a denaturing agent such as 2M urea, 0.8M guanidine hydrochloride, and 0.01% sodium lauryl sulfate (the above-mentioned protein nucleic acid). enzyme). These properties make it clear that this enzyme is very useful for protein fragmentation. In addition, this enzyme is
It has a strong transpeptidation activity and catalyzes the reaction represented by the following formula (Formula 1).
95).

[0003]

Embedded image A-Asn-B + C → A-Asn-C + B

In the formula, A represents an amino acid or peptide or a derivative thereof or hydrogen, Asn represents an asparagine residue, B represents an amino acid or peptide or a derivative thereof or NH ₂ , and C represents an amino acid or peptide or a derivative thereof. Represents a derivative. It is clear that the present enzyme is useful for enzymatic peptide synthesis utilizing this property.

[0005]

[Problems to be Solved by the Invention] Asparaginyl endopeptidase is purified from jack bean seeds and is commercially available. However, its gene structure and amino acid sequence are still unknown. Further, it does not disclose an industrially advantageous production method of asparaginyl endopeptidase. The gene for asparaginyl endopeptidase can be used for the genetic engineering production of the enzyme and is used for measuring the degree of expression in vivo. Also, the amino acid sequence corresponding to the DNA sequence is useful for making an antibody. The object of the present invention is the gene for asparaginyl endopeptidase,
To provide its DNA sequence and its amino acid sequence.

[0006]

SUMMARY OF THE INVENTION In general terms, the first aspect of the present invention relates to the asparaginyl endopeptidase gene. The second invention of the present invention relates to the gene of the first invention, which is characterized in that it is selected from the group consisting of DNA sequences represented by SEQ ID NOs: 1 to 8 in the sequence listing. The third invention of the present invention relates to the gene of the first invention and is characterized in that it is hybridizable with the gene of the second invention.

The present invention will be specifically described below. For example, the following method is used for cloning the cDNA encoding asparaginyl endopeptidase. First, highly purified purified asparaginyl endopeptidase,
The partial amino acid sequence is determined. This may be determined directly from the N-terminus of the protein by the Edman degradation method,
Alternatively, the protein may be fragmented using a protease, separated and purified, and each sequenced. From this partial amino acid sequence, a synthetic DNA having an appropriate length is prepared according to the fluctuation of the sequence and used as a primer or a probe. Next, RNA is extracted from a tissue rich in asparaginyl endopeptidase, for example, jack bean seeds, and poly (A) RNA is purified from this by using, for example, a cellulose carrier to which oligo (dT) is bound.
Using this as a template (template), reverse transcriptase is allowed to act to synthesize cDNA, which is then connected to a plasmid or phage vector by a method such as the Okayama-Berg method or the Gubler-Hoffmann method (Gubler-Hoffmann method) and introduced into the host. And create a cDNA library.

As a method for obtaining the cDNA encoding the desired asparaginyl endopeptidase, for example, the following two methods can be applied. One of them is a method of directly screening a cDNA library using the previously synthesized DNA as a probe. Further, when a part of the target cDNA is obtained, it is effective to use the DNA fragment as a probe. That is, first, the library is cultured on a plate, the grown colonies or plaques are transferred to a nitrocellulose or nylon membrane, and the DNA is fixed to the membrane by denaturing treatment. This membrane is kept warm in a solution containing probe DNA labeled in advance with ³² P or the like to form a hybrid between the DNA on the membrane and the probe DNA (hereinafter, this operation is abbreviated as hybridization). The temperature to be kept is set slightly lower than the Tm (melting temperature) of the probe used as a guide. After hybridization, non-specific adsorption is washed away, and clones that hybridize with the probe are identified by autoradiography or the like. This operation is repeated until a single clone is obtained. Among the clones thus obtained, the cDNA encoding the protein of interest
Has been inserted.

Another method is to amplify the cDNA encoding the target asparaginyl endopeptidase by the PCR method (polymerase-chain-reaction method) using the cDNA synthesized from jack bean seed poly (A) RNA as a template. Is. The primers used at this time are synthetic DNAs deduced from the partial amino acid sequence of asparaginyl endopeptidase, or the synthetic DN.
A and a random primer (mixture of all sequences), a common primer having a known sequence with the synthetic DNA, and the like are considered. However, in order to use the common primer, it is necessary to add a region where the common primer anneals to the end of the target DNA. For this, a known method using DNA ligase may be used. Further, in order to efficiently amplify the target DNA, it is effective to carry out two-step PCR. That is, first, PC with appropriate primers
This is a method in which R is performed, and then the second PCR is performed using the amplified product as a template and a primer encoding a sequence further inside than the primer used in the first time. P
Regarding the CR method, a gene amplification kit containing a tuck polymerase and an automatic gene amplification device are commercially available from Takara Shuzo, and a desired DNA region may be amplified using these.

The nucleotide sequence of the obtained cDNA is determined to confirm whether it is the desired cDNA. In the case of a clone obtained by hybridization, if the recombinant is Escherichia coli, it is cultured in a test tube or the like and the plasmid is extracted according to a conventional method. This is cleaved with a restriction enzyme to remove the inserted fragment, subcloned into an M13 phage vector or the like, and the nucleotide sequence is determined by the dideoxy method. When the recombinant is a phage, the nucleotide sequence can be basically determined by the same steps. In the case of cDNA obtained by the PCR method, the amplified DNA is purified by agarose gel electrophoresis or the like to make the ends blunt. This is M
13) Introduce into a phage vector etc. to determine the nucleotide sequence. For basic experimental methods from culture to nucleotide sequencing, see, for example, Molecular Cloning a Laboratory Manual (Molecular Cloning AL
aboratory Manual) [1982, issued by Cold Spring Harbor Laboratory, T.A. Maniatis (T.
Maniatis) et al.] Etc.

The determined nucleotide sequence can be compared with the partial amino acid sequence and molecular weight of asparaginyl endopeptidase, N-terminal analysis, etc. to determine its gene structure and amino acid sequence. When the obtained cDNA does not contain all of the asparaginyl endopeptidase coding region, a synthetic DNA primer was prepared based on the obtained nucleotide sequence to amplify the insufficient region by PCR, or the obtained region was obtained. By further screening the cDNA library using the cDNA fragment as a probe, the nucleotide sequence of the entire coding region can be determined.

The obtained asparaginyl endopeptidase gene is ligated to an expression vector so that it can be expressed in an appropriate host cell, for example, Escherichia coli, introduced into the host cell, and cultivated to obtain asparaginyl. Polypeptides having endopeptidase activity can be produced. Depending on the expression system used, the polypeptide expressed in the transformant may be insoluble (inclusion body (incclus
ion body)]. In this case, the insoluble matter can be recovered and solubilized under mild conditions such as urea, and then the denaturing agent can be removed to restore the activity.

The expression can be confirmed by measuring the asparaginyl endopeptidase activity.
For example, as described in JP-A-3-160997,
As a substrate, the formula shown in SEQ ID NO: 9 in the sequence listing (Formula 2):
DNP-L-Pro-L-Glu-L-Ala-LA
For example, solubilization of cell extract or inclusion body of recombinant Escherichia coli in a system for measuring the activity of asparaginyl endopeptidase containing sn-NH ₂ (DNP is 2,4-dinitrophenyl) and containing dithiothreitol, EDTA, etc. Add the mixture and keep warm at 37 ° C. At this time, the substrate is hydrolyzed, and the formula (Formula 3) shown in SEQ ID NO: 10 of the sequence listing:
P-L-Pro-L-Glu-L-Ala-L-Asn
Asparaginyl endopeptidase activity can be measured by measuring the amount of

A conventional chromatography technique is used for purifying the polypeptide having asparaginyl endopeptidase activity from the culture of the transformant. For example, if the desired polypeptide is solubilized by crushing the cultured bacterial cells, the supernatant can be subjected to chromatography such as hydrophobicity, ion exchange, gel filtration, or affinity chromatography to obtain the desired polypeptide. When the expression product is accumulated as an insoluble matter, the cells are disrupted, the precipitate is recovered, and solubilized with a denaturing agent such as urea. Then, after removing the denaturing agent and carrying out refolding, the desired polypeptide can be obtained by the above-mentioned chromatography.

An example of determining the structure of the asparaginyl endopeptidase gene will be described below. The determined N-terminal amino acid sequence of asparaginyl endopeptidase is shown in SEQ ID NO: 11 of the sequence listing. Synthetic DNA (HASN-1) shown in SEQ ID NO: 12 of the sequence listing, which corresponds to the 5 ′ → 3 ′ direction of the mRNA encoding the polypeptide corresponding to the amino acid numbers 7 to 14 of the sequence, and the same amino acid Synthetic DNA (HASN-2) shown in SEQ ID NO: 13 of the sequence listing, which corresponds to the 5 ′ → 3 ′ direction of mRNA encoding the polypeptide corresponding to the portions of Nos. 15 to 22.
Are each synthesized and purified by a DNA synthesizer. Then cd
Using NA as a template, HASN-1 and random 8
Primary PCR is performed using the mer primer to amplify the DNA. Next, using the amplified DNA as a template, HAS
Secondary PC using N-2 and random 8mer primer
R is performed to prepare a specifically amplified DNA fragment (Rn8-63) of about 800 bp on the N-terminal side of asparaginyl endopeptidase. The base sequence of this fragment is shown in SEQ ID NO: 14 of the sequence listing.

A cDNA library is prepared from the cDNA, and positive plaques are isolated from the cDNA library using the above Rn8-63 as a probe. The isolated plaques are λ _AEP 101, λ _AEP 102, λ _AEP 10
3, λ _AEP 104, λ _AEP 107 respectively,
λ _AEP 101 contains a 1.7 kb DNA fragment (101), λ
_AEP 102 has a 1.3 kb DNA fragment (102), λ
_AEP 103 has a 1.8 kb DNA fragment (103), λ
_AEP 104 contains a 1.4 kb DNA fragment (104), λ
_A 1.8 kb DNA fragment (107) is inserted into _AEP 107. 101, 102, 103, 104, 107
The nucleotide sequences of are shown in SEQ ID NOs: 15 to 19 of the sequence listing, respectively. Rn of each restriction map and the above probe
The restriction enzyme map of 8-63 is shown in FIGS. 1 and 2. These 6
Among the DNA fragments of the seeds, as shown in FIG. 1, 102, 104, R
The overlapping parts of the DNA sequence of n8-63 are in good agreement. A DNA sequence (ASN-
1) is shown in SEQ ID NO: 20 in the sequence listing.

Next, the cDNA is transferred to cDNA of Amersham.
Λgt10 using the A cloning system λgt10
It is introduced into the EcoRI site of and used as a template for PCR. Rn8-63 base numbers 73 to 92, 40 to 5
PCR primers, ASNN-1, AS complementary to 9
NN-2 is synthesized respectively. ASNN-1, ASNN
The DNA sequence of -2 is shown in SEQ ID NOs: 21 and 22 of the sequence listing, respectively.

Next, primary PCR was performed using ASNN-1 and λgt10 primer (Foward) (manufactured by Takara Shuzo Co., Ltd.), and then secondary PCR was performed using ASNN-2 and the λgt10 primer described above to detect asparaginyl endopeptidase. A cDNA encoding the N-terminal portion is amplified. The sequence of the amplified DNA (ASN-N) is shown in SEQ ID NO: 2 in the sequence listing.
3 shows. By analyzing the DNA sequences of ASN-1 and ASN-N described above, the entire base sequence of the cDNA (ASN) shown in SEQ ID NO: 24 of the sequence listing including the gene encoding asparaginyl endopeptidase can be determined. AS
The N restriction enzyme map is also shown in FIG. The results of the N-terminal analysis of asparaginyl endopeptidase are combined to determine the region encoding the asparaginyl endopeptidase in ASN.

The region is shown in SEQ ID NO: 1 in the sequence listing. The DNA sequence similar to the DNA sequence of SEQ ID NO: 1 is 10
DNA of SEQ ID NO: 1, which is also present in 1, 103, and 107
Sequences corresponding to the sequences are shown in SEQ
4 shows. The DNA sequences represented by SEQ ID NOs: 1 to 4 are regions sufficient for active expression of asparaginyl endopeptidase.

A plasmid encoding the entire length of the asparaginyl endopeptidase gene can be constructed from the obtained cDNA fragment. For example, the ASN-N DNA fragment and a part of the Rn8-63 DNA fragment having a portion homologous thereto can be mixed, and after the heat denaturation, an extension reaction is carried out with a DNA polymerase to connect the ASN-N and Rn8-63 DNA fragments. In the same manner, λ is added to the 3'side of the resulting DNA fragment.
_{The AEP} 102 EcoRI fragment can be ligated. Using this as a template, PCR was performed using, for example, ASNFw and ASNRv primers shown in SEQ ID NOs: 29 and 30, respectively.
By carrying out the reaction, the DNA encoding the full length can be amplified (Fig. 3).

This DNA fragment is used, for example, in the plasmid pTV1.
It is possible to obtain a plasmid ASN-ful-pTV118N encoding the full-length asparaginyl endopeptidase gene by incorporating it into 18N (Fig. 4).

Using this ASN-ful-pTV118N, a plasmid expressing the asparaginyl endopeptidase polypeptide from which the signal-like sequence has been removed can be constructed.
For example, the ASNMNF and ASNMNR primers shown in SEQ ID NOS: 31 and 32 of the sequence listing are
PCR was carried out using pTV118N as a template to introduce an NcoI site between the signal sequence and the N-terminus of the mature protein, and then an NcoI-AccIII fragment was prepared, and this fragment was added to the AccIII site of the EcoRI fragment of λ _AEP 104. By ligation, a DNA encoding the full length can be prepared from the N-terminus of the mature protein (Fig. 5
7). The DNA can also be obtained, for example, by carrying out PCR using ASN-ful-pTV118N as a template with a set of ASNMNF primer and ASNRv primer represented by SEQ ID NO: 30 in the sequence listing. The plasmid containing the DNA encoding the full length from the N terminus was designated as pUC18-ASN.

A DNA fragment containing the DNA encoding the full length from the N-terminal was excised from pUC18-ASN, and a plasmid for expression was constructed in the plasmid pET3b (Novagen), and the plasmid was named pEASN201 (Fig. 8).

PEASN201 contains the DNA represented by SEQ ID NO: 5 in the sequence listing, and the DNA corresponds to the base numbers 229 to 1551 of ASN represented by SEQ ID NO: 24 in the sequence listing and encodes the entire length of asparaginyl endopeptidase. To do.

DNA encoding polypeptides of various lengths can be prepared using pUC18-ASN. For example, after cutting with a suitable restriction enzyme just downstream of the stop codon of the asparaginyl endopeptidase-encoding region of the plasmid, exonuclease can be allowed to act to remove the base encoding the C-terminal polypeptide. ,
By incorporating this into an expression plasmid, expression plasmids encoding various polypeptides in which the C-terminal region of asparaginyl endopeptidase has been deleted can be prepared. In order to construct an expression vector encoding this deletion polypeptide, a DNA encoding the deletion gene is amplified by PCR and introduced into an expression plasmid, or a region encoding asparaginyl endopeptidase is added. It can be constructed by cleaving at a suitable restriction enzyme site inside to delete it and introducing it into an expression plasmid.

For example, pUC18-ASN was replaced with Nsp7524V and XbaI.
The DNA fragment obtained by cleavage with pET3b is introduced into
pEASN305 can be constructed (Figure 9).

PEASN305 contains the DNA shown in SEQ ID NO: 6 in the sequence listing, and this DNA corresponds to the portion of base numbers 229 to 1380 of ASN shown in SEQ ID NO: 24 in the sequence listing, and the amino acid shown in SEQ ID NO: 24. It encodes a polypeptide of 384 residues which corresponds to amino acid numbers 1 to 384 of the sequence. Similarly, replace pUC18-ASN with HincII and HincII.
DNA fragments cut out with SnaBI, HincII and SspI, HincII and HindIII were respectively pET3d (Novagen)
Introduced into pEASN304 (Fig. 10), pEASN303, pEASN302,
You can build pEASN301.

PEASN304 contains the DNA shown in SEQ ID NO: 7 in the sequence listing, and this DNA corresponds to the portion of base numbers 229 to 1122 of ASN shown in SEQ ID NO: 24 in the sequence listing, and the amino acid shown in SEQ ID NO: 24. Encodes a polypeptide of 298 residues corresponding to amino acid numbers 1-298 of the sequence. pEASN303 is a DN corresponding to the base numbers 229 to 855 of ASN shown in SEQ ID NO: 24 in the sequence listing.
The DNA contains A and encodes a polypeptide of 209 residues corresponding to amino acids 1 to 209 of the amino acid sequence shown in SEQ ID NO: 24. pEASN302 contains a DNA corresponding to the portion of base numbers 229 to 768 of ASN shown in SEQ ID NO: 24 in the sequence listing, and the DNA is a 180-residue polypeptide corresponding to 1 to 180 of the amino acid sequence of SEQ ID NO: 24. To code. pEASN301 is SEQ ID NO: 2 in the sequence listing
4 includes a DNA corresponding to the portion of base numbers 229 to 720 of ASN shown in 4, and the DNA encodes a polypeptide of 164 residues corresponding to amino acid numbers 1 to 164 of the amino acid sequence shown in SEQ ID NO: 24.

Further, PCR was carried out using pUC18-ASN as a template with the primer set shown in SEQ ID NOs: 33 and 34 and the primer set shown in SEQ ID NOs: 35 and 36.
After that, the amplified DNA fragments are collected, mixed and heat-denatured, and then annealed by slow cooling. Then, PCR is performed again with the primer set shown in SEQ ID NOs: 34 and 36 in the sequence listing. And then digesting with an appropriate restriction enzyme to obtain a DNA fragment into which a site-specific mutation has been introduced. By this operation, the codon corresponding to the 272nd Tyr residue in the amino acid sequence represented by SEQ ID NO: 24 in the sequence listing can be changed to a stop codon. This mutated DNA fragment can be transferred to ASN gene and then introduced into pET3d to construct pEASN404 (FIGS. 11 to 13). pEASN404 contains the DNA shown in SEQ ID NO: 8 of the sequence listing, and the DNA corresponds to the base numbers 229 to 1041 of ASN shown in SEQ ID NO: 24 of the sequence listing, and the amino acid of the amino acid sequence shown in SEQ ID NO: 24. It encodes a polypeptide of 271 residues corresponding to the numbers 1-271.

When the obtained genes are used as probes for hybridization under strict conditions, all of the genes similar to the present enzyme, which are expected to have similar enzyme activity although the sequences are slightly different from the present enzyme. Can be expected to get. The strict conditions mentioned here mean the following conditions. That is, a nylon membrane having DNA immobilized thereon was treated with 6 × SSC (1 × SSC is sodium chloride 8.7).
6 g, sodium citrate 4.41 g dissolved in 1 liter of water), 1% sodium laurin sulfate, 10
0 μg / ml salmon sperm DNA, 5 x Denharz
dt ^' s) (containing bovine serum albumin, polyvinylpyrrolidone, and ficoll at a concentration of 0.1% each) at 65 ° C. for 20 hours.

Escherichia coli for expression, such as Escherichia coli BL21 (DE3), is transformed with these plasmids, and the transformant is cultured under appropriate conditions to accumulate the polypeptide in the form of inclusion body in E. coli. .. Purification of the polypeptide is performed, for example, as follows. After culturing the recombinant Escherichia coli in a medium such as L-medium to collect the cells, ultrasonication is performed to obtain a disrupted cell solution. After centrifuging the disrupted cell suspension, the precipitate is collected and washed with a solution containing an appropriate surfactant,
For example, inclusion bodies of the target polypeptide can be solubilized and refolded by solubilizing with a denaturing agent such as urea and removing the denaturing agent under appropriate conditions.

In this way, 7 kinds of polypeptides can be obtained. Thus, polypeptide 210 was obtained from recombinant E. coli containing the expression plasmid pEASN201.
It contains the amino acid sequence of a 40 residue asparaginyl endopeptidase.

Similarly, the polypeptide 305 has 384 residues, the polypeptide 304 has 298 residues, the polypeptide 303 has 209 residues, the polypeptide 302 has 180 residues, and the polypeptide 301 has 164 residues. Polypeptide 404 comprises the amino acid sequence of a 271 residue asparaginyl endopeptidase.

FIG. 14 shows the relationship between each expressed polypeptide and the asparaginyl endopeptidase activity. That is,
Polypeptides 210, 305, 304, 404 were found to be active, but polypeptides 303, 302, 301
No activity was observed in. That is, the polypeptide 404 is the minimum unit of polypeptide required for activity,
The DNA shown in SEQ ID NO: 8 of the sequence listing that encodes the polypeptide is the minimum unit of DNA required for activity expression.

As described in detail above, the present invention clarified the gene and the primary structure of asparaginyl endopeptidase, and made it possible to provide a method for genetically engineering the gene.

[0036]

EXAMPLES Examples of the present invention will be shown below, but these do not limit the present invention.

Example 1 Cloning of asparaginyl endopeptidase cDNA (1-1) Determination of partial amino acid sequence of asparaginyl endopeptidase <Purification of asparaginyl endopeptidase> 500 g of commercially available jack bean meal (manufactured by Sigma) was added to 1 mM dithio. The supernatant was obtained by homogenization with 20 mM acetate buffer (pH 5.0) containing threitol and 1 mM EDTA and centrifugation. The supernatant was dialyzed against the above buffer solution, and the precipitate formed again was removed by centrifugation to obtain SP-Toyopearl 65.
Ion exchange chromatography was performed with 0M (manufactured by Tosoh Corporation). 60% of the active fraction adsorbed on the SP-Toyopearl 650M column and eluted with 0.5M sodium chloride
After saturated ammonium sulfate precipitation, it was dialyzed against a 20 mM acetate buffer (pH 5.0) containing 0.1 M sodium chloride, 1 mM dithiothreitol and 1 mM EDTA. Next, chromatography was performed using Toyopearl (manufactured by Tosoh Corporation) having paramercury benzoic acid immobilized thereon, and 1M sodium chloride, 50
A fraction eluted with an acetate buffer (pH 5.0) containing mM dithiothreitol and 1 mM EDTA was excluded to have a molecular weight of 10.0.
Toyopearl HW-55 after concentrating with an ultrafiltration membrane of 00
Gel filtration with S (manufactured by Tosoh Corporation) was performed. Gel filtration is 0.2M sodium chloride, 1 mM dithiothreitol, 1
20 mM acetate buffer (pH 5.2) containing mM EDTA was used, and the eluted active fractions were equilibrated with the same buffer. Egg white cystatin was added to immobilized Sepharose CL4B (Pharmacia). After being sufficiently washed with the same buffer, it contains 5 mM of the polypeptide A shown in SEQ ID NO: 25 in the sequence listing, 1 mM dithiothreitol, and 1 mM EDTA.
Elution was performed with 0 mM acetate buffer (pH 5.2). The eluted active fraction was concentrated at the same time as polypeptide A was removed with an ultrafiltration membrane having an exclusion molecular weight of 10,000. The enzyme preparation thus obtained was single by sodium lauryl sulfate-polyacrylamide gel electrophoresis.

<N-terminal sequence analysis> N-terminal sequencing is
A model 477A gas phase peptide sequencer manufactured by Applied Biosystems was used. Using about 100 pmol of purified asparaginyl endopeptidase, the sequencer was determined from the N-terminal by the Edman degradation method using the above sequencer, and the amino acid sequence of about 25 residues could be determined. The result is shown in SEQ ID NO: 11 of the sequence listing.

(1-2) Amplification of asparaginyl endopeptidase cDNA by PCR <Extraction and purification of mRNA and cDNA synthesis> Bean seeds were harvested and immediately frozen and stored in liquid nitrogen. Ultra Disperser (ULT) with 50 mM Tris-HCl buffer (pH 9.0) containing 30% sodium lauryl sulfate and 30 ml water-saturated phenol.
RA-DISPERSER) (made by Yamato Scientific Co., Ltd.) was extracted 20 times for 10 seconds. Centrifuge the milled solution at 10,000 g for 5 minutes, take the upper aqueous layer, and add 10 ml of water-saturated phenol, 1
0 ml of chloroform: isoamyl alcohol (24:
1) was added, and the mixture was stirred well and centrifuged at 10,000 g for 5 minutes to obtain an upper aqueous layer. 1/4 volume of 10 in this water layer
M lithium chloride was added, and the mixture was kept at 0 ° C. for 2 hours and then 20,000
The RNA precipitate was recovered by centrifugation at 0 g for 20 minutes.
10 ml of this RNA purified water (autoclaved)
And then dissolved in 1/10 volume of 3M sodium acetate,
After adding 25 ml of ethanol and holding at -20 ° C for 1 hour,
RNA was recovered by centrifugation at 20,000 g for 20 minutes. This RNA was suspended in 70% ethanol, recovered by the same centrifugation operation, and then dried under reduced pressure (hereinafter, this operation is abbreviated as ethanol precipitation). This gave 1.8 mg of total RNA. Of this, 1 mg is oligotex-dT
By treating with 30 (Takara Shuzo), 27 μg of poly (A) RNA was obtained. From 5 μg of this poly (A) RNA, a cDNA synthesis kit manufactured by Amersham was used to synthesize cDNA with a random hexamer primer to obtain about 1 μg.

<Synthesis of primers and amplification by PCR>
From the asparaginyl endopeptidase N-terminal amino acid sequence (represented by SEQ ID NO: 11 in the sequence listing) determined in Example (1-1), two types of primers (HASN-1, HSN) were used.
ASN-2) was designed and synthesized. The respective sequences are shown in SEQ ID NOS: 12 and 13 of the sequence listing. HASN-1 is N
M corresponding to the portion of amino acid numbers 7 to 14 in the terminal sequence
23mer corresponding to the 5 '→ 3'direction of RNA, HAS
N-2 is the mRN corresponding to the portion of amino acid numbers 15 to 22 downstream of the portion corresponding to HASN-1 in the N-terminal sequence.
23-mer synthetic DNA corresponding to the 5 '→ 3' orientation of A
Is. CD prepared as above as template
Transfer about 10 ng (1 μl) NA to a 0.5 ml tube,
1 of 10 μl in GeneAmp ^TM kit (Takara Shuzo)
0x Concentration Amplification Buffer, 16 μl of 1.25 mM dNT
P mixture, 2 μl of 0.5 μg / μl HASN-1 primer, 4 μl of 0.1 μg / μl random 8mer primer, 0.5 μl of 5 units / μl AmpliTac ^™ , and sterilized water to add 100 μl of solution I chose After 100 μl of mineral oil (manufactured by Sigma) was overlaid on this reaction solution, an amplification reaction was carried out by an automatic gene amplifier thermal cycler (manufactured by Takara Shuzo). The reaction conditions were 94 ° C. for 30 seconds (denaturation) → 55 ° C. for 2 minutes (primer annealing) → 72 ° C. for 1 minute (synthesis reaction), 40 cycles. Next, using 1 μl of this reaction solution as a template, 2 μl of 0.5 μg / μl HASN-2 primer was used in place of HASN-1, and PCR was performed again under the same conditions as above. After the reaction, the upper layer mineral oil was removed, and 5 μl of the reaction solution was subjected to agarose gel electrophoresis, followed by D with ethidium bromide.
Approximately 800 bp on the N-terminal side that was specifically amplified by staining NA
The asparaginyl endopeptidase cDNA band was identified. The DNA fragment amplified by this PCR was named Rn8-63.

<Determination of amplified DNA base sequence> The rest of the PCR reaction solution was concentrated by ethanol precipitation, and the entire amount was subjected to agarose gel electrophoresis. After staining with ethidium bromide, the portion containing the desired Rn8-63 DNA fragment was cut out from the gel under UV irradiation. This was extracted and purified from the gel using an Easy Trap kit manufactured by Takara Shuzo. This DNA fragment is a DNA produced by Takara Shuzo
It was blunt-ended using a blunting kit, mixed with a restriction enzyme HincII digestion product of plasmid pUC18, and ligated using a ligation kit manufactured by Takara Shuzo. A part of this ligation reaction solution was used to transform Escherichia coli JM109 strain and pUC1 having an Rn8-63 DNA fragment amplified by PCR at the HincII site.
A recombinant E. coli containing 8 was obtained. The obtained recombinant Escherichia coli was cultivated by liquid culture, and plasmid DNA was prepared by the alkaline lysis method. The obtained plasmid DNA was designated as pASN
It was named Rn8-63. The entire nucleotide sequence of the insert fragment in the pASNRn8-63 plasmid was determined by the dideoxy method. The sequence is shown in SEQ ID NO: 14 of the sequence listing. In addition, the base numbers 1 to 23 in the sequence are sequences derived from the primer HASN-2, and the base numbers 737 to 753 are sequences derived from the random primer. In addition, Rn8-63 is contained in this pASNRn8-63 plasmid.
The DNA fragment was inserted in the HincII site of pUC18 in the direction opposite to the direction of the lacZ gene.

(1-3) Screening from cDNA library <Screening by hybridization> Oligo (dT) was prepared from 4 μg of poly (A) RNA described in Example (1-2) using a cDNA synthesis kit manufactured by Amersham.
CDNA was synthesized by the primers. Next, Amersham's cDNA cloning system λgt10
Was used to package the cDNA incorporated into the restriction enzyme EcoRI site of λgt10 into a lambda phage to prepare a cDNA library. However, the EcoRI linker shown in SEQ ID NO: 26 of the Sequence Listing was manufactured by Takara Shuzo Co., Ltd., and the packaging was GIGAPACK GOLD manufactured by Stratagene. The cd
E. coli C600hf with NA library as host bacterium
Using 1 strain, 16 pieces of 14 cm x 10 cm square petri dish
About 30,000 plaques were formed per sheet. That is, phages were added to 0.2 ml of C600hf1 culture solution which had been cultured overnight at 37 ° C. in L medium containing 4 mg / ml maltose.
The solution (0.1 ml) was mixed and kept at 37 ° C. for 15 minutes. To this, 8 ml of soft agar (agarose added to L medium to a final concentration of 0.6%, treated with an autoclave and kept at 50 ° C.) was added, spread on L medium-plate, and solidified 37 The temperature was kept at 10 ° 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 manufactured by Amersham (trade name: Hybond-N) was brought into contact with the plate surface for 30 seconds, and this was applied to the plate.
5 minutes on filter paper soaked in 5M sodium hydroxide, 1.5M sodium chloride solution (denaturation), 0.5M Tris-HCl buffer (pH 7.0), on filter paper soaked in 1.5M sodium chloride solution After 5 minutes (neutralization) treatment, 2 × S
Rinse with SC and dry on filter paper. The second filter was similarly prepared. However, the contact time between the plate and the nylon membrane was 2 minutes. The dried filter was irradiated with ultraviolet rays from a UV lamp for 5 minutes to immobilize DNA on the membrane.

As a hybridization probe, the DNA fragment Rn8-6 described in Example (1-2).
3 was used. About 100 ng of Rn8-63 DNA fragment was labeled with ³² P using a random primer DNA labeling kit manufactured by Takara Shuzo. Using the whole amount of this probe and the filter prepared above, 6 × SSC, 1% sodium lauryl sulfate, 100 μg / ml salmon sperm DN
65 ° C in a solution of about 100 ml containing A and 5 × Denhartz
Hybridization was performed overnight. Then room temperature 6
The filters were washed in × SSC for 10 minutes. Further, 1 at room temperature in 2 × SSC, 0.1% sodium lauryl sulfate.
After being washed twice for 0 minutes and twice for 10 minutes in 0.2 × SSC and 0.1% sodium lauryl sulfate, the filter was transferred onto a filter paper to remove excess water. This was attached to Whatman 3MM filter paper, an amplification paper was applied thereto, and autoradiography was performed overnight at -70 ° C. As a result, 5 positive signals were obtained.

Plaques at positions corresponding to these signals were agar together with 0.2 ml of SM solution [sodium chloride 5.
8g, magnesium sulfate, 7H ₂ O 2g, 1M Tris-
50 ml of hydrochloric acid buffer (pH 7.5) and 5 ml of 2% gelatin were dissolved in water to make the total volume 1 liter]. These phage solutions were diluted with SM solution, plated (about 300 plaques / φ9 cm round petri dish), and hybridized in the same manner as above. As a result, a single plaque could be isolated for all clones. These clones were _designated λ _AEP 101, λ _AEP 10
2, λ _AEP 103, λ _AEP 104, λ _AEP 107.

<Determination of positive clone insertion fragment nucleotide sequence> Recombinant λ phage λ _AEP 101, λ _AEP
102, λ _AEP 103, λ _AEP 104, λ _AEP 107
Was subjected to liquid culture using the Escherichia coli L87 strain as a host, and about 40 μg of λDNA was prepared from 40 ml of the culture solution. 20 μg of the obtained λDNA was used as a restriction enzyme RcoR
After digestion with I, a part of the digest was subjected to agarose gel electrophoresis to determine the size of the insert fragment. As a result, λ _AEP 101 has 1.7 kb, λ _AEP 102 has 1.3 kb, and λ _AEP 10
1.8 kb for 3 and 1.4 kb for λ _AEP 104, λ _AEP 1
A DNA fragment having a size of 1.8 kb was inserted into 07, and the respective DNA fragments were 101, 102, 103, 10
It was named 4,107.

Next, the remaining reaction solution previously digested with EcoRI was subjected to agarose gel electrophoresis. After staining with ethidium bromide, the portion containing the DNA fragment of interest was cut out from the gel under UV irradiation. This was extracted and purified from the gel using an Easy Trap Kit manufactured by Takara Shuzo. These Ec
The oRI fragment was mixed with M13mp18 phage vector digested with EcoRI, and ligation reaction was performed using a ligation kit manufactured by Takara Shuzo. A part of this ligation reaction solution was used to transform Escherichia coli JM109 strain to obtain recombinants in which each EcoRI fragment was inserted.

The obtained recombinant phage was transformed into Escherichia coli JM10.
Liquid culture was performed using 9 strains as host bacteria, and RF DNA was prepared by the alkaline lysis method. A part of this RF DNA was digested with the restriction enzyme EcoRI and subjected to agarose gel electrophoresis to confirm that the target DNA fragment had been inserted. Further, a part of the obtained RF DNA was cleaved with several kinds of restriction enzymes and analyzed by agarose gel electrophoresis. The results are shown in FIGS. 1 and 2. That is, 101 in FIGS.
It is a restriction enzyme map of 102, 103, 104, 107 etc.

It was also found that two types of clones were obtained depending on the orientation of the M13mp18 phage inserted into the EcoRI site. The clones in which the orientations of the inserts were reversed were M _AEP 101-5 and M _AEP 101-2, respectively.
M _AEP 102-4, M _AEP 102-3, M _AEP 103-
3 and M _AEP 103-2, M _AEP 104-2 and M _AEP 10
4-22, M _AEP 107-2 and M _AEP 107-4.

In order to determine the entire nucleotide sequences of these inserts, a Takara-Kiro sequence deletion kit manufactured by Takara Shuzo Co., Ltd. was used to analyze M _AEP 101-5 and M _AEP 101-2.
For the 8 clones except for, the insert fragments were digested from the side of the sequencing primer to obtain DNs of various sizes.
A derivative having A deleted was prepared. Also, M _AEP 101
-5 and _MAEP 101-2 are derivatives in which DNAs of various sizes have been deleted by cutting at the appropriate restriction enzyme site in the insert and the appropriate restriction enzyme site in the M13mp18 multicloning site. It was created. Suitable ones of these derivatives, M _AEP 101-5, M _AEP 10
1-2, M _AEP 102-4, M _AEP 102-3, M _AEP
103-3, M _AEP 103-2, M _AEP 104-2, M
_AEP 104-22, M _AEP 107-2, M _AEP 107-
4 was infected with Escherichia coli JM109 strain and cultured respectively.
sDNA was prepared. DN by the dideoxy method
By performing A sequence and connecting them, λ _AEP 101, λ _AEP 102, λ _AEP 103, λ _AEP
The entire base sequence of the EcoRI insert fragment of 104, λ _AEP 107 was determined. These are shown in SEQ ID NOs: 15 to 19 of the sequence listing. These sequences are shown after removing the sequence derived from the EcoRI linker in each insert.

A homology search of the nucleotide sequences of these five types of insert fragments revealed that λ _AEP 102 and λ _AEP
The overlapping portion of the insert fragment of 104 completely matched, and also the overlapping portion of the Rn8-63 DNA fragment of Example (1-2) matched. This group of sequences are joined together to form asparaginyl endopeptidase ASN-1.
I named it. The base sequence of ASN-1 is shown in SEQ ID NO: 20 in the sequence listing. The other three inserts, including ASN-1, had 60-90% homology, but were not perfectly matched. Also, these λ _AEP 101 and λ _AEP 10
3. When the nucleotide sequence of the λ _AEP 107 insert fragment was translated into an amino acid sequence, a sequence very similar to the N-terminal sequence determined in Example (1-1) was found, but it did not completely match. It was In addition, the DNA sequence from the position corresponding to the N-terminal amino acid sequence determined in Example (1-1) to the position of the stop codon in the reading frame is SEQ ID NO: 2 in the sequence listing,
3 and 4 respectively. This portion is a portion considered to be a sufficient position for asparaginyl endopeptidase activity.

(1-4) Cloning of the N-terminal portion of asparaginyl endopeptidase ASN-1 ASN-1 obtained by joining the nucleotide sequences determined in Examples (1-2) and (1-3) is asparagus. Since the N-terminal portion of ginyl endopeptidase was deleted, a cDNA encoding the N-terminal portion was amplified by the PCR method and cloned as shown below.

<Preparation of template DNA> Example (1
-2), about 100 ng of the cDNA prepared in 2), the EcoRI manufactured by Amersham, shown by SEQ ID NOs: 21 and 22 of the sequence listing, respectively.
250 pmol of the adapter was mixed, and a ligation reaction was performed using a ligation kit manufactured by Takara Shuzo. The ligation reaction solution was purified by ethanol precipitation,
Half the amount of DNA using Takara Shuzo Mega Label Kit
Phosphorylation of the 5'end of the
Mix with 0.5 μg of digested gt10 restriction enzyme EcoRI,
Ligation reaction was performed using a ligation kit manufactured by Takara Shuzo Co., Ltd. to prepare a template DNA.

<Synthesis of N-terminal partial amplification primer and P
Amplification by CR> In the base sequence existing in the positions of base numbers 73 to 92 and the positions of base numbers 40 to 59 of the Rn8-63 DNA fragment shown in SEQ ID NO: 8 of the sequence listing, which was determined in Example (1-2) Two complementary primers (ASNN
-1, ASNN-2) were synthesized. The respective sequences are shown in SEQ ID NOs: 21 and 22 of the sequence listing.

A 1/10 volume (1 μl) of the DNA prepared as described above was used as a template in a 0.5 ml tube, and 10 μm in the GeneAmp ^™ kit (Takara Shuzo) was used.
l 10 times concentration amplification buffer, 16 μl 1.25 mM
dNTP mixed solution, 1 μl of 0.1 μg / μl ASNN
-1 primer, 1 μl of 0.1 μg / μl λgt10
A primer (Forward) (manufactured by Takara Shuzo Co., Ltd.) and 0.5 μl of 5 units / μl AmpliTac ^™ were added, and sterilized water was further added to obtain a 100 μl solution. 100 for this reaction
After overlaying μl of mineral oil (manufactured by Sigma), automatic gene amplification device thermal cycler (manufactured by Takara Shuzo)
The amplification reaction was carried out. Regarding the reaction conditions, 25 cycles of 94 ° C. for 30 seconds (denaturation) → 55 ° C. for 2 minutes (primer annealing) → 72 ° C. for 1 minute (synthesis reaction) were performed.

Next, using 1 μl of this reaction solution as a template, 1 μl of 0.1 μg / μl was used instead of ASNN-1.
PC using ASNN-2 primer under the same conditions as above
R was done again. After the reaction, the upper layer mineral oil was removed, and 5 μl of the reaction solution was subjected to agarose gel electrophoresis, and the DNA was stained with ethidium bromide. I confirmed the band. The DNA fragment amplified by this PCR was named ASN-N.

<Determination of amplified DNA base sequence> The rest of the PCR reaction solution was concentrated by ethanol precipitation, and the whole amount was subjected to agarose gel electrophoresis. After staining with ethidium bromide, the portion containing the desired ASN-N DNA fragment was cut out from the gel under UV irradiation. This was extracted and purified from the gel using an Easy Trap Kit manufactured by Takara Shuzo. This DNA fragment was blunt-ended using a DNA branding kit manufactured by Takara Shuzo, mixed with a restriction enzyme HincII digestion product of plasmid pUC18, and subjected to a ligation reaction using a ligation kit manufactured by Takara Shuzo. A part of this ligation reaction solution was used to transform Escherichia coli JM109 strain to obtain recombinant Escherichia coli containing pUC18 having the ASN-N DNA fragment amplified by PCR at the HincII site.

The recombinant Escherichia coli obtained was cultivated by liquid culture, and plasmid DNA was prepared by the alkaline lysis method. The entire nucleotide sequence of the insert ASN-N of this plasmid DNA was determined by the dideoxy method. The base sequence is shown in SEQ ID NO: 23 in the sequence listing. The sequence is
Sequences from the EcoRI adapter have been removed and shown. A homology search of the obtained ASN-N base sequence and ASN-1 base sequence revealed that the overlapping portions were in good agreement. These sequences were put together and named ASN. The base sequence is shown in SEQ ID NO: 24 in the sequence listing. Further, when this was translated into an amino acid sequence, a sequence completely identical to the N-terminal sequence determined in Example (1-1) was recognized.
The DNA sequence from the position corresponding to the N-terminal amino acid sequence determined in Example (1-1) to the position of the stop codon in the open reading frame is shown in SEQ ID NO: 1 of the sequence listing. This part encodes asparaginyl endopeptidase D
Sequences that are NA regions and are similar to the DNA sequences represented by SEQ ID NOS: 2 to 4 are shown.

Example 2 Construction of Plasmid Expressing Asparaginyl Endopeptidase Polypeptide (2-1) Construction of Plasmid Encoding Full-Length Asparaginyl Endopeptidase Gene pASNRn8-obtained in Example (1-2) 63 plasmid DN
A was digested with HindIII and subjected to agarose gel electrophoresis, and a HindIII fragment of about 800 bp containing Rn8-63 was cut out and purified. About 10 ng of each of the HindIII fragment and the ASN-N DNA fragment obtained in Example (1-4) was mixed with 16 μl of 1.25 mM dNTP buffer in 10 μl of 10 times concentration amplification buffer in GeneAmp ^™ kit. Mix the liquids and add water to bring the total volume to 9
The volume was 7.5 μl. Set this in a thermal cycler, keep it at 94 ℃ for 10 minutes, and from 94 ℃ to 37 ℃ for 30 minutes.
Slowly cool for 15 minutes, incubate at 37 ℃ for 15 minutes, then 0.5 μl
Add 5 units / μl AmpliTac ^™ of
The extension reaction was performed for a minute. Example (1-3) was added to this reaction solution.
Approximately 10 ng of EcoRI fragment of λ _AEP 102 obtained in
The mixture was kept at 10 ° C. for 10 minutes, gradually cooled from 94 ° C. to 37 ° C. for 30 minutes, kept at 37 ° C. for 15 minutes, and then extended at 72 ° C. for 3 minutes. 20 pmol each of ASNF _W primer shown in SEQ ID NO: 29 of the sequence listing and ASNR _v primer shown in SEQ ID NO: 30 of the sequence listing and 0.5 μl of 5 units / μl AmpliTac ^™ were added to this reaction solution at 94 ° C. for 30 seconds →
2 cycles of 55 ° C for 15 minutes → 72 ° C for 1.5 minutes
Five cycles were performed to amplify the DNA containing the entire length of the asparaginyl endopeptidase gene.

ASNF _W was determined in Example (1-4),
ASN base number 123 shown in SEQ ID NO: 24 in the sequence listing
Is a 27-mer synthetic DNA having a BamHI recognition sequence on the 5'side thereof, and ASNR _V contains a sequence complementary to the nucleotide sequence of ASN base numbers 1602 to 1620. It is a 28-mer synthetic DNA having a SphI recognition sequence on its side. The PCR reaction solution was concentrated by ethanol precipitation, digested with BamHI and SphI, subjected to agarose gel electrophoresis, and a fragment of about 1500 bp was cut out and purified (Fig. 3). This DNA fragment was introduced into the BamHI and SphI sites of plasmid pTV118N (Takara Shuzo) to obtain plasmid ASN-ful-pTV118N (FIG. 4). This plasmid DNA contains a nucleotide sequence encoding the entire length of the asparaginyl endopeptidase gene.

(2-2) Construction of plasmid expressing polypeptide from asparaginyl endopeptidase mature N-terminus Plasmid DNA ASN-ful-pTV obtained in Example (2-1)
A PCR reaction (25 cycles) was carried out once under the same conditions as in Example (1-4) using 10 ng of 118N as a template. As the primers, the ASN-MNF primers and ASN-MNR shown in SEQ ID NOS: 31 and 32 of the sequence listing, respectively.
A primer was used. The ASN-MNF primer has base numbers 229 to 2 of ASN shown in SEQ ID NO: 24 of the sequence listing.
Equivalent to 47, the 234th G is set to T, and the 237th A is set.
25-mer synthetic DN containing a sequence mutated to T and containing a sequence capable of forming an NcoI recognition sequence on the 5'side thereof
It is A. The ASN-MNR primer is a 24 mer synthetic DNA containing a sequence complementary to the sequence obtained by mutating the T at the 642nd position of the sequence of ASN base numbers 628 to 651 to C. The PCR reaction solution was concentrated by ethanol precipitation, digested with NcoI and subjected to agarose gel electrophoresis to cut out a fragment of about 420 bp for extraction and purification. This DNA fragment was introduced into the NcoI site of plasmid pTV119N (Takara Shuzo) in the same direction as the lacZ gene on the plasmid to obtain plasmid pTASN-N (FIG. 5).

Ec of λ _AEP 104 obtained in Example (1-3)
oRI fragment was transformed into EcoRI of plasmid pUC18 (Takara Shuzo)
A plasmid pUC18-104 was obtained by introducing the plasmid into the site in the opposite direction of the lacZ gene on the plasmid (FIG. 6). The plasmid pTASN-N obtained above was digested with NcoI, blunt-ended with Klenow fragment, and further digested with AccIII. The reaction mixture was subjected to agarose gel electrophoresis to give about 34
The 0 bp fragment was excised and extracted and purified. This DNA fragment was introduced into the AccIII and SmaI sites of plasmid pUC18-104 to obtain plasmid pUC18-ASN (FIG. 7).

Plasmid pUC18-ASN was digested with XbaI and ApaLI
After digestion with Klenow fragment, blunt end,
Agarose gel electrophoresis was performed to cut out a fragment of about 1300 bp, which was then extracted and purified. This DNA fragment was transformed into plasmid pE
It was introduced in the same direction as the T ₇ promoter on plasmid BamHI site T3b in was blunted with Klenow fragment to construct plasmid PEASN201 (Figure 8). This plasmid encodes a polypeptide in which 19 amino acid residues shown in SEQ ID NO: 37 of the sequence listing are added to the N-terminal side of the 440 amino acid residue polypeptide of the amino acid sequence shown in SEQ ID NO: 24 of the sequence listing. pEASN201
E. coli BL21 (DE3) introduced with E. coli BL21
It was named (DE3) / pEASN201.

(2-3) Construction of a plasmid expressing a C-terminal deletion polypeptide of asparaginyl endopeptidase From the plasmid pUC18-ASN obtained in Example (2-2) Genes encoding polypeptides of various lengths were excised to construct a plasmid expressing the C-terminal deletion polypeptide.

<Construction of pEASN305> Plasmid pUC18-ASN
Was digested with XbaI and Nsp7524V, blunt-ended with Klenow fragment, subjected to agarose gel electrophoresis, and a fragment of about 1120 bp was cut out and purified. This DNA
The fragment was introduced into pET3b in the same manner as when pEASN201 was constructed in Example (2-2) to construct plasmid pEASN305 (FIG. 9). This plasmid has 19 amino acid residues shown in SEQ ID NO: 37 in the sequence listing at the C-terminal on the N-terminal side of a 384-residue polypeptide corresponding to amino acid numbers 1-384 in the amino acid sequence shown in SEQ ID NO: 24 in the sequence listing. On the side is included a DNA encoding a polypeptide to which 4 residues shown in SEQ ID NO: 38 of the Sequence Listing are added.

<Construction of pEASN304> Plasmid pUC18-ASN
Was digested with HincII to obtain a DNA fragment of about 920 bp,
After plasmid pET3d was digested with NcoI and BamHI and blunt-ended with Klenow fragment, it was introduced in the same direction as the T ₇ promoter on the flasmid to construct plasmid pEASN304 (FIG. 10). In this plasmid, the 8 residues shown in SEQ ID NO: 39 of the sequence listing are N-terminal to the C residue of the polypeptide of 298 residues corresponding to amino acid numbers 1-298 of the amino acid sequence shown in SEQ ID NO: 24 of the sequence listing. It contains a DNA encoding a polypeptide having 20 residues shown in SEQ ID NO: 40 in the sequence listing added on the terminal side.

<Construction of pEASN303, pEASN302, pEASN301>
About 510 bp fragment obtained by digesting plasmid pUC18-ASN with Klenow fragment after digestion with HincII and HindIII, about 560 bp fragment obtained by digestion with HincII and SspI, and about 650 bp obtained by digestion with HincII and SnaBI. The fragment was introduced into pET3d in the same manner as the construction of pEASN304 to construct plasmids pEASN301, pEASN302 and pEASN303, respectively. The plasmid pEASN301 corresponds to amino acid numbers 1-164 of the amino acid sequence shown in SEQ ID NO: 24 of the sequence listing, the plasmid pEASN302 corresponds to amino acid numbers 1-180, and the plasmid pEASN303 corresponds to amino acid numbers 1-209. 8 residues shown in SEQ ID NO: 39 of the sequence listing are shown in FIG.
It includes DNA encoding a polypeptide with 0 residues added.

<Construction of pEASN404> Plasmid pUC18-ASN
Using 1 ng of the template, two kinds of PCR reactions (25 cycles) were performed once under the same conditions as in Example (1-4). As a primer, a set of MASN-P1 primer and M4 primer (manufactured by Takara Shuzo) shown in SEQ ID NOs: 33 and 34 of the sequence listing, and MUTR1 primer and RV primer (manufactured by Takara Shuzo) shown in SEQ ID NOs: 35 and 36 of the sequence listing. ) I went with the group. Here, the MASN-P1 primer is the 1041st T in the sequence of base numbers 1030 to 1051 of the base sequence of ASN shown in SEQ ID NO: 24 of the sequence listing.
Is a C sequence, and the 1044th C is a sequence complementary to a G sequence mutated to G. The other three types of primers are the base sequence of plasmid pUC18 or a similar base sequence.

Each PCR reaction product was subjected to agarose gel electrophoresis for extraction and purification. 5 ng of each fragment was mixed in 50 μl water, and the mixture was gradually cooled to room temperature after 5 minutes at 94 ° C. PCR was performed again with the same conditions using 2 μl of this mixture as a template and M4 primer and RV primer.
The reaction was performed once. The PCR reaction product was concentrated by ethanol precipitation, digested with SphI and HpaI, and subjected to agarose gel electrophoresis to cut out a fragment of about 930 bp, which was then extracted and purified. This DNA fragment is S of M13mp19 (Takara Shuzo)
It was introduced into the phI and HincII sites to obtain MMASN1 (FIG. 11). This MMASN1 phage was transformed into E. coli JM1
Liquid culture was carried out using the 09 strain as a host to prepare ssDNA. This was subjected to DNA sequencing by the dideoxy method, and it was confirmed that the MASN1 primer had a mutation.

The obtained MMASN1 phage DNA was labeled with A
It was digested with ccIII and HincII, subjected to agarose gel electrophoresis, and a fragment of about 560 bp was excised and extracted and purified. This D
The plasmid pTASN-N whose NA fragment was obtained in Example (2-2)
Plasmid pTMA introduced into AccIII and HincII sites of
SN204 was obtained (Fig. 12). This pTMASN204 is NcoI
Partial digestion with agarose gel electrophoresis
The 0 bp fragment was cut out and extracted and purified. P this DNA fragment
The plasmid pEASN404 was introduced into the NcoI site of ET3d in the same direction as that of the T ₇ promoter on the plasmid.
Was constructed (Fig. 13). This plasmid contains N of a 271 residue polypeptide corresponding to amino acid numbers 1-271 of the amino acid sequence of ASN shown in SEQ ID NO: 24 of the sequence listing.
D encoding a polypeptide with Met added to the terminal side
Including NA.

Plasmids pEASN305, pEASN304, pEASN303, pEASN302, pEASN30 constructed as described above
1. E. coli BL21 (DE3) was transformed with pEASN404. The obtained transformants were transformed into E. coli BL21 (DE3) / pEASN305 and E. coli BL21 (DE), respectively.
3) / pEASN304, E. coli BL21 (DE3) / pEASN30
3, E. coli BL21 (DE3) / pEASN302, E. coli BL
21 (DE3) / pEASN301, E. coli BL21 (DE3)
It was named / pEASN404.

Example 3 Expression of Asparaginyl Endopeptidase Polypeptide in Escherichia coli Escherichia coli BL21 (DE3) / pEASN2 obtained in Example 2
01, 305, 304, 404, 303, 302, 301 respectively 1
A 500 ml L-medium containing 00 µg / ml of ampicillin was inoculated and shake-cultured at 37 ° C. The turbidity during the culture is O. D.
_{When 660} = 0.5, IPTG (isopropyl-β-D-thio-galactopyranoside) was added to a final concentration of 1 mM, and the mixture was shake-cultured at 37 ° C overnight. The culture solution was centrifuged to collect the cells, and 30 ml of 0.1 M NaCl,
After suspending in 10 mM Tris-HCl buffer containing 1 mM EDTA, the cells were disrupted by ultrasonic treatment. This was centrifuged to collect the precipitate, which was suspended in 30 ml of a 1 M sucrose aqueous solution and
The precipitate was recovered by centrifugation at 7,000 g for 15 minutes. This was suspended in 40 ml of Triton X-100 containing 10 mM EDTA and left at 4 ° C. overnight. The precipitate was recovered by centrifugation, 25 ml of 8M urea, 10 mM 2-mercaptoethanol, 30 mM Na
Dissolve in 30 mM Tris-HCl buffer containing Cl and gradually remove urea and 2-mercaptoethanol by dialysis,
This was used as a solubilized solution of recombinant protein inclusion bodies.

A part of the solubilized solution of the recombinant protein inclusion body thus obtained was dialyzed against a 30 mM sodium phosphate buffer (pH 5.9) containing 30 mM NaCl, and the precipitate was removed by centrifugation to collect the supernatant. As a sample solution for activity measurement, the activity was measured as follows. 50 including 5 mM EDTA
mM sodium acetate buffer (pH 5.0) 20 μl, 50
mM DTT 5 μl, 0.1 mM formula (Formula 2): DNP-L
-Pro-L-Glu-L-Ala-L-Asn-NH
₂ 10 μl (shown as SEQ ID NO: 9 in Sequence Listing), 0.0
10 µl of 1 mM DNP-Ser (internal standard substance) and 5 µl of an appropriately diluted sample solution were kept warm at 37 ° C for 20 minutes, and the reaction was stopped by adding 5 µl formic acid. A part of this reaction solution was analyzed by high performance liquid chromatography using an ODS column, and the generated formula (formula 3): DN
P-L-Pro-L-Glu-L-Ala-L-Asn
The amount (shown in SEQ ID NO: 10 in the sequence listing) is determined by DNP-Se.
It was quantified by comparison with the amount of r. Results of activity measurement E. coli BL21
(DE3) / pEASN201, pEASN305, pEASN304, pEASN404
In each activity measurement sample solution obtained from the culture of
Asparaginyl endopeptidase activity was seen,
No asparaginyl endopeptidase activity was found in each activity measurement sample solution obtained from the cultures of Escherichia coli BL21 (DE3) / pEASN303, 302, 301, and the polypeptide produced by Escherichia coli BL21 (DE3) / pEASN404 was It was the smallest unit of activity.

[0074]

From the above results, the present invention clarifies the nucleotide sequence and amino acid sequence of the asparaginyl endopeptidase gene, makes it possible to provide the asparaginyl endopeptidase gene, and exhibits the asparaginyl endopeptidase activity. Provided is an industrially advantageous method for producing a genetically engineered polypeptide. It has also become possible to synthesize a probe DNA or a PCR primer DNA based on this base sequence, or to prepare an antibody based on the amino acid sequence.

[Sequence list]

SEQ ID NO: 1 Sequence Length: 1323 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Sequence: GAAGTGGGAA CCCGGTGGGC GGTTCTCGTG GCTGGTTCAA ACGGTTATGG AAATTATAGG 60 CATCAAGCGG ATGTGTGCCA TGCTTACCAG TTGCTGATAA AA AACATTGTGG TGTTTATGTA TGATGATATA GCTTATAACG CCATGAATCC CAGACCCGGA 180 GTCATCATCA ACCATCCTCA GGGGCCAGAC GTGTATGCTG GTGTACCTAA GGATTACACC 240 GGTGAGGACG TAACACCTGA GAACCTATAT GCTGTCATTC TTGGGGACAA GAGTAAAGTT 300 AAAGGTGGAA GTGGCAAGGT GATCAACAGC AATCCGGAGG ATAGGATATT TATATTCTAC 360 TCCGATCATG GAGGTCCCGG AGTTCTTGGG ATGCCAAACG CACCATTCGT TTATGCCATG 420 GATTTTATTG ATGTTTTGAA GAAGAAACAT GCAAGTGGAG GCTACAAGGA GATGGTTATA 480 TACATAGAAG CTTGTGAGAG TGGGAGCATA TTTGAGGGTA TAATGCCCAA GGATCTGAAT 540 ATTTATGTGA CAACTGCGTC AAATGCACAA GAGAACAGTT TTGGAACTTA TTGTCCTGGG 600 ATGAATCCTC CTCCACCAGA AGAGTACGTA ACTTGCCTGG GGGATTTATA CAGCGTTTCT 660 TGGATGGAAG ATAGTGAGAC TCACAATCTA AAAAGGGAAA CGGTACAACA GCAATACCAG 720 TCGGTA AGGA AACGGACTTC AAATTCTAAC AGCTATAGGT TTGGTTCTCA TGTGATGCAA 780 TACGGTGACA CTAACATTAC TGCTGAAAAG CTTTACTTGT ACCATGGTTT TGATCCTGCC 840 ACCGTGAACT TTCCTCCACA CAACGGCAAC CTAGAAGCTA AAATGGAAGT TGTTAACCAG 900 AGAGATGCAG AGCTTTTGTT CATGTGGCAA ATGTATCAGA GATCAAACCA TCAACCGGAA 960 AAGAAGACTC ACATCCTGGA ACAGATTACA GAGACAGTGA AGCATAGGAA TCATTTGGAT 1020 GGCAGTGTGG AATTGATTGG AGTTTTGTTG TATGGACCAG GAAAAAGTTC TTCGGTTCTA 1080 CATTCCGTGA GGGCTCCTGG TCTGCCCCTA GTTGATGATT GGACATGCTT GAAATCTATG 1140 GTTAGAGTGT TCGAAACTCA CTGTGGGTCA CTGACTCAGT ATGGCATGAA ACACATGCGG 1200 GCATTCGGCA ACGTTTGCAA CAGCGGCGTT TCTAAGGCCT CCATGGAGGA GGCTTGTAAG 1260 GCAGCTTGCG GTGGCTATGA CGCTGGGTTA TTATATCCAT CAAACAGG GGTAGGGATTA CG: GGTAGGGTA TACATACGCAT AAACAGG ATATAGTGCT 1320: TGA1323 Type: cDNA to mRNA Sequence: GACGAGGGGA CCAGGTGGGC CGTTTTAATT GCTGGTTCCA ATGGTTACTG GAATTACAGG 60 CATCAGTCTG ATGTATGCCA TGCGTATCAA GTGCTGAGGA AAGGTGGTCT GAAAGAAGAA 1 20 AATATTGTTG TGTTTATGTA TGATGACATT GCTTTCCATA AAGAAAACCC ACGGCCTGGA 180 ATCATCCTTA ACAGTCCACA TGGAGATGAT GTTTACAAAG GAGTACCTAA GGATTACGTT 240 GGTAAAGATG TTACTGTTAA CAACTTTTTA GCTGCTATAC TTGGAAATAA GTCAGCTATT 300 ACCGGCGGTA GTGGGAAGGT TGTCAATAGT GGTCCCAATG ATCATATATT TATATTCTAC 360 TCTGATCATG GGGGTCCGGG AGTGCTTGGG ATGCCTACTA GTCCGTTCTT GTATGCATCT 420 GATCTGATTG AAGTCTTGAA GAAGAAGCAT GCTTCTGGAA CATATAAAAG CCTTGTATTT 480 TATCTAGAGG CATGTGAATC TGGGAGTATC TTTGAAGGTC TTCTTCCTGA AGGTCTAAAT 540 ATCTATGCAA CAACAGCTGC AAATGCAAAA GAAAGCAGTT GGGGAACATA TTGTCCTGGG 600 GAGTCTCCTA GTCCTCCCCC AGAATATGAA ACCTGCCTGG GTGACCTGTA CAGTGTTGCT 660 TGGATGGAAG ACAGTGACAT ACACAATTTA CGAACAGAAA CTCTACATCA ACAATACGAA 720 TCGGTCAAAG CAAGGACTAT CAATGGAAAT TCAATTTATG GTTCTCACGT GATGCAGTAT 780 GGTGACATAG AGCTTAGCAA AAATAATCTC TTCCTATATT TGGGTACAAA TCCTGCAAAT 840 GATAATTTTA CTTTTGTGGA TAAAAACTCG TTGGTGCCAC CTTCAAAAGC AGTAAACCAA 900 CGTGATGCTG ATCTCGTCCA TTTCTGGGAT AAGTTCCGCA AAGCTCCTGA GGGTTCTGCT 960 AGGAAAGCTG CAGCCA GGAA ACAAGTTCTG GAAGCAATGT CTCACAGAAT GCATATAGAT 1020 GACAGCATGA AACTTATTGG GAAGCTCTTA TTTGGCATTG AAAAGGGCCC AGAAGTGCTC 1080 AGCAGTGTTA GACCTGCTGG GCAAGCACTT GTTGATGATT GGGACTGCCT TAAAACACTG 1140 GTTAGGACTT TTGAGACACA TTGCGGATCA CTGTCTCAGT ATGGGATGAA GCATATGAGG 1200 TCCTTTGCAA ACTTCTGCAA CGCTGGTATT CAGAAGGAGC AAATGGCTGA GGCCTCAGCA 1260 CAAGCGTGCG TCAGTATTCC TGCAACTCCC TGGAGTTCAT TGCACAGGGG TTTCAGTGCA 1320 TAA 1323 SEQ ID NO: 3 sequence length Length: 1323 Sequence type: Nucleic acid Number of strands: Double-strand topology: Linear Sequence type: cDNA to mRNA Sequence: ATTCCCGGTA CCAGATGGGC CATCCTAATC GCCGGCTCCA ATGGCTACTG GAATTACAGG 60 CATCAGGCTGATTACTGTCA TGCGTATCAA ATACTGAGGA AAGGTGGCTT AAATATTA 120A GTCATCATTA ACAAACCAGA TGGGGATGAT GTTTATGAAG GAGTTCCAAA GGATTACACT 240 GGCGACGATG TTACTGCTGA TAACTTCTAT GCTGTTATAC TTGGAAATAA ATCAGCACTT 300 ACAGGTGGCA GTGGGAAGGT TGTGAACAGT GGCCCTGATG ATCGTATATT CATATTCA TCTAT TG GAGGTCCAGG AGTTCTTGGG ATGCCTGCTG GTCCTTTCTT ATATGCATCT 420 GATCTGATTG AAGTCTTGAA GAAAAAACAT GCTTCTGGAA CGTATAAAAG CCTTGTATTT 480 TATCTTGAGG CATGTGAATC TGGGAGTATC TTTGAAGGCC TTCTTCCTGA AGATATCAAT 540 ATTTATGCAA CAACAGCTTC CAATGCAGAA GAAAGCAGCT GGGGAACATA TTGCCCAGGC 600 GAGGATCCCA GTCCTCCCCC AGAATATTCA ACTTGCTTGG GTGACGAGTA CAGTGTTGCT 660 TGGATGGAAG ACAGTGACAG GAAGAATTTG CGAACAGAAA CTTTGCACCA ACAATATGAA 720 TTGGTTAAAG AGAGGACTAT TAACGGAAGT ATATACCATA GCTCTCACGT GATGCAGTAT 780 GGTGATATAA GTCTCAGCGA TGATGTTCTC TTCCTATATT TGGGTACAAA TCCTGCTAAT 840 GATAATTTTA CCTATGTGGA TGAGAACTCC TTGAGGTCAC CTTCAAAAGC AATCAGCCAA 900 CGTGTTGCTG ATCTCATCCA TTTTTGGGAG AAGTTCCGCA AAGCTCCTGA GGGTTCTACC 960 AGGAAAGATG CTGCTCAGAA ACAATTTCTG GAAGTAATGT CTCACAGAAT GCATATAGAC 1020 AACAGTGTGA AAATTATTGG GAGTCTTTTA TTTGGCATTG AAAAGGGTCC AGAAGTACTC 1080 AATGCTGTTA GACCGGCTGG AATGGCACTT GTTGATGACT GGGACTGCCT GAAAAATATG 1140 GTAAGGACTT TTGAGACATA TTGTGGATCC TTGTCTCAGT ATGGGATGAA ATATATGAGG 1200 TCCTTTGCAA ACATCTGCAA TG CAAGAATT AAGAATGACC AAATGGCTGA TGCCTCAGCA 1260 CAAGCTTGTG TCAGTATTCC TGCCAATCCC TGGAGTTCTC TGCAAAGGGG TTTCAGTGCA 1320 TAA 1323 SEQ ID NO: 4 Sequence length: 1323 Sequence type: Nucleic acid Chain number: Double-stranded topology: Linear c sequence mRNA: Sequence CTTCCCGGTA CCAGGTGGGC CATCCTTCTC GCCGGTTCCA ATGGCTACTG GAATTACAGG 60 CATCAGGCTG ATATTTGTCA TGCGTATCAA ACACTGAGGA AAGGTGGCCT GAAAGAAGAA 120 AATATTATTG TTTTTATGTA TGATGACATT GCGTTCAATG AGGAGAACCC AAGACCTGGG 180 GTCATCATTA ACAAACCAGA TGGGACTGAT GTTTATGAAG GAGTTCCAAA GGATTACACT 240 GGCGAAGATG TTACTGCTGA AAACTTCTAC GCTGTTTTAC TTGGAAATAA GTCGGCACTT 300 AGCGGTGGCA GTGGGAAGGT TGTGAACAGT GGCCCCGATG ATCGTATATT TGTATTCTAT 360 AGTGATCATG GAGGTCCAGG AGTTCTCGGG ATGCCTGCCG GTCCTTACTT ATATGCATCT 420 GATCTGAATG ATGTCTTGAA GAAAAAACAT GCTTCCGGAA CATATAAAAG CCTAGTATTT 480 TATCTTGAGG CATGTGAATC TGGGAGTATC TTTGAAGGCC TTCTTCCTGA AGATGTCAAT 540 ATTTATGCAA CAACAGCTTC GAATGCAGAA GAAAGCAGCT GGGGAACATA TTGCCCCCGTGG CC AGAATATTCA ACTTGCTTGG GAGACCTGTA CAGTGTTGCT 660 TGGATGGAAG ACAGTGACAG ACACAATTTG CGAACTGAAA CTTTGAACCA ACAATATAAA 720 TTGGTTAAAG AGAGGACCAT TAGTGGAGGT TCATACTATG GCTCTCACGT GATGCAGTAT 780 GGTGATATAG GGCTCAGCGA TGATGTTCTC TTCCTATATT TGGGTACAAA TCCTGCTAAT 840 GATAATTTTA CCTTTGTCGA TGAAAACTCC TTGAGGTCAC CTTCAAAAGC AGTCAACCAA 900 CGGGATGCTG ATCTCATCCA TTTCTGGGAG AAGTTCCGCA AAGCTCCTGA GGGTTCTCCC 960 GAGAAAAATG CTGCTCAGAA ACAAGTTGTG GAAGTAATGT CTCACAGGAT GCATATAGAC 1020 AACGGTGTGG AACTTATTGG GAAGCTTTTA TTTGGCATTG AAAAGGGTCC AAAAGTACTG 1080 GATGCTGTTA GACCGGCTGG AATGGCACTT GTTGATGACT GGGACTGCCT GAAAACCATG 1140 GTAAGGACAT TTGAGACATA TTGTGGATCC TTGTCTCAGT ATGGGATGAA ACATATGAGG 1200 TCCTTTGCAA ACATCTGCAA CGCAGGAATT AAGAATGACC AAATGGCCGA TGCCTCAGCA 1260 CAAGCTTGTG TCAGTATTCC TGCCAATTCC TGGAGTTCTC TGCAAAGGGG TTTCAGTGCA 1320 TAA 1323 SEQ ID NO: 5 length of sequence: 1323 SEQ type: nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: GAAGTTGGT A CCCGGTGGGC GGTTCTCGTG GCTGGTTCAA ACGGTTATGG AAATTATAGG 60 CATCAAGCGG ATGTGTGCCA TGCTTACCAG TTGCTGATAA AGGGTGGAGT CAAAGAAGAA 120 AACATTGTGG TGTTTATGTA TGATGATATA GCTTATAACG CCATGAATCC CAGACCCGGA 180 GTCATCATCA ACCATCCTCA GGGGCCAGAC GTGTATGCTG GTGTACCTAA GGATTACACC 240 GGTGAGGACG TAACACCTGA GAACCTATAT GCTGTCATTC TTGGGGACAA GAGTAAAGTT 300 AAAGGTGGAA GTGGCAAGGT GATCAACAGC AATCCGGAGG ATAGGATATT TATATTCTAC 360 TCCGATCATG GAGGTCCCGG AGTTCTTGGG ATGCCAAACG CACCATTCGT TTACGCCATG 420 GATTTTATTG ATGTTTTGAA GAAGAAACAT GCAAGTGGAG GCTACAAGGA GATGGTTATA 480 TACATAGAAG CTTGTGAGAG TGGGAGCATA TTTGAGGGTA TAATGCCCAA GGATCTGAAT 540 ATTTATGTGA CAACTGCGTC AAATGCACAA GAGAACAGTT TTGGAACTTA TTGTCCTGGG 600 ATGAATCCTC CTCCACCAGA AGAGTACGTA ACTTGCCTGG GGGATTTATA CAGCGTTTCT 660 TGGATGGAAG ATAGTGAGAC TCACAATCTA AAAAGGGAAA CGGTACAACA GCAATACCAG 720 TCGGTAAGGA AACGGACTTC AAATTCTAAC AGCTATAGGT TTGGTTCTCA TGTGATGCAA 780 TACGGTGACA CTAACATTAC TGCTGAAAAG CTTTACTTGT ACCATGGTTT TGATCCTGCC 840 ACCGTGAACT TTCCTCCACA CAACGGCA AC CTAGAAGCTA AAATGGAAGT TGTTAACCAG 900 AGAGATGCAG AGCTTTTGTT CATGTGGCAA ATGTATCAGA GATCAAACCA TCAACCGGAA 960 AAGAAGACTC ACATCCTGGA ACAGATTACA GAGACAGTGA AGCATAGGAA TCATTTGGAT 1020 GGCAGTGTGG AATTGATTGG AGTTTTGTTG TATGGACCAG GAAAAAGTTC TTCGGTTCTA 1080 CATTCCGTGA GGGCTCCTGG TCTGCCCCTA GTTGATGATT GGACATGCTT GAAATCTATG 1140 GTTAGAGTGT TCGAAACTCA CTGTGGGTCA CTGACTCAGT ATGGCATGAA ACACATGCGG 1200 GCATTCGGCA ACGTTTGCAA CAGCGGCGTT TCTAAGGCCT CCATGGAGGA GGCTTGTAAG 1260 GCAGCTTGCG GTGGCTATGA CGCTGGGTTA TTATATCCAT CAAACACAGG ATATAGTGCT 1320 TGA 1323 SEQ ID NO: 6 Sequence length: 1152 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence: GAAGTTGGTA CCCGGTGGGC GGTTCTCGTG GCTGGTTCAA ACGGTTATGG AAAGCATAGG 60 CATT ATGTGTGCCA TGCTTACCAG TTGCTGATAA AGGGTGGAGT CAAAGAAGAA 120 AACATTGTGG TGTTTATGTA TGATGATATA GCTTATAACG CCATGAATCC CAGACCCGGA 180 GTCATCATCA ACCATCCTCA GGGGCCAGAC GTGTATGCTG GTGTACCTAA GGATTACACC 240 GGTGAGGACG TA GAACCTATAT GCTGTCATTC TTGGGGACAA GAGTAAAGTT 300 AAAGGTGGAA GTGGCAAGGT GATCAACAGC AATCCGGAGG ATAGGATATT TATATTCTAC 360 TCCGATCATG GAGGTCCCGG AGTTCTTGGG ATGCCAAACG CACCATTCGT TTACGCCATG 420 GATTTTATTG ATGTTTTGAA GAAGAAACAT GCAAGTGGAG GCTACAAGGA GATGGTTATA 480 TACATAGAAG CTTGTGAGAG TGGGAGCATA TTTGAGGGTA TAATGCCCAA GGATCTGAAT 540 ATTTATGTGA CAACTGCGTC AAATGCACAA GAGAACAGTT TTGGAACTTA TTGTCCTGGG 600 ATGAATCCTC CTCCACCAGA AGAGTACGTA ACTTGCCTGG GGGATTTATA CAGCGTTTCT 660 TGGATGGAAG ATAGTGAGAC TCACAATCTA AAAAGGGAAA CGGTACAACA GCAATACCAG 720 TCGGTAAGGA AACGGACTTC AAATTCTAAC AGCTATAGGT TTGGTTCTCA TGTGATGCAA 780 TACGGTGACA CTAACATTAC TGCTGAAAAG CTTTACTTGT ACCATGGTTT TGATCCTGCC 840 ACCGTGAACT TTCCTCCACA CAACGGCAAC CTAGAAGCTA AAATGGAAGT TGTTAACCAG 900 AGAGATGCAG AGCTTTTGTT CATGTGGCAA ATGTATCAGA GATCAAACCA TCAACCGGAA 960 AAGAAGACTC ACATCCTGGA ACAGATTACA GAGACAGTGA AGCATAGGAA TCATTTGGAT 1020 GGCAGTGTGG AATTGATTGG AGTTTTGTTG TATGGACCAG GAAAAAGTTC TTCGGTTCTA 1080 CATTCCGTGA GGGCTCCTGG TCTGCCCCTA GTTGAT GATT GGACATGCTT GAAATCTATG 1140 GTTAGAGTGT TC 1152 SEQ ID NO: 7 Sequence length: 894 Sequence type: Nucleic acid Strand number: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence: GAAGTTGGTA CCCGGTGGGC GGTTCTCGTG GCTGGTTCAA ACGGTTATGG AAATTATAGG 60 CATCAAGCGG ATGTGTGCCA TGCTTACCAG TTGCTGATAA AGGGTGGAGT CAAAGAAGAA 120 AACATTGTGG TGTTTATGTA TGATGATATA GCTTATAACG CCATGAATCC CAGACCCGGA 180 GTCATCATCA ACCATCCTCA GGGGCCAGAC GTGTATGCTG GTGTACCTAA GGATTACACC 240 GGTGAGGACG TAACACCTGA GAACCTATAT GCTGTCATTC TTGGGGACAA GAGTAAAGTT 300 AAAGGTGGAA GTGGCAAGGT GATCAACAGC AATCCGGAGG ATAGGATATT TATATTCTAC 360 TCCGATCATG GAGGTCCCGG AGTTCTTGGG ATGCCAAACG CACCATTCGT TTACGCCATG 420 GATTTTATTG ATGTTTTGAA GAAGAAACAT GCAAGTGGAG GCTACAAGGA GATGGTTATA 480 TACATAGAAG CTTGTGAGAG TGGGAGCATA TTTGAGGGTA TAATGCCCAA GGATCTGAAT 540 ATTTATGTGA CAACTGCGTC AAATGCACA GAGAACAGTT TTGGAACTTA TTGTCCTGGG 600 ATGAATCCTC CTCCACCAGA AGAGTACGTA ACTTGCCTGG GGGATTTATA CAGCGTTTGA TAG ATAGTGGATG AATCTA AAAAGGGAAA CGGTACAACA GCAATACCAG 720 TCGGTAAGGA AACGGACTTC AAATTCTAAC AGCTATAGGT TTGGTTCTCA TGTGATGCAA 780 TACGGTGACA CTAACATTAC TGCTGAAAAG CTTTACTTGT ACCATGGTTT TGATCCTGCC 840 ACCGTGAACT TTCCTCCACA CAACGGCAAC CTAGAAGCTA AAATGGAAGT TGTT 894 SEQ ID NO: 8 SEQ Length: 813 SEQ types: the number of nucleic acid strands: double strand Topology: linear sequence type: cDNA-to mRNA sequences: GAAGTTGGTA CCCGGTGGGC GGTTCTCGTG GCTGGTTCAA ACGGTTATGG AAATTATAGG 60 CATCAAGCGG ATGTGTGCCA TGCTTACCAG TTGCTGATAA AGGGTGGAGT CAAAGAAGAA 120 AACATTGTGG TGTTTATGTA TGATGATATA GCTTATAACG CCATGAATCC CAGACCCGGA 180 GTCATCATCA ACCATCCTCA GGGGCCAGAC GTGTATGCTG GTGTACCTAA GGATTACACC 240 GGTGAGGACG TAACACCTGA GAACCTATAT GCTGTCATTC TTGGGGACAA GAGTAAAGTT 300 AAAGGTGGAA GTGGCAAGGT GATCAACAGC AATCCGGAGG ATAGGATATT TATATTCTAC 360 TCCGATCATG GAGGTCCCGG AGTTCTTGGG ATGCCAAACG CACCATTCGT TTACGCCATG 420 GATTTTATTG ATGTTTTGAA GAAGAAACAT GCAAGTGGAG GCTACAAGGA GATGGTTATA 480 TACATAGAAG CTTGTGAGAG TAGGAGCATA GGTA TAATGCCCAA GGATCTGAAT 540 ATTTATGTGA CAACTGCGTC AAATGCACAA GAGAACAGTT TTGGAACTTA TTGTCCTGGG 600 ATGAATCCTC CTCCACCAGA AGAGTACGTA ACTTGCCTGG GGGATTTATA CAGCGTTTCT 660 TGGATGGAAG ATAGTGAGAC TCACAATCTA AAAAGGGAAA CGGTACAACA GCAATACCAG 720 TCGGTAAGGA AACGGACTTC AAATTCTAAC AGCTATAGGT TTGGTTCTCA TGTGATGCAA 780 TACGGTGACA CTAACATTAC TGCTGAAAAG CTC 813 SEQ ID NO: 9 Length of sequence: 4 types of sequences : Number of amino acid chains: 1 chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1 E 2,4-dinitrophenyl-L-proline 4 E L-asparaginamide SEQ ID NO: 10 Sequence Length: 4 Sequence Type: Amino Acid Number of Chains: 1 Strand Topology: Linear Sequence Type: Peptide Sequence Characteristics: 1 E 2,4-dinitrophenyl-L-proline SEQ ID NO: 11 Sequence length: 25 Sequence type: Amino acid Number of chains: 1 strand Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment Sequence characteristics: 6 E Unknown amino acid sequence: Glu Val Gly Thr Arg Xaa Ala Val Leu Val Ala Gly Ser Asn Gly 1 5 10 15 Tyr Gly Asn Tyr Arg His Gln Ala Asp Val 20 25 SEQ ID NO: 12 Sequence length: 23 Sequence type: Nucleic acid Number of strands: 1 Main chain Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence: GCNGTNCTNG TNGCNGGNAG NAA 23 SEQ ID NO: 13 Sequence length: 23 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence: GGNTAYGGNA AYTAYMGNCA YCA 23 SEQ ID NO: 14 Sequence length: 753 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear sequence type: cDNA to mRNA sequence: GGTTATGGTA ATTATCGGCA TC AAGCGGAT GTGTGCCATG CTTACCAGTT GCTGATAAAA 60 GGTGGAGTCA AAGAAGAAAA CATTGTGGTG TTTATGTATG ATGATATAGC TTATAACGCC 120 ATGAATCCCA GACCCGGAGT CATCATCAAC CATCCTCAGG GGCCAGACGT GTATGCTGGT 180 GTACCTAAGG ATTACACCGG TGAGGACGTA ACACCTGAGA ACCTATATGC TGTCATTCTT 240 GGGGACAAGA GTAAAGTTAA AGGTGGAAGT GGCAAGGTGA TCAACAGCAA TCCGGAGGAT 300 AGGATATTTA TATTCTACTC CGATCATGGA GGTCCCGGAG TTCTTGGGAT GCCAAACGCA 360 CCATTCGTTT ATGCCATGGA TTTTATTGAT GTTTTGAAGA AGAAACATGC AAGTGGAGGC 420 TACAAGGAGA TGGTTATATA CATAGAAGCT TGTGAGAGTG GGAGCATATT TGAGGGTATA 480 ATGCCCAAGG ATCTGAATAT TTATGTGACA ACTGCGCCAA ATGCACAAGA GAACAGTTTT 540 GGAACTCATT GTCCTGGGAT GAATCCTCCT CCACCAGAAG AGTACGTAAC TTGCCTGGGG 600 GATTTATACA GCGTTTCTTG GATGGAAGAT AGTGAGACTC ACAATCTAAA AAGGGAAACG 660 GTACAACAGC AATACCAGTC GGTAAGGAAA CGGACTTCAA ATTCTAACAG CTATAGGTTT 720 GGTTCTCATG TGATGCCTAT AATTCCCGTA GCC 753 SEQ ID NO: 15 length of sequence: 1742 type of sequence : Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA to mRNA SEQ: CTATATGATG TGATATGATA TTTTAGTTTA AGAGAGGGGA GAGGATACTC TTCTTTCATT 60 CCAACTACTC TTCTTCAATT ACCATGAACC GCTTTCCGAT CCTCTTTCTC ATCGCCACCC 120 TCCTCGTCTT CGTCTCCGGT GACCATGACG ATGTTCTCCG ATTGCCCTCC GAAGCTTCCA 180 GATTCTTCCA AGCACCCGAG GAAAAC GAC GAG GGG ACC AGG TGG GCC GTT TTA ATT 236 Asp Glu Gly Thr Arg Trp Ala Val Leu Ile 1 5 10 GCT GGT TCC AAT GGT TAC TGG AAT TAC AGG CAT CAG TCT GAT GTA 281 Ala Gly Ser Asn Gly Tyr Trp Asn Tyr Arg His Gln Ser Asp Val 15 20 25 TGC CAT GCG TAT CAA GTG CTG AGG AAA GGT GGT CTG AAA GAA GAA 326 Cys His Ala Tyr Gln Val Leu Arg Lys Gly Gly Leu Lys Glu Glu 30 35 40 AAT ATT GTT GTG TTT ATG TAT GAT GAC ATT GCT TTC CAT AAA GAA 371 Asn Ile Val Val Phe Met Tyr Asp Asp Ile Ala Phe His Lys Glu 45 50 55 AAC CCA CGG CCT GGA ATC ATC CTT AAC AGT CCA CAT GGA GAT GAT 416 Asn Pro Arg Pro Gly Ile Ile Leu Asn Ser Pro His Gly Asp Asp 60 65 70 GTT TAC AAA GGA GTA CCT AAG GAT TAC GTT GGT AAA GAT GTT ACT 461 Val Tyr Lys Gly Val Pro Lys Asp Tyr Val Gly Lys Asp Val Thr 75 80 85 GTT AAC AAC TTT TTA GCT GCT ATA CTT GGA AAT AAG TCA GCT ATT 506 Val Asn Asn Phe Leu Ala Ala Ile Leu Gly Asn Lys Ser Ala Ile 90 95 100 ACC GGC GGT AGT GGG AAG GTT GTC AAT AGT GGT CCC AAT GAT CAT 551 Thr Gly Gly Ser Gly Lys Val Val Asn Ser Gly Pro Asn Asp His 105 110 115 ATA TTT ATA TTC TAC TCT GAT CAT GGG GGT CCG GGA GTG CTT GGG 596 Ile Phe Ile Phe Tyr Ser Asp His Gly Gly Pro Gly Val Leu Gly 120 125 130 ATG CCT ACT AGT CCG TTC TTG TAT GCA TCT GAT CTG ATT GAA GTC 641 Met Pro Thr Ser Pro Phe Leu Tyr Ala Ser Asp Leu Ile Glu Val 135 140 145 TTG AAG AAG AAG CAT GCT TCT GGA ACA TAT AAA AGC CTT GTA TTT 686 Leu Lys Lys Lys His Ala Ser Gly Thr Tyr Lys Ser Leu Val Phe 150 155 160 TAT CTA GAG GCA TGT GAA TCT GGG AGT ATC TTT GAA GGT CTT CTT 731 Tyr Leu Glu Ala Cys Glu Ser Gly Ser Ile Phe Glu Gly Leu Leu 165 170 175 CCT GAA GGT CTA AAT ATC TAT GCA ACA ACA GCT GCA AAT GCA AAA 776 Pro Glu Gly Leu Asn Ile Tyr Ala Thr Thr Ala Ala Asn Ala Lys 180 185 190 GAA AGC AGT TGG GGA ACA TAT TGT CCT GGG GAG TC T CCT AGT CCT 821 Glu Ser Ser Trp Gly Thr Tyr Cys Pro Gly Glu Ser Pro Ser Pro 195 200 205 CCC CCA GAA TAT GAA ACC TGC CTG GGT GAC CTG TAC AGT GTT GCT 866 Pro Pro Glu Tyr Glu Thr Cys Leu Gly Asp Leu Tyr Ser Val Ala 210 215 220 TGG ATG GAA GAC AGT GAC ATA CAC AAT TTA CGA ACA GAA ACT CTA 911 Trp Met Glu Asp Ser Asp Ile His Asn Leu Arg Thr Glu Thr Leu 225 230 235 CAT CAA CAA TAC GAA TCG GTC AAA GCA AGG ACT ATC AAT GGA AAT 956 His Gln Gln Tyr Glu Ser Val Lys Ala Arg Thr Ile Asn Gly Asn 240 245 250 TCA ATT TAT GGT TCT CAC GTG ATG CAG TAT GGT GAC ATA GAG CTT 1001 Ser Ile Tyr Gly Ser His Val Met Gln Tyr Gly Asp Ile Glu Leu 255 260 265 AGC AAA AAT AAT CTC TTC CTA TAT TTG GGT ACA AAT CCT GCA AAT 1046 Ser Lys Asn Asn Leu Phe Leu Tyr Leu Gly Thr Asn Pro Ala Asn 270 275 280 GAT AAT TTT ACT TTT GTG GAT AAA AAC TCG TTG GTG CCA CCT TCA 1091 Asp Asn Phe Thr Phe Val Asp Lys Asn Ser Leu Val Pro Pro Ser 285 290 295 AAA GCA GTA AAC CAA CGT GAT GCT GAT CTC GTC CAT TTC TGG GAT 1136 Lys Ala Val Asn Gln Arg As p Ala Asp Leu Val His Phe Trp Asp 300 305 310 AAG TTC CGC AAA GCT CCT GAG GGT TCT GCT AGG AAA GCT GCA GCC 1181 Lys Phe Arg Lys Ala Pro Glu Gly Ser Ala Arg Lys Ala Ala Ala 315 320 325 AGG AAA CAA GTT CTG GAA GCA ATG TCT CAC AGA ATG CAT ATA GAT 1226 Arg Lys Gln Val Leu Glu Ala Met Ser His Arg Met His Ile Asp 330 335 340 GAC AGC ATG AAA CTT ATT GGG AAG CTC TTA TTT GGC ATT GAA AAG 1271 Asp Ser Met Lys Leu Ile Gly Lys Leu Leu Phe Gly Ile Glu Lys 345 350 355 GGC CCA GAA GTG CTC AGC AGT GTT AGA CCT GCT GGG CAA GCA CTT 1316 Gly Pro Glu Val Leu Ser Ser Val Arg Pro Ala Gly Gln Ala Leu 360 365 370 GTT GAT GAT TGG GAC TGC CTT AAA ACA CTG GTT AGG ACT TTT GAG 1361 Val Asp Asp Trp Asp Cys Leu Lys Thr Leu Val Arg Thr Phe Glu 375 380 385 ACA CAT TGC GGA TCA CTG TCT CAG TAT GGG ATG AAG CAT ATG AGG 1406 Thr His Cys Gly Ser Leu Ser Gln Tyr Gly Met Lys His Met Arg 390 395 400 TCC TTT GCA AAC TTC TGC AAC GCT GGT ATT CAG AAG GAG CAA ATG 1451 Ser Phe Ala Asn Phe Cys Asn Ala Gly Ile Gln Lys Glu Gln Met 405 410 415 GCT GAG GCC TCA GCA CAA GCG TGC GTC AGT ATT CCT GCA ACT CCC 1496 Ala Glu Ala Ser Ala Gln Ala Cys Val Ser Ile Pro Ala Thr Pro 420 425 430 TGG AGT TCA TTG CAC AGG GGT TTC AGT GCA TAATTCCTAG AATGTACTAC 15 Trp Ser Ser Leu His Arg Gly Phe Ser Ala 435 440 ATTAAAGAAC AATGTATAAT CATTGTGGCA GAATGCTTTA TCATTGTGTG AAATTTATAT 1606 GCTGTACTTC GGTCATGATT TCTTATACCA ACATTGTAAA TACAAATGGG ACGCTGGGAA CTTT 1AAACATAAAAGAA CATAAAAAAAAGAAACATAAAAAAAAAAAAAAAAAAGAAAGAAACATAAAAAAAAAAAAAAAA Number of: Double-stranded Topology: Linear Sequence type: cDNA to mRNA Sequence: CAT GGA GGT CCC GGA GTT CTT GGG ATG CCA AAC GCA CCA TTC GTT 45 His Gly Gly Pro Gly Val Leu Gly Met Pro Asn Ala Pro Phe Val 1 5 10 15 TAT GCC ATG GAT TTT ATT GAT GTT TTG AAG AAG AAA CAT GCA AGT 90 Tyr Ala Met Asp Phe Ile Asp Val Leu Lys Lys Lys His Ala Ser 20 25 30 GGA GGC TAC AAG GAG ATG GTT ATA TAC ATA GAA GCT TGT GAG AGT 135 Gly Gly Tyr Lys Glu Met Va l Ile Tyr Ile Glu Ala Cys Glu Ser 35 40 45 GGG AGC ATA TTT GAG GGT ATA ATG CCC AAG GAT CTG AAT ATT TAT 180 Gly Ser Ile Phe Glu Gly Ile Met Pro Lys Asp Leu Asn Ile Tyr 50 55 60 GTG ACA ACT GCG TCA AAT GCA CAA GAG AAC AGT TTT GGA ACT TAT 225 Val Thr Thr Ala Ser Asn Ala Gln Glu Asn Ser Phe Gly Thr Tyr 65 70 75 TGT CCT GGG ATG AAT CCT CCT CCA CCA GAA GAG TAC GTA ACT TGC 270 Cys Pro Gly Met Asn Pro Pro Pro Pro Glu Glu Tyr Val Thr Cys 80 85 90 CTG GGG GAT TTA TAC AGC GTT TCT TGG ATG GAA GAT AGT GAG ACT 315 Leu Gly Asp Leu Tyr Ser Val Ser Trp Met Glu Asp Ser Glu Thr 95 100 105 CAC AAT CTA AAA AGG GAA ACG GTA CAA CAG CAA TAC CAG TCG GTA 360 His Asn Leu Lys Arg Glu Thr Val Gln Gln Gln Tyr Gln Ser Val 110 115 120 AGG AAA CGG ACT TCA AAT TCT AAC AGC TAT AGG TTT GGT TCT CAT 405 Arg Lys Arg Thr Ser Asn Ser Asn Ser Tyr Arg Phe Gly Ser His 125 130 135 GTG ATG CAA TAC GGT GAC ACT AAC ATT ACT GCT GAA AAG CTT TAC 450 Val Met Gln Tyr Gly Asp Thr Asn Ile Thr Ala Glu Lys Leu Tyr 140 145 150 TTG TAC CAT GGT TTT GAT CCT GCC ACC GTG AAC TTT CCT CCA CAC 495 Leu Tyr His Gly Phe Asp Pro Ala Thr Val Asn Phe Pro Pro His 155 160 165 AAC GGC AAC CTA GAA GCT AAA ATG GAA GTT GTT AAC CAG AGA GAT 540 Asn Gly Asn Leu Glu Ala Lys Met Glu Val Val Asn Gln Arg Asp 170 175 180 GCA GAG CTT TTG TTC ATG TGG CAA ATG TAT CAG AGA TCA AAC CAT 585 Ala Glu Leu Leu Phe Met Trp Gln Met Tyr Gln Arg Ser Asn His 185 190 195 CAA CCG GAA AAG AAG ACT CAC ATC CTG GAA CAG ATT ACA GAG ACA 630 Gln Pro Glu Lys Lys Thr His Ile Leu Glu Gln Ile Thr Glu Thr 200 205 210 GTG AAG CAT AGG AAT CAT TTG GAT GGC AGT GTG GAA TTG ATT GGA 675 Val Lys His Arg Asn His Leu Asp Gly Ser Val Glu Leu Ile Gly 215 220 225 GTT TTG TTG TAT GGA CCA GGA AAA AGT TCT TCG GTT CTA CAT TCC 720 Val Leu Leu Tyr Gly Pro Gly Lys Ser Ser Ser Val Leu His Ser 230 235 240 GTG AGG GCT CCT GGT CTG CCC CTA GTT GAT GAT TGG ACA TGC TTG 765 Val Arg Ala Pro Gly Leu Pro Leu Val Asp Asp Trp Thr Cys Leu 245 250 255 AAA TCT ATG GTT AGA GTG TTC GAA ACT CAC TGT GGG TCA CTG ACT 810 Lys Ser Met Val Arg Val Phe Glu Thr His Cys Gly Ser Leu Thr 260 265 270 CAG TAT GGC ATG AAA CAC ATG CGG GCA TTC GGC AAC GTT TGC AAC 855 Gln Tyr Gly Met Lys His Met Arg Ala Phe Gly Asn Val Cys Asn 275 280 285 AGC GGC GTT TCT AAG GCC TCC ATG GAG GAG GCT TGT AAG GCA GCT 900 Ser Gly Val Ser Lys Ala Ser Met Glu Glu Ala Cys Lys Ala Ala 290 295 300 TGC GGT GGC TAT GAC GCT GGG TTA TTA TAT CCA TCA AAC ACA GGA 945 Cys Gly Gly Tyr Asp Ala Gly Leu Leu Tyr Pro Ser Asn Thr Gly 305 310 315 TAT AGT GCT TGATTTTGGG TTGTGCACAC AAATTAAAAG CCTTTAACAA 994 Tyr Ser Ala GTTAAACAGC CAAGTTGATG ATGTGCTGCT TCTGAATTGC GTTCTCCCTT CTGGTTTCTG 1054 CTGCTTGTGT TAAATTTGTT CCCTGTAATT AAATTAGCCA AATAGCACAC GTGTAATCGG 1114 GCAGCTAACC CAGTGCAACA CACTCTATTA TTTCTATTGA GAGTGGTTAG GAGAGAATGC 1174 ATATATCGAT CACGTGTATA TAAATGCAGT GCCTTTTCAT AAAATAAGTA ATTAAGTATT 1234 AGTCTATTAA AAAAAA 1250 SEQ ID NO: 17 Sequence length: 1830 Sequence type: Nucleic acid Strand number: Double strand Topology: Linear Sequence type: cDNA to m RNA sequence: CGCTATCGCA ACCCTCGTCT CCGGTGGCCG TGACCTCGCC GGGGACTTCC TCCGGTTGCC 60 CTCCCAACTT GACACCGATC ACAAC ATT CCC GGT ACC AGA TGG GCC ATC CTA ATC 115 Ile Pro Gly Thr Arg TGC ALA Ile Leu Ile 1CC ATC GAT GCC TCC AGT GAT ATT 160 Ala Gly Ser Asn Gly Tyr Trp Asn Tyr Arg His Gln Ala Asp Ile 15 20 25 TGT CAT GCG TAT CAA ATA CTG AGG AAA GGT GGC CTG AAA GAA GAA 205 Cys His Ala Tyr Gln Ile Leu Arg Lys Gly Gly Leu Lys Glu Glu 30 35 40 AAT ATT ATT GTT TTT ATG TAT GAT GAC ATT GCT TTC AAT GAG AAC 250 Asn Ile Ile Val Phe Met Tyr Asp Asp Ile Ala Phe Asn Glu Asn 45 50 55 AAC CCA AAA CCT GGA GTC ATC ATT AAC AAA CCA GAT GGG GAT GAT 295 Asn Pro Lys Pro Gly Val Ile Ile Asn Lys Pro Asp Gly Asp Asp 60 65 70 GTT TAT GAA GGA GTT CCA AAG GAT TAC ACT GGC GAC GAT GTT ACT 340 Val Tyr Glu Gly Val Pro Lys Asp Tyr Thr Gly Asp Asp Val Thr 75 80 85 GCT GAT AAC TTC TAT GCT GTT ATA CTT GGA AAT AAA TCA GCA CTT 385 Ala Asp Asn Phe Tyr Ala Val Ile Leu Gly Asn Lys Ser Ala Leu 90 95 100 ACA GGT GGC AGT GGG AAG GTT GTG AAC AGT GGC CCT GAT GAT CGT 430 Thr Gly Gly Ser Gly Lys Val Val Asn Ser Gly Pro Asp Asp Arg 105 110 115 ATA TTC ATA TTC TAT AGT GAT CAT GGA GGT CCA GGA GTT CTT GGG 475 Ile Phe Ile Phe Tyr Ser Asp His Gly Gly Pro Gly Val Leu Gly 120 125 130 ATG CCT GCT GGT CCT TTC TTA TAT GCA TCT GAT CTG ATT GAA GTC 520 Met Pro Ala Gly Pro Phe Leu Tyr Ala Ser Asp Leu Ile Glu Val 135 140 145 TTG AAG AAA AAA CAT GCT TCT GGA ACG TAT AAA AGC CTT GTA TTT 565 Leu Lys Lys Lys His Ala Ser Gly Thr Tyr Lys Ser Leu Val Phe 150 155 160 TAT CTT GAG GCA TGT GAA TCT GGG AGT ATC TTT GAA GGC CTT CTT 610 Tyr Leu Glu Ala Cys Glu Ser Gly Ser Ile Phe Glu Gly Leu Leu 165 170 175 CCT GAA GAT ATC AAT ATT TAT GCA ACA ACA GCT TCC AAT GCA GAA 655 Pro Glu Asp Ile Asn Ile Tyr Ala Thr Thr Ala Ser Asn Ala Glu 180 185 190 GAA AGC AGC TGG GGA ACA TAT TGC CCA GGC GAG GAT CCC AGT CCT 700 Glu Ser Ser Trp Gly Thr Tyr Cys Pro Gly Glu Asp Pro Ser Pro 195 200 205 CCC CCA GAA TAT TCA ACT TGC TTG GGT GAC GAG TAC AGT GTT GCT 745 Pro Pro Glu Tyr Ser Thr Cys Leu Gly Asp Glu Tyr Ser Val Ala 210 215 220 TGG ATG GAA GAC AGT GAC AGG AAG AAT TTG CGA ACA GAA ACT TTG 790 Trp Met Glu Asp Ser Asp Arg Lys Asn Leu Arg Thr Glu Thr Leu 225 230 235 CAC CAA CAA TAT GAA TTG GTT AAA GAG AGG ACT ATT AAC GGA AGT 835 His Gln Gln Tyr Glu Leu Val Lys Glu Arg Thr Ile Asn Gly Ser 240 245 250 ATA TAC CAT AGC TCT CAC GTG ATG CAG TAT GGT GAT ATA AGT CTC 880 Ile Tyr His Ser Ser His Val Met Gln Tyr Gly Asp Ile Ser Leu 255 260 265 AGC GAT GAT GTT CTC TTC CTA TAT TTG GGT ACA AAT CCT GCT AAT 925 Ser Asp Asp Val Leu Phe Leu Tyr Leu Gly Thr Asn Pro Ala Asn 270 275 280 GAT AAT TTT ACC TAT GTG GAT GAG AAC TCC TTG AGG TCA CCT TCA 970 Asp Asn Phe Thr Tyr Val Asp Glu Asn Ser Leu Arg Ser Pro Ser 285 290 295 AAA GCA ATC AGC CAA CGT GTT GCT GAT CTC ATC CAT TTT TGG GAG 1015 Lys Ala Ile Ser Gln Arg Val Ala Asp Leu Ile His Phe Trp Glu 300 305 310 AAG TTC CGC AAA GCT CCT GAG GGT TCT ACC AGG AAA GAT GCT GCT 1060 Lys Phe Arg Lys Ala Pro Glu G ly Ser Thr Arg Lys Asp Ala Ala 315 320 325 CAG AAA CAA TTT CTG GAA GTA ATG TCT CAC AGA ATG CAT ATA GAC 1105 Gln Lys Gln Phe Leu Glu Val Met Ser His Arg Met His Ile Asp 330 335 340 AAC AGT GTG AAA ATT ATT GGG AGT CTT TTA TTT GGC ATT GAA AAG 1150 Asn Ser Val Lys Ile Ile Gly Ser Leu Leu Phe Gly Ile Glu Lys 345 350 355 GGT CCA GAA GTA CTC AAT GCT GTT AGA CCG GCT GGA ATG GCA CTT 1195 Gly Pro Glu Val Leu Asn Ala Val Arg Pro Ala Gly Met Ala Leu 360 365 370 GTT GAT GAC TGG GAC TGC CTG AAA AAT ATG GTA AGG ACT TTT GAG 1240 Val Asp Asp Trp Asp Cys Leu Lys Asn Met Val Arg Thr Phe Glu 375 380 385 ACA TAT TGT GGA TCC TTG TCT CAG TAT GGG ATG AAA TAT ATG AGG 1285 Thr Tyr Cys Gly Ser Leu Ser Gln Tyr Gly Met Lys Tyr Met Arg 390 395 400 TCC TTT GCA AAC ATC TGC AAT GCA AGA ATT AAG AAT GAC CAA ATG 1330 Ser Phe Ala Asn Ile Cys Asn Ala Arg Ile Lys Asn Asp Gln Met 405 410 415 GCT GAT GCC TCA GCA CAA GCT TGT GTC AGT ATT CCT GCC AAT CCC 1375 Ala Asp Ala Ser Ala Gln Ala Cys Val Ser Ile Pro Ala Asn Pro 420 425 43 0 TGG AGT TCT CTG CAA AGG GGT TTC AGT GCA TAATTCCTGA AAATGCACAC 1425 Trp Ser Ser Leu Gln Arg Gly Phe Ser Ala 435 440 TAATGAACAC CAAAAGTATG ATTACTCTTA CATTATGTTT TATGGCTGTA TGTATATATA 1485 TGTTGTACAA TGGTCATAAT ATTTTGCCCT GCCTTGTAAT TACAATAGGG GCAATACTAG 1545 GAGTCCAAAG GGTCTTTACA TTGTAGTTCC GCACATAGAT ACTTCAACTT CGTTTATATA 1605 ATCCATTTCT TCTGAGGTTG TATATTTGCA ATTTGCAAGA AAAGCAATTA CAGGGGATCG 1665 TGACATTCAC GGCATAAGGT GGGATAATGG ACCAATTGCT TTGGAATGTC TACTATGACA 1725 AGGGCATGAG GTCATGCATG TATGCCAATG CATAGAACTA ATGTGTATTT TTATTCAGTA 1785 GACTATTGCC AAAA 145AACTAAAAAAAAAAGTAA Sequence type: cDNA to mRNA Sequence: AG GAT TAC ACC GGT GAG GAC GTA ACA CCT GAG AAC CTA TAT GCT 44 Asp Tyr Thr Gly Glu Asp Val Thr Pro Glu Asn Leu Tyr Ala 1 5 10 GTC ATT CTT GGG GAC AAG AGT AAA GTT AAA GGT GGA AGT GGC AAG 89 Val Ile Leu Gly Asp Lys Ser Lys Val Lys Gly Gly Ser Gly Lys 15 20 25 GTG ATC AAC AGC AAT CCG GAG GAT AGG ATA TTT ATA TTC TAC TCC 134 Val Ile Asn Ser Asn Pro Glu Asp Arg Ile Phe Ile Phe Tyr Ser 30 35 40 GAT CAT GGA GGT CCC GGA GTT CTT GGG ATG CCA AAC GCA CCA TTC 179 Asp His Gly Gly Pro Gly Val Leu Gly Met Pro Asn Ala Pro Phe 45 50 55 GTT TAT GCC ATG GAT TTT ATT GAT GTT TTG AAG AAG AAA CAT GCA 224 Val Tyr Ala Met Asp Phe Ile Asp Val Leu Lys Lys Lys His Ala 60 65 70 AGT GGA GGC TAC AAG GAG ATG GTT ATA TAC ATA GAA GCT TGT GAG 269 Ser Gly Gyr Tyr Lys Glu Met Val Ile Tyr Ile Glu Ala Cys Glu 75 80 85 AGT GGG AGC ATA TTT GAG GGT ATA ATG CCC AAG GAT CTG AAT ATT 314 Ser Gly Ser Ile Phe Glu Gly Ile Met Pro Lys Asp Leu Asn Ile 90 95 100 TAT GTG ACA ACT GCG TCA AAT GCA CAA GAG AAC AGT TTT GGA ACT 359 Tyr Val Thr Thr Ala Ser Asn Ala Gln Glu Asn Ser Phe Gly Thr 105 110 115 TAT TGT CCT GGG ATG AAT CCT CCT CCA CCA GAA GAG TAC GTA ACT 404 Tyr Cys Pro Gly Met Asn Pro Pro Pro Pro Glu Glu Tyr Val Thr 120 125 130 TGC CTG GGG GAT TTA TAC AGC GTT TCT TGG ATG GAA GAT A GT GAG 449 Cys Leu Gly Asp Leu Tyr Ser Val Ser Trp Met Glu Asp Ser Glu 135 140 145 ACT CAC AAT CTA AAA AGG GAA ACG GTA CAA CAG CAA TAC CAG TCG 494 Thr His Asn Leu Lys Arg Glu Thr Val Gln Gln Gln Tyr Gln Ser 150 155 160 GTA AGG AAA CGG ACT TCA AAT TCT AAC AGC TAT AGG TTT GGT TCT 539 Val Arg Lys Arg Thr Ser Asn Ser Asn Ser Tyr Arg Phe Gly Ser 165 170 175 CAT GTG ATG CAA TAC GGT GAC ACT AAC ATT ACT GCT GAA AAG CTT 584 His Val Met Gln Tyr Gly Asp Thr Asn Ile Thr Ala Glu Lys Leu 180 185 190 TAC TTG TAC CAT GGT TTT GAT CCT GCC ACC GTG AAC TTT CCT CCA 629 Tyr Leu Tyr His Gly Phe Asp Pro Ala Thr Val Asn Phe Pro Pro 195 200 205 CAC AAC GGC AAC CTA GAA GCT AAA ATG GAA GTT GTT AAC CAG AGA 674 His Asn Gly Asn Leu Glu Ala Lys Met Glu Val Val Asn Gln Arg 210 215 220 GAT GCA GAG CTT TTG TTC ATG TGG CAA ATG TAT CAG AGA TCA AAC 719 Asp Ala Glu Leu Leu Phe Met Trp Gln Met Tyr Gln Arg Ser Asn 225 230 235 CAT CAA CCG GAA AAG AAG ACT CAC ATC CTG GAA CAG ATT ACA GAG 764 His Gln Pro Glu Lys Lys Thr His Ile L eu Glu Gln Ile Thr Glu 240 245 250 ACA GTG AAG CAT AGG AAT CAT TTG GAT GGC AGT GTG GAA TTG ATT 809 Thr Val Lys His Arg Asn His Leu Asp Gly Ser Val Glu Leu Ile 255 260 265 GGA GTT TTG TTG TAT GGA CCA GGA AAA AGT TCT TCG GTT CTA CAT 854 Gly Val Leu Leu Tyr Gly Pro Gly Lys Ser Ser Ser Val Leu His 270 275 280 TCC GTG AGG GCT CCT GGT CTG CCC CTA GTT GAT GAT TGG ACA TGC 899 Ser Val Arg Ala Pro Gly Leu Pro Leu Val Asp Asp Trp Thr Cys 285 290 295 TTG AAA TCT ATG GTT AGA GTG TTC GAA ACT CAC TGT GGG TCA CTG 944 Leu Lys Ser Met Val Arg Val Phe Glu Thr His Cys Gly Ser Leu 300 305 310 ACT CAG TAT GGC ATG AAA CAC ATG CGG GCA TTC GGC AAC GTT TGC 989 Thr Gln Tyr Gly Met Lys His Met Arg Ala Phe Gly Asn Val Cys 315 320 325 AAC AGC GGC GTT TCT AAG GCC TCC ATG GAG GAG GCT TGT AAG GCA 1034 Asn Ser Gly Val Ser Lys Ala Ser Met Glu Glu Ala Cys Lys Ala 330 335 340 GCT TGC GGT GGC TAT GAC GCT GGG TTA TTA TAT CCA TCA AAC ACA 1079 Ala Cys Gly Gly Tyr Asp Ala Gly Leu Leu Tyr Pro Ser Asn Thr 345 350 355 GGA TAT AGT GCT TGATTTTGGG TTGTGCACAC AAATTAAAAG CCTTTAACAA 1131 Gly Tyr Ser Ala 360 GTTAAACAGC CAAGTTGATG ATGTGCTGCT TCTGAATTGC GTTCTCCCTT CTGGTTTCTG 1191 CTGCTTGTGT TAAATTTGTT CCCTGTAATT AAATTAGCCA AATAGCACAC GTGTAATCGG 1251 GCAGCTAACC CAGTGCAACA CACTCTATTA TTTCTATTGA GAGTGGTTAG GAGAGAATGC 1311 ATATATCGAT CACGTGTATA TAAATGCAGT GCCTTTTCAT AAAATAAGTA ATTAAGTATT 1371 AGTCTATTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1431 AAAAAAAAAA AAAAAAAAAA AA 1453 SEQ ID NO: 19 Sequence length: 1749 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: cDNA to mRNA Sequence: CAATTCCAAA CCCATACGCC ACCACCACTC CCTCTCACCA AATTATTCTT TATTTAAATC 60 CACTTCCCCT CTTTCCCTTG TTAATCACTC CAACTCTTACGCT CCTTTTCCAA TACTCCTCCT TCTCACCGCC ATCGCAACCC TCGTTTCTGG TAGCCGTAAC 180 CTCGCCGGAG ATTTCCTCCG ATTGCCTTCC CAACCTGACA CCGATGACAA C 231 CTT CCC GGT ACC AGG TGG GCC ATC CTT CTC GCC GGT TCC AAT GGC 276 Ar Leu Aru Gly 276 Leu Tru sn Gly 1 5 10 15 TAC TGG AAT TAC AGG CAT CAG GCT GAT ATT TGT CAT GCG TAT CAA 321 Tyr Trp Asn Tyr Arg His Gln Ala Asp Ile Cys His Ala Tyr Gln 20 25 30 ACA CTG AGG AAA GGT GGC CTG AAA GAA GAA AAT ATT ATT GTT TTT 366 Thr Leu Arg Lys Gly Gly Leu Lys Glu Glu Asn Ile Ile Val Phe 35 40 45 ATG TAT GAT GAC ATT GCG TTC AAT GAG GAG AAC CCA AGA CCT GGG 411 Met Tyr Asp Asp Ile Ala Phe Asn Glu Glu Asn Pro Arg Pro Gly 50 55 60 GTC ATC ATT AAC AAA CCA GAT GGG ACT GAT GTT TAT GAA GGA GTT 456 Val Ile Ile Asn Lys Pro Asp Gly Thr Asp Val Tyr Glu Gly Val 65 70 75 CCA AAG GAT TAC ACT GGC GAA GAT GTT ACT GCT GAA AAC TTC TAC 501 Pro Lys Asp Tyr Thr Gly Glu Asp Val Thr Ala Glu Asn Phe Tyr 80 85 90 GCT GTT TTA CTT GGA AAT AAG TCG GCA CTT AGC GGT GGC AGT GGG 546 Ala Val Leu Leu Gly Asn Lys Ser Ala Leu Ser Gly Gly Ser Gly 95 100 105 AAG GTT GTG AAC AGT GGC CCC GAT GAT CGT ATA TTT GTA TTC TAT 591 Lys Val Val Asn Ser Gly Pro Asp Asp Arg Ile Phe Val Phe Tyr 110 115 120 AGT GAT CAT GGA GGT CCA GGA GTT CTC GGG ATG CCT GCC GGT CCT 636 Ser Asp His Gly Gly Pro Gly Val Leu Gly Met Pro Ala Gly Pro 125 130 135 TAC TTA TAT GCA TCT GAT CTG AAT GAT GTC TTG AAG AAA AAA CAT 681 Tyr Leu Tyr Ala Ser Asp Leu Asn Asp Val Leu Lys Lys Lys His 140 145 150 GCT TCC GGA ACA TAT AAA AGC CTA GTA TTT TAT CTT GAG GCA TGT 726 Ala Ser Gly Thr Tyr Lys Ser Leu Val Phe Tyr Leu Glu Ala Cys 155 160 165 GAA TCT GGG AGT ATC TTT GAA GGC CTT CTT CCT GAA GAT GTC AAT 771 Glu Ser Gly Ser Ile Phe Glu Gly Leu Leu Pro Glu Asp Val Asn 170 175 180 ATT TAT GCA ACA ACA GCT TCG AAT GCA GAA GAA AGC AGC TGG GGA 816 Ile Tyr Ala Thr Thr Ala Ser Asn Ala Glu Glu Ser Ser Trp Gly 185 190 195 ACA TAT TGC CCA GGG GAG TAT CCC AGT CCT CCC CCA GAA TAT TCA 861 Thr Tyr Cys Pro Gly Glu Tyr Pro Ser Pro Pro Pro Glu Tyr Ser 200 205 210 ACT TGC TTG GGA GAC CTG TAC AGT GTT GCT TGG ATG GAA GAC AGT 906 Thr Cys Leu Gly Asp Leu Tyr Ser Val Ala Trp Met Glu Asp Ser 215 220 225 GAC AGA CAC AAT TTG CGA ACT GAA ACT TTG AAC CAA CAA TAT AAA 951 Asp Arg His Asn Leu Arg Thr Glu Thr Leu Asn Gln Gln Tyr Lys 230 235 240 TTG GTT AAA GAG AGG ACC ATT AGT GGA GGT TCA TAC TAT GGC TCT 996 Leu Val Lys Glu Arg Thr Ile Ser Gly Gly Ser Tyr Tyr Gly Ser 245 250 255 CAC GTG ATG CAG TAT GGT GAT ATA GGG CTC AGC GAT GAT GTT CTC 1041 His Val Met Gln Tyr Gly Asp Ile Gly Leu Ser Asp Asp Val Leu 260 265 270 TTC CTA TAT TTG GGT ACA AAT CCT GCT AAT GAT AAT TTT ACC TTT 1086 Phe Leu Tyr Leu Gly Thr Asn Pro Ala Asn Asp Asn Phe Thr Phe 275 280 285 GTC GAT GAA AAC TCC TTG AGG TCA CCT TCA AAA GCA GTC AAC CAA 1131 Val Asp Glu Asn Ser Leu Arg Ser Pro Ser Lys Ala Val Asn Gln 290 295 300 CGG GAT GCT GAT CTC ATC CAT TTC TGG GAG AAG TTC CGC AAA GCT 1176 Arg Asp Ala Asp Leu Ile His Phe Trp Glu Lys Phe Arg Lys Ala 305 310 315 CCT GAG GGT TCT CCC GAG AAA AAT GCT GCT CAG AAA CAA GTT GTG 1221 Pro Glu Gly Ser Pro Glu Lys Asn Ala Ala Gln Lys Gln Val Val 320 325 330 GAA GTA ATG TCT CAC AGG ATG CAT ATA GAC AAC GGT GTG GAA CTT 1266 Glu Val Met Ser His Arg Met His Ile Asp Asn Gly Val Glu Leu 335 340 345 A TT GGG AAG CTT TTA TTT GGC ATT GAA AAG GGT CCA AAA GTA CTG 1311 Ile Gly Lys Leu Leu Phe Gly Ile Glu Lys Gly Pro Lys Val Leu 350 355 360 GAT GCT GTT AGA CCG GCT GGA ATG GCA CTT GTT GAT GAC TGG GAC 1356 Asp Ala Val Arg Pro Ala Gly Met Ala Leu Val Asp Asp Trp Asp 365 370 375 TGC CTG AAA ACC ATG GTA AGG ACA TTT GAG ACA TAT TGT GGA TCC 1401 Cys Leu Lys Thr Met Val Arg Thr Phe Glu Thr Tyr Cys Gly Ser
380 385 390 TTG TCT CAG TAT GGG ATG AAA CAT ATG AGG TCC TTT GCA AAC ATC 1446 Leu Ser Gln Tyr Gly Met Lys His Met Arg Ser Phe Ala Asn Ile 395 400 405 TGC AAC GCA GGA ATT AAG AAT GAC CAA ATG GCC GAT GCC TCA GCA 1491 Cys Asn Ala Gly Ile Lys Asn Asp Gln Met Ala Asp Ala Ser Ala 410 415 420 CAA GCT TGT GTC AGT ATT CCT GCC AAT TCC TGG AGT TCT CTG CAA 1536 Gln Ala Cys Val Ser Ile Pro Ala Asn Ser Trp Ser Ser Leu Gln 425 430 435 AGG GGT TTC AGT GCA TAATTCCTGA AAATGCACAC TAGTGAAGAC CAAAGTATGA 1591 Arg Gly Phe Ser Ala 440 TTGCTGTTAC ATTATGTTTA ATGGTTGTAT GTATACACAT GCTGGACACT GGTCATGATG 1651 TTTTGTCCTG CCTTGTAAAT ACAATAGGGA CACTACTAGG AGTTGAAAGG GTCTTTACAT 1711 TGTAGTTTCG CACATAGATA CTTCAACTTC GTTTATAT 1749 SEQ ID NO: 20 SEQ length: 1640 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Sequence: GGTTATGGTA ATTATCGGCA TCAAGCGGAT GTGTGCCATG CTTACCAGTT GCTGATAAAA 60 GGTGGAGTCA AAGAAGAAAA CATTGTGGTG TTTATGTATG ATGATATAGC ATTTATAACG GAATCCCA GACCCGGAGT CATCATCAAC CATCCTCAGG GGCCAGACGT GTATGCTGGT 180 GTACCTAAGG ATTACACCGG TGAGGACGTA ACACCTGAGA ACCTATATGC TGTCATTCTT 240 GGGGACAAGA GTAAAGTTAA AGGTGGAAGT GGCAAGGTGA TCAACAGCAA TCCGGAGGAT 300 AGGATATTTA TATTCTACTC CGATCATGGA GGTCCCGGAG TTCTTGGGAT GCCAAACGCA 360 CCATTCGTTT ATGCCATGGA TTTTATTGAT GTTTTGAAGA AGAAACATGC AAGTGGAGGC 420 TACAAGGAGA TGGTTATATA CATAGAAGCT TGTGAGAGTG GGAGCATATT TGAGGGTATA 480 ATGCCCAAGG ATCTGAATAT TTATGTGACA ACTGCGTCAA ATGCACAAGA GAACAGTTTT 540 GGAACTTATT GTCCTGGGAT GAATCCTCCT CCACCAGAAG AGTACGTAAC TTGCCTGGGG 600 GATTTATACA GCGTTTCTTG GATGGAAGAT AGTGAGACTC ACAATCTAAA AAGGGAAACG 660 GTACAACAGC AATACCAGTC GGTAAGGAAA CGGACTTCAA ATTCTAACAG CTATAGGTTT 720 GGTTCTCATG TGATGCAATA CGGTGACACT AACATTACTG CTGAAAAGCT TTACTTGTAC 780 CATGGTTTTG ATCCTGCCAC CGTGAACTTT CCTCCACACA ACGGCAACCT AGAAGCTAAA 840 ATGGAAGTTG TTAACCAGAG AGATGCAGAG CTTTTGTTCA TGTGGCAAAT GTATCAGAGA 900 TCAAACCATC AACCGGAAAA GAAGACTCAC ATCCTGGAAC AGATTACAGA GACAGTGAAG 960 CATAGGAATC ATTTGGATGG CAGTGTGGAA TTGATTGGAG TTTTGTTGTA TGGACCAGGA 1020 AAAAGTTCTT CGGTTCTACA TTCCGTGAGG GCTCCTGGTC TGCCCCTAGT TGATGATTGG 1080 ACATGCTTGA AATCTATGGT TAGAGTGTTC GAAACTCACT GTGGGTCACT GACTCAGTAT 1140 GGCATGAAAC ACATGCGGGC ATTCGGCAAC GTTTGCAACA GCGGCGTTTC TAAGGCCTCC 1200 ATGGAGGAGG CTTGTAAGGC AGCTTGCGGT GGCTATGACG CTGGGTTATT ATATCCATCA 1260 AACACAGGAT ATAGTGCTTG ATTTTGGGTT GTGCACACAA ATTAAAAGCC TTTAACAAGT 1320 TAAACAGCCA AGTTGATGAT GTGCTGCTTC TGAATTGCGT TCTCCCTTCT GGTTTCTGCT 1380 GCTTGTGTTA AATTTGTTCC CTGTAATTAA ATTAGCCAAA TAGCACACGT GTAATCGGGC 1440 AGCTAACCCA GTGCAACACA CTCTATTATT TCTATTGAGA GTGGTTAGGA GAGAATGCAT 1500 ATATCGATCA CGTGTATATA AATGCAGTGC CTTTTCATAA AATAAGTAAT TAAGTATTAG 1560 TCTATTAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1620 AAAAAAAAAA AAAAAAAAAA 1640 SEQ ID NO: 21 SEQ length: 20 sequence types: the number of nucleic acid strand: single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: AACACCACAA TGTTT TCTTC 20 SEQ ID NO: 22 Sequence length: 20 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: TTTATCAGCA ACTGGTAAGC 20 Sequence Number: 23 Sequence Length: 329 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: cDNA to mRNA Sequence: TGCTTGTTCA CTACTATATA GCATAGAGTT CTCTTGTGTG AGTGACAAGT TGCGATTTCC 60 CTTTAGCTGA AATGATGGTT TCTGCTCTCCTCTCGTGT ATAGAC ATC TGATGTTGGT GATGCTGAGT TTGCACGGTA CTGCAGCGAG GTTGAACCGG 180 AGGGAGTGGG ACTCGGTCAT TCAGTTACCG ACCGAACCGG TGGATGATGA AGTGGGAACC 240 CGGTGGGCGG TTCTCGTGGC TGGTTCAA AC GGTAGTACCAAAA GCATTAGTGAA ATTATAGGCA TCAAGCCATG GTTATGGAA ATTATAGGCA TCAAGCGGCT 300GT Sequence type: cDNA to mRNA Sequence: TGCTTGTTCA CTACTATATA GCATAGAGTT CTCTTGTGTG AGTGACAAGT TGCGATTTCC 60 CTTTAGC TGA AATGATGGTT TCTGCTCTCT TCTCAGCAAA AACACGTGGT GTAGCCTTGT 120 ATTATGGTCA TGATGTTGGT GATGCTGAGT TTGCACGGTA CTGCAGCGAG GTG GCA GCA GCA GCA GCA GTG GCA GTG GCA GTG GCA GTG GLA Ala Gly Ser Asn Gly Tyr Gly Asn 5 10 15 TAT AGG CAT CAA GCG GAT GTG TGC CAT GCT TAC CAG TTG CTG ATA 327 Tyr Arg His Gln Ala Asp Val Cys His Ala Tyr Gln Leu Leu Ile 20 25 30 AAA GGT GGA GTC AAA GAA GAA AAC ATT GTG GTG TTT ATG TAT GAT 372 Lys Gly Gly Val Lys Glu Glu Asn Ile Val Val Phe Met Tyr Asp 35 40 45 GAT ATA GCT TAT AAC GCC ATG AAT CCC AGA CCC GGA GTC ATC ATC 417 Asp Ile Ala Tyr Asn Ala Met Asn Pro Arg Pro Gly Val Ile Ile 50 55 60 AAC CAT CCT CAG GGG CCA GAC GTG TAT GCT GGT GTA CCT AAG GAT 462 Asn His Pro Gln Gly Pro Asp Val Tyr Ala Gly Val Pro Lys Asp 65 70 75 TAC ACC GGT GAG GAC GTA ACA CCT GAG AAC CTA TAT GCT GTC ATT 507 Tyr Thr Gly Glu Asp Val Thr Pro Glu Asn Leu Tyr Ala Val Ile 80 85 90 CTT GGG GAC AAG AGT AAA GTT AAA GGT GGA AGT GGC AAG GTG ATC 552 Leu Gly Asp Lys Ser Lys Val Lys Gly Gly Ser Gly Lys Val Ile 95 100 105 AAC AGC AAT CCG GAG GAT AGG ATA TTT ATA TTC TAC TCC GAT CAT 597 Asn Ser Asn Pro Glu Asp Arg Ile Phe Ile Phe Tyr Ser Asp His 110 115 120 GGA GGT CCC GGA GTT CTT GGG ATG CCA AAC GCA CCA TTC GTT TAT 642 Gly Gly Pro Gly Val Leu Gly Met Pro Asn Ala Pro Phe Val Tyr 125 130 135 GCC ATG GAT TTT ATT GAT GTT TTG AAG AAG AAA CAT GCA AGT GGA 687 Ala Met Asp Phe Ile Asp Val Leu Lys Lys Lys His Ala Ser Gly 140 145 150 GGC TAC AAG GAG ATG GTT ATA TAC ATA GAA GCT TGT GAG AGT GGG 732 Gly Tyr Lys Glu Met Val Ile Tyr Ile Glu Ala Cys Glu Ser Gly 155 160 165 AGC ATA TTT GAG GGT ATA ATG CCC AAG GAT CTG AAT ATT TAT GTG 777 Ser Ile Phe Glu Gly Ile Met Pro Lys Asp Leu Asn Ile Tyr Val 170 175 180 ACA ACT GCG TCA AAT GCA CAA GAG AAC AGT TTT GGA ACT TAT TGT 822 Thr Thr Ala Ser Asn Ala Gln Glu Asn Ser Phe Gly Thr Tyr Cys 185 190 195 CCT GGG ATG AAT CCT CCT CCA CCA GAA GAG TAC GTA ACT TGC CTG 867 Pro Gly Met Asn Pro Pro Pro Pro Glu Glu Tyr Val Thr Cys Leu 200 205 210 GGG GAT TTA TAC AGC GTT TCT TGG ATG GAA GAT AGT GAG ACT CAC 912 Gly Asp Leu Tyr Ser Val Ser Trp Met Glu Asp Ser Glu Thr His 215 220 225 AAT CTA AAA AGG GAA ACG GTA CAA CAG CAA TAC CAG TCG GTA AGG 957 Asn Leu Lys Arg Glu Thr Val Gln Gln Gln Tyr Gln Ser Val Arg 230 235 240 AAA CGG ACT TCA AAT TCT AAC AGC TAT AGG TTT GGT TCT CAT GTG 1002 Lys Arg Thr Ser Asn Ser Asn Ser Tyr Arg Phe Gly Ser His Val 245 250 255 ATG CAA TAC GGT GAC ACT AAC ATT ACT GCT GAA AAG CTT TAC TTG 1047 Met Gln Tyr Gly Asp Thr Asn Ile Thr Ala Glu Lys Leu Tyr Leu 260 265 270 TAC CAT GGT TTT GAT CCT GCC ACC GTG AAC TTT CCT CCA CAC AAC 1092 Tyr His Gly Phe Asp Pro Ala Thr Val Asn Phe Pro Pro His Asn 275 280 285 GGC AAC CTA GAA GCT AAA ATG GAA GTT GTT AAC CAG AGA GAT GCA 1137 Gly Asn Leu Glu Ala Lys Met Glu Val Val Asn Gln Arg Asp Ala 290 295 300 GAG CTT TTG TTC ATG TGG CAA ATG TAT CAG AGA TCA AAC CAT CAA 1182 Glu Leu Leu Phe Met Trp Gln Met Tyr Gln Arg Ser Asn His Gln 305 310 315 CCG GAA AAG AAG ACT CAC ATC CTG GAA CAG ATT ACA GAG ACA GTG 1227 Pro Glu Lys Lys Thr His Ile Leu Glu Gln Ile Thr Glu Thr Val 320 325 330 AAG CAT AGG AAT CAT TTG GAT GGC AGT GTG GAA TTG ATT GGA GTT 1272 Lys His Arg Asn His Leu Asp Gly Ser Val Glu Leu Ile Gly Val 335 340 345 TTG TTG TAT GGA CCA GGA AAA AGT TCT TCG GTT CTA CAT TCC GTG 1317 Leu Leu Tyr Gly Pro Gly Lys Ser Ser Ser Val Leu His Ser Val 350 355 360 AGG GCT CCT GGT CTG CCC CTA GTT GAT GAT TGG ACA TGC TTG AAA 1362 Arg Ala Pro Gly Leu Pro Leu Val Asp Asp Trp Thr Cys Leu Lys 365 370 375 TCT ATG GTT AGA GTG TTC GAA ACT CAC TGT GGG TCA CTG ACT CAG 1407 Ser Met Val Arg Val Phe Glu Thr His Cys Gly Ser Leu Thr Gln 380 385 390 TAT GGC ATG AAA CAC ATG CGG GCA TTC GGC AAC GTT TGC AAC AGC 1452 Tyr Gly Met Lys His Met Arg Ala Phe Gly Asn Val Cys Asn Ser 395 400 405 GGC GTT TCT AAG GCC TCC ATG GAG GAG GCT TGT AAG GCA GCT TGC 1497 Gly Val Ser Lys Ala Ser Met Glu Glu Ala Cys Lys Ala Ala Cys 410 415 420 GGT GGC TAT GAC GCT GGG TTA TTA TAT CCA TCA AAC ACA GGA TAT 1542 Gly Gly Tyr Asp Ala Gly Leu Leu Tyr Pro Ser Asn Thr Gly Tyr 425 430 435 AGT GCT TGATTTTGGG TTGTGCACAC AAATTAAAAG CCTTCAGCAATT ATGTGCTGCT TCTGAATTGC GTTCTCCCTT CTGGTTTCTG CTGCTTGTGT 1658 TAAATTTGTT CCCTGTAATT AAATTAGCCA AATAGCACAC GTGTAATCGG GCAGCTAACC 1718 CAGTGCAACA CACTCTATTA TTTCTATTGA GAGTGGTTAG GAGAGAATGC ATATATCGAT 1778 CACGTGTATA TAAATGCAGT GCCTTTTCAT AAAATAAGTA ATTAAGTATT AGTCTATTAA 1838 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1898 AAAAAAAAAA AA 1910 SEQ ID NO: 25 length of sequence: 4 types of sequences : Number of amino acid chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 4 E L-asparaginamide SEQ ID NO: 26 Sequence length: 10 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCGAATTCGG 10 SEQ ID NO: 27 Sequence length : 24 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: AATTCGAGGA TCCGGGTACC ATGG 24 SEQ ID NO: 28 Sequence length: 20 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: CCATGGTACC CGGATCCTCG 20 SEQ ID NO: 29 Sequence length: 27 Sequence type: Nucleic acid Number of strands: 1 Strand topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: NO Sequence: CCGGATCCTA TGGTCATGAT GTTGGTG 27 SEQ ID NO: 30 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single-strand topology : Linear sequence type: Other nucleic acid (synthetic DNA) Antisense YES Sequence: CGCGCATGCG AAGCAGCACA TCATCAAC 28 SEQ ID NO: 31 Sequence Length: 25 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid (Synthetic DNA) Antisense: NO Sequence : GCCATGGAAG TTGGTACCCG GTGGG 25 SEQ ID NO: 32 Sequence length: 24 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: ATCCATGGCG TAAACGAATG GTGC 24 SEQ ID NO: 33 Sequence length: 22 Sequence type: Nucleic acid Number of strands: 1 strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Antisense: YES Sequence: GGTACAACTA GAGCTTTTCA GC 22 SEQ ID NO: 34 Sequence length: 17 Sequence type: Nucleic acid Number of strands: 1 Strand Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GTTTTCCCAG TCACGAC 17 SEQ ID NO: 35 Sequence Length: 20 Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid (synthetic DNA) Sequence: GGCCAGTGCC TAGCTTACAT 20 SEQ ID NO: 36 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single-strand Topology: Linear Type Sequence type: other nucleic acid (synthetic DNA) Sequence: CAGGAAACAG CTATGAC 17 SEQ ID NO: 37 Sequence length: 19 Sequence type: Amino acid Number of chains: 1 strand Topology: Linear Sequence type: Peptide fragment Type: N-terminal fragment Sequence: Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Pro Arg 1 5 10 15 Gly Ser Pro Met SEQ ID NO: 38 Sequence length: 4 Sequence type: Amino acid Number of chains: 1 Main chain Topology: Linear Sequence type: Peptide Fragment type: C-terminal fragment SEQ ID NO: 39 Sequence length: 8 Sequence type: Amino acid Number of chains: 1 strand Topology: Linear Sequence type: Peptide Fragment type: N-terminal fragment SEQ ID NO: 40 sequence length: 20 sequence type: amino acid number of chains: 1 chain topology: linear sequence type: peptide fragment type: C-terminal fragment sequence: Asp Pro Ala Ala Asn Lys Ala Arg Lys Glu Ala Glu Leu Ala Ala 1 5 10 15 Ala Thr Ala Glu Gln 20

[Brief description of drawings]

FIG. 1 shows ASN, ASN-1, Rn8-63, 104,
It is a figure which shows the restriction enzyme map of ASN-N and 102, and each relationship.

FIG. 2 is a diagram showing a restriction enzyme map of ASNs, 101, 103, and 107, and their relationships.

FIG. 3 is a diagram showing a part of a construction process of ASN-ful-pTV118N.

FIG. 4 is a diagram showing the construction of ASN-ful-pTV118N together with FIG. 3.

FIG. 5 shows the construction of pTASN-N.

FIG. 6 shows the construction of pUC18-104.

FIG. 7 shows the construction of pUC18-ASN.

FIG. 8 shows the construction of pEASN201.

FIG. 9 shows the construction of pEASN305.

FIG. 10 shows the construction of pEASN304.

FIG. 11 is a diagram showing the construction of MMASN1.

FIG. 12 shows the construction of pTMASN204.

FIG. 13 shows the construction of pEASN404.

FIG. 14 shows the relationship between each expressed polypeptide and activity.

Front Page Continuation (72) Inventor Shinichi Ishii 2-4-20 Sugano, Ichikawa City, Chiba Prefecture (72) Inventor Yukichi Abe 40-48 Omagari, Hiroshima-cho, Sapporo-gun, Hokkaido

Claims (3)

[Claims] 1. An asparaginyl endopeptidase gene. 2. The gene according to claim 1, which is selected from the group consisting of DNA sequences represented by SEQ ID NOs: 1 to 8 in the sequence listing. 3. The gene according to claim 1, which is hybridizable with the gene according to claim 2.