JPH07163373A - Multi-cloning vector, manifestation vector and production of foreign protein - Google Patents

Abstract

PURPOSE:To obtain the subject vector having a promoter region capable of functioning in eucaryotic cells, a multi-cloning site used for introducing a foreign protein structural gene, and recognition site for a specific restriction enzyme, and capable of expressing the foreign protein in a high yield. CONSTITUTION:On introducing a foreign protein structural gene governed by a promoter in down stream of the promoter region of a vector having the promoter capable of functioning in eucaryotic cells, this vector is constituted by forming a ligated portion having a sequence of -ACATGN- by ligating a tip of the foreign protein structural gene cleaved at a restriction enzyme recognition site having a sequence of from 5' side -N'CATGN- (where N is an arbitrary base; N' is a complimentary base to N) to an end of the vector cleaved at a restriction enzyme recognition site having a sequence of from 5' side -ACATGT-, by making the ATG an initiation site for translation, and by making a base next to the ATG as an arbitrary base in combining the cleaved end of restriction enzyme recognition site of the vector side and the cleaved tip of the restriction enzyme recognition site of the foreign protein structural gene side.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a multicloning vector capable of producing a heterologous protein by the fission yeast Schizosaccharomyces pombe (hereinafter referred to as S. pombe), and an expression vector into which a heterologous protein gene is introduced. , A method of constructing the expression vector, S. The present invention relates to a pombe transformant and a method for producing a heterologous protein using the transformant. INDUSTRIAL APPLICABILITY According to the present invention, the expression of genes of various heterologous proteins can be made efficient and the production cost of the produced peptide or protein can be reduced.

[0002]

2. Description of the Related Art Production of a heterologous protein using gene recombination technology is carried out by Escherichia coli (hereinafter referred to as E. coli), Saccharomyces cerevisiae, or Bacillus (Bacillus).
cillus) microorganisms such as genus, animal cells (including insect cells),
It has been actively carried out using plant cells. Polypeptides derived from various organisms are considered to be targets, and many have been industrially produced and used as pharmaceuticals and the like.

However, the method using prokaryotes is not always effective for all polypeptides, and it is not always easy to reproduce complicated post-translational modifications of eukaryote-derived proteins or the same three-dimensional structures as natural bodies. Absent. In fact, it is known that the production and the activity of the products produced in prokaryotic cells are heterogeneous, and thus their use in medicines is rejected (Proc.Natl.Acad.Sci.USA, 86. , 3428-34
32 (1989)). Also, E. Unique endotoxin is present in E. coli and may be a contaminant in the final product.

The method using animal cells or plant cells is more difficult to handle than microorganisms, the culture is costly, and the resulting cell concentration is low, resulting in poor production efficiency. Therefore, the most ideal organism for producing a heterologous protein, in particular, a polypeptide derived from eukaryote is the yeast which is a microorganism and a eukaryote. Yeast has a well-established culturing method and is convenient in that it can express the genetic information of eukaryotes. Furthermore, it has been used in the fermentation and food industries for a long time, the safety for human body has been established, and it is characterized by not containing endotoxin.

[0005]

Among the yeasts, S.
Pombe is believed to have properties closer to animal cells than Saccharomyces cerevisiae. Therefore, as a host for expressing a heterologous protein, S. It is expected that by using the pombe, a gene product similar to that in the animal cell, which is closer to the natural form, can be obtained. The culturing methods also have a lot in common with yeasts, and the knowledge known for other yeasts can be easily applied. Therefore, using methods of microbiology and recombinant DNA technology, S. Obviously, it is advantageous to use a method for heterologous protein production using pombe.

However, S. Studies on gene recombination using pombe are described in E. coli and Saccharomyces
It lags far behind S. cerevisiae, and there are few studies on gene expression. For example, JP-A-61-181
There are only 397, JP-A-2-283288, and JP-A-4-63596. This is because there is no expression vector that has a strong promoter, is stably present in the bacterial cell, and is optimal and convenient for gene transfer.

The present inventors have proposed an expression vector capable of solving the above problems (see Japanese Patent Laid-Open No. 15380/1993). However, it cannot be said that the expression vector is sufficient for expressing a heterologous protein gene without excess or deficiency, and further improvement of the expression vector is required.

[0008]

Means for Solving the Problems As a result of investigations in view of the above points, the present inventor has found that the heterologous protein gene of interest can be replaced by modifying the translation initiation site of the heterologous protein gene into a restriction enzyme recognition site. We have constructed an expression vector that can express without deficiency. The present invention relates to S. po
A multi-cloning vector capable of producing a heterologous protein by mbe, an expression vector into which a heterologous protein gene is introduced, a method for constructing the expression vector, and an S. The present invention relates to a pombe transformant and a method for producing a heterologous protein using the transformant,
It is the following invention.

(1) A vector having a promoter region capable of functioning in eukaryotic cells and having a multi-cloning site for introducing a heterologous protein structural gene controlled by the promoter downstream of the promoter region The restriction enzyme recognition site for introducing a heterologous protein structural gene at the tip of the multi-cloning site of the vector has -ACAT from the 5'side.
A multi-cloning vector which is a restriction enzyme recognition site having a sequence GT-.

(2) A promoter region capable of functioning in a eukaryotic cell, and a heterologous protein structural gene under the control of the promoter region downstream of the promoter region.
It has a sequence of ACATGN- (N is an arbitrary base), ATG in this sequence is the translation initiation site of the heterologous protein structural gene, and this sequence recognizes a restriction enzyme when the heterologous protein structural gene is introduced into the vector. An expression vector, which is a sequence derived from a site.

(3) In the method of introducing a heterologous protein structural gene controlled by a promoter downstream of the promoter region of a vector having a promoter region capable of functioning in eukaryotic cells, from the 5'side to -ACAT
A restriction enzyme recognition site having a sequence of -N'CATGN- (N is an arbitrary base, N'is a complementary base to N) from the 5'side at the end of the vector cleaved at the restriction enzyme recognition site having the sequence GT- The ends of the heterologous protein structural gene cleaved by the above are ligated to form a ligation part of the sequence -ACATGN-, and the ATG of the ligation part is used as the translation initiation site of the heterologous protein structural gene, and the restriction enzyme recognition cleavage part on the vector side. Expression in a eukaryotic cell characterized by setting the base next to ATG to an arbitrary base selected from A, T, G, and C by a combination of the end and the end of a restriction enzyme recognition cleavage site on the side of a structural gene of a heterologous protein Of possible expression vectors.

(4) A transformant comprising Schizosaccharomyces pombe carrying the above expression vector or a recombinant of the expression vector and a yeast vector having an origin of replication.

(5) A method for producing a heterologous protein, which comprises culturing the above transformant, producing and accumulating the heterologous protein in the culture, and collecting the heterologous protein.

S. In introducing a heterologous protein structural gene into a multi-cloning vector that can function in pombe, the gene containing the heterologous protein structural gene and the multi-cloning vector are each treated with a restriction enzyme, cut, and ligated to form an expression vector. To build. In the present invention, at this time, the gene containing the heterologous protein structural gene and the restriction enzyme recognition site of the multi-cloning vector each contain ATG, and the end (5 ′ end) of the gene containing the heterologous protein structural gene after cleavage and the multi-cloning vector Connect the ends (3 'end),
The ATG present at either end is used as the translation initiation site of the heterologous protein structural gene. For example, when the tip of a gene containing a heterologous protein structural gene contains ATG, the corresponding anti-coding strand portion of the end of the multi-cloning vector contains TAC. On the contrary, when the end of the multi-cloning vector contains ATG, TAC is contained in the anti-coding strand at the tip of the gene containing the corresponding heterologous protein structural gene.

In order to allow the introduction of various heterologous protein structural genes, the following bases of ATG are A, T, G,
It is preferable that the above construction means can be applied to any of C. Therefore, a combination of a restriction enzyme recognition cleavage site on the multi-cloning vector side and a restriction enzyme recognition cleavage site on the heterologous protein structural gene side is selected, and the next base of ATG can be any of A, T, G and C. It is preferable to adopt the means. A multi-cloning vector into which various heterologous protein structural genes can be introduced, to which such means can be applied, has a multi-cloning site which is a heterologous protein structural gene introduction site.

The restriction enzyme recognition site serving as the heterologous protein structural gene introduction site of the multi-cloning vector of the present invention is a restriction enzyme recognition site of -ACATGT- from the 5'side. This restriction enzyme recognition site is cleaved from the 5'side with a restriction enzyme forming an end called -A or -ACATG, and a heterologous protein structural gene having a tip corresponding to the end is ligated. As a restriction enzyme for this, Afl
III or NspI is used. Further, the formation of the sequence -ACATGT- at the vector-side ligation site can be performed by modifying the vector having various other ligation sites (for example, by using the PCR method).

The heterologous protein structural gene having a tip corresponding to the vector side end is one excised with a restriction enzyme from the gene containing the heterologous protein structural gene, and the restriction enzyme corresponds to the vector side end of -A or -ACATG. It is a restriction enzyme that can form a tip. A restriction enzyme that cleaves a heterologous protein structural gene is -N'CATGN- (N
Is an arbitrary base and N'is a restriction enzyme which recognizes a restriction enzyme recognition site of N) and forms a tip of CATGN- or N-. Restriction enzymes for this purpose include Nco I, Bsp HI, Afl III, Ns
p I or Sph I is used. If this restriction enzyme recognition site does not exist at the tip of the heterologous protein structural gene, it is possible to arrange the restriction enzyme recognition site at the tip of the heterologous protein structural gene using, for example, site-directed mutagenesis or PCR. it can.

By combining the vector-side end-A and the heterologous protein structural gene-side tip CATGN- or the vector-side terminal-ACATG and the heterologous protein structural gene-side tip N-, the combination of -ACATGN
A connecting portion is formed. ATG in that sequence serves as the translation initiation site of the heterologous protein structural gene. Moreover, AT
The N next to G can be made arbitrary by the combination of the restriction enzymes as described above, whereby the restriction of the structural gene of the heterologous protein that can be used is completely eliminated. As a combination of restriction enzymes for this purpose, the following combinations are particularly preferable.

Afl is used as a restriction enzyme for cutting the vector.
III or Nsp I and Nco I as a restriction enzyme for cleaving a gene containing a heterologous protein structural gene,
Bsp HI, Afl III, Nsp I, or S
Table 1 shows an example of means for using ph I and combining them to make the next base of ATG any of A, T, G, and C. All of these restriction enzymes recognize the 6-base palindromic sequence, and the 1'from the 5'end of the sense strand at this recognition site
Cut between the 2nd and 2nd bases or between the 5th and 6th bases, and between the 1st and 2nd bases or the 5th and 6th bases from the 3'end of the anticoding strand. It is a kind of restriction enzyme. In this case, as shown in the structure after ligation in Table 1, the sequence of 6 bases at the ligation site was 5'-ACAT.
GN-3 ′, and by selecting the combination, the N next to the translation initiation codon ATG can be any base of A, T, G, and C. In Table 1, N
Represents any of A, T, G, and C (provided that N in the anticoding chain in the table corresponds to N in the coding chain).

[0020]

[Table 1]

The multi-cloning vector and expression vector of the present invention are introduced (or introduced).
It contains a promoter region that controls the expression of a heterologous protein structural gene. This promoter controls the expression of the heterologous protein structural gene introduced downstream thereof. This promoter is capable of functioning in eukaryotic cells, and specifically, S. It must be able to function in pombe cells.

The above promoter is S. It is capable of functioning in the pombe and promotes transcription of the introduced heterologous protein structural gene. Examples of this promoter include an alcohol dehydrogenase gene promoter, a human cytomegalovirus gene promoter, and a human chorionic gonadropin α gene promoter. Especially those that strongly promote transcription of promoters derived from animal cell viruses (R. Toyama et a
l., FEBS Lett, 268, 217-221 (1990)). Such preferred promoters include promoters derived from animal cell viruses, particularly promoters of human cytomegalovirus gene.

The vector of the present invention may further contain, if necessary,
It may have a drug resistance gene such as an antibiotic resistance gene, a signal peptide coding gene for secreting a heterologous protein extracellularly, and other various genes. In addition, E. It is also possible to incorporate a promoter capable of functioning in prokaryotic cells such as E. coli, a drug resistance gene and the like into a shuttle vector.

It is necessary for an expression vector to have an origin of replication for expression in cells. However, the above-mentioned multicloning vector or expression vector of the present invention does not necessarily have to have a replication origin. The origin of replication can be introduced after the construction of the expression vector. Also,
After inserting an expression vector having no replication origin into a cell, the replication origin can be automatically introduced into the expression vector in the cell. Means for introducing these replication origins are known. For example, a vector having a replication origin capable of functioning in yeast (hereinafter referred to as yeast vector) can be incorporated into the expression vector of the present invention (see JP-A-5-15380). Further, the expression vector of the present invention and the yeast vector can be put into one cell to automatically fuse the both in the cell. Since such a replication origin introducing means can be adopted, the expression vector of the present invention may or may not have a replication origin. Similarly, the multi-cloning vector of the present invention may or may not have an origin of replication. However,
In all cases, the vector ultimately requires an origin of replication for the expression of the expression vector to occur in the cell.

Incorporation of a drug resistance gene such as an antibiotic resistance gene into a vector is usually essential for cloning and for use as a marker. It is preferable that the vector of the present invention also has an antibiotic resistance gene and a promoter that promotes its transcription (hereinafter referred to as a second promoter). This second promoter is
It is preferable that the transcription promoting activity is lower than that of the promoter that promotes the transcription of the heterologous protein structural gene. Moreover, as the second promoter, a promoter derived from an animal cell virus, particularly SV4
The 0 early promoter is preferred. The antibiotic resistance gene controlled by the second promoter may be a usual one, but a neomycin resistance gene is particularly preferable in the present invention.

In the present invention, the expression level of a heterologous protein can be increased by using an expression vector having an antibiotic resistance gene. For this purpose, the second promoter needs to have a lower transcription-promoting activity than the promoter controlling the heterologous protein structural gene.
For example, S. cerevisiae harboring an expression vector having the SV40 early promoter and the neomycin resistance gene controlled by it as described above. The case of culturing pombe will be described as an example. This S. When the pombe transformant is cultured in a G418 (neomycin) -containing medium, the copy number of the expression vector increases in the cells depending on the concentration of G418 in the medium. Therefore, by increasing the G418 concentration, the copy number of the expression vector can be increased in the cells, and as a result, the expression amount of the heterologous protein can be increased. At this time, if the activity of the second promoter is higher than that of the promoter governing the heterologous protein structural gene, expression will be sufficient because a sufficient neomycin resistance protein (enzyme) is produced even if the copy number of the expression vector is small. It is not necessary to increase the copy number of the vector, and it is not possible to increase the expression level of the target heterologous protein.

FIG. 4 shows a restriction enzyme digestion map of pTL2M prepared in Examples as an example of the multicloning vector of the present invention. This pTL2M is approximately 5,000 bp
(More accurately, about 5,000 ± 200 bp), a promoter region of the human cytomegalovirus gene (hCMV), and an untranslated region (5′-UT) that connects the promoter region and the multicloning site (MCS).
R), and M for introducing a heterologous protein structural gene
It has CS in this order in the clockwise direction. The specific restriction enzyme recognition site described above is present at the MCS tip. On the other hand, it further has an SV40 promoter region and a neomycin resistance gene (Nm ^R ) in this order in the counterclockwise direction from the vicinity of the tip of the hCMV promoter region. Also,
Further, in the downstream region of Nm ^R , as described above, E. coli
Has an ampicillin resistance gene (Ap ^R ) which is a drug resistance gene capable of functioning in prokaryotic cells such as SV40 terminator (2 places), and an origin of replication functioning in prokaryotic cells ( Ori), and the downstream untranslated region (3′-UTR).

The untranslated region (5'-UTR) is a sequence existing between the end of the promoter region (usually TATA sequence) and the translation initiation codon ATG. The length of this untranslated region often affects translational activity. The length of this part of the vector in the present invention is 20-200.
It is preferably bp. More preferably 30 to 10
It is 0 bp. In the illustrated pTL2M, it is about 56 bp.

The illustrated pTL2M itself does not have an origin of replication capable of functioning in eukaryotic cells. To use this pTL2M, it is necessary to introduce a replication origin capable of functioning in eukaryotic cells. A sequence having a replication origin capable of functioning in eukaryotic cells and a vector having the sequence are known, and a replication origin can be introduced into pTL2M by using this. S. Known vectors having an origin of replication that can function in pombe cells include, for example, pA.
There are U5 and pAL7 (K.Okazaki et.al., Nucleic Aci
ds Res., 18,6485-6489 (1990), K.Okayama et.al., Molec.
ular Cellular Biol., 3,280-289 (1983)). These yeast vectors have automatic replication sequences (ars) and stabilizing sequences (st).
b), and further has a selection marker and an antibiotic resistance gene. By incorporating this yeast vector into pTL2M, S. A multi-cloning vector is obtained which can replicate in pombe cells. Similarly, by incorporating this yeast vector into an expression vector in which a heterologous protein structural gene has been introduced into a multi-cloning vector,
S. An expression vector is obtained which can replicate in pombe cells. A method for constructing an expression vector by incorporating this yeast vector is described in Japanese Patent Laid-Open No. 15380/1993 related to the inventions of the present inventors, in addition to the above-mentioned documents.

In general, it is often difficult to insert a large vector into a cell. The pTL2M itself is a relatively large vector, and it is relatively difficult to incorporate the yeast vector into the pTL2M and insert it into a yeast cell. As a means for solving this, there is a method in which the above yeast vector (usually using linear DNA cleaved with a restriction enzyme) and the multicloning vector or expression vector of the present invention are separately put into the same cell. in this case,
The yeast vector is automatically incorporated into the multi-cloning vector or expression vector in the cell, and the vector having the replication origin as described above is automatically constructed.
In the examples described below, S. Transformation of pombe was performed.

General techniques for constructing multicloning and expression vectors are well known and are described, for example, in the literature “J. Sambrook et al.,“ Molecular Cloning 2n.
d ed. ", Cold Spring Harbor Laboratory Press (1989)
"It is described in. The multi-cloning vector and expression vector of the present invention can be constructed by the method described above using this general method. S. cerevisiae used in the present invention as a host for expression vectors. As the pombe strain,
For example, ATCC 38399 (leu1-32h-)
Or ATCC 38436 (ura4-294h-, etc. are mentioned, These are American Type Culture Collection (American Type Culture Collection)
Available from.

The expression vector was used to transform S. Methods for transforming pombe are known, for example, the lithium acetate method (K. Okazaki et al., Nucleic Acids Res., 18,6485-6489).
(1990)). A transformant of pombe is obtained. A medium for culturing the transformant is known, and a nutrient medium such as YPD medium (MDRose et al., "Metho
ds In Yeast Genetics ", Cold Spring Harbor Laborator
y Press (1990)) or minimal medium such as MB medium (K.Okaz
aki et al., Nucleic Acids Res., 18,6485-6489 (1990))
Etc. can be used. Cultivation of transformants is usually 1
It is carried out at 6 to 42 ° C., preferably 25 to 37 ° C. for 8 to 168 hours, preferably 48 to 96 hours. Both shaking culture and stationary culture are possible, but stirring and aeration may be added as necessary.

As a method for isolating and purifying the protein produced in the culture, known methods such as salting out or solvent precipitation, which utilize the difference in solubility, dialysis, ultrafiltration or gel electrophoresis, etc., can be used. Utilizing difference in molecular weight, utilizing difference in charge such as ion exchange chromatography, utilizing specific affinity such as affinity chromatography, utilizing difference in hydrophobicity such as reverse phase high performance liquid chromatography And a method of utilizing a difference in isoelectric points such as an isoelectric focusing method.

As a method for confirming the isolated / purified protein, a known Western blotting method or activity measuring method can be mentioned. The structure of the purified protein can be clarified by amino acid analysis, amino terminal analysis, primary structure analysis and the like.

[0035]

The present invention will be described in more detail with reference to the following examples. However, the technical scope of the present invention is not limited by these examples.

In the following, Example 1 shows the preparation of the vector pRL2M which is the basis of the vector construction of the present invention, and Example 2 shows the preparation of the vector pTL2M before the introduction of the heterologous protein structural gene using the pRL2M. This vector pTL2M is a multi-cloning vector of the present invention, and the examples below show this vector pTL2M.
This is an example in which various heterologous protein structural genes were introduced into and the expression was tested. In addition, Example 3 shows this vector pTL.
S. The production of pombe transformants is shown, and Example 4 shows the preparation (for control) of this transformant cell extract. Example 5 shows an example in which various tests were conducted particularly for the production of human lipocortin I.

[Example 1] [Preparation of vector pRL2M] The plasmid pcD4B (see JP-A-5-15380) prepared by a known method was digested with the restriction enzyme Sac I, and the end was T4 DN.
It is blunted with A polymerase and further restricted with Bam H restriction enzyme.
After digestion with I, the nucleic acid fraction was collected by phenol extraction and ethanol precipitation. Furthermore, after agarose gel electrophoresis, the glass beads method ("DNA manufactured by Asahi Glass Co., Ltd."
Approximately 45 by using "PREP" (trade name), and so on
The DNA corresponding to 00 base pairs was purified.

Separately, a vector pc containing the human lipocortin I gene (cDNA), which was also prepared by a known method.
After digesting D4lipoI (see JP-A-5-15380) with the restriction enzymes XmnI and BamHI,
The nucleic acid fraction was collected by phenol extraction and ethanol precipitation. Further, after agarose gel electrophoresis, the DNA corresponding to about 1300 base pairs was purified by the glass bead method.

Both DNAs were ligated with a ligation kit (sold by Takara Shuzo Co., Ltd.), and then E. coli DH5
(Toyobo Co., Ltd.) was transformed. A vector is prepared from the obtained transformant, and the desired vector pRL is obtained.
Transformants with 2 L (Fig. 1) were screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

This lipocortin I expression vector pRL2
L was digested with restriction enzymes Eco RI and Hind III, extracted with phenol, precipitated with ethanol, and then a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method. Separately, a known plasmid pUC19 was digested with restriction enzymes Eco RI and Hind III, extracted with phenol, precipitated with ethanol, and then subjected to polyacrylamide gel electrophoresis to give about 6
The band corresponding to 0 base pairs was cut out and extracted and purified from the gel.

After ligation of these two fragments,
Target vector pRL obtained by transforming Escherichia coli DH5
2M (Figure 2) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

[Example 2] [Preparation of vector pTL2M] Using pRL2M as a template, oligodeoxyribonucleotide 5'-TTGACTAG
PCR using Taq polymerase with TTATTAATAGTA-3 'and oligodeoxyribonucleotide 5'-CTAGAATTCACATGTTTGAAAAAGTGTCTTTATC-3' as synthetic primers
The target fragment was amplified by. The ends were adjusted with restriction enzymes Spe I and Eco RI, phenol extraction and ethanol precipitation were performed, and then about 60 by agarose gel electrophoresis.
The band corresponding to 0 base pairs was cut out and purified by the glass bead method.

Separately, pRL2M was digested with restriction enzymes Spe I and Eco RI, extracted with phenol and precipitated with ethanol. Then, a band corresponding to about 4500 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method. After ligation of these two fragments, Escherichia coli DH5 was transformed with the desired vector pTL2M.
(FIG. 3) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme cleavage map, the target vector was confirmed. Figure 4 shows a restriction enzyme digestion map of pTL2M.
Shown in.

[Example 3] [Introduction of pTL2M into yeast] A leucine-requiring strain of pombe, ATCC 38399 (leu1-32h
-) Was grown in a minimal medium to 0.8 × 10 ⁷ cells / ml. After the cells were collected and washed, the cells were suspended in 0.1 mol lithium acetate (pH 5.0) so that the number of cells was 10 ⁹ cells / ml, and the cells were incubated at 30 ° C. for 60 minutes. Then, in 100 μl of the above suspension, 1 μg of the DNA obtained by cleaving pAL7 with Pst I and 2 μg of the vector pTL2M obtained in Example 1 were added to TE (Tris buffer containing EDTA) 1
Dissolve in 5 μl and add 290% 50% PEG4000
After adding μl and mixing well, the mixture was incubated at 30 ° C. for 60 minutes, 43 ° C. for 15 minutes, and room temperature for 10 minutes in that order.
PEG4000 was removed by centrifugation and suspended in 1 ml of culture solution.

Of this, 100 μl was taken and 900 μl
The culture solution was added and incubated at 32 ° C. for 30 minutes. 300 μl of this was spread on the minimal agar medium.
After incubating at 32 ° C. for 3 days, the transformants were subjected to G41
The cells were transferred to a plate containing 8 and further cultured at 32 ° C. for 5 days. The obtained product was the desired transformant.

[Example 4] [Cultivation of transformant and preparation of cell extract] Example 3
2% glucose, 1% yeast extract, 2% peptone, and G418 at 200 μg / ml.
The cells were cultured in 50 ml of a liquid culture medium containing 32 ° C. for 3 days. About 10 ⁸ cells were collected and washed from the culture solution, and 5
Suspended in 0 mM Tris-HCl buffer (pH 7.5),
Ultrasonic crushing was performed. Cell extract (supernatant) by centrifugation
Got This cell extract was used in the following examples as a negative control (control) for the analysis performed by SDS-polyacrylamide gel electrophoresis (4-20% polyacrylamide gel manufactured by TEFCO, hereinafter referred to as SDS-PAGE).

[Example 5] [Expression of human lipocortin I] Known (JP-A 5-153
80), using the vector pcD4lipoI as a template for oligodeoxyribonucleotide 5'-ATGCCATGGCAATGGTATCAGAAT.
T-3 'and oligodeoxyribonucleotide 5'-AGCCAG
The target fragment was amplified by PCR using Taq polymerase with TATACACTCCGCTA-3 'as a synthetic primer. After adjusting the ends with restriction enzymes Nco I and Bam HI, extracting with phenol and precipitating with ethanol, a band corresponding to about 1400 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method.

Separately, pTL2M was digested with the restriction enzyme Afl II.
After digestion with I and Bam HI, phenol extraction and ethanol precipitation, agarose gel electrophoresis showed about 500
The band corresponding to 0 base pairs was cut out and purified by the glass bead method. After ligation of both fragments,
Target vector pTL obtained by transforming Escherichia coli DH5
2L (Figure 5) were screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in the third embodiment, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. At this time, protease (12 micrmol PMS) was added to the cell extract.
F, 25 micromol leupeptin, 5 micromol E-64) is present. When examined by SDS-PAGE, a band not found in the yeast extract containing the control pTL2M at a molecular weight of 36,000 was confirmed, and Western blotting was performed using an anti-human lipocortin I antibody (rabbit). As a result, only this band was specifically stained (FIG. 6). FIG. 6 is an SDS-PAGE observation diagram and a Western blot observation diagram showing the expression of human lipocortin I, and 1-4 in the figure represent the following.

1; S. pombe (pTL2M) cell extract, 2; pombe (pTL2L) cell extract, 3; pombe (pTL2M) cell extract, 4; Pombe (pTL2L) cell extract.

Western blotting was carried out using ¹²⁵ I-Protein A as a secondary antibody, and quantification was carried out using natural lipocortin I as a standard. The expressed lipocortin occupied 50 wt% of the total soluble protein. all right.

[Human lipocortin I expressed in fission yeast
Purification of the above] The crude extract obtained above was added to an affinity column using a monoclonal antibody that recognizes only the calcium-binding form of human lipocortin I as a ligand, 1 mM calcium chloride, 50 mM Tris-HCl (pH 7.5),
Adsorbed with 0.15 mol sodium chloride and 2 mmol E
GTA, 50 mM Tris-HCl (pH 7.5), 0.
By eluting with 15 molar sodium chloride, S
A single band of recombinant human lipocortin I was obtained on DS-PAGE (Fig. 7). FIG. 7 is an SDS-PAGE observation diagram showing the purification of human lipocortin I, and A and B in the figure represent the following.

A; S. Pombe (pTL2L) crude extract, B; purified human lipocortin I.

[Phospholipase A2 (PLA2) Inhibitory Activity] Recombinant human lipocortin I purified in the above Example
The PLA2 inhibitory activity by B.Rothhut et al.
ut, et al, Biochem.Biophys.Res.Commun.117,878-884 (19
83)). That is, the inhibitory activity of human lipocortin I against the activity of the bee venom PLA2 was examined using E. coli labeled with oleic acid containing tritium as a substrate.
The result is shown in FIG. From these results, it was found that the inhibitory activity exhibited by human placenta-derived lipocortin I, the degree of inhibition, and the mode of inhibition corresponded. Figure 8 shows protein mass and P
It is a graph which shows the relationship of the activity of LA2.

[Binding ability with F-actin] The purified human lipocortin I obtained in the above-mentioned example was used in the presence of calcium in the presence of F.
− The method of H. Hayashi et al.
(H. Hayashi et al, Biochem.Biophys.Res.Commun.146,91
2-919 (1987)). Similar to human placenta-derived lipocortin I, 50 wt in the presence of 1 mM calcium
% Of human lipocortin I bound to F-actin, and no binding was observed in the presence of 1 mM EGTA (FIG. 9). FIG. 9 shows SDS-PA showing actin-binding activity.
It is a GE observation figure, and 1-4 in the figure show the following.

1; centrifugation residue in the presence of 1 mM EGTA, 2; centrifugation supernatant in the presence of 1 mM EGTA, 3; centrifugation residue in the presence of 1 mM calcium chloride, 4; centrifugation supernatant in the presence of 1 mM calcium chloride .

[Amino acid composition] The amino acid composition of the recombinant human lipocortin I obtained in the above Example was analyzed by an amino acid analyzer (JEOL JLC-360). The results are shown in Table 2. It was found to be in agreement with the composition expected from the DNA base sequence.

[0058]

[Table 2]

[Amino-Terminal Amino Acid Sequence] The determination of the amino acid sequence from the amino terminus of the recombinant human lipocortin I obtained in the above Example was carried out by the gas phase sequencer (SHIMAD).
ZU PSQ-1), but no PTH-amino acid was detected. Therefore, recombinant human lipocortin I was completely decomposed by V8 protease (Boehringer), each fragment was separated by ODS C18 column (HPLC), and the amino-terminal fragment was selected by examining the amino acid composition of each fragment. Next, an acylamino acid-releasing enzyme was allowed to act on the peptide. The result is shown in FIG.

FIG. 10 is a graph showing the elution amount by HPLC, FIG. 10a is a graph showing the elution amount of the amino terminal fragment, and FIG. 10b is a fragment after the amino acid fragment is reacted with an acylamino acid-releasing enzyme. 3 is a graph showing the elution amount of As shown in FIG. 10b, a peak appeared at an early elution time, and the elution position of the amino terminal fragment moved.

When the former peak was collected and hydrolyzed to examine the amino acid composition, it was Ala, and when acetyl Ala was eluted by HPLC, it coincided with this peak as shown in FIG. 10b. The latter peak was analyzed by a sequencer and found to be Met-Val-Ser-Glu. Therefore, the amino terminal amino acid sequence of recombinant human lipocortin I is considered to be Acetyl-Ala-Met-Val-Ser-Glu as in the natural form.

[Confirmation of copy number of human lipocortin I expression vector] The transformant prepared in Example was cultured and
× 10 ⁷ cells were obtained. The culture method was the same as that of Example 4, except that the concentration of G418 in the medium was 0, 25, 50, 100, and 200 μg / ml. The cells were disrupted using glass beads, SDS and phenol, and total DNA was extracted. Restriction enzyme Eco
0.8% after digestion with RI and Hind III
After agarose gel electrophoresis, transferred to a nylon membrane, Eco RI-Hin of human lipocortin I gene
Southern hybridization was performed using the dIII fragment as a probe. The result is shown in FIG. FIG. 11 is a graph showing the relationship between the G418 concentration and the copy number of pTL2L. As shown in the figure, the vector copy number in the cells increased depending on the G418 concentration in the medium, and the maximum vector copy number was 200.
It turned out to rise to about a copy.

[Example 6] [Expression of rat arginase] Rat liver arginase cDNA provided by Professor Masataka Mori of Kumamoto University School of Medicine.
Vector pARGr-2 containing the full length [S. Kawamoto et a
l., Biochem.Biophis.Res.Commun., 136,955-961 (1986)
], The inserts were digested with restriction enzymes Eco RI and P
It was cut out with stI and subcloned into the EcoRI-PstI site of the commercially available vector Bluescript.

Using this vector as a template, oligodeoxyribonucleotide 5'-GACTCATGAGCTCCAAGCCAAA
GCC-3 'and oligodeoxyribonucleotide 5'-TTCC
Using CAGTCACGACGTTGTA-3 'as a synthetic primer, Taq
The target fragment was amplified by PCR using a polymerase. The ends were adjusted with the restriction enzymes Bsp HI and Xba I, and after phenol extraction and ethanol precipitation, a band corresponding to about 1300 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

Separately, pTL2M was digested with the restriction enzyme Afl II.
After digestion with I and Xba I, extraction with phenol, precipitation with ethanol, agarose gel electrophoresis of about 5000
The band corresponding to the base pair was cut out and purified by the glass bead method. After ligation of these two fragments, Escherichia coli DH5 was transformed with the desired vector pTL2.
R (FIG. 12) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in Example 3, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. SDS
When the expression was confirmed by -PAGE, a clear band not found in the control pTL2M was detected at a position near the molecular weight of 35,000 corresponding to rat arginase. The expression level was 30 to 50 wt% of the total cell protein as measured by a densitometer. Moreover, it was confirmed to be rat arginase by performing Western blotting using an antibody specific to rat arginase.

[Example 7] [Expression of human IL-6] Using a known vector pUC19 containing the full-length human IL-6 cDNA as a template, oligodeoxyribonucleotide 5'-ATCGCATGCCAGTACCCCCA.
GGAGAAGA-3 'and oligodeoxyribonucleotides
The target fragment was amplified by PCR using Taq polymerase with 5'-TGAAAATCTTCTCTCATCCG-3 'as a synthetic primer. Restriction enzymes Sph I and Hind I
After adjusting the end with II, phenol extraction and ethanol precipitation, a band corresponding to about 1000 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

Separately, pTL2M was digested with the restriction enzyme Afl II.
Digested with I and Hind III, phenol extracted,
Approximately 5 after agarose gel electrophoresis after ethanol precipitation
A band corresponding to 000 base pairs was cut out and purified by the glass bead method. After ligation of these two fragments, E. coli DH5 was transformed with the desired vector p
TL26m (FIG. 13) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in Example 3, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. SDS
-When the expression was confirmed by PAGE, human IL-
A clear band not found in the control pTL2M was detected at a position near the molecular weight of 21,000 corresponding to 6. The expression level was about 10 wt% of the total cell protein as measured by a densitometer. Human IL
Western blotting was performed using a -6-specific antibody, and it was confirmed to be human IL-6.

[Example 8] [Expression of rat NDP kinase α-form] A vector pNDPKα containing the full-length rat NDP kinase α-form cDNA, which was provided by Dr. Shigemichi Kimura of the Institute of Gerontology, Tokyo, was treated with restriction enzymes Nco I and Hind III. After digestion, phenol extraction and ethanol precipitation, a band corresponding to about 600 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method. Separately, pTL2M was digested with restriction enzymes Afl III and Hind III, extracted with phenol and precipitated with ethanol. Then, a band corresponding to about 5000 base pairs was cut out by agarose gel electrophoresis and purified by a glass bead method.

After ligation of these two fragments,
Target vector pTL obtained by transforming Escherichia coli DH5
2-Nα (FIG. 14) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in the third embodiment, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. SDS
-When the expression was confirmed by PAGE, rat ND
A clear band not found in the control pTL2M was detected at a position near the molecular weight of 17,000 corresponding to the P-kinase α-form. The expression level was 30 to 50 wt% of the total cell protein as measured by a densitometer. Further, Western blotting was carried out using an antibody specific to rat NDP kinase α-form, and it was confirmed to be rat NDP kinase α-form.

[Example 9] [Expression of rat NDP kinase β-form] A vector pNDPKβ containing the full-length rat NDP kinase β-form cDNA, which was provided by Dr. Shigemichi Kimura of the Institute of Gerontology, Tokyo, was treated with restriction enzymes Nco I and EcoRI. After digestion, phenol extraction and ethanol precipitation, a band corresponding to about 700 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

Separately, pTL2M was digested with the restriction enzyme Afl II.
After digestion with I and Eco RI, phenol extraction and ethanol precipitation, agarose gel electrophoresis gave about 500
The band corresponding to 0 base pairs was cut out and purified by the glass bead method. After ligation of both fragments,
Target vector pTL obtained by transforming Escherichia coli DH5
2-Nβ (FIG. 15) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in the third embodiment, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. SDS
-When the expression was confirmed by PAGE, rat ND
A clear band not found in the control pTL2M could be detected at a position near the molecular weight of 17,000 corresponding to the P-kinase β-form. The expression level was 30 to 50 wt% of the total cell protein as measured by a densitometer. Further, Western blotting was carried out using an antibody specific to rat NDP kinase β-form, and it was confirmed to be rat NDP kinase β-form.

[Example 10] [Expression of human serum albumin] Human serum albumin cDNA, donated by National Institute of Preventive Health, Gene Bank
Using the vector pILMALB5 containing A as a template, oligodeoxyribonucleotide 5'-AGACCATGGATG
The target fragment was amplified by PCR using Taq polymerase with CACACAAGAGTGAGGT-3 'and oligodeoxyribonucleotide 5'-CAGGAAACAGCTATGACCAT-3' as synthetic primers. Restriction enzymes Nco I and Hind
After adjusting the ends with III, phenol extraction and ethanol precipitation, a band corresponding to about 1800 base pairs was cut out by agarose gel electrophoresis and purified by the glass bead method.

Separately, pTL2M was digested with the restriction enzyme Afl II.
Digested with I and Hind III, phenol extracted,
Approximately 5 after agarose gel electrophoresis after ethanol precipitation
A band corresponding to 000 base pairs was cut out and purified by the glass bead method. After ligation of these two fragments, E. coli DH5 was transformed with the desired vector p
TL2Bm (FIG. 16) was screened. From the confirmation of the partial base sequence and the construction of the restriction enzyme map, the target vector was confirmed.

As in Example 3, S. Pombe was transformed and a cell extract was prepared in the same manner as in Example 4. SDS
When the expression was confirmed by -PAGE, a clear band which was not found in the control pTL2M was detected at a position near the molecular weight of 69,000 corresponding to human serum albumin. The expression level was about 30 wt% of the total cell protein as measured by a densitometer. Further, it was confirmed to be human serum albumin by performing Western blotting using an antibody specific to human serum albumin.

[0079]

INDUSTRIAL APPLICABILITY In the present invention, any protein can be expressed at a high expression level, so that the efficiency of heterologous protein production can be significantly increased. Further, as seen in the case of human lipocortin I described above, the expression level occupies 50 wt% of the bacterial protein, and its molecular weight, amino acid composition, amino acid sequence near the amino terminus, etc. are lipocortin I derived from human placenta (Nature 320). , 77-81 (1986)), and the degree of PLA2 inhibitory activity and the ability to bind to actin are similar to those of the above-mentioned natural type. It has become possible to obtain a protein that is almost the same as that of 1.

[Brief description of drawings]

FIG. 1 is a structural diagram of vector pRL2L.

FIG. 2 is a block diagram of the vector pRL2M.

FIG. 3 is a block diagram of the vector pTL2M.

FIG. 4 Restriction enzyme digestion map of vector pTL2M

FIG. 5: Construction diagram of expression vector pTL2L

FIG. 6 is an SDS-PAGE and Western blot observation diagram.

FIG. 7: SDS-PAGE observation diagram

FIG. 8 is a graph showing PLA2 inhibitory activity.

FIG. 9 is an SDS-PAGE observation diagram.

FIG. 10 is a graph showing HPLC elution.

FIG. 11 is a graph showing changes in the copy number of pTL2L.

FIG. 12: Construction diagram of expression vector pTL2R

FIG. 13: Construction diagram of expression vector pTL26m

FIG. 14: Construction diagram of expression vector pTL2-Nα

FIG. 15: Construction diagram of expression vector pTL2-Nβ

FIG. 16: Construction diagram of expression vector pTL2Bm

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1: 645) (C12P 21/02 C12R 1: 645)

Claims (14)

[Claims] 1. A vector having a promoter region capable of functioning in a eukaryotic cell, and having a multi-cloning site for introducing a heterologous protein structural gene controlled by the promoter downstream of the promoter region, The restriction enzyme recognition site for introducing a heterologous protein structural gene at the tip of the multi-cloning site of the vector is -ACATGT- from the 5'side.
A multi-cloning vector having a restriction enzyme recognition site having the following sequence. 2. The vector further has a second promoter region and an antibiotic resistance gene controlled by the second promoter, and the transcription promoting activity of the second promoter is higher than that of the first promoter. Is also low
The multi-cloning vector according to claim 1. 3. A promoter region capable of functioning in a eukaryotic cell, and a heterologous protein structural gene under the control of the promoter region downstream of the promoter region, wherein the heterologous protein structural gene front end is from the 5 ′ side to −AC.
ATGN- (N is an arbitrary base), the ATG in this sequence is the translation initiation site of the heterologous protein structural gene, and this sequence recognizes the restriction enzyme when the heterologous protein structural gene is introduced into the vector. An expression vector, which is a sequence derived from a site. 4. A sequence of -ACATGN- is linked to a vector-side end of -A and a heterologous protein structural gene end of CATGN-, or a -ACATGN vector-side end and a heterologous protein structural gene side of N-. The expression vector according to claim 3, which is a sequence formed by ligation with a tip. 5. The expression vector according to claim 3, which has an untranslated region between the promoter terminal and the translation initiation site of the heterologous protein structural gene, and the length of the untranslated region is 20 to 200 bp. 6. The vector further has a second promoter region and an antibiotic resistance gene controlled by the second promoter, and the transcription promoting activity of the second promoter is higher than that of the first promoter. Is also low
The expression vector according to claim 3, 4 or 5. 7. A method of introducing a heterologous protein structural gene under the control of a promoter region downstream of a promoter region of a vector having a promoter region capable of functioning in a eukaryotic cell, from the 5'side to -ACATGT-
Cleavage at the restriction enzyme recognition site having a sequence from the 5'side to -N'CATGN- (N is an arbitrary base, N'is a complementary base to N) at the end of the vector cleaved at the restriction enzyme recognition site having the sequence The ends of the heterologous protein structural genes described above are ligated to form a ligated portion of the sequence -ACATGN-,
The ATG at the connecting portion is used as the translation initiation site of the heterologous protein structural gene, and the base next to the ATG is A by the combination of the end of the restriction enzyme recognition cleavage part on the vector side and the tip of the restriction enzyme recognition cleavage part on the heterologous protein structural gene side. A method for constructing an expression vector capable of being expressed in a eukaryotic cell, which comprises using any base selected from T, G, and C. 8. The restriction enzyme that cuts the restriction enzyme recognition site on the vector side is Afl III or Nsp I, and the restriction enzymes that cuts the restriction enzyme recognition site on the heterologous protein structural gene side are Nco I, Bsp HI, Afl III, Ns.
The method for constructing an expression vector according to claim 7, which is pI or SphI. 9. The vector further has a second promoter region and an antibiotic resistance gene controlled by the second promoter, and the transcription promoting activity of the second promoter is higher than that of the first promoter. Is also low
A method for constructing the expression vector according to claim 7 or 8. 10. The method according to claim 3, 4, 5 having no replication starting point.
Alternatively, a method for constructing an expression vector, which comprises constructing an expression vector having a replication origin by artificially or automatically recombining the expression vector of 6 with a yeast vector having a replication origin. 11. A transformant comprising Schizosaccharomyces pombe carrying the expression vector of claim 3, 4, 5 or 6 or a recombinant of the expression vector and a yeast vector having a replication origin. 12. A method for producing a heterologous protein, which comprises culturing the transformant according to claim 11, allowing the heterologous protein to be produced and accumulated in the culture, and collecting the heterologous protein. 13. The transformant according to claim 11, which comprises Schizosaccharomyces pombe carrying the expression vector according to claim 6 or a recombinant of the expression vector and a yeast vector having a replication origin. 12 manufacturing methods. 14. The method according to claim 13, wherein the transformant is cultured in a culture medium containing an antibiotic corresponding to the antibiotic resistance gene contained in the expression vector.