JPH0568570A - Proctase b 1gene - Google Patents

Abstract

PURPOSE:To efficiently mass-produce proctase B useful as an antiphlogistic enzyme pharmaceutical, a digestive enzyme pharmaceutical, etc., by culturing a cell transduced with a expression vector containing a proctase B gene having a specific amino acid sequence and collecting the resultant product from the culture. CONSTITUTION:A microorganism Aspergillus.niger DBD-0406 (FERM P-2737) capable of producing proctase B is cultured at 28 deg.C in a culture medium for 32hr and collagen is then added to further carry out culture for 12hr. Hyphae are subsequently collected and pulverized in liquid nitrogen to purify an RNA according to a conventional method. The purified RNA is treated with an oligodt column to isolate an mRNA, which is then used to synthesize a cDNA and prepare a cDNA library. The resultant cDNA library is cloned by plaque hybridization with a probe to select a positive clone. A DNA is recovered from the clone and treated with a restriction enzyme. Thereby, the objective proctase B gene capable of coding a specific amino acid sequence is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a proctase B gene, a vector containing the gene, a host cell transformed with the vector, and a method for producing proctase B using the cell. More specifically, it relates to a gene encoding Proctase B, which is an Aspergillus niger-derived acidic protease, a vector containing the gene, a host cell transformed with the vector, and a method for producing Procutase B using the cell.

[0002]

BACKGROUND OF THE INVENTION Proctase is used as an enzyme for hydrolyzing proteins as a component of anti-inflammatory enzyme preparations, digestive enzyme preparations and the like. However, Proctase produced by Aspergillus niger strain is mainly used. Is composed of a procutase A and a procutase B component, and the gene of the procutase A has already been cloned by the present inventors, and the recombinant has been successfully expressed in a host cell. However, regarding Proctase B, no report has been made that the structure was determined by isolating and purifying Proctase B.

Further, in recent years, a gene has been isolated by the development of recombinant DNA technology, and by introducing the gene into a host cell, a technique for mass-producing the target enzyme has become possible, and the gene of the enzyme of commercial interest is available. Production by recombination is also being considered and implemented.

Proctase B is a typical aspartic acid type protease among conventionally known ordinary acid proteases and has a property different from that of proctase A (Japanese Patent Publication No. 43-4436). The structural gene of Protase B existing in the chromosome of Aspergillus niger strain was cloned, and DN encoding Proctase B was cloned.
It is an industrially important task to determine the A base sequence, create a host cell containing such a DNA sequence, and use the cell to produce the protease.

[0005]

[Means for Solving the Problems] The present inventors have found that Aspergillus niger var. Macrosporu
Focusing on the acidic protease produced by (s),
Previously, intensive studies were conducted to find a method for easily producing the protease with respect to Proctase B, which has a property different from that of Proctase A cloned by the present inventors. As a result, a gene encoding the protease was successfully cloned and successfully expressed in a host,
The present invention has been completed.

[0006] The present invention relates to a proctase B gene encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing, a proctase B gene containing a base sequence encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing, and a vector containing the gene. ,
It is intended to provide a host cell transformed with the vector and a method for producing Proctase B, which comprises culturing the cell and collecting Procidase B from the culture.

Proctase B having an acidic protease activity according to the method of the present invention is Aspergillus niger strain, particularly Aspergillus niger strain.
var. macrosuporus) DBD-0406. Also, the proctase B gene of the present invention can be obtained from this strain. This strain can be obtained from the Institute of Microbiology and Industrial Technology, National Institute of Advanced Industrial Science and Technology, by Micro Engineering Research Institute No. 2737 (ATCC
No. 16513).

The nucleotide sequence of the gene for Proctase B enzyme cloned from Aspergillus niger strain and the amino acid sequence of Proctase B deduced therefrom are shown in SEQ ID NO: 1 of the sequence listing. In the table, the upper row shows the entire nucleotide sequence of the cloned gene, of which the 63rd nucleotide to the 1244th nucleotide encodes a polypeptide consisting of 394 amino acids (lower row).

The N-terminal of this amino acid sequence contains a signal sequence and a pro sequence from Met at position -69 to Ala at position -1 and the active enzyme is amino acid positions 1 to 325.
Up to position 325 residues of the polypeptide. Hereinafter, the present invention will be described step by step.

A. Preparation of Proctase B Specific DNA Probe In order to clone the gene or cDNA of Procutase B using a DNA probe, information on the amino acid sequence is required. Then, based on the amino acid sequence determined by the group of the present inventors, the procedure and the result will be described below.

Purified proctase B was digested with trypsin, the purified peptide was separated by gel filtration and liquid chromatography, and the amino acid composition of three peptide fragments was analyzed to determine the amino acid sequence Ed.
Applied biosystem using the man method (AB
It was determined by a peptide sequencer manufactured by I). The N-terminal amino acid sequence of purified Proctase B was also determined in the same manner (see FIG. 1).

The longer the specific base sequence of a DNA probe, the more reliable the screening will be. Therefore, the polymerase chain reaction (PCR) method using the total DNA of the Proctase B-producing bacterium as a template will make it possible to detect several hundred base pairs. A DNA having a specific sequence was amplified and used. The PCR method has an advantage that if the nucleotide sequences at both ends are known, the DNA can be obtained even if the sequence between them is unknown.

In the case of the present invention, although the entire base sequence of Proctase B is unknown, it was revealed that the partial amino acid sequence of this enzyme has a high homology with penicillopepsin (see FIG. 2), and therefore PCR Part D obtained by the method
The length of NA is also estimated to some extent. Therefore, we selected two regions with higher homology to the amino acid sequence of penicillopepsin from the determined partial amino acid sequences, and reduced the degeneracy of the nucleotide sequence to 16 or less for the mixture of oligonucleotides containing all possible nucleotide sequences. Several pieces were synthesized and used as PCR primers (Fig. 3).

Next, the Proctase B-producing bacterium Aspergillus niger Var Macrosporus
niger var. macrosporus) DBD-0406 (ATCC No. 16513) was used for a PCR reaction as a template, and a DNA of about 480 base pairs was amplified by the combination of primer a-1 and primer b-1. It was confirmed by electrophoresis using an agarose gel. The DNA band was cut out from the gel, extracted, and then labeled with ³² P to be used as a probe.

B. Cloning of Proctase B Gene Using the cDNA obtained above and the λgt10 cloning kit from Amersham, a cDNA library was prepared by a conventional method. Plaques produced by infecting Escherichia coli with the phage library were transferred to a nylon membrane, subjected to alkali denaturation and neutralization treatment, and then hybridized with the above probe. As a result, 5 hybridization-positive plaques were obtained from about 14,000 clones. This plaque was taken, similarly subjected to the secondary screening, and cloned.

C. Determination of nucleotide sequence For one of the clones obtained above, phage DNA was prepared and digested with restriction enzyme EcoR1
It was subcloned by inserting it into the EcoR1 site of the 13mp18 vector. Double-stranded plasmid DNA was prepared using Escherichia coli JM103, deletion mutants of various lengths were prepared from the inserted DNA using ExoIII nuclease, and the nucleotide sequence was determined using a DNA sequencer from ABI. As a result, the obtained clone contained 1446 base pairs and had an open reading frame of 1184 bases as shown in SEQ ID NO: 1 in the sequence listing. As a result, it was revealed that Proctase B was biosynthesized as a precursor of 394 amino acid residues, and had a signal sequence and a pro sequence at positions -69 to -1 at its N-terminal, and active enzyme was at positions 1 to 325. It was found to consist of up to 325 amino acid residues.

D. Expression in Escherichia coli It was confirmed that the gene for Proctase B obtained as described above can be expressed in E. coli as follows. That is, using Aspergillus niger cDNA as a template, P
A nucleotide sequence encoding a proctase B precursor (amino acids -49th to 325th of SEQ ID NO: 1 in the Sequence Listing) was amplified by the CR method, and the expression vector pKK23 was obtained.
3-2 (Clontech) was used as a recombinant plasmid. After culturing the E. coli transformed with the expression plasmid thus prepared, it was lysed and SD
S electrophoresis and Western blotting were performed. As a result, it was confirmed that the procutase B precursor protein was produced by Escherichia coli using the anti-proctase B antiserum prepared from rabbits using purified procutase B as an antigen.

[0018]

The present invention will be described in more detail by way of examples, which should not be construed as limiting the invention.

Example 1 Determination of partial amino acid sequence of Proctase B 200 μl containing purified Proctase B (2.0 mg), TPCK-trypsin (40 μg) and 50 mM N-ethylmorpholine acetate buffer (pH 8.0)
After reacting the solution at 37 ° C. for 6 hours, high performance liquid chromatography (column: TSKgel ODS120T, flow rate: 0.8 ml / min, solvent system: 0.1% trifluoroacetic acid-0 to 50% acetonitrile {gradient,
30 minutes}), and the three peptide fragments are lyophilized and subjected to amino acid composition analysis (Applied Biosystems: ABI, model 420A), and then Ed.
The amino acid sequence was determined by a peptide sequencer manufactured by ABI using the man method. The N-terminal amino acid sequence of purified Proctase B was also determined in the same manner. The results are shown in FIG. All of these sequences are Penicilliu
Since homologous sequences were found in the sequence of penicillopepsin, which is an acidic protease of m janthinellum, it was presumed that the sequences have the positional relationship as shown in FIG.

Example 2 Preparation of cDNA and cDNA Library Aspergillus niger (Propertase B-producing bacterium)
illus niger) in CES-Mu3 medium at 28 ° C. and 220 ° C.
Cultured at rpm. The composition of CES-Mu3 is glucose (2.0%), glycerin (1.0%), polypeptone (1.0%), yeast extract (0.3).
%), Monopotassium phosphate (0.05%), and pH 6.0. After culturing for 32 hours, collagen was added to 0.5% and further culturing was continued for 12 hours. Then, the mycelium was collected, 2 g of the mycelium was crushed in liquid nitrogen, and RNA was purified by a conventional method. From 1.9 mg of the obtained RNA, 64 μg of messenger R was treated with oligo dT column.
I got NA. A cDNA library was prepared by synthesizing cDNA using the Amersham cDNA synthesis system and λgt10 cloning kit.

Example 3 Synthesis of DNA Primer As described above, since the amino acid sequences of the present enzyme and penicillopepsin are highly homologous, the underline a and b in FIG. 2 were used as primers, and all cDNAs of Aspergillus niger were used.
When polymerase chain reaction (PCR) is performed using A as a template, it is expected that a DNA of about 480 bases will be amplified. Therefore, a mixture of oligonucleotides containing all possible nucleotide sequences predicted from the underlined a and b amino acid sequences was synthesized and used as a PCR primer. Where a is the sense strand sequence 1
Six pools of mixture 2 pool, b were synthesized by dividing the sequence of the antisense strand into 16 pools of mixture 4 pool (FIG. 3). The synthesis of the oligonucleotide was performed using a Synthesizer model 381A manufactured by ABI.

Example 4 Amplification of Specific DNA Probe by PCR Method A PCR was carried out using a synthetic oligonucleotide mixture as a primer and the entire cDNA obtained from Aspergillus niger as a template. For PCR, the Gene Amp (registered trademark) DNA Amplification Reagent Kit manufactured by Takara Shuzo was used. Template DNA is 100 ng / ml, primer mixture is 10 each
μM, 94 ° C (1 minute), 55 ° C (2 minutes), 72 ° C (3
Min) for 35 cycles. As a result, it was confirmed by electrophoresis using an agarose gel that a DNA of about 480 base pairs was specifically amplified in any of the 8 combinations of the primers a2 and b4. Of the combination of a-1 and b-1, which produced the most amount, a DNA band was cut out from an agarose gel, extracted, and labeled with ³² P and used as a probe. Amersham's Multiprime® for labeling
A DNA labeling system and {α- ³² P} dCTP were used.

Example 5 Gene Cloning of Proctase B The phage library obtained in Example 2 was infected with Escherichia coli, spread on L-agar medium, and cultured at 37 ° C., and the resulting plaques were then washed with Amersham nylon. Transferred to a membrane, the DNA was denatured with 0.5 M NaOH and 1.5 M NaCl, and neutralized with 0.5 M Tris-HCl buffer (pH 7) and 1.5 M NaCl.
Then, it was hybridized with the PCR probe of Example 4. The hybridization is 6
Double concentration of SSC (0.15M NaCl, 0.015M
Sodium citrate, pH 7.0, Amersham (Am
ersham) Hybridization buffer and D
65 with NA probe approximately 5 x 10 5 cpm / ml
It was carried out at ℃ for 2 hours. After this, 6 times SSC (0.1% S
(Including DS) at 65 ° C. for 30 minutes, followed by washing twice with 2 × concentration of SSC and once with 1 × of SSC. Furthermore,
After lightly washing with 2 times SSC containing no SDS, it was air-dried and subjected to autoradiography (-80 ° C, overnight). As a result, 5 hybridization-positive plaques were obtained from about 14,000 clones. This plaque was taken, secondarily screened and cloned in the same manner.

Example 6 Determination of nucleotide sequence LB was obtained by infecting E. coli with the clone obtained in Example 5.
After culturing in the medium and lysing, centrifugation was performed to obtain a supernatant phage lysate. 5 μg each of RNase A and D was added to this lysate.
After adding NaseI and incubating at 37 ° C. for 30 minutes, an equal amount of 20% polyethylene glycol and 2M sodium chloride solution was added and incubated on ice for 1 hour. Then, centrifuge to remove the supernatant and deposit 0.5m
l of SM buffer was added and suspended, and the suspension was centrifuged to collect the supernatant. 10 mM each of EDTA and SDS in the supernatant,
It was added to 0.1% and kept at 60 to 65 ° C for 15 minutes. After phenol extraction and phenol-chloroform extraction, the aqueous layer was precipitated with isopropanol to obtain phage D.
I got NA. This DNA was digested with restriction enzyme EcoR1 and subjected to agarose gel electrophoresis.
The nucleotide sequence of the clone (1.4 kb) having a long NA was determined. Cut out the insert DNA and M1
It was inserted into the EcoR1 site of 3mp18 vector, introduced into Escherichia coli JM103, and double-stranded plasmid DNA was prepared according to a conventional method. ExoIII for the inserted DNA
Various deletion mutants were prepared using nuclease, and then M13 single-stranded DNA was prepared using JM103,
The nucleotide sequence was determined using a DNA sequencer manufactured by ABI. The ExoIII deletion was performed using the Takara Shuzo Kilosequence Kit.

As a result, the obtained clone contained 1446 base pairs, and as shown in SEQ ID NO: 1 in the sequence listing, 118 clones were formed.
4-base open reading frame (Open rea
dingframe). The amino acid sequences of the N-terminal of tryptase B and the digested fragment of trypsin were all encoded by this cDNA without any single residue difference. Also,
A 69-residue signal peptide and prosequence are present at the N-terminus.

Example 7 Construction of Expression Plasmid Using the cloned Proctase B cDNA as a template, the sense primer AGAGACCATGGCGCCGG-
CTCCTACTCGCAAG, antisense primer-AGAGAAAGCTTACTAAGCCTG-A-
PCR was performed in the same manner as in Example 4 using GCGGCGAATCC. After amplification, NcoI / Hin of the vector
To insert at dIII site, NcoI site for sense primer and Hind for antisense primer
The III site is incorporated. The amplified reaction solution was extracted with chloroform and subjected to agarose gel electrophoresis.
The kb DNA was isolated. After digestion with NcoI and HindIII, the pKK233-2 vector (Clontech,
Clontech) was inserted into the NcoI and HindIII sites to construct an expression plasmid for a proctase B precursor (amino acid numbers 21 to 394 of SEQ ID NO: 1 in the sequence listing) (FIG. 4).

Example 8 Expression of Proctase B Precursor by Escherichia coli M9CA containing 50 μg ampicillin of Escherichia coli HB101 transformed with the plasmid constructed in Example 7.
Incubate for 1.5 hours at 37 ℃ in the medium, and then culture with IPTG (Isopropy
l β-D-Thiogalactopyranoside, final concentration 0.1 mM) was added, and the mixture was further cultured for 7 hours. After collecting the bacteria, 2% SDS / 0.
Suspended in 125 M Tris-HCl (pH 6.8) /0.2% bromphenol blue solution, boiled for 5 minutes to lyse, and after SDS-electrophoresis, Western blotting was performed. It was confirmed that Proctase B precursor that specifically reacts with serum was produced.

[0028]

INDUSTRIAL APPLICABILITY According to the present invention, a gene encoding Proctase B was cloned, the recombinant was successfully expressed in a host cell, and the structure of the enzyme was clarified. Furthermore, according to the present invention, industrially useful Proctase B can be efficiently mass-produced using a recombinant host cell.

SEQ ID NO: 1 Sequence length: 1447 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin organism name: Aspergillus niger var. Macrosporus Strain name: DBD -0406 Sequence GGCTTCTTTG TCTGGTTCTA TCTCTTCTCC GAACTCTCTT GCTTGACATT CTCGTGGTCAA 61 AATG GTC GTC TTC AGC AAA ACC GCT GCC CTC GTT CTG GGT CTG TCC TCC 110 Met Val Val Phe Ser Lys Thr Ala Ala Leu Val Leu G65-55 GTC TCT GCG GCG CCG GCT CCT ACT CGC AAG GGC TTC ACC ATC AAC 158 Ala Val Ser Ala Ala Pro Ala Pro Thr Arg Lys Gly Phe Thr Ile Asn -50 -45 -40 CAG ATT GCC CGG CCT GCC AAC AAG ACC CGC ACC ATC AAC CTG CCA GGC 206 Gln Ile Ala Arg Pro Ala Asn Lys Thr Arg Thr Ile Asn Leu Pro Gly -35 -30 -25 ATG TAC GCC CGT TCC CTG GCC AAG TTT GGC GGT ACG GTG CCC CAG AGC 254 Met Tyr Ala Arg Ser Leu Ala Lys Phe Gly Gly Thr Val Pro Gln Ser -20 -15 -10 GTG AAG GAG GCT GCC AGC AAG GGT AGT GCC GTG ACC ACG CCC CAG AAC 302 Val Lys Glu Ala Ala Ser Lys Gly Ser Ala Val Thr Thr Pro Gln Asn -5 -1 1 5 10 AAT GAC GAG GAG TAC CTG ACT CCC GTC ACT GTC GGA AAG TCC ACC CTC 350 Asn Asp Glu Glu Tyr Leu Thr Pro Val Thr Val Gly Lys Ser Thr Leu 15 20 25 CAT CTG GAC TTT GAC ACC GGA TCT GCA GAT CTC TGG GTC TTC TCG GAC 398 His Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp Val Phe Ser Asp 30 35 40 GAG CTC CCT TCC TCA GAG CAG ACC GGT CAC GAT CTG TAC ACG CCT AGC 446 Glu Leu Pro Ser Ser Glu Gln Thr Gly His Asp Leu Tyr Thr Pro Ser 45 50 55 TCC AGC GCG ACC AAG CTG AGC GGC TAC ACT TGG GAC ATC TCC TAC GGT 494 Ser Ser Ala Thr Lys Leu Ser Gly Tyr Thr Trp Asp Ile Ser Tyr Gly 60 65 70 75 GAC GGC AGC TCA GCC AGC GGA GAC GTG TAC CGG GAT ACT GTC ACT GTC 542 Asp Gly Ser Ser Ala Ser Gly Asp Val Tyr Arg Asp Thr Val Thr Val 80 85 90 GGC GGT GTC ACC ACC AAC AAG CAG GCT GTT GAA GCA GCC AGC AAG ATC 590 Gly Gly Val Thr Thr Asn Lys Gln Ala Val Glu Ala Ala Ser Lys Ile 95 100 105 AGC TCC GAG TTC GTT CAG GAC ACG GCC AAT GAC GGC CTT TTG GGA CTG 638 Ser Ser Glu Phe Val Gln Asp Thr Ala Asn Asp Gly Leu Leu Gly Leu 110 115 120 GCC TTT AGC TCC ATT AAC ACT GTC CAG CCC AAG GCG CAG ACC ACC TTC 686 Ala Phe Ser Ser Ile Asn Thr Val Gln Pro Lys Ala Gln Thr Thr Phe 125 130 135 TTC GAC ACC GTC AAG TCC CAG CTG GAC TCT CCC CTT TTC GCC GTG CAG 734 Phe Asp Thr Val Lys Ser Gln Leu Asp Ser Pro Leu Phe Ala Val Gln 140 145 150 155 CTG AAG CAC GAC GCC CCC GGT GTT TAC GAC TTT GGC TAC ATC GAT GAC 782 Leu Lys His Asp Ala Pro Gly Val Tyr Asp Phe Gly Tyr Ile Asp Asp 160 165 170 TCC AAG TAC ACC GGT TCT ATC ACC TAC ACG GAT GCC GAT AGC TCC CAG 830 Ser Lys Tyr Thr Gly Ser Ile Thr Tyr Thr Asp Ala Asp Ser Ser Gln 175 180 185 GGC TAC TGG GGC TTC AGC ACC GAC GGC TAC AGT ATC GGT GAC GGC AGC 878 Gly Tyr Trp Gly Phe Ser Thr Asp Gly Tyr Ser Ile Gly Asp Gly Ser 190 195 220 TCC AGC TCC AGC GGC TTC AGC GCC ATT GCT GAC ACC GGT ACC ACC CTC 926 Ser Ser Ser Ser Gly Phe Ser Ala Ile Ala Asp Thr Gly Thr Thr Leu 205 210 215 ATC CTC CTC GAT GAC GAA ATC GTC TCC GCC TAC TAC GAG CAG GTT TCT 974 Ile Leu Leu Asp Asp Glu Ile Val Ser Ala Tyr Tyr Glu Gln Val Ser 220 225 230 235 GGC GCT CAG GAG AGC GAG GAA GCC GGT GGC TAC GTT TTC TCT TGC TCG 1022 Gly Ala Gln Glu Ser Glu Glu Ala Gly Gly Tyr Val Phe Ser Cys Ser 240 245 250 ACC AAC CCC CCT GAC TTC ACT GTC GTG ATT GGC GAC TAC AAG GCC GTT 1070 Thr Asn Pro Pro Asp Phe Thr Val Val Ile Gly Asp Tyr Lys Ala Val 255 260 265 GTT CCG GGC AGG TAC ATC AAC TAC GCT CCC ATC TCG ACT GGC AGC TCC 1118 Val Pro Gly Arg Tyr Ile Asn Tyr Ala Pro Ile Ser Thr Gly Ser Ser 270 275 280 ACC ACC TGC TTT GGC GGT ATC CAG AGC AAC AGC GGT CTG GGA CTG TCC ATC 1166 Thr Cys Phe Gly Gly Ile Gln Ser Asn Ser Gly Leu Gly Leu Ser Ile 285 290 295 CTG GGC GAT GTG TTC TTG AAG AGC CAG TAC GTG GTC TTC AAC TCT GAG 1214 Leu Gly Asp Val Phe Leu Lys Ser Gln Tyr Val Val Phe Asn Ser Glu 300 305 310 315 GGC CCT AAG CTG GGA TTC GCC GCT CAG GCT TAG ATCAACCACT GAAGTGGAGT 1267 Gly Pro Lys Leu Gly Phe Ala Ala Gln Ala *** 320 325 CTATAATCTG CTGATTGATC CCTCGACGAT GAACTACATG TGGAAATGTA TAGCAGACGA 1327 GGGTGATGGT GATGATGTTG ATTTGATGA T GACCCGTACA TACTTGATGA AGCTCGGTAC 1387 ATATGCAAAT GTGACTGTAT CTATGTGAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1447

SEQ ID NO: 2 Sequence length: 17 Sequence type: Amino acid Topology-: Linear Sequence type: Protein Fragment type: N-terminal fragment Origin Biological name: Aspergillus niger var. Macrosporus Strain name: DBD-0406 Sequence Ser Ile / Lys Ser Ala Val Thr Thr Pro Gln Asn Asn Asp Glu Glu Tyr Leu Thr 1 5 10 15

SEQ ID NO: 3 Sequence length: 24 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Aspergillus niger var. Macrosporus Strain name: DBD-0406 Array Ser Thr Leu His Leu Asp Phe Asp Thr Gly Ser Ala Asp Leu Trp Val 1 5 10 15 Phe Ser Asp Glu Leu Pro Ser Ser 20

SEQ ID NO: 4 Sequence length: 16 Sequence type: Amino acid Topology :: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Aspergillus niger var. Macrosporus Strain name: DBD-0406 Sequence His Asp Ala Pro Gly Val Tyr Asp Phe Gly Tyr Ile Asp Asp Ser Lys 1 5 10 15

SEQ ID NO: 5 Sequence length: 5 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide Fragment type: Intermediate fragment Origin Biological name: Aspergillus niger var. Macrosporus Strain name: DBD-0406

SEQ ID NO: 6 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 7 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA

SEQ ID NO: 8 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes

SEQ ID NO: 9 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes

SEQ ID NO: 10 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology-: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes

SEQ ID NO: 11 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology :: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: Yes

[0040]

[Brief description of drawings]

FIG. 1: A partial amino acid sequence of Proctase B is shown. Figure 2: Shows a comparison of the amino acid sequences of penicillopepsin (top) and Proctase B (bottom). Figure 3: Shows the sequences of PCR primers. FIG. 4: shows the construction method of the plasmid pMP-10 of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI Technical display location // (C12N 15/57 C12R 1: 685) (C12N 1/21 C12R 1:19) (C12N 9 / 62 C12R 1:19)

Claims (9)

[Claims] 1. A proctase B gene encoding the amino acid sequence set forth in SEQ ID NO: 1 in the sequence listing. 2. A proctase B gene containing a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 1 in the Sequence Listing. 3. A procutase B precursor enzyme containing 394 amino acid residues from amino acid numbers -69 to 325 in the amino acid sequence of SEQ ID NO: 1 in the sequence listing. 4. The gene according to claim 1, which comprises 1182 base pairs from base number 63 to 1244 of the base sequence shown in SEQ ID NO: 1 in the sequence listing. 5. The gene according to claim 1, which comprises 325 amino acid residues from amino acid Nos. 1 to 325 of the amino acid sequence shown in SEQ ID No. 1 in the sequence listing. 6. The gene according to claim 1, which comprises 975 base pairs of base numbers 270 to 1244 of the base sequence shown in SEQ ID NO: 1 of the sequence listing. 7. An expression vector into which the base sequence encoding the proctase B according to claim 2 has been incorporated. 8. A cell into which the expression vector according to claim 7 has been introduced. 9. A method for producing Proctase B, which comprises culturing the cell according to claim 8 and collecting Proctase B from the culture.