JPH08168380A - Dna fragment for vector plasmid highly expressible in yeast - Google Patents

Abstract

PURPOSE: To obtain the DNA fragment containing a specific range of upstream on 5' side of glyceroaldehyde-3-phosphatedihydrogenase structural gene of a yeast and extraneous gene and capable of expressing extraneous gene in remarkably high efficiency by introducing to yeast. CONSTITUTION: This new DNA fragment comprises a non-translation region up to either base pair of about 350-750 base pairs of further upstream on 5' side than about 150 base pairs on 5" side of glyceroaldehyde-3-phosphite dehydrogenase and extraneous gene. The DNA fragment comprises integrating into a vector in a state capable of expressing in a yeast, transforming vector in a vector plasmid and culturing the transformer. The fragment is capable of expressing polypeptide or protein coded by extraneous gene, etc., in remarkably high efficient compared with the case in which conventional promoter is used. The DNA fragment is obtained by treating nucleic DNA of Saccharomyces yeast XS16-5C with restriction enzyme and binding the treated DNA to a vector and cloning the prepared gene library.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a promoter for a gene (operon) that controls glyceraldehyde-3-phosphate dehydrogenase (GAP-DH), one of the glycolytic enzymes of the yeast Saccharomyces. And a useful target peptide gene. More specifically, the present invention relates to a DNA fragment comprising an untranslated region 5 'further upstream than the base codon of the GAP-DH structural gene by 150 base pairs and a foreign heterologous gene.

[0002]

2. Description of the Related Art Pharmaceutically useful peptides, such as somatostatin, insulin, growth hormone, various interferons, influenza virus, hepatitis B virus protein, thymosin α, have been hitherto used by genetic engineering techniques.
Many substances such as ₁ , β-endorphin, α-neoendorphin, secretin, urokinase, and plasminogen activator are produced by microorganisms or animal cells.

Many of these are prokaryotic (pr) cells as hosts.
E. coli, which is an okaryote, is used, but in recent years, attention has been paid to the use of a yeast, which is a nucleated cell, as a host. This is due to the fact that the yeast culture conditions are simple, the division rate is high, the safety is high, and the genetic biochemical analysis is particularly performed more frequently for Saccharomyces yeast. However, there are few examples of foreign heterologous peptide production in yeast, and α-interferon, HB hepatitis surface antigen protein,
It is the level found in α-neoendorphin.

On the other hand, a foreign heterologous gene, for example, a chemically synthesized gene, a complementary DNA (cDNA) gene obtained from mRNA by reverse transcriptase, or a gene obtained from chromosome by appropriate treatment is to be expressed. Requires the presence of a promoter region suitable for the host used. Since the quality of a promoter greatly affects the expression of a target peptide gene, expression plasmid vectors have been developed using various promoters.

[0005]

However, the reported foreign gene expression vector using yeast as a host is not sufficiently efficient in expressing a desired foreign gene as compared with a vector using Escherichia coli as a host.

[0006]

Means for Solving the Problems The present inventors have developed glyceraldehyde-3-phosphate dehydrogenase (G), which is an enzyme that controls the glycolysis system of yeast Saccharomyces.
We focused on the fact that AP-DH) was produced in large amounts in yeast cells, and studied the use of the operon promoter gene encoding this enzyme, and proceeded with research. As a result, they found that this promoter was very useful for the production of 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. The present invention was completed by performing yeast transformation using the obtained yeast and separating and analyzing the transformed yeast.

The DNA fragment of the present invention and the yeast vector into which the DNA fragment has been inserted are prepared as follows.

A DNA fragment corresponding to 1.9 kb to 2.2 kb obtained by cleaving the nuclear DNA of Saccharomyces yeast XS 16-5C strain [cir ⁰ ] with a restriction enzyme HindIII is used as a suitable plasmid vector, for example, plasmid pBR.
322 was cloned into the HindIII site to obtain a yeast gene library. A yeast DNA fragment having a length of 2.1 kb is obtained from the gene library, and the DNA fragment has one restriction enzyme SalI and one restriction enzyme H.
A clone having a fragment cleaved at one site with paI was isolated. This plasmid uses the Maxam-Gilbert method (PNAS74, 560) for the inserted DNA fragment.
-564 (1977)), and the result was determined by Holland et al., J. Biol.
Chem. 254,9839 (1979) ", it can be confirmed that the desired GAP-DH gene has been inserted.

The plasmid into which the GAP-DH gene has been inserted in this manner is digested with a specific restriction enzyme, and the DNA fragment containing the gene is converted into a yeast vector having a selectable marker, preferably an Escherichia coli-yeast shuttle for convenience of operation. Insert into vector.

Next, the reading frame is fitted to the restriction enzyme SalI cleavage site near the C-terminus of the GAP-DH gene in this vector so that the reading frame fits.
A fragment containing the desired peptide gene of interest is inserted. The yeast is transformed with the plasmid containing the GAP-DH gene and the target peptide gene thus obtained.

[0011] The target peptide gene includes both an exogenous heterologous peptide gene and a peptide gene other than the GAP-DH gene in yeast (a homologous peptide gene).

Here, the foreign heterologous peptide gene includes a gene in which a part of the GAP-DH gene and the foreign gene are fused.

In the thus transformed yeast,
It was confirmed that the GAP-DH gene promoter was very excellent in expressing the target peptide gene.

That is, 150 base pairs 5 'upstream of the GAP-DH structural gene disclosed by Holland et al.
BC 254 , 9839-9845, 1979, specifically p. 9842), by using a region containing an untranslated region further 5 ′ upstream as a promoter region.
It can be said that extremely high heterologous peptide or protein productivity was observed.

It has also been recognized that the productivity of the target peptide in yeast is significantly increased by adding the 3'-terminal untranslated region of the GAP-DH gene downstream of the target peptide gene in the plasmid. The effect of the addition of the 3'-terminal untranslated region is described in Japanese Patent Application No.
This is described in detail in US Pat.

The method of using the 3'-terminal untranslated region as described above, a plasmid into which this region is inserted, and its use are also included in the scope of the present invention.

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

In a specific example, the alpha neoendorphin (αNE) gene is used as the target peptide gene, and GAP-DH promoter-GAP-DH structural gene-αNE gene (abbreviated as pGAP-GAP-αNE).
And pGAP-GAP-αNE-3′GAP (GAP
A method for producing a target substance (αNE) as a GAP-αNE hybrid protein from a sequence such as a GAP-αNE hybridized protein).
It is also possible to directly produce the target peptide by adding a target gene, for example, a gene encoding a peptide such as interferon, with an appropriate nucleotide sequence from the position of the -DH promoter. Thus, the GAP-DH promoter can be widely used for the expression of foreign genes and the production of heterologous and homologous peptides other than alpha neoendorphin.

[0019]

[Example] 1. Cloning of GAP-DH gene (see FIG. 1) Saccharomyces yeast XS16-5C [cir ⁰ ] (S
accaromyces cerevisiae X
S16-5C MATa leu2 his3tryp
1 [sir ⁰ ]) in 1 liter of YPD medium (1% yeast extract, 2% polypeptone, 2% glucose) at 30 ° C., 2
After culturing for 4 hours, the obtained cells are cryo
r) et al. (Cryer et al., Isola).
Tion of yeast DNA, Methods
in Cell Biology, Vol. 12, A
Cademic Press, New York, 19
75, pp 39-44).

50 μg of yeast DNA was added to 100 units of Hi
Cleavage was performed by heating at 37 ° C. for 2 hours using ndIII. The reaction solution was separated by 0.8% agarose gel electrophoresis, and the Hind corresponding to 1.9 kb to 2.2 kb was obtained.
The DNA fragment according to III was separated and purified. On the other hand, 0.5μ
g of pBR322 using 1 unit of HindIII, in a TA buffer (33 mM Tris acetate, pH 7.9, 66 mM potassium acetate, 10 mM magnesium acetate, 0.5 mM DT).
Cleavage was carried out at 37 ° C. for 1 hour in T). Next, both these DNAs were added to a buffer for T4 DNA ligase (20 mM Tris-HCl, pH 7.5, 10 mM Mg
Cl ₂ , 10 mM DTT, 0.5 mM ATP), add 2 units of T4 DNA ligase, and add 1 unit at 15 ° C.
The reaction was carried out for 8 hours. 10 μl of this reaction solution was treated with 0.3 ml of CaCl ₂ . In addition, E. coli JA221 was used for transformation to obtain an ampicillin-resistant clone. Of these, 4 clones sensitive to tetracycline
00 were selected and the plasmid DNA was analyzed by the alkali denaturation method.

According to the analysis results of Holland et al. Already reported, the 2.1 kb fragment containing the GAP-DH gene has one HpaI and SalI cleavage site, respectively. The 2.1 kb HindIII fragment was cut by SalI into two fragments of 0.14 kb and 1.96 kb, and cut by HpaI into two fragments of 1.39 kb and 0.71 kb, respectively.

The present inventors examined the above-mentioned 400 clones and found that one clone having a plasmid having the same restriction enzyme cleavage site as that of the fragment containing the GAP-DH gene was obtained. . The plasmid of this clone was designated as pYgap87.

The sticky ends of the pYgap87 near the SalI cleavage site obtained by HindIII cleavage are labeled with γ- [ ³² P] -ATP and polynucleotide kinase, and the DNA nucleotide sequence is determined according to the Maxam-Gilbert method. did. The results were compared with the report of Holland et al., And pYgap87 was GAP-DH.
We confirmed that we had the gene. The E. coli K-12 strain transformed with this pYgap87 is E. coli K-12. coli
SBM151 , deposited with the Research Institute of Microbial Industry (MIC) of the National Institute of Advanced Industrial Science and Technology, and deposited number FERM P-6762.
(Accession number FERM of international deposit based on the Budapest Treaty
BP-382).

2. pYE237 plasmid vector
The pYE237 vector is a plasmid in which the SalI cleavage point has been eliminated from the pYE227 vector disclosed in Japanese Patent Application No. 56-167615, and was prepared as follows.

5 μg of pYE227 was added to 10 units of Sal
Using I, the reaction was carried out in a TA buffer at 37 ° C. for 1.5 hours for cleavage.

Subsequently, by heating at 65 ° C., Sal
After inactivation of I, 4 dNTPs were added at 0.3 mM;
Mercaptoetal was added to a concentration of 8 mM, and 1 unit of T was added.
Reaction was carried out at 37 ° C. for 30 minutes using 4 DNA polymerase to eliminate sticky ends generated by SalI digestion. After completion of the reaction, phenol extraction was performed once, and DNA was precipitated with 2 volumes of ethanol. 20 μl T of DNA precipitate
After dissolving in 4 DNA ligase buffer, 5 units of T4 DNA
The reaction was carried out using ligase at 15 ° C. for 18 hours for binding.

The reaction solution was used as a donor DNA according to a conventional method.
E. coli JA221 was transformed to obtain an ampicillin-resistant clone. DNA was separated and analyzed from these clones to obtain pYE237 in which the SalI cleavage site had disappeared from pYE227.

3. pYE1201 yeast-E. coli shuttle
Preparation of Better (see FIG. 2) 5 μg of pYE237 was reacted with 20 units of HindIII in a TA buffer at 37 ° C. for 1 hour. Thereafter, the reaction of the restriction enzyme was carried out in a TA buffer at 37 ° C. for 1 hour.
On the other hand, the previously obtained plasmid pYgap87 5μ
g was cut with 20 units of HindIII, and then a 2.1 kb DNA fragment (GAP-
DH gene) and DNA was eluted from the agar piece and purified. This 2.1 kb DNA fragment and Hi of pYE237
The ndIII-digested DNA was dissolved in 20 μl of DNA ligase buffer, and 1 unit of T4 DNA ligase was added thereto.
The reaction was performed for 16 hours. 0.3 ml of this reaction solution (10 μl)
Was treated with CaCl ₂ . E. FIG. E. coli JA221 and transformation was performed.

From the ampicillin-resistant transformant, plasmid DNA was isolated and analyzed by a conventional method, and pYE
It was confirmed that 1201 was obtained.

The yeast (XS16-5C) strain transformed with this plasmid was Saccharomyces.
cerevisiae SBM321, and deposited with the Japan Fine Art Institute as deposit number FERM P-6766 (accession number FERM BP-381 of the international deposit based on the Budapest Treaty).

[0031] 4. Plasmid DNA containing αNE gene
Preparation of (pαNE5′-SalI-c) Nucleic Acid Research 10
ΑNE according to the method of 1741 to 1754 (1982)
A gene (EcoRI-BamHI DNA fragment) was prepared.

The αNE gene consisting of 46 base pairs thus obtained was ligated with EcoRI / BamHI of pBR322.
The fragment was inserted into the larger fragment (4 kb) digested with both restriction enzymes to obtain pYαNE5 ′. Next, to align the reading frame with the SalI site in the GAP-DH gene and insert it, 5 ′
-Plasmid pYαNE5 '(SalI) into which a SalI linker consisting of GGGTCGACCC-3' was inserted-
I got c. 5 μg of pYαNE5 ′ was added to 10 units of Eco.
It cut | disconnected by heating at 37 degreeC for 1 hour in TA buffer using RI. After heating at 65 ° C, 0.3 mM dAT
P, dTTP and 8 mM of 2-mercaptoethanol were added, respectively, and reacted at 37 ° C. for 30 minutes to obtain Ec.
The sticky end of oRI was filled. After completion of the reaction, phenol extraction was performed once, and then DNA was recovered by ethanol precipitation. On the other hand, 1 μg of the SalI linker was added at 25 nmo in 50 mM Tris-HCl, pH 7.5, 10 mM MgCl _2.
The mixture was reacted at 37 ° C. for 45 minutes using 1 ATP and 10 units of polynucleotide kinase to phosphorylate the 5 ′ end. These DNAs were mixed, and used as a T4 DNA ligase buffer, and 5 units of T4 DNA ligase was added and reacted at 15 ° C. for 18 hours. 10 μl of this reaction solution was added to 0.3 ml of Ca
Cl ₂ treated E. E. coli JA221 and transformation was performed. Plasmid DNA was separated and analyzed from the ampicillin-resistant transformant according to a conventional method, and pYαNE5′-S
all-c was obtained.

5. Alpha neoendorphin (αN
E) Insertion of gene (see FIG. 2) 5 μg of pYE1201 in 20 units of BamHI, 20
The fragment was cleaved into two fragments with a unit of SalI, separated by agar electrophoresis, and the large fragment was eluted from the agar for purification. On the other hand, the fragment containing the αNE gene is pαNE5′-SalI-c.
50 μg for 100 units of BamHI, 100 units of S
After digestion with alI, DN corresponding to 50 base pairs separated by electrophoresis on 5% polyacrylamide gel
It was obtained by elution and purification of the A fragment. Dissolve the above two DNA fragments in 20 μl of DNA ligase buffer and add 2 units of T
4DNA ligase was added and reacted at 15 ° C for 16 hours.
10 μl of this reaction solution was treated with 0.3 ml of CaCl ₂ treated with E. E. coli JA221 and transformation was performed, and the ampicillin-resistant transformant was transformed into plasmid DN by a conventional method.
A was separated and analyzed to obtain pYαNE53.

That is, the promoter region for producing α-neoendorphin as a fusion protein with a part of the GAP-DH enzyme in the present plasmid is a 5 ′ terminal disclosed by Holland et al. This indicates that the gene contains an untranslated region 5 'further upstream than the start codon (150 base pairs upstream). There is no known example in which a heterologous peptide or protein is expressed very efficiently in yeast using such a promoter region.

6. P of the 3 'terminal region of the GAP-DH gene
Addition to YαNE53 (see FIG. 3) A specific example of adding the 3 ′ terminal region of the GAP-DH gene to pYαNE53 downstream of the αNE gene in which pAP is incorporated is shown below.

10 μg of pYαNE53 is added to 30 units of B
After cleavage with amHI, 67 mM Tris-HCl (pH 8.8), 6.7 mM using 1 unit of T4 DNA polymerase
MgCl ₂ , 10 mM _2- mercaptoethanol 1
6.6 mM ammonium sulfate, 6.7 μM EDTA, 0.
The reaction was carried out at 37 ° C. for 30 minutes in a buffer (T4 DNA polymerase buffer) containing 3 mM dNTPs.
The sticky ends generated by the I cleavage were filled. On the other hand, the 3'-terminal region of GAP-DH gene was obtained from pYE1201 at 10 μg.
Of pYE1201 was digested with 30 units of SalI, and 37 ° C. in the same buffer using 1 unit of T4 DNA polymerase.
The reaction was allowed to proceed for 30 minutes to fill in the sticky ends generated by SalI digestion. Then, cut each with 30 units of PstI and pY
From αNE53, a fragment containing the αNE gene, pYE12
From 01, a fragment containing the 3 'terminal region of the GAP-DH gene was separated and purified by agarose gel electrophoresis. Then, both fragments were ligated by reacting with 5 units of T4 DNA ligase in 20 μl of T4 DNA ligase buffer at 15 ° C. for 17 hours. Using 10 μl of this reaction solution, a transformant was obtained by the above method, and plasmid DNA was separated and analyzed from it to confirm that pYαNE155 was obtained.

[0037] 7. Transformation and culture of yeast Plasmid pYE1201, obtained as described above,
pYαNE53 and pYαNE155 were purchased from Beggs J .;
D. , Nature, Vol. 27 5 104 (19
78) according to the method described in (78).
S. cerevisiae XS16-5C). After the obtained transformant was cultured with shaking in YPD medium at 30 ° C. for 24 hours, the cells were collected by centrifugation. 0.5 ml of cold acetone was added to the cells obtained from 1 ml of the culture solution, and -2
After leaving at 0 ° C. for 1 to 24 hours, it was freeze-dried. The dried cells were suspended in a 70% formic acid solution containing 5 mg / ml of cyanogen bromide and reacted in the dark for 24 hours. This sample was lyophilized, αNE was eluted with 0.1 ml of 0.1N acetic acid, neutralized with 0.1 ml of 1.3 M Tris-HCl (pH 8.0), and used as an RIA sample. RIA is N.A. Minamino
et al, BBRC Vol. 102 226 (1
981). The results are shown in Table 1.
It was shown to. In the case of pYαNE53 (GAP-DH), a hybrid protein using the GAP-DH promoter and binding αNE immediately after its N-terminal (323 amino acid residues)
In the absence of the 3'terminal region of the gene), 2 million molecules were expressed per cell. Furthermore, the present inventors have studied the GAP-DH in the experiment for modifying the promoter region in the present invention.
The production amount of α-neoendorphin was about 300 Gbp and 480 Gbp upstream of the start codon of the structural gene as the promoter region.
It was found that the production amount was reduced to 1/10 (1/10) of the production amount when about 900 base pairs 5 ′ upstream of the initiation codon of the -DH structural gene was used as the promoter region.

GAP-DH was added to the above-mentioned pYαNE53.
A gene with an added 3 ′ terminal region (pYαNE15
For 5), 20 million molecules were expressed per cell, which was about 10 times that of -pYαNE53. About 2 μg / ml production was observed per culture solution. The stability of the plasmid was examined.
Complete GAP-DH when cultured in D medium)
10-20% for plasmid pYE1201 with
And was quite unstable. However, a plasmid (pYαNE53, pYα
In the case of NE155), the stability was somewhat improved.

[0039]

[Table 1]

[Brief description of drawings]

FIG. 1 shows the cloning of the GAP-DH gene.

FIG. 2 shows a method for constructing a plasmid containing a GAP-DH gene and incorporating an αNE gene.

FIG. 3 shows a method for constructing a plasmid in which the 3′-terminal untranslated site of the GAP-DH gene is added downstream of the αNE gene on the plasmid pYαNE53 obtained in FIG.

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[Procedure amendment]

[Submission date] May 25, 1994

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Fig. 4

[Correction method] Added

[Correction content]

FIG. 4 is a diagram showing Hin from the gene library in FIG.
It is a figure which shows the restriction enzyme cut map of the DNA fragment cut out by dIII.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Name of item to be corrected] Fig. 4

[Correction method] Added

[Correction content]

[Figure 4]

Claims (6)

[Claims] 1. A glyceraldehyde-3-phosphate dehydrogenase structural gene of yeast, 5 ′ upstream of which is about 15
A DNA fragment consisting of an untranslated region from 0 base pairs to the 5 ′ upstream side and a foreign gene. 2. The untranslated region further comprises about 35 bases upstream of about 150 base pairs 5 ′ upstream of the structural gene.
The DNA fragment according to claim 1, which is a region up to any base pair of 0 to 750 base pairs. 3. The untranslated region comprises about 75 base pairs upstream of about 150 base pairs 5 ′ upstream of the structural gene.
The DNA fragment according to claim 1, which is a region of up to 0 base pairs. 4. About 15 upstream of the 5'-side of the yeast glyceraldehyde-3-phosphate dehydrogenase structural gene.
A yeast expression plasmid comprising a DNA fragment consisting of an untranslated region from 0 base pairs to the 5 ′ upstream side and a foreign gene. 5. The untranslated region further comprises about 35 bases upstream of about 150 base pairs 5 ′ upstream of the structural gene.
The plasmid according to claim 4, which is a region from 0 to 750 base pairs to any base pair. 6. The untranslated region further comprises about 75 base pairs upstream of about 150 base pairs 5 ′ upstream of the structural gene.
The plasmid according to claim 4, which is a region of up to 0 base pairs.