JPS5974987A - Yeast developable vector plasmid utilizing promotor for gene controlling glycolytic enzyme and method for utilizing the same - Google Patents

Abstract

PURPOSE:To produce the aimed useful peptide in high yield, by utilizing a promoter of an operon controlling 3-phosphoglycerokinase. CONSTITUTION:A plasmid containing a promoter for a gene controlling 3-phosphoglycerokinase (PGK) and the aimed peptide is prepared, and a yeast transformed by the above-mentioned plasmid is cultivated. alpha-Neoendorphin (alphaNE) gene is used as the aimed peptide gene, and the aimed substance (alphaNE) is produced as a PGK-alphaNE hybrid protein from the sequence such as PGK promoter- PGK structure gene-alphaNE gene. A gene capable of coding the aimed gene, e.g. a peptide such as interferon, is added to a site at a distance of a suitable base sequence from the position of the PGK promoter, and the aimed peptide can be directly produced.

Description

Detailed Description of the Invention The present invention provides a promoter of a gene (operon) controlling 3-phosphoglycerokinase (hereinafter abbreviated as PGK), which is one of the glycolytic enzymes of the yeast Satucharomyces, and a useful target peptide. This invention relates to a yeast vector that is linked to a gene. Furthermore, the present invention relates to a method for producing a useful target peptide in high yield from yeast using the promoter of the operon controlling the above-mentioned PGK.

Until now, genetic engineering techniques have been used to produce pharmaceutically useful peptides such as somatostatin, insulin, growth hormone, various interferons, influenza virus and hepatitis B virus proteins, Simon α1, β-endorphin, α-neoendorphin, secretin, Many substances such as urokinase and plasminogen activators are now produced in microorganisms or animal cells.

Many of these host prokaryotic cells (prokaryotic cells).
We use Escherichia coli, which is a type of nucleated cell (e).
The use of yeast, which is known as ukaryoute, as a host is attracting attention. This is due to the fact that yeast culture conditions are simple, the rate of division is high, and safety is high, and in particular, more genetic and biochemical analyzes have been performed on Satucharomyces yeast. However, there are few examples of foreign peptide production in yeast, and the only examples are α-interferon, HB hepatitis surface antigen protein, and α-neoendorphin.

On the other hand, a complementary DNA obtained from a foreign heterologous gene, such as a chemically synthesized gene, or mRNA using reverse transcriptase.
A (cDNA) To express a gene, a gene obtained from a chromosome by appropriate treatment, or a homologous peptide gene contained in yeast but produced in small amounts, it is necessary to use a promoter region suitable for the host used. Existence is essential.

Since the quality of the promoter greatly influences the expression of the desired peptide gene, expression plasmid vectors using various promoters have been developed.

However, the expression vectors for foreign genes that have been reported to use yeast as hosts cannot be said to have sufficient efficiency in expressing the target foreign genes compared to vectors that use E. coli as hosts. The inventors discovered that 3-phosphoglycerokinase (P
Focusing on the fact that GK) is produced in large quantities in yeast cells,
We proceeded with our research by considering the use of the promoter gene of the operon that encodes this enzyme. As a result, we discovered that this promoter is extremely useful for producing heterologous or homologous peptides in yeast, and created a yeast vector in which a heterologous or homologous peptide gene was linked downstream of this promoter, and used this vector. Transform yeast using
The present invention was completed by isolating and analyzing transformed yeast.

The PGK gene of Zatsucharomyces yeast has already been
It has been cloned by Eman et al. (Hitze
man, R. A. , Clarke, L. , &carbo
n, j. j. Biol, Chem, 255:12073
(1980)). According to them, the PGK gene consists of approximately 3.1 kb of D
It exists in the NA fragment, and this fragment is further cleaved at one site by EcoR1, two sites by Sall, and one site by Bg1■.

In addition, the nucleotide sequence near the 5' end of the PGK gene and a portion upstream thereof was published by Dobson et al. (Dohson, M.J.
tal. , Nucleic Acids Res. 10
:2625-2637 (1982)).

In addition, the 4th GIM (Fourth
International Symposium o
n Genetics of Industrial M
icroganisms: June 6-11, 19
82, Kyoto, Japan) and Goeddel (G
Enentech, Inc. has made an oral announcement, but the detailed manufacturing method has not been disclosed. Therefore, the inventors
This study specifically demonstrates that the PGK gene was cloned into a monomer by the method described below, and that the PGK gene promoter is effective for producing homologous or heterologous peptides in yeast.

A method for producing yeast vectors according to the present invention will be described below.

First, nuclear DNA of Zatucharomyces yeast XS16-5c strain
is cut with restriction yeast Hind■. Among the obtained fragments, a DNA fragment having a length of about 3.1 kb was cloned into the Hind■ cut point of pBR322 to obtain a yeast gene library (E. coli) 301 clone. Next, one plasmid having a DNA fragment that is cleaved at one site with EcoRI, two sites with SalI, and one site with Bg1ll among the cloned DNA fragments of approximately 3.1 kb is obtained. This plasmid was extracted using the Maxman-Gilbert method (PNAS
74:560-564 (1977)), and the base sequence was determined by Hitzeman et al. and Dobson et al.
By comparing the results with those of et al., it can be confirmed that the target PGK gene is present.

The plasmid into which the PGK gene has been inserted is cut with a specific restriction enzyme, and the DNA fragment containing the gene is inserted into a yeast vector having a selection marker, preferably an E. coli-yeast shuttle vector for convenience of operation.

Next, two Sa in the PGK gene in this vector
At the Sa1I cleavage site on the N-terminal side of the 1I cleavage point,
In order to match the reading frame, the purpose obtained in advance is BamH containing the peptide gene.
Insert the DNA fragment cut with I and SalI. Yeast is transformed with the thus obtained plasmid containing the PGK gene and the target peptide gene.

The purpose is to include both an exogenous heterologous peptide gene and a peptide gene (homologous peptide gene) other than the PGK gene in yeast as a peptide gene.

A part of the base sequence of the PGK gene (the part near the 5' end and part upstream thereof, and the base sequence corresponding to positions 270 to 400 of the amino acid sequence) has been shown by Dobson et al., but the entire base sequence is unknown. Therefore, when trying to obtain the desired peptide by inserting the same or different gene into the structural gene region, it is necessary to be creative (because if the reading frame does not match, the desired peptide will not be obtained). (cannot be obtained). Therefore, of the two SalI cleavage points in the PGK gene, the SslI cleavage point (a site corresponding to about 240 amino acid residues from the n-terminus), which is closer to the N-terminus, has a reading mark.
Three target genes (α-neoendorphin gene in the example of the present invention) are cloned by shifting the frame by one. If we shift the reading frame one by one in this way, we should be able to obtain one of the three genes whose reading frame matches the reading frame from the beginning of the PGK gene. In fact, in this way, the inventors constructed a plasmid vector (pYαNE61-
C) has been obtained, and the above method has been implemented correctly. The target peptide can be produced by transforming yeast with the plasmid thus obtained and culturing it.

In the transformed yeast, the PGK gene promoter was found to be extremely effective in expressing the target peptide gene.

The present invention will be explained in more detail by the following examples.

In a specific example, the alpha neoendorphin (αNE) gene is used as the target peptide gene, and the target substance (
The method for producing αNE) is described, but the target gene is
For example, the target peptide can be directly produced by adding a gene encoding a peptide such as interferon. As described above, the PCK promoter can be widely used for the expression of foreign genes and for the production of heterologous and homologous peptides other than alpha neoendorphin.

Example 1. Cloning of PGK gene (see Figure 1) PGK
The gene has already been cloned by Hitzeman et al. (R.A.Hitzeman, L.Clar
Keand J. Carbon: J. Biol. Che
m. 255 12073, 1980), is present in an approximately 3.1 kb fragment that is cleaved with Hind■, one site with ECoR■, two sites with SalI, and one site with Bgl■.

It was also cloned by Dobson et al. (Nuc
leic Acid Research 10, 2625
, 1982), part of the base sequence and part of the amino acid sequence are shown. Therefore, an attempt was made to select from the Hind ■ cleavage fragment gene bank based on the results of Hitzeman et al.

Satucharomyces yeast XS16-5C [cir°] (S
accharomyces cerevisiao
16-5C: MATa leu 2 his3 trp
1 (cir°), Suntory Biomedical Research Institute preserved strain)
The cells obtained by culturing 2lYPD medium (1% yeast extract, 2% polypeptone, 2% glucose) at 30°C for 24 hours were cultured using the method of Cryer et al.
Cell Biology, Vol. 12.39-44
, 1975). this DNA
50 μg was incubated at 37°C for 2 hours using 100 units of Hind■.
It was cut by heating for an hour. The reaction solution was separated by 0.8% agarose gel electrophoresis, and 2.9 kb to 3
．． A DNA fragment corresponding to 2 kb was obtained. On the other hand, 0.5μ
pBR322 was cleaved using 1 unit of Hind 1 in a TA buffer at 37° C. for 1 hour.

Next, both DNAs were dissolved in 20 μl of T4 DNA ligase buffer, 2 units of T4 DNA ligase was added, and 15 μl of T4 DNA ligase was added.
The reaction was carried out at ℃ for 18 hours. Using this reaction solution as donor DNA, E. The clone was transformed into E.coli JA221 to obtain an ampicillin-resistant clone.

Among them, 301 clones sensitive to tetracycline were selected, and their plasmid DNA was analyzed using the alkaline denaturation method (Nucleic Acid Research).
ch7.6.1979). Hitzem
According to the results of an et al., approximately 3.1 kb containing the PGK gene
The fragment contains EcoRI (1 site) SalI (2 sites) B
There is one (1) cutting point.

As a result of testing 301 clones, one clone was obtained that had a plasmid having the above-mentioned restriction enzyme cleavage site. The plasmid of this clone was named pYpgk301.

The base sequence of the part corresponding to the upstream part of the PGK gene on pYpgk301 was determined according to the Maxam-Gilbert method, and the base sequence was compared with the base sequence of the upstream part of the PGK gene reported by Dobson et al.
It was confirmed that pgk301 has the PGK gene. E. coli K- transformed with this pYpgk301
The 12 strains were named E. coli SBM 152 and deposited with the Institute of Microbiology, Agency of Industrial Science and Technology (Feiken), with deposit number FE.
RM P-6763 has been obtained.

2. Construction of pYE237 plasmid vector pYE2
37 The vector is a plasmid obtained by deleting the Sal■ break point from the pYE227 vector disclosed in Japanese Patent No. 56-167615, and was produced as follows.

TA of 5 μg pYE227 using 10 units of SalI
The mixture was reacted in a buffer solution at 37°C for 1.5 hours and then cleaved.

Subsequently, SalI was inactivated by heating at 65°C, and then four types of dNTPs were added to 0.3mM and 2-mercaptoequenol was added to 80mM, and 1 unit of T4DN was added.
A polymerase was used to react at 37°C for 30 minutes, and Sa
The sticky ends generated by II cleavage were eliminated. After the reaction was completed, phenol extraction was performed once, and DN was extracted with 2 volumes of ethanol.
A was precipitated.

After dissolving the DNA precipitate in 20 μl of T4 DNA ligase buffer, incubate at 15°C with 5 units of T4 DNA ligase.
They were bound by reacting for a period of time.

Using the reaction solution as donor DNA, E. coli JA
221 to obtain an ampicillin-resistant clone. DNA was isolated and analyzed from these clones, and pYE
pYE237 with the SalI cleavage site deleted from 227
I got it.

3. Construction of pYE1301 yeast-E. coli shuttle vector (see Figure 2) 5 μg of pYE237 was cut with 20 units of Hind■, and 5 μg of pYpgk301 was digested with 20 units of H
After cutting with ind■, 3.1 was determined by agar electrophoresis.
Isolate and purify the Kb DNA fragment (PGK gene) and dissolve it in 20 μl of DNA ligase buffer.
1 unit of T4 DNA ligase was added and reacted at 15°C for 16 hours. Add 10 μl of this reaction solution to 0.3 ml of CaCl.
2 treated E. In addition to coli TA221, transformation was performed. Plasmid DNA was separated and analyzed from the transformant according to a conventional method to obtain pYE1302.

This plasmid vector pYE1301 was used to transform Saccharomyces yeast strain XS16-5C, and the transformant was transformed into Saccharomyces cerevisi.
It was named aeSBM331 and deposited at the National Institute of Fine Technology.

(Deposit number: FERM P-6767) 4 Preparation of plasmid vector pYENE61c into which αNE gene was inserted (see Figure 2) Cut 5 μg of pYE1301 into 3 fragments with 20 units of BamHI and 20 units of SalI and perform agar electrophoresis. After separation, the largest fragment was eluted from agar and purified. On the other hand, the fragment containing the αNE gene is pα
50 μg each of NE-SalI-a, -b, -c at 1
After cutting with 00 units of BamHI and 100 units of SalI, the DNA fragments containing the αNE gene (corresponding to 48 base pairs, 49 base pairs, and 50 base pairs, respectively) were separated by electrophoresis using a 5% polyacrylamide gel. D.N.
A fragment) was isolated and purified, and the DNA fragments containing the three types of αNE genes and the previous pYE1301
The Hind■ fragment was dissolved in 20 μl of DNA ligase buffer, 2 units of T4 DNA ligase was added, and the mixture was reacted at 15° C. for 16 hours. Add 10 μl of this reaction solution to 0.3 ml of Ca.
Cl2-treated E. In addition to coli JA221, transformation was performed. Plasmid DNA was isolated and analyzed from the obtained ampicillin-resistant transformant, and pYαNE61-a
, -b, -c were obtained.

Plasmids pYE1301 and pYαNE6 obtained above
1-a, -b, -c from Beggs. J. D. Nature
e, Vol. 275, 104 (1978).
isiae XS16-5C). The obtained transformant was cultured with shaking in YPD (1% yeast extract, 2% polypeptone, 2% glucose) medium at 30° C. for 24 hours, and then the bacterial cells were collected by centrifugation. 0.5 ml of cold acetone was added to the bacterial cells obtained from 1 ml of the culture solution, and after standing at -20°C for 1 to 24 hours, the acetone was removed under vacuum. 5 mg of dried bacterial cells obtained in this way
/ml cyanogen bromide in a 70% formic acid solution,
The reaction was allowed to proceed in the dark for 4 hours. After freeze-drying this sample, 0.1
Elute αNE with 0.1 ml of 0.1 N acetic acid and add 0.1 ml of 1.
After neutralization with 3M Tris-HCl buffer, it was used as a sample for radioimmunoassay (RIA). RIA is N. Minamin
o et al. BBRC Vol 102 226 (
It was carried out according to the method described in (1981).

The results are shown in Table 1. High αNE productivity was observed in pYαNE6.1-c. pYαNE61-a, -b had the same level of radioimmunoreactivity as pYE1301, which did not contain the αNE gene, as a control. Therefore, the reading is that pαNE-Sal■-c is this Sa of PGK.
It was found that this matched the lI cleavage site. pYαNE
The αNE productivity of 61-c was approximately 2 million molecules per single cell. This was approximately the same productivity as the case using the GAP-DH gene, which was conducted separately.

[Brief explanation of the drawing]

Figures 1 to 3 are diagrams showing a method of constructing a plasmid containing the PGK gene and incorporating the αNE gene. Figure 1 shows the cloning of the PGK gene. FIG. 2 shows a method for constructing a plasmid containing the PGK gene and incorporating the αNE gene. Figure 3 shows a method for constructing a plasmid to which the 3'-end untranslated site of the PGK gene is added. Patent applicant Suntory Ltd. agent Patent attorney Kyozo Yuasa (and 4 others) Procedural amendment October 19, 1980 Commissioner of the Japan Patent Office Kazuo Wakasugi 1. Display of the case 1984 Patent Application No. 184292 2 Name of the invention Yeast expression vector plasmid using a glycolytic enzyme-controlled gene promoter and its method of use 3 Person making the amendment Relationship to the case Patent applicant address name (190) Suntory Stock Company 4 Agent Address Room 206, Shin-Otemachi Building, 2-2-1 Otemachi, Chiyoda-ku, Tokyo (Telephone: 270-6641-6) Name (2770) Patent Attorney Kyozo Yuasa [Details of the invention in the specification subject to the 5th amendment [brief explanation] and [brief explanation of the drawing]
Contents of the supplement to Figures 2 and 3 of the drawings (1) The statement in the specification is corrected as follows. Page Line Before correction After correction 6
4-5 Above mentioned... (Deleted)
and 7 5-6 and amino acids (deletion)
Base sequence corresponding to the sequence... 10-11 From the beginning of reading Frame of from the beginning of reading Reading frame 13 5 Hind■ Hind■
13 TA■ JA22114
2 pYαNE61c pYαNE61c
18 E. Coli E. coli
17 8-9 Figure 3 is... (Deleted)
shows. (2) Figure 3 in the drawings will be deleted and Figure 2 will be corrected as shown in the attached drawings. that's all

Claims (1)

[Scope of Claims] 1) A plasmid containing a promoter of a gene controlling 3-phosphoglycerokinase and expressing a target peptide gene in yeast. 2) A method of producing a target peptide by preparing a plasmid containing the promoter of the gene controlling 3-phosphoglycerokinase and the target peptide gene, and culturing yeast cells transformed with this plasmid.