JPH0475586A - Proctase a gene - Google Patents

Abstract

PURPOSE:To readily produce proctase A having different properties from those of conventionally known aspartic protease by culturing a host transformed with a plasmid containing a gene to code proctase A. CONSTITUTION:A vector containing a gene to code proctase A having acidic protease activity is constituted. Then a host transformed with the vector is cultured and proctase A having acidic protease activity is collected from the culture mixture. The produced proctase A having acidic protease activity is produced by Aspergillus niger var. macrosporus DBD-0406 and the above- mentioned gene is obtained from the strain. The strain is deposited as FERM P-2,737 (ATCC No.16,513). Since this enzyme is acidic protease of pepstatin insusceptibility, has protein hydrolyzing ability and hardly release an amino acid, the enzyme is considered to be endo type protease.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention provides proctase A derived from Aspergillus niger.
The present invention relates to a gene encoding the above polypeptide, a vector containing the gene, and a method for producing the polypeptide using a host transformed with the vector.

(Prior art and problems to be solved by the invention) Protase is an enzyme that hydrolyzes proteins and is used as a component of anti-inflammatory enzyme preparations, digestive enzyme preparations, etc.
It is composed of several components, and there are no reports yet on the isolation and purification of each enzyme as a single enzyme, and the determination of its structure.

In addition, with the recent development of recombinant DNA technology, genes have been isolated and introduced into host cells, making it possible to mass-produce desired enzymes. are also being considered and implemented.

Protase A is a new protease that has different properties from aspartic acid proteases among conventional acidic proteases (Japanese Patent Publication No. 43-4
436). The structural gene of protase A present in the chromosome of Aspergillus niger strain is cloned, the DNA base sequence encoding protase A is determined, and the protease is produced in host cells containing recombinant DNA containing this DNA sequence. This is an important industrial issue.

However, the present inventors have discovered that the Aspergillus niger strain produces
var, macros,. We focused on the acidic protease produced by Rus.
As a result of intensive research to find a method for easily producing the protease, the present invention was completed by successfully cloning the gene encoding the protease and expressing it in a host. Therefore, the present invention involves culturing a gene encoding protase A having acidic protease activity; a vector containing the gene; and a host transformed with the vector; and collecting protase A having acidic protease activity from this culture. It is an object of the present invention to provide a method for producing protase A characterized by the following.

Protase A having acid grotease activity produced by the method of the present invention is ASp6rgil lusn
iger var-macrosporus DBD-
0406, and the genes of the present invention can be obtained from this strain. This strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, and was submitted to the Microbiological Research Institute No. 2737 (ATCC No.
, 16513).

Although this enzyme is a pepstatin-insensitive acidic protease and has proteolytic ability, it is considered to be an endoprotease because it releases almost no amino acids.

FIG. 1 shows the base sequence of the protase A enzyme gene cloned from the Aspergillus niger strain and the amino acid sequence of protase A deduced from the base sequence. The upper row shows the entire base sequence of the cloned gene, of which a polypeptide consisting of 282 amino acids (lower row) is encoded by nucleotides 614th to 1462nd. The base sequence of this part is cD
NA is also exactly the same and does not contain introns.

At the N-terminus of this amino acid sequence, 59
It contains a signal sequence and a prosequence up to position ^sn, and there is an insertion of 11 amino acid residues from position 99 to position 109. The active enzyme consists of a polypeptide consisting of 39 residues from positions 60 to 98, and 1 residue from positions 110 to 282.
It has a polypeptide consisting of 73 residues.

Below, the content of the present invention will be explained step by step.

a. Preparation of a specific DNA probe for protase A In order to clone the gene or cDNA of protase A using a DNA probe, information on the amino acid sequence is required, and based on the amino acid sequence determined by the inventor's group. However, the procedure and results are described below. Purified protase A is reduced and aminoethylated or reduced and pyridylethylated, decomposed with lysyl endopeptidase, trypsin, chymotrypsin, v8 protease, etc., the resulting peptide is separated by gel filtration and liquid chromatography, and structural analysis is performed. The entire amino acid sequence was determined using an Applied Biosystems (ABI) peptide sequencer using the Edman method. As a result, as shown in Figure 2, protase A is a protein with a total number of residues of 212 consisting of two chains, a light chain (39 residues) and a heavy chain (173 residues), and disulfide bonds are , heavy chain Cys45-Cys69, Cy
It was applied to s57-Cys140. The N-terminus of the heavy chain was pyroglutamic acid.

The longer the specific base sequence of a DNA probe, the more reliable the screening becomes. DNA with the sequence was amplified and used. The advantage of the PCR method is that if the base 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 was unknown, two regions with a small number of base sequence degeneracy predicted from the amino acid sequence were selected and a mixture of oligonucleotides containing all possible base sequences was synthesized. and used as a primer for PCH.

That is, the amino acid sequence from position 41 to position 46 of the N-terminus of the heavy chain is Trp -Tyr -Glu -Trp-Tyr
-Pro, but if we remove the third letter of the proline codon, eight 17-base sequences corresponding to this amino acid sequence can be predicted. Also, from the heavyweight 149th place to 15th
Amino acid sequence at position 4 Met-Asp-11e-Gl
From u-11;In-Asp, 24 17-base sequences are predicted, excluding the third letter of the aspartic acid codon at position 154. Use these two locations as primers
If CR is performed, 341 base pairs of DNA should be amplified using the protase A gene as a template if no intron exists. Therefore, the N-terminus primer was the coding strand sequence at positions 41 to 46 of the heavy chain, and the C-terminus primer was the anti-strand sequence at positions 149 to 154. All possible sequences were synthesized as a mixture and used. .

24 types of C-terminal primers can be predicted, but in order to increase the concentration of specific primers in the mixture, they were synthesized into two pools of 12 types each and subjected to PCH.

Protase A-producing bacterium Aspergillus niger macrosporus (Aspergillus nig
er var, macrosporus DBD-04
06 (ATCC No. 15513)) as a template, the combination of primer 1 and primer 3 (see Example 1) resulted in approximately 350 base pairs of DNA.
It was confirmed by electrophoresis using agarose gel that A was amplified. A DNA band was cut out from the gel, and after extraction, it was labeled with 32p and used as a probe.

b. DNA plotting Total DNA obtained from Aspergillus niger was treated with restriction enzyme E.
Cut with coRI and Bam and perform Southern hybridization using the above probe.
hybridization method) [Journal Fist of Molecular Biology (J, Mo1.Biol
), vol. 98, 503-517 (1975)].

As a result, a hybridizing DNA fragment (EcoRl
10kb fragment, BamHI 3kb fragment, EcoRl
+BamHI 3kb fragment) was obtained.

Preparation of C0 gene library From the above results, the protase A gene has a 3kb B
Since it is thought that am is encoded in the old fragment, Ba
The previously digested total DNA was fractionated by sucrose density gradient centrifugation, and a fragment containing 2 to 4 kb was taken, blunt-ended with T4 DNA polymerase, ligated with an EcoRI adapter, and inserted into the EcoR1 site of λgtlO. This DN
A was packaged with phage protein to create a gene library.

d. Gene cloning of protase A. Infect Escherichia coli with the phage obtained above and spread it on a lambda agar medium.
After culturing at 7°C, the resulting plaques were treated with Amer jam.
The DNA was transferred to a nylon membrane (shaIll), denatured with alkali, neutralized with Tris-HCl buffer (pH 7), and then hybridized with the glove described above.

Hybridization was carried out using 6 times the concentration of 5SC (0°15
M NaCl210.015M Sodium Citrate, p
H7,0), Amersham hybridization buffer and DNA probe approx.
The test was carried out at 65°C for 12 hours using X 10' cpm/mQ. After this, 6 times S S C (0,1% S
The cells were then washed twice with 2x SSC and once with 1x SSC at 65°C for 30 minutes. After further washing with 2x SSC that does not contain SDS,
It was air-dried and subjected to autoradiography (-80°C, overnight). As a result, nine hybridization-positive plaques were obtained from approximately 40,000 clones. This plaque was taken and a secondary screening was performed in the same manner.
Cloned.

e. Determination of the nucleotide sequence For one of the clones obtained in step d above, phage DNA was prepared and digested with the restriction enzyme EcoRI to obtain M
Subcloning was performed by inserting into the EcoR1 site of the 13mp18 vector. Double-stranded plasmid DNA was prepared using E. coli JMI03, and deletion mutants of various lengths were created using EXOIII nuclease for the inserted DNA, and the base sequences were determined using a DNA sequencer manufactured by AB1. As a result, the obtained clone contained 2714 base pairs, and as shown in FIG.
It had an eading frame). The amino acid sequences of the two polypeptide chains of procase A were all encoded by this gene without a single residue difference. Additionally, there is a 59-residue signal peptide and prosequence at the N-terminus, and an 11-residue insertion between the light chain and the heavy chain.

In addition, in order to confirm the presence of introns, Nm and C-terminal oligonucleotide primers were synthesized based on the gene base sequence, and the entire base sequence of the CDNA was obtained using the PCH method. c by reverse transcriptase from menosenger RNA obtained from the protase-producing bacterium Aspergillus niger.
DNA was synthesized and used as a template for PCH. P
The DNA amplified by CH was inserted into the M13 vector, and the DNA base sequence was determined. As a result, it was confirmed that the entire gene sequence was transcribed into cDNA and that it did not contain introns.

f. Expression by E. coli The protase A gene obtained as described above was
It was confirmed as follows that it could be expressed in E. coli. Protase A precursor (amino acids 1-282) was produced by the PCH method using Aspergillus niger cDNA as a template.
and (amino acids 16-282) were respectively amplified, and the former was added to the expression vector pKK
233-2 (Clontic, C1ontech
) into the recombinant plasmid pMP-1, the latter of which is an expression vector pKT containing a secreted signal peptide.
287 (Clontech) to create recombinant plasmid pMP-2; D of the nucleotide sequence encoding procase A precursor 1-282)
For NA amplification, Nco I/H of the vector after amplification
To insert into the indn site, a sense primer with a BspI site and an antisense primer with a Hindl1 site were used. In addition, for amplification of the DNA of the base sequence encoding procase A precursor (16-282), primers having both sense 1 and antisense PstI sites were used, and the primers were inserted into the PstI site of the vector. After culturing Escherichia coli transformed with the expression plasmid thus prepared, the cells were lysed and subjected to SDS electrophoresis and Western blotting. the result,
It was confirmed using antiserum prepared from mice using purified protase A as an antigen that procase A protein was produced by Escherichia coli using any of the expression plasmids described above.

Further, the present invention will be explained in detail with reference to Examples, but the present invention is not limited by the Examples.

Example I Synthesis of DNA Primer An oligonucleotide (17 mar) was synthesized based on the amino acid sequence of a major procase A enzyme. That is, the sequence Trp-Tyr − from position 41 to position 46 from the N-terminus of the heavy chain
A mixture of eight base sequences predicted from lu -Trp -Tyr -Pro, T-G-G-T-A-T/C
-(, -A-A/G-T-G-GT-^-T/C-C-
C (primer 1) and the sequence Met-Asp-11e-Glu- from position 149 to position 154.
- A mixture of 24 anti-strand base sequences predicted from Asp, 12 each, T-C
-T/C-T-G-T/C-T-C-A/G/T-A-
T-A-T-C-C-^-T (primer 2) and T-C
-T/C-T-G-T/C-T-C-A/ni/T-A-
T-G-T-C-CA-T (primer 3) was synthesized. Oligonucleotide synthesis was performed using a DNA synthesizer (Synthesizer) model 38 manufactured by AB1.
1A) row (1, the purification method of synthetic NA is described in the literature [
Musketeer Chemical Experiment Course I.

According to Gene Research Methods 1, pi-33, 1986J.

Aspergillus niger var, ma
The nuclear DNA of C. crosporus DBD-0406 was prepared as follows. Cultivate the above strain for 24 hours and make 24g
The cells were collected, frozen in liquid nitrogen, and then treated with polytron in liquid nitrogen to obtain a powder. This was mixed with 60mQ of buffer (50mMEDTA, 0.2% SDS, 1flQ
/mQ DEPC), heated at 68°C for 15 minutes, and then cooled to room temperature.

10. Centrifuge at OOOrpm for 10 minutes to obtain 70 mQ of upper groove. The supernatant was added with 8M potassium acetate (pH 8,0) at O''C14,4mQ and left at 0°C for 1 hour.
,000 rpm for 15 minutes and separate the upper groove.

Add 70 mQ of isopropyl alcohol to this supernatant,
After centrifuging to obtain a pellet and washing it once with ethanol,
Pour the pellet into 10ml buffer (50mM EDTA,
0.2% SDS.

Pronase K (final concentration 300 μg/raQ) was suspended at 37°C for 6 hours, washed twice with phenol, precipitated with ethanol, and dialyzed against TES-L at 23°08A xmo/m (2 3.5mL total 4mg DN
I got an A.

Polymerase chain reaction (PCR) was performed using a synthetic oligonucleotide mixture as a primer and DNA obtained from Aspergillus niger as a template. PCR is performed using Takara Shuzo's GeneAmp"DNA Amp.
A Reagent Kit was used. Template DNA is 1100n/++12, primer mixture is each IOμM 194℃ (1 minute), 55℃ (2 minutes)
, 72°C (3 minutes) for 35 cycles. As a result, from the combination of primer 1 and primer 3, approximately 35
Since a 0 base pair DNA was obtained, this was isolated by electrophoresis using agarose gel, and was labeled with 3IP and used as a probe. The label uses Amersham's Multiprime DNA labeling.
system and [,-”R3dCTP were used.

Example 4 DNA plotting DNA obtained from Aspergillus niger was treated with restriction enzymes EcoRI, BamHI, and EcoR respectively.
After cutting with +BaaH1, electrophoresing on agarose gel, transferring to Amersham nylon membrane, UV irradiation, Southern hybridization using the above probe.
hybridizati. . Law) [Journal of Molecular Biology (J, No. 1. Biol.
), vol. 98, 503-517 (1975)]. Hybridization was performed using 6x concentrated SS
C (0,15M NaC0, 0,015M sodium citrate, pH 7,0), 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.5% SDS, 10% dextran Sulfuric acid, 011m
g/m (2) in a solution of salmon sperm DNA overnight at 42°C. Washing was carried out at 65°C with 2x concentration of 5SC (0.1% 5DS
) for 15 and 30 minutes, 0.5x 5SC (0.1% 5
DS) for 30 minutes, rinsed with 2x SSC without SDS, air-dried, and then performed autoradiography.

As a result, a hybridizing DNA fragment (EcoRl
10kb fragment, BamHl 3kb fragment, EcoRl
+BamHl 3 kb fragment) was obtained.

Example 5 Aspergillus niger
lu Aspergillus niger DNA 300μg
was digested with BamHI, and the fragments were fractionated by sucrose density gradient centrifugation to obtain fragments of 2 to 4 kb. This DNA fragment 1
μg was blunt-ended with T4 DNA polymerase using Takara Shuzo's DNA Planting Kiratra, treated with phenol, and then precipitated with ethanol. This DNA fragment contains Ec
Ec of λgtlo after ligation of oR1 adapter.

It was inserted into the R1 site. This DNA was packaged into phage particles using AmaJam's puff caging kit to create a gene library.

Example 6 Gene cloning of Protase A E. coli was infected with the phage obtained above, plated on L-agar medium, and cultured at 37°C.
A+n5rsha+m) Transfer to nylon membrane plan, 0
．． Denature DNA with 5M NaOH, 1,5M NaCl, 0.5M) Lis-HCl buffer (pH 7)
, 1.51 After neutralization with NaCl2, hybridization was performed with the above-mentioned Zub. Hybridization was carried out in 6-fold concentrated SSC (0.15M N
aC4,0,015M Sodium Citrate pH7-
0), Amersham hybridization buffer and DNA probe approximately 5X
The test was carried out at 65°C for 2 hours using 10'cpm/mQ.

This was followed by 30 min at 65°C with 5 sc of 6@ (containing 0.1% SDS), followed by 2 min with 2x SSC.
Washed once with 1x SSc. After washing lightly with 2x SSC not containing SDS, it was air-dried and subjected to autoradiography (-80°C 1-night). As a result, nine hybridization-positive plaques were obtained from approximately 40,000 clones. Take this plaque,
Secondary screening was performed in the same manner and cloned.

Example 7 Determination of base sequence For one of the clones obtained in Example 6, L
- After culturing on agar medium and lysis, the phages were extracted with SM/(Fur).
A solution containing 20% polyethylene glycol, 2M sodium chloride in M buffer was added and incubated on ice for 1 hour. Centrifuge to remove the upper groove and reduce the precipitate to 0.7
D suspended in 5 mQ L-broth and suspended in L-gloss
After adding 0.75 m4 of E52 and mixing, the mixture was centrifuged and the upper groove was removed. Add 13 μg of 0.1 mg/+ni2 proteinase and 32 μβ of 10% SDS to the upper groove and incubate for 5 minutes at room temperature.
Leave it for a minute. Add 130 μg of 3M potassium acetate,
Incubate at 88°C for 20 min, then on ice for 10 min.
Cooled for minutes. After centrifugation, the upper groove was precipitated with Improper Tools to obtain phage DNA. DNA with restriction enzyme EcoRI
and inserted into the EeoR1 site of the M13mp18 vector, and inserted into E. coli JM103 to prepare double-stranded plasmid DNA according to a conventional method. Various deletion mutants were created using EXOm nuclease for the inserted DNA, single-stranded DNA of M13 was prepared using JM103, and the base sequence was determined using an ABI DNA sequencer. Takara Shuzo's Kiroshifence kit was used for deletion using ExolH.

Example 3 Cloning and nucleotide sequence determination of cDNA N-terminal and C-terminal oligonucleotide primers were synthesized based on the nucleotide sequence of the gene, and PC was used in the same manner as in Example 2.
cDNA was amplified using Method B. Metsenger RN obtained from the proctase-producing bacterium Aspergillus niger
Synthesize cDNA from A using reverse transcriptase and convert it into PC
It was used as a template for H. DNA amplified by PCH
was inserted into the Sma 1 site of M2S, and the DNA base sequence was determined in the same manner as in Example 7. As a result, it was confirmed that the gene sequence completely matched the cDNA and did not contain any introns.

Example 9 Preparation of polyclonal antibody to protase A 100 μg of isolated and purified protase A was dissolved in 1 m+2 physiological saline, and Freund's complete adjuvant manufactured by Gyudon Co., Ltd. was used as an adjuvant.

Furthermore, five male Bath/c mice from Shizuoka Kyodo were used. For each mouse, mix 10 μg of protase A (0°1 mf2) and adjuvant O, 1 mQ.
It is administered into the peritoneal cavity of sBa1b/c mice in the form of il1nWaLer. Sensitization is carried out a total of 40 times every week, and one week after the fourth sensitization, blood is collected from the artery and left on a petri dish at room temperature for 2 hours. Put the upper groove into the centrifuge tube,
Centrifuge at 250 rpm for 10 minutes, and use the supernatant as proctase A antiserum.

Example 10 Construction of expression plasmid Aspergillus niger
iger) (1) Using c DNA as a template, sense
Primer CT-CT-G-T-C-A-T-G
-A-A-G-T-T-C-T-C-T-A-C-C
-A-T-C-C-T-T, 77th sense ply?
-C-T-C-T-C-A-A-G-C-T-T -
A-T-T-A-C-A-C-G-T-A-C-G-T
PCR was performed in the same manner as in Example 3 using -G-A-C-AC-T-G-A-C-G-C-T. After agarose gel electrophoresis, approximately 0.8 kb ODNA was isolated, and after BspHI and old nd■ digestion, pKK233-
2 vector (Clontech) into the Ncol and H4ndn1 sites to construct an expression plasmid for procase A precursor (1-282).

In addition, sense primer C-T-C-T-G-C-T
-G-C-A-G-C-T-C-T-G-G-C-T-
G-C-T-C-C-TC-T-C-A-C-T, antisense primer C-T-C-T-C-C-TmG
-C-A-G-C-T-A-T-Ding-A-G-A-C (
Knee T-A-G-G-T-G-A-C-A-GiT-C
- Perform PCB using A-C-G-C-T and D
NA was isolated, digested with Pstl, and inserted into the Pst1 site of pKT287 vector (Clontech, Inc.) to create a secreted expression plasmid for proctase A precursor (16-282). Figures 3 and 4.

Example 11 Escherichia coli HBIOI transformed with the plasmid prepared in Example 10 of Protase A precursor using Escherichia coli was cultured in MQCA medium containing 50 μg ampicillin at 37° C. for 1.5 hours, and IPTG (final concentration 0 21 mM) was added. The cells were further cultured for 7 hours. After collecting bacteria, 2
%S D S 10.125M Tris-HCl (pH 6
-8) Suspend in 10.2% mercaptoethanol/lO% glycerol 10% bromophenol blue solution, boil for 5 minutes to lyse, perform 5DS-electrophoresis, and perform Western blotting to prepare the cells in Example 9. The production of a protase A precursor that specifically reacts with the protase A antibody was confirmed.

(Effects of the Invention) The present invention enables mass production of industrially useful protase A.

[Brief explanation of drawings]

1st r! ! J: Shows the nucleotide sequence of the protase A enzyme gene cloned from the Aspergillus niger strain and the amino acid sequence of protase A deduced from it. Figure 2: Shows the amino acid sequence of active protase A. FIG. 3 shows the method for constructing the plasmid pMP-1 of the present invention. Section 4-2 shows the method for constructing the plasmid pMP-2 of the present invention.

Claims (6)

[Claims] (1) [There is a gene sequence] A gene that encodes the amino acid sequence represented by (2) A polypeptide consisting of a peptide consisting of 39 residues from position 60 to position 98 in the amino acid sequence according to claim (1) and a peptide consisting of 173 residues from position 110 to 282. [There is a gene sequence] A gene that encodes the amino acid sequence represented by. (3) [There is a gene sequence] gene represented by the base sequence of (4) [There is a gene sequence] A gene represented by the nucleotide sequence. (5) A vector comprising the gene according to any one of claims (1) to (4). (6) A method for producing a polypeptide, which comprises culturing a host transformed with the plasmid according to claim (5), and collecting a polypeptide having acidic protease activity from the culture.