JPH07107980A - Production of mold protein disulfide isomerase in high efficiency - Google Patents

Abstract

PURPOSE:To provide a process for the efficient production of a mold protein disulfide isomerase and provide a genetic system for the production. CONSTITUTION:This invention relates to a recombinant DNA produced by combining the 3' terminal of a DNA containing a promoter region originated from Bacillus brevis with a DNA coding for a mold protein disulfide isomerase; a transformed Bacillus brevis containing the recombinant DNA; and a process for the production of a mold protein disulfide isomerase using the transformant.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a fungal PDI by Bacillus brevis carrying a DNA encoding a protein disulfide isomerase derived from mold as a heterologous gene (hereinafter sometimes referred to as mold PDI). Production method, and the DNA and its D
Regarding Bacillus brevis holding NA.

[0002]

BACKGROUND OF THE INVENTION Humicola insolens (humicola
protein disulfide isomerase (PDI) derived from one strain of insolens ) is highly stable and can be used in a wide temperature range as compared to PDI derived from mammals and yeasts, and thus is useful for protein refolding. It can be used advantageously [JP-A-4-13525
4]. The gene encoding the PDI has already been cloned, and the entire base sequence and the entire amino acid sequence deduced therefrom have been clarified [JP-A-5-4401].
4].

PDI was first discovered by Anfinsen et al. As an enzyme that catalyzes the formation of disulfide bonds [J. Bi
ol.Chem., 238 , 628, (1963)], in vitro ,
It acts on ribonucleases of reduced or scrambled type (inactive by recombination of multiple disulfide bonds in the molecule into non-natural type), promotes correct disulfide bond formation, and has an action of becoming active type.

The industrial applicability of PDI may be applied to the refolding of recombinant proteins produced in Escherichia coli and the like. Here, refolding means
It means that a protein that does not show its original physiological activity due to the wrong way of applying a disulfide bond is converted to an active form by re-establishing the correct disulfide bond. Recent rapid advances in genetic engineering technology have led to the large-scale synthesis of eukaryotic proteins in prokaryotes, but the recombinant proteins thus obtained lack the correct disulfide bonds or have incorrect disulfide bonds. You may have a person. Therefore, the utility value of PDI is expected to be applied to the refolding of recombinant proteins produced in Escherichia coli or yeast, and mass production of recombinant mold PDI having excellent stability by genetic engineering is desired. It is rare.

To date, many recombinant DNA studies have been carried out using E. coli , and many heterologous proteins have already been produced in E. coli. However, in this method, since the produced heterologous protein is accumulated in the microbial cells, it not only takes a lot of time and labor to extract the target product from the microbial cells and to purify it from the extract, but It is not easy to obtain the substance in pure form in perfect form.

On the other hand, Bacillus genus has been industrially used as a bacterium for producing various extracellular enzymes since ancient times. Among these extracellular enzymes, Bacillus amyloliquefaciens ( Ba
α-amylase gene of Cillus amyloliquefaciens (I. Palva et.al., Gene, 22 , 229 (1983)), penicillinase gene of Bacillus licheniformis (S. Chang et.al., Molecular Cloning a)
nd Gene Regulation in Bacilli, Academic Press, 659,
(1982)) and Bacillus subtilis
Α-amylase gene (H. Yamazaki et.al., J. Bacter
iol., 156 , 327 (1983)) and the like have already been cloned, and secretory production of heterologous proteins using these promoters or signal peptides has been reported.

Bacillus subtilis is mainly used as a host for the secretory production of the above-mentioned heterologous protein in Bacillus, but since this microorganism produces a large amount of extracellular protease, it is secreted by recombinant DNA technology. The produced heterologous protein undergoes degradation, and its accumulated amount is significantly reduced. On the other hand, Utaka et al., Bacillus brevis 47 (Japanese Unexamined Patent Publication No. 60-5807) produces almost no protease outside the cells.
4, JP-A-62-201583; FERM P-722.
4) as a host, one of the major extracellular proteins of this strain, MWP (Middle Wall Protein) (H. Yamagata et.al.,
J. Bacteriol., 169 , 1239 (1987), Norihiro Tsukakoshi, Journal of Japanese Society of Agricultural Chemistry, 61, 68 (1987)), and α-amylase (JP-A-62-201583, H). .Yamagata et.al., J. Bacterio
l., 169 , 1239 (1987)) and Butapepsinogen (Cormorant ▲ High ▼
Shigezo, Proceedings of the 62th Annual Meeting of the Japanese Society of Agricultural Chemistry p8
37-p838; Norihiro Tsukakoshi, Journal of the Japanese Society of Agricultural Chemistry, 61,
68 (1987)) has been successfully produced.

[0008] Cormorants also isolated Bacillus brevis HPD31 (FERM BP-1087), a strain that does not produce extracellular protease of Bacillus brevis,
Using this as a host, thermostable α-amylase (JP-A-63-
56277, Abstracts of Annual Meeting of Japan Society for Agricultural Chemistry 1987, p27) Human EGF (H. Yamagata et.al.Proc.Nat
l.Acad. USA, 86 , 3589 (1989), JP-A-2-31682)
Has succeeded in high secretory production of.

Moreover, Kajino et al. Isolated a mutant strain Bacillus brevis 31-OK capable of stably accumulating a heterologous protein secreted outside the cells, and succeeded in secretory production of human growth hormone using this strain as a host. ing. Regarding PDI, bovine PDI is produced by Escherichia coli or the like (Japanese Patent Laid-Open No. 64-2).
0086), and production of human PDI by Bacillus brevis (JP-A-3-206887) and the like have already been reported.

[0010]

As described above, production of PDI has been attempted using Escherichia coli and Bacillus bacteria as hosts, or yeast, animal cells, etc. as hosts, but both of these require complicated purification and production. It is hard to say that you can be satisfied in terms of sex. Therefore, it is an object of the present invention to provide a host-vector system that enables efficient production of mold PDI, and to provide an efficient method for producing mold PDI using them.

[0011]

[Means for Solving the Problems] Utaka et al. Have developed a host-vector system for the production of a heterologous protein, and found that Bacillus brevis can be advantageously used as a host. It has been proposed for use in the production of heterologous proteins. On the other hand, as a result of intensive research to create recombinant plasmids capable of efficiently expressing PDI-encoding DNA in these host strains, Bacillus
We have found that the use of a Brevis-derived promoter in certain embodiments is effective in the expression of mold PDI.

[0012] Therefore, the above-mentioned problem is solved by recombinant DN in which DNA encoding mold PDI is ligated to the 3'end of DNA containing the promoter region derived from Bacillus brevis.
Solved by the use of A. That is, according to the present invention, mold PDI is collected from the culture by culturing the recombinant DNA, Bacillus brevis carrying the recombinant DNA and the Bacillus brevis in a nutrient medium. An efficient method for producing mold PDI is provided.

[0013]

Specific Embodiments As DNA encoding fungi PDI, in addition to DNA directly isolated from fungi, known fungus P
Any of the chemically synthesized DNAs based on the amino acid sequence of DI (Japanese Patent Application No. 5-44014) may be used. A promoter derived from any strain belonging to B. brevis can be used as long as it functions as Bacillus brevis (hereinafter sometimes abbreviated as "B. brevis"). For example, as a concrete example, B. Brevis 47 (FER
MP-7224) or B. Brevis HPD31 (F
The promoter of the major extracellular protein gene of ERM BP-1087) can be mentioned. The DNA containing the promoter region needs to have an SD sequence, a translation initiation codon, etc. in addition to the above promoter, and may further contain a part of the major extracellular protein gene.

DN containing these promoter regions
To attach the DNA encoding mold PDI to the 3'end of A, D that had been excised from the chromosome of B. brevis
DNA encoding mold PDI may be bound to the 3'end of NA. Mold PDI may be accumulated inside or outside the cells of B. brevis, but mold PD may be accumulated outside the cells.
DNA containing a promoter region when accumulating I
It is necessary that the region encoding the signal peptide is also included at the 3'-terminal side of. Examples of the signal peptide include signal peptides of major extracellular proteins of Brevis 47 or Brevis HPD31. In particular, the signal peptide of Brevis 47 MWP (Middle Wall Protein) is one example.

A mold PD containing the above promoter region
Examples of the expression vector used for expressing the I gene include pHY500, pNU200 (JP-A-2-31682,
Cormorant Takataka Shigezo, Journal of the Japanese Society of Agricultural Chemistry, 61,669 (19
87)) and the like. A method for constructing a mold PDI expression plasmid using these expression vectors and the above DNA is described in, for example, Molecular Clonig 2nd. Ed., A Laborator.
y Manual (Cold Spring Harbor Laboratory (1990)), known in the art, and a recombinant DNA of interest (mold PDI expression plasmid) can be constructed according to these methods. Preferable examples of such a mold PDI expression plasmid include pNU211L4PDI and the like prepared in Examples described later. The host used for constructing the plasmid may be any of microorganisms belonging to Escherichia coli, Bacillus subtilis, and B. brevis. For example, Escherichia coli HB101, E. coli JM109, Bacillus subtilis RM141 (J. Bacteriol., 158 , 1).
054 (1984)), B.I. Brevis 47 (FERM P-72
24) and the like.

The host used for gene expression is
Mold P produced by expression of the above-mentioned plasmids including B. brevis 47 (FERM P-7224), B. brevis 47-5 (FERM BP-1664) and B. brevis HPD31 (FERM BP-1087).
Any one can be used as long as the host itself does not significantly affect DI. Particularly preferred is B. brevis 31-OK (FERM P-1327) obtained by mutation treatment of B. brevis HPD31.

To transform host B. brevis with the above-mentioned mold PDI expression plasmid, for example, the method of Takahashi et al. (J. Bacteriol., 156 , 1130 (1983)) or the method by electroporation (H. Takagi et al. , Agric.Bi
ol. Chem., 53, 3099-3100 (1989)) and the like. The transformants thus created produce a large amount of mold PDI by culturing them in a nutrient medium, and most of them are secreted outside the cells.

The medium used for culturing contains a carbon source, a nitrogen source and, if necessary, inorganic salts. Alternatively, the culture may be carried out using a synthetic medium mainly containing sugar and inorganic salts. When using a auxotrophic strain, it is desirable to add nutrients required for its growth to the medium. further,
If necessary, antibiotics and antifoaming agents may be added to the medium. As the culture condition, the initial pH of the medium is 5.0 to 9.
It is adjusted to 0, preferably 6.5 to 7.5. The culture temperature is usually 15 ° C to 42 ° C, preferably 24 to 37 ° C, and the culture time is generally 16 to 360 hours, preferably 24.
~ 144 hours.

After the completion of the culture, in order to collect the mold PDI from the culture, the cells and the supernatant are separated by, for example, centrifugation or filtration. Mold PDI produced outside the cells was
The desired mold PDI can be obtained by purification by a commonly used protein purification method, for example, salting out, isoelectric precipitation, gel filtration, ion exchange, various chromatographies such as reverse layer, and the like.

The obtained mold PDI can be quantified by an enzyme immunoassay using an anti-mold PDI antibody.
In addition, the refolding activity of scrambled ribonuclease was measured (Method in Enzymology 107 , 281-294 (1
984)) can also quantify the mold PDI.

[0021]

INDUSTRIAL APPLICABILITY According to the present invention, a host-vector system capable of efficiently producing mold PDI is provided. That is,
By using Bacillus brevis as a host and using a recombinant DNA in which a DNA encoding a mold PDI is ligated to the 3'end of a DNA containing a promoter region derived from Bacillus brevis as a mold PDI expression plasmid, PDI can be mass-produced efficiently. Mass-produced mold PDI is a protein disulphide isomerase (P
Compared to DI), it is highly stable and can be used in a wide temperature range, so that it can be advantageously used for protein refolding.

[0022]

The present invention will be described in more detail with reference to the following examples, but the present invention should not be limited thereto.

Example 1. Construction of mold PDI expression vector
And preparation of transformants MW, one of the major cell wall proteins of Bacillus brevis 47
C among the signal peptides of P (Middle Wall Protein)
Plasmid pBR-AN2 containing a terminal region
257876) was treated with the restriction enzymes NcoI and XbaI to obtain an approximately 3.4 kb [NcoI-XbaI] fragment.
In order to ligate the signal peptide of MWP and the mold PDI, the chemically synthesized DNA adapters (SEQ ID NOs: 1 and 2) were ligated with T4 DNA ligase,
An approximately 3.4 kb [PvuII-XbaI] fragment was obtained. The plasmid pBluePDI-2 (Japanese Patent Application No. 5-4401) was added to this.
About 1.4 kb encoding the mold PDI isolated from 4)
Inserting the [PvuII-XbaI] fragment into plasmid pB
R-AN2-PDI was obtained.

The plasmid pBR-AN2-PDI was treated with restriction enzymes ApaLI and XbaI to obtain about 1.4 kb [Ap
The aLI-XbaI] fragment was isolated. On the other hand, a plasmid pNU211L4 having an improved signal sequence in which 3 Leu were added to the hydrophobic region of the MWP signal sequence was treated with the restriction enzymes ApaLI and XbaI, and the obtained plasmid was about 4.2.
kb [ApaLI-XbaI] fragment and about 1.4 kb [A
The paLI-XbaI] fragment was ligated with T4 DNA ligase, and the mold PDI expression plasmid pNU211L4PD was ligated.
I was constructed (Fig. 1). In addition, the plasmid pNU211
The nucleotide sequence of the insert containing the PDI coding sequence in L4PDI and the amino acid sequence encoded thereby are shown in SEQ ID NO: 3.

Using this plasmid, Bacillus brevis 31-OK (FERM P-13274) was transformed by the method of Takahashi et al. (J. Bacteriol., 156, 1130 (1983)). A stable strain was isolated from the obtained transformants, and this was transformed into Bacillus brevis 31-OK (pNU2
11L4PDI).

Example 2. Cultivation of transformants and mold P
Secretory production of DI The transformant Bacillus brevis 31 obtained in Example 1
-OK (pNU211L4PDI) and its control Bacillus brevis 31-OK (pNU211) were polypeptone 3%, yeast extract 0.2%, glucose 3%,
_{MgSO 4 · 7H 2 O 0.01%} , CaCl 2 · 7H
₂ O 0.01%, MnSO ₄ .4H ₂ O 0.001
_{%, FeSO 4 · 7H 2 O} 0.001%, ZnSO 4
7H ₂ O 0.0001% and erythromycin 1
The cells were cultured in a medium consisting of 0 mg / L (pH 7.2) at 30 ° C for 2 days.

The above culture solution was centrifuged and the supernatant was added to Lae
Following SDS-polyacrylamide gel electrophoresis according to the method of mmli (Nature, 227, 680 (1970)), and staining with Coomassie Brilliant Blue R250, the polypeptide having mold PDI activity showed a band at a molecular weight of about 59000 daltons. Indicated. Also, Burnette's method (Anal.Bioch
Em., 112, 195 (1981)), Western blotting was performed using an anti-PDI antibody. As a result, mold PDI was observed in the culture supernatant of Bacillus brevis 31-OK (pNU211L4PDI), and its molecular weight was Is a natural mold PDI
It showed a value slightly smaller than that.

When the refolding action of scrambled ribonuclease was measured on the culture supernatant,
PDI activity value of 500 mg per 1 L of culture supernatant (bovine P
DI conversion) is shown.

Example 3. Purification of mold PDI 1 L of the culture supernatant obtained in Example 2 was subjected to ultrafiltration (Filtron,
After pretreatment with Minisette Omega, 10kcutoff), D
EAE-Sephacel anion exchange column chromatography (Pharmacia) and phenyl-Toyopearl hydrophobic chromatography (Tosoh) were purified in this order to obtain 80 mg of a purified sample of mold PDI (FIG. 2).

Example 4. Protein Chemical Properties of Mold PDI The following protein chemical properties of the purified mold product of mold PDI obtained in Example 3 were examined.

(1) Molecular weight As a result of examination by 8% polyacrylamide gel electrophoresis containing 0.1% SDS, the purified mold of fungus showed one band and was found to be a pure product. Its molecular weight was found to be 59.00 based on the mobility of markers of known molecular weight that were simultaneously run.
Calculated as 0. In addition, a value of a molecular weight of about 120,000 was obtained by gel filtration chromatography.

(2) Amino Acid Composition After hydrolyzing in 6N hydrochloric acid at 110 ° C. for 24 hours, it was analyzed with a Picotag system for amino acid composition analysis manufactured by Waters. Regarding cystine, mold PDI was previously oxidized with formic acid and then hydrolyzed with 6N hydrochloric acid, and quantified as cystine acid. The obtained amino acid composition values are shown in Table 1.

[0033]

[Table 1]

(3) N-terminal amino acid sequence Protein sequencer by pulse liquid automatic Edman degradation method (Applied Biosystems, 473A type)
As a result of examining the N-terminal amino acid sequence of the purified standard by using, the sequence of the purified standard was identical to that of the natural mold PDI (Table 2).

[0035]

[Table 2]

Example 5. Mold PDI activity measurement method The refolding action of scrambled ribonuclease was measured for the purified mold preparation of mold PDI obtained in Example 3. Scrambled ribonuclease (Sc-R
Nase) in the literature [Methods in Enzymology, 107, 281 (19
84)], and prepared using bovine pancreatic ribonuclease. The activity of RNase is reported in the literature [J. Biochem. 108 , 846 (199
0)], and measurement was performed using yeast ribonucleic acid as a substrate.

50 mM sodium phosphate buffer (pH 7.
5) S in 1 mM ethylenediaminetetraacetic acid (EDTA)
c-RNase 5 μM and dithiothreitol 5 μM were added, and mold PDI was added to make 50 μL.
Let react for minutes. Before and after the reaction, 5 μL of each sample was taken and combined with 495 μl of ribonucleic acid (0.1 mg / ml), and the amount of decomposed ribonucleic acid was measured by the increase in absorbance at 260 nm. The PDI activity was defined as the rate of increase in this absorbance per minute.

Example 6. Properties of mold PDI The following properties of the purified mold product of PDI obtained in Example 3 were examined.

(1) Substrate specificity In the same manner as above, 5 ng of mold PDI was allowed to act using scrambled chicken egg white-derived lysozyme and bovine reduced pancreas-derived aprotinin. As a result, RNase
Similar to the above, recovery of lysozyme and aprotinin activity was found, and the degree was similar to that of natural mold PDI.
Was reacted with mold PDI5ng of the present invention to insulin [Biochem. J.., 1 59, 377-384 (1976)], the reduction of insulin was observed (Fig. 3).

(2) Specific activity and enzyme reaction rate parameter The specific activity was measured using a scrambled ribonuclease as a substrate for the enzyme. Further, Km and Vmax were obtained by the Lineweaver-Burk reciprocal plotting method. The specific activity, Km value and Vmax value of the mold PDI of the present invention were similar to those of the native mold PDI (Table 3).

[0041]

[Table 3]

(3) Temperature stability 5 ng each of the mold PDI of the present invention and natural mold PDI is heated in a phosphate buffer pH 7.5 at 30 to 90 ° C. for 30 minutes,
After that, the remaining activity was measured. The results are shown in Fig. 4. In the figure, the activity is shown as 100% without heating. From FIG. 4, the mold PDI of the present invention showed heat resistance equivalent to that of the natural mold PDI.

(4) pH stability For Sc-RNase, 5 ng each of the mold PDI of the present invention and natural mold PDI was added to a Briton-Robinson buffer solution pH.
Warm in 5-7 at 30 ° C. for 30 minutes, then pH 7.
The residual activity after returning to 5 was measured. PH is all active
The activity of 7.5 was expressed as 100%. Results are shown in FIG. The mold PDI of the present invention showed pH stability similar to that of the natural mold PDI.

(5) Effect of Guanidine Hydrochloride Concentration In a 50 mM phosphate buffer (pH 7.5) and 5 μM dithiothreitol, 5 ng of each PDI was incubated at 30 ° C. for 5 minutes to activate PDI, and then guanidine hydrochloride was added. Salt 0
0.8M and substrate scrambled ribonuclease (S
c-RNase) was added and refolded.
The reactivated RNase activity was determined by measuring the amount of ribonucleic acid decomposed by increasing the absorbance at 260 nm. By this method, S by PDI in the presence of guanidine hydrochloride
The refolding of c-RNase was comparable to the native form (Figure 6).

Reference Example 1. Contains the human growth hormone gene
No plasmid pNU200-GH Construction Ikehara et al (Ikehara et al., Proc.Natl.Acad.Sci.USA., 81, 5956-
5960 (1984)), the DNA encoding the human growth hormone (HinfI-SalI fragment) was isolated from the plasmid pGH-L9 (Ikehara et al., Ibid.).
A synthetic linker for ligating the MWP signal peptide and hGH in frame was ligated to the fragment with T4 DNA ligase and treated with restriction enzymes NcoI and SalI to obtain a 596 bp NcoI-SalI fragment. One of the major membrane proteins of Bacillus brevis 47 MWP (Middle
Plasmid pBR containing the C-terminal region of Wall Protein)
-27 bp from AN2 (JP-A-2-257876)
The NcoI-SalI fragment was removed and the
Insertion of 6 bp NcoI-SalI fragment into plasmid p
BR-AN2-GH was obtained.

The plasmid pBR-AN2-GH was digested with restriction enzymes ApaLI and HindIII to obtain 656 bp Ap.
The aLI-HindIII fragment was isolated. On the other hand, plasmid pNU200 (Cormorant Shigezo, Journal of Japan Society for Agricultural Chemistry, 6
1,669-676 (1987)), the restriction enzyme Ap
cleaved with aLI-HindIII, and the large fragment and the above 656 bp ApaLI, HindIII fragment were T4 DNA.
Bacillus brevis 47 (FERM BP-1664, IF was used according to the method of Takahashi et al. (See J. Bacteriol., 156, 1130 (1983)) using the reaction solution ligated with ligase.
O 14698) was transformed. A plasmid was isolated from the resulting erythromycin-resistant transformant,
This was named pNU200-GH.

The plasmid pNU200-GH was transformed into T
The parent strain Bacillus brevis HPD by the method of akahashi and others
31 (JP-A-63-56277, FERM BP
-1087) introduced, Bacillus brevis HPD31
(PNU200-GH) was obtained. The transformant was designated as T2.
The cells were diluted and suspended in a medium, smeared on a T2 plate, and then exposed to ultraviolet rays ( ² J / m ² ). After culturing at 30 ° C. overnight, the obtained colonies were inoculated into 5YC medium and cultured at 30 ° C. for 6 days. The culture broths on the 3rd and 6th days were collected, and the amount of hGH contained in the culture supernatant was measured by the enzyme immunoassay. At this time, one strain in which the amount of hGH in the culture supernatant increased from the 3rd day to the 6th day was selectively isolated. When the mutant strain and the parent strain are compared, the amount of hGH in the culture supernatant of the parent strain is 3
The resulting mutant strain showed a remarkable difference in that the amount of hGH increased even after the third day, whereas the amount decreased from the day to the sixth day. In order to remove the plasmid from the mutant strain, the mutant strain was repeatedly subcultured in T2 medium containing no erythromycin, and a strain that was sensitive to erythromycin and did not produce hGH was easily obtained. This strain was named Bacillus brevis 31-OK.

Bacillus brevis 31-OK (FERM
P-13274) has the mycological properties of the parent strain Bacillus
It exhibited almost the same properties as Brevis HPD31 (see Japanese Patent Laid-Open No. 63-562777) except that the action on a specific heterologous protein is different. The main properties are shown below.

Physiological Properties Reduction of Nitrate-Production of VP Test Acetoin-Production of Indole-Production of Hydrogen Sulfide-TSI Agar-Lead Acetate Agar + Utilization of Citric Acid + Utilization of Inorganic Nitrogen Source Nitrate-Ammonium Salt + Pigment Production (King's medium) -Urease-oxidase + acylamidase-catalase + OF test No decomposition Decomposition of gelatin-Generation of acid from glucose-Xylose decomposition-Lactose decomposition-Maltose decomposition-Arginine hydrolysis-Growth pH 5 .5-8.5

Morphological properties Cell size Liquid medium; 0.9-1.2x
3.9 to 5.8 μm agar medium; 0.9 to 1.2 × 2.9 to 4.2 μm cell shape bacillus cell polymorphism presence / absence motility presence / absence

Reference Example 2. Humicola Insolence
Preparation of mRNA-derived cDNA library Fumicola insolens (FERM BP-1291
1) Guanidine isothiocyanate method (Chirgvin, JV et al., Biochemistor ) from bacterial cells (hereinafter referred to simply as "mold")
y , 18 , 5294 (1979)) was used to extract total RNA and
Oligo dT cellulose column chromatography from A
(Pharmacia) was used to purify poly (A) RNA. Gubler, U and Hoffman, B. using this poly (A) RNA as a template.
CDNA was prepared by the method of J. ( Gene , 25 , 263 (1983)), and EcoRI / NotI adapter (cDNA Synthesis
Kit, Pharmacia), and then λZAPII (Strata
cloned into the EcoRI site of
A library was created.

Reference Example 3. Mold P by RT-PCR method
Using specific amplification RNA PCR Kit (Takara Shuzo) of DI gene , mold PDI
The gene was specifically amplified. Details of the operation followed the manufacturer's protocol. CDNA was synthesized using total RNA extracted from mold (Reference Example 2) as a template and random hexamers as primers. Next, using the obtained cDNA as a template, a part of the amino acid sequence of mold PDI (Lys Asp Thr
Phe Asp Asp Phe Ile and Glu Val Gly His Gln Gln C
The oligonucleotide synthesized based on ys) was used as a primer to specifically amplify the PDI gene. The sequences of the primers are shown below.

[0053]

[Chemical 1]

The primer was synthesized by a DNA synthesizer (Applied Biosystems). PCR
DNA of about 0.2 kbp was specifically amplified by the method.
When this DNA fragment was digested with the restriction enzyme EcoRI, it was cut into two fragments of about 50 bp and 150 bp.
Each of these DNA fragments was transformed into E of plasmid pUC118.
cloned into the coRI site and plasmid pUC11
8ΔPDI-1 and pUC118ΔPDI-2 were made. E. coli JM109 (Messin
g, J., Gene, 33 , 119 (1985)), and clone JM109 / pUC11 containing the desired plasmid.
8ΔPDI-1 and JM109 / pUC118ΔPDI
-2 was selected. Then, the obtained JM109 / pUC
118ΔPDI-1 and JM109 / pUC118ΔP
From DI-2, alkali method [Birnboim, HC and Doly,
J., Nucl. Acids Res., 11 , 1513 (1979)], the plasmids pUC118ΔPDI-1 and pUC118ΔP.
DI-2 was extracted and purified.

Reference Example 4. Black containing mold PDI gene
Isolation of DNA and determination of its nucleotide sequence Escherichia coli XL-1 Blue was infected with λZAPII containing the cDNA obtained in Reference Example 2 to form a total of about 4000 transformed plaques on LB agar medium. This was transferred onto a nylon filter (Amersham, Hybond-N), and the recombinant DNA exposed and denatured in 0.5N NaOH solution was UV-fixed on the filter (Maniatis).
Et al., Molecular Cloning 2nd Ed.Cold Spring Harbor L
aboratory, 1989).

On the other hand, the plasmid pUC described in Reference Example 3
A DNA fragment obtained by cleaving 118ΔPDI-2 with a restriction enzyme EcoRI is a DNA Tailing Kit (Boehringer).
Was labeled with digoxigenin according to the manufacturer's protocol and used as a probe. From the filter on which the recombinant DNA was immobilized, a clone that reacted with the labeled probe was searched using the DIG-ELISA Detection Kit (Boehringer) according to the manufacturer's protocol. The insert fragment of the positive clone λZAPIPDI-2 obtained by this method was used for in vivo excision method (Stratage).
ne company) plasmid Bluescript SK (-) (Stratage
ne)) and cloned into plasmid pBlueP
DI-2 was prepared. E. coli XL-1 Blue was transformed with the plasmid pBluePDI-2, and the plasmid pBluePDI-2 was extracted and purified from the obtained recombinant XL-1 Blue / pBluePDI-2 by the above-mentioned alkaline method. CDNA contained in this plasmid
The portion had a total length of about 1.7 kbp.

[0057] dideoxy method the nucleotide sequence of the cDNA portion (Sanger, F., Et al., Proc.Natl.Acad.Sci., 74, 5463 ( 197
7)). The determined nucleotide sequence and the entire amino acid sequence deduced from it are shown in SEQ ID NO: 3 in the sequence listing.

[0058]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence CATGGCTTTC GCTTCGGATG TTGTCCAG 28

SEQ ID NO: 2 Sequence Length: 24 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence CTGGACAACA TCCGAAGCGA AAGC 24

SEQ ID NO: 3 Sequence length: 1755 Sequence type: Nucleic acid Number of strands: Duplex topology: Linear Sequence type: cDNA to mRNA Origin: Humicola insolens Direct origin: Plasmid pBluePDI-2 Characteristics: DNA encoding protein disulphide isomerase of Humicola insolens

Sequence CGCATACCTG CAGGGTATCA ATTGGAATTC AATTCCTTGG TTGCGGTGAT 50 TCCTCCTATC TGTCCTTTTC TGCCTTTACT TTAAGTAGCC CAGAAACAGG 100 AACATCGCG ATG CAT AAG GCC CAG AAG TTC GCG CTC GGC CTG CTT 145 Mu Leu Gly Leu Ghe Leu Phe Ala GCG GCA GTT GCC ACA GCT TCG GAT GTT GTC CAG CTG AAG 190 Ala Ala Ala Ala Val Ala Thr Ala Ser Asp Val Val Gln Leu Lys -5 1 5 AAG GAC ACC TTC GAC GAC TTC ATC AAG ACG AAT GAC CTT GTT CTC 235 Lys Asp Thr Phe Asp Asp Phe Ile Lys Thr Asn Asp Leu Val Leu 10 15 20 GCC GAA TTC TTC GCG CCG TGG TGC GGT CAC TGC AAG GCT CTC GCC 280 Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala 25 30 35 CCC GAG TAC GAG GAG GCT GCG ACC ACA CTG AAG GAG AAG AAC ATC 325 Pro Glu Tyr Glu Glu Ala Ala Thr Thr Leu Lys Glu Lys Asn Ile 40 45 50 AAG CTC GCC AAG GTG GAC TGC ACA GAG GAG ACG GAC CTC TGC CAA 370 Lys Leu Ala Lys Val Asp Cys Thr Glu Glu Thr Asp Leu Cys Gln 55 60 65 CAA CAT GGT GTT GAG GGC TAC CCG ACT CTC AAG GTC TTC CGC GGC 415 Gln His Gly Val Glu Gly Tyr Pro Thr Leu Lys Val Phe Arg Gly 70 75 80 CTT GAC AAC GTC TCC CCC TAC AAG GGC CAG CGC AAG GCT GCT GCT 460 Leu Asp Asn Val Ser Pro Tyr Lys Gly Gln Arg Lys Ala Ala Ala 85 90 95

ATC ACC TCG TAC ATG ATC AAG CAG TCT CTG CCC GCC GTG TCC GAG 505 Ile Thr Ser Tyr Met Ile Lys Gln Ser Leu Pro Ala Val Ser Glu 100 105 110 GTC ACG AAG GAC AAC CTG GAG GAG TTC AAG AAG GCC GAC AAG GCC 550 Val Thr Lys Asp Asn Leu Glu Glu Phe Lys Lys Ala Asp Lys Ala 115 120 125 GTC CTT GTC GCC TAT GTG GAT GCT TCC GAC AAG GCG TCC AGT GAG 595 Val Leu Val Ala Tyr Val Asp Ala Ser Asp Lys Ala Ser Ser Glu 130 135 140 GTT TTC ACC CAG GTC GCC GAG AAG CTG CGC GAC AAC TAC CCG TTC 640 Val Phe Thr Gln Val Ala Glu Lys Leu Arg Asp Asn Tyr Pro Phe 145 150 155 GGC TCC AGC AGC GAT GCT GCG CTG GCC GAG GCT GAG GGC GTC AAG 685 Gly Ser Ser Ser Asp Ala Ala Leu Ala Glu Ala Glu Gly Val Lys 160 165 170 GCT CCC GCT ATC GTC CTT TAC AAG GAC TTT GAT GAG GGC AAG GCG 730 Ala Pro Ala Ile Val Leu Tyr Lys Asp Phe Asp Glu Gly Lys Ala 175 180 185 GTC TTC TCC GAG AAG TTC GAG GTG GAG GCG ATC GAG AAG TTC GCC 775 Val Phe Ser Glu Lys Phe Glu Val Glu Ala Ile Glu Lys Phe Ala 190 195 200 AAG ACG GGC GCC ACC CCG CTC A TT GGC GAG ATT GGC CCC GAA ACC 820 Lys Thr Gly Ala Thr Pro Leu Ile Gly Glu Ile Gly Pro Glu Thr 205 210 215 TAC TCC GAC TAC ATG TCG GCC GGC ATC CCT CTG GCC TAC ATT TTC 865 Tyr Ser Asp Tyr Met Ser Ala Gly Ile Pro Leu Ala Tyr Ile Phe 220 225 230

GCC GAA ACG GCC GAG GAG CGG AAG GAG CTC AGC GAC AAG CTC AAG 910 Ala Glu Thr Ala Glu Glu Arg Lys Glu Leu Ser Asp Lys Leu Lys 235 240 245 CCG ATC GCC GAG GCT CAG CGC GGC GTC ATT AAC TTT GGT ACT ATT 955 Pro Ile Ala Glu Ala Gln Arg Gly Val Ile Asn Phe Gly Thr Ile 250 255 260 GAC GCC AAG GCT TTT GGT GCC CAC GCC GGC AAC CTG AAC CTG AAG 1000 Asp Ala Lys Ala Phe Gly Ala His Ala Gly Asn Leu Asn Leu Lys 265 270 275 ACC GAC AAG TTC CCC GCC TTC GCC ATC CAG GAG GTC GCC AAG AAC 1045 Thr Asp Lys Phe Pro Ala Phe Ala Ile Gln Glu Val Ala Lys Asn 280 285 290 CAG AAG TTC CCC TTC GAT CAG GAG AAG GAG ATC ACC TTC GAG GCG 1090 Gln Lys Phe Pro Phe Asp Gln Glu Lys Glu Ile Thr Phe Glu Ala 295 300 305 ATC AAG GCT TTC GTC GAC GAC TTT GTC GCC GGT AAG ATC GAG CCC 1135 Ile Lys Ala Phe Val Asp Asp Phe Val Ala Gly Lys Ile Glu Pro 310 315 320 AGC ATC AAG TCG GAG CCG ATC CCT GAG AAG CAG GAG GGC CCC GTC 1180 Ser Ile Lys Ser Glu Pro Ile Pro Glu Lys Gln Glu Gly Pro Val 325 330 335 ACC GTC GTC GTT GCC AAG AAC TAC AAT GAG ATC GTC CTG GAC GAC 1225 Thr Val Val Val Ala Lys Asn Tyr Asn Glu Ile Val Leu Asp Asp 340 345 350 ACC AAG GAT GTG CTG ATT GAG TTC TAC GCC CCG TGG TGC GGC CAC 1270 Thr Lys Asp Val Leu Ile Glu Phe Tyr Ala Pro Trp Cys Gly His 355 360 365

TGC AAG GCC CTG GCT CCC AAG TAC GAG GAG CTC GGC GCC CTG TAT 1315 Cys Lys Ala Leu Ala Pro Lys Tyr Glu Glu Leu Gly Ala Leu Tyr 370 375 380 GCC AAG AGC GAG TTC AAG GAC CGG GTC GTC ATC GCC AAG GTT GAT 1360 Ala Lys Ser Glu Phe Lys Asp Arg Val Val Ile Ala Lys Val Asp 385 390 395 GCC ACG GCC AAC GAC GTT CCC GAT GAG ATC CAG GGA TTC CCC ACC 1405 Ala Thr Ala Asn Asp Val Pro Asp Glu Ile Gln Gly Phe Pro Thr 400 405 410 ATC AAG CTG TAC CCG GCC GGT GCC AAG GGT CAG CCC GTC ACC TAC 1450 Ile Lys Leu Tyr Pro Ala Gly Ala Lys Gly Gln Pro Val Thr Tyr 415 420 425 TCT GGC TCG CGC ACT GTC GAG GAC CTC ATC AAG TTC ATC GCC GAG 1495 Ser Gly Ser Arg Thr Val Glu Asp Leu Ile Lys Phe Ile Ala Glu 430 435 440 AAC GGC AAG TAC AAG GCC GCC ATC TCG GAG GAT GCC GAG GAG ACG 1540 Asn Gly Lys Tyr Lys Ala Ala Ile Ser Glu Asp Ala Glu Glu Thr 445 450 455 TCG TCC GCA ACC GAG ACG ACC ACC GAG ACG GCC ACC AAG TCG GAG 1585 Ser Ser Ala Thr Glu Thr Thr Thr Glu Thr Ala Thr Lys Ser Glu 460 465 470 GAG GCT GCC AAG GAG AC G GCG ACG GAG CAC GAC GAG CTC TAG 1627 Glu Ala Ala Lys Glu Thr Ala Thr Glu His Asp Glu Leu *** 475 480 485 AAGACTTGTC GTACATGTAT TTTACGAAAT GCTTTCTTGG GTTTTTCATT 1677 AGGGACCATA GGCACGCGGA TCCAGGGGCCGGTTGGC

SEQ ID NO: 4 Sequence length: 31 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGGAATTC AAR GAY ACN TTY GAY GAY TTY AT 31 Lys Asp Thr Phe Asp Asp Phe Ile 5

SEQ ID NO: 5 Sequence length: 28 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GGGAATTC TC NAC NCC RTG YTG YTG RCA 28 Glu Val Gly His Gln Gln Cys 5

[Brief description of drawings]

FIG. 1 shows the plasmid pNU obtained in Example 1.
The construction procedure of 211-L4-PDI is shown.

FIG. 2 shows an SDS-polyacrylamide gel electrophoresis pattern of a polypeptide having PDI activity according to Example 3.

FIG. 3 shows the substrate specificity of PDI obtained in Example 6. 3-1 shows refolding of s-RNase. 3-2 shows refolding of s-lysozyme. 3-3 shows refolding of s-aprotinin. 3-4 indicates reduction of insulin.

FIG. 4 shows the temperature stability of the mold PDI obtained in Example 6.

FIG. 5 shows the pH stability of the mold PDI obtained in Example 6.

FIG. 6 shows the reactivity of mold PDI obtained in Example 6 in the presence of guanidine hydrochloride.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display area // (C12N 15/09 ZNA C12R 1: 645) (C12N 1/21 C12R 1:08) (C12N 9/90 C12R 1:08) C12R 1: 645) (72) Inventor Tsutomu Kajino, 1st 41st Yokomichi Nagakute-cho, Aichi-gun, Aichi-gun Toyota Central Research Institute Co., Ltd. (72) Inventor Masanai Hirai Aichi 1 in 41 Chuo-dori, Nagakute-machi, Aichi-gun, Toyota Central Research Institute Co., Ltd. (72) Inventor Chie Ikiba 1-41 in Nagakute-machi, Nagakute-cho, Aichi-gun 1 Chuo-dori Research Center, Toyota (72) ) Inventor Osamu Osamu 41, Nagachote, Nagakute-cho, Aichi-gun, Aichi Prefecture, 1st side street, Toyota Central Research Institute Co., Ltd. (72) Inventor Yukio Yamada Nagakute, Aichi-gun Oaza Nagakute-shaped side street No. 41 land of 1 Co., Ltd. Toyota Central Research Institute in (72) inventor cormorant ▲ high ▼ 3 of the heavy three Nagoya, Aichi Prefecture Meito-ku 1-chome address 24-cho Uesono

Claims (4)

[Claims] 1. Bacillus brevi
s ) A recombinant DNA in which a DNA encoding a mold protein disulphide isomerase is bound to the 3'end of a DNA containing a promoter region derived from s ). 2. The recombinant D according to claim 1, wherein the DNA encoding mold protein disulfide isomerase encodes the amino acid sequence shown in SEQ ID NO: 3.
NA. 3. Bacillus brevis carrying the recombinant DNA according to claim 1 or 2. 4. A method for producing mold protein disulfide isomerase, which comprises culturing Bacillus brevis according to claim 3 in a nutrient medium and collecting mold protein disulfide isomerase from the culture.