JP2000078977A - Stress-responsive gene promoter - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new promotor consisting of a gene promoter of the 2-deoxyglucose-6-phosphate dephosphorylase gene, from Saccharomyces cerevisiae, having a specific base sequence and useful e.g. for gene expression vectors. SOLUTION: This promoter is a new stress-responsive gene promotor of the 2-deoxyglucose-6-phosphate dephosphorylase gene (DOG2), from Saccharomyces cerevisiae, consisting of a DNA having a base sequence shown by the formula or a DNA having a base sequence wherein at least one base is (are) deleted, substituted or added in the base sequence shown by the formula and having stress-responsive promotor activity, and useful e.g. for the preparation of expression vectors for production of heterologous proteins using Saccharomyces cerevisiae as host cells. This promoter is obtained by screening a genomic DNA library from Saccharomyces cerevisiae using oxidative stress responsiveness as an indicator.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

SUMMARY OF THE INVENTION The present invention provides a stress-responsive promoter for the 2-deoxyglucose-6-phosphate phosphatase gene (DOG2) of Saccharomyces cerevisiae, a recombinant vector containing the promoter, The present invention relates to a transformant containing a recombinant vector and a method for producing a polypeptide using the transformant.

[0002]

BACKGROUND ART Yeast Saccharomyces cerevisiae (Sac
(charomayces cerevisiae) has long been used for brewing beer, wine, sake, etc. and in the manufacture of bread.
It is widely used in basic research as a model system for higher eukaryotes because of its advantages, such as protein modification that closely resembles higher organisms and generational change occurring in a short period of time, making analysis easier. I have. With the recent advances in molecular genetic analysis technology, analysis at the gene level has also progressed rapidly. In 1996, the entire nucleotide sequence of the genome was finally elucidated.

[0003] Saccharomyces cerevisiae, unlike Escherichia coli, hardly forms an inclusion body in cells and has a function of adding a sugar chain to a protein similarly to higher organisms. In fact, hepatitis B virus surface antigen protein and the like are industrially produced using the yeast.

In general, when a heterologous protein is produced using Saccharomyces cerevisiae as a host, the expression vector may be YEp type, YRp type, YCp type, pYAC type, or YIp type.
A type vector or the like is used. Here, an important point for efficiently producing a heterologous protein is to use a strong promoter, for example, an ADH (alcohol dehydrogenase) gene promoter, a PGK (phosphoglycerate kinase) gene promoter, a GAP (glycelase). Glycolytic genes such as aldehyde 3-phosphate dehydrogenase) gene promoter and MFα1
Sex pheromone gene promoters such as gene promoters have been used. However, since these promoters cause constitutive gene expression, it is necessary to express the target protein for the purpose of expressing a cytotoxic protein that inhibits the growth of the host cell itself or for analyzing the function of the protein. If it is desired to express at a specific stage, it is difficult to use.

[0005] Therefore, GAL (galactose metabolism) gene promoter, PHO5 (inhibitory acid phosphatase) gene promoter, SUC2 (invertase) gene promoter, CUPI
(Metallothionein) gene promoter, and HSP30 (He
Promoters that cause inducible gene expression, such as the at Shock Protein 30) gene promoter, have been developed. However, the conditions for inducing these promoters are
Glucose deficiency and galactose addition in the gene promoter, phosphate deficiency in the PHO5 gene promoter, glucose deficiency in the SUC2 gene promoter, copper addition in the CUPI gene promoter, and a sharp rise in temperature in the HSP30 gene promoter It is. Therefore, in order to set these induction conditions, in the GAL, PHO5, SUC2 gene promoter, a step of collecting yeast cells, a step of washing the cells,
In addition, a step of re-inoculating the cells into the induction medium is required. In the case of the CUPI gene promoter, a step of removing copper must be added to the step of purifying the target protein, and the operation is complicated. Further, in the case of the HSP30 gene promoter, when culturing is performed on a large scale, it is difficult to rapidly raise the temperature of the entire medium.

[0006] As described above, production of a heterologous protein by a genetic recombination method using Saccharomyces cerevisiae as a host has many problems in practical use as compared with its industrial utility value.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a stress-responsive promoter for the 2-deoxyglucose-6-phosphate phosphatase gene (DOG2 gene) of Saccharomyces cerevisiae, a recombinant vector containing the promoter, An object of the present invention is to provide a transformant containing a recombinant vector, and a method for producing a polypeptide using the transformant.

[0008]

Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, oxidative stress, osmotic stress, and glucose starvation stress have been obtained from a genomic DNA library of Saccharomyces cerevisiae. And succeeded in isolating a gene responsive to stress, and completed the present invention. That is, the present invention
It is a promoter containing the following DNA of (a) or (b).

(A) a DNA comprising the nucleotide sequence represented by SEQ ID NO: 1; (b) a DNA comprising a sequence in which at least one base has been deleted, substituted or added in the nucleotide sequence represented by SEQ ID NO: 1, and DNA Having Stress-Responsive Promoter Activity Further, the present invention is a promoter that hybridizes with the above DNA under stringent conditions and contains a DNA having a stress-responsive promoter activity. Here, examples of the stress include oxidative stress, osmotic stress, and glucose starvation stress.

[0010] Further, the present invention is a gene expression cassette containing the above promoter. Furthermore, the present invention is an expression vector containing the above-mentioned gene expression cassette. Furthermore, the present invention is a recombinant vector in which a gene encoding any polypeptide is incorporated into the above expression vector.

Furthermore, the present invention is a transformant containing the above-mentioned recombinant vector. Further, the present invention is the method for producing a polypeptide, which comprises culturing the transformant and collecting the polypeptide from the obtained culture. Hereinafter, the present invention will be described in detail.

[0012]

BEST MODE FOR CARRYING OUT THE INVENTION The stress-responsive promoter of the present invention is a promoter isolated from a genomic DNA library of Saccharomyces cerevisiae using oxidative stress response as an index. The promoter is a promoter of a 2-deoxyglucose-6-phosphate phosphatase gene (DOG2 gene), and expression of a polypeptide in yeast cells by an expression vector of the present invention incorporating the promoter is conventionally known. In contrast to the expression vector, it can be induced by various stress loads such as oxidative stress, osmotic stress, and glucose starvation stress. Hereinafter, the stress-responsive promoter of the present invention is referred to as DOG2.
It is called a promoter.

[0013] The DOG2 promoter of the present invention can be isolated as follows. 1. Isolation of Saccharomyces cerevisiae-derived DOG2 promoter (1) Preparation of Saccharomyces cerevisiae genomic library Can be prepared. For example, cells obtained by liquid culture,
After collection by centrifugation, the cells are suspended in a suitable buffer (eg, 1 M sorbitol-0.1 M EDTA (pH 7.5)), and the cell wall is treated with a cell wall lytic enzyme such as Zymolyase 100T.
The cells are further treated with SDS to lyse the cells, and then the nucleic acid is extracted. Then, after degrading the RNA by ribonuclease treatment,
Genomic DNA can be prepared by alcohol precipitation.

A genomic DNA library can be prepared, for example, as follows. First, genomic DNA
Is digested with restriction enzymes such as restriction enzymes Sau3AI, PvuII, HincII, and StuI, treated with chloroform, and the DNA fragment is recovered by ethanol precipitation. After adding a suitable linker (e.g., NotI linker) to the obtained DNA fragment, pET vector
(Novagen) and the like. Next, the method of M. Snyder et al. [N. Burns et al .: Gen.
es Dev., 8: 1087-1105 (1994)].
A cassette composed of the lacZ gene deleted from the initiation codon, the ampicillin resistance gene, and the LEU2 gene was introduced using a transposon (Tn3), and the yeast genomic DNA fragment containing the cassette was transformed into a leucine-requiring diploid yeast cell. And randomly inserting it into the yeast chromosome.

The genomic DNA library includes YEp13, YEp2
4. It can also be prepared by ligating the genomic DNA fragment to a plasmid vector such as YCp50 or pRS415, and transforming the fragment into Saccharomyces cerevisiae.

(2) Cloning of the DOG2 Promoter of the Present Invention The DOG2 promoter of the present invention can be cloned from a colony bank of the genomic DNA library of Saccharomyces cerevisiae prepared in (1) above. That is, the cells are cultured on a YPD agar medium, and after the culture,
Replicate the cells on the filter. Again, the replica cells were cultured on a YPD agar medium supplemented with lipid peroxide (such as tert-butyl hydroperoxide) as oxidative stress,
When the colony became about 1 mm, the filter was immersed together with the cells in liquid nitrogen to destroy the cell wall, and then X-gal (5-bromo-4-chloro-3) was examined to confirm the presence or absence of reporter gene lacZ expression. -Indolyl-β-D-galactoside) on an agar medium (e.g., YPD medium) and screen colonies corresponding to replica bacteria that showed a blue color with X-gal and β-galactosidase infiltrated from the cell walls. I do. Then, a DNA region involved in the induction of reporter gene expression in the obtained yeast cells is rescued from the yeast. The method is the method of M. Snyder et al.
[N. Burns et al .: Genes Dev., 8: 1087-1105 (1994)]. That is, the target yeast is YIp5
Transform with YIp5 introduced into the cell undergoes homologous recombination at the ampicillin resistance gene portion of the cassette composed of the lacZ gene, the ampicillin resistance gene, and the LEU2 gene. Since there is a restriction enzyme site NsiI in YIp5, Ns
When digested with iI, a linear fragment containing the NsiI site in the genome and the NsiI in YIp5 is obtained. When Escherichia coli is transformed with this fragment cyclized with DNA ligase, rescuing of a gene fragment having a promoter region that has induced the expression of the reporter gene lacZ in the cassette in the yeast cells can be performed.

(3) Determination of Nucleotide Sequence The determination of the nucleotide sequence of the DNA fragment involved in oxidative stress responsiveness obtained in the above (2) was carried out by transposons and yeast involved in oxidative stress as shown in FIG. After rescuing the DNA in Escherichia coli, the plasmid is purified, and the obtained plasmid is used as a template, and a sequence complementary to the sequence in the lacZ + LUE2 + ampC cassette (for example, a sequence complementary to a partial sequence of the lacZ gene). Using 5'-CGTTGTAAAACGACGGGATCCCCCT-3 '(SEQ ID NO: 2)) as a primer, a method such as Maxam-Gilbert chemical modification, or an automatic base sequencer by the dideoxynucleotide chain termination method of Sanger et al. (For example, ABI manufactured by Perkin Elmer) PRISM 310 Genetic Analyzer
Etc.). Homology search of the base sequence of the cloned gene using oxidative stress response as an index can be performed using a homology search program such as FESTA or BLAST from a database such as the GenBank / EMBL nucleic acid database.

(4) Amplification of the DOG2 Promoter of the Present Invention by PCR The promoter region of the DOG2 gene partially sequenced in (3) above is a database (Stanford) in which the entire nucleotide sequence of Saccharomyces cerevisiae is stored.
The DNA can be obtained by amplifying by PCR using a genomic DNA of Saccharomyces cerevisiae as a template, using a primer designed based on the nucleotide sequence of the 5 'upstream region of the DOG2 gene available from the University of Saccharomyces genome database. Here, as a primer that can be used for PCR, 5′-sense primer represented by the base sequence of 5′-AACGAATATATATAGATG-3 ′ (SEQ ID NO: 4) and
5′-CATTTTTTTTTTTTATTTTTTTGACG-3 ′ (SEQ ID NO: 5) 3 ′ antisense primer and the like. However, the present invention is not limited to these primers.

SEQ ID NO: 1 shows the nucleotide sequence of a DNA fragment having a stress-responsive promoter activity of the present invention. As long as it has a stress-responsive promoter activity, at least one base in the nucleotide sequence is deleted or substituted. , And mutations such as addition may occur. In addition, a DNA that hybridizes with the stress-responsive promoter gene under stringent conditions and has a stress-responsive promoter activity is also included in the promoter gene of the present invention. The stringent conditions are, for example, a sodium concentration of 0 to 800 mM, preferably 700 to 750 mM,
The temperature is 30 to 70 ° C, preferably 40 to 50 ° C.

In order to introduce a mutation into DNA, Kunkel
A known method such as a method or a gapped duplex method or a method similar thereto can be employed. For example, a mutagenesis kit using site-directed mutagenesis (for example, Mutant-K
(TAKARA) or Mutant-G (TAKARA)) or TAKARA LA PCR in vitro Mutagenesi
Mutations can be introduced using the s-series kit. Once the nucleotide sequence of the DNA fragment of the present invention is determined, thereafter, by chemical synthesis, or by PCR using a cDNA or genomic DNA containing the DNA fragment of the present invention as a template,
Alternatively, the DNA fragment of the present invention can be obtained by hybridization using a DNA fragment having the nucleotide sequence as a probe.

2. Stress responsiveness of the DOG2 promoter of the present invention. After culturing the yeast strain derived from the blue colony obtained in the above under normal culture conditions, oxidative stress, osmotic stress, after exposure to various stresses such as glucose starvation stress, crush cells, and centrifuged supernatant β-galactosidase activity can be measured using a commercially available β-galactosidase activity kit
(Manufactured by STRATAGENE) or the like and can be examined.

3. Construction of the expression vector of the present invention DO obtained in
It can be constructed by ligating the G2 promoter to an appropriate plasmid vector. Here, the vector is a highly stable YIp-based vector of the chromosome insertion type.
(E.g. YIp5), YE present extrachromosomally as a plasmid
p-based vectors (e.g., YEp13), YRp-based vectors (e.g.,
YRp7), YCp-based vectors (eg, YCp50) and the like.

In the expression vector, a gene that complements the auxotrophy of the host, such as LEU2 and HIS3 for amino acid requirement, and LEU2 and HIS3 for nucleic acid requirement, for convenient detection of transformants.
It is desirable to link a marker gene such as URA3.
In addition, a terminator of the yeast ARG3 gene can be ligated to regulate transcription termination. Further, in the expression vector, a drug resistance marker gene of Escherichia coli and a replication origin can be ligated so as to be used as a shuttle vector with Escherichia coli for rescue of the transgene. As described above, a group of genes such as the promoter, terminator, and marker of the present invention contained in the expression vector of the present invention is referred to as a gene expression cassette.

4. Production of Polypeptide Using the Recombinant Expression Vector of the Present Invention Using the recombinant vector for expression of the present invention, a target polypeptide can be produced using Saccharomyces cerevisiae as a host. The above 2. A recombinant vector can be prepared by ligating (inserting) a gene encoding a target polypeptide into the expression vector obtained in the above. That is, to insert a gene encoding a polypeptide of interest into the vector of the present invention, first, the purified DNA is cut with an appropriate restriction enzyme, and inserted into a restriction enzyme site or a multiple cloning site of the vector of the present invention. And then ligating to a vector.

A transformant can be obtained by introducing the obtained recombinant vector into Saccharomyces cerevisiae by a method such as lithium method, protoplast method, and electroporation method. When a gene that complements auxotrophy is used as a marker gene, the target transformant is selected by selecting cells that have grown in a medium from which the auxotrophic substance has been removed or a medium to which the drug has been added. When a resistance gene is used as a marker gene, a target transformant can be obtained by selecting cells that have grown in a medium to which a drug has been added.

Next, the desired transformant can be obtained by culturing the obtained transformant and collecting it from the culture. The method for culturing the transformant of the present invention is performed according to a usual method used for culturing Saccharomyces cerevisiae. As a medium for culturing the transformant obtained using Saccharomyces cerevisiae as a host, a medium containing a carbon source, nitrogen source, inorganic salts, and the like that Saccharomyces cerevisiae can utilize, and a medium that can efficiently culture the transformant If so, either a natural medium or a synthetic medium may be used.

As the carbon source, carbohydrates such as glucose, fructose, sucrose, maltose, dextrin, raffinose and inositol, organic acids such as acetic acid and citric acid, and alcohols such as ethanol and glycerol are used. Examples of the nitrogen source include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic salts or ammonium salts of organic acids or other nitrogen-containing compounds, peptone, meat extract, corn steep liquor, casamino acid, NZ An amine or the like is used.

As the inorganic substance, potassium (II) phosphate, potassium (II) phosphate, calcium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, zinc sulfate, cobalt chloride and the like are used. Can be The culture is usually performed at 28 ° C. for 10 to 16 hours under aerobic to microaerobic conditions such as shaking culture, aeration stirring culture, or stationary culture. During the culture period, the pH is adjusted using an inorganic or organic acid or alkali solution.

After the culture, if the desired polypeptide is produced in the cells, the cells are disrupted. On the other hand, when the target polypeptide is produced outside the cells, the culture solution is used as it is, or the cells are removed by centrifugation or the like to obtain a supernatant. Then, by using a general biochemical method used for isolation and purification of the polypeptide, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc., alone or in appropriate combination, the above culture The target polypeptide can be isolated and purified therefrom. Examples of the target polypeptide include insulin, growth hormone, interferon, and other useful polypeptides used in the pharmaceutical field, the food field, and the like.

[0030]

The present invention will be described below in more detail with reference to Examples, but it should not be construed that the present invention is limited thereto. Example 1 Cloning of Oxidative Stress Responsive Gene from Saccharomyces cerevisiae (1) Preparation of Saccharomyces cerevisiae Genomic DNA Genome of Saccharomyces cerevisiae in accordance with a conventional method
DNA was prepared. That is, Saccharomyces cerevisiae S288C strain (California State University Berkeley, East Georgia)
The netic Stock was obtained from Center), YPD medium (1% yeast extract, peptone 2%, glucose 2%, after culturing until the A ₆₁₀ = 0.5 to 1.0 at 28 ℃ at pH 6.0), 10 minutes at 3,000 × g The cells were collected by centrifugation. The obtained cells were suspended in 1 ml of 1 M sorbitol-0.1 M EDTA (pH 7.5), and then suspended.
μl of 2.5 mg / ml Zymolyase100T was added and incubated at 37 ° C. for 1 hour. Next, the cells were centrifuged at 5,000 xg for 2 minutes, and the cells were removed from 50 µl of 50 mM Tris-HCl (pH 7.4) -20 mM EDTA.
Suspended in water. Add 70 μl of 10% SDS solution, mix and at 65 ° C
Incubated for 30 minutes. Then, 25 μl of 5 M potassium acetate (pH 5.0) was added, and the mixture was left on ice for 1 hour. 10,000 ×
After centrifugation at g for 5 minutes, the supernatant was transferred to a sterilized centrifuge tube, an equal volume of isopropyl alcohol was added, and the mixture was allowed to stand at room temperature for 5 minutes. After centrifugation at 10,000 xg for 10 minutes, remove the supernatant,
The remaining white precipitate was dried in a desiccator. Next, the suspension was suspended in 200 μl of TE (10 mM Tris-HCl (pH 7.4), 1 mM EDTA), 1 μl of a 1 mg / ml ribonuclease A solution was added, and the mixture was incubated at 37 ° C. for 30 minutes. 200 μl of 3M sodium acetate (pH 5.2) and 0.2 ml of isopropyl alcohol were added, mixed well, and centrifuged at 10,000 × g for 10 minutes to remove the supernatant. The precipitate was dried in a desiccator and then dissolved in 40 μl of TE to obtain genomic DNA.

(2) Preparation of genomic DNA library Using the genomic DNA obtained in (1) above, the method of M. Snyder et al. [N. Burns et al., Genes Dev., 8,1087-1105 (199)
4)], the genome D of Saccharomyces cerevisiae
An NA library was prepared. That is, 5 μg of genomic DN
A was cleaved with 10 units of restriction enzyme Sau3AI to about 2-3 kb, and then blunted with 8 units of T4 DNA polymerase (TAKARA). Then, the reaction solution was treated with phenol / chloroform and then precipitated with ethanol to give 50 μl.
Was dissolved in a TE buffer solution to obtain a genomic DNA fragment. here,
The chain length of the cut genomic DNA was confirmed by subjecting a part of the digested product of the genomic DNA to 1% agarose gel electrophoresis.

Next, a solution having the following reaction composition was prepared, and a NotI adapter was added to the genomic DNA fragment. Genomic DNA fragment solution 4 μl Sterile water 1 μl 10 × ligase buffer 1 μl 10 mM ATP 1 μl NotI adapter 1 μl (100 ng) T4 DNA ligase (TAKARA) 2 μl (8 units) Total volume 10 μl

The above reaction solution was incubated at 16 ° C. for 24 hours to add NotI adapters to both ends of the genomic DNA fragment. Then, after phenol / chloroform treatment, a genomic DNA fragment having a length of 2 to 3 kbp was obtained by ethanol precipitation. A genomic DNA fragment (50 ng) having NotI restriction enzyme sites at both ends obtained as described above, a pET vector
(Novagen) at the NotI site with 2 units of T4 DNA ligase (TA
(Made by KARA). The reaction was performed at 16 ° C. for 24 hours. After transformation of the entire ligation reaction solution (10 μl) into an E. coli strain, LB containing 50 μg / ml kanamycin was used.
The medium was inoculated. And cultured overnight at 37 ° C., scraped transformant colonies obtained was maintained as 18 pools (including a total of 10 ⁵ recombinants). Next, as shown in FIG. 1, the lacZ gene in which the start codon of E. coli was deleted, the marker gene for yeast LEU2 gene,
Escherichia coli marker gene ampC cassette (lacZ + LE
U2 + ampC cassette), and after transformation of a DNA fragment consisting of a Tn3-derived repeat sequence necessary for the transfer of the DNA fragment, 50 μg / m
LB containing 1 kanamycin and 50 μg / ml ampicillin
The medium was inoculated. After culturing overnight at 37 ° C., the resulting transformed colonies were scraped off, and a plasmid was prepared from the obtained colonies.

Next, la is randomly added to the yeast genome fragment.
The plasmid containing the cZ + LEU2 + ampC cassette was digested with NotI. The obtained NotI digest was purified by the lithium acetate method.
Diploid yeast Saccharomyces cerevisiae YPH274 strain (a /
α, ura3-52 / ura3-52, lys2-801 / lys2-801, ade2-101 / ade
2-101, trp1-Δ1 / trp1-Δ1, his3-Δ200 / his3-Δ200, le
After transformation into u2-Δ1 / leu2-Δ1), a strain capable of growing on a medium lacking leucine was selected, and a colony bank of about 3,000 strains was prepared.

(3) Cloning of Oxidative Stress Responsive Gene From the colony bank obtained in the above (2), a clone whose expression is increased in response to lipid peroxide present in the medium is replaced with the expression product β X-gal was degraded by the action of -galactosidase and isolated by selecting the strain that turned blue. That is, from the colony bank,
The cells were inoculated on a YPD agar medium so as to have 0 copies, cultured at 28 ° C., and when the colonies became about 1 mm in size, they were replicated on a nylon filter. Then 0.6 mM tert-
Place a replica filter on YPD agar medium containing butyl hydroperoxide, further culture at 28 ° C until colonies become about 1 mm, immerse the replica filter in liquid nitrogen after culture, crush the outer wall of the cells, and remove X-gal. The resultant was placed on a YPD medium containing the cells to obtain 20 blue-colored colonies. Next, a similar selection was performed on a YPD agar medium containing 0.06 mM and 0.6 mM tert-butyl hydroperoxide, and five colonies with a darker blue color were obtained at 0.6 mM than at 0.06 mM. Further, as a third screening, a colony in which expression was not induced without addition was searched, and finally one colony (SET8 strain) was obtained.

(4) Determination of Nucleotide Sequence In the SET8 strain obtained in (3) above, the nucleotide sequence of a DNA fragment involved in the oxidative stress-inducing property of β-galactosidase was determined as shown in FIG. Was determined by constructing a plasmid for That is, the obtained yeast SET8 strain was cultured in 50 ml of YPD medium at 28 ° C. for 16 hours, and then collected by centrifugation, and the obtained cells were cut with PvuI.
After transformation with YIp5 by the lithium acetate method, the cells were seeded on a medium lacking leucine and uracil, and transformants were selected.
DNA was extracted from the obtained transformant, and the obtained extract was cut with NsiI and ligated with T4 DNA ligase. Escherichia coli DH1 was transformed with this fraction, and a recombinant which became an ampicillin-resistant strain was selected.

Next, a plasmid was extracted from this recombinant, and the resulting plasmid was used as a template to prepare a primer (Pr) 5'-CGTTGTAAAAC for the minus strand of the reporter gene lacZ.
Using GACGGGATCCCCCT-3 ′ (SEQ ID NO: 2), the nucleotide sequence of the 408 bp upstream region adjacent to the lacZ gene was determined by the dideoxy method (SEQ ID NO: 3). When a homology search of the obtained nucleotide sequence was performed, it was consistent with the sequence of the DOG2 gene encoding 2-deoxyglucose-6-phosphate phosphatase.

Example 2 Stress Responsiveness of the DOG2 Gene Promoter The stress responsiveness of the DOG2 gene promoter (DOG2 promoter) obtained in Example 1 (2) was examined. That is, after culturing the SET8 strain obtained in Example 1 using an YPD medium at 28 ° C. until A ₆₁₀ = 0.8 to 1.0, ter
What added t-butyl hydroperoxide (t-BHP) to a final concentration of 0.6 mM, 2-deoxyglucose (2-deGl
c) added to a final concentration of 0.1%, NaCl added to a final concentration of 0.3M, sorbitol added to a final concentration of 0.6M, 2-deGlc to a final concentration of
0.1% and sorbitol to a final concentration of 0.6M, glucose-free medium (YP
The cells were further cultured for 1 hour under conditions such as those transferred to a medium (yeast extract 1%, peptone 2%, pH 6.0), those shifted at 37 ° C., those directly cultured in a YPD medium (control), and the like. The cells were collected by centrifugation at xg for 10 minutes.

The collected cells were washed with physiological saline, and then washed with 10 mM
The cells were suspended in a potassium phosphate buffer (pH 7.0) and disrupted by a brown homogenizer using glass beads at 0 ° C. for 3 minutes. Next, the cell suspension was centrifuged at 14,000 rpm for 15 minutes to recover the supernatant, and the β-galactosidase activity in the supernatant was measured using a β-galactosidase kit (STRATAGENE
(Manufactured by the company). The results are shown in FIG. From these results, it was found that the expression of the DOG2 promoter was not induced by heat shock, and was induced by not only oxidative stress but also osmotic stress and glucose starvation stress.

Example 3 Construction of Expression Vector pDOG2-galZ Vector (1) Isolation of DOG2 Promoter The DOG2 gene and the gene from the Saccharomyces cerevisiae whole nucleotide sequence database (Stanford University, Samaromyces genome database) The nucleotide sequence of the region adjacent to was obtained, and about 500b (SEQ ID NO: 1) of the 5 'upstream region of the DOG2 gene translation initiation codon was examined in detail.
(Stress response e) found upstream of genes encoding heat shock stress proteins such as HSP26 and HSP26
lement) 5'-AGGGG-3 'and specific base sequences (TATAbox etc.) found in various promoters.

Therefore, about 500 b upstream from the translation initiation codon was regarded as a promoter region, and this region was amplified by PCR. Here, as a template, genomic DNA prepared from Saccharomyces cerevisiae strain YPH250 (obtained from California State University Berkeley, Yeast Genetic Stock Center) by the same procedure as in Example 1 was used, and the primer was 5 ′
As a primer, -50 from the start codon of the DOG2 gene
Sequence 5'-AACGAATATATATAGAT present at position 0 to -483 bp
The one having G-3 '(SEQ ID NO: 4) was used as a 3' primer, a sequence 5'-CATTTTTTTTTTTTATTTTTTTGACG-3 '(SEQ ID NO: 5) existing at a position of -23 to +3 bp from the start codon. The PCR reaction was performed for 20 cycles, each cycle consisting of 94 ° C. for 3 seconds and 35 ° C. for 3 seconds. After the reaction product was subjected to 1% low-melting point agarose gel electrophoresis, a band giving a molecular weight corresponding to about 500 bp was extracted from the gel. .

(2) Construction of Expression Vector Using the promoter region of the DOG2 gene isolated in (1) above, a vector for expressing a foreign protein was constructed. That is, the PCR fragment obtained in the above (1), Sm
aI digested promoter search vector pMC1871 (Pharma
cia), and the ligation was transformed into E. coli DH1 strain. Next, the plasmid was purified from 10 colonies of the obtained many transformants, and a strain having the plasmid into which the DOG2 promoter was inserted in the forward direction was selected by PCR after PCR.

Here, as the 5 'primer, 5'-GGAATTCCCAACGAATATA-3' in which 9 bases out of 19 bases are a vector-derived sequence so that amplification is performed only when the DOG2 promoter is ligated in the forward direction. (SEQ ID NO: 6), and as the 3 ′ primer, the same primer 5′-CATTTTTTTTTTT as used for the amplification of the DOG2 promoter
TATTTTTTTGACG-3 ′ (SEQ ID NO: 5) was used. 94 PCR reactions
Twenty-five cycles were performed at 3 ° C. for 3 seconds, 72 ° C. for 3 seconds, and 35 ° C. for 3 seconds, and a band appearing at about 500 bp confirmed that the DOG2 promoter was in the forward direction.

Next, a strain having a plasmid in which the DOG2 promoter was ligated in the forward direction was cultured to prepare a large amount of the plasmid. Next, this DOG2 promoter and β
-Restriction enzyme B for galactosidase gene expression cassette region
excised with amHI, YRSp vector pRS413 (STRATA
GENE) (pDOG2-galZ).

Example 4 Expression of Heterologous Gene Using Expression Vector of the Present Invention Saccharomyces cerevisiae YPH499 (MATa, ura3-52, l) was expressed with the expression vector pDOG2-galZ prepared in Example 3.
ys2-801, ade2-101, trp1-Δ63, his3-Δ200, leu2-Δ1)
Was transformed according to the lithium method. After pre-cultivating the transformed recombinant yeast in 2 ml of YPD medium at 30 ° C. for 2 days,
The cells were cultured in the same medium (500 ml) at 28 ° C. until A ₆₁₀ = 1.0.

Thereafter, the bacterial solution was divided into six 50 ml portions,
0.6 mM tBHP, 0.1% 2-deGlc,
0.3M NaCl, 0.3M KCl, 0.6M sorbitol, respectively
Alternatively, a medium containing both 0.1% 2-deGlc and 0.6M sorbitol, a medium without glucose (YP medium (1% yeast extract, 2% peptone, pH 6.0)), and the temperature is shifted to 37 ° C. The culture was further cultured for 1 hour under the same conditions as those cultured in a YPD medium (control) and collected. After harvest, the cells were washed with physiological saline, suspended in 10 mM potassium phosphate buffer (pH 7.0), and crushed with a brown homogenizer using glass beads at 0 ° C. for 3 minutes. Next, the cell suspension was centrifuged at 14,000 rpm for 15 minutes, and the supernatant was used to confirm an increase in β-galactosidase activity. β-galactosidase activity is STRATAGEN
The measurement was performed using a β-galactosidase kit manufactured by E Company.
FIG. 4 shows the results. These results revealed that the DOG2 promoter was not expressed by heat shock and was induced not only by oxidative stress but also by osmotic stress and glucose starvation.

[0047]

Industrial Applicability According to the present invention, there are provided a stress-responsive promoter of Saccharomyces cerevisiae, a recombinant vector containing the promoter, a transformant containing the recombinant vector, and a method for producing a polypeptide using the transformant. You.

[0048]

[Sequence List] SEQUENCE LISTING <110> MARUHA Co. <120> Stress responsible promoter <160> 6 <170> PatentIn Ver. 2.0 <210> 1 <211> 500 <212> DNA <213> Saccharomyces cerevisiae <400> 1 aacgaatata tatagatgta aaacatatgg acaagcaaaa agtcgaatta tgtatgtcat 60 tttaggtact gaagaggtaa gatttttttt gagtttttct tcgaagatgg ttgtgtggtt 120 atatgttaat cttccttagc gcaaaacact tccatcaact gtatttcgtt ggaatgcttt 180 gtattcagtt ttgtatcatt atctttaatc acaattgcgt caggatgtaa gaactacgta 240 atgatcttat tatttctcgt agagaatagt tccgtagatt gaatacgctc cgtcattatt 300 tttaaatgtg gggaaggggt aattctcgag gatttttcaa aaacttaaaa tgcgctggca 360 acatcttctt tggtgaaaac aaatgctaaa aggagactaa gagtactttt tgttattcac 420 tatagtatta gccaacacgt tatcgataca tttactgcta tatacataaa aaatttacgt 480 caaaaaaata aaaaaaaaaa 500 <210> 2 <211> 828 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide as a primer used for sequencing oxidative stress responsible gene. <400> 2 CGTTGTAAAA CGACGGTC CCCCT 25 <210> 3 <211> 408 <212> DNA <213> Saccharomyc es cerevisiae <220> <221> CDS <222> (81) .. (407) <400> tatagtatta gccaacacgt tatcgataca tttactgcta tatacataaa aaatttacgt 60 caaaaaaata aaaaaaaaaa atg cca caa ttt tca gta gat ctt tgt ctt ttthe Met Pro Gln Asp Leu Cys Leu Phe 1 5 10 gac cta gat ggg act att gtc agc aca aca act gca gcg gaa agt gcc 161 Asp Leu Asp Gly Thr Ile Val Ser Thr Thr Thr Ala Ala Glu Ser Ala 15 20 25 tgg aaa aaa tta tgc cgt cag cat ggg gtt gat cct gtt gag tta ttc 209 Trp Lys Lys Leu Cys Arg Gln His Gly Val Asp Pro Val Glu Leu Phe 30 35 40 aag cat tcc cat ggt gca aga tca caa gaa atg atg aag aaa ttt ttt 257 Lys His Ser His Gly Ala Arg Ser Gln Glu Met Met Lys Lys Phe Phe 45 50 55 cca aaa ttg gac aat acc gat aat aaa ggt gtt ctt gcg tta gaa aag 305 Pro Lys Leu Asp Asn Thrh Asp Asn Lys Gly Val Leu Ala Leu Glu Lys 60 65 70 75 gat atg gca gat aat tat ttg gac aca gta agc ctt atc cct ggt gca 353 Asp Met Ala Asp Asn Tyr Leu Asp Thr Val Ser Leu Ile Pro Gly Ala 80 85 90 gag aat tta ttg tta tcg tta gat gta gat act gag act caa aaa aag 401 Glu Asn Leu Leu Leu Ser Leu Asp Val Asp Thr Glu Thr Gln Lys Lys 95 100 105 tta cct g 408 Leu Pro <210> 4 <211> <212> DNA <213> Artificial Sequence <220> < 223> Designed oligonucleotide based on the sequence of promoter region of DOG2 gene. <400> 4 aacgaatata tatagatg 18 <210> 5 <211> <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based on the sequence of promoter region of DOG2 gene. <400> 5 catttttttt ttttattttt ttgacg 36 <210> 6 <211> <212> DNA <213> Artificial Sequence <220> <223> Designed oligonucleotide based on the sequence of promoter region of DOG2 gene. <400> 6 ggaattccca acgaatata 19

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[Sequence List Free Text]

[0050]

[SEQ ID NO: 2] Oligonucleotide used as a primer for determining the base sequence of oxidative stress responsive gene.

[0051]

[SEQ ID NO: 4] Oligonucleotide designed based on the sequence of the promoter region of DOG2 gene.

[0052]

[SEQ ID NO: 5] Oligonucleotide designed based on the sequence of the promoter region of DOG2 gene.

[0053]

[SEQ ID NO: 6] Oligonucleotide designed based on the sequence of the promoter region of DOG2 gene.

[Brief description of the drawings]

FIG. 1 is a diagram showing a procedure for screening a clone containing an oxidative stress responsive gene.

FIG. 2 is a diagram showing a rescue procedure of a plasmid for determining a base sequence of an oxidative stress-responsive gene.

FIG. 3 is a diagram showing the stress responsiveness of the promoter of the present invention.

FIG. 4 is a diagram showing the stress responsiveness of expression of a target gene in the expression vector of the present invention.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification FI theme coat ゛ (Reference) (C12N 1/19 C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Hikaru Kimura 81 (72) Inventor Nobuyuki Sato 16-2 Wadai, Tsukuba-shi, Ibaraki Pref. Maruha Corporation Central Research Laboratory F-term (reference) 4B024 AA03 CA03 DA12 FA02 4B064 AG01 CA06 CA19 CC24 DA16 4B065 AA80X AA80Y AB01 AC07 AC20 BA02 CA24

Claims (8)

[Claims] 1. A promoter comprising the following DNA (a) or (b): (a) DNA consisting of the base sequence represented by SEQ ID NO: 1 (b) consisting of a sequence in which at least one base is deleted, substituted or added in the base sequence represented by SEQ ID NO: 1, and having a stress responsiveness DNA with promoter activity 2. A promoter that hybridizes with the DNA according to claim 1 under stringent conditions and contains a DNA having a stress-responsive promoter activity. 3. The promoter according to claim 1, wherein the stress is oxidative stress, osmotic stress, or glucose starvation stress. A gene expression cassette comprising the promoter according to any one of claims 1 to 3. 5. An expression vector comprising the gene expression cassette according to claim 4. 6. A recombinant vector in which a gene encoding any polypeptide is incorporated into the expression vector according to claim 5. A transformant comprising the recombinant vector according to claim 6. 8. A method for producing a polypeptide, comprising culturing the transformant according to claim 7, and collecting the polypeptide from the resulting culture.