JPH06245777A - Plasmid and manifesting polypeptide usable with filamentous fungus and yeast and production of polypeptide using the same - Google Patents

Abstract

PURPOSE:To obtain a plasmid having a DNA fragment necessary for manifesting a useful protein or peptide by using filamentous fungi or yeast as a host and to provide production of a useful protein or peptide utilizing the plasmid. CONSTITUTION:A plasmid for manifesting a polypeptide has a promoter functioning with filamentous fungi and yeast, has a terminator of a mono-and a diacylglycreol lipase gene of Penicillium camembertii as a terminator, has a DNA range replicable and selectable with Escherichia coli and is usable with filamentous fungi and yeast. A polypeptide is produced by using the plasmid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plasmid having a DNA fragment necessary for expressing a useful protein or peptide using a filamentous fungus or yeast as a host, and a method for producing a useful protein or peptide using the plasmid. is there.

[0002]

BACKGROUND ART Filamentous fungi secrete a large amount of many useful enzymes such as various hydrolases that hydrolyze starch, proteins, lipids, cellulose, etc. to the outside of the cells. It has been used. Therefore,
The safety of many strains is widely recognized, as it is supported by the old history and research results of fermentation technology such as culture methods for filamentous fungi and methods for separating and purifying product proteins. Moreover, since it is a eukaryote, it has a mechanism of glycosylation of proteins like higher organisms such as mammals.

For these reasons, it was expected that filamentous fungi would be used as a host for high production of heterologous proteins.
In genetic engineering technology, the production amount of a target protein is determined by factors such as promoters, terminators and other factors related to transcription, factors related to translation such as types of amino acid codons, glycosylation to proteins, and intracellular expression of expressed proteins. It is affected by many factors such as posttranslational factors such as the mode of presence (including movement in the secretory process), factors of gene copy number, factors relating to the stability of expressed proteins such as protease derived from the host. The most important and easily controllable of these is the choice of promoter.

From this point of view, examples of utilizing a strong promoter in filamentous fungi include, for example, the promoter of the glucoamylase gene derived from Aspergillus niger [Bi
otechnology, 5, 368 (1987)], promoter of cellobiohydrolase I gene from Trichoderma reesei [Biotechnology, 7, 596 (1989)], promoter of α-amylase gene from Aspergillus oryzae [Biotechnology, 6, 1419 (1988)].

However, Penicillium
No example has been reported so far in which a promoter derived from the genus m was used for high expression of a heterologous protein.

[0006] In addition, the host to be used can usually be used only for a donor of the promoter for one promoter, and an example in which one promoter efficiently functions with a plurality of filamentous fungi as hosts, or further filamentous fungi There was no heterologous protein expression plasmid using a promoter available in both yeast and yeast hosts.

[0007] One of the advantages of such an expression plasmid that can utilize a plurality of filamentous fungi and yeast as hosts is that the host can be arbitrarily selected depending on the kind of the target polypeptide. That is, the production efficiency of the target polypeptide in each host, the stability of the target polypeptide in each host or in the culture solution, and the easy purification of the target polypeptide from each host cell or culture solution. The expression host can be arbitrarily selected in consideration of factors such as sex.

[0008] Another advantage is that a protein engineering research method for elucidating the structure-function relationship of a desired polypeptide and for modifying the function can be performed more efficiently and in a shorter time.

[0009] In general, gene manipulation techniques using filamentous fungi as hosts can be expected to produce large amounts of expression and secretion, but on the other hand, slow growth rate, low transformation frequency, polynuclear, chromosome-integrated transformation, etc. For that reason, it is complicated and takes a long time. On the other hand, gene manipulation technology using yeast as a host generally cannot be expected to produce a large amount of expression, but is simple and short-term because of its fast growth rate, high transformation frequency, and the availability of extranuclear autonomous replicating vectors. Can be done between.

Therefore, site-specific mutations and random mutations were introduced in vitro, the properties of the mutant polypeptide translated from the mutant gene were investigated, and the mutant polypeptide was produced in a large amount, and its higher-order structure was investigated. In the so-called protein engineering research such as determining the value or putting it into practical use, it is possible to rapidly perform it by properly using yeast and filamentous fungi as expression hosts.

In particular, when attempting to introduce random mutations and screen for a desired mutant gene, it is virtually impossible to use only filamentous fungi as a host, the transformation frequency is high, and an extranuclear autonomous replicating vector is used. It is essential to use an available host.

Bilirubin oxidase (hereinafter referred to as BO) is an enzyme classified by the International Union of Biochemistry (IUB) Enzyme Committee as Enzyme No. EC 1.3.3.5. BO is a genus Myrothecium, Trichoderma (Trich
It is an enzyme that has been reported to be produced by microorganisms such as the genus Oderma and the genus Coprinas, and catalyzes a reaction that acts on bilirubin and changes through biliverdin to an almost colorless product. That is, it is an enzyme that acts on bilirubin having an absorption maximum near 460 nm and catalyzes a reaction of converting it into an almost colorless product via biliverdin.In particular, the enzyme derived from the genus Milocesium has a It has been revealed in detail (Japanese Patent Publication No. 60-12032).

BO is widely used as a diagnostic reagent for liver diseases and the like, which is used for quantifying bilirubin in body fluids.
Alternatively, it is also used for eliminating bilirubin in serum in order to solve the interference effect of bilirubin, which is a problem when performing various biochemical tests using serum. Furthermore, application to medicines, detergents, wastewater treatments, etc. is also attempted.

[0014]

Thus, there was a need for a transformation plasmid applicable to both yeast and filamentous fungi as an expression host. Furthermore, there are many points in which the productivity of important enzymes such as BO, which are widely used in the industry at present, should be improved. On the other hand,
At present, the creation of functionally modified BO is highly expected as a way to expand further applications in the future.

The present inventors have found that the promoters of the mono- and diacylglycerol lipase genes of Penicillium camambertii function strongly not only in this strain but also in other filamentous fungi and yeast, and the useful protein And a new expression plasmid was constructed to express the peptide in these hosts.
In addition, the present invention was completed by succeeding in producing useful proteins (for example, BO) and peptides of interest using the expression plasmid by filamentous fungi and yeast.

[0016]

The novel expression plasmid of the present invention has a promoter and terminator that function in filamentous fungi and yeast, and further has a DNA region capable of replication and selection in Escherichia coli.

The promoter may be one that functions in filamentous fungi and yeast. Specifically, the β-glucosidase gene of Aspergillus niger [Mol. Ge
n. Gent., 194, 494 (1984)], Rhizopus nibeus glucoamylase gene [Agric. Biol. Chem., 50, 95.
7 (1986)], aspartic proteinase gene of Rhizopus nibeus [Agric. Biol. Chem., 54, 17]
71 (1990)], mono- and diacylglycerol lipase genes of Penicillium camemberti [Gene, 10
3, 61 (1991)].

More preferably, promoters of mono- and diacylglycerol lipase genes of Penicillium camemberti can be mentioned.

The terminator should not be selected as strictly as the promoter, but specifically, the terminators of the mono- and diacylglycerol lipase genes of Penicillium camemberti are preferably used.

As a more specific example, the plasmid pY1 represented by the restriction map of FIG. 1 can be shown.

In this plasmid, two restriction enzyme recognition sites are provided at the junction between the promoter and the terminator in order to facilitate insertion of the DNA fragment encoding the desired polypeptide to be expressed between the promoter and the terminator. It has been introduced. In addition, it is easy to insert the expression cassette consisting of [promoter-DNA fragment encoding the desired polypeptide-terminator] into the transformation plasmid for the strain selected as the expression host after inserting the desired polypeptide. Therefore, another restriction enzyme recognition site is introduced.

Construction of this plasmid can be carried out, for example, as follows. Site-directed mutagenesis at both ends of the promoter region and terminator region of the gene [Proc. Natl. Acad. Sci. USA, 82, 488 (1985)]
After introducing an appropriate restriction enzyme recognition site by, cut out the promoter region, terminator region with a restriction enzyme,
It can be obtained by isolating it as a DNA fragment and inserting both fragments in the same order as the original gene into an E. coli vector such as pUC19.

The DNA fragments in the promoter region and terminator region can be obtained by PCR [Science, 230, 1350 (198
5)] can be used for amplification.

The present invention also discloses a method for producing enzymes, hormones, physiologically active peptides or proteins using filamentous fungi and yeast as hosts. This is accomplished, for example, by the following steps.

(1) Construction of expression cassette (plasmid pY1
(Insertion of DNA fragment encoding target polypeptide into (2) Construction of transformation-expression plasmid (insertion of expression cassette into transformation vector) (3) Introduction of transformation-expression plasmid into host strain ( 4) Cultivation of transformants The following is a step-by-step description.

(1) Construction of expression cassette Restriction enzyme XbaI site upstream of the start codon of the DNA encoding the polypeptide of interest and restriction enzyme downstream of the stop codon
BamHI sites are introduced by the site-directed mutagenesis method or the PCR method, and the DNA fragments having these restriction enzyme attachment sites at both ends are inserted into the XbaI and BamHI sites of the plasmid pY1. DNA encoding the polypeptide of interest is
The gene or cDNA obtained by cloning or the one obtained by chemical synthesis may be used. Further, even a gene containing an intron can be used as long as it has a sequence in the intron that causes splicing in the host.

(2) Transformation-Construction of Expression Plasmid In order to introduce the expression cassette constructed in (1) into host filamentous fungi or yeast cells, a plasmid having a marker gene necessary for selection of transformants, that is, transformation Insert the expression cassette into the vector. The transformation vector is appropriately selected according to each host. In the case of filamentous fungi, since co-transformation is possible, this step is not always necessary when the filamentous fungus is used as a host.

(3) Introduction of transformation-expression plasmid into host strain Using the transformation-expression plasmid obtained in (2), filamentous fungi [eg Mol. Gen. Gent., 218, 99
(1989)] or yeast [eg J. Bacteriol., 153,
163 (1983)].

When carrying out co-transformation, the DNA fragment of the expression cassette obtained in (1) or a mixture of the plasmid having the expression cassette and the transformation plasmid is used in place of the transformation-expression plasmid.

(4) Cultivation of transformant The transformant obtained in (3) is cultured under an appropriate medium and culture conditions to produce the desired polypeptide. In this case, it is preferable to carry out the culture under the medium and culture conditions in which the promoter functions most. The produced recombinant target polypeptide is qualitatively and quantitatively determined by an appropriate method, and is optionally isolated and purified.

The desired polypeptide may be any polypeptide, and examples thereof include BO derived from Milocesium bergaria MT-1.

The present invention will be described in detail below with reference to examples. In the present specification, unless otherwise stated, the gene manipulation method is in accordance with the publication [eg “Molecular Cloning”, Second Edition, edited by Sunbrook, Frisch, Maniatis, Cold Spring Harbor Laboratories (1989)]. went.

[0033]

【Example】

Example 1 Construction of plasmid pY1 Mono- and diacylglycerol lipases of Penicillium sp. Gene, 2.0-kb
The HindIII fragment was used as an E. coli vector pUC19 [Toyobo, Gen
e, 33, 103 (1985)] and inserted into the HindIII site of plasmids pLGG300 and pLGG100 [Gene, 103, 61 (1991)].
Was used as the material. Both of these plasmids have 2.0-kb HindI
The II fragments are inserted in opposite directions.

The mono- and diacylglycerol lipase genes can be obtained by a method known from Penicillium camemberti [Gene, 103, 61 (1991)].

The promoter region was amplified by PCR using pLGG300 as a template. As an upstream primer for PCR reaction,

M4 ′: 5′-GTTTTCCCAGTCACGACGTTGTA-3 ′ As a downstream primer,

Xba: 5'-AGACGCATGATGTCTAGAGGTCAATGGC-3 '

Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using a Gene AMP ^™ kit (manufactured by Perkin Elmer Japan Co., Ltd.) and a thermal cycler (DNA amplification device) of the same company. The composition of the reaction solution is as follows.

H ₂ O 63.0 μl [10x] Reaction buffer 10.0 μl dNTPs, Mix., 1.25 mM 16.0 μl Upstream primer LGP-a, 20 μM 5.0 μl Downstream primer LGP-b, 20 μM 5.0 μl pLGG300, 2 ng / μl 0.5 μl AmpliTaq ^TM DNA polymerase 0.5 μl

The conditions of the thermal cycler are as follows. 94 ° C. 0.5 minutes 55 ° C. 2.0 minutes 72 ° C. 0.5 minutes After 50 cycles of the reaction under these conditions, the reaction was further incubated at 72 ° C. for 7 minutes.

The reaction solution was subjected to 1.0% low melting point agarose electrophoresis to isolate and purify the amplified DNA fragment of about 0.6-kb. This DNA fragment was treated with restriction enzymes HindIII and XbaI and cloned into pUC19 which was also treated with both enzymes to obtain pPXba.

Next, the terminator region was cloned. After treating pLGG100 with restriction enzymes DraI and HincII, the digestion mixture was subjected to 1.0% low-melting point agarose electrophoresis, and the terminator region of about 0.48-kb DraI / HincII was used.
The fragment was isolated and purified. This DNA fragment was cloned into the SmaI site of pUC9 to obtain pT7.

Next, the promoter and terminator regions were ligated. After treating pT7 with restriction enzymes BamHI and EcoRI, the digestion mixture was subjected to 1.0% low-melting point agarose electrophoresis, and the terminator region of about 0.49-kb BamHI / EcoRI was used.
The fragment was isolated and purified. This DNA fragment was cloned into pPXba which was also treated with the restriction enzymes BamHI and EcoRI to obtain pY1 (FIG. 2).

Example 2 Milocesium bergaria MT-1
Construction of the BO expression cassette of p. P.-BO (Japanese Patent Application No. 4-34126) was used as a material. The cDNA of this BO is Milocesium bergaria MT-1.
It can be obtained by a publicly known method from (Ministry of Industrial Technology Article 653). To facilitate the insertion of this cDNA between the promoter and the terminator of pY1, a restriction enzyme XbaI site was introduced 5 bp upstream of the translation initiation codon using the PCR method. As an upstream primer for PCR reaction,

B-a: 5′-ATTTTCTAGACATCATGTTCAAACACACACTTGG-3 ′ As a downstream primer,

B-c: 5'-CACCTTGAAGTCGACAAGATGGATG-3 '

Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using a Gene AMP ^™ kit (manufactured by Perkin Elmer Japan Co., Ltd.) and a thermal cycler (DNA amplification device) of the same company. The composition of the reaction solution is as follows.

H ₂ O 63.0 μl [10x] Reaction buffer 10.0 μl dNTPs, Mix., 1.25 mM 16.0 μl Upstream primer Ba, 20 μM 5.0 μl Downstream primer Bc, 20 μM 5.0 μl pUC-BO, 2 ng / μl 0.5 μl AmpliTaq ^TM DNA polymerase 0.5 μl

The conditions of the thermal cycler are as follows. 93 ℃ 1.0 min 50 ℃ 1.0 min 72 ℃ 1.0 min After 50 cycles of reaction under these conditions, at 72 ℃
Incubated for 7 minutes.

The reaction solution was subjected to 1.0% low melting point agarose electrophoresis, and the amplified DNA fragment of about 1.4-kb was isolated and purified. This DNA fragment was treated with the restriction enzymes XbaI and SalI and cloned into pUC19 which was also treated with both enzymes.
I got BXS1.

Next, pBXS1 and pUC-BO were treated with restriction enzymes EcoRI and NcoI, and the digestion mixture was subjected to 1.0% low-melting point agarose electrophoresis to obtain about 0.98-kb EcoRI / NcoI derived from pBXS1.
The fragment and an approximately 3.6 kb EcoRI / NcoI fragment derived from pUC-BO were isolated and purified. By ligating the two DNA fragments obtained, a plasmid containing the BO expression cassette
We obtained pBXba1 (Fig. 3).

Further, after treating pBXba1 with a restriction enzyme Xba1, the digestion mixture was subjected to 1.0% low melting point agarose electrophoresis to give about 1.9
The -kb Xba1 fragment was isolated and purified. This DNA fragment is the X of pY1
It was cloned into the baI site to obtain pBC1 (Fig. 3).

Example 3 Milocesium bergaria MT-1
In order to facilitate the introduction of the BO expression cassette into the expression transformation vector of Aspergillus oryzae, the transformation vector pSTA14 for Aspergillus oryzae [Mol. Gen. Genet., 218, 99-104 (198
9)] was modified as follows to prepare pN3 and pN4.

After treating pSTA14 with the restriction enzyme HindIII, the digestion mixture was subjected to 1.0% low-melting point agarose electrophoresis, and niaD
An approximately 5.5-kb HindIII fragment containing the gene was isolated and purified.
This DNA fragment was blunted at both ends using a DNA branching kit (Takara Shuzo), and then subcloned into the restriction enzyme SmaI site of pUC19 to obtain pN3 and pN4 (FIG. 4). Both of these plasmids have an approximately 5.5-kb HindIII fragment inserted in the opposite direction.

Next, in order to construct a transformation-expression plasmid, pBC1 was treated with the restriction enzyme HindIII, and the digestion mixture was treated with
The sample was subjected to 1.0% low-melting point agarose electrophoresis to isolate and purify a BO expression cassette of about 2.9-kb HindIII fragment. This DNA fragment was cloned into the HindIII site of pN3,
BC1 was obtained (Fig. 4).

Next, using this pNBC1, Aspergillus oryzae AO1.1 strain [Mol. Gen. Genet., 218, 99-104 (19
89)] by the method of Uncles et al. [Mol. Gen. Genet., 21
8, 99-104 (1989)]. Randomly picked up 10 transformants (PNB-1 to PNB-10) that had grown in the selection medium, and 100 ml of soybean oil medium (3% soybean oil, 0.5% yeast extract, 0.3% NaNO ₃ , 0.1%
_{K 2 HPO 4, 0.05% KCl} , 0.05% MgSO 4 · 7H 2 O, 0.003% Cu
SO ₄ .5H ₂ O, 0.001% FeSO ₄ .12H ₂ 0) was inoculated and shake-cultured at 30 ° C. for 4 days or 10 days, and the obtained culture was filtered to remove bacterial cells.

The BO activity of the obtained culture broth was measured by the following method, and the results are shown in Table 1.

Method for measuring BO activity In 250 ml of 0.2 M Tris-HCl buffer containing 1 mM of ethylenediaminetetraacetic acid, 5 mg of reagent bilirubin (manufactured by Wako Pure Chemical Industries)
Is dissolved and 2 ml of this is reacted with 0.2 ml of enzyme solution at 37 ° C.
The decrease in nm absorbance is measured.

[0059]

[Table 1]

The strain A-1 is a transformant obtained by using the transformation vector pN3. Cultivation of Milocesium bergaria MT-1 was carried out under optimum conditions for BO production according to Japanese Patent Publication No. 60-12031.

Thus, while the transformant with pN3 hardly produces BO, transformant 1 with pNBC1
It can be seen that 6 out of 0 strains produce a large amount of BO. The most productive strain showed about twice the productivity of the BO-producing bacterium Milocesium bergaria MT-1 so far.

The culture filtrates of these transformants were subjected to SDS-polyacrylamide gel electrophoresis, and it was confirmed that a protein having the same molecular weight as that of BO of Milocesium bergaria MT-1 was secreted. Furthermore, by Western blotting analysis, it was confirmed that this protein reacts with an antibody against BO of Myroscesium bergaria MT-1.

Example 4 Milocesium bergaria MT-1
Expression of BO in Penicillium camambertii In order to construct a transformation-expression plasmid for Penicillium camambertii, pBC1 was digested with the restriction enzyme HindIII.
After treatment with, the digestion mixture was subjected to 1.0% low-melting point agarose electrophoresis to isolate and purify the BO expression cassette of about 2.9-kb HindIII fragment. This DNA fragment was cloned into the HindIII site of the transformation vector pH4 for Penicillium camemberti described below to obtain pHBC1 (FIG. 5).

Construction of transformation plasmid pH1 The plasmids pLGG300 and pLGG100 described in Example 1
Both plasmids of [Gene, 103, 61 (1991)] are about 2.0-k.
b HindIII fragments are inserted in opposite directions. The mono- and diacylglycerol lipase genes are prepared by the method known from Penicillium camembertii [Gene,
103, 61 (1991)].
As the drug resistance marker gene, a plasmid pHph0 (manufactured by Boehringer Mannheim) having a hygromycin B phosphotransferase gene of Escherichia coli was used.

7 from the promoter region and translation initiation codon of the mono- and diacylglycerol lipase genes
A DNA fragment containing up to the amino acid residue was amplified by PCR using pLGG300 as a template. As an upstream primer for PCR reaction,

LGP-a: 5′-CAAGCTTCCGGGAGTAAATTTTC-3 ′ As a downstream primer, LGP-b: 5′-TGTCGACCTGTGAAGAAAGAGAGACGCAT-3 ′

Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using a Gene AMP ^™ kit (manufactured by Perkin Elmer Japan Co., Ltd.) and a thermal cycler (DNA amplification device) of the same company. The composition of the reaction solution is as follows.

H ₂ O 62.5 μl [10x] Reaction buffer 10.0 μl dNTPs, Mix., 1.25 mM 16.0 μl Upstream primer LGP-a, 20 μM 5.0 μl Downstream primer LGP-b, 20 μM 5.0 μl pLGG300, 2 ng / μl 1.0 μl AmpliTaq ^TM DNA polymerase 0.5 μl

The conditions of the thermal cycler are as follows. 93 ℃ 1.0 min 55 ℃ 1.0 min 72 ℃ 1.0 min

After 50 cycles of the reaction under these conditions,
It was further incubated at 72 ° C for 7 minutes.

The reaction solution was subjected to 1.0% low melting point agarose electrophoresis, and the amplified DNA fragment of about 0.6-kb was isolated and purified. This DNA fragment was treated with restriction enzymes HindIII and SalI and cloned into pUC19 which was also treated with both enzymes to obtain pP2.

The terminator region was then cloned downstream of the hygromycin B phosphotransferase gene at pHph0 as follows.

After treating pLGG100 with restriction enzymes DraI and HincII, the digestion mixture was subjected to 1.0% low-melting point agarose gel electrophoresis to obtain a terminator region of about 0.48-kb DraI / HincI.
The I fragment was isolated and purified. This DNA fragment is Sph of pHph0
Cloning into the aI site gave pT6.

Next, a DNA fragment containing the promoter region of the mono- and diacylglycerol lipase genes and the 7th amino acid residue from the translation initiation codon was used as described below to obtain the N-terminal of the protein in the hygromycin B phosphotransferase gene of pT6. It was cloned into the restriction enzyme site present in the coding region.

After treating pP2 with the restriction enzymes HinndIII and SalI, the digestion mixture was subjected to 1.0% low melting point agarose gel electrophoresis to isolate and purify about 0.57-kb HindIII / SalI fragment. This DNA fragment was cloned into pT6 which was also treated with the restriction enzymes HindIII and SalI to obtain pH1.

The pH1 thus obtained has the amino acid sequence from the translation initiation codon of hygromycin B phosphotransferase protein encoded by pHph0 up to the 10th residue from the translation initiation codons of mono- and diacylglycerol lipases. It has a DNA encoding a fusion type hygromycin B phosphotransferase protein in which the amino acid sequence up to the 7th residue is replaced. The promoters and terminators of the penicillium camembertii mono- and diacylglycerol lipase genes are arranged upstream and downstream of the DNA encoding the fused hygromycin B phosphotransferase protein, respectively.

Construction of transformation plasmid pH4 In order to facilitate the insertion of the mono- and diacylglycerol lipase genes into pH1, pH1 was modified to make pH4 as follows. After treating pH1 with the restriction enzyme HindIII, the digestion mixture was subjected to 1.0% low melting point agarose electrophoresis to isolate and purify an approximately 2.2-kb HindIII fragment containing the fusion drug resistance gene.

After blunting both ends of this DNA fragment using a DNA branching kit (Takara Shuzo), pU
By subcloning into the restriction enzyme SmaI site of C19, pH4 was obtained.

In this plasmid, an approximately 2.2-kb HindIII fragment was inserted in the opposite directions. 2 at pH 1
There is one restriction enzyme HindIII site that was present at one site.

[0080]

Next, using this pHBC1, penicillium
Camembertti was transformed by the method described below.

100 μl of a spore suspension of Penicillium camemberti (2 × 10 ⁸ / ml) was inoculated into 100 ml of glucose / peptone medium (manufactured by Eiken Chemical Co., Ltd.), shake-cultured at 30 ° C. for 48 hours, and then cultured. The product was filtered and the bacterial cells were collected. 10 ml of this fungus body
The washed bacterial cells were collected by suspending them in the protoplast-forming buffer [0.8 M NaCl, 10 mM phosphate buffer (pH 6.0)] and filtering. The washed cells were suspended in a protoplast formation buffer containing 5 mg / ml of Rising Enzyme (Sigma Product number L-2265, manufactured by Sigma), and 30
After shaking at ℃ for 2 hours, filter with glass filter 3G2,
The filtrate was subjected to centrifugation at 2000 rpm for 5 minutes to obtain protoplasts as a precipitate.

This precipitate was added to 10 ml of sorbitol solution [1.
2M sorbitol, 50 mM CaCl ₂ , 10 mM Tris-HCl buffer (pH 7.5)] and suspended at 2000 rpm for 5 minutes to collect the precipitate. After repeating this operation again, the precipitate was
The solution was suspended in a sorbitol solution at a concentration of × 10 ⁸ / ml to obtain a protoplast solution. Then 50 μl of protoplast solution
4 μl of pH 1 solution (1 μg / μl) and 6.75 μl of PEG solution [50% PEG4000, 50 mM CaCl ₂ , 10 mM Tris-HCl buffer (pH 7.5)] were added and mixed, and the mixture was allowed to stand on ice for 30 minutes.

Next, 0.5 ml of PEG solution was added and mixed, and
1.0 ml sorbitol solution was added and mixed. This mixture 3
00 μl of PDS containing 17 ml of 1.5% agarose [0.25% soybean oil, 0.25% monoolein, 1.2M sorbitol, 2.4
% Potato dextrose broth (manufactured by Difco)], and 3.0 ml of PDS containing 0.7% agarose preliminarily kept at 48 ° C. was poured to solidify after mixing.

After standing at 30 ° C. for 24 hours, 613 μg / ml hygromycin B (manufactured by Sigma) and P containing 0.7% agarose
DS 3.0 ml was overlaid, left at 30 ° C for 4 days, and 12 colonies (PHB-1 to PHB-10) that appeared on the plate were picked up at random and 100 ml of soybean oil medium (3% large Soybean oil, 0.5% yeast extract, 0.3% NaNO ₃ , 0.1% K ₂ HPO ₄ ,
_{0.05% KCl, 0.05% MgSO 4} · 7H 2 O, 0.003% CuSO 4 · 5H
₂ O, 0.001% FeSO ₄ .12H ₂ 0) and shake-cultured at 30 ° C. for 7 or 10 days, and the obtained culture was filtered to remove bacterial cells.

Table 2 shows the results of measuring the BO activity of the obtained culture broth.

[0087]

[Table 2]

The H-1 strain is a transformant obtained by using the transformation vector pH4. Thus, it can be seen that the transformants with pH4 hardly produce bilirubin oxidase, whereas most of the 12 transformants with pHBC1 produce clearly large amounts of bilirubin oxidase. The most productive strain showed about 5.4 times the productivity of the conventional bilirubin oxidase-producing bacterium Milosecium bergaria MT-1.

The culture filtrates of these transformants were subjected to SDS-polyacrylamide gel electrophoresis, and it was confirmed that a protein having the same molecular weight as that of bilirubin oxidase of Milocesium bergaria MT-1 was secreted. did. Furthermore, by Western blotting analysis,
It was confirmed that this protein reacts with the antibody against bilirubin oxidase of Milocesium velcaria MT-1.

Example 5 Milocesium bergaria MT-1
Expression of BO in yeast Saccharomyces cerevisiae Transformation into yeast Saccharomyces cerevisiae-
To construct an expression plasmid, pBC1 was digested with HindII restriction enzyme.
After treatment with I, the digestion mixture was subjected to 1.0% low melting point agarose electrophoresis to isolate and purify a BO expression cassette of about 2.9-kb HindIII fragment. This DNA fragment was used as a transformation vector pL1 for Saccharomyces cerevisiae [Biosci.
Biotech. Biochem., 56, 315-319 (1992)] HindIII
It was cloned into the site to obtain pLBC2 (Fig. 6).

Next, using this pLBC2, Saccharomyces cerevisiae SHY2 (ATCC 44770) was transformed by the method of Ito et al. [J. Bacteriol., 153, 163-168 (1983)]. Pick up one transformant that has grown on the selection medium, and use the minimum medium containing 20 mg / ml uracil, 20 mg / ml tryptophan, and 20 mg / ml histidine [2% glucose, 0.67% Yeast Nitrogenn Base w / o amino acid
(Difco)] 50 ml and inoculated with shaking at 30 ° C. for 24 hours. 1 ml of the obtained culture solution was added to YPGal medium (2% galactose, 2% polypeptone, 1%) containing 0.003% CuSO ₄ .5H ₂ O.
% Yeast extract), the mixture was cultivated with shaking at 30 ° C. for 72 hours, and the resulting culture was centrifuged to remove bacterial cells.

The BO activity of the obtained culture broth was measured. As a result, in the case of a transformant having the plasmid pLBC2,
It was 0.032 U / ml. As a control, the Saccharomyces cerevisiae SHY2 strain having pL1 was cultured in the same manner as above, and the BO activity was measured and found to be 0.004 U / ml.

Thus, it can be seen that the transformant with pL1 hardly produces BO, whereas the transformant with pLBC2 clearly produces BO. Also, when Western blotting analysis was performed on the culture filtrate of this transformant, a band was detected at a position at which this protein had a molecular weight similar to that of BO of Milosecium velcaria MT-1.

[0094]

Industrial Applicability According to the present invention, industrially useful enzymes, hormones, physiologically active peptides or proteins can be produced in large quantities by filamentous fungi. At the same time, it can be expressed in yeast, and it becomes easy to apply a protein engineering method for the purpose of creating a functionally modified polypeptide, which is a great contribution to the industry.

[Brief description of drawings]

FIG. 1 shows a restriction enzyme map of plasmid pY1.

FIG. 2 shows the procedure for constructing plasmid pY1.

FIG. 3 shows the construction procedure of plasmid pBC1.

FIG. 4 shows the procedure for constructing plasmid pNBC1.

FIG. 5 shows the procedure for constructing plasmid pHBC1.

FIG. 6 shows the procedure for constructing plasmid pLBC2.

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[Procedure amendment]

[Submission date] January 25, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0091

[Correction method] Change

[Correction content]

Next, using this pLBC2, Saccharomyces cerevisiae SHY2 (ATCC 44770)
By the method of J. Ito [J. Bacteriol. , 15
3, 163-168 (1983)]. Pick one transformant that has grown on the selection medium. Up, minimum medium [2 containing uracil 20 mg / L, tryptophan 20 mg / L, histidine 20 mg / L
% Glucose, 0.67% Yeast Nitroge
nn Base w / o amino acid (Di
fco)] 50 ml and inoculated with shaking at 30 ° C. for 24 hours. 1 ml of the obtained culture solution was added to 0.003% C
YPGal medium containing _{uSO 4 · 5H 2 O (2} % galactose, 2% polypeptone, 1% yeast extract) 50 ml
The cells were inoculated with the strain and cultured at 30 ° C. for 48 hours with shaking.

Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area (C12N 9/06 C12R 1:80) (C12N 9/06 C12R 1: 865)

Claims (6)

[Claims] 1. A filamentous filament having a promoter that functions in filamentous fungi and yeast, having as a terminator the terminators of the mono- and diacylglycerol lipase genes of Penicillium camembertii, and having a DNA region capable of replication and selection in Escherichia coli. A plasmid for expressing a polypeptide that can be used in fungi and yeast. 2. The plasmid according to claim 1, wherein the promoter is a promoter of the mono- and diacylglycerol lipase genes of Penicillium camembertii. 3. The plasmid according to claim 1, wherein the terminator is a terminator of Penicillium camembertiy mono- and diacylglycerol lipase genes. 4. The plasmid according to claim 1, which has a DNA encoding a polypeptide between a promoter and a terminator. 5. The plasmid according to claim 4, wherein the polypeptide is bilirubin oxidase. 6. A method for producing a polypeptide, which comprises introducing the plasmid according to claim 4 into a filamentous fungus or yeast using a transformation vector and culturing the obtained transformant.