JP2743207B2 - Method for producing CPB-I, recombinant plasmid and transformed yeast used for the method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to an anticoagulant (calphobinden, hereinafter referred to as CPB-I) obtained from human tissues including human placenta by genetic recombination. The present invention relates to a production method, and a recombinant plasmid and a transformed yeast used for the method.

[Conventional technology]

Until now, heparin, heparin cofactor II, antithrombin II
I, αI-antitrypsin and the like are known. To date, only heparin has been put to practical use as an anticoagulant, but it has not been satisfactory because of problems such as side effects and limited use.

Under these circumstances, the Applicant has determined that CP from human placenta
We succeeded in separating and purifying BI (originally referred to as PCI), and filed a patent application (Japanese Patent Application Laid-Open No. 62-1704023). further,
The present applicant has prepared a monoclonal antibody specific to CPB-I (JP-A-63-123395), and subsequently used this antibody as a probe to extract CPB from a human placenta cDNA library.
The gene fragment encoding BI was cloned and successfully expressed in Escherichia coli by genetic recombination technology (JP-A-64-20095).

Thus, CPB-expressed and purified by Escherichia coli
I was confirmed to have an anticoagulant activity almost equivalent to the original CPB-I purified from the placenta. However, Escherichia coli, which is a host for expression in this case, is known to produce a particularly large amount of pyrogens (pyrogens). In the purification of the target CPB-I, such an operation of removing pyrogens and the like is required. It was feared that it would be complicated.

In general, when a specific foreign gene is expressed by a gene recombination technique, the expression level of a target peptide varies greatly depending on what is used as a host cell for expression and what is used as an expression promoter. It is known. Reports on the expression of foreign genes by various genetic recombinations to date have gradually revealed a generally efficient expression system. However, an expression system that is usually considered to have a high expression level may not show favorable results for the expression of a specific foreign gene due to the effect of the expression product of the target foreign gene on the host cell of expression. I knew it. On the contrary, it is also known that there is an expression system that is very compatible with a specific foreign gene and that enables expression of an extremely high amount.

Therefore, in pursuing research for the purpose of industrial production of CPB-I, it is more efficient and more stable.
It has been desired to find an expression system suitable for the production of CPB-I capable of producing E. coli.

[Object of the invention]

Under such circumstances, the present inventors have repeated studies on the production of CPB-1 by genetic recombination. As a result, by using yeast as a host and using a specific expression system, CPB-1 having a desired physiological activity can be obtained. The present inventors have succeeded in obtaining a transformed yeast which produces I efficiently and stably, and have found an efficient method for producing CPB-I, thereby completing the present invention.

That is, an object of the present invention is to produce an anticoagulant substance CPB-I using a gene recombination technique,
Yeast Recombinant Plasmid and Transformed Yeast for Very Efficient Production of I
-I to provide a manufacturing method.

[Structure and effect of the invention]

The structure of the recombinant plasmid of the present invention that achieves the above object is a shuttle vector having a yeast-derived gene and an Escherichia coli plasmid-derived gene, and further comprises a Pho5 promoter, a CPB-I-cDNA and a GAP-DH terminator. It is characterized by having an I-expressing gene fragment.

In the present invention, the gene expressed by the gene recombination technique: CPB-I-cDNA was previously cloned by the present inventors, and details thereof are described in JP-A-64-20095.
No., published in Japanese Unexamined Patent Publication No.

This CPB-I-cDNA is a 1566 bp gene fragment including a gene fragment encoding a peptide consisting of 319 amino acids (CPB-I). It has been confirmed that the CPB-I structural gene encoded thereby encodes the following amino acid sequence.

Therefore, a gene fragment encoding the above amino acid sequence is used for construction of the CPB-I expression plasmid in the present invention. As a specific nucleotide sequence of the gene fragment encoding CPB-I, a gene fragment containing a DNA consisting of the following nucleotide sequence is listed as one sequence.

As a promoter used in a yeast expression system reported so far, an ADH1 (alcohol dehydrogenase) promoter [Hetzeman et al., Nature, Vol. 293, p71
7-722 (1981)], Pho5 (repressive acid phosphatase) promoter [Miyanohara et al., Proc. Natl. Acad. Sci.
A, Vol. 80, p1-5 (1983)], PGK1 (phosphoglucokinase) promoter [Hitzman et al., Science, Vol.
9, p620-625 (1983)], GAP-DH (glyceroaldehyde phosphate dehydrogenase) promoter [Bi
tter et al., Gene, Vol. 32, p264-274 (1983)]. Among the yeast promoters listed here,
It is known that the GAP-DH promoter has a strong promoter activity and, as a result, efficiently expresses a foreign gene.

In the expression of the CPB-I according to the present invention, although the expression system using the GAP-DH promoter yielded fairly good results in terms of the amount of expression, in terms of the stability of expression by subculture of the transformant, It was confirmed that the problem remained.

For that purpose, various CPB-I expression systems were constructed by combining with various promoters, and the examination was carried out in terms of both the expression level of CPB-I by the obtained transformants and the stability of expression by subculture. However, it was confirmed that when the expression system using the plasmid of the present invention was used to express CPB-I, very good results were obtained.

That is, in the yeast expression system according to the present invention, Pho5
It is characterized by using a promoter and a GAP-DH terminator, and uses a shuttle vector having yeast and E. coli genes. In particular, it is preferable to use two origins, 2μori and ars1, and a selectable marker gene as yeast-derived genes.

As the yeast selectable marker gene, genes that produce various amino acids, for example, a leucine-producing gene, a histidine-producing gene, a tryptophan-producing gene, and the like are used. When such an amino acid production selectable marker gene is used, a yeast requiring the amino acid is used as a host yeast for transformation.

On the other hand, as the E. coli-derived gene, an origin that enables autonomous propagation of a plasmid in E. coli and a selectable marker gene that functions in E. coli are used. As the selectable marker for Escherichia coli, various drug resistance genes, for example, an ampicillin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene and the like are used.
As such an E. coli-derived gene fragment having both the origin and the drug resistance gene, for example, E. coli plasmid pBR3
It is possible to use gene fragments from 22.

An example of such a shuttle vector for exogenous gene expression is the shuttle vector pPS1 shown in FIG. This shuttle vector contains the Pho5 promoter and GAP
Between the terminators, there are XhoI and BamHI sites as integration sites for foreign genes.

The CPB-I expression plasmid of the present invention contains a CPB-
It is constructed by incorporating I-cDNA. The binding of both genes is carried out by a conventional method, but by using a synthetic linker or the like, it is possible to bind two DNA fragments having different restriction enzyme recognition sites (cut by the restriction enzyme) at the ends. Such a synthetic linker can be a commercially available one, or can be prepared by a DNA synthesizer depending on the purpose.

By introducing such a CPB-I expression plasmid into yeast as a host by a conventional method, the CPB-I of the present invention can be obtained.
An expression transformed yeast is obtained. A typical method for transforming yeast is a method of transforming yeast into protoplasts and introducing a plasmid (Hinnen et al., Proc. Natl. Acad. Sci. USA, V.
ol. 75, p1929 (1978)], a method for transforming yeast with an alkali metal [Kimura et al., J. Bacteriol., Vol. 153, p163 (198)
3)].

Representative examples of host yeast include Saccharomyces cerevisi
ae AH22 [a leu2 his4 Can1] (Miyako Kenjo No. 312) and the like. As the host yeast, those suitable for the yeast selection marker gene used for the expression plasmid are used. Further, in a normal host yeast, since the Pho5 promoter has a suppressed promoter activity in the presence of phosphate, use an improved yeast host cell so as not to suppress the Pho5 promoter activity even in the presence of phosphate. It is also possible. Such yeasts include Saccharomy
ces cerevisiae AH22 pho80 (Microtechnical Laboratory No. 509). With such an improved strain, the function of the Pho5 promoter is not suppressed even in the presence of phosphate, and CP
It becomes possible to express BI.

Normal yeast host, Ph in the presence of phosphate
In a transformant using yeast that suppresses the function of the o5 promoter, the yeast is first grown to exponential growth in a medium containing phosphoric acid, and then collected by centrifugation, and then contains no phosphate. By culturing in a medium, the yeast that has grown to the desired number of cells allows the Pho5 promoter to function,
-I can be expressed all at once.

In addition, as the medium, a medium in which a yeast selection marker is utilized is used. For example, the above Saccharomyces cere
When using visiae AH22 (a leu2 his4 Can1),
When the host yeast is a leucine-histidine auxotroph and any of its amino acids is supplemented by a recombinant plasmid, a synthetic medium containing the other defective amino acid, such as a minimal bulk holder medium (Higashie et al., J.Bact
eriol., 113, p727-738, PRBurkholder, Am. J. Bot., 30,
p206 (1943)].

In order to increase the number of cells per unit medium and improve the expression level of CPB-I, instead of the above-described selective synthetic medium, a nutrient-rich medium containing a yeast extract called a semi-synthetic medium Can be used. Usually, when such a non-selective medium is used, the number of cells increases, but the yeast from which the plasmid has been removed also proliferates, and as a result, the expression level often decreases. In the body, such a phenomenon is not confirmed, and even in a non-selective medium, the expression level is equivalent to that of the selective medium.

In the case of large-scale culture, the number of yeast cells is multiplied by stepwise culture.First, culture is performed using a selective medium on the scale of several liters, and then this is tens to ten times using a nutrient-rich semisynthetic medium. It can be cultured on a scale of several hundred liters.

According to such a method for producing CPB-I, CPB-I can be expressed as the most abundant protein contained in the yeast cell lysate, and the target CPB-I can be extremely efficiently used in subsequent purification. Can be purified.

As a method for purifying CPB-I, CPB-I can be purified very efficiently by applying ordinary purification means.

Analysis of the amino terminus of the yeast-derived CPB-I thus obtained revealed that CPB-I purified from the placenta was used.
As in the above, it was confirmed that the amino terminus had undergone acetylation. From this, it was produced by the method of the present invention.
It is presumed that the physiological activity of CPB-I is not at all inferior to the natural one.

The present invention enables CPB-I having such excellent properties to be expressed in a very large amount by a transformed yeast, and is particularly excellent in the production of CPB-I on an industrial level. Technology.

 Hereinafter, the present invention will be described in more detail with reference to examples.

Example (1) Preparation of CPB-I Gene Cloning was performed using phage from a human placenta cDNA library, and the expression product was screened using an anti-CPB-I mouse monoclonal antibody. As a result, an almost full-length cDNA spanning 1566 bp was obtained, and this gene fragment contained a CPB-
It was confirmed to contain the gene encoding I (see FIG. 1). The cloning of the cDNA of CPB-I is described in further detail in JP-A-64-20095.

(2) Preparation of shuttle vector pPS1 for yeast A yeast-Escherichia coli shuttle vector pAM82 (JP-A-59-36699) having an acid phosphatase (Pho5) promoter as a promoter for exogenous gene expression was obtained by using a restriction enzyme.
It was treated with Xho I and Pvu II and subjected to 2% agarose gel electrophoresis to obtain a gene fragment of about 9.8 kbp containing the Pho5 promoter and others.

On the other hand, a Hind III fragment of the GAP-DH gene (J. Biol. Chem.,
Vol. 255, No. 6, p2596-2605 (1980)]
Plasmid pBR-GAP integrated at the II site was replaced with the restriction enzyme Sa.
Treatment with lI and EcoR V yielded a gene fragment containing the GAP terminator (Sal I-EcoR V fragment). This gene fragment was cloned into SalI-Sma of the cloning vector pUC19.
A plasmid pUC-GAPter having a GAP terminator was incorporated at the I site. This plasmid is replaced with the restriction enzyme Sal I
After cleavage by digestion with DNA polymerase (Klenow fragment), the cut was filled in.
The I linker was introduced and circularized again. Next, this plasmid was treated with a restriction enzyme PstI, and reacted with a DNA polymerase in the same manner as described above, and then an XhoI linker was introduced. This results in a BamHI site and X just upstream of the GAP terminator.
Plasmid pUC-GAPter (BamH I,
Xho I). This plasmid was treated with a restriction enzyme RsaI, and a gene fragment of about 1.4 kbp containing a GAP terminator was obtained by agarose electrophoresis. This is further restricted enzyme
After treatment with XhoI, the same operation as above was performed to obtain an RsaI-XhoI fragment containing a GAP terminator.

This gene and the Xho I-Pvu derived from pAM82 prepared above
The II fragment was ligated using T4 DNA ligase to obtain the yeast-E. Coli shuttle vector pPS1. This shuttle vector has a Pho5 promoter and a GAP terminator as expression control genes for foreign genes, and further functions as a yeast-E. Coli shuttle vector.
And leucine production gene (Leu2) as ampicillin resistance gene and pBR322 orig
This is a vector for high expression of a foreign gene having in (see FIG. 2).

(3) Construction of CPB-I expression plasmid The CPB-I-cDNA obtained in the above (1) was digested with the restriction enzyme NcoI.
And Nc containing all the structural genes of CPB-I
o An I-SacI fragment was obtained (see FIG. 1).

In order to incorporate the CPB-I-cDNA Nco I-Sac I fragment into the Xho I-BamHI site of the shuttle vector pPS1,
The two different synthetic linkers shown in FIG. 3 were made as follows. DNA synthesizer (Applied Biosystems 381
The following four types of DNAs were synthesized using A).

The above DNA1 and DNA2 were mixed and annealed to obtain a synthetic linker shown as Linker A in FIG. This linker joins two DNA fragments having different cuts cut with restriction enzymes Xho I and Nco I. The base sequence of the linker as a whole is originally based on the Pho5 It is designed to be very close to the DNA sequence of the reading sequence.

Further, DNA3 and DNA4 were mixed and annealed to obtain a synthetic linker shown as Linker B in FIG. This synthetic linker joins two DNA fragments cut with restriction enzymes SacI and BamHI, and the DNA sequence in the middle thereof has restriction enzyme recognition sequences of restriction enzymes HindIII and XhoI.

CPB-I-cDNA Nco I-Sac I gene fragment, synthetic linker A, synthetic linker B and shuttle vector pPS1
Four types of gene fragments obtained by treating with restriction enzymes Xho I and BamH I were mixed and reacted with T4 DNA ligase.

The reaction solution was subjected to phenol treatment and ethanol precipitation, and then used to transform E. coli HB101 competent cells. The plasmid DNA of the resulting transformed clone was recovered, treated with an appropriate restriction enzyme, and analyzed for a cleavage pattern from agarose gel electrophoresis, whereby CPB was introduced into the XhoI and BamHI sites of the shuttle vector pPS1.
The desired CPB incorporating the I-cDNA Nco I-Sac I fragment
An E. coli clone having the -I expression plasmid pAPCPB-I (see FIG. 4) was obtained. Plasmid pAPCPB-I was prepared from this clone according to a conventional method.

(4) Preparation of transformed yeast expressing CPB-I As a host yeast, Saccharomyces cerevisiae AH22P
Using ho80 (Microtechnical Co., Ltd. No. 508), this was used for YPD medium (2
% Polypeptone, 1% yeast extract, 2% glucose), inoculated into 100 ml, cultured at 30 ° C. to 5-7 × 10 ⁶ , centrifuged, collected, washed, and suspended in 1 ml of lithium solution. After shaking at 30 ° C for 1 hour, 1/10
0 μm) and about 2 μg of the recombinant DNA (plasmid pAPCPB-
I) was added and shaken at 30 ° C. for 30 minutes. 0.7 ml of a polyethylene glycol solution was added thereto, and the mixture was allowed to stand at 30 ° C. for 90 minutes. After heat treatment at 42 ° C. for 5 minutes, the plate was washed twice with sterilized water.
The cells were suspended in 0.1 ml of a minimal medium (0.7% yeast nitrogen-based amino acid, 2% glucose, 20 μg / ml histidine, 2% agar) and applied to the minimal medium plate.
After culturing at 30 ° C., leucine non-requiring colonies were obtained. The colony was cultured in a bulk holder minimum medium containing 20 μg / ml histidine to obtain a transformed yeast Saccharomyces cerevisiae pAPCPB-I.

(5) Expression of CPB-I by yeast The transformed yeast was cultured with shaking at 30 ° C for 3 days, and the yeast cells were collected by centrifugation (3500 rpm, 5 minutes).
The suspension was suspended in a lysis solution (25 mM EDTA-25 mM Tris-HCl buffer (pH 7.4)) in the amount of a culture solution, mixed with glass beads, and subjected to physical shock to the yeast to disrupt the yeast. This was centrifuged to remove the glass beads and the yeast fragment, and a centrifuged supernatant was obtained.

For this supernatant, the activity of CPB-I was measured by ELISA using an anti-CPB-I monoclonal antibody. This ELIS
A consists of the following operations.

First, an anti-CPB-I mouse monoclonal antibody as a primary antibody was diluted to 10 μg / ml with a coating buffer [0.05 M sodium carbonate buffer (pH 9.6)] in a polystyrene 96-well ELISA microplate. To 100μ
Inject each time. Let stand at 25 ° C for 2 hours, and add PBS-Tween (PBS1
After washing with 0.5 ml of Tween 20), 100 μl of the CPB-I standard solution and the specimen are injected into each well, and the mixture is reacted at 25 ° C. overnight. Then, after washing with PBS-Tween, horseradish peroxidase-labeled Fab 'anti-CPB-
Antibody I is diluted with PBS-Tween, injected into each well at 100 μl, and reacted at 25 ° C. for 2 hours or more. After washing three times,
The OPD solution is added as an enzyme substrate solution in 100 μl increments. After reacting at 25 ° C. for 30 minutes in a dark place, 4.5 MH ₂ SO ₄ is added by 50 μl each and mixed with a mixer to stop the reaction. The absorbance at 492 nm is measured, and a calibration curve is determined from the absorbance of the CPB-I standard to determine the amount of CPB-1 in each sample.

As a result, it was confirmed that about 150 mg of CPB-I was obtained from one yeast culture. Also, from the results and the results of measuring the total solubilized proteins in the yeast crushed liquid, in the present invention, about 40% of the total solubilized proteins contained CP.
It has been found possible to obtain BI.

Next, this yeast crushed liquid was analyzed by SDS-polyacrylamide gel electrophoresis. That is, the sample was electrophoresed on a 10% polyacrylamide gel, and stained with Commercial Brilliant Blue (CBB). As a result, a band of CPB-I (molecular weight: about 34,000), which was extremely large in amount, was confirmed as compared with bands of other yeast-derived proteins. FIG. 5 shows a schematic diagram of the result.

(6) Production of CPB-I by mass cultivation on an industrial scale The transformed yeast obtained in the above (4) was cultured in a minimal medium.
The culture was performed at 30 ° C. on a scale of 2. This was then inoculated into 30 minimal media and cultured at 30 ° C. for 71 hours.
Semi-synthetic medium (40 g sucrose, 5 yeast extract per medium)
g, ammonium sulfate 5g, magnesium sulfate heptahydrate
0.5 g, 0.025 ml of polyoxyethylene polyoxypropylene ether as an antifoaming agent) at 30 ° C. for 24 hours.
The amount of CPB-I present in the yeast cells after the final culture was determined by the ELI method.
As measured by SA, even in a semi-synthetic medium without selectivity, the expression level was the same as that in a selective synthetic medium (minimum medium).
The presence of CPB-I was confirmed.

This indicates that the transformed strain has extremely high expression stability even in subculture.

Further, the expression level of CP at the same level as the expression level obtained in the expression experiment on the small scale in (4) above.
It was confirmed that the expression of BI was maintained.

(7) Large-scale purification of CPB-I Recombinant yeast was collected from the culture solution cultured in large quantities using a 0.1 μm membrane filter, and subjected to physical stimulation by a French press-type cell disrupter to obtain yeast. The fungus was disrupted. Then, filtration was performed, and the obtained crude extract was concentrated by an ultrafilter. After acetic acid was added thereto to perform isoelectric precipitation (pH 4.5), the formed precipitate was removed by centrifugation, and the pH of the supernatant was adjusted to 7.0 with ammonia.
Subsequently, it was subjected to anion exchange chromatography using QAE-Toyopearl 550C (manufactured by Tosoh Corporation). That is,
10 mM phosphate buffer (pH 7.4) containing 70 mM sodium chloride
The crude extract was applied to the column equilibrated in, washed with the same buffer, and eluted with a 10 mM phosphate buffer (pH 7.4) containing 300 mM sodium chloride. The fraction containing the obtained CPB-I was concentrated by an ultrafilter, and then subjected to gel filtration using a TSKG3000 column (manufactured by Tosoh Corporation) equilibrated with 10 mM phosphate buffer (pH 7.4) containing 10 mM sodium chloride. went. afterwards
The fraction containing CPB-I was re-contained in 1 containing 70 mM sodium chloride.
QAE Toyopearl 5 equilibrated with 0 mM phosphate buffer (pH 7.4)
After applying to 50C and washing, the residue was eluted with a linear gradient of sodium chloride from 70 mM to 300 mM to obtain purified CPB-I.

(8) Physical properties of CPB-I produced by yeast (A) N-terminal amino acid sequence analysis: 5.6 mg of yeast-derived CPB-I was dissolved in 0.2 M Tris-HCl buffer (pH 8.2) containing 6 M guanidine, After adding 5 mg of dithiothreitol and reacting at room temperature for 30 minutes, iodoacetic acid 5
0 mg was added and reacted at room temperature for 30 minutes to obtain S-carboxymethyl-CPB-I.

20 mM Tris-HCl buffer (pH 7.4) containing 2 M urea
, And 100 μg of trypsin was added thereto, followed by digestion at 37 ° C. for 24 hours. Then, the peptide was separated by reverse phase chromatography.

Using Cosmosil 5 C ₁₈ 300 Å (diameter 10 mm × length 250 mm, manufactured by Hanoi Chemical Co., Ltd.) as a chromatographic column,
Using a 0.1% trifluoroacetic acid solution and an 80% acetonitrile solution containing 0.1% trifluoroacetic acid as an eluent, elution was performed at a flow rate of 1 ml / min by a linear concentration gradient.

Detection was by UV absorption at 214 nm. Amino acid composition analysis was performed on each of the obtained peptides using a picodag amino acid analyzer (manufactured by Millipore Limited), and a fraction containing the N-terminal peptide was identified. It was confirmed that this N-terminal peptide was composed of arginine and five amino acids of glutamic acid, alanine, valine and leucine.

Subsequently, as a result of amino acid sequence analysis, the N-terminal amino group was blocked. Further, as a result of analysis by a FAB-MASS analyzer (JMS-D300 manufactured by JEOL Ltd.), the molecular weight was 627.

From the above results, it was determined that the N-terminus was acetylated and had the following sequence.

Acetyl-Ala-Gln-Val-Leu-Arg (B) Comparison of anticoagulant activity with placenta-extracted CPB-I The anticoagulant activities of the present yeast-derived CPB-I and human placenta-derived CPB-I were compared. That is, 100 μg of a 0.5 mg / ml PT reagent (Rioplastin, manufactured by Mochida Pharmaceutical Co., Ltd.) and 100 μm of various concentrations of the sample were mixed, and 3 minutes later, 2 μl of physiological saline was added.
A 200-fold diluted standard solution was added, and the blood coagulation time was measured.

As can be seen from Table 1 comparing the anticoagulant effects of the yeast-derived CPB-I and the human placenta-derived CPB-I, the yeast-derived CPB-I
BI and human placenta-derived CPB-I showed almost the same prolongation effect of blood coagulation time (PT).

[Brief description of the drawings]

FIG. 1 shows the CPB-I cloned earlier by the applicant.
FIG. 2 is a view showing the entire base sequence of cDNA. Translation start codon AT
Numbers were assigned with A of G as the first. FIG. 2 is a diagram showing the structure of the shuttle vector pPS1 used in the present invention. FIG. 3 is a diagram showing the structure of a synthetic linker used in the construction of plasmid pAPCPB-I. FIG. 4 shows the CPB-I expression plasmid pAPCPB-I of the present invention.
FIG. 3 is a diagram showing the structure of FIG. FIG. 5 is a schematic diagram showing a pattern of polyacrylamide gel electrophoresis of a disrupted yeast solution, showing the expression of CPB-I by the transformant of the present invention. Lane 1 is a molecular weight marker from the top, 200K, 97K, 68K, 43K, 29K, 18.4K, 14.3K
Indicates Dalton. Lanes 2 to 4 show the migration patterns of the crushed liquid of the transformant for CPB-I production of the present invention. Lane 5
Fig. 2 shows the electrophoresis pattern of a disrupted solution of the host yeast having no plasmid of the present invention (negative control).

Continuation of the front page (51) Int.Cl. ⁶ Identification symbol FI (C12P 21/02 C12R 1: 865) (72) Inventor Akio Iwasaki 1-1-14 Tokiwadaira, Matsudo City, Chiba Prefecture Hasegawa Residence 302 (72) Invention Person Makoto Suda 2-17-43 Noguchicho, Higashimurayama-shi, Tokyo Kowa Higashimurayamaso 206

Claims (6)

(57) [Claims] 1. A shuttle vector having a yeast-derived gene and an Escherichia coli plasmid-derived gene, further comprising a Ph.
A recombinant plasmid having a CPB-I expression gene fragment consisting of an o5 promoter, CPB-I-cDNA and a GAP-DH terminator. 2. The recombinant plasmid according to claim 1, wherein the yeast-derived gene has 2 μori, ars1, and a selectable marker gene for transformed yeast. 3. The gene derived from Escherichia coli is plasmid pBR322.
The recombinant plasmid according to claim 1, which has an origin of origin and a drug resistance gene. 4. A transformed yeast obtained by introducing the recombinant plasmid according to claim 1 into yeast. 5. The transformed yeast according to claim 4, wherein the host yeast is Saccharomyces cerevisiae. 6. A method for producing CPB-I, comprising culturing the transformed yeast according to (4) or (5) and collecting CPB-I from the culture.