JPH0678767A - Mutant type gal4, its dna and method for enhancing expression of foreign protein with the same mutant type gal4 - Google Patents

Abstract

PURPOSE:To provide a method for enabling a foreign protein under the control of a GAL1, GAL7 or GAL10 promoter to further enhance and improve its expression as compared with the case of employing no or a wild type GAL4 by using a mutant type GAL4. CONSTITUTION:The mutant type GAL4 described in sequence No.1 (the sequence table is omitted) is obtained by adding Asp to a position between Asp in the 863-position and Val in the 864-position from the N-terminal of a wild type GAL4. Sequence No.2 (the sequence table is omitted) can be exemplified as the DNA capable of coding the mutant type GAL4. Thereby, the expression of a foreign protein can be enhanced by transducing an expression unit of the mutant type GAL4 and a unit capable of expressing the foreign protein under the control of a GAL1, GAL7 or GAL10 promoter into a yeast and culturing the resultant yeast.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mutant GAL4 useful for enhancing the expression of a heterologous protein, its DNA and a method for enhancing the expression of the heterologous protein.

[0002]

BACKGROUND ART The transcriptional activation of yeast galactose metabolism genes, that is, the GAL1, GAL7 or GAL10 promoter is controlled by the positive transcriptional regulatory gene GAL4 and the negative transcriptional regulatory gene GAL80, which is activated by the addition of D-galactose. It is an inducible promoter.

It has been reported that the transcriptional activation of the GAL gene by GAL4 can be used to enhance the expression of a heterologous protein under the control of a GAL promoter (Japanese Patent Laid-Open No. 63-94969, PCT International Publication WO88 /
02779). On the other hand, the structure and function of GAL4 at the protein level are being analyzed, and DNA sequence and GAL4
The site of interaction with 80 has also been elucidated. That is, GAL4 consists of 881 amino acid residues, and in the absence of galactose, GAL80 is 2 at the C-terminal of GAL4.
6 amino acid residues (region 856 to 881 from N terminus)
And suppresses the activity of GAL4 (“Gene expression and repression I” Maruzen, 219-245 (1990)). Further, although wild-type GAL4 requires induction of D-galactose, various mutant GAL4s which do not require induction of D-galactose by adding, deleting or adding amino acids in the above region are also known. ing. [Nucl.
Acid, Res. 18, 5213-5217 (199
0), Cell, 50, 143-146 (1987),
Genetics, 125, 21-28 (199
0)].

[0004]

The inventors of the present invention have made extensive studies in view of the above circumstances, and as a result, by using the newly obtained mutant GAL4, wild type GAL4 was obtained.
The inventors have found that the expression of a heterologous protein under the control of the GAL1, GAL7 or GAL10 promoter can be further enhanced / improved as compared with the case of using AL4 or the case of not using GAL4, and the present invention has been completed.

[0005]

SUMMARY OF THE INVENTION The present invention is a wild type GAL.
863th Asp and 864th V from the N terminus of 4
Mutant G of SEQ ID NO: 1 in which Asp is added between al
AL4, DNA encoding the same, and method for enhancing expression of heterologous protein using the same, that is, mutant GAL4
Expression unit and GAL1, GAL7 or GA
This is a method for enhancing the expression of a heterologous protein, which comprises introducing a unit for expressing a heterologous protein into yeast under the control of the L10 promoter and culturing the yeast. (1) Mutant GAL4 The mutant GAL4 of the present invention is As at the 863rd position from the N-terminus of wild-type GAL4 (consisting of 881 amino acid residues).
Asp (in SEQ ID NO: 1, between p and the 864th Val (ie, the 865th Val in SEQ ID NO: 1))
864th) is added, and other sequences are the same as the wild type. Therefore, it consists of 882 amino acid residues. The entire nucleotide sequence of wild-type GAL4 in yeast has been determined [Allen et al., Molecular.
and Cellular Biology, 4, 26
0-267 (1984)] in the present invention.
The base sequence of AL4 can include all base sequences encoding the amino acid sequence of SEQ ID NO: 1, and the newly inserted A
The base encoding sp is not particularly limited and is arbitrary.
Examples of the DNA sequence encoding the mutant GAL4 include those described in SEQ ID NO: 2.

Examples of the method for preparing mutant GAL4 include a method in which the necessary DNA sequence of wild-type GAL4 is cleaved / deleted by treatment with a restriction enzyme, and a desired DNA sequence is synthesized and then replaced. . (2) Expression of heterologous protein (i) Unit for expressing heterologous protein Units for expressing heterologous protein are GAL1 and GA
A DNA encoding a heterologous protein is placed under the control of the L7 or GAL10 promoter. The heterologous protein is not particularly limited, but specifically, pro-urokinase (JP-A-60-180591), modified pro-urokinase (JP-A-63-146789, JP-A-3-87180, JP-A-3-87180, JP-A-3-87180, JP-A-3-87180 and JP-A-3-87180).
87181), tissue plasminogen activator (t-PA; JP-A-59-183693, 59-42).
321), hepatitis B virus surface antigen (HBsAg; JP-A-59-36698), PreS-HBsAg (JP-A-56-63995), interferon-γ (JP-A-61-108397), colony formation stimulating factor (special feature). Kai-61-199787), human serum albumin
8-56684, 58-90515, 58-150
517, 63-74493, and JP-A-2-10429.
0), antithrombin-III (JP-A-63-4489).
8), chimeric antibody (PCT International Publication WO91 / 0664
9) etc. are illustrated. It may also be an active fragment or derivative of these heterologous proteins.

The unit for expressing a heterologous protein of the present invention can be obtained by introducing a DNA encoding the heterologous protein into an expression system usually used in yeast except that the GAL1, GAL7 or GAL10 promoter is used. A preferred embodiment is a vector in which a GAL1 promoter, a signal sequence, a DNA encoding a heterologous protein, a terminator, a homologous region, a marker gene for selection and the like are arranged (PCT International Publication WO91.
/ 06649).

(Ii) GAL4 Expression Unit The GAL4 expression unit can be obtained by introducing (1) the DNA encoding the mutant GAL4 into an expression system usually used in the yeast of the present invention. As a preferred embodiment, a vector in which a promoter, (1) DNA encoding mutant GAL4, terminator, homologous region, marker gene for selection and the like are arranged is exemplified. Examples of the promoter include GAL1 promoter, GAP-DH promoter and the like. GA as a terminator
P-DH terminator, PHO5 terminator, etc. are illustrated. Examples of the homologous region include amino acid or nucleic acid synthesis genes, and specifically, LEU2, HIS4,
Examples include TRP1 and URA3. As the marker gene for selection, the above-mentioned amino acid or nucleic acid synthesis genes can be used, and also genes expressing antibiotic resistance and the like can be mentioned. For example, ampicillin, cyclohexide, G
-418, chloramphenicol, bleomycin or hygromycin B, and the like, which confers resistance to antibiotics.

The unit (i) expressing a heterologous protein and the unit (ii) expressing GAL4 may be arranged on the same vector. In this case, according to the known method,
(Ii) The GAL4 expression unit is ligated to the upstream side of the GAL1 promoter or the downstream side of the terminator in the unit (i) expressing the heterologous protein. (Iii) Introduction into yeast In the present invention, yeast, particularly Saccharomyces or Pichia, is used as a host. Preferably, an auxotrophic strain or an antibiotic sensitive strain can be used. Saccharomyces which is an auxotrophic strain and a G418 sensitive strain
Cerevisiae (Saccharomyces cerev
isiae ) AH22 strain (a, his4, leu2, c
an1) or auxotrophic strain UNHB derived from AH22
(A, lue2, his4, ura3, can1) and the like are preferably used.

Examples of the formation conversion method include a method of directly introducing a plasmid into a host cell and a method of integrating the plasmid into a yeast chromosome. The former is performed by a known method. Specific examples include the protoplast ethylene glycol method and the electroporation method. The latter is carried out, for example, by the following method, and in the present invention, a heterologous protein-producing yeast in which the plasmid is thus integrated into the yeast chromosome is more preferably used.

In the latter method, the plasmid contains a partial DNA sequence of a gene existing in the host yeast chromosome (for example, LEU2, HIS4, TRP1, URA3, ribosomal DNA gene, etc.). Due to the homologous sequence,
The entire plasmid or its linear fragment can be stably introduced into the host chromosome by homologous recombination. For example, a plasmid containing a naturally occurring sequence in a yeast chromosomal gene and a heterologous protein gene can be stably integrated at the locus of the chromosomal gene.

[0012] Hereinafter, a method for incorporating a chimeric antibody as a heterologous protein will be specifically described.
This example shows one suitable means, and is not limited to this method. The homologous sequence sites can be replaced by selective repeats. As a host, Saccharomyces cerevisiae ( Saccharomyces cerevisiae), which is a leucine- and histidine-requiring mutant and is a G418-sensitive strain,
yces cerevisiae ) AH22 strain (a strain having a mutation in leucine synthesis gene LEU2 and histidine synthesis gene HIS4) or an AH22-derived uracil auxotrophic mutant strain is used.

First, LEU2, a gene for making leucine non-auxotrophic, is transformed with a plasmid having a sequence homologous to the chromosomal sequence of the host yeast cell. The obtained transformant has a plasmid containing a heterologous protein coding region inserted at the LEU2 gene site on the chromosome, and is a strain that does not require leucine, that is, can grow even in a leucine-free medium.

Next, using this transformant as a host, a plasmid having URA3, a gene for making uracil non-auxotrophic, as a sequence homologous to the chromosomal sequence of the host yeast cell (including, of course, a heterologous protein coding region) Transform with. The obtained transformant has a plasmid containing a heterologous protein coding region inserted at the URA3 gene site on the chromosome, and is a strain that does not require uracil, that is, can grow even in a uracil-free medium. At this point, the heterologous protein, the gene of interest for expression, is introduced at two sites, URA3 and LEU2.

Then, using the transformant that has become non-auxiliary for uracil and leucine as a host, HIS4 is transformed with a plasmid having a sequence homologous to the chromosomal sequence of the host yeast cell. This plasmid contains the mutant GAL4 coding region. The obtained transformant has a plasmid containing a mutant GAL4 coding region inserted at the HIS4 gene site on the chromosome, and is not histidine auxotrophic. Therefore, this transformant contains the heterologous protein gene at two sites in the URA3 and LEU2 gene sites on the chromosome and the mutant GAL4 gene at one site in the HIS4 gene site on the chromosome. At this time, the order of insertion does not matter.

If many kinds of auxotrophic mutant strains can be obtained as a host, or if a strain showing sensitivity to many kinds of antibiotics can be obtained, a plurality of useful genes can be introduced into a region corresponding thereto. Thus, the target gene can be inserted into multiple regions on the host chromosome. The genes integrated into these chromosomes are stably maintained without loss, and by incorporating a plurality of genes, it becomes possible to obtain a large amount of the target product.

The transformant is cultured in a known medium. Y
PD medium [1% yeast extract (Difco
2% bactopeptone (Difco), 2% glucose], YPG medium [1% yeast extract,
2% bactopeptone, 2% galactose] and the like. Culturing is usually performed at 15 to 43 ° C. (preferably about 30 ° C.) for about 20 to 100 hours, and aeration and stirring can be added if necessary.

For example, in the case of intracellular expression, cells expressing a heterologous protein through genetic manipulation are treated by a known method such as a freeze-thaw method, a glass bead method, a high pressure method, an ultrasonic treatment method and an enzyme treatment method. , Can be collected as an extract fraction containing a heterologous protein. In addition, a heterologous protein such as a chimeric antibody can be obtained from the culture supernatant if it is expressed extracellularly (secretion expression). The heterologous protein can be purified by a known method. For example, fractionation treatment, ultrafiltration, gel filtration, ion exchange chromatography, affinity chromatography, adsorption chromatography, density gradient centrifugation, dialysis and the like can be mentioned.

Further, a heterologous protein preparation can be prepared by applying a known preparation technique.

[0020]

EXAMPLES Hereinafter, specific examples of the present invention will be described.
The present invention is not limited to this. Materials and Methods 1) Construction of GAL4 transfer unit 1-1. The plasmid DNA GAL4 gene was ligated to the SmaI site of pUC19.
pKI1 cloned in the same direction as the c operator
Prepared from 01. The base sequence of the gene is LOCU registered in the international DNA database GenBank.
S: According to YSCGAL4. GAL1 promoter,
GAP-DH promoter and GAP-DH terminator were cloned into pGAL19,
Prepared from pMB018 and pMM016. In addition, yeast HIS4, URA3 and LEU2 genes are pMB0
35, pMB046 and pMB051. G
Chimeric L and H chain transcription units (Head-to-Head) using the AL1 promoter were prepared from PMB039. The above-mentioned plasmid was purified by CsCl density gradient centrifugation in the presence of EtBr.

Reference Example 1 (Construction of plasmid pKI101 carrying GAL4 gene) S. cerevisiae GRF18 pho80
cir0 (α, leu2-3, 112, his3-1
Chromosomal DNA of 1,15, met2) is AflII and Ba
GAL consisting of 3.6 kbp after double digestion with nII
A 5'-overhanging 3'-overhanging DNA fragment containing 4 genes and their promoters was obtained. At this time, S. cerevisia
The entire nucleotide sequence of the GAL4 gene of e has been determined [A
llen et al., Molecular and Cellu
lar Biology, 4, 260-267 (198)
4)], the 21-mer G4-1 (SEQ ID NO: 3) which is the complementary strand up to the base sequence number 2945 in the report.
And 21-mer G4-2 (SEQ ID NO: 4), which is a complementary strand of nucleotide sequence numbers 659 to 679, as a synthetic probe, Southern hybridization was carried out,
The DNA fragment of interest was recovered.

After blunting with Klenow enzyme, p
It was inserted into the SmaI site of UC19. E. coli
After transformation using HB 101 competent cells, screening by colony hybridization was performed to obtain pK which is a plasmid containing the target DNA fragment.
I101 was obtained. The DNA sequence was partially determined by the dideoxy reaction. As a result, the sequence starts from T of SEQ ID NO: 7 of Allen et al. Immediately after CCC of the SmaI site of the universal cloning site of pUC19, and it is possible to read to C of 55, which is completely consistent with their report. did.

The DNA sequence was partially determined using RV and the above-mentioned two kinds of synthetic probes. As a result, when RV was used as a primer, the nucleotide sequence number 3599 of Allen et al. Was located 5 ′ from immediately after GGG at the SmaI site of the universal cloning site of pUC19.
I was able to read from number G to number 3552, which was completely consistent with their report. When G4-1 was used as a primer, it was possible to read from G at 2930 to C at 2861, which was completely consistent with their report. When G4-2 is used as a primer, you can read from T at 651 to T at 601.
It was in perfect agreement with their report.

As a result, it was clarified that pKI101 contains the GAL4 gene in the forward direction with respect to the lac promoter of pUC19. Reference Example 2 pGAL19, pMM016, pMB035, pMB0
46, pMB051 is PCT international publication 091/0664
9 are described.

Reference Example 3 (Construction of pMM018) pMB007 is described in PCT International Publication 091/06649. pYN019 is a plasmid carrying the GAP-DH promoter. The GAP-DH promoter is described by J. Biol. Chem. , 254, 12,
5466 (1979); Biol. Chem. , 2
54, 19, 9839 (1979); JP-A-63-84.
498 or a method similar thereto.

The mHSA signal (signal) sequence is described in JP-A-2-167095 (that is, the DNA and amino acid sequences set forth in SEQ ID NO: 5). The transcription unit of the H chain is composed of the GAP-DH promoter (derived from pYN019) and the chimeric H chain DNA (pMB007).
Origin (step 1) and then downstream of GA
It was prepared by ligating a P-DH terminator (derived from pMM016) (step 2). The thus-constructed plasmid containing the chimeric H chain transcription unit (H-unit) was designated as pMB018 (FIG. 11).
reference).

Reference Example 4 (construction of pMB039) Materials and method 1. The chimeric A-7 Fab expression vector, pMB023, which uses the plasmid DNA GAL1 promoter, is described in PCT International Publication WO91 / 06649 described in Reference Example 2. Yeast HI
PYeHis4 containing S4 gene is Gene, 18 , 4
7-59 (1982).

2. Linker DNA The NotI linker [NEB. # 11
25] was used. 3. Enzymes Restriction enzymes are manufactured by Takara Shuzo or BRL, CIAP is manufactured by Boehringer, and T4-DNA polymerase is manufactured by Phar.
macia (# 27-0710-02) to T4-DN
A ligase manufactured by Takara Shuzo was used.

4. Other Transformation of E. coli E. coli HB101 frozen competent cells (Takara Shuzo). Other general methods followed the book. Result 1. Subcloning of HIS4 gene Among the yeast HIS4 genes contained in pYeHIS4,
The 4.75 kb restriction map from XhoI to EcoRI is in good agreement with the YSCHIS4 sequence of the GenBank DNA database.

About 4.5 kb between NcoI and SphI sites
Since the fragment from HIS4 gene to the 3'non-coding region is included in the fragment of p.
And digested with SphI, treated with T4-DNA polymerase to convert both ends into blunt ends, and a fragment of about 4.5 kb was isolated. This fragment was ligated with pUC19 which had been digested with EcoRI and SmaI and filled in with Klenow PolI to obtain pUCHIS (see FIG. 12).

2. Construction of Fab expression vector Double digestion of pMB023 with BalI and ClaI gives an approximately 4.5 kb fragment containing the Fab transcription unit and part of the G-418 resistance gene. pUCHIS
Has an NdeI site as a unit site.
Therefore, pUCHIS was digested with NdeI to form a linear chain,
Filling in with Klenow PolI and inserting a NotI linker created pMB035. On the other hand, pMB023 was double-digested with BalI and ClaI and filled in with Klenow PolI, and then a Fab transcription unit of about 4.5 kb was isolated. A NotI linker was added to this fragment to
Ligated with tI digested pMB035. Plasmid DNA was extracted from the obtained transformant by miniprep and X
As a result of screening by digesting with hoI, the transcription unit of the L-chain and the HIS4 gene were arranged in tandem,
The vector pMB039 for expression of chimera A-7 Fab (hereinafter also simply referred to as chimera Fab) in which the -unit and the H-unit are present head-to-head was obtained (see FIG. 12).

1-2. In order to bind the GAP-DH terminator downstream of the structural gene of GAL4 in constructing the transcription unit (promoter / GAL4 / terminator) of synthetic DNA GAL4,
Four types of single-stranded DNA including the following wild type and mutant type
(SEQ ID NO: 7: wild type, sense; SEQ ID NO: 8: wild type, antisense; SEQ ID NO: 9: mutant type, sense; SEQ ID NO: 1)
0: mutant type, antisense). Cohesive ends at the MluI and SalI sites are introduced at the 5'end and 3'end of the synthetic DNA so that the synthetic DNA can be easily introduced.

The sense DNA sequence is translated into the amino acid sequence described in the sequence listing by using the synthesized DNA. 1-3. DNA nucleotide sequence analysis The DNA nucleotide sequence was determined by the plasmid nucleotide sequence analysis method. The analysis kit is USB Sequenas
e Ver2.0 (TOYOBO, # 70771, d
CTP type) was used. [Α- ³² P] -dCTP is
Amersham (PB10205, ~ 3000C
i / mmol) was used. GA for sequence analysis primer
Synthesized 17-mer single-stranded DNA for confirming the nucleotide sequence of the DNA encoding the 3'terminal region of L4
(SEQ ID NO: 11) and RV primer (SEQ ID NO: 12, # 3830A manufactured by Takara Shuzo) were used.

1-4. Other restriction enzymes, T4 DNA ligase, Klenow enzyme and E. coli.
The E. coli HB101 frozen competent cell manufactured by Takara Shuzo was used. The general method follows the book. 2) Preparation of transformant producing chimeric A-7 Fab 2-1. Yeast In order to integrate the chimeric Fab expression vector and the GAL4 high expression vector prepared in the yeast into the yeast chromosome, the yeast Saccharomyces ce, which is relatively rich in the integration site, is used.
UNHB strains (a, c) derived from revisiae AH22
an1, leu2, his4, ura3) were used. After obtaining a single colony by the usual method, uracil (20m
g / L), leucine (30 mg / L) and histidine (201 mg / L) were confirmed as auxotrophy and used as hosts. The strain is YPD agar [2% bactopeptone, 2%
% Bacto-yeast extract, 2% glucose,
It was grown on 2% Noble agar) and stored at 4 ° C. until use.

2-2. Transformation method of yeast Transformation was performed by the protoplast method. In order to improve the transformation efficiency, the plasmid DNA to be introduced was linearized by digesting it at a unique site existing at one site in the selectable marker gene. The selection of transformants was performed by using the auxotrophy as an index. The selection plate is an SD plate (0.
7% yeast nitrogen base W / O amino acid, 2% glucose, 2% noble agar) was used as a base, and the required amino acid was added thereto. The amount of amino acid added [uracil (20 mg / L), leucine (30 mg / L) and histidine (20 mg / L)] was in accordance with the written manual. Arbitrarily 60 from the obtained transformants
~ 120 clones were spread on selection plates to serve as master plates and stored at 4 ° C until use.

2-3. Primary screening of chimeric Fab-producing clones YP of various preserved bacteria from master plate using toothpick
Test tube containing D medium (2 ml) (FALCON, # 2
057) was inoculated and shake-cultured at 30 ° C. for 3 days, the culture supernatant was replaced with YPG medium (2 ml) and shake-cultured for 3 days. Alternatively, each preserved bacterium can be directly analyzed from the master plate.
A test tube (FALCON, # containing PG medium (2 ml)
2057) and inoculated with shaking at 30 ° C. for 3 days.

2-4. Culture of chimeric Fab-producing clone YN
After culturing in B medium for 2 days, the cells were collected and YPD, YPG, YPG
ly (3% glycerol as carbon source) and YPGly
Using Lac (3% glycerol + 2% potassium lactate as carbon source) medium (30 ml), small-scale culture was carried out at 30 ° C. for about 3 days. The degree of yeast growth is OD540.
nm was used as an index.

2-5. Quantification of chimeric Fab The quantification of secreted chimeric Fab in the medium was determined by chimera A-
A 7-Fab purified product was used as a standard and the sandwich ELISA method described below was performed. A goat anti-human γ1 chain antibody (TAGO Inc., # 4100) that specifically recognizes the CH1 region of human γ1 chain was used as the primary antibody, and a POD-labeled goat anti-human κ chain antibody (TAGO Inc., was used as the secondary antibody. ，
# 4506) was used. In this assay, the chimeric Fab was unaffected by free κ chains present in the culture supernatant.
Can be accurately quantified.

3) Construction of Expression Vector from Transcription Unit In this study, in order to increase the production of chimeric A-7 Fab, two types of expression vectors (GAL4) were added to the yeast chromosome.
And the chimera A-7 Fab) must be incorporated. Therefore, we decided to design each expression vector according to the following policy. The GAL4 expression vector is a GAL4 expression vector in which GAL4 itself is used as a chimeric A-7 Fab expression vector.
Considering that it functions as a positive transcriptional regulator of 1 promoter, it was prepared as a transcription unit lacking a signal sequence so as to be accumulated in cells. Further, in order to maximize the effect of GAL4, it was decided not to use the original promoter of GAL4 itself but to use a high expression promoter as the promoter of the expression vector.

A constitutive GAP-DH promoter which does not require activation of a promoter (addition of an inducer, etc.) and operates with the growth of yeast, and an inducible GAL1 promoter which operates the promoter once yeast has grown. Decided to use. On the other hand, the chimeric A-7 Fab is secreted to the outside of the cells and processed exactly as a mature protein,
Since it must be secreted, it was created as a transcription unit with a signal sequence.

An auxotrophic gene marker was used to introduce the integrated expression vector into the yeast chromosome. Yeast Saccharomyces cerevisiae
UNHB strain (a, can1, leu2, his4, ur
a3) has three auxotrophic genes, but GA
Not only the L4 expression vector but also the chimeric A-7 Fab expression vector must be incorporated. Chimera A-7F
The construction of both expression vectors was proceeded by using leu2 and ura3 as the ab expression vector and his4 as the integration site of the GAL4 expression vector.

3-1. G using the GAL1 promoter
Construction of transcription unit of AL4 Construction of transcription unit of mutant GAL4 using GAL1 promoter was carried out according to the following method. GAL4 was isolated from pKI101 in which GAL4 was subcloned except for the TATA box and the 3'terminal region of the structural gene, and the GAL1 promoter (GAL1 derived from pGAL19 was isolated.
1P) was connected upstream to prepare pKT200 (Fig. 1).
Step 1). By this operation, GAL4 is connected to the GAL1 promoter at the position of the upstream-53 base (47th of SEQ ID NO: 13) of the structural gene (99th and later of SEQ ID NO: 13).

Furthermore, from pMM016, GAP-DH terminator (GAP-DHT) and synthetic DNA ( S.
O.- m, which constitutes the 3'terminal region of mutant GAL4 (see SEQ ID NOs: 9 and 10), was ligated to the downstream portion of GAL4 of pKT200 to construct a mutant GAL4 transcription unit (Fig. 1, step 2). This was named pKT203.
The transcription unit of wild type GAL4 is also p
Synthetic DNA ( SO , constituting the 3'terminal region of wild-type GAL4 constitutes downstream of GAL4 of KT200 (SEQ ID NOS: 7, 8)
(Refer to FIG. 2)) and GAP-DH terminator were connected to each other and named pKT207 (FIG. 2).

3-2. Construction of transcription unit of GAL4 using GAP-DH promoter Construction of transcription unit of mutant GAL4 using GAP-DH promoter was carried out according to the following method. 3'from pKI101 in which GAL4 is subcloned
The structural gene was isolated except for the terminal region, and the GAP-DH promoter derived from the expression vector pMB018 of the chimeric H chain was connected upstream to prepare pKT201 (FIG. 3, step 1). By this operation, GAL4 is connected to the GAP-DH promoter at the upstream-53 base (42nd of SEQ ID NO: 14) position of its structural gene (95th and later of SEQ ID NO: 14).

Furthermore, DNA fragments of the 3'end of GAL4 and the GAP-DH terminator region were prepared from the pKT203 prepared above (FIG. 1), and GAL4 of pKT201 was prepared.
The transcription unit of GAL4 was constructed by connecting it to the downstream part (FIG. 3, step 2). This was named pKT204. 3-3. Construction of GAL4 expression vector using GAL1 promoter An auxotrophic gene marker was introduced into the previously prepared GAL4 transcription unit in order to incorporate the expression vector into the yeast chromosomal gene. Yeast UNHB strain (a, can1,
Within the three auxotrophic gene markers (leu2, his4, ura3), the his4 gene was used as the integration site of the GAL4 expression vector.

The mutant GAL4 expression vector is HIS4
To pMB035 where the gene has already been cloned,
GAL4 using the GAL1 promoter prepared in FIG.
It was prepared by introducing the transcription unit (derived from pKT203) of (FIG. 4). This allows HIS4 and GAK4
Two types of expression vectors having the same transcription unit and the opposite transcription unit were obtained. PMB048 and pMB04, respectively
It was named 7.

On the other hand, the wild type GAL4 expression vector
HIS4 and GAL4 so that suppression of AL4 expression does not occur
Was prepared by using the mutant GAL4 expression vector pMB047 whose transcription unit was in the reverse direction and replacing the 3'terminal structural portion of the mutant GAL4 transcription unit. This is pK
It was named T210 (Fig. 5). 3-4. GAL4 using GAP-DH promoter
Construction of expression vector of GAL4 expression vector was prepared by introducing the transcription unit (derived from pKT204) of GAL4 using the GAP-DH promoter prepared in FIG. 3 into pMB035 in which the HIS4 gene has already been cloned (FIG. 6). As a result, an expression vector in which the transcription units of HIS4 and GAL4 were only in the reverse direction was obtained. This is pMB
It was named 049.

On the other hand, the wild type GAL4 expression vector was prepared by using the wild type GAL4 expression vector pKT207 by substituting the 3'end portion of the mutant GAL4 transcription unit. This was named pKT211 (FIG. 7). 3-5. Construction of integrated chimeric A-7 Fab expression vector using GAL1 promoter The leu2 and ura3 genes were used for the integration site of the chimeric A-7 Fab expression vector to construct the expression vector.

Transcriptional unit of integrated chimera A-7 Fab with GAL1 promoter (chimeras L- and H-
(The chain transcription unit is head-to-head), pMB
Prepared from 039 (Fig. 8). The auxotrophic gene marker LEU2 required for integration into the yeast chromosome is
Integrated chimeric A-7F with PGK promoter
Prepared from the ab expression vector pMB051. pMB0
By replacing the 39 HIS4 gene with the LEU2 gene of pMB051, an expression vector of an integrated chimeric A-7 Fab having a chimeric L chain and two types of GAL1 promoters in which the transcription units of LEU2 were in the forward and reverse directions was obtained. These were named pMB054 and pMB055, respectively.

Similarly, the auxotrophic gene marker URA
3 is an integrated chimera A having a PGK promoter
-7 Fab expression vector pMB046 (FIG. 8 right). The HIS4 gene of pMB039 was transformed into pMB046
Chimera L by substituting the URA3 gene of
An expression vector was obtained in which the chain and the transcription unit of URA3 were oriented in the forward direction. This was named pMB053.

Results 1) Preparation of transformant by yeast transformation Yeast UNHB strain (a, leu2, his4, can1)
Chimeric Fab expression vector, pMB053,
pMB054, pMB055, and GAL4 [wild type (pKT210 and pKT211) and mutant type (pMB0
47 and pMB049)] The construction of the high expression vector was completed. In the chimeric Fab expression vector, transcription units of chimeric L and H chains are composed head-to-head. In addition, pMB053 and pMB053 and p
MB054 has the URA3 and LEU2 genes, respectively. On the other hand, each GAL4 expression vector has the HIS4 gene. pKT210 (wild type) and pMB
047 (mutant type) constitutively expresses GAL4 in cells, whereas pKT211 (wild type) and pMB04
9 (mutant type) is designed to inducibly express GAL4 by addition of D-galactose.

The method for incorporating each expression vector into the yeast UNHB strain was according to the method shown in FIG. First, the chimeric Fab expression vector was incorporated into yeast, and stable and high-producing clones were selected from the obtained transformants by primary and secondary screening, and used as the mother strain into which the GAL4 expression vector was introduced. GAL4 expression vectors are four types of expression vectors shown in Table 1. In FIG. 9, the name on the top of each clone is a stable and high-performance clone obtained as a result of the primary screening.

2) Introduction of chimera A-7 Fab expression vector Chimera F in culture supernatant by sandwich ELISA method
When the ab concentration was measured, the production amount of each transformant obtained was the result shown in FIG. Chimeric Fab production is
It was observed that the number of sites of the integrated chimeric Fab expression vector increased proportionally. this is,
It was also observed in a production study of chimeric A-7 Fab using the PGK promoter. In addition, two types of expression vectors (pMB054 and pMB055) were incorporated into the leu2 site. Comparing the amounts of chimeric Fabs produced by the transformants obtained, pMB054 was introduced into pMB05.
A clone having a higher production amount than that in which 5 was introduced was observed.

3) Introduction of GAL4 expression vector GAL4 [wild type (pKT210 and pKT211) and mutant type (pMB047 and pMB049)] high expression vector (Table 1) is a chimeric Fab high producing clone L20U1.
It was integrated into the his4 site of 7 strains (Fig. 10). Comparing the appearance rates of the transformants, it was confirmed that the expression vector using the GAL1 promoter (pKT210 and pMB047) was obviously lower than the expression vector using the GAP-DH promoter.

[0055]

[Table 1] 3-1. Introduction of wild-type GAL4 expression vector As a result of the primary screening from the obtained transformants, a high-producing clone was re-cultured and chimeric Fa
The reproducibility of b production was examined (Table 2). As a result, a chimeric Fab high-producing strain could be obtained by introducing the GAL4 high expression vector into yeast in which the chimeric Fab expression vector using the GAL1 promoter was incorporated into the chromosome. Transformant into which a GAL4 high expression vector using the inducible GAL1 promoter was introduced (pKT210-2
In 3), a clone that is stable and highly produced by the induction of D-galactose (about 10.3 times more than the L20U17 strain)
was gotten. In the wild type GAL4 expression system, there was no great difference in the production amount between the clones using the inducible GAL1 and the constitutive GAP-DH promoter (Table 3).

The GAL1 promoter should originally be an inducible promoter that does not operate in the absence of D-galactose (culture in YPD medium). In the clone expressed under the wild type GAL4 using the inducible GAL1 promoter, the chimeric Fab was obtained in the absence of D-galactose.
Has not been observed. However, the configuration type GAP-
Production of chimeric Fab was observed in clones expressed under wild type GAL4 using the DH promoter (for example, pKT211-25 etc.).

3-2. Introduction of Mutant GAL4 Expression Vector Similar to wild-type GAL4, the amount of chimeric Fab produced could be increased in the mutant GAL4 expression vector (Table 2). In the wild type GAL4 (GAL4) expression system, there was no great difference in the production amount between the clones using the inducible GAL1 and the constitutive GAP-DH promoter (GAP-DHp), but the expression of the mutant GAL4 (GAL4-m). In the system, the clone using the GAL1 promoter (GAL1p) clearly increased the production amount (Table 3). Transformant into which a mutant GAL4 high expression vector using the inducible GAL1 promoter was introduced (pMB047-4)
, A stable and high-producing clone (17.3 times higher than the L20U17 strain) was obtained by the induction of D-galactose. In addition, in the clone into which the mutant GAL4 was introduced,
L20U was already obtained without induction with D-galactose.
Clones with more than 17 strains (pMB049
-34 etc.) exists.

On the other hand, production of the chimeric Fab was observed in the YPD medium even in the transformant in which the mutant GAL4 was introduced,
It was observed when any expression vector was introduced regardless of inducible and constitutive promoters (Table 2). Also,
The transformant obtained by introducing the mutant GAL4 expression vector had a higher overall chimeric Fab production amount than the case of introducing the wild type GAL4.

[0059]

[Table 2]

[0060]

[Table 3] 4) Characteristics of transformants Table 4 shows the results of the amount of chimeric Fab produced by the high-producing clones cultured on a small scale in each medium in order to investigate the characteristics of the obtained transformants. The degree of growth differs depending on the carbon source that is utilized in each clone, and YPD, YPGlyLac, Y
It became difficult to grow in the order of PG and YPGly medium.

A comparison of the production amount of chimeric Fabs shows that
Although the small-scale culture is reduced as a whole as compared to the test-tube culture, the culture results in the YPG medium used to activate the GAL1 promoter showed a high total production,
It reached about 7.5 to 10.5 times that of the mother strain L20U17 strain into which the AL4 expression vector was introduced. On the other hand, by culturing in YPD medium, a clone expressing the mutant GAL4 under the control of the constitutive GAP-DH promoter (pMB049-
34 and 49) showed almost the same production as when the induction was performed in YPG medium. Moreover, the production amount of the mother strain L20U17 cultured in YPG medium was far higher (about 5.0 to 11.2 times). This production amount is the same as that of the integrated chimeric Fa using the constitutive PGK promoter created previously.
b clone into which the expression vector was introduced (ura3 and his)
(4 sites) is almost equal to the production amount.

[0062]

[Table 4]

[0063]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 882 Sequence type: Amino acid Topology: Linear Sequence type: Protein Met Lys Leu Leu Ser Ser Ile Glu Gln Ala Cys Asp Ile Cys Arg 5 10 15 Leu Lys Lys Leu Lys Cys Ser Lys Glu Lys Pro Lys Cys Ala Lys 20 25 30 Cys Leu Lys Asn Asn Trp Glu Cys Arg Tyr Ser Pro Lys Thr Lys 35 40 45 Arg Ser Pro Leu Thr Arg Ala His Lys Thr Glu Val Glu Ser Arg 50 55 60 Leu Glu Arg Lys Glu Gln Leu Phe Leu Leu Ile Phe Pro Arg Glu 65 70 75 Asp Lys Asp Met Ile Leu Lys Met Asp Ser Leu Gln Asp Ile Lys 80 85 90 Ala Leu Leu Thr Gly Leu Phe Val Gln Asp Asn Val Asn Lys Asp 95 100 105 Ala Val Thr Asp Arg Leu Ala Ser Val Glu Thr Asp Met Pro Leu 110 115 120 Thr Leu Arg Gln His Arg Ile Ser Ala Thr Ser Ser Ser Glu Glu 125 130 135 Ser Ser Asn Lys Gly Gln Arg Gln Leu Thr Val Ser Ieu Asp Ser 140 145 150 Ala Ala His His Asp Asn Ser Thr Ile Pro Leu Asp Phe Met Pro 155 160 165 Arg Asp Ala Leu His Gly Phe Asp Trp Ser Glu Glu Asp Asp Met 170 175 180 Ser Asp Gly Leu Pro Phe Leu Lys Thr Asp Pro Asn Asn Asn Gly 185 190 195 Phe Phe Gly Asp Gly Ser Leu Leu Cys Ile Leu Arg Ser Ile Gly 200 205 210 Phe Lys Pro Glu Asn Tyr Thr Asn Ser Asn Val Asn Arg Leu Pro 215 220 225 Thr Met Ile Thr Asp Arg Tyr Thr Leu Ala Ser Arg Ser Thr Thr 230 235 240 Ser Arg Leu Leu Gln Ser Tyr Leu Asn Asn Phe His Ser Tyr Cys 245 250 255 Pro Ile Val His Ser Pro Thr Leu Met Met Leu Tyr Asn Asn Gln 260 265 270 Ile Glu Ile Ala Ser Lys Asp Gln Trp Gln Ile Leu Phe Asn Cys 275 280 285 Ile Leu Ala Ile Gly Ala Trp Cys Ile Glu Gly Glu Ser Thr Asp 290 295 300 Ile Asp Val Phe Tyr Tyr Gln Asn Ala Lys Ser His Leu Thr Ser 305 310 315 Lys Val Phe Glu Ser Gly Ser Ile Ile Leu Val Thr Ala Leu His 320 325 330 Leu Leu Ser Arg Tyr Thr Gln Trp Arg Gln Lys Thr Asn Thr Ser 335 340 345 Tyr Asn Phe His Ser Phe Ser Ile Arg Met Ala Ile Ser Leu Gly 350 355 360 Leu Asn Arg Asp Leu Pro Ser Ser Phe Ser Asp Ser Ser Ile Leu 365 370 375 Glu Gln Arg Arg Arg Ile Trp Trp Ser Val Tyr Ser Trp Glu Ile 380 385 390 Gln Leu Ser LeuLeu Tyr Gly Arg Ser Ile Gln Leu Ser Gln Asn 395 400 405 Thr Ile Ser Phe Pro Ser Ser Val Asp Asp Val Gln Arg Thr Thr 410 415 420 Thr Gly Pro Thr Ile Tyr His Gly Ile Ile Gln Thr Ala Arg Leu 425 430 435 Leu Gln Val Phe Thr Lys Ile Tyr Gln Leu Asp Lys Thr Val Thr 440 445 450 Ala Glu Lys Ser Pro Ile Cys Ala Glu Lys Cys Leu Met Ile Cys 455 460 465 Asn Glu Ile Glu Glu Val Ser Arg Gln Ala Pro Lys Phe Leu Gln 470 475 480 Met Asp Ile Ser Thr Thr Ala Leu Thr Asn Leu Leu Lys Glu His 485 490 495 Pro Trp Leu Ser Phe Thr Arg Phe Glu Leu Lys Trp Lys Gln Leu 500 505 510 Ser Leu Ile Ile Tyr Val Leu Arg Asp Phe Phe Thr Asn Phe Thr 515 520 525 Gln Lys Lys Ser Gln Leu Glu Gln Asp Gln Asn Asp His Gln Ser 530 535 540 Tyr Glu Val Lys Arg Cys Ser Ile Met Leu Ser Asp Ala Ala Gln 545 550 555 Arg Thr Val Met Ser Val Ser Ser Tyr Met Asp Asn His Asn Val 560 565 570 Thr Pro Tyr Phe Ala Trp Asn Cys Ser Tyr Tyr Leu Phe Asn Ala 575 580 585 Val Leu Val Pro Ile Lys Thr Leu Leu Ser Asn Ser Lys Ser Asn 590 595 600 Ala Glu Asn Asn Glu Thr Ala Gln Leu Leu Gln Gln Ile Asn Thr 605 610 615 Val Leu Met Leu Leu Lys Lys Leu Ala Thr Phe Lys Ile Gln Thr 620 625 630 Cys Glu Lys Tyr Ile Gln Val Leu Glu Glu Val Cys Ala Pro Phe 635 640 645 Leu Leu Ser Gln Cys Ala Ile Pro Leu Pro His Ile Ser Tyr Asn 650 655 660 Asn Ser Asn Cys Ser Ala Ile Lys Asn Ile Val Gly Ser Ala Thr 665 670 675 Ile Ala Gln Tyr Pro Thr Leu Pro Glu Glu Asn Val Asn Asn Ile 680 685 690 Ser Val Lys Tyr Val Ser Pro Gly Ser Val Gly Pro Ser Pro Val 695 700 705 Pro Leu Lys Ser Gly Ala Ser Phe Ser Asp Leu Val Lys Leu Leu 710 715 720 Ser Asn Arg Pro Pro Ser Arg Asn Ser Pro Val Thr Ile Pro Arg 725 730 735 Ser Thr Pro Ser His Arg Ser Val Thr Pro Phe Leu Gly Gln Gln 740 745 750 Gln Gln Leu Gln Ser Leu Val Pro Leu Thr Pro Ser Ala Leu Phe 755 760 765 Gly Gly Ala Asn Phe Asn Gln Ser Gly Asn Ile Ala Asp Ser Ser 770 775 780 Leu Ser Phe Thr Phe Thr Asn Ser Ser Asn Gly Pro Asn Leu Ile 785 790 795 Thr Thr Gln Thr Asn Ser Gln Ala Leu Ser Gln Pro Ile Ala Ser 800 805 810 Ser Asn Val His Asp Asn Phe Met Asn Asn Glu Ile Thr Ala Ser 815 820 825 Lys Ile Asp Asp Gly Asn Asn Ser Lys Pro Leu Ser Pro Gly Trp 830 835 840 Thr Asp Gln Thr Ala Tyr Asn Ala Phe Gly Ile Thr Thr Gly Met 845 850 855 Phe Asn Thr Thr Thr Met Asp Asp Asp Val Tyr Asn Tyr Leu Phe 860 865 870 Asp Asp Glu Asp Thr Pro Pro Asn Pro Lys Lys Glu 875 880 SEQ ID NO: 2 Sequence Length: 2646 Sequence Type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid DNA sequence ATG AAG CTA CTG TCT TCT ATC GAA CAA GCA TGC GAT ATT TGC CGA 45 CTT AAA AAG CTC AAG TGC TCC AAA GAA AAA CCG AAG TGC GCC AAG 90 TGT CTG AAG AAC AAC TGG GAG TGT CGC TAC TCT CCC AAA ACC AAA 135 AGG TCT CCG CTG ACT AGG GCA CAT CTG ACA GAA GTG GAA TCA AGG 180 CTA GAA AGA CTG GAA CAG CTA TTT CTA CTG ATT TTT CCT CGA GAA 225 GAC CTT GAC ATG ATT TTG AAA ATG GAT TCT TTA CAG GAT ATA AAA 270 GCA TTG TTA ACA GGA TTA TTT GTA CAA GAT AAT GTG AAT AAA GAT 315 GCC GTC ACA GAT AGA TTG GCT TCA GTG GA G ACT GAT ATG CCT CTA 360 ACA TTG AGA CAG CAT AGA ATA AGT GCG ACA TCA TCA TCG GAA GAG 405 AGT AGT AAC AAA GGT CAA AGA CAG TTG ACT GTA TCG ATT GAC TCG 450 GCA GCT CAT CAT GAT AAC TCC ACA ATT CCG TTG GAT TTT ATG CCC 495 AGG GAT GCT CTT CAT GGA TTT GAT TGG TCT GAA GAG GAT GAC ATG 540 TCG GAT GGC TTG CCC TTC CTG AAA ACG GAC CCC AAC AAT AAT GGG 585 TTC TTT GGC GAC GGT TCT CTC TTA TGT ATT CTT CGA CGT ATT GGC 630 TTT AAA CCG GAA AAT TAC ACG AAC TCT AAC GTT AAC AGG CTC CCG 675 ACC ATG ATT ACG GAT AGA TAC ACG TTG GCT TCT AGA TCC ACA ACA 720 TCC CGT TTA CTT CAA AGT TAT CTC AAT AAT TTT CAC CCC TAC 765 CCT ATC GTG CAC TCA CCG ACG CTA ATG ATG TTG TAT AAT AAC CAG 810 ATT GAA ATC GCG TCG AAG GAT CAA TGG CAA ATC CTT TTT AAC TGC 855 ATA TTA GCC ATT GGA GCC TGG TGT ATA GAG GGG GAA TCT ACT GAT 900 GAT GTT TTT TAC TAT CAA AAT GCT AAA TCT CAT TTG ACG AGC 945 AAG GTC TTC GAG TCA GGT TCC ATA ATT TTG GTG ACA GCC CTA CAT 990 CTT CTG TCG CGA TAT ACA CAG TGG AGG GAG AAA ACA AAT ACT AGC 1035 TAT AAT TTT C AC AGC TTT TCC ATA AGA ATG GCC ATA TCA TTG GGC 1080 TTG AAT AGG GAC CTC CCC TCG TCC TTC AGT GAT AGC AGC ATT CTG 1125 GAA CAA AGA CGC CGA ATT TGG TGG TCT GTC TAC TCT TGG GAG ATC 1170 CAA TTG TCC CTG CTT TAT GGT CGA TCC ATC CAG CTT TCT CAG AAT 1215 ACA ATC TCC TTC CCT TCT TCT GTC GAC GAT GTG CAG CGT ACC ACA 1260 ACA GGT CCC ACC ATA TAT CAT GGC ATC ATT GAA ACA GCA AGG CTC 1305 TTA CAA GTC TTC ACA AAA TAT GAA CTA GAC AAA ACA GTA ACT 1350 GCA GAA AAA AGT CCT ATA TGT GCA AAA AAA TGC TTG ATG ATT TGT 1395 AAT GAG ATT GAG GAG GTT TCG AGA CAG GCA CCA AAG TTT TTA CAA 1440 ATG GAT ATT TCC ACC ACC GCT CTA ACC AAT TTG TTG AAG GAA CAC 1485 CCT TGG CTA TCC TTT ACA AGA TTC GAA CTG AAG TGG AAA CAG TTG 1530 TCT CTT ATC ATT TAT GTA TTA AGA GAT TTT TTC ACT AAT TTT ACC 1575 CAG AAA AAG TCA CAA CTA GAA CAG GAT CAA AA GAT CAT CAA AGT 1620 TAT GAA GTT AAA CGA TGC TCC ATC ATG TTA AGC GAT GCA GCA CAA 1665 AGA ACT GTT ATG TCT GTA AGT AGC TAT ATG GAC AAT CAT AAT GTC 1710 ACC CCA TAT TTT GCC TGG AAT TGT TCT TA T TAC TTG TTC AAT GCA 1755 GTC CTA GTA CCC ATA AAG ACT CTA CTC TCA AAC TCA AAA TCG AAT 1800 GCT GAG AAT AAC GAG ACC GCA CAA TTA TTA CAA CAA ATT AAC ACT 1845 GTT CTG ATG CTA TTA AAA AAA CTG GCC ACT TTT AAA ATC CAG ACT 1890 TGT GAA AAA TAC ATT CAA GTA CTG GAA GAG GTA TGT GCG CCG TTT 1935 CTG TTA TCA CAG TGT GCA ATC CCA TTA CCG CAT ATC AGT TAT AAC 1980 AAT AGT AAT GGT AGC GCC ATT AAA AAT ATT GTC GGT TCT GCA ACT 2025 ATC GCC CAA TAC CCT ACT CTT CCG GAG GAA AAT GTC AAC AAT ATC 2070 AGT GTT AAA TAT GTT TCT CCT GGC TCA GTA GGG CCT TCA CCT GTG 2115 CCA TTG AAA TCA GGA GCA AGT TTC AGT GAT CTA GTC AAG CTG TTA 2160 TCT AAC CGT CCA CCC TCT CGT AAC TCT CCA GTG ACA ATA CCA AGA 2205 AGC ACA CCT TCG CAT CGC TCA GTC ACG CCT TTT CTA GGG CAA CAG 2250 CAA CAG CTG CAA TCA TTA GTG CCA CTG ACC CCG TCT GCT TTG TTT 2 GGC GCC AAT TTT AAT CAA AGT GGG AAT ATT GCT GAT AGC TCA 2340 TTG TCC TTC ACT TTC ACT AAC AGT AGC AAC GGT CCG AAC CTC ATA 2385 ACA ACT CAA ACA AAT TCT CAA GCG CTT TCA CAA CCA ATT GCC TCC 24 30 TCT AAC GTT CAT GAT AAC TTC ATG AAT AAT GAA ATC ACG GCT AGT 2475 AAA ATT GAT GAT GGT AAT AAT TCA AAA CCA CTG TCA CCT GGT TGG 2520 ACG GAC CAA ACT GCG TAT AAC GCG TTT GGA ATC ACT ACA GGG ATG 2565 TTT AAT ACC ACT ACA ATG GAT GAT GAT GTA TAT AAC TAT CTA TTC 2610 GAT GAT GAA GAT ACC CCA CCA AAC CCA AAA AAA GAG 2646 SEQ ID NO: 3 Sequence length: 21 Sequence type: Nucleic acid Number of strands: single strand Topology: Linear Sequence type: Other nucleic acid Synthetic probe DNA Sequence CGTCCAACCA GGTGACAGTG G 21 SEQ ID NO: 4 Sequence length: 21 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear sequence type : Other nucleic acids Synthetic probe DNA sequence CATGTCAAGG TCTTCTCGAG G 21 SEQ ID NO: 5 Sequence length: 54 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence ATG AAG TGG GTT ACT TTC ATC TCT TTG TTG TTC TTG TTC GCT AAG 45 Met Lys Try Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ala Lys 5 10 15 GTT TCA GCT 54 Val Ser Ala SEQ ID NO: 6 Sequence Length: 10 Sequence Type: Nucleic Acid Number of Strands: Double Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic Linker DNA Sequence AGCGGCCGCT 10 SEQ ID NO: 7 Sequence Length: 107 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Sequence C GCG TTT GGA ATC ACT ACA GGG ATG TTT AAT ACC ACT ACA ATG GAT 46 Ala Phe Gly Ile Thr Thr Gly Met Phe Asn Thr Thr Thr Met Asp 1 5 10 15 GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC CCA 94 Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro Pro Asn Pro 20 25 30 AAA AAA GAG TAAG 107 Lys Lys Glu SEQ ID NO: 8 Sequence length: 107 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Type of sequence: Other nucleic acid Synthetic DNA sequence TCGACTTACT CTTTTTTTGG GTTTGGTGGG GTATCTTCAT CATCGAATAG ATAGTTATAT 60 ACATCATCCA TTGTA GTGGT ATTAAACATC CCTGTAGTGA TTCCAAA 107 SEQ ID NO: 9 Sequence length: 110 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence C GCG TTT GGA ATC ACT ACA GGG ATG TTT AAT ACC ACT ACA ATG GAT 46 Ala Phe Gly Ile Thr Thr Gly Met Phe Asn Thr Thr Thr Met Asp 1 5 10 15 GAT GAT GTA TAT AAC TAT CTA TTC GAT GAT GAA GAT ACC CCA CCA AAC 94 Asp Asp Val Tyr Asn Tyr Leu Phe Asp Asp Glu Asp Thr Pro Pro Asn 20 25 30 CCA AAA AAA GAG TAAG 110 Pro Lys Lys Glu SEQ ID NO: 10 Sequence Length: 107 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Type of sequence: Other nucleic acid Synthetic DNA sequence TCGACTTACT CTTTTTTTGG GTTTGGTGGG GTATCTTCAT CATCGAATAG ATAGTTATAT 60 ACATCATCAT CCATTGTAGT GGTATTAAAC ATCCCTGTAG TGATTCCAAA 110 SEQ ID NO: 11 Sequence length: 17 Sequence type: Nucleic acid Chain topology: Number of single-stranded chains: One Type of sequence: Other nucleic acids Synthetic DNA Sequence AATGAAATCA CGGCTAG 17 Sequence number: 12 Sequence length: 17 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Sequence CAGGAAACAG CTATGAC 17 SEQ ID NO: 13 Sequence Length: 131 Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Other nucleic acid Vector DNA sequence CTATACTTTA ACGTCAAGGA GAAAAAACCC CGGATCACCG CGGATCGTAA TGCACGCCAA 60 TCATTTTAGA GAGGACAGAG AAGCAAGCCT CCTGAAAG ATG AAG CTA CTA CTA CCT CTG Lys Leu Leu Ser Ser 15 ATC GAA CAA GCA TGC 131 Ile Glu Gln Ala Ser 10 SEQ ID NO: 14 Sequence length: 127 Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Other nucleic acid vector DNA sequence TTTTTTTTAG TTTTAAAACA CCAAGAACTT AGTTTCGGGG ATCGTAATGC ACGCCATCAT 60 TTTAGAGAGG ACAGAGAAGC AAGCCTCCTG AAAG ATG AAG CTA CTG TCT TCT 112 Met Lys Leu Leu Ser Ser 1 5 ATC GAA CAA GCA TGC 127 Ile Glu n Ala Ser 10

[Brief description of drawings]

FIG. 1 Mutant GAL4 using GAL1 promoter
FIG. 3 is a diagram showing the construction of the transcription unit of FIG.

FIG. 2 Wild type GAL4 using GAL1 promoter
FIG. 3 is a diagram showing the construction of the transcription unit of FIG.

FIG. 3: Mutant GA using GAP-DH promoter
The figure which shows the construction of the transcription unit of L4.

FIG. 4 Mutant GAL4 using the GAL1 promoter
The figure which shows the construction of an expression vector.

FIG. 5: Wild type GAL4 using GAL1 promoter
The figure which shows the construction of an expression vector.

FIG. 6: Mutant GA using GAP-DH promoter
The figure which shows the construction of an L4 expression vector.

FIG. 7: Wild-type GA using GAP-DH promoter
The figure which shows the construction of an L4 expression vector.

FIG. 8: Construction of an integrated chimeric A-7 Fab expression vector with the GAL1 promoter.

FIG. 9 shows the introduction of a high expression vector into yeast.

FIG. 10 shows the relationship between the production amount of chimeric Fab and the transformant in the primary screening.

FIG. 11: Chimera F using GAP-DH promoter
The figure which shows the construction of pMB018 which has the transcription unit of the ab H chain.

FIG. 12 is a diagram showing the construction of pMB039 having an L chain transcription unit (L-unit) and an H chain transcription unit (H-unit).

[Explanation of symbols]

Description of abbreviations GAL1 P: GAL1 promoter GAP-DH P: GAP-DH promoter GAP-DH T: GAP-DH terminator S. O. -M: mutant synthetic DNA S. O. : Wild type synthetic DNA Ori: Origin of replication Amp ^r : Ampicillin resistance gene HSA1: Human serum albumin 1 (N-terminal part) HSA2: Human serum albumin 2 (C-terminal part) mHSA signal (signal): Mutant human serum albumin secretion Signal VL: Mouse L chain variable region CK: Human L chain constant region VH: Mouse H chain variable region FD: Human H chain constant region (CH1)

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display area // (C12N 15/81 C12R 1: 865)

Claims (3)

[Claims] 1. The mutant GAL4 according to SEQ ID NO: 1, wherein Asp is added between Asp at the 863th position and Val at the 864th position from the N terminus of wild type GAL4. 2. A DNA encoding the mutant GAL4 of claim 1. 3. A heterologous protein comprising introducing the mutant GAL4 expression unit of claim 1 and a unit expressing a heterologous protein under the control of the GAL1, GAL7 or GAL10 promoter into yeast and culturing the yeast. Method of enhancing expression.