JP3556965B2 - Plasmid for polypeptide secretion and expression usable in filamentous fungi and yeast and method for producing polypeptide using the same - Google Patents

Description

[0001]
[Industrial applications]
The present invention relates to a plasmid having a DNA fragment necessary for secretory expression of useful proteins and peptides using a filamentous fungus or yeast as a host, and a method for producing useful proteins and peptides using the same.
[0002]
[Prior art]
In genetic engineering technology, the production amount of the target protein is determined by factors related to transcription such as promoter and terminator, factors related to translation such as amino acid codon type, addition of sugar chain to protein, and intracellular expression of expressed protein. It is influenced by many factors, such as factors involved in post-translation such as the mode of existence (including movement in secretory processes), factors relating to the copy number of genes, and factors relating to the stability of expressed proteins such as host-derived proteases.
[0003]
Examples of using a promoter in a filamentous fungus for high expression of a heterologous protein include promoters of a glucoamylase gene derived from Aspergillus niger (Biotechnology, 5, 369 (1987)) and a promoter of a cellobiohydrolase I gene derived from Trichoderma reesei ( Biotechnology, 7, 596 (1989)) and the promoter of the alpha-amylase gene derived from Aspergillus oryzae (Biotechnology, 6, 1419 (1988)). However, there has been no example in which a promoter derived from the genus Penicillium was used for high expression of a heterologous protein.
[0004]
In addition, the host to be used is usually one in which only a donor of the promoter can be used for one promoter, and an example in which one promoter functions efficiently with a plurality of filamentous fungi as a host, or both a filamentous fungus and yeast. There was no plasmid available for expression of the heterologous protein using the promoter available in the host.
[0005]
Furthermore, when a heterologous protein is produced using genetic engineering technology, secretion of the heterologous protein outside the cells is also an important factor in order to facilitate downstream processes such as isolation and purification. A method of introducing a signal peptide of a secretory protein derived from a host organism to be used or a signal peptide that functions in the organism upstream of a target polypeptide is used, or fusion of a secretory protein and a target polypeptide. A method of expressing the protein is also used.
[0006]
However, examples using Aspergillus nidulans as a host and Aspergillus niger-derived glucoamylase as a signal required for secretion (Biotechnology, 5, 369 (1987), Biotechnology, 5, 713 (1987)), Aspergillus awamori as a host, Example using glucoamylase derived from Aspergillus awamori as a signal required for secretion (Biotechnology, 8, 435 (1990)), Trichoderma reesei as a host, and cellobiohydrolase I derived from Trichoderma reesei as a signal required for secretion. There is only an example used (Biotechnology, 7, 596 (1989)), and there is a fraction usable in a plurality of filamentous fungi and yeasts of different genera. Example of success in the secretion expression of a heterologous protein by using the signals necessary for did not exist so far.
[0007]
[Problems to be solved by the invention]
Thus, if a transformation plasmid that can be applied to both filamentous fungi and yeast as an expression host and that can promote the secretion of the expressed heterologous protein outside the cells can be created, the host Can be used properly, thereby enabling efficient production of many physiologically active substances which are present only in trace amounts in nature, and further promoting the application of these substances to pharmaceuticals.
[0008]
[Means for Solving the Problems]
The present inventors have found that the promoters and signals required for secretion of the mono- and diacylglycerol lipase genes of Penicillium camembertii function not only in the present strain but also in other filamentous fungi and yeasts. Thus, while constructing a novel expression plasmid for secretory expression in a plurality of filamentous fungi and yeast, and using the secretion expression plasmid to successfully secrete and produce desired useful proteins and peptides by the filamentous fungus and yeast, The present invention has been completed.
[0009]
The novel secretory expression plasmid of the present invention has a promoter that functions in filamentous fungi and yeast, a signal necessary for secretion, a terminator, and a DNA region that can be replicated and selected in Escherichia coli.
[0010]
The promoter may be any as long as it functions in filamentous fungi and yeast, and specifically, the β-glucosidase gene of Aspergillus niger (Mol. Gen. Gent., 194, 494 (1984)), Rhizopus Niveus glucoamylase gene (Agric. Biol. Chem., 50, 957 (1986)), Rhizopus niveus aspartic proteinase gene (Agric. Biol. Chem., 54, 1771 (1990)), Penicillium camembergii Mono- and diacylglycerol lipase genes (Gene, 103, 61 (1991)).
[0011]
More preferably, the promoters of the mono- and diacylglycerol lipase genes of Penicillium camembergii are mentioned.
[0012]
The signal required for secretion may be any signal as long as it functions in filamentous fungi and yeast. Specifically, the prepro region, prepro and subsequent N-terminal region, prepro region of mono and diacylglycerol lipase of Penicillium camembertii And the coding region of the mature protein or the prepro region and the coding region of the mature protein with one amino acid residue deleted at the C-terminus.
[0013]
The terminator should not be selected as strictly as the promoter, but specific examples include terminators of the mono- and diacylglycerol lipase genes of Penicillium camambertii.
[0014]
And the novel secretion expression plasmid of the present invention is also provided with the following conditions.
[0015]
(1) In order to facilitate the insertion of a DNA fragment encoding the desired polypeptide to be expressed between the signal required for secretion and the terminator, three restrictions are required at the junction between the signal required for secretion and the terminator. Enzyme recognition site has been introduced.
[0016]
(2) An expression cassette consisting of [promoter-signal necessary for secretion-DNA fragment encoding precursor or mature form of target polypeptide-terminator] after insertion of the target polypeptide is constructed and expressed. Another restriction site has been introduced to facilitate insertion of the cassette into the transformation plasmid for the strain selected as the expression host.
[0017]
(3) It is possible to construct an expression cassette having a signal necessary for secretion of an arbitrary length using an appropriate restriction enzyme site in the coding region of mono- and diacylglycerol lipase. In this case, the signal necessary for secretion of an arbitrary length includes, for example, a prepro region, a prepro and N-terminal region, a prepro and a mature region, and the like.
[0018]
(4) The amino acid sequence at the junction between the signal required for secretion and the precursor or mature form of the target polypeptide may be an artificial amino acid sequence due to the joining of DNA fragments, or may be Lys-Arg or the like. May be introduced intentionally, or the Lys-Arg sequence exists at the third and second positions from the C-terminal of mono- and diacylglycerol lipase, and processing may occur at this position. Since it is known, it is also possible to use this sequence at the junction between the signal required for secretion and the precursor or mature form of the target polypeptide.
[0019]
The construction of the novel secretory expression plasmid of the present invention can be performed, for example, as follows.
[0020]
Appropriate restriction enzyme recognition sites at both ends of the promoter, coding region and terminator region of the gene by site-directed mutagenesis (Proc. Natl. Acad. Sci. USA, 82, 488 (1985)). After the introduction, the promoter, coding region and terminator region are cut out with restriction enzymes, isolated as DNA fragments, and both fragments are inserted into an E. coli vector, for example, pUC19, in the same order as the original gene.
[0021]
DNA fragments of the promoter, the coding region, and the terminator region can be amplified by the PCR method (Science, 230, 1350 (1985)).
[0022]
The present invention further discloses a method for producing enzymes, hormones, bioactive peptides or proteins using filamentous fungi and yeast as hosts, and can be achieved, for example, by the following steps.
[0023]
(1) Construction of an expression cassette (insertion of a DNA fragment encoding the precursor or mature form of the target polypeptide into plasmid pY4 ')
(2) Transformation-construction of expression plasmid (insertion of expression cassette into transformation vector)
(3) Transformation-introduction of the expression plasmid into the host strain
(4) Culture of transformant
The description will be given in order below.
[0024]
(1) Construction of an expression cassette (insertion of a DNA fragment encoding the precursor or mature form of the target polypeptide into plasmid pY4 ')
Appropriate restriction enzyme sites are introduced upstream of the N-terminal amino acid codon of the DNA encoding the precursor or mature form of the target polypeptide and downstream of the stop codon by site-directed mutagenesis or PCR, respectively. Is inserted into an appropriate restriction enzyme site of plasmid pY4 '.
[0025]
In this case, the amino acid sequence selected as a signal necessary for secretion, ie, the amino acid sequence of the prepro region, prepro and N-terminal region or prepro region of mono and diacylglycerol lipase and the mature form, and the precursor or maturation of the polypeptide to be inserted The amino acid sequence of the body is noted to match the reading frame.
[0026]
This is possible by making full use of primer design and site-specific mutation at the time of PCR, and the DNA encoding the target polypeptide precursor or mature form may be a gene or cDNA obtained by cloning. Alternatively, a gene obtained by chemical synthesis may be used, and a gene containing an intron may be used as long as the intron has a sequence that causes splicing in a host.
[0027]
(2) Transformation-construction of expression plasmid (insertion of expression cassette into transformation vector)
In order to introduce the expression cassette constructed in (1) into a host filamentous fungus or yeast cells, the expression cassette is inserted into a plasmid having a marker gene necessary for selecting a transformant, that is, a transformation vector. A transformation vector is appropriately selected according to each host. In the case of a filamentous fungus, co-transformation is possible, so this step is not necessarily required when the filamentous fungus is used as a host.
[0028]
(3) Transformation-introduction of the expression plasmid into the host strain
Using the transformation-expression plasmid obtained in (2), a filamentous fungus (for example, Mol. Gen. Gent., 218, 99 (1989)) or a yeast (for example, J. Bacteriol., 153, 163) by a known method. (1983)). When performing co-transformation, a mixture of the plasmid having the expression cassette DNA fragment or the expression cassette obtained in (1) and the transformation plasmid is used instead of the transformation-expression plasmid.
[0029]
(4) Culture of transformant
The transformant obtained in (3) is cultured in an appropriate medium and culture conditions to produce the desired polypeptide. In this case, it is preferable to carry out the culture in a medium / culture condition in which the promoter functions most. The produced recombinant polypeptide of interest is qualitatively and quantitatively determined by an appropriate method, and is isolated and purified as necessary.
[0030]
The polypeptide of interest may be of any type, and examples include zarpesin derived from Sarcophaga peregrina.
[0031]
Hereinafter, the present invention will be described in detail with reference to Examples. In this specification, unless otherwise specified, the genetic manipulation method is a written book (for example, “Molecular Cloning”, 2nd edition, edited by Sambrook, Frisch, Maniacis, Cold Spring Harbor Laboratories, (1989)). Was performed according to
[0032]
【Example】
Example 1
Plasmid pY4 ' Building
Plasmids pLGG100 and pLGG300 (pLGG300 (pLGG300) in which a 2.0-kb HindIII fragment of the mono- and diacylglycerol lipase gene of Penicillium camellumii U-150 strain were inserted into the HindIII site of Escherichia coli vector pUC19 (Toyobo, Gene, 33, 103 (1985)). Both plasmids used a 2.0-kb HindIII fragment inserted in the opposite direction) and an approximately 1.0-kb cDNA fragment (Gene, 103, 61 (1991)).
[0033]
The mono- and diacylglycerol lipase genes and cDNAs were identified as Penicillium camembertii U-150 strain (Penicillium sp. U-150, FERM P-10452. This strain was later identified as Penicillium camembertii). (Appl. Microbiol. Biotechnol., 34, 720 [1991])) and a known method (Gene, 103, 61 (1991)).
[0034]
First, the intron-free mono- and diacylglycerol lipase genes were constructed. pLGG300 was partially digested with restriction enzymes NcoI and XhoI, and the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to remove the region containing two introns, the 0.73-kb XhoI / NcoI fragment. The kb XhoI / NcoI fragment was isolated and purified. This DNA fragment was ligated with a 0.63-kb XhoI / NcoI fragment derived from cDNA to obtain pLGR80 (FIG. 1).
[0035]
The DNA fragment containing the promoter and the coding region was amplified by PCR using pLGR80 as a template. As an upstream primer for a PCR reaction,
M4 ': 5'-GTTTTCCCAGTCACGACGTTGTA-3'
As a downstream primer,
Sal: 5'-GCTTGATCTAAAATTGTCGACCCTCTTGAATG-3 '
Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using Gene Amp^TM  kit (manufactured by PerkinElmer Japan) using a thermal cycler (DNA amplification device) of the company. The composition of the reaction solution is as follows.
[0036]
H₂O 63.0 μl
[10x] Reaction buffer 10.0 μl
dNTPs, Mix. , 1.25 mM 16.0 μl
Upstream primer M4 ', 20 μM 5.0 μl
Downstream primer Sal, 20 μM 5.0 μl
pLGG300, 2 ng / μl 0.5 μl
AmpliTaq^TMDNA polymerase 0.5 μl
The conditions of the thermal cycler are as follows.
[0037]
0.5 minutes at 94 ° C
55 ° C 2.0 minutes
72 ° C 0.5 minutes
After 50 cycles of the reaction under these conditions, the mixture 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 1.6-kb was isolated and purified. This DNA fragment was treated with restriction enzymes HindIII and SalI, and cloned into pUC19 also treated with both enzymes to obtain pPMsal.
[0038]
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 to isolate and purify an approximately 0.48-kb DraI / HincII fragment, which is a terminator region. This DNA fragment was cloned into the SmaI site of pUC9 to obtain pT7.
[0039]
Further, the promoter and coding region were linked to the terminator region. After treating pT7 with the restriction enzymes BamHI and EcoRI, the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis, and the about 0.49-kb BamHI / EcoRI fragment, which is a terminator region, was isolated and purified. This DNA fragment was cloned into an approximately 4.3-kb EcoRI / BamHI fragment obtained by partially digesting pPMSal with the restriction enzymes BamHI and EcoRI to obtain pY4 '(FIG. 2).
[0040]
Example 2
Zapescin expression cassette from Sarcoferga peregrina pS2 Building
The Zapesin gene can be obtained by culturing NIH-Sap-4 cells, which are a cell line derived from the fly fly embryo, by a known method (J. Biol. Chem., 263, 17117-17121 (1988)). It is. In order to insert the DNA encoding the zapesin precursor between the preprocoding regions of the penicillium camembertii mono- and diacylglycerol lipases of pY4 'and the terminator, a DNA fragment was prepared by PCR. As an upstream primer for a PCR reaction,
S2-a: 5'-ATCCGTCGACTCGCCTGCTGCAGCTGCTTGA-3 '
As a downstream primer,
Sb: 5'-AATAGGATCCCTTAATTGCGACATACGCAG-3 '
Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using Gene Amp^TM  kit (manufactured by PerkinElmer Japan) using a thermal cycler (DNA amplification device) of the company. The composition of the reaction solution is as follows.
[0041]
H₂O 62.5 μl
[10x] Reaction buffer 10.0 μl
dNTPs, Mix. , 1.25 mM 16.0 μl
Upstream primer S2-a, 20 μM 5.0 μl
Downstream primer S-b, 20 μM 5.0 μl
Template, 1 ng / μl 1.0 μl
AmpliTaq^TMDNA polymerase 0.5 μl
The conditions of the thermal cycler are as follows.
[0042]
94 ° C 1.0 min
45 ° C 2.0 minutes
2.0 seconds at 72 ° C
After 25 cycles of the reaction under these conditions, the mixture 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 0.22-kb was isolated and purified.
[0043]
Next, this DNA fragment was treated with the restriction enzymes SalI and BamHI, and pY4 ′ was partially digested with the restriction enzymes XhoI and BamHI, and then the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a PCR product. A certain about 0.22-kb SalI / BamHI fragment and a about 3.8-kb XhoI / BamHI fragment derived from pY4 ′ were isolated and purified. By ligating the two obtained DNA fragments, a plasmid pS2 'was obtained.
[0044]
Further, the deletion of an unnecessary base (cytosine) found at the junction between the prepro coding region of mono- and diacylglycerol lipase and the coding region of the precursor zapesin in pS2 ′ was carried out by using Mutan-K (Takara Shuzo). The plasmid pS2 was obtained by a specific mutagenesis method (FIG. 3).
[0045]
Example 3
Zapescin expression cassette from Sarcoferga peregrina pS3 Building
To insert the DNA encoding the zapesin precursor between the prepro- and N-terminal coding regions of the mono- and diacylglycerol lipases of Penicillium camembertii in pY4 'and the terminator, a DNA fragment was prepared by PCR. As an upstream primer for a PCR reaction,
S3-a: 5'-ATCCGAATTCTCGCCTGCTGCAGCTGCTTGA-3 '
As a downstream primer, Sb was synthesized in the same manner as in Example 2, and a PCR reaction was performed. The reaction solution was subjected to 1.0% low melting point agarose electrophoresis, and the amplified DNA fragment of about 0.22-kb was isolated and purified.
[0046]
Next, this DNA fragment was treated with restriction enzymes EcoRI and BamHI, and pY4 ′ was partially digested with restriction enzymes EcoRI and BamHI. The digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a PCR product. A certain about 0.22-kb EcoRI / BamHI fragment and a about 3.9-kb EcoRI / BamHI fragment derived from pY4 ′ were isolated and purified. The resulting two DNA fragments were ligated to obtain a plasmid pS3 (FIG. 4).
[0047]
Example 4
Zapescin expression cassette from Sarcoferga peregrina pS4 Building
In order to insert the DNA encoding the zapesin precursor between the prepro and mature protein coding regions of penicillium camembertii mono- and diacylglycerol lipases of pY4 'and the terminator, the DNA fragment was obtained by PCR method as in Example 2. Was prepared.
[0048]
Next, this DNA fragment and pY4 ′ were treated with restriction enzymes SalI and BamHI, and the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a PCR product of about 0.22-kb SalI / BamHI fragment and An approximately 4.7-kb SalI / BamHI fragment derived from pY4 ′ was isolated and purified. The plasmid pS4 was obtained by ligating the two obtained DNA fragments (FIG. 5).
[0049]
Example 5
Zapescin expression cassette from Sarcoferga peregrina pS5 Building
In order to insert the DNA encoding mature zapesin between the prepro- and mature protein coding regions of penicillium camembertii mono- and diacylglycerol lipase of pY4 'and the terminator, its DNA fragment was prepared by PCR. As an upstream primer for a PCR reaction,
S5-a: 5'-CTACGTCGACGCCCACTTGCGATTATTATTGAG-3 '
As a downstream primer, Sb was synthesized in the same manner as in Example 2, and a PCR reaction was performed. The reaction solution was subjected to 1.0% low-melting point agarose electrophoresis, and the amplified DNA fragment of about 0.13-kb was isolated and purified.
[0050]
Next, this DNA fragment and pY4 ′ were treated with restriction enzymes SalI and BamHI, and the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a PCR product of about 0.13-kb SalI / BamHI fragment and An approximately 4.7-kb SalI / BamHI fragment derived from pY4 ′ was isolated and purified. The resulting two DNA fragments were ligated to obtain a plasmid pS5 '.
[0051]
Further, the deletion of an unnecessary base (5′-GTCGAC-3 ′) found at the junction of the coding region of mature mono- and diacylglycerol lipase and the coding region of mature zapesin in pS5 ′ was carried out in the same manner as in Example 2. Plasmid pS5 was obtained by site-directed mutagenesis (FIG. 6).
[0052]
Example 6
▲ 1 ▼Transformation plasmid pH1 Building
Both plasmids pLGG300 and pLGG100 [Gene, 103, 61 (1991)] described in Example 1 have about 2.0-kb HindIII fragments inserted in opposite directions. These mono- and diacylglycerol lipase genes can be obtained by a method known from Penicillium camembertii [Gene, 103, 61 (1991)]. As a drug resistance marker gene, a plasmid pHph0 (manufactured by Boehringer Mannheim) having a hygromycin B phosphotransferase gene of Escherichia coli was used.
[0053]
DNA fragments including the promoter region of the mono- and diacylglycerol lipase genes and the 7th amino acid residue from the translation initiation codon were amplified by PCR using pLGG300 as a template. As an upstream primer for a PCR reaction,
LGP-a: 5'-CAAGCTTCCGGGAGTAAATTTTC-3 '
As a downstream primer,
LGP-b: 5'-TGTCGACCTGTGAAGAAGAGAGAGACGCAT-3 '
Was synthesized using a cyclone plus DNA synthesizer (Milligen Biosearch). The PCR reaction was performed using Gene Amp^TM  kit (manufactured by PerkinElmer Japan) using a thermal cycler (DNA amplification device) of the company. The composition of the reaction solution is as follows.
[0054]
H₂O 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^TMDNA polymerase 0.5 μl
The conditions of the thermal cycler are as follows.
[0055]
93 ° C 1.0 min
55 ° C 1.0 min
72 ° C 1.0 minutes
After 50 cycles of the reaction under these conditions, the mixture 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 singly used. Separated 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.
[0056]
Next, the terminator region was cloned downstream of the hygromycin B phosphotransferase gene at pHph0 as follows. After treating pLGG100 with restriction enzymes DraI and HincII, the digested mixture was subjected to 1.0% low melting point agarose electrophoresis to isolate and purify about 0.48-kb DraI / HincII fragment which is a terminator region. The fragment was cloned into the SmaI site of pHph0 to give pT6.
[0057]
Furthermore, a DNA fragment containing the promoter region of the mono- and diacylglycerol lipase gene and the seventh amino acid residue from the translation initiation codon is present in the N-terminal coding region of the protein in the hygromycin B phosphotransferase gene of pT6 as follows. Cloned into a restriction enzyme site. After treating pP2 with restriction enzymes HindIII and SalI, the digestion mixture was subjected to 1.0% low melting point agarose electrophoresis to isolate and purify an approximately 0.57-kb HindIII / SalI fragment, and to subject this DNA fragment to the same restriction. Cloning into pT6 treated with the enzymes HindIII and SalI gave pH1.
[0058]
The thus-obtained pH1 has the amino acid sequence from the translation initiation codon of the hygromycin B phosphotransferase protein encoded by pHph0 to the tenth residue from the translation initiation codon of the mono- and diacylglycerol lipase, seven residues from the translation initiation codon. It has a DNA encoding a fusion type hygromycin B phosphotransferase protein that has been replaced with the amino acid sequence up to the eye. The promoter and terminator 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.
[0059]
▲ 2 ▼Transformation plasmid pH 4 Building
To facilitate insertion of the mono- and diacylglycerol lipase genes into pH1, pH1 was modified as follows to produce pH4. 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. Both ends were blunt-ended using a DNA branching kit (manufactured by Takara Shuzo Co., Ltd.), and then subcloned into the restriction enzyme SmaI site of pUC19 to obtain pH4.
[0060]
In this plasmid, an approximately 2.2-kb HindIII fragment is inserted in the opposite direction to each other, and one restriction enzyme HindIII site existing at pH 1 is one site.
[0061]
▲ 3 ▼Expression of Zarpesin from Sarcoferga peregrina in Penicillium camembergii
In order to construct a transformation-expression plasmid for Penicillium camembergii, pS2, pS3, pS4 and pS5 were treated with the restriction enzyme HindIII, and the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a Zapescin expression cassette. About 1.3, 1.4, 2.2, 2.1-kb HindIII fragments were isolated and purified, respectively. These DNA fragments were cloned into the HindIII site of the transformation vector pH4 for Penicillium camembergii to obtain pSH2, pSH3, pSH4 and pSH5 (FIG. 7).
[0062]
Using these pSH2, pSH3, pSH4, and pSH5, Penicillium camembergii was transformed by the method described below.
[0063]
Penicillium camembertii spore suspension (2 × 10⁸/ Ml) was inoculated into 100 ml of glucose-peptone medium (manufactured by Eiken Chemical Co., Ltd.) and cultured with shaking at 30 ° C. for 48 hours. The culture was filtered to collect the cells. The cells were suspended in 10 ml of a protoplast buffer [0.8 M NaCl, 10 mM phosphate buffer (pH 6.0)] and filtered to collect washed cells. The washed cells were suspended in a protoplast buffer containing 5 mg / ml of a rising enzyme (Sigma Product number L-2265, manufactured by Sigma), shaken at 30 ° C. for 2 hours, and filtered through a glass filter 3G2. The filtrate was subjected to centrifugation at 2,000 rpm for 5 minutes to obtain protoplasts as a precipitate, and the precipitate was subjected to 10 ml of a sorbitol solution [1.2 M sorbitol, 50 mM CaCl 2₂, 10 mM Tris-HCl buffer (pH 7.5)], and centrifuged at 2,000 rpm for 5 minutes to collect a precipitate. After repeating this operation again, the precipitate was⁸/ Ml in a sorbitol solution to obtain a protoplast solution.
[0064]
Next, 4 μl of DNA solution (1 μg / μl), 6.75 μl of PEG solution: 50% PEG 4000, 50 mM CaCl were added to 50 μl of protoplast solution.₂, 10 mM Tris-HCl buffer (pH 7.5)], left to stand on ice for 30 minutes, added and mixed with 0.5 ml of PEG solution, and further added and mixed with 1.0 ml of sorbitol solution. 300 μl of this mixture was mixed with 17 ml of 1.5% agarose-containing PDS [0.25 / soy oil, 0.25% monoolein, 1.2 M sorbitol, 2.4% potato dextrose broth (manufactured by Difco) ], And 3.0 ml of PDS containing 0.7% agarose, which had been kept at 48 ° C in advance, was poured and solidified after mixing. After standing at 30 ° C. for 24 hours, 3.0 ml of PDS containing 613 μg / ml hygromycin B (manufactured by Sigma) and 0.7% agarose were overlaid, left at 30 ° C. for 4 days, and colonies appearing on the plate 10 strains were randomly picked up and 100 ml of a soybean oil medium (3% soybean oil, 0.5% yeast extract, 0.3% NaNO₃, 0.1% K₂HPO₄, 0.05% KCl, 0.05% MgSO₄・ 7H₂O, 0.003% CuSO₄・ 5H₂O, 0.001% FeSO₄・ 12H₂O), and cultured with shaking at 30 ° C. for 6 days. The resulting culture was filtered to remove the cells.
[0065]
The resulting culture was subjected to Western blotting analysis using a rabbit anti-serpescin antibody, and the strains SH2-9, SH3-2, and SH4, which had the highest zapesin productivity among the pSH2, pSH3, pSH4, and pSH5 transformants, respectively, were obtained. 5 shows the results of Western blotting of the SH5-3 culture solution (FIG. 8).
[0066]
Here, the H-1 strain is a transformant obtained by using the transformation vector pH4, and secretion of a protein having the same antigenicity and molecular weight as serpesin is not observed in the H-1 culture solution. However, in the SH2-9, SH3-2, SH4-5, and SH5-3 cultures, secretion of a protein having the same antigenicity and molecular weight as zapesin was observed.
[0067]
These were thought to be penicillium camembertii expressed zapesins, and their productivity was estimated to be about 0.5, 5, 10, and 7 mg / l, respectively. The highest production of zapesin was observed when a gene encoding a zapesin precursor was inserted between the terminator and the prepro- and mature protein coding regions of mono- and diacylglycerol lipases of Penicillium camembergii.
[0068]
SH4-5 is obtained by introducing a gene in which a DNA fragment encoding a precursor zapesin is linked downstream of a prepro region of mono- and diacylglycerol lipase and a DNA encoding a mature protein, and the produced zapesin is the original zapesin. It is considered that the fragment was cleaved at the processing site.
[0069]
On the other hand, for SH5-3, a gene in which a DNA fragment encoding a mature zapesin was ligated downstream of a DNA encoding a mature protein in which the prepro regions of mono- and diacylglycerol lipases and one C-terminal amino acid residue had been deleted was introduced. It is presumed that the produced zapesin was processed at the Lys-Arg site present at the junction of the fusion protein.
[0070]
In the culture medium of SH4-5 and SH5-3, secretion of a protein having a molecular weight corresponding to a fusion protein of mono- and diacylglycerol lipase and zapesin, which seems to remain unprocessed at about 46 and 43 kDa, respectively, was observed. Was seen.
[0071]
Example 7
Purification of recombinant zapesin and determination of its antibacterial activity
First, 200 ml of the SH4-5 strain culture was treated by ion exchange chromatography using a CM cellulose column according to the method of JP-A-4-335883, and the antibacterial activity of each of the obtained fractions against Staphylococcus aureus was determined. It was measured. Next, the active fraction was purified by adsorption, separation and fractionation using HPLC reverse phase chromatography according to the method of JP-A-63-185997 to obtain about 1.4 mg of a purified sample. (About 7 mg / l). The obtained sample was subjected to SDS polyacrylamide electrophoresis, and the gel was stained with silver (FIG. 9).
[0072]
As is clear from the figure, the purified sample showed only one band at the same molecular weight position as natural type zapesin, and no other bands were observed, confirming that the purified sample was sufficiently purified. The specific activity of the sample with respect to native zerpesin was measured (FIG. 10).
[0073]
As a result, the antibacterial activity of the purified sample was almost the same as that of natural zapesin, indicating that the recombinant zapesin was secreted and expressed by introducing the zapesin gene into the Penicillium camembergii U-150 strain.
[0074]
Example 8
▲ 1 ▼Plasmid pN3 Building
The transformation vector pSTA14 for Aspergillus oryzae [Mol. Gen. Genet. , 218, 99-104 (1989)] was modified as follows to prepare pN3. After treating pSTA14 with the restriction enzyme HindIII, the digestion mixture was subjected to 1.0% low melting point agarose electrophoresis,niaDAn approximately 5.5-kb HindIII fragment containing the gene was isolated and purified. This DNA fragment was blunt-ended at both ends using a DNA branching kit (manufactured by Takara Shuzo Co., Ltd.) and subcloned into the restriction enzyme SmaI site of pUC19 to obtain pN3 (FIG. 11).
[0075]
▲ 2 ▼Expression of Zapescin from Sarcophaga peregrina in Aspergillus oryzae
In order to construct a transformation-expression plasmid, pS4 obtained in Example 4 was treated with a restriction enzyme HindIII, and the digested mixture was subjected to 1.0% low-melting-point agarose electrophoresis to obtain a Zapescin expression cassette of about 2. The 2-kb HindIII fragment was isolated and purified. This DNA fragment was cloned into the HindIII site of the transformation plasmid pN3 for Aspergillus oryzae to obtain pSN4 (FIG. 12).
[0076]
Next, this pSN4 was used to transform Aspergillus oryzae AO1.1 strain (Mol. Gen. Genet., 218, 99-104 (1989)) into the method of Ankles et al. (Mol. Gen. Genet., 218, 99-). 104 (1989)). Ten transformants (SN4-1 to SN4-10) grown on the selection medium were randomly picked up, and each 100 ml of soybean oil medium (3% soybean oil, 0.5% yeast extract, 0.3% % NaNO₃, 0.1% K₂HPO₄, 0.05% KCl, 0.05% MgSO₄・ 7H₂O, 0.003% CuSO₄・ 5H₂O, 0.001% FeSO₄・ 12H₂0), and cultured with shaking at 30 ° C. for 4 days. The resulting culture was filtered to remove cells, and the resulting culture was subjected to Western blotting analysis.
[0077]
As a result, secretion of a protein having the same antigenicity and molecular weight as zapesin was observed in the culture solution, and the zapesin productivity was estimated to be about 3 mg / l in the highest producing strain (SN4-3). .
[0078]
Example 9
Expression of Zarpescin from Sarcoferga peregrina in Saccharomyces cerevisiae
In order to construct a transformation-expression plasmid for the yeast Saccharomyces cerevisiae, pS4 obtained in Example 4 was treated with a restriction enzyme HindIII, and the digestion mixture was subjected to 1.0% low-melting-point agarose electrophoresis, and a Zapesin expression cassette was prepared. Approximately 2.2-kb HindIII fragment was isolated and purified. This DNA fragment was cloned into the HindIII site of the transformation vector pL1 for Saccharomyces cerevisiae (Biosci. Biotech. Biochem., 56, 315-319 (1992)) to obtain pSL4 (FIG. 13).
[0079]
Next, using this pSL4, Saccharomyces cerevisiae SHY2 (ATCC 44770) was transformed by the method of Ito et al. (J. Bacterol., 153, 163-168 (1983)). One transformant grown on the selection medium is picked up, and the minimum medium containing 2 mg / ml of uracil, 20 mg / ml of tryptophan, and 20 mg / ml of histidine (2% glucose, 0.67% East Nitrogen Base Base) is picked up. 50 ml of w / o amino acid (Difco) was inoculated and cultured with shaking at 30 ° C. for 24 hours. 1 ml of the obtained culture solution is mixed with 0.003% CuSO₄・ 5H₂50 ml of YPGa1 medium (2% galactose, 2% polypeptone, 1% yeast extract) containing O was inoculated and cultured with shaking at 30 ° C. for 48 hours, and the obtained culture was centrifuged to remove cells, thereby obtaining The obtained culture solution was concentrated 20-fold using an ultrafiltration membrane (Ultra-free C3LGC, manufactured by Nippon Millipore) and subjected to Western blotting analysis.
[0080]
As a result, secretion of a protein having the same antigenicity and molecular weight as zapesin was observed in the concentrated culture solution, and the zapesin productivity was estimated to be about 0.02 mg / l.
[0081]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, it became possible to produce a large amount of industrially useful enzymes, hormones, bioactive peptides or proteins outside the cells by filamentous fungi. It can also be expressed by yeast at the same time, which facilitates the application of protein engineering techniques for creating functionally modified polypeptides, greatly contributing to industry.
[0082]
[Brief description of the drawings]
FIG. 1 shows the procedure for constructing plasmid pLGr80.
FIG. 2 shows a procedure for constructing plasmid pY4 ′.
FIG. 3 shows a procedure for constructing a plasmid pS2.
FIG. 4 shows the procedure for constructing plasmid pS3.
FIG. 5 shows the procedure for constructing plasmid pS4.
FIG. 6 shows a procedure for constructing plasmid pS5.
FIG. 7 shows a restriction map of plasmids pSH2, pSH3, pSH4 and pSH5.
FIG. 8 shows zapsin productivity of various transformants.
FIG. 9 shows an SDS polyacrylamide electrophoretogram of the recombinant zapesin.
FIG. 10 is a diagram showing the measured specific activity of recombinant zapesin with respect to natural zapesin.
FIG. 11 shows the procedure for constructing plasmid pN3.
FIG. 12 shows a restriction map of plasmid pSN4.
FIG. 13 shows a restriction map of plasmid pSL4.

Claims (2)

A restriction enzyme SalI and a BamHI fragment portion of a gene encoding a zapesin precursor derived from a cell line of a Sentime fly embryo, a promoter located upstream of a gene encoding mono- and diglycerol lipase derived from Penicillium camembertii FERMP-10452; and the plasmid obtained by inserting between the mono- and diglycerol lipase consisting prepro and Mature gene encoding region and terminator region, a transformant obtained by introducing the filamentous fungus was cultured in a nutrient medium, culturing A method for producing Zapesin, comprising accumulating Zapesin in an object and collecting the same. 2. The method for producing zerpescin according to claim 1, wherein the filamentous fungus is Penicillium camembertii or Aspergillus oryzae.

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