JPH0638771A - Method for expressing human protein disulfide isomerase gene and method for producing polypeptide by co-expression with the gene - Google Patents

Abstract

(57) [Summary] [Purpose] Protein disulfide isomerase (PD
I) Provide expression of a gene and co-expression of the gene with a foreign gene encoding a useful polypeptide. The present invention relates to a method for producing PDI by incorporating a novel ligation gene consisting of a DNA encoding a human serum albumin prepro sequence and a human PDI gene into an expression vector, transforming a host cell, and expressing it.
And a method for producing the polypeptide by co-expressing a transformant containing the linked gene capable of co-expression and a foreign gene encoding a useful polypeptide. [Effect] A large-scale production method of human PDI has been established, and co-expression with the human PDI gene in the same cell has made it possible to improve the production efficiency of useful polypeptides.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to the expression of a gene encoding a protein disulfide isomerase which promotes the formation of higher-order structure of a polypeptide by catalyzing the exchange reaction of disulfide bond in the polypeptide.
The present invention further relates to coexpression of the gene with a foreign gene encoding a useful polypeptide.

[0002]

RELATED ART Reconstitution of denatured proteins in vitro (Ref
From the results of the olding) experiment, it is known that isomerization reaction of disulphide bond and isomerization of proline peptide are known as reactions that control the folding rate of polypeptide (Freedman, Cell 57 , 1069-1072, 1989; Fisher &
Schmid, Biochemistry 29 , 2205-2212, 1990), peptidylprolyl cis-trans isomerase (PPI) for the latter and protein disulfide isomerase (PDI) for the former as enzymes that catalyze these slow reactions in the folding reaction. Thioredoxin etc. have been found. In vitro experiments have shown that these enzymes promote the rate of remodeling of denatured proteins, and the inactive protein produced in vitro is engineered in vitro.
It is expected to be used for reconstruction in o (Schein, Bio
/ Technology 7 , 1141-1148,1989; Shigezo Utaka, Journal of the Japanese Society of Agricultural Chemistry 64 , 1035-1038, 1990).

PDI is soluble and relatively easily isolated from mammalian liver and its catalytic properties have been investigated in detail. PDI catalyzes the exchange reaction of thiol / disulfide bond and can form, isomerize, or reduce disulfide bond of protein substrate (Free.
dman, Cell 57 , 1069-1072, 1989). PDI is In vitr
In o, it promotes intramolecular formation of disulfide bonds and exchange reactions such as proteins consisting of a single domain such as RNase and proteins consisting of multiple domains such as serum albumin, or such as immunoglobulin and procollagen. It is known to promote reactions such as intermolecular disulfide bond formation of proteins having subunit structures (Freedman, Nature 329 , 196, 1987).

Mammalian-derived PDI usually exists as a homodimer of a polypeptide having a molecular weight of about 57,000 and has a pI value (pI 4.2 to 4.3) of extremely high acidity.

With respect to rat liver-derived PDI, its gene was isolated, the amino acid sequence of PDI was deduced from the nucleotide sequence of the gene, and PDI has an intramolecular overlapping structure consisting of two types of homology units. It is shown. Two
One of the homology units of the species was found to be homologous to the amino acid sequence of thioredoxin and is believed to have a similar active site amino acid sequence (Edma
n et al., Nature 317 , 267-270, 1985). Thioredoxin, or reduction of the disulfide bonds of insulin in in vivo, it is possible to promote the exchange reaction of disulfide bonds of RNase, it has been suggested that the same function and PDI in the protein folding process in the in vivo (Pigict & Schuster, Proc . Natl . Acad . Sci .
USA 83 , 7643-7647, 1986).

The abundance of PDI in vivo varies depending on the type of tissue and the stage of cell differentiation, but there is a correlation between this and the presence of a specific secreted protein, and , PDI is abundantly localized inside the endoplasmic reticulum, which is known to pass through during protein secretion. Therefore, PDI is also formed intracellularly in the formation of a disulfide bond of a newly synthesized secretory protein. It is estimated to be involved in. This means that γ-gliadin biosynthesis was carried out as a model system using a cell-free protein synthesis system, and at this time, formation of a disulfide bond coupled to translation of γ-gliadin was observed only in the endoplasmic reticulum fraction washed out of PDI. It rarely happens, but is supported by the result that the addition of PDI restores the ability to form disulfide bonds (Bulleid & Freedman, Nature , 335 649-65.
1, 1988).

With respect to PDI, evidence has been obtained that it is involved not only in the formation of disulfide bonds but also in other post-translational modification reactions of proteins. For example, PDI is
Prolyl that hydroxylates the proline residue of procollagen-
Asn-X-Ser / Thr
Glycosylation site binding protein (Pihlajaniemi
.. et al, EMBO J 6 , 643-649 1987; Geetha-Habib e
al., Cell 54 , 1053-1060 1988), and also thyroid hormone-binding protein (triiodo-L-thyronine binding
protein) (Cheng et al . J. Biol . Chem . 262 , 11221-
11227, 1987) and the like, suggesting multifunctionality in the protein modification reaction of the PDI molecule. In addition to these facts, a molecular species different from the PDI molecule but having homology in the amino acid sequence has been found. Examples of them include a sequence homologous to the amino acid sequence that is considered to be the active site of PDI and were found to catalyze the isomerization of disulfide bonds (Follitropin) and lutropin (Lu
tropin) and (Boniface & Reiche
rt et al ., Science 247 , 61-64, 1990), an enzyme and molecule that hydrolyzes phosphatidylinositol 4,5-bisphosphate into 1,2-diacylglycerol and inositol 1,4,5-triphosphate. Phospholipase C, which has a region having homology with PDI, is also known (Bennettet al., Nature 334 , 268-270, 1988),
The involvement of PDI and PDI-like molecules in a very wide range of intracellular and extracellular life phenomena is considered.

Although a wide range of actions have been suggested as described above, the main effect of PDI is to catalyze the isomerization of intramolecular and intermolecular disulfide bonds, and thus the protein (and assembly) having a natural higher-order structure. Body) is believed to cause.
However, often almost stoichiometric amounts of PDI are required to achieve optimal reaction rates. Therefore,
When the disulfide isomerase activity is low, the rate of disulfide isomerization within and between protein molecules is low,
Therefore, it is expected that the efficiency of forming a protein having an appropriate disulfide bond is low. It is possible to think that one of the causes of the formation of insolubilized molecular assemblies in various Eukaryote-derived proteins (especially secretory proteins) is the disulfide isomerase activity. Escherichia coli contains thioredoxin as a disulfide reductase, and thioredoxin is stronger than PDI as a disulfide reductase, but its efficiency as an isomerase is not good. On the other hand, since intramolecular disulfide bonds are frequently found in secreted proteins, it is expected that disulfide bond activity via disulfide isomerization is high in cells or tissues with high secretory ability. This is strongly suggested by the comparison of the relative PDI mRNA contents of the tissues (in the order of liver> pancreas, kidney>lung> testis, spleen>heart> brain) (Edman et al., N.
ature 314 , 267-270, 1985).

Also, when the reduced environment is provided as a field for protein synthesis, the formation of disulfide bonds required for proper folding will be inhibited. Such environments occur, for example, in prokaryotic cells that lack compartments. Given these points, factors involved in disulfide bond formation and the environment that enables them may differ between prokaryotic cells and eukaryotic cells. When recombinant proteins are used to produce useful proteins (most of which are secretory proteins), it is necessary to allow the formation of disulfide bonds under conditions suitable for the proteins. To do this, an environment (appropriate compartment) in the host cell must be realized, and a large amount of disulfide-forming (disulfide isomerizing) enzyme with high affinity for that environment (compartment) must be present. Will.

[0010] These two points are considered to be the most important points to be noted in efficiently producing a protein having a disulfide bond using recombinant DNA technology.

However, up to now, there has been no in vivo system capable of causing a large amount of protein disulfide isomerase in an appropriate compartment and coexisting with a useful protein of interest to act on it.

[0012]

[Problems to be Solved by the Invention] Although the utilization of the denatured protein for reconstitution in vitro or the utilization of the secretory protein in the cell for improving the production rate is considered, the enzyme Obtained was limited to direct purification from organs. Expression of PDI derived from other species in various cells has not yet been made, and means for producing by genetic engineering, means for increasing the production efficiency by coupling with genes of other useful polypeptides, etc. have not been established. It was

The present invention relates to a DNA encoding a human serum albumin prepro sequence for expressing human PDI and human PDI.
A linked gene consisting of a gene, an expression vector capable of expressing the linked gene in a host, a transformant obtained by transforming a host with the vector, and a recombinant human PDI by expressing the linked gene in the transformant It aims at providing the manufacturing method of this, and recombinant PDI.

Furthermore, the present invention provides a transformant containing the linked gene that can be coexpressed and a foreign gene encoding a polypeptide to be produced, and human PDI and the foreign gene in the transformant. It is an object to provide a method for producing the polypeptide by co-expressing it.

[0015]

[Means for Solving the Problems] As a result of intensive studies aimed at achieving the above object, the present inventors have prepared a gene in which a DNA encoding a human serum albumin prepro sequence and a human protein disulfide isomerase gene are linked. Then, the present invention was completed by finding an expression vector incorporating this.

The details of the present invention will be described below.

A clone encoding human protein disulfide isomerase (abbreviated as "PDI") cDNA is human liver λgt11.
It is isolated from the cDNA library and the human placenta λgt11 cDNA library (Clontech) as follows.

Human liver and human placenta λgt11 cDNA library were phage-infected into Escherichia coli, propagated, and the phage DNA was immobilized on a filter. On the other hand, human proline 4-hydroxylase (same protein as PDI) cDNA
243 of [Pihlajaniemi, T. et al. (1987) EMBO J. 6 , 643]
40 mer corresponding to the complementary strand of the 282nd base sequence
Positive clones are screened by hybridization using the synthetic oligomer DNA of 1. as a probe, the phage DNA is digested with EcoRI, and the resulting insert DNA of about 150 bp is used as a probe for PDI cDNA screening. This probe is used to screen the phage DNA immobilized on the filter to separate positive clones.

The plurality of positive clones thus obtained were digested with EcoRI to obtain EcoRI insert DNA fragments, a restriction enzyme map was prepared for the insert of each clone, and the result was compared with the restriction enzyme map by Pihlajaniemi et al. It was speculated that the liver-derived clone (pHPDIl6) and the placenta-derived clone (pHPDIp4) covered the entire length of human PDI cDNA.

As a result of determining the DNA base sequences of both clones, these clones were found to have the full length 2454 shown in SEQ ID NO: 1.
It was found to encode a human PDI cDNA consisting of base pairs. The amino acid sequence deduced from the base sequence was as shown in SEQ ID NO: 1. In the sequence, the mature protein is considered to be composed of 491 amino acids from Asp ¹ to Leu ⁴⁹¹ , and the peptide consisting of 17 amino acids preceding Asp ¹ is considered to represent a signal peptide.

The present invention provides a ligated gene for expressing and producing PDI, which comprises a DNA encoding a human serum albumin gene prepro sequence and the human PDI gene.

The ligated gene can be usually prepared by placing a DNA encoding the prepro sequence upstream of the PDI gene as shown in FIG. 1C.
However, the leader sequence for transporting human PDI into an appropriate compartment (which is considered to be the endoplasmic reticulum) does not need to be limited to the prepro sequence of human serum albumin, and may be other signal sequence or prepro sequence. Good.

Specifically, the clones pHPDIl6 and p
HPDIp4 DNA was digested with EcoRI / PstI and PstI / BamHI,
DNA fragments of about 490 bp and about 1.3 kbp were obtained, and both fragments were EcoR
Ligated to the I / BamHI digested plasmid vector pUC119 (phPD
IEB), Kunkel method [Kunkel, TA (1985) Proc.Natl.Acad.S
ci. USA 82 , 488], a restriction enzyme NaeI cleavage site was introduced at the boundary between the PDI signal sequence on the cDNA and the PDI body (phPDINae), and PDI was digested with NaeI / Hind III.
A PDI DNA fragment of about 1.7 kb containing no signal sequence is obtained.

On the other hand, pUC119 was digested with EcoRI, XhoI linker: 5′-AATTCTCGAG GAGCTCTTAA-5 ′ was ligated to this, and XhoI / BamHI digested, and a human serum albumin (hereinafter referred to as “HSA”) prepro sequence Connect
(pUC 119 Sig), StuI / Hind III digested 3.2 kb DNA
Fragments are obtained (methods of HSA prepro sequence synthesis are given in the Examples below).

1.7 kb DNA fragment from phPDINae and pUC 119
The 3.2 kb DNA fragment from Sig was ligated (phPDILy1) and EcoRI
Digestion, blunting with Klenow fragment, and BamHI digestion were sequentially carried out to connect the human PDI main body downstream of the HSA prepro sequence (Fig. 2) to obtain a leader sequence-modified ligated gene.

The method for producing the ligated gene of the present invention and the arrangement between the constituent genes are not limited to the above-mentioned methods, and all those having the ability to express PDI are included. Also, it will be apparent that analogs of the linking gene are outside the scope of the invention, but can be readily made from the corresponding gene from other animals besides humans.

The invention is also shown in SEQ ID NO: -2.
The ligated gene comprising a nucleotide sequence encoding the 4th to + 491st amino acid sequences is provided. In this case, a gene having substantially the same action as this base sequence, for example, a derivative of the base sequence based on the degeneracy of the genetic code, is included in the present invention. For example, according to an embodiment of the present invention,
The present invention provides the linked gene consisting of the entire base sequence shown in SEQ ID NO: 2.

The present invention further provides a replicable expression vector capable of expressing the linking gene of the present invention in a host.

The vector for incorporating the ligated gene of the present invention is capable of expression in a host and has replication ability. In general, vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with the host. Vectors usually carry a marker sequence and a replication site that allow phenotypic selection in transformed cells.

Examples of the vector for constructing the expression vector of the present invention include, for example, Japanese Patent Application Laid-Open No. 2-1173 by the present applicant.
The plasmid pJDB-ADH-HSA-A (see FIG. 1-C) disclosed in Japanese Patent Publication No. 84 is used. This plasmid contains the HSA cDNA and also contains yeast alcohol dehydrogenase I (ADH
I) It contains a promoter, ADH terminator, ampicillin resistance gene (Amp ^r ), and Leu2 gene.
Therefore, this plasmid is digested with XhoI, blunted with Klenow fragment and digested with BamHI to remove the HSA cDNA. The expression plasmid (pAHhPDILy1) can be obtained by dephosphorylating the 5'end of the obtained about 8 kb DNA fragment and then ligating the above-mentioned ligation gene of the present invention. Of course, it is also possible to use another type of vector that can perform the equivalent function of expressing the linked gene of the present invention.

The present invention further provides a transformant obtained by transforming a host with the expression vector of the present invention.

Examples of the host include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and yeast, and a host capable of secreting mature PDI through processing is preferable. The preferred host is yeast. As a host yeast, Saccharomyces ce
revisiae, etc., and the yeast AH22 strain is particularly preferably used for the production of the transformant of the present invention. It will be appreciated that eukaryotic cells other than yeast (eg animal cells) may also be used as hosts, although this is outside the scope of the invention. Transfer of the expression vector into the host cell is carried out by a conventional method, and for example, it can be easily carried out by a calcium chloride treatment method, a protoplast (or spheroplast) -polyethylene glycol method, an electroporation method and the like. When the expression vector is pAHhPDILy1, the target transformant is obtained by screening the obtained bacterial cells on an SD (-Leu) plate.

Therefore, the present invention also provides a method for producing recombinant human PDI by expressing the ligated gene of the present invention in the transformant prepared as described above.
According to the embodiment of the present invention, the production method of the present invention includes the following steps.

That is, the step of constructing a replicable expression vector capable of expressing the ligated gene of the present invention in a host, the step of transforming a host with the expression vector to obtain a transformant, and the expression of the ligated gene The step of culturing the transformant to secrete the recombinant human PDI under the condition that allows it, and the step of recovering the recombinant PDI are included.

When yeast is used as a host, human P
The DI precursor protein is processed to secrete recombinant human PDI as a gene product. If a microorganism other than yeast such as Escherichia coli or Bacillus subtilis is used as the host, unprocessed human PD
I precursor protein will be obtained.

Recombinant human PDI was obtained by separating cells from the culture medium by centrifugation, disrupting the cells if necessary, and then subjecting the concentrated solution concentrated by, for example, ultrafiltration to hydrophobic column chromatography. Can be easily purified and isolated. The hydrophobic column that can be used for this chromatography is not limited to a particular one, but for example, a TSK-gel Phenyl-5PW hydrophobic column (manufactured by Tosoh Corporation) can be used, in which case recombinant human PDI is KCl.
It can be eluted by a linear concentration gradient from 0.85M to 0M ammonium sulfate in borate buffer containing pH 8.0 (4th).
Figure). From SDS electrophoresis analysis (Fig. 5), recombinant human P
DI has a molecular weight of about 55 kDa, and it was also confirmed that the recombinant human PDI obtained had PDI activity by quantifying the degree of rearrangement of scrambled ribonuclease A as an index (see below. See example).

The recombinant human PDI produced by the method of the present invention had the N-terminal amino acid changed from Asp to Gly as compared with the natural human PDI. Therefore,
The present invention also provides a recombinant human PDI consisting of the amino acid sequence of Gly ¹ ... Leu ^{491 represented} by SEQ ID NO: 3 consisting of ⁴⁹¹ amino acids.

The present invention further provides for co-expressable human PD
Provided is a transformant containing a ligated gene consisting of an I gene and a DNA encoding a human serum albumin prepro sequence, and a foreign gene encoding a polypeptide to be produced.

The ligated gene and the foreign gene in the transformant may be on the same genome or on different genomes as long as they can co-express each other.
For transformation of host cells, for example, the ligated gene and the foreign gene are integrated into the same or different vectors, and the resulting vector is treated with calcium chloride, protoplast (or spheroplast) -polyethylene glycol method, electroporation. It can be carried out by introducing it into a host by a conventional method such as a method.

The polypeptide encoded by the foreign gene directly exerts the catalytic action of the amplified and expressed PDI (ie, promotes formation of disulfide bond in the polypeptide, exchange reaction, etc.), Any kind of polypeptide may be used as long as it has a disulfide bond in its structure. Furthermore, the present invention is also applicable to the case where the effect of the amplified and expressed PDI activity is exerted on a protein involved in gene expression, polypeptide folding, transport, etc., which indirectly increases the productivity. Applied. According to an embodiment of the invention, the invention also provides that the foreign gene is human serum albumin (HSA).
Provides a gene encoding

As used herein, the term "polypeptide" is meant to include short and long chain peptides and proteins.

Examples of the host include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast and animal cells. In particular, a host capable of secreting a mature polypeptide through post-translational modification or processing, such as a eukaryotic cell, is preferable, and yeast is particularly preferable.

The present invention further comprises coexpressing the human PDI gene and a foreign gene encoding another polypeptide in the transformant to produce the polypeptide, and recovering the polypeptide. A method for producing a polypeptide is provided.

According to an embodiment of the present invention, when HSA and PDI are co-expressed in an arbitrary medium in a yeast obtained by transforming HSA-producing yeast with a human PDI expression plasmid, HS compared to when expressed
The amount of A secreted increased by about 60% on average (Fig. 8).

Although not intending to be bound by theory, it can be considered as follows regarding the increase in HSA secretion amount by coexpression.

HSA is a protein having 17 disulfide bonds, and the presence of a stoichiometric amount of PDI in an in vitro reconstitution experiment from a denatured protein promotes the formation of its higher-order structure. It is known that

HSA is secreted as a soluble molecule by the yeast HIS23 strain, but HSA is secreted in the cells of the same strain.
Molecules have been detected. When intracellular HSA was analyzed by SDS electrophoresis, it showed the same behavior as a normal HSA molecule as a single band on the gel in the presence of a reducing agent, while it showed a higher molecular weight in the absence of the reducing agent. Detected as a large discontinuous band group, which behaves distinctly from normal HSA. These results are presumed to be caused by the incomplete formation of intramolecular disulfide bonds in HSA molecules existing in cells. On the other hand, in cells in which PDI was co-expressed, the HSA molecule was found to be exogenous PDI by SDS electrophoresis in the absence of intracellular HSA reducing agent.
Intracellular H obtained from yeast that does not co-express cDNA
Since it is detected as a more organized band as compared with the SA sample, PDI promotes the formation of normal disulfide bonds in the HSA molecule and more efficiently assists the formation of the higher-order structure of the HSA molecule. Presumed. As a result, for example, it is considered that HSA having an unstable structure is less associated with intracellular association and degraded by protease, so that the amount of secretion is increased.

In addition, comparing the amount of HSA mRNA present in cells of the HIS23 strain with and without PDI coexpression, by Northern blotting, the amount of HSA mRNA increased when the PDI gene was expressed. ing. This suggests not only the possibility that PDI acts directly on the HSA molecule, but also the influence on the transcription level of the HSA gene. However, a large amount of human PDI was secreted from yeast by the fusion of the leader sequence of human serum albumin, which is involved in intracellular transport through the membrane translocation process to the endoplasmic reticulum, and the increased amount of HSA was correlated with each other. Therefore, it seems simpler to think that PDI coexisted with HSA in the endoplasmic reticulum and directly acted on HSA was the main factor that increased the production level of HSA. Furthermore, HIS2
Looking at the amounts of HSA and PDI secreted by the three strains, PD
I is secreted several times as much as HSA, and the level of human PDI detected in cells is also high.
It is presumed that the amount of PDI required for exhibiting the promoting effect on the in vitro reconstitution is sufficiently secured in the yeast endoplasmic reticulum. This also seems to support PDI acting directly on HSA.

As described above, in the case of HSA, the co-expression of PDI has the effect of increasing the secretion amount, and the effect is P
Since it is highly likely that DI is directly involved in the formation of higher-order structure of HSA, it is more generally the same for all secretory proteins in which the formation of disulfide bonds contributes to the formation and stabilization of higher-order structure. It is considered that the similar increase effect of the secretory amount can be expected by highly amplifying and expressing PDI in cells.

The present invention will be further described by the following examples, but the present invention is not limited to these examples.

[0051]

[Examples] Human PDI (protein disulphide isomerase) cDNA clones
Over emissions of human liver λgt11cDNA library (Clontech, Inc.) about 100,
000 clones in LB medium containing 0.2% maltose (1%
Escherichia coli Y1090 strain culture solution cultured overnight at 37 ° C with bactotryptone, 1% NaCl and 0.5% yeast extract).
Mix with 500 μl, add 5 μl of 1M MgCl ₂ and add 37 ° C.
The phages were infected with E. coli by heating for 10 minutes. Add 50 ml of LB upper layer agar medium (LB medium, 10 m
23 cm after mixing with M MgCl ₂ and 0.7% agarose)
Seed on LB agar in x23 cm plates. After solidifying the upper layer agar medium, it was cultured overnight at 37 ° C. to grow the phage. The obtained phage was filtered (Hybond-N, Amersh
am company) and transfer to alkaline solution (0.5N NaOH and 0.15M
Place on a 3MM filter paper (Whatman) soaked in NaCl) with the phage attachment side facing up for 1 minute, and then neutralize solution [1M
It was placed on the same filter paper soaked in Tris-HCl (pH 7.5) and 1.5 M NaCl for 1 minute. Further filter the 2 × SSC solution (20
× SSC = 3M NaCl and 0.3M trisodium citrate)
After washing with water and air-drying, UV irradiation was performed for 2 minutes to fix the phage DNA to the filter. Using the filter thus obtained, human PDI cDNA was screened according to the following procedure.

As a probe, human proline 4-hydroxylase (the same protein as PDI) cDNA [Pihlajaniemi, T.
et al. (1987) EMBO J., 6 , 643], from the 243rd to the second.
40mer oligomer DNA (5'-TGGCGTCCACCTTGGCCAACCTGATCTCGGAACCT) corresponding to the complementary strand of the 82nd base sequence
TCTGC-3 ') is an automated DNA synthesizer (Applied Biosyste
The one synthesized by ms model 380B) was used.

Synthetic DNA (20 pmoles) was added to 50 mM Tris-HCl
(PH 7.5); 10 mM MgCl ₂ , 5 mM dithiothreitol, 10
0 μCi [γ- ³² P] ATP (up to 3000 Ci / mmol, Amersham
And 5 units of T4 polynucleotide kinase (Takara Shuzo) at 37 ° C. for 60 minutes to phosphorylate the 5 ′ end. Pre-hybridization solution [5 × Denhardt's solution (100 × Denhardt's solution = 2% bovine serum albumin, 2% Ficoll 400 and 2% polyvinylpyrrolidone), 1M NaCl, 50 mM Tris-HCl (pH 7.5), 10 mM EDTA]
(pH8.0), 0.1% sodium dodecyl sarcosinate, and 20 μg / ml sonicated salmon sperm DNA]
After soaking for 1 hour at 37 ° C, it was soaked in a hybridization solution (a solution containing about 10 ⁶ cpm / ml of the above labeled DNA in a prehybridization solution) at 37 ° C for 15 hours. Wash the filter with 2x SSC solution at room temperature and add 2 more
X-ray film (XAR-5, Kodak) after washing with SSC, 0.1% sodium dodecyl sarcosinate solution at 42 ℃ for 30 minutes
Exposed at -80 ° C overnight. As a result of developing the film, eight positive signals were obtained in the primary screening.
The phages at the positions corresponding to these signals were cut as gel slices from the plate, and 1 ml of SM buffer [100 mM NaCl, 10 mM MgCl ₂ , 50 mM Tris-HCl (pH 7.5) was used.
And 0.01% gelatin] and allowed to stand overnight at 4 ° C. to recover the phage in the gel in the solution. The thus-obtained 8 types of primary screening positive phages were subjected to secondary screening under the same conditions as in the primary screening, and as a result, only one remained as a positive clone. This clone was further subjected to a third screening and completely isolated as a single positive clone.

The phage DNA of the obtained positive clone was
der et al. [Leder, P., Tiemeir, D. & Enquist L. (1
977) Science 196 , 175]. A 1/5 volume of the obtained phage DNA was added to a solution [100 mM Tris-HCl (pH 7.
5), 100 mM NaCl, 6 mM MgCl ₂ , 6 mM mercaptoethanol, 0.1% gelatin, 20 μg / ml ribonuclease A and 20 units of EcoRI (Nippon Gene)] 50 μl
After digestion at 37 ° C. for 1 hour, electrophoresis on 0.8% agarose gel revealed that this positive clone contained an insert DNA of about 150 bp. Glass powder (Ge
The insert DNA was separated and purified using ne Clean ^™ , Bio-101. About 20 ng of the recovered DNA fragment and about 100 ng of pUC19 vector digested with EcoRI were ligated to each other by reacting them in a mixed solution of 20 μl of solution A and 4 μl of solution B for 16 hours at 16 ° C. A recombinant plasmid was obtained. Using 10 μl of this reaction solution, the Mandel method [Mandel, M. & Higa, A. (1970) J. Mol. Bio
l. 53, 154 E. coli TG1 strain transformed by.
The resulting transformant was treated with L containing 25 μg / ml ampicillin.
Incubate in 100 ml of B medium at 37 ℃ overnight, and use the alkaline lysis method [Birn
boim, HC & Doly J. (1979) Nucleic Acids Res. 7 , 15
13] to purify the plasmid DNA. 10 μg of this plasmid DNA was added to a solution [100 mM Tris-HCl (pH 7.5), 100 mM
NaCl, 6 mM MgCl ₂ , 6 mM mercaptoethanol, 0.1%
After digestion with gelatin and 100 units of EcoRI (Nippon Gene Co., Ltd.) in 200 μl at 37 ° C. for 1 hour, phenol extraction and ethanol precipitation were performed, and the mixture was concentrated and subjected to 0.8% agarose gel electrophoresis. An insert DNA of about 150 bp was recovered with glass powder and used as a probe for screening PDI cDNA described below.

To obtain a clone containing the full-length human PDI cDNA, the human liver λgt11 cDNA library (Clon
tech) and about 50,000 clones of human placenta λgt11 cDNA library (the same company) were screened. Filters on which the phage DNAs of both libraries were immobilized were prepared in the same manner as the above procedure. About 100 ng of the above 150 bp human PDI cDNA fragment was added to [α- ³² P] dCTP.
(> 400Ci / mmol, Amersham) and a nick-translation kit (Company) were used for this screening. Both of the above filters were immersed in a prehybridization solution at 60 ° C for 1 hour, and then immersed in a hybridization solution (a solution containing about 10 ⁶ cpm / ml of the labeled DNA in the prehybridization solution) at 60 ° C for 15 hours. This filter was washed with 2 × SSC solution at room temperature and further with 0.5 × SSC, 0.1% sodium dodecyl sarcosinate solution at 65 ° C. for 1 hour, and then X-ray film (XAR-5, Kodak) -80 Exposed overnight at ° C. As a result of film development, 6 positive signals were obtained from the liver cDNA library and 5 positive signals from the placenta cDNA library. By subjecting these to further secondary and tertiary screening, 4 positive clones from the liver cDNA library and 3 positive clones from the placenta cDNA library were finally isolated. E of the obtained 7 clones
After subcloning the coRI insert DNA fragment into the EcoRI site of the plasmid vector pUC19 according to the same method as described above, a restriction map of the inserts of 7 clones was prepared. As a result, four of the liver cDNAs and two of the placental cDNAs overlap each other,
Moreover, one of the clones derived from the liver (pHPDIl6) and one clone derived from the placenta (pHPDIp4) covered the full-length human PDI cDNA of interest, and these clones and Pihlajaniemi et al. It was expected from the comparison of restriction maps of clones. About both clones M13 SEQUENC
ING KIT (Toyobo Co., Ltd.), M13 Sequencing Kit (Takara Shuzo) and automatic DNA sequencer (370A, Applied)
The DNA base sequence was determined by Biosystems. Pihl
Both clones have a total length of 24 by comparison with the data of ajaniemi and others.
It was revealed to encode a human PDI cDNA consisting of 54 base pairs (SEQ ID NO: 1).

Construction of yeast expression plasmids for human PDI Two clones, pHPDIl, encoding the above human PDI cDNA.
Based on 6 and pHPDIp4, a plasmid for expression of human PDI in yeast was constructed by the following procedure (Fig. 1, A, B and C).

PHPDI16 DN prepared by the alkaline lysis method
About 1 μg of A solution [10 mM Tris-HCl (pH 7.5), 100 mM NaC
l, 6 mM MgCl ₂ , 6 mM mercaptoethanol, 0.1% gelatin, 10 units EcoRI (Nippon Gene) and 10
Unit PstI (Company)] After digesting in 20 μl for 1 hour at 37 ℃,
Electrophoresis was performed on 8% agarose gel and the 5 end of PDI cDNA was detected.
A DNA fragment of about 490 bp in length from EcoRI to PstI was separated and purified with glass powder. On the other hand, pHPDIp4 DNA
About 1 μg was added to the solution [10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 6
mM MgCl ₂ , 6 mM mercaptoethanol, 0.1% gelatin, 10 units PstI (Nippon Gene) and 10 units
BamHI (manufactured by the same company)] After digesting in 20 μl at 37 ° C. for 1 hour, a DNA fragment having a length of about 1.3 kb of the BamHI portion was separated and purified from PstI at the 3 ′ end of PDI cDNA in the same manner. Both DNA fragments recovered in this way were approximately 50 ng and EcoRI and
Linearized plasmid vector pUC119 digested with BamHI
About 20 ng of DNA is DNA Ligation Kit A from Takara Shuzo
The solution was ligated by reacting in 25 μl of solution and 5 μl of solution B at 16 ° C. for 15 hours. E. coli MV1190 strain competent cells were transformed by the calcium method using 10 μl of this reaction solution. E. coli can be used in X-Gal plates (50 μm) with a diameter of 90 mm.
g / ml 5-bromo-4-chloro-3-indolyl-β-
D-galactopyranoside, 80 μg / ml isopropyl-β
-D-thiogalactopyranoside, 25 μg / ml ampicillin, LB medium and 1.5% agar). After culturing overnight at 37 ° C., the obtained white colonies were picked up, plasmid DNA was prepared by the alkaline lysis method, and analysis using restriction enzymes was performed to select a transformant carrying the desired plasmid. The resulting plasmid was named phPDIEB.

Based on phPDIEB, the Kunkel method [Kunkel,
TA. (1985) Proc. Natl. Acad. Sci. USA. 82 , 488], a restriction enzyme NaeI cleavage site was introduced at the boundary between the PDI signal sequence on the cDNA and the body of PDI. phPDIE
Escherichia coli BW313 strain competent cells were transformed by the calcium method with B DNA. A single colony of the obtained transformant was added to 2 × YT containing 150 μg / ml of ampicillin.
Medium (1.6% bactotryptone, 0.5% NaCl and 1%
Bacto yeast extract) was precultured overnight at 37 ° C. 1 ml of this culture was inoculated into 50 ml of 2 × YT medium containing 150 μg / ml of ampicillin and further cultured at 37 ° C.
When the turbidity (OD ₆₀₀ ) reached about 0.3, M13KO7 phage was added at about moi = 2 and the mixture was allowed to stand at 37 ° C for 30 minutes for infection. Kanamycin was added to this to a concentration of 70 μg / ml and shake culture was carried out at 37 ° C. for 20 hours. The culture solution was centrifuged, and 1/5 volume of 2.5 M NaCl and 20% polyethylene glycol # 6000 solution was added to the resulting supernatant, and the mixture was stirred at room temperature.
Let stand for 15 minutes. The precipitate obtained by centrifugation was dissolved in 5 ml of TE buffer [10 mM Tris-HCl, 1 mM EDTA (pH 8.0)], an equal volume of neutralized phenol was added, and the mixture was stirred and centrifuged to collect an aqueous layer. An equal volume of chloroform was added to this, and the mixture was stirred and centrifuged to collect the aqueous layer. In the resulting solution
Add 1/10 volume of 3M sodium acetate and 2.5 volumes of ethanol, stir, stir at -80 ° C for 30 minutes and centrifuge to DN.
A was collected as a precipitate. This was washed with 70% ethanol, dried under reduced pressure, and then dissolved in 100 μl of TE buffer. Single-stranded DNA derived from phPDIEB containing dU prepared by the above method
The desired mutation, that is, the NaeI site was introduced using the following procedure. Mutagenesis synthetic oligonucleotide (5'-C
GGGGGCGCCGGCGCGC-3 ', Takara Shuzo)] 10 pmol solution [10
0 mM Tris-HCl (pH8.0), 10 mM MgCl ₂ , 7 mM dithiothreitol, 1 mM ATP and 10 units of T4 polynucleotide kinase (Takara Shuzo Co., Ltd.)] After reacting in 10 μl at 37 ° C for 15 minutes, heat at 70 ° C for 10 minutes T4 polynucleotide kinase was inactivated. 0.2 pmol of single-stranded DNA derived from the above phPDIEB
And 1 μl of annealing buffer (Site-directed mu
Tagenesissystem Mutan ^TM -K, Takara Shuzo Co., Ltd.) was added sterile water to make the final volume 10 μl, 1 μl of which was mixed with 1 μl of the phosphorylated mutation-introducing synthetic oligonucleotide solution, and allowed to stand at 65 ° C. for 15 minutes and 37 ° C. for 15 minutes. After that, 25 μl of extension buffer (Mutan ^TM -K, the same company), 60 units of E. coli DNA ligase (Mutan ^TM -K, company) and 1 unit of T4 DNA polymerase (Mutan ^TM -K, company) were added at 25 ° C. The complementary strand was synthesized by reacting for a time. 3 μl of this solution
0.2M EDTA (pH8.0) was added and the complementary strand synthesis was stopped by heating at 65 ° C for 5 minutes. The obtained DNA solution 3
μl was mixed with 30 μl of Escherichia coli BMH71-18mutS competent cell, and the mixture was allowed to stand in ice for 30 minutes, 42 ° C. for 45 seconds and ice for 1 minute. Add 300 μl of SOC medium (2% bactotryptone, 0.5% yeast extract, 10 mM NaCl, 2.5
mM KCl, 10 mM MgSO ₄ , 10 mM MgCl ₂ and 20 mM glucose) were added, and the mixture was shaken at 37 ° C. for 1 hour. Another 10 μl of M13K
After adding O7 phage and allowing it to stand at 37 ℃ for 30 minutes, it contains 150 μg / ml ampicillin and 70 μg / ml kanamycin.
1 ml of × YT medium was added and shaken at 37 ° C. for 20 hours. The obtained culture broth was centrifuged, 20 μl of the supernatant was collected, and E. coli MV was collected.
Mix with 80 μl of 1190 culture solution, warm at 37 ° C for 10 minutes, and then
LB plates containing g / ml of ampicillin were plated and incubated overnight at 37 ° C. Of the resulting transformants, the target
M13 SEQUENCI that retains the NaeI site-introduced plasmid
It was identified by DNA nucleotide sequence analysis using NG KIT (Toyobo Co., Ltd.). This plasmid was named phPDINae.

PhPDINae DNA 2 prepared by alkaline lysis method
μg to a solution [10 mM Tris-HCl (pH8.0), 20 mM NaCl, 7 mM
MgCl ₂ , 7 units of NaeI (Nippon Gene Co., Ltd.) and 10 units of Hind III (Takara Shuzo Co., Ltd.)] Digested in 30 μl at 37 ° C. for 4 hours, and electrophoresed on 0.8% agarose gel to give a DNA fragment of about 1.7 kb in length. Separated and purified with powder. Construction of a plasmid pUC119Sig in which a DNA fragment encoding a prepro sequence of human serum albumin with a codon frequently used in yeast was cloned was carried out by the following procedure (Fig. 1A).

1 μg of the plasmid vector pUC119 DNA was dissolved in a solution [100 mM Tris.HCl (pH7.5), 10 mM MgCl ₂ , 50 mM NaCl.
And 12 units of EcoRI (Nippon Gene Co.) were digested in 20 μl at 37 ° C. for 1 hour and then heated at 70 ° C. for 5 minutes to inactivate the enzyme. Then, 38 μl of sterilized water and 1 unit of bacterial alkaline phosphatase (Takara Shuzo Co., Ltd.) were added and incubated at 37 ° C. for 1 hour, followed by phenol extraction, and the obtained aqueous layer was used for ethanol precipitation to recover DNA. This DNA
And an equimolar amount of an XhoI linker containing an XhoI site composed of the 5'-AATTCTCGAG GAGCTCTTAA-5 'sequence [66 mM Tris-HCl (pH 7.5), 6.6 mM MgCl ₂ , 10 mM
Dithiothreitol, 0.1 mM ATP and 300 units T4
DNA ligase (Takara Shuzo)] Incubated in 30 μl at 16 ° C. overnight. E. coli JM107 strain competent cells were transformed with 10 μl of this solution according to the calcium method, and 50 μg /
The LB plate containing ml of ampicillin was spread and incubated at 37 ° C. overnight. A plasmid DNA was prepared from the obtained colonies using an alkaline lysis method and subjected to restriction enzyme analysis to selectively obtain the plasmid DNA in which the desired XhoI linker was inserted into the pUC119 EcoRI site.

Four oligonucleotides having the following sequences: 1.5'- TCGAGAATTCATGAAGTGGGTTACCTTCATCTCTTTGTTGTT-3 ', 2.5'- AACAAGAACAACAAAGAGATGAAGGTAACCCACTTCATGAATTC-3', 3.5'-CTTGTTCTCTTCTGCTAA 4. -3 'automated DNA synthesizer (Applied Biosystems, Model 38
0B). About 30 pmol of each of these was added to 25 μl of a solution [50 mM Tris.HCl (pH 7.6), 10 mM MgCl ₂ , 5 mM dithiothreitol, 0.2 mM ATP and 6 units of T4 polynucleotide kinase (Takara Shuzo)] at 37 ° C. The 5'end was phosphorylated by reacting for a period of time. Mix the solution containing the obtained oligonucleotide (total 100 μl) 100
After leaving it in a water bath at ℃ for 5 minutes, it was left to cool at room temperature for annealing. To this, 600 units of T4 DNA ligase (Takara Shuzo Co., Ltd.) was added and the mixture was kept at 16 ° C. overnight to ligate the fragments to give a double-stranded fragment. The double-stranded DNA was deproteinized by phenol extraction and recovered by ethanol precipitation.

1 μg of the above-mentioned XhoI linker-introduced vector plasmid was added to a solution [100 mM Tris.HCl (pH 7.5), 10 m
M MgCl ₂ , 100 mM NaCl, 10 units BamHI (Nippon Gene) and 12 units XhoI (Takara Shuzo)] 37 in 20 μl
After digesting at 1 ° C. for 1 hour, phenol extraction was performed, and DNA was recovered from the obtained aqueous layer by ethanol precipitation. An equimolar amount of double-stranded DNA fragment obtained by ligating this DNA with the above-mentioned four oligonucleotides was added to a solution [66 mM.
The mixture was kept at 16 ° C. overnight in 30 μl of Tris-HCl (pH 7.5), 6.6 mM MgCl ₂ , 10 mM dithiothreitol, 0.1 mM ATP and 300 units of T4 DNA ligase (Takara Shuzo). Using 10 μl of this solution, Escherichia coli JM107 strain competent cells were transformed according to the calcium method, spread on an LB plate containing 50 μg / ml ampicillin, and kept at 37 ° C. overnight. The resulting colonies were subjected to nucleotide sequence analysis of their retained plasmid DNA to select transformants having the desired recombinant plasmid. This plasmid was named pUC119Sig.

Plasmid pUC119S prepared by the above procedure
ig DNA was prepared by the alkaline lysis method. 2 μg of this DNA
Solution [10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 7 mM MgCl
₂ , 8 units of StuI (Nippon Gene) and 10 units of
Hind III (Takara Shuzo)], digested at 37 ° C for 4 hours, and then subjected to 0.8% agarose gel electrophoresis.
The fragments were separated and purified with glass powder. Approximately 50 ng of the 1.7 kb DNA fragment derived from phPDINae thus obtained and pUC1
About 50 ng of the 3.2 kb DNA fragment derived from 19 Sig was reacted in Takara Shuzo Co., Ltd. ligation kit A solution 30 μl B solution 6 μl at 16 ° C. for 30 minutes, and then 10 μl was used by the calcium method to determine Escherichia coli strain HB101.
Competent cells (Takara Shuzo) were transformed to 50 μl /
LB plates containing ml ampicillin were plated. Colonies obtained by allowing this plate to stand overnight at 37 ° C were used to prepare plasmid DNA using the alkaline lysis method, and analysis using restriction enzymes was carried out to detect the presence of human serum albumin in the downstream of the prepro sequence of human. A recombinant plasmid in which the PDI body was connected (Fig. 2) was selected and obtained. This plasmid was named phPDILy1.

In order to express the leader sequence-modified PDI obtained as described above under the control of the yeast alcohol dehydrogenase I gene promoter, a human PDI expression plasmid was constructed by the following procedure. 7 μl of the above phPDILy1 DNA prepared by the alkaline lysis method was used as a solution [100 mM Tris.HCl (pH8.0), 100 mM NaCl, 7 mM MgCl ₂
And 40 units of EcoRI (Nippon Gene)] 100 μl
After digesting at 37 ° C. for 2 hours, an equal volume of a phenol / chloroform mixed solution (a mixed solution of saturated phenol and chloroform) was added and stirred, and the aqueous layer was recovered after centrifugation. This phenol / chloroform extraction was repeated, and 1/10 volume of 3M sodium acetate (pH 5.3) and 2.5 volume of ethanol were added to the obtained aqueous layer and mixed, and the mixture was allowed to stand at -40 ° C for 2 hours. The precipitate obtained by centrifugation was washed with 70% ethanol and dried under reduced pressure, and 50 μl of Klenow buffer (Kilo-Sequence Deletion K
It, Takara Shuzo Co., Ltd.), 4 units of Klenow fragment buffer (Takara Shuzo Co., Ltd.) was added, and the mixture was allowed to react at 37 ° C. for 45 minutes to smooth the EcoRI cleaved portion. This solution was subjected to phenol / chloroform extraction twice, and the resulting aqueous layer was diluted with 1/10 volume of 3M sodium acetate (pH 5.3) and
2.5 volumes of ethanol was added and mixed, and the mixture was allowed to stand at -40 ° C for 1 hour. The precipitate obtained by centrifugation was washed with 70% ethanol and dried under reduced pressure, and 50 μl of Klenow buffer (for Kilo-Sequence)
Deletion Kit, Takara Shuzo) 4 units of Klenow f
A ragment (Takara Shuzo) was added and reacted at 37 ° C for 45 minutes to smooth the EcoRI cleavage portion. This solution was extracted twice with phenol / chloroform, and 1/10 volume of 3M sodium acetate (pH 5.3) and 2.5 volume of ethanol were added to the obtained aqueous layer and mixed, and the mixture was allowed to stand at -40 ° C for 1 hour. . The precipitate obtained by centrifugation was washed with 70% ethanol and dried under reduced pressure, and the solution [10 mM Tris · HCl (pH8.0), 60 mM was added.
40 μl of NaCl, 7 mM MgCl ₂ and 10 units of BamHI (Nippon Gene Co., Ltd.) and reacted at 37 ° C. for 3 hours. The obtained DNA solution is subjected to 0.8% agarose gel electrophoresis,
The 1.8 kb DNA fragment was separated and purified with glass powder. On the other hand, pJDB-ADH-HSA-A prepared by alkaline lysis method
(Japanese Patent Laid-Open No. 2-117384) 5 μl of DNA in a solution [10 mM Tris
HCl (pH 8.0), 100 mM NaCl, 7 mM MgCl ₂ and 24 units Xh
oI (Takara Shuzo)] After digesting in 100 μl at 37 ° C for 2 hours, phenol / chloroform extraction was performed twice, and the resulting aqueous layer was
1/10 volume of 3 M sodium acetate (pH 5.3) and 2.5 volumes of ethanol were added and mixed, and the mixture was stood still at -40 ° C for 2 hours and then centrifuged to collect DNA as a precipitate. The DNA precipitate was washed with 70% ethanol and dried under reduced pressure, and 50 μl of Klenow buffer (Kilonow buffer) was added.
-Sequence Deletion Kit. Takara Shuzo Co., Ltd.), 4 units of Klenow fragment (Takara Shuzo Co., Ltd.) was added, and the reaction was allowed to proceed at 37 ° C for 45 minutes to smooth the XhoI cleavage site. This solution was extracted with phenol / chloroform twice, and the resulting aqueous layer contained 1/10 volume of 3M sodium acetate (pH
5.3) and 2.5 volumes of ethanol are added and mixed at -40 ° C.
After standing for 1 hour, the DNA was recovered as a precipitate by centrifugation.
The obtained DNA was washed with 70% ethanol, dried under reduced pressure, and dissolved in 40 μl of a solution [10 mM Tris-HCl (pH8.0), 60 mM NaCl, 7 mM MgCl ₂ and 10 units of BamHI (Nippon Gene)], and 37 ° C 75 Digested for a minute. Add 10 μl of 2M Tris to this solution.
HCl (pH 8.0), 110 μl sterile water and 1 unit E. coli
Alkaline phosphatase derived from C75 strain (Takara Shuzo) was added and mixed, and the mixture was heated at 60 ° C. for 1 hour to carry out 5 ′ dephosphorylation of the enzyme cleavage site. 1/10 volume of the resulting solution
3M sodium acetate (pH 5.3) and 2.5 volumes of ethanol were added and mixed, and the mixture was left standing at -40 ° C for 1 hour. DNA was collected as a precipitate by centrifugation, dried under reduced pressure, and dissolved in 20 μl TE to 0.8%.
It was subjected to agarose gel electrophoresis. A DNA fragment of about 8 kb was recovered using glass powder. Approximately 50 ng of phPDILy1-derived 1.8 kb DNA fragment and pJDB obtained as described above.
-Approximately 50 ng of 8 kb DNA fragment derived from ADH-HSA-A was mixed with 30 μl of DNA ligation kit A solution (Takara Shuzo) and 6 μl of solution B to prepare 16
Both DNAs were ligated by reacting at 2.5 ° C for 2.5 hours. The obtained DNA
Escherichia coli C600 strain was transformed with 10 μl of the solution by the calcium method, plated on an LB plate containing 50 μl / μl of ampicillin, and cultured at 37 ° C. overnight. A plasmid DNA is prepared from the obtained colonies by the alkaline lysis method and subjected to restriction enzyme analysis to obtain a modified leader sequence downstream of the target alcohol hydrogenase I promoter.
Transformants harboring the PDI-ligated plasmid were selected. The PDI expression plasmid constructed in this way was transformed into pA
It was named HhPDILy1. In addition, as a result of this construction, mature PD
The N-terminal amino acid of I was changed from Asp to Gly.

On the other hand, a control plasmid for human PDI expression experiment was prepared by the following procedure. Add 5 μl of pJDB-ADH-HSA-A DNA prepared by the alkaline lysis method to a solution [10 mM Tris-
HCl, 100 mM NaCl, 7 mM MgCl ₂ , 24 units of XhoI (Takara Shuzo) and 29 units of BamHI (Nippon Gene)] 100
After digestion in μl for 2 hours at 37 ° C, phenol / chloroform extraction was performed twice, and 1/10 volume of 3M sodium acetate (pH5.3) and 2.5 volume of ethanol were added to the obtained water tank and mixed.
After standing at 40 ° C for 2 hours, the DNA was collected as a precipitate by centrifugation. This DNA was washed with 70% ethanol and dried under reduced pressure.
50 μl of Klenow buffer (Kilo-Sequence Deletion Kit,
The product was dissolved in Takara Shuzo Co., Ltd., 4 units of Klenow fragment (Takara Shuzo Co., Ltd.) was added, and the mixture was allowed to react at 37 ° C. for 45 minutes to smooth the XhoI and BamHI cleavage sites. This solution was extracted twice with phenol / chloroform. To the resulting aqueous layer was added 1/10 volume of 3M sodium acetate (pH 5.3) and 2.5 volumes of ethanol and mixed, and the mixture was left standing at -40 ° C for 1 hour and then centrifuged. The DNA was recovered as a precipitate. 20μ after dried under reduced pressure
Dissolve in 1 ml of TE and apply 0.8% agarose electrophoresis to
The 8 kb DNA fragment was recovered with glass powder. Got
Approximately 50 ng of DNA fragment is mixed with 30 μl of A solution and 6 μl of B solution of Takara Shuzo DNA Ligation Kit and reacted at 16 ° C overnight.
It was circularized by self-ligation. E. coli 101 strain competent cells (Takara Shuzo) were transformed with 10 μl of this DNA solution by the calcium method, plated on an LB plate containing 50 μl / ml of ampicillin, and cultured at 37 ° C. overnight. A plasmid DNA was prepared from the obtained colony by the alkaline lysis method and subjected to restriction enzyme analysis to selectively obtain the desired control plasmid. The resulting plasmid is pAH
I named it.

Expression of Human PDI by Yeast Human PDI expression plasmid pAHhPDIL constructed by the above procedure
Y1 was used to express human PDI in yeast by the method described below.

YPD plate (2% bactopeptone, 1% yeast extract, 2% glucose and 1.5% agar)
A single colony of the yeast AH22 strain cultivated above was inoculated into 5 ml of YPD medium (2% bactopeptone, 1% yeast extract and 2% glucose) and cultivated with shaking at 30 ° C. for 24 hours. 0.9 ml of this preculture liquid was inoculated into 45 ml of YPD medium, shake-cultured at 30 ° C., and when the OD ₆₀₀ (turbidity) reached about 0.5, low-speed centrifugation was performed to collect the bacteria as a precipitate. The obtained cells were suspended in 3 ml of 0.2 M LiSCN, and 1 ml of the suspension was centrifuged to collect the cells. 50 μl of this cell
% PEG # 4000, 10 μl of LiSCN, and 10 μl of pAHhPDILy1 DNA solution (27 μl of DNA) prepared by the alkaline lysis method were added, mixed by pipetting, and allowed to stand at 30 ° C. overnight.
To this, 1 ml of sterilized water was added and suspended, and the cells were collected as a precipitate by centrifugation. Suspend the cells in 100 μl of sterilized water, and use SD (-Leu) plate [SD (-Leu) medium (0.67% bactonitrogen base, 2% glucose, 20 mg / l adenine,
Uracil, tryptophan, histidine, arginine, methionine, 30 mg / l tyrosine, isoleucine, lysine, 50 mg / l phenylalanine, 100 m
g / l aspartic acid, same glutamic acid, 150mg / l
Valine, 200 mg / l threonine and 375 mg / l serine (these amino acids are from Wako Pure Chemical Industries)) and 1.5%
Agar] and cultured at 30 ° C. The transformant obtained on the 5th day of culture was inoculated into 5 ml of SD (-Leu) medium and cultured with shaking at 30 ° C for 2 days. 100 μl of this preculture liquid is added to 5 ml of YPD.
The medium was inoculated and cultured with shaking at 30 ° C. for 24 hours. The obtained culture solution
1.5 ml was centrifuged to collect 500 μl of the supernatant, an equal volume of ethanol was added thereto, and the mixture was mixed and allowed to stand in ice for 1 hour.
This was centrifuged to collect the secretion product from the yeast cells in the medium as a precipitate, which was dried under reduced pressure. 10 μl of the obtained precipitate
Sample buffer for SDS-PAGE (125 mM Tris-HCl (pH 6.8)
, 4% SDS, 20% glycerin, 10% β-mercaptoethanol and 0.01% bromophenol blue), boiled for 5 minutes and electrophoresed on SDS-PAG plate 10/20 (Daiichi Pure Chemicals) . Stain this gel with a staining solution (0.15%
After staining with Coomassie Brilliant Blue, 10% acetic acid and 40% methanol), it was immersed in a decolorizing solution (10% acetic acid and 40% methanol) to visualize the expressed product. At this time, as a control, the above-mentioned pAH was used as a starting point, and a culture medium sample obtained by the same operation as that for the above-mentioned pAHhPDILy1 was simultaneously run. Phosphorylase b as a molecular weight standard
(Molecular weight 94.000), bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000) and α-lactalbumin (14,000) were used (3rd
Figure). As a result, an expression product having a molecular weight of about 55K could be found. This molecular weight was in agreement with the molecular weight of mature PDI, and it was expected that the desired human PDI was expressed and secreted. Therefore, for the purpose of investigating the protein chemical properties of the expressed secretion, large-scale culture was carried out by the following procedure.

A single colony of the yeast AH22 strain carrying pAHhPDILy1 was inoculated into 80 ml of SD (-Leu) medium and cultured at 30 ° C. for 2 days with shaking. The resulting pre-culture liquid was added to each of 4 ml of YPD.
Phosphate medium (YPD medium, 6 g / l Na ₂ HPO ₄ and 3 g / l KH ₂ PO ₄ , pH 7.0) was inoculated at 30 ° C.
Shaking culture was performed for 4 hours. The culture was centrifuged to collect the supernatant, which was used for purification of the secreted expression product described below.

Isolation of recombinant human PDI from the medium and its characterization 4 l of transformed yeast culture medium obtained as described above
The Millipore-Millitan ultrafilter (excluded molecular weight 30,00
0) and concentrated 40 times (100 ml), then use TSK-gel Phe
Human PDI was isolated by a nyl-5PW hydrophobic column.
The hydrophobic column was equilibrated with 10 mM boric acid-10 mM KCl buffer pH 8.0 containing 0.85 M ammonium sulfate and 0.05% NaN _3.
Elution was performed by forming a linear concentration gradient into the same buffer without ammonium sulfate in 25 minutes. The flow velocity at this time is 2
ml / min. The results are shown in FIG. FIG. 5 shows an SDS electropherogram of isolated human PDI.
As shown in the figure, human PDI was separated into almost single components by hydrophobic column chromatography,
Moreover, it was revealed that the PDI activity was retained. It can be seen that the ultraviolet absorbing substance derived from the YPD medium can be removed extremely efficiently by this chromatography.

Measurement of PDI activity PDI activity is measured by a scrambled ribonuclease A (RNase A) prepared by a method involving reduction, denaturation and reoxidation.
This was done by observing the promoting effect on the reconstruction of. The degree of reconstitution of ribonuclease A was quantified using the degree of recovery of its enzyme activity as an index. The specific method is shown below.

Preparation of scrambled RNase A: 120 mg R
Nase A was dissolved in 3 ml of 0.1 M Tris-HCl buffer (pH 8.6) containing 6 M guanidine hydrochloride and 0.15 M dithiothreitol, and then reduced at room temperature for 15 hours under a nitrogen stream. The reducing agent was removed by a Sephadex G-25 column (15 mmφ × 38 cm) equilibrated with 0.01 N HCl. Guanidine hydrochloric acid was added to the desalted product so as to have a final concentration of 6 M, Tris was further added to adjust the pH to 9.0, and an S—S bond exchange reaction was carried out in the dark at 4 ° C. for 14 days. This sample was stored at -80 ° C and used as scrambled RNase A.

Measurement of PDI activity: 55 mM with nitrogen substitution
Prepare 20 μl of phosphate buffer (pH 7.5) to which 10 μl of 1M dithiothreitol was added.
7.5) Add 420 μl and leave at 30 ° C for 5 minutes and a half. To this, add 50 μl of the scrambled RNase solution and incubate at 30 ° C for 15 minutes and a half. Here, 1.945 ml of deaerated 50 mM Tris-HCl, 5 mM magnesium chloride, 25 mM potassium chloride in buffer pH 7.5, 50 μl of yeast RNA solution (10 mM Tris-HCl buffer pH 7.5 / 1 mM EDTA, absorbance at 280 nm (Adjusted to 80) is added to a 1 cm square quartz cell, and the temperature is equilibrated to 45 ° C. with stirring. At this time 26
Make sure that the absorbance at 0 nm does not change. From the scrambled RNase A solution treated with dithiothreitol, take 5 μl and mix with the solution in the cell.
Measure the absorbance at 260 nm every minute for 2 minutes. PDI activity is 26
It can be calculated from the initial rate of change in absorbance at 0 nm.

The human PDI expression plasmid pAHhPDILy1
Transformation of yeast HIS23 strain using the human PDI expression plasmid pAHhPDILy1 according to the following procedure, HSA producing yeast HIS23 strain [Japanese Patent Application No. 2-57]
No. 885 / Microtech Lab. No. 11351 (FERM P-113
8)] was transformed.

YPD plate (2% bactotryptone,
HSA expressing yeast HIS23 cultivated on 1% Bacto yeast extract, 2% glucose and 1.5% agar)
A single colony of the strain was inoculated into 5 ml of YPD medium (2% bactotryptone, 1% yeast extract and 2% glucose) and cultured at 30 ° C. for 24 hours with shaking. 1 ml of this culture solution was inoculated into 50 ml of YPD medium and shake-cultured at 30 ° C. When the OD ₆₀₀ (turbidity) reached about 0.5, the cells were collected as a precipitate by low speed centrifugation. To the collected cells, 46 μl of 50% polyethylene glycol # 4000, 10
μl LiSCN and alkaline lysis [Birnboim, H.
C. & Doly, J. (1979) Nucleic AcidsRes . 7 , 151
Human PDI expression plasmid pAHhPDILy1 DN prepared in 3.]
10 μl of A solution (about 20 μg of DNA) was added, mixed by pipetting, and allowed to stand at 30 ° C. overnight. To this
After suspending by adding ml of sterilized water, cells were collected as a precipitate by centrifugation. The cells were suspended in 100 μl of sterilized water, and then SD (-His, -Leu) plate [SD (-H
is, -Leu) medium (0.67% bactonitrogen base, 2% glucose, 20 mg / l adenine, uracil, tryptophan, arginine, methionine,
30 mg / l tyrosine, isoleucine, lysine, 5
0 mg / l phenylalanine, 100 mg / l aspartic acid, glutamic acid, 150 mg / l valine, 20
0 mg / l threonine and 375 mg / l serine (the above amino acids are manufactured by Wako Pure Chemical Industries, Ltd.) and 1.5% agar] were seeded and cultured at 30 ° C. On the 5th day of culture, transformants were obtained as colonies on the plate.

Obtained transformant (pAHhPDILy1 / HIS23)
The expression of PDI was examined by the following procedure. At this time, as a control experiment, the transformant (pAH / HIS23) obtained by using the control plasmid pAH obtained by removing the PDI cDNA portion from pAHhPDILy1 was used below. The colonies on the plate were inoculated into 5 ml of SD (-His, -Leu) medium and cultured at 30 ° C for 2 days with shaking. This pre-culture liquid 1
After inoculating 5 μl of YPD medium with 5 μl of the culture, shake culture at 30 ° C. for 24 hours, centrifuge 1.5 ml of the obtained culture solution to collect 500 μl of the supernatant, add an equal volume of ethanol to the mixture, and mix 1 on ice. Let stand for hours. This was subjected to centrifugation to collect the expressed secretion product from yeast in the medium as a precipitate, which was dried under reduced pressure by a centrifugal evaporator. 1 for the obtained precipitate
0 μl of SDS-PAGE sample buffer [62.5 mM T
ris-HCl (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 0.005% bromophenol blue and 20
% Glycerin] and boiled for 5 minutes, then SDS-PAG
Electrophoresis was performed on Plate 4 / 20-1010 (manufactured by Daiichi Pure Chemicals Co., Ltd.). The gel after electrophoresis was stained with a staining solution (0.15% Coomassie Brilliant Blue, 10% acetic acid and 40% methanol) and then immersed in a decolorizing solution (10% acetic acid and 40% methanol) to visualize the expressed secretion product in the medium. At this time, phosphorylase b (molecular weight
94,000 daltons, bovine serum albumin (67,000), ovalbumin (43,000), carbonic anhydrase (30,000), soybean trypsin inhibitor (20,000) and α-lactalbumin (14,000) were used (6th
Figure). As a result, expression and secretion of PDI having a molecular weight of about 55,000 Dalton was detected in the yeast HIS23 strain transformed with pAHhPDILy1.

Effect of Human PDI on HSA Expression and Secretion Using the co-expression system of HSA and PDI in yeast described above, the effect of human PDI on HSA expression and secretion was examined by the following procedure.

Yeast HIS23 strains transformed with the control plasmid pAH and the human PDI expression plasmid pAHhPDILy1, respectively, 5 independent SD colonies of 5 pAH / HIS23 strains and 5 pAHhPDILy1 / HIS23 strains each, 5 ml of SD (-His, -Leu) The medium was inoculated and precultured at 30 ° C. for 24 hours. This pre-culture liquid 10
0 μl of each was inoculated into 5 ml of YPD medium and incubated at 30 ° C for 2
After shaking culture for 4 hours, SDS-PAGE samples were prepared from each culture medium by the method described in the previous section.
AGE was performed (Fig. 7). Using the obtained gel,
HSA secretion of each strain is measured by densitometer (IMAGE ANALYS
Quantification by IS SYSTEM, manufactured by Tefco Co., Ltd., and changes in the expression and secretion amount of HSA due to PDI co-expression were examined (No. 8).
Figure). As a result, pAH / HIS23 strain averaged 0.93 mg /
In addition, pAHhPDILy1 / HIS23 strain also had 1.50 mg / l HS.
Human PD in the yeast HIS23 strain that secretes A
Co-expression of I showed an average increase in HSA secretion of about 60%.

[0078]

INDUSTRIAL APPLICABILITY The present invention was the first to establish means for mass production of human PDI by using a ligating gene consisting of a DNA encoding a human serum albumin prepro sequence and a human protein disulfide isomerase gene. Therefore, this method can be used as a large-scale and inexpensive means for promoting the activation of a protein in which higher-order structure formation is incomplete due to cross-linking of S—S bonds or the like. It is considered to be effective mainly for the activation of inactive proteins produced by genetic engineering, and by coupling the expression of this enzyme with the expression of other useful polypeptides, it is It has become possible to increase production efficiency. In addition, it can be used as a research reagent.

[0079]

[Sequence list]

SEQ ID NO: 1 Sequence length: 2454 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin: Human liver or placenta λgt 11 cDNA library (Cl
ontech) Sequence GAATTCCGGG GGCGACGAGA GAAGCGCCCC GCCTGATCCG TGTCCGAC ATG CTG CGC 57 Met Leu Arg -15 CGC GCT CTG CTG TGC CTG GCC GTG GCC GCC CTG GTG CGC GCC GAC GCC 105 Arg Ala Leu Leu Cys Leu Ala Aru Ala Valu Ala Ala -10 -5 1 CCC GAG GAG GAG GAC CAC GTC CTG GTG CTG CGG AAA AGC AAC TTC GCG 153 Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys Ser Asn Phe Ala 5 10 15 GAG GCG CTG GCG GCC CAC AAG TAC CTG CTG GTG GAG TTC TAT GCC CCT 201 Glu Ala Leu Ala Ala His Lys Tyr Leu Leu Val Glu Phe Tyr Ala Pro 20 25 30 TGG TGT GGC CAC TGC AAG GCT CTG GCC CCT GAG TAT GCC AAA GCC GCT 249 Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys Ala Ala 35 40 45 50 GGG AAG CTG AAG GCA GAA GGT TCC GAG ATC AGG TTG GCC AAG GTG GAC 297 Gly Lys Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys Val Asp 55 60 65 GCC ACG GAG GAG TCT GAC CTG GCC CAG CAG TAC GGC GTG CGC GGC TAT 345 Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg Gly Tyr 70 75 80 CCC ACC ATC AAG TTC TTC AGG AAT GGA GAC ACG GCT TCC CCC AAG GAA 393 Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro Lys Glu 85 90 95 TAT ACA GCT GGC AGA GAG GCT GAT GAC ATC GTG AAC TGG CTG AAG AAG 441 Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu Lys Lys 100 105 110 CGC ACG GGC CCG GCT GCC ACC ACC CTG CCT GAC GGC GCA GCT GCA GAG 489 Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly Ala Ala Ala Glu 115 120 125 130 TCC TTG GTG GAG TCC AGC GAG GTG GCT GTC ATC GGC TTC TTC AAG GAC 537 Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe Phe Lys Asp 135 140 145 GTG GAG TCG GAC TCT GCC AAG CAG TTT TTG CAG GCA GCA GAG GCC ATC 585 Val Glu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala Glu Ala Ile 150 155 160 GAT GAC ATA CCA TTT GGG ATC ACT TCC AAC AGT GAC GTG TTC TCC AAA 633 Asp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val Phe Ser Lys 165 170 175 TAC CAG CTC GAC AAA GAT GGG GTT GTC CTC TTT AAG AAG TTT GAT GAA 681 Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys Phe Asp Glu 180 185 190 GGC CGG AAC AAC TTT GAA GGG GAG GTC ACC AAG GAG AAC CTG CTG GAC 729 Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn Leu Leu Asp 195 200 205 210 TTT ATC AAA CAC AAC CAG CTG CCC CTT GTC ATC GAG TTC ACC GAG CAG 777 Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe Thr Glu Gln 215 220 225 ACA GCC CCG AAG ATT TTT GGA GGT GAA ATC AAG ACT CAC ATC CTG CTG 825 Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile Leu Leu 230 235 240 TTC TTG CCC AAG AGT GTG TCT GAC TAT GAC GGC AAA CTG AGC AAC TTC 873 Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu Ser Asn Phe 245 250 255 AAA ACA GCA GCC GAG AGC TTC AAG GGC AAG ATC CTG TTC ATC TTC ATC 921 Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile Phe Ile 260 265 270 GAC AGC GAC CAC ACC GAC AAC CAG CGC ATC CTC GAG TTC TTT GGC CTG 969 Asp Ser Asp His Thr Asp Asn Gln Arg Ile Leu Glu Phe Phe Gly Leu 275 280 285 290 AAG AAG GAA GAG TGC CCG GCC GTG CGC CTC ATC ACC CTG GAG GAG GAG 1017 Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu Glu Glu 295 300 305 ATG ACC AAG TAC AAG CC C GAA TCG GAG GAG CTG ACG GCA GAG AGG ATC 1065 Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu Arg Ile 310 315 320 ACA GAG TTC TGC CAC CGC TTC CTG GAG GGC AAA ATC AAG CCC CAC CTG 1113 Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro His Leu 325 330 335 ATG AGC CAG GAG CTG CCG GAG GAC TGG GAC AAG CAG CCT GTC AAG GTG 1161 Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val Lys Val 340 345 350 CTT GTT GGG AAG AAC TTT GAA GAC GTG GCT TTT GAT GAG AAA AAA AAC 1209 Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys Lys Asn 355 360 365 370 GTC TTT GTG GAG TTC TAT GCC CCA TGG TGT GGT CAC TGC AAA CAG TTG 1257 Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Gln Leu 375 380 385 GCT CCC ATT TGG GAT AAA CTG GGA GAG ACG TAC AAG GAC CAT GAG AAC 1305 Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His Glu Asn 390 395 400 ATC GTC ATC GCC AAG ATG GAC TCG ACT GCC AAC GAG GTG GAG GCC GTC 1353 Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu Ala Val 405 410 415 AAA GTG CAC AGC TTC CCC ACA CTC AAG TTC TTT CCT GCC AGT GCC GAC 1401 Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser Ala Asp 420 425 430 AGG ACG GTC ATT GAT TAC AAC GGG GAA CGC ACG CTG GAT GGT TTT AAG 1449 Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly Phe Lys 435 440 445 450 AAA TTC CTG GAG AGC GGT GGC CAG GAT GGG GCA GGG GAT GAT GAC GAT 1497 Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp Asp Asp 455 460 465 CTC GAG GAC CTG GAA GAA GCA GAG GAG CCA GAC ATG GAG GAA GAC GAT 1545 Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu Glu Asp Asp 470 475 480 GAT CAG AAA GCT GTG AAA GAT GAA CTG TAA TACGCAAAGC CAGACCCGGG 1595 Asp Gln Lys Ala Val Lys Asp Glu Leu * 485 490 CGCTGCCGAG ACCCCTCGGG GGCTGCACAC CCAGCAGCAG CGCACGCCTC CGAAGCCTGC 1655 GGCCTCGCTT GAAGGAGGGC GTCGCCGGAA ACCCAGGGAA CCTCTCTGAA GTGACACCTC 1715 ACCCCTACAC ACCGTCCGTT CACCCCCGTC TCTTCCTTCT GCTTTTCGGT TTTTGGAAAG 1775 GGATCCATCT CCAGGCAGCC CACCCTGGTG GGGCTTGTTT CCTGAAACCA TGATGTACTT 1835 TTTCATACAT GAGTCTGTCC AGAGTGCTTG CTACCGTGTT CGGAGTCTCG CTGCCTCCCT 1895 CCCGCGGGAG GTTTCTCCTC TTTTTGAAAA TTCCGTCTGT GGGATTTTTA GACATTTTTC 1955 GACATCAGGG TATTTGTTCC ACCTTGGCCA GGCCTCCTCG GAGAAGCTTG TCCCCCGTGT 2015 GGGAGGGACG GAGCCGGACT GGACATGGTC ACTCAGTACC GCCTGCAGTG TCGCCATGAC 2075 TGATCATGGC TCTTGCATTT TTGGGTAAAT GGAGACTTCC GGATCCTGTC AGGGTGTCCC 2135 CCATGCCTGG AAGAGGAGCT GGTGGCTGCC AGCCCTGGGG CCCGGCACAG GCCTGGGCCT 2195 TCCCCTTCCC TCAAGCCAGG GCTCCTCCTC CTGTCGTGGG CTCATTGTGA CCACTGGCCT 2255 CTCTACAGCA CGGCCTGTGG CCTGTTCAAG GCAGAACCAC GACCCTTGAC TCCCGGGTGG 2315 GGAGGTGGCC AAGGATGCTG GAGCTGAATC AGACGCTGAC AGTTCTTCAG GCATTTCTAT 2375 TTCACAATCG AATTGAACAC ATTGGCCAAA TAAAGTTGAA ATTTTACCCA CCCAAAAAAAAA 2435 AAAAAAAAAA CCCGAATTC 2454 Type: Other nucleic acid (semi-synthetic DNA) sequence ATG AAG TGG GTT ACC TTC ATC TCT TTG TTG 30 Met Lys Trp Val Thr Phe Ile Ser Leu Leu -20 -15 TTC TTG TTC TCT TCT GCT TAC TCT AGA GGT GTT TTC AGA AGG GGC GCC 78 Phe Leu Phe Ser Ser Ala Tyr Ser Arg Gly Val Phe Arg Arg Gly Ala -10 -5 1 CCC GAG GAG GAG GAC CAC GTC CTG GTG CTG CGG AAA AGC AAC TTC GCG 126 Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys Ser Asn Phe Ala 5 10 15 GAG GCG CTG GCG GCC CAC AAG TAC CTG CTG GTG GAG TTC TAT GCC CCT 174 Glu Ala Leu Ala Ala His Lys Tyr Leu Leu Val Glu Phe Tyr Ala Pro 20 25 30 TGG TGT GGC CAC TGC AAG GCT CTG GCC CCT GAG TAT GCC AAA GCC GCT 222 Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys Ala Ala 35 40 45 50 GGG AAG CTG AAG GCA GAA GGT TCC GAG ATC AGG TTG GCC AAG GTG GAC 270 Gly Lys Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys Val Asp 55 60 65 GCC ACG GAG GAG TCT GAC CTG GCC CAG CAG TAC GGC GTG CGC GGC TAT 318 Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg Gly Tyr 70 75 80 CCC ACC ATC AAG TTC TTC AGG AAT GGA GAC ACG GCT TCC CCC AAG GAA 366 Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro Lys Glu 85 90 95 TAT ACA GCT GGC AGA GAG GCT GAT GAC ATC GTG AAC TGG CTG AAG AAG 414 Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu Lys Lys 100 105 110 CGC ACG GGC CCG GCT GCC ACC ACC CTG CCT GAC GGC GCA GCT GCA GAG 462 Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly Ala Ala Ala Glu 115 120 125 130 TCC TTG GTG GAG TCC AGC GAG GTG GCT GTC ATC GGC TTC TTC AAG GAC 510 Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe Phe Lys Asp 135 140 145 GTG GAG TCG GAC TCT GCC AAG CAG TTT TTG CAG GCA GCA GAG GCC ATC 558 Val Glu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala Glu Ala Ile 150 155 160 GAT GAC ATA CCA TTT GGG ATC ACT TCC AAC AGT GAC GTG TTC TCC AAA 606 Asp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val Phe Ser Lys 165 170 175 TAC CAG CTC GAC AAA GAT GGG GTT GTC CTC TTT AAG AAG TTT GAT GAA 654 Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys Phe Asp Glu 180 185 190 GGC CGG AAC AAC TTT GAA GGG GAG GTC ACC AAG GAG AAC CTG CTG GAC 702 Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn Leu Leu Asp 195 200 205 210 TTT ATC AAA CAC AAC CAG CTG CCC CTT GTC ATC GAG TTC ACC GAG CAG 750 Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe Thr Glu Gln 215 220 225 ACA GCC CCG AAG ATT TTT GGA GGT GAA ATC AAG ACT CAC ATC CTG CTG 798 Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile Leu Leu 230 235 240 TTC TTG CCC AAG AGT GTG TCT GAC TAT GAC GGC AAA CTG AGC AAC TTC 846 Phe Leu Pro Lys Ser Val Ser Asp Try Asp Gly Lys Leu Ser Asn Phe 245 250 255 AAA ACA GCA GCC GAG AGC TTC AAG GGC AAG ATC CTG TTC ATC TTC ATC 894 Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile Phe Ile 260 265 270 GAC AGC GAC CAC ACC GAC AAC CAG CGC ATC CTC GAG TTC TTT GGC CTG 942 Asp Ser Asp His Thr Asp Asn Gln Arg Ile Leu Glu Phe Phe Gly Leu 275 280 285 285 290 AAG AAG GAA GAG TGC CCG GCC GTG CGC CTC ATC ACC CTG GAG GAG GAG 990 Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu Glu Glu 295 300 305 ATG ACC AAG TAC AAG CCC GAA TCG GAG GAG CTG ACG GCA GAG AGG ATC 1038 Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu Arg Ile 310 315 320 AC A GAG TTC TGC CAC CGC TTC CTG GAG GGC AAA ATC AAG CCC CAC CTG 1086 Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro His Leu 325 330 335 ATG AGC CAG GAG CTG CCG GAG GAC TGG GAC AAG CAG CCT GTC AAG GTG 1134 Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val Lys Val 340 345 350 CTT GTT GGG AAG AAC TTT GAA GAC GTG GCT TTT GAT GAG AAA AAA AAC 1182 Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys Lys Asn 355 360 365 370 GTC TTT GTG GAG TTC TAT GCC CCA TGG TGT GGT CAC TGC AAA CAG TTG 1230 Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Gln Leu 375 380 385 GCT CCC ATT TGG GAT AAA CTG GGA GAG ACG TAC AAG GAC CAT GAG AAC 1278 Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His Glu Asn 390 395 400 ATC GTC ATC GCC AAG ATG GAC TCG ACT GCC AAC GAG GTG GAG GCC GTC 1326 Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu Ala Val 405 410 415 AAA GTG CAC AGC TTC CCC ACA CTC AAG TTC TTT CCT GCC AGT GCC GAC 1374 Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser Ala Asp 420 425 430 AGG ACG GTC ATT GAT TAC AAC GGG GAA CGC ACG CTG GAT GGT TTT AAG 1422 Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly Phe Lys 435 440 445 450 AAA TTC CTG GAG AGC GGT GGC CAG GAT GGG GCA GGG GAT GAT GAC GAT 1470 Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp Asp Asp 455 460 465 CTC GAG GAC CTG GAA GAA GCA GAG GAG CCA GAC ATG GAG GAA GAC GAT 1518 Leu Glu Asp Leu Glu Ala Glu Glu Pro Asp Met Glu Glu Asp Asp 470 475 480 GAT CAG AAA GCT GTG AAA GAT GAA CTG 1545 Asp Gln Lys Ala Val Lys Asp Glu Leu 485 490 SEQ ID NO: 3 Sequence length: 491 Sequence type: Amino acid topology : Type of linear sequence: Protein sequence Gly Ala Pro Glu Glu Glu Asp His Val Leu Val Leu Arg Lys Ser Asn 1 5 10 15 Phe Ala Glu Ala Leu Ala Ala His Lys Tyr Leu Leu Val Glu Phe Tyr 20 25 30 Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala Pro Glu Tyr Ala Lys 35 40 45 Ala Ala Gly Lys Leu Lys Ala Glu Gly Ser Glu Ile Arg Leu Ala Lys 50 55 60 Val Asp Ala Thr Glu Glu Ser Asp Leu Ala Gln Gln Tyr Gly Val Arg 65 70 75 80 Gly Tyr Pro Thr Ile Lys Phe Phe Arg Asn Gly Asp Thr Ala Ser Pro 85 90 95 Lys Glu Tyr Thr Ala Gly Arg Glu Ala Asp Asp Ile Val Asn Trp Leu 100 105 110 Lys Lys Arg Thr Gly Pro Ala Ala Thr Thr Leu Pro Asp Gly Ala Ala 115 120 125 Ala Glu Ser Leu Val Glu Ser Ser Glu Val Ala Val Ile Gly Phe Phe 130 135 140 Lys Asp Val Glu Ser Asp Ser Ala Lys Gln Phe Leu Gln Ala Ala Glu 145 150 155 160 Ala Ile Asp Asp Ile Pro Phe Gly Ile Thr Ser Asn Ser Asp Val Phe 165 170 175 Ser Lys Tyr Gln Leu Asp Lys Asp Gly Val Val Leu Phe Lys Lys Phe 180 185 190 Asp Glu Gly Arg Asn Asn Phe Glu Gly Glu Val Thr Lys Glu Asn Leu 195 200 205 Leu Asp Phe Ile Lys His Asn Gln Leu Pro Leu Val Ile Glu Phe Thr 210 215 220 Glu Gln Thr Ala Pro Lys Ile Phe Gly Gly Glu Ile Lys Thr His Ile 225 230 235 240 Leu Leu Phe Leu Pro Lys Ser Val Ser Asp Tyr Asp Gly Lys Leu Ser 245 250 255 Asn Phe Lys Thr Ala Ala Glu Ser Phe Lys Gly Lys Ile Leu Phe Ile 260 265 270 Phe Ile Asp Ser Asp His Thr Asp As n Gln Arg Ile Leu Glu Phe Phe 275 280 285 Gly Leu Lys Lys Glu Glu Cys Pro Ala Val Arg Leu Ile Thr Leu Glu 290 295 300 Glu Glu Met Thr Lys Tyr Lys Pro Glu Ser Glu Glu Leu Thr Ala Glu 305 310 315 320 Arg Ile Thr Glu Phe Cys His Arg Phe Leu Glu Gly Lys Ile Lys Pro 325 330 335 His Leu Met Ser Gln Glu Leu Pro Glu Asp Trp Asp Lys Gln Pro Val 340 345 350 Lys Val Leu Val Gly Lys Asn Phe Glu Asp Val Ala Phe Asp Glu Lys 355 360 365 Lys Asn Val Phe Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys 370 375 380 Gln Leu Ala Pro Ile Trp Asp Lys Leu Gly Glu Thr Tyr Lys Asp His 385 390 395 400 Glu Asn Ile Val Ile Ala Lys Met Asp Ser Thr Ala Asn Glu Val Glu 405 410 415 Ala Val Lys Val His Ser Phe Pro Thr Leu Lys Phe Phe Pro Ala Ser 420 425 430 Ala Asp Arg Thr Val Ile Asp Tyr Asn Gly Glu Arg Thr Leu Asp Gly 435 440 445 Phe Lys Lys Phe Leu Glu Ser Gly Gly Gln Asp Gly Ala Gly Asp Asp 450 455 460 Asp Asp Leu Glu Asp Leu Glu Glu Ala Glu Glu Pro Asp Met Glu Glu 465 470 475 480 Asp Asp Asp Gln Lys Ala Val Lys As p Glu Leu 485 490

[Brief description of drawings]

FIG. 1A shows a construction process diagram of the expression plasmid pAHhPDILy1.

FIG. 1B shows a construction process diagram of the expression plasmid pAHhPDILy1.

FIG. 1C shows a construction process diagram of the expression plasmid pAHhPDILy1.

FIG. 2 is a diagram showing a boundary between HSA prepro sequence and PDI on a human expression plasmid.

[Fig. 5] Fig. 3 shows the expressed and secreted crude recombinant human PD.
1 is a photograph showing the results of SDS electrophoresis of I, in which lane 1 shows a molecular weight marker, lane 2 shows pAH / AH22 (control), and lane 3 shows pAHhPDILy1 / AH22.

FIG. 4 is a diagram showing separation of recombinant human PDI by hydrophobic column chromatography.

FIG. 7 is a diagram showing the results of SDS electrophoresis of purified recombinant human PDI. In the figure, the lower numbers represent the fraction numbers of the hydrophobic column chromatography shown in FIG.
M represents a molecular weight marker.

FIG. 6 shows human PDI in yeast HIS23.
2 is an electrophoretic photograph showing the expression of

FIG. 7: Human PDI in yeast HIS23
3 is an SDS-electrophoresis photograph showing HSA secretion by co-expression with and HSA.

FIG. 8 is a diagram showing the results of quantifying the amount of HSA secreted by a densitometer using the SDS electrophoretic gel of FIG. 7.

[Procedure amendment]

[Submission date] March 2, 1993

[Procedure Amendment 1]

[Document name to be amended] Statement

[Name of item to be corrected] Brief description of the drawing

[Correction method] Change

[Correction content]

[Brief description of drawings]

FIG. 1A shows the expression plasmid pAHhPD.
The construction process drawing of ILyl is shown.

FIG. 1B. Expression plasmid pAHhPD.
The construction process drawing of ILyl is shown.

FIG. 1C shows the expression plasmid pAHhPD.
The construction process drawing of ILyl is shown.

FIG. 2 shows HSA on human expression plasmid.
It is a figure which shows the boundary part of a prepro sequence and PDI.

FIG. 3 is a photograph showing the results of SDS electrophoresis of crude recombinant human PDI expressed and secreted, wherein lane 1 is a molecular weight marker and lane 2 is pAH / AH2.
2 (control), lane 3 is pAHhPDILyl
/ A22 is shown.

FIG. 4 is a diagram showing separation of recombinant human PDI by hydrophobic column chromatography.

FIG. 5: SDS of purified recombinant human PDI
It is a figure which shows an electrophoresis result, In the figure, the lower number shows the fraction number of the hydrophobic column chromatography shown in FIG. 4, and M shows a molecular weight marker.

FIG. 6 shows human P in yeast HIS23.
It is an electrophoresis photograph which shows the expression of DI.

FIG. 7: Human P in yeast HIS23
SDS showing HSA secretion by co-expression of DI and HSA
It is an electrophoretic photograph.

FIG. 8 is a diagram showing the results of quantifying HSA secretion using a densitometer using the SDS electrophoretic gel of FIG.

[Procedure Amendment 2]

[Document name to be corrected] Drawing

[Correction target item name] All drawings

[Correction method] Change

[Correction content]

[Figure 3]

[Figure 4]

[Figure 5]

FIG. 1A

[Figure 6]

FIG. 1B

[FIG. 1C]

[Fig. 2]

[Figure 7]

[Figure 8]

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Internal reference number FI technical display location C12N 15/12 C12P 21/02 C 8214-4B // G01N 30/00 8310-2J (C12N 1 / 19 C12R 1: 865) (C12N 9/90 C12R 1: 865) (C12P 21/02 C12R 1: 865) (72) Inventor Masanori Suzuki 1-3-3 Nishitsurugaoka, Oimachi, Iruma-gun, Saitama Tonen Corporation Inside the research institute

Claims (15)

[Claims] 1. A ligated gene for expressing human protein disulfide isomerase, which comprises a DNA encoding a human serum albumin prepro sequence and a human protein disulfide isomerase gene. 2. -24th to +4 shown in SEQ ID NO: 2.
DN encoding a human serum albumin prepro sequence for expression of human protein disulfide isomerase, which comprises a nucleotide sequence encoding the 91st amino acid sequence
A connecting gene consisting of A and the human protein disulfide isomerase gene. 3. The base sequence shown in SEQ ID NO: 2
The ligated gene according to claim 2, wherein the ligated gene comprises the 1st to 1545th sequences. 4. A replicable expression vector capable of expressing the ligated gene according to any one of claims 1 to 3 in a host. 5. A transformant obtained by transforming a host with the expression vector according to claim 4. 6. The transformant according to claim 5, wherein the host is yeast. 7. A method for producing a recombinant human protein disulfide isomerase, which comprises expressing the ligated gene according to any one of claims 1 to 3 in the transformant according to claim 5 or 6. 8. A replicable expression vector capable of expressing the ligated gene according to any one of claims 1 to 3 in a host is constructed, and the host is transformed with the expression vector to obtain a transformant. 8. The method, wherein the transformant is cultured under the condition that the linked gene can be expressed to secrete recombinant human protein disulfide isomerase, and the recombinant human protein disulfide isomerase is recovered. The method described. 9. The secreted recombinant human protein disulfide isomerase is separated and recovered by hydrophobic column chromatography.
The method described. 10. The first to the third sequences shown in SEQ ID NO: 3 obtained by the method according to any one of claims 7 to 9.
A recombinant human protein disulfide isomerase consisting of the amino acid sequence at position 491. 11. A transformant comprising the ligated gene according to any one of claims 1 to 3 capable of coexpression and a foreign gene encoding a polypeptide to be produced. 12. The transformant according to claim 11, which is a transformed yeast. 13. The transformant according to claim 11, wherein the foreign gene is a gene encoding human serum albumin. 14. The polypeptide according to any one of claims 11 to 13, wherein the human protein disulfide isomerase gene and a foreign gene encoding a polypeptide to be produced are co-expressed in the transformant. And a method for producing a polypeptide, which comprises recovering the polypeptide. 15. The method of claim 14, wherein the polypeptide is human serum albumin.