JP3012908B2 - Fusion protein of dihydrofolate reductase-antiallergic pentapeptide multimer (I) - Google Patents

Description

The present invention relates to dihydrofolate reductase (hereinafter abbreviated as DHFR) and aspartic acid (Asp) -serine (Ser) -aspartic acid which is an antiallergic peptide A fusion protein (hereinafter, fusion protein) containing a multimer having a repeating unit of a peptide (hereinafter abbreviated as DSDGK) having five amino acid sequences of (Asp) -glycine (Gly) -lysine (Lys) as arginine as an intervening amino acid (Abbreviated as (I).)
Recombinant plasmid which enables large-scale production of E. coli, Escherichia coli containing the plasmid, DHFR-DSDGK multimeric fusion protein (I), method for producing DHFR-DSDGK multimeric fusion protein (I), production of DSDGK multimer It relates to the production method and the production method of DSDGK. The present invention is effectively used in fields such as fermentation engineering and the pharmaceutical industry.

[Conventional technology]

DSDGK is a type of anti-allergic peptide, and is expected to be used as a preventive and therapeutic agent for allergic rhinitis, allergic dermatitis, bronchial asthma and other allergic diseases caused by chemical mediators It is an interesting bioactive peptide (Japanese Patent Laid-Open No. 1-31).
No. 6398).

As a method for producing an oligopeptide having a small number of amino acids, a chemical synthesis method is generally known, but in recent years, a method which can be easily produced by a gene recombination technique has been developed. For example, Iwakura et al. Prepared a plasmid pTP70-1 capable of expressing DHFR of Escherichia coli in large amounts (JP-A-63-267276), and variously modified the nucleotide sequence encoding the vicinity of the carboxyl-terminal amino acid of DHFR. Thus, a useful polypeptide was produced in Escherichia coli in the form of a fusion protein with DHFR. Examples thereof include somatostatin (JP-A-1-144977, JP-A-1-144955) and bradykinin (JP-A-1-252289).

In addition, Iwakura and colleagues aimed to increase the efficiency of DHFR production by using the rrnB gene, which is known as an efficient
A recombinant plasmid pTH104-4 was prepared using H70-1 (Japanese Patent Laid-Open No. 1-144979), and a recombinant plasmid pLEK1 capable of expressing a DHFR-leucine enkephalin fusion protein was also prepared using the recombinant plasmid. ing. Since the fusion protein obtained here also retains the enzyme activity of DHFR, its purification is easily carried out by utilizing the properties and activity of DHFR. The target peptide is obtained by subjecting the fusion protein to bromocyan treatment or trypsin treatment and then purifying it by HPLC (Japanese Patent Laid-Open Publication No.
252290).

In these examples, the promoter region and the terminator region are modified in order to increase the production amount of the target polypeptide, but a polypeptide in which a plurality of the target polypeptides are linked in tandem is produced. It is also performed (Japanese Patent Application Laid-Open No. 61-31090).

[Problems to be solved by the invention]

An object of the present invention is to produce DSDGK in a large amount by a genetic engineering technique.For this purpose, an attempt is made to produce an effective plasmid incorporating a gene encoding a multimer having DSDGK as a repeating unit. is there. This plasmid must satisfy the following requirements.

(I) that it can be produced as a fusion protein (I) containing DSDGK repeatedly; (ii) that it expresses a large amount of the fusion protein (I) gene; and (iii) that the fusion protein (I) that is produced ) Is easy to purify. (Iv) It is easy to separate DSDGK from the fusion protein (I). [Means for Solving the Problem] Using the dihydrofolate reductase gene of Escherichia coli, we found that the above problems could be solved, and according to the findings, we found that DHFR and DSDGK
The present invention was completed by preparing a recombinant plasmid into which a gene encoding a fusion protein (I) consisting of a multimer of the above was incorporated.

That is, the present invention provides a set comprising a base sequence encoding a (dihydrofolate reductase)-(aspartic acid-serine-aspartic acid-glycine-lysine) n fusion protein (n is 2 to 10) having the following amino acid sequence: On the replacement plasmid.

(Wherein _ represents an intervening amino acid Arg) In the fusion protein (I) of dihydrofolate reductase-antiallergic pentapeptide multimer in the recombinant plasmid, the fusion protein wherein the antiallergic pentapeptide is a dimer (I) has the following amino acid sequence.

(Wherein _ represents an intervening amino acid between dihydrofolate reductase and an anti-allergic pentapeptide) Examples of the base sequence encoding the fusion protein (I) in which the anti-allergic pentapeptide is a dimer are as follows: Base sequence.

(In the formula, _ indicates a nucleotide sequence encoding an amino acid that mediates dihydrofolate reductase and antiallergic pentapeptide.) Further, fusion of dihydrofolate reductase-antiallergic pentapeptide multimer in the above-mentioned recombinant plasmid The fusion protein (I) in which the anti-allergic pentapeptide is a trimer in the protein (I) has the following amino acid sequence.

(Wherein _ has the same meaning as described above) Examples of the base sequence encoding the fusion protein (I) in which the anti-allergic pentapeptide is a trimer include the following base sequences.

(Wherein _ has the same meaning as described above.) Further, the above-mentioned antiallergic pentapeptide is a dimer, and the recombinant plasmid containing the nucleotide sequence encoding the fusion protein (I) is stably replicated in E. coli. ,
Escherichia coli which confers trimethoprim resistance and ampicillin resistance to the host and includes those having a size of 4655 base pairs.

Furthermore, the recombinant plasmid containing the nucleotide sequence encoding the fusion protein (I) in which the anti-allergic pentapeptide is a trimer is stably replicated in Escherichia coli and has trimethoprim resistance and ampicillin resistance in the host Escherichia coli. And having a size of 4670 base pairs.

The above-mentioned recombinant plasmid includes the recombinant plasmid pBBK7DA having the nucleotide sequence shown in FIG.

Further, the above-mentioned recombinant plasmid includes a recombinant plasmid pBBK5.6TA having the nucleotide sequence shown in FIG.

Furthermore, the present invention resides in Escherichia coli transformed with the above recombinant plasmid.

The present invention further relates to a dihydrofolate reductase-Arg-antiallergic pentapeptide dimer fusion protein having the following amino acid sequence:

(Wherein _ has the same meaning as described above.) Further, the present invention relates to a fusion protein of dihydrofolate reductase-Arg-antiallergic pentapeptide trimer having the following amino acid sequence.

(Wherein _ has the same meaning as described above.) The present invention further comprises culturing the above-mentioned transformed Escherichia coli and, from the cultured cells, the above-mentioned dihydrofolate reductase-antiallergic pentapeptide dimer fusion protein (I) Or a method for producing a dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (I), which comprises collecting a dihydrofolate reductase anti-allergic pentapeptide trimer fusion protein (I). is there. The step of collecting the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (I) comprises:
The method comprises a step of sequentially purifying from a cell-free extract of the cultured cells using streptomycin treatment, ammonium sulfate precipitation, and mesotrixate binding affinity chromatography.

The present invention further provides an anti-allergic pentapeptide comprising an amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine by enzymatically treating the fusion protein (I) of dihydrofolate reductase-antiallergic pentapeptide multimer. Is to collect.

And as an enzyme used for the above-mentioned enzyme treatment, trypsin can be mentioned.

The details of the present invention are described below in (1) Preparation of new recombinant plasmid, (2) transformed E. coli, (3) DHFR-DSDG
A process for producing the K multimer fusion protein (I), (4) the obtained DHFR-DSDGK multimer fusion protein (I),
(5) DSDGK multimer production process and (6) DSDGK production process will be described in this order.

(1) Preparation of New Recombinant Plasmid i) Preparation of DNA Encoding DSDGK Multimer Preparation of DNA encoding DSDGK multimer is performed by chemically synthesizing single-stranded DNA and annealing both strands.
When the target double-stranded DNA is long, it may be prepared by dividing it into several pieces on the way.

The DNA encoding the DSDGK multimer has cohesive ends linked to the vector at both ends, and arginine, an intervening amino acid for cleaving the bond between DHFR and the DSDGK multimer, is inserted between the DHFR coding region and the closer one. Base sequence to encode,
It is a structure containing a base sequence encoding a DSDGK multimer and a base sequence that directs the termination of translation.

The sticky end is the base sequence of the cut surface generated by the action of the restriction enzyme, and when using plasmid pMEK2, D
It is convenient to use a cut surface by BamHI at the end closer to the HFR coding region and a cut surface by XhoI at the opposite end.

Intervening amino acids between DHFR and DSDGK multimers include:
For example, those having arginine at the carboxy terminus such as arginine, isoleucine-glutamic acid-glycine-arginine [recognition sequence of factor tene (FXa)] can be used, but arginine is preferable.

The polymerization degree of the multimer of DSDGK is preferably from a dimer to a decamer. In addition, as specific examples described later, dimers and trimers are exemplified.

ii) Insertion of DNA encoding DSDGK multimer into vector plasmid As a vector for expressing DNA encoding DSDGK multimer, a vector capable of expressing DHFR is used.
An example thereof is the recombinant plasmid pMEK2 (see JP-A-1-252289) already prepared by Iwakura et al. pME
K2 is stably retained in E. coli, and E. coli containing pMEK2 has been deposited with the Japan Institute of Fine Arts and Chemicals as FERM BP-1816. p
MEK2 can be separated and purified from E. coli containing pMEK2 in accordance with a usual plasmid separation method and used. The double-stranded DNA of the above i) was ligated to a DNA fragment obtained by digesting the purified pMEK2 with BamH I and Xho I using T4-DNA ligase, and the desired fusion protein (I) was transformed into E. coli cells. A new recombinant plasmid that can be expressed is obtained.

iii) Base sequence of novel plasmid FIG. 1 shows a novel recombinant plasmid pB which is an example of the present invention.
The entire nucleotide sequence of BK7DA is shown. Of the double-stranded circular DNA, only the base sequence of one of the DNA strands that encodes the genetic information is used as the only cleavage site for the restriction enzyme ClaI present only in the plasmid, 5'-ATCGAT-3 'and the first "A "Is counted as number 1 and described in the direction from the 5 'end to the 3' end. pBBK7DA is 4655 base pairs in size and can confer trimethoprim and ampicillin resistance to the host Escherichia coli. pBBK7DA is a 41 base pair containing the larger DNA fragment (4614 base pairs) obtained by digesting pMEK2 (4640 base pairs) with BamH I and Xho I and the DSDGK dimer. Has a structure in which chemically synthesized DNA is bound. In FIG. 1, the sequence from the 533rd position to the 573rd position is a sequence derived from chemically synthesized DNA.
Incidentally, the entire nucleotide sequence of pMEK2 has already been elucidated by the present inventors (see JP-A-1-252289), and the sequence from the 533th to the 573th of the nucleotide sequence shown in FIG. This is the structure replaced by the 26 base pair sequence shown in FIG.

The terminal sequence of the chemically synthesized double-stranded DNA contains restriction enzyme B
Sticky end generated upon cleavage with amHI, 5'-GATC-
3 ′ (sequences from 533 to 536 in FIG. 1) and sticky ends generated upon cleavage with the restriction enzyme Xho I,
3'-AGCT-5 '(sequence complementary to the sequence from position 574 to position 577 in FIG. 1). Since a portion derived from pMEK2 has a sticky end site generated by BamH I and Xho I cleavage, heterologous DNA can be introduced in a predetermined direction, and a fusion gene with the DHFR gene can be easily prepared.

The sequence from the 57th to the 569th in FIG. 1 encodes a DHFR-DSDGK dimer fusion protein (I) in which a DSDGK dimer is bound to the DHFR carboxyl terminal side via arginine.

Upstream of the sequence encoding the DHFR-DSDGK dimer fusion protein (I), there is a sequence that allows efficient expression of the DHFR-DSDGK dimer fusion protein (I) gene (Japanese Patent Application Laid-Open (JP-A) 63). -267276).

Immediately, the 43rd to 50th sequence is called the SD sequence, which is used for efficient translation.
The second is the consensus transcription promoter, which contributes to efficient transcription. Therefore, when introduced into E. coli, pBBK7DA produces a large amount of DHFR-DSDGK dimer fusion protein (I). The produced DHFR-DSDGK dimer fusion protein (I) accumulates about 20% of the bacterial cell protein in a soluble state in the bacterial body. Trimethoprim is an inhibitor of this DHFR, but Escherichia coli having pBBK7DA accumulates a large amount of the DHFR-DSDGK dimer fusion protein (I) in the cells, and thus becomes resistant to trimethoprim.

FIG. 2 shows the entire nucleotide sequence of a novel recombinant plasmid pBBK56TA which is another example of the present invention. As in FIG. 1, only the base sequence of one of the double-stranded circular DNAs encoding the genetic information is recognized by the restriction cleavage site of the restriction enzyme Cla I, which is present only in the plasmid, and 5'-ATCGAT. −
The first "A" of 3 'is counted as number 1 and described from the 5' end to the 3 'end. pBBK56TA is 4670 base pairs in size and BamH of pMEK2 (consisting of 4640 base pairs).
The larger DNA fragment (4
Consists of 614 base pairs. ) And a 59 bp chemically synthesized DNA containing a base sequence encoding the DSDGK trimer. In FIG. 2, the sequence from the 533rd position to the 588th position is a sequence derived from chemically synthesized DNA.

Sequences 57 to 584 in FIG. 2 encode a DHFR-DSDGK trimer fusion protein (I) in which a DSDGK trimer is bound to the carboxyl terminal side of DHFR via arginine.

pBBK56TA contains a large amount of DHFR-
Other than producing DSDGK trimer fusion protein (I)
It has the same features as BBK7DA.

pBBK7DA and pBBK56TA can be prepared according to Examples 1 and 2, respectively, but the present invention is not limited by the method for preparing a recombinant plasmid.

(2) Transformed Escherichia coli The recombinant plasmid prepared in the above (1) is incorporated into Escherichia coli according to a conventional method. Transformed E. coli show resistance to trimethoprim and ampicillin. Escherichia coli containing this plasmid, DHFR-
As a result of efficient expression of the DSDGK multimeric fusion protein (I) gene, a large amount of the DHFR-DSDGK multimeric fusion protein (I) is accumulated in a soluble state in the cells. Escherichia coli containing this plasmid was transformed into YT + Ap medium (5 g of NaCl, 8 g of tryptone, 5 g of yeast extract,
And a liquid medium containing 100 mg of sodium ampicillin. ), The DHFR-DSDGK multimeric fusion protein (I) accumulates to about 20% of the bacterial cell protein when cultured at 37 ° C. until the stationary phase. When the cultured cells are suspended in an appropriate buffer such as a phosphate buffer and crushed by a French press method or an ultrasonic crushing method, and this is separated into a supernatant and a precipitate by centrifugation, almost all DHFR −DSDG
The K multimeric fusion protein (I) is recovered in the supernatant. Escherichia coli containing pBBK7DA among the novel recombinant plasmids is described in Microtechnological Research Institute No. 2391 (FERM BP-2391).
And E. coli containing pBBK56TA have been deposited as Microtechnical Research Institute Deposit No. 2390 (FERM BP-2390), respectively.

(3) DHFR-DSDGK Multimer Fusion Protein (I) Production Step The DHFR-DSDGK multimer fusion protein (I) of the present invention
The production process comprises: i) culturing the cells, ii) disrupting the cells, ii)
i) streptomycin treatment, iv) ammonium sulfate precipitation, and v)
It consists of 5 steps of mesotrixate (MTX) binding affinity chromatography.

i) Culture of bacterial cells Culture of Escherichia coli containing the plasmid of the present invention is performed using YT +
It can be cultured in an Ap medium (a liquid medium containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 50 mg of sodium ampicillin in 1 L of medium). As the medium, in addition to this, bacterial cells such as ST + Ap medium (a liquid medium containing 2 g of glucose, 1 g of disodium phosphate, 5 g of polypeptone, 5 g of yeast extract and 50 mg of ampicillin sodium in 1 L of medium). Any medium can be used as long as it is a growth medium, but YT + Ap medium is desirable for production of the DHFR-DSDGK multimeric fusion protein (I). Escherichia coli containing the plasmid of the present invention is inoculated into a medium, and cultured at 37 ° C. until late log phase or stationary phase. The accumulation amount of the fusion protein (I) of the DHFR-DSDGK multimer in the cells varied depending on the culture temperature. As far as the examination was conducted, the accumulation amount was larger as the culture temperature was higher. The cultured cells are collected by centrifugation at 4,700 × g. Medium 1L
Yields 2 to 4 g of wet cells. It is preferable to perform the collection and subsequent operations at a low temperature (4 ° C. is desirable between 0 ° C. and 10 ° C.) unless otherwise specified.

The bacterial cells obtained by crushing the culture in ii) bacteria, per culture medium 1L, the buffer 1 [0.mM disodium ethylenediaminetetraacetate (EDTA · Na ₂₎ and 14 mM 2-mercaptoethanol at a rate of 10m Containing 10 mM potassium phosphate buffer (pH 7.0)], and disrupt the cells using a French press or an ultrasonic disrupter. The cell lysate is centrifuged at 27,000 × g for 20 minutes to obtain a supernatant (hereinafter, referred to as a cell-free extract).

iii) Streptomycin treatment 1.5% (W / V) streptomycin sulfate is slowly added to the cell-free extract while stirring with a stirrer.
After stirring for 20 minutes, the solution treated with streptomycin was
Centrifuge at 00 × g for 20 minutes to obtain a supernatant.

iv) Ammonium sulfate precipitation 0.8 to 0.9 with respect to 1 volume of the supernatant obtained by the above operation.
Of saturated ammonium sulfate solution is added slowly with stirring with a stirrer. After stirring for 20 minutes, the solution subjected to the ammonium sulfate precipitation treatment is centrifuged at 27,000 × g for 20 minutes to obtain a supernatant.

v) MTX binding affinity chromatography To the supernatant obtained by the above operation, 25 m of MTX binding agarose gel (manufactured by Sigma) previously equilibrated with buffer 1 is added. After standing for 10 minutes to adsorb to the gel, the gel is packed into a column. The column was buffered with buffer 1, buffer 1 containing 1 M KCl, buffer 2 containing 1 M KCl [10 mM potassium phosphate buffer containing 0.1 mM disodium ethenediaminetetraacetate (EDTA.Na ₂ ), 14 mM 2-mercaptoethanol] Liquid (pH
8.5). ] In order. The elution of the fusion protein (I) of the DHFR-DSDGK multimer was carried out using a solution of buffer 1 containing 1 M KCl and 3 mM folic acid adjusted to pH 9.3 with 2N NaOH,
The eluate is fractionated by a fixed amount using a fraction collector. The DHFR activity of the fractionated eluate is measured, and the fraction containing the enzyme activity is collected. The resulting solution is dialyzed against buffer 1. At this stage, DH with 90% or more purity
The fusion protein (I) of FR-DSDGK multimer is obtained.

By the above operation, the fusion protein (I) of the DHFR-DSDGK multimer can be purified with good reproducibility. According to the present invention, the purification of the DHFR-DSDGK multimeric fusion protein (I) can be performed within 3 days including culturing, and a uniform protein preparation can be obtained with a recovery of about 80%. .

The DHFR enzyme activity was measured using a reaction solution [50 mM potassium phosphate buffer (pH 7.0) containing 0.05 mM dihydrofolate, 0.06 mM NADPH, and 12 mM 2-mercaptoethanol. ] 1m
In a cuvette, add the sample to it, 340 nm at 25 ° C.
The measurement is performed by measuring the change in absorbance with time. Enzyme activity is expressed in units, and the amount of enzyme required to reduce 1 micromol of dihydrofolate per minute under the above reaction conditions is defined as 1 unit. This measurement can be easily performed using a spectrophotometer.

(4) The obtained DHFR-DSDGK multimer fusion protein (I) FIG. 3 shows an example of the DHFR-DSDGK multimer fusion protein (I) as a DHFR-DSDGK dimer fusion protein contained in pBBK7DA DNA of the part encoding (I)
The sequence and the amino acid sequence of the protein made therefrom are shown. DHFR-DSDGK dimer fusion protein (I)
Is a novel protein consisting of 171 amino acids. From the amino terminal side, the sequence from the 1st to the 159th is
This is a [Cys-152 (wild) → Glu-152] sequence in which one amino acid substitution has occurred in the wild-type DHFR of Escherichia coli. The 162nd to 171st positions are fusion proteins of the DSDGK dimer (I).
Is an array of The 160th isoleucine (Ile) is pMEK
This is a sequence generated to match the reading frame of genetic information when a gene encoding a DSDGK dimer is introduced into the BamHI site of No. 2. The 161st amino acid, the amino acid immediately before the sequence of the DSDGK dimer, is arginine (Arg). Therefore, the DSDGK dimer can be cut out by treating the DHFR-DSDGK dimer fusion protein (I) with arginyl endopeptidase. DHFR-DSD
The molecular weight of the GK dimer fusion protein (I) is 19,299.

FIG. 4 shows DHFR-D contained in pBBK5.6TA as another example of the fusion protein of DHFR-DSDGK multimer (I).
The DNA sequence of the portion encoding the fusion protein (I) of the SDGK acid mer and the amino acid sequence of the protein produced therefrom are shown. DHFR-DSDGK trimer fusion protein (I) is a novel protein consisting of 176 amino acids. The sequence from position 1 to position 171 counted from the amino terminal side is the fusion protein of the DHFR-DSDGK dimer (I)
DHFR-DSDGK trimer fusion protein (I) has the same structure as DHFR-DSDGK dimer fusion protein (I) with another DSDGK bound to the carboxyl terminal side of DHFR-DSDGK dimer fusion protein (I). The molecular weight of the DHFR-DSDGK trimer fusion protein (I) is 19,801.

The DHFR-DSDGK dimer fusion protein (I) and the DHFR-DSDGK trimer fusion protein (I)
At the carboxy-terminal side of
Despite having a structure in which a DSDGK dimer and a DSDGK trimer are bound, it has DHFR enzyme activity. For this reason, Escherichia coli was transformed with DHFR-DSDGK dimer fusion protein (I) or DHF.
When a large amount of R-DSDGK trimer fusion protein (I) is produced, it becomes resistant to trimethoprim, an inhibitor of DHFR.

(5) Production process of DSDGK multimer In the above-mentioned (4) DHFR-DSDGK multimer fusion protein (I), the amino acid immediately before the DSDGK multimer is arginine (Arg). When the DHFR-DSDGK multimeric fusion protein (I) is treated with arginyl endopeptidase, the DSDGK multimer is cut out from the DHFR-DSDGK multimeric fusion protein (I). The DSDGK multimer is obtained by purifying the enzyme-treated degradation product by HPLC.

(6) DSDGK production process The DSDGK multimer obtained in the above (5) is treated with trypsin or treated with lysyl endopeptidase.
Get. In addition, it is also possible to directly obtain DSDGK without passing through the DSDGK multimer by allowing trypsin to act on the fusion protein (I) of the DHFR-DSDGK multimer obtained in (4) and purifying the degradation product by HPLC. .

〔Example〕

Next, the present invention will be described in more detail by way of examples. However, the present invention is not limited by these examples.

(Example 1) Preparation of pBBK7DA DNA encoding DSDGK dimer includes Is chemically synthesized using a chemical synthesizer (Milligen, Model 7500) according to the phosphomidide method, and purified with an oligonucleotide purification cartridge (Applied Biosystems).
2 m (containing about 0.1 μg of DNA) was added thereto, and the mixture was added to a reaction solution for kaination (50 mM Tris-HCl pH 7.8, 10
mM MgCl ₂ , 5 mM dithiothreitol, 0.5 mM ATP)
μ and 1 μ of T4 polynucleotide kinase (10 units / μ, manufactured by Takara Shuzo) were added, and the mixture was incubated at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end. This was once incubated at 80 ° C. and then gradually cooled to anneal both DNAs, thereby obtaining the following double-stranded DNA (hereinafter, referred to as DNA1).

About 1 μg of the purified pMEK2 was cut with 20 units of BamHI (Takara Shuzo) and 20 units of XhoI (Takara Shuzo), and then separated by 0.85% agarose gel electrophoresis. Cut out the gel containing the DNA fragment of about 4.6 kilobase pairs,
The DNA was recovered from the gel (this is hereinafter referred to as DNA2). B
The operation of DNA cleavage with amH I and Xho I is described in “Molecular
Cloning: A Laboratory Manual "[T. Maniatis, EFFrit
sch, J. Sambrook, eds. Cold Spring Harbor Laboratory
(1982). ] Was carried out according to the method described in the above. all
20 μl of ligase reaction solution (10 mM Tris
-HCl pH7.4,5mM MgCl _2, 10mM dithiothreitol, 5m
MATP), 10 μl of DNA1 and 1 μl of T4-DNA
Add ligase (Takara Shuzo 300 units / μ)
The DNA was ligated at room temperature for 19 hours. This reaction product was subjected to a transformation method (Maniati, supra).
s et al. ), E. coli (Takara Shuzo Ec)
oli HB101 competent cell). The treated cells were added to a nutrient agar medium (1 L of medium) containing 100 mg / ampicillin sodium and 5 mg / trimethoprim.
Contains 2 g glucose, 1 g dipotassium phosphate, 5 g yeast extract, 5 g polypeptone, and 15 g agar. ) By incubating at 37 ° C for 24 hours
Dozens of colonies could be obtained. Six of these colonies were selected from 2 m of YT + Ap medium (5 g of medium in 1 L of medium).
NaCl, 8g tryptone, 5g yeast extract, and 100m
Contains g of ampicillin sodium. ) At 37 ° C overnight. Each of the culture solutions is placed in an Eppendorf centrifuge tube (15 m capacity), and centrifuged by an Eppendorf centrifuge (5
414S) and centrifuged at 12,000 rpm for 10 minutes, and the cells were collected as a precipitate. To this, 0.1m sample preparation solution for electrophoresis [0.0709M Tris-HCl pH6.8, 2%
(W / V) sodium lauryl sulfate (SDS), 11% (V / V
V) glycerin, 5% (V / V) 2-mercaptoethanol, and 0.045% (W / V) bromophenol blue. Was added, and the cells were suspended. The cells were kept in boiling water for 5 minutes to dissolve the cells. The sample processed this way is SD
S-polyacrylamide gel electrophoresis [UKLaemmli; N
ature, vol.227, p680 (1970). ]. Lactalbumin (molecular weight 14,20)
0), trypsin inhibitor (molecular weight 20,100), trypsinogen (molecular weight 24,000), carbonic anhydrase (molecular weight 29,000), glyceraldehyde-3-phosphate dehydrogenase (molecular weight 36,000), egg albumin (molecular weight 45,000), and bovine serum albumin (Molecular weight 66,0
Using the mixture (00) (DALTON MARK VII-L manufactured by Sigma), the mixture was electrophoresed on a concentration gradient gel (manufactured by Daiichi Kagaku) having an acrylamide concentration of 10 to 20%. As a result, it was revealed that a protein having an estimated size was produced in one of the six colonies. This colony was cultured in YT + Ap medium, and the method of Tanaka and Weisbium [T,
Tanaka, B. Weisblum; J. Bacteriology vol. 121, p. 354 (19
75). And the plasmid was prepared. The resulting plasmid was named pBBK7DA. pBBK7DA is the Bam of pMEK2
The sequence between the HI and Xho I sites should have replaced the synthetic DNA. The synthetic DNA contains the restriction enzyme Xho I
Since the sequence 5'-CTCGAC-3 ', which is recognized and cleaved, is included, the cleavage of pBBK7DA with XhoI was successful. In addition, EcoRI of pBBK7DA (471 in FIG. 1)
The DNA of about 400 nucleotides obtained by digestion with Sal- (sequences 872-877 in FIG. 1) and Sal I (sequences 872-877 in FIG. 1) was subjected to a dideoxy method using M13 phage [J. Messing; , vol.101, p20 (198
3). ], The nucleotide sequence was determined in the direction from EcoR I to Sal I. As a result, the sequence from the 471st position to the 872th position in the entire base sequence of pBBK7DA shown in FIG. 1 was confirmed.

Also, about 4.6 kilobase pairs of DNA obtained by BamHI-XhoI digestion of pBBK7DA is EcoR I, Pst I, Hind III, Hpa
I, Pvu II, Bgl II, Cla I (these are Takara Shuzo) and Aat
As a result of a cleavage experiment using a restriction enzyme using II (manufactured by Toyobo Co., Ltd.), about 4.
It was shown to be exactly the same as 6 kilobase pairs.

From the above results, the entire nucleotide sequence of pBBK7DA was determined as shown in FIG.

(Example 2) DHFR-DSDGK dimer fusion protein produced by E. coli containing pBBK7DA (I) The amino acid sequence of the DHFR-DSDGK dimer fusion protein (I) produced by E. coli containing pBBK7DA is DHFR- DSDGK
This can be predicted from the nucleotide sequence of the dimeric fusion protein (I) gene. Since the sequence from the 57th position to the 569th position in FIG. 1 encodes the DHFR-DSDGK dimer fusion protein (I), the amino acid sequence was estimated using the triplet code table. As a result, the amino acid sequence shown in FIG. 3 was obtained. Then, using E. coli containing pBBK7DA, DHFR-DSDGK dimer fusion protein (I) was separated and purified and identified as follows.

Purification of DHFR-DSDGK dimer fusion protein (I) A. Bacterium amount used: 15.9 g / 3 L wet culture B. Enzyme purification step: shown in Table 1 below (in the table, cell-free extract, Represents streptomycin treatment, and represents mesotrixate-bound affinity chromatography.

To measure the DHFR enzyme activity, 1 m of the reaction solution [0.05 mM dihydrofolate, 0.06 mM NADPH, 12 mM 2-mercaptoethanol, 50 mM potassium phosphate buffer (pH 7.0)] was placed in a cuvette, and the sample was placed in the cuvette. In addition, the measurement was performed by measuring the time change of the absorbance at 340 nm at 25 ° C. Enzyme activity is expressed in units and is 1 minute per minute under the above reaction conditions.
The amount of enzyme required to reduce micromolar dihydrofolate was defined as one unit.

The obtained DHFR-DSDGK dimer fusion protein (I)
Was subjected to SDS-polyacrylamide gel electrophoresis (Example 1).
The method described in. ) Showed approximately 22,000 single protein bands and the resulting DHFR-DSDGK
The dimeric fusion protein (I) preparation was shown to be homogeneous.

(Example 3) Separation of DSDGK dimer from fusion protein (I) of purified and separated DHFR-DSDGK dimer 31 ml of purified and homogenized DHFR-DSDG obtained in Example 2
The solution of the K dimer fusion protein (I) was concentrated with an ultrafiltration membrane (YM5, 25 mm in diameter, manufactured by Amicon), dialyzed against EDTA · Na ₂ -free buffer solution 1, and treated with 6 m DHFR-DSDGK dimer. A body fusion protein (I) concentrate was obtained. This is 27.6mg / m
Of DHFR-DSDGK dimer at different concentrations (I)
Was included. 20 μl of the DHFR-DSDGK dimer fusion protein (I) concentrate was added to a 1 M Tris-HCl buffer (pH 8.
0) 10 μ, purified water 130 μ, 1 unit / m of arginyl endopeptidase (Takara Shuzo) 60 μ were added, and 37
Incubated overnight at ° C. After the reaction, this reaction solution 25μ
And a high-performance liquid chromatography system (Hitachi 655
YMC-ODS-5 column (diameter 4.6, manufactured by Yamamura Chemical Co., Ltd.)
× 250 mm). Elution was performed by applying a gradient of acetonitrile (0% to 10%) in 0.1% trifluoroacetic acid. 0% acetonitrile from 0 to 5 minutes, 0% to 10% from 5 minutes to 35 minutes
A linear gradient of acetonitrile was applied. as a result,
An elution curve as shown in FIG. 5 was obtained. Approximately after sample injection
The peak fraction after 28 minutes was separated, and a small amount of water was added to the separated eluate, followed by lyophilization to remove the solvent and obtain a peptide. After the obtained peptide was subjected to acid hydrolysis, one-fourth of the peptide was used for amino acid analysis. As a result, aspartic acid was 1.8 nm
ole, serine, glycine, and lysine are each 0.9 n
Each mole was detected. Amino acid composition was consistent with that of the DSDGK dimer. From these results, the purified and homogenized DHFR-
The DSDGK dimer fusion protein (I) was treated with arginine endopeptidase and then separated using high performance liquid chromatography, which revealed that the DSDGK dimer can be recovered with a yield of about 19%. DHFR-DSDGK
The purification yield of the dimeric fusion protein (I) is about 43% and the separation of the DSDGK dimer from the DHFR-DSDGK dimer fusion protein (I) is about 19%. From that
The isolation yield of DSDGK from the cell-free extract was about 8%.

Example 4 Separation of DSDGK from Separated and Purified DHFR-DSDGK Dimer Fusion Protein (I) 20 μl of the DHFR-DSDGK Dimer Fusion Protein (I) Concentrate Used in Example 3 was Eppendorf Centrifuge Tube (1.5m
), And 80 μl of acetone was added thereto. Then, the mixture was placed in a freezer at -20 ° C for 30 minutes to precipitate the DHFR-DSDGK dimer fusion protein (I). This was centrifuged at 12,000 rpm for 2 minutes using an Eppendorf centrifuge (Model 5414S) to remove the supernatant, and 10 µM of 1 M Tris-HCl buffer (pH 8.0), 90 µP was purified. Water, 10μ
Of TPCK-trypsin (Sigma) was added, and the mixture was incubated at 37 ° C. overnight. After the reaction, 10 μm of this was taken and a YMC-ODS-5 column (Yamamura Chemical Co., 4.6 × 250
mm). Elution was performed under the same conditions as in Example 3. As a result, an elution curve as shown in FIG. 6 was obtained. The peak about 8 minutes after sample injection is DSDGK. Using the DSDGK solution of known concentration obtained by chemical synthesis as a standard, the DSDG obtained from the peak height of the chromatogram was used.
When the amount of K was determined, it was 1.8 μg. From this, the recovery rate when DSDGK is obtained by trypsinizing the fusion protein (I) of the DHFR-DSDGK dimer is calculated to be about 69%.

(Example 5) Preparation of pBBK56TA As DNA encoding DSDGK trimer, Were chemically synthesized using a chemical synthesizer (Model 7500, manufactured by Milligen) according to the phosphoramidite method, and purified using an oligonucleotide purification cartridge (manufactured by Applied Biosystems). DNA). 180 µl of a reaction solution for chiration (50 mM Tris-HCl pH 7.8, 10 mM MgCl ₂ , 5 mM dithiothreitol, 0.5 mM ATP) and T4 polynucleotide kinase (Takara Shuzo) 10 units / μ), and incubated at 37 ° C. for 30 minutes.
The 5 'end was phosphorylated. This was once incubated at 80 ° C. and then gradually cooled to anneal both DNAs to obtain the following double-stranded DNA (hereinafter referred to as DNA
Call it 3. ).

To 10 µ of DNA3, 20 µ of DNA1, 20 µ of ligase reaction solution, and 1 µ of T4-DNA ligase (all of which are described in Example 1)
Described above) and ligation reaction of DNA was carried out at room temperature for 19 hours. Then, Escherichia coli was transformed, the transformant was cultured, and SDS-polyacrylamide gel electrophoresis using the cultured cells was performed according to the method described in Example 1.

As a result, production of a protein having a size estimated for four colonies was observed. Therefore, one of these colonies was cultured in YT + Ap medium, and
A plasmid was prepared according to the method of isblum. The resulting plasmid was named pBBK56TA. pBBK56TA is pMEK2
Should have a structure in which the sequence between the BamH I and Xho I sites has been replaced with synthetic DNA. Synthetic DNA contains restriction enzyme X
Since a sequence recognized and cleaved by hoI was included, 5'-CTCGAG-3 ', when pBBK56TA was cleaved with XhoI, it was indeed cleaved. In addition, EcoRI of pBBK56TA (second
Sequences 471-476 in the figure and Sal I (887-892 in FIG. 2)
The nucleotide sequence of the DNA of about 400 nucleotides obtained by the cleavage by the second sequence) was determined in the direction from EcoRI to SalI according to the dideoxy method using M13 phage. As a result, the sequence from the 471st position to the 887th position in the entire base sequence of pBBK56TA shown in FIG. 2 was confirmed.

Also, about 4.6 kilobase pairs of DNA obtained by BamHI-XhoI digestion of pBBK56TA is EcoR I, Pst I, Hind III, Hp
a I, Pvu II, Bgl II, Cla I (both from Takara Shuzo) and Aat
As a result of a cleavage experiment using a restriction enzyme using II (manufactured by Toyobo Co., Ltd.), about 4.
It was shown to be exactly the same as 6 kilobase pairs.

From the above results, the entire nucleotide sequence of pBBK56TA could be determined as shown in FIG.

(Example 6) DHFR-DSDGK trimer fusion protein produced by E. coli containing pBBK56TA (I) The amino acid sequence of DHFR-DSDGK trimer fusion protein (I) produced by E. coli containing pBBK56TA is DHFR- DSDG
This can be predicted from the nucleotide sequence of the K trimer fusion protein (I) gene. The sequence from position 57 to position 584 in FIG. 2 is a DHFR-DSDGK trimer fusion protein (I)
Therefore, the amino acid sequence was estimated using the triplet code table. As a result, the amino acid sequence shown in FIG. 4 was obtained. Then, using Escherichia coli containing pBBK56TA, DHFR-DSDGK trimer fusion protein (I) was separated and purified and identified as follows.

Purification of DHFR-DSDGK trimer fusion protein (I) A. Amount of bacterial cell used: 28.4 g / 3 L culture solution in wet weight B. Enzyme purification step: shown in Table 2 below. (In the table, cell-free extract, streptomycin treatment and mesotrixate binding affinity chromatography are shown.)

The method for measuring the DHFR enzyme activity and the definition of the activity follow Example 2.

The obtained DHFR-DSDGK trimer fusion protein (I)
Was subjected to SDS-polyacrylamide gel electrophoresis (Example 1).
The method described in. ) Showed approximately 21,500 single protein bands and the resulting DHFR-DSDGK
The trimeric fusion protein (I) preparation was shown to be homogeneous.

Example 7 Separation of DSDGK Trimer from Separated and Purified DHFR-DSDGK Trimer Fusion Protein (I) The 56 m purified DHFR-DSDGK trimer fusion protein (I) obtained in Example 6 ) Was concentrated using an ultrafiltration membrane (YM5, 25 mm in diameter, manufactured by Amicon) and 6.5 m of DHFR-DSD
A GK trimer fusion protein (I) concentrate was obtained. It contained the fusion protein (I) of the DHFR-DSDGK trimer at a concentration of 65 mg / m.

To 10 µ of the DHFR-DSDGK trimer fusion protein (I) concentrate, 10 µ of 1 M Tris-HCl buffer (pH 8.0), 140 µ of purified water, and 60 µ of arginyl endopeptidase (manufactured by Takara Shuzo) at 1 unit / m. In addition, the mixture was incubated at 37 ° C. overnight. After the reaction, take 30 µ of the reaction solution, and use a high-performance liquid chromatography device (Hitachi type 655) to prepare Y
Separation was performed using an MC-ODS-5 column (4.6 × 250 mm, manufactured by Yamamura Chemical Co., Ltd.). Elution was performed under the same conditions as in Example 3. As a result, an elution curve as shown in FIG. 7 was obtained. The peak fraction at about 34 minutes after sample injection was separated, a small amount of water was added to the separated eluate, and lyophilization was performed to remove the solvent and obtain a peptide. The obtained peptide was hydrolyzed with an acid,
Was used for amino acid analysis. As a result, aspartic acid
3.1 nmole, serine, glycine and lysine are each 1.6
n moles were detected. Amino acid composition was consistent with that of the DSDGK trimer. From this result, the purified DHFR-DSDG
The KDG trimer protein was treated with arginyl endopeptidase and then separated using high performance liquid chromatography, showing that the DSDGK trimer could be recovered with a yield of about 17%. The purification yield of the DHFR-DSDGK trimer fusion protein (I) is about 90%, and the yield of separation of the DSDGK trimer from the DHFR-DSDGK trimer fusion protein (I) is about 17%. % From the cell-free extract
The yield of DGK trimer is calculated to be about 15%.

Example 8 Separation of DSDGK from Separated and Purified DHFR-DSDGK Trimer Fusion Protein (I) 10 μl of DHFR-DSDGK Trimer Fusion Protein (I) Concentrate Used in Example 7 was Eppendorf Centrifuge Tube (1.5m
Volume), and 40 μl of acetone was added thereto. It was then placed in a freezer at -20 ° C for 30 minutes to precipitate the DHFR-DSDGK trimer fusion protein (I).

This was centrifuged at 12,000 rpm for 2 minutes using an Eppendorf centrifuge (model 5414S) to remove the supernatant, and 20 μl of 8 M urea was added to the precipitate to dissolve it. Further 80
μM 0.1M Tris-HCl buffer (pH 8.0), 10μ 0.5
% Trypsin (Difco) was added and incubated at 37 ° C overnight. After the reaction, take 20μ of the solution and use a high performance liquid chromatography system (Hitachi type 655) to perform YMC-
Separation was carried out using an ODS-5 column (4.6 × 250 mm, manufactured by Yamamura Chemical Co., Ltd.). Elution was performed under the same conditions as in Example 3. as a result,
An elution curve as shown in FIG. 8 was obtained. About 9 minutes after the sample injection, the peak fraction was separated, and after the separated elution, a small amount of water was added and lyophilized to remove the solvent to obtain a peptide. The obtained peptide was subjected to acid hydrolysis and then used for amino acid analysis of 1 / 1.6. As a result, 9.2 nmol of aspartic acid
e, serine, glycine and lysine were detected at 4.7 nmole each. Amino acid composition was consistent with that of DSDGK. From these results, the purified DHFR-DSDGK trimer fusion protein (I) was treated with trypsin, and then separated using high performance liquid chromatography, yielding about
It was found that 42% could recover DSDGK. DHFR-DSDGK
Since the yield of purification of the trimeric fusion protein (I) is about 90% and the yield of separation of DSDGK from the DHFR-DSDGK trimeric fusion protein (I) is 42%, The yield of DSDGK from the cell extract is calculated to be about 38%.

〔The invention's effect〕

As described above, the novel recombinant plasmid of the present invention comprises DH
E. coli harboring this plasmid encodes the fusion protein (I) of FR-DSDGK multimer.
The DGK multimeric fusion protein (I) is produced and accumulated in a large amount in a soluble state. Furthermore, the produced DHFR-DSDGK multimeric fusion protein (I) retains the DHFR enzyme activity and can be easily purified. According to the purification method of the present invention, the fusion protein (I) of DHFR-DSDGK multimer can be purified quickly and efficiently.
Alternatively, trypsin is directly allowed to act on the thus obtained DHFR-DSDGK multimeric fusion protein (I),
Alternatively, DSDGK multimers can be cut out with an appropriate enzyme such as arginyl endopeptidase, and then trypsin is allowed to act thereon, and DSDGK can be isolated and purified by high performance liquid chromatography. Purified DSDGK is useful as a therapeutic agent for allergic diseases. Therefore, the present invention has provided an advantageous means for producing DSDGK useful as a therapeutic drug for allergic diseases by gene manipulation technology.

[Brief description of the drawings]

FIG. 1 and FIG. 2 are diagrams showing the entire nucleotide sequences of pBBK7DA and pBBK56TA, respectively. Only the nucleotide sequence of one of the DNA strands encoding the genetic information of the double-stranded DNA is shown in FIG.
It is described in a direction from less than 5 'to less than 3'. The symbols in the figure represent nucleic acid bases, A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. The numbers in the figure indicate the recognition and cleavage sites of the restriction enzyme Cla I, which are present at one place each in pBBK7DA and pBBK56TA, 5′-ATCGAT-3 ′,
The first number "A" is counted as the first number. FIG. 3 and FIG. 4 show DHFR-DSDGK dimer fusion protein (I) present in pBBK7DA and pBBK56TA, respectively.
FIG. 1 shows the nucleotide sequence of the portion encoding the fusion protein (I) of DHFR-DSDGK trimer and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A represents adenine, C represents cytosine, G represents guanine, T represents thymine, Gly represents glycine, Ala represents alanine, Val represents valine, and Leu represents leucine. , Ile is isoleucine, Ser is serine, Thr is threonine, Cys is cysteine, Met is methionine, Asp is aspartic acid, Asn is asparagine, Glu is glutamic acid, Gln
Is glutamine, Arg is arginine, Lys is lysine,
His indicates histidine, Phe indicates phenylalanine, Tyr indicates tyrosine, Trp indicates tryptophan, and Pro indicates proline. The numbers in the figure indicate the numbers of methionine, which is the amino terminal amino acid of the fusion protein (I), as number 1. FIG. 5 and FIG. 7 show that DHFR-DSDGK dimer fusion protein (I) and DHFR-DSDGK trimer fusion protein (I) were treated with arginyl endopeptidase, respectively, and the reverse phase column was used. 3 shows a chromatogram obtained by separation with a high-performance liquid chromatography device. The horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units. Arrows in the figure are DSDGK dimer and DSD, respectively.
The position where GK trimer is eluted is shown. FIGS. 6 and 8 show DHFR-DSDGK dimer fusion protein (I) and DHFR-DSDGK trimer fusion protein (I), respectively.
Is a chromatogram obtained by treating a sample with trypsin and separating it with a high-performance liquid chromatography apparatus using a reversed-phase column. The horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units. Each arrow in the figure indicates the position where DSDGK is eluted.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C07K 1/22 C07K 19/00 7/06 C12N 1/21 19/00 9/02 C12N 1/21 C12P 21/02 C 9 / 02 A61K 37/02 C12P 21/02 37/50 // (C12N 1/21 C12R 1:91) (C12P 21/02 C12R 1:19) (72) Inventor Hiroshi Izutsu 48th Wadadai, Tsukuba, Ibaraki, Hitachi Kasei Kogyo Co., Ltd., Tsukuba Development Laboratory Co., Ltd. (72) Inventor Kazuhiko Ohara, 48, Wadai, Tsukuba, Ibaraki Prefecture Hitachi Chemical Co., Ltd. Tsukuba Development Laboratory Co., Ltd. (56) References JP-A-1-316398 (JP, A)

Claims (16)

(57) [Claims] 1. A fusion protein of (dihydrofolate reductase)-(aspartic acid-serine-aspartic acid-glycine-lysine) n having the following amino acid sequence, wherein n is 2
To 10). (Wherein _ indicates an intervening amino acid Arg) 2. The fusion protein according to claim 1, wherein the fusion protein is (aspartic acid-
The recombinant plasmid according to claim 1, comprising a dimer of (serine-aspartic acid-glycine-lysine) and having the following amino acid sequence. (In the formula, _ has the same meaning as described above.) 3. The recombinant plasmid according to claim 2, wherein the nucleotide sequence encoding the fusion protein is the following nucleotide sequence. (Wherein _ indicates a base sequence encoding an intervening amino acid Arg) 4. The method according to claim 1, wherein the fusion protein is (aspartic acid-
The recombinant plasmid according to claim 1, comprising a trimer of (serine-aspartic acid-glycine-lysine) and having the following amino acid sequence. (In the formula, _ has the same meaning as described above.) 5. The recombinant plasmid according to claim 4, wherein the nucleotide sequence encoding said fusion protein is the following nucleotide sequence. (Where _ has the same meaning as described above) 6. The recombinant plasmid according to claim 2, wherein the recombinant plasmid is stably replicated in Escherichia coli, confers trimethoprim resistance and ampicillin resistance to the host Escherichia coli, and has a size of 4655 base pairs. . 7. The recombinant plasmid according to claim 4, wherein the recombinant plasmid stably replicates in Escherichia coli, imparts trimethoprim resistance and ampicillin resistance to the host Escherichia coli, and has a size of 4670 base pairs. . 8. The recombinant plasmid according to claim 2, wherein the recombinant plasmid is a recombinant plasmid pBBK7DA having the nucleotide sequence shown in FIG. 9. The recombinant plasmid according to claim 4, wherein the recombinant plasmid is a recombinant plasmid pBBK5.6TA having the nucleotide sequence shown in FIG. 10. Escherichia coli transformed with the recombinant plasmid according to claim 1. 11. A fusion protein of dihydrofolate reductase-Arg-antiallergic pentapeptide dimer having the following amino acid sequence: (In the formula, _ has the same meaning as described above.) 12. A fusion protein of dihydrofolate reductase-Arg-antiallergic pentapeptide trimer having the following amino acid sequence: (In the formula, _ has the same meaning as described above.) 13. A method for producing a fusion protein, comprising culturing the Escherichia coli according to claim 10, and collecting the fusion protein according to claim 1 from the cultured cells. 14. The method according to claim 13, wherein the step of collecting the fusion protein comprises a step of purifying the cell-free extract of the cultured cells using a streptomycin treatment, ammonium sulfate precipitation, and mesotrixate binding affinity chromatography in that order. Production method. 15. A fusion protein of dihydrofolate reductase-Arg-antiallergic pentapeptide dimer according to claim 11 or 12, or dihydrofolate reductase-Arg-
Enzymatic treatment of the fusion protein of the anti-allergic pentapeptide trimer gives
A method for producing an anti-allergic pentapeptide comprising an amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine, comprising collecting an anti-allergic pentapeptide comprising an amino acid sequence of aspartic acid-glycine-lysine. 16. The method according to claim 15, wherein the enzyme is trypsin.