JPH04117286A - Dihydrofolate reductase-antiallergic pentapeptide polymer fused protein (ii) - Google Patents

Abstract

NEW MATERIAL:A recombinant plasmid containing a base sequence encoding dihydrofolate reductase-antiallergic peptide polymer fused protein (II). EXAMPLE:A recombinant plasmid wherein an antiallergic pentapeptide, a fused protein (II) of dimer contains a base sequence encoding an amino acid sequence shown by the [formula (underline shows amino acids existing between dihydrofolate reductase and antiallergic pentapeptide). USE:Production of DSDGK. PREPARATION:A DNA encoding DSDGK polymer obtained by chemically synthesizing a single strand DNA and annealing both chains is inserted into a vector plasmid capable of manifesting DHFR polymer.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to dihydrofolate reductase (hereinafter abbreviated as DHFR) and an antiallergic peptide aspartic acid (Asp)-serine (Ser)-aspartic acid. (A+
A novel recombinant method that enables large-scale production of a fusion protein (I[) containing a multimer of a peptide (hereinafter abbreviated as DSDGK) consisting of a 5-amino acid sequence of p)-glycine (Gly)-lysine (Lys) Plasmid, Escherichia coli containing the plasmid, DHFR-DSDGK multimer fusion protein (I['), DHFR-DSDGK multimer fusion protein award (I[) production method, DSDGK multimer production method, and DSDG It concerns the manufacturing method.

The present invention can be effectively utilized in fields such as fermentation engineering and pharmaceutical industry.

[Conventional technology]

DSDGK is a type of anti-allergic peptide,
It is an interesting bioactive peptide that is expected to be used as a prophylactic and therapeutic agent for allergic rhinitis, allergic dermatitis, bronchial asthma, and other allergic diseases caused by chemical mediators (Japanese Patent Application Laid-Open No. −
No. 316398).

Chemical synthesis methods are generally known as methods for producing oligopeptides with a small number of amino acids, but in recent years, methods have been developed that allow easy production using genetic recombination technology. For example, Iwakura et al. created a plasmid ρTP70-1 capable of expressing a large amount of DHPR in E. coli (Japanese Patent Application Laid-open No. 63-267276), and made various modifications to the base sequence encoding the vicinity of the carboxyl-terminal amino acid of DI (FR). DH
It was produced in Escherichia coli in the form of fusion protein (II) with PR. An example of this is somatostatin (Unexamined Japanese Patent Publication No. 1-1-1
44977, JP-A-1-144995), and bradykinin (JP-A-1-252289).

In addition, in order to increase the production efficiency of Kano PR, Iwakura et al. used a recombinant plasmid pTP1 using the rrnB gene, which is known as an efficient terminator region, and pTP70-1.
04-4 (Japanese Patent Application Laid-open No. 1-144979), and using it, we are also producing a recombinant plasmid pLEK1 that can express DHPR-leucine enkephalin fusion protein (II). Since the fusion protein (I[) obtained here also retains the enzymatic activity of D)IPR, its purification can be easily performed using the properties and activity of DHPR. Furthermore, the target peptide has been obtained by subjecting the fusion protein (II) to bromo cyanide treatment or trypsin treatment and then purifying it by IPLC (Japanese Patent Application Laid-Open No. 1-252290).

In these examples, the promoter region and terminator region are modified in order to increase the production amount of the target polypeptide, but it is not possible to produce a polypeptide in which multiple target polypeptides are linked in tandem. It has also been done (Japanese Unexamined Patent Publication No. 31090/1983).

[Problem to be solved by the invention]

An object of the present invention is to produce DSDGK in large quantities by genetic engineering techniques, and for this purpose, it is an attempt to create an effective plasmid incorporating a gene encoding a repeat multimer of DSDGK. This plasmid must meet the following requirements.

(i) Fusion protein containing repeating DSDGK (I
I) can be produced as (ii)
) The gene for the fusion protein (II) can be expressed in large quantities. (ij) The produced fusion protein (II) can be easily purified. (iv) The separation of DSDGK from the fusion protein (II) can be carried out easily. [Means for solving the problem] As a result of intensive research, the present inventors found that the above problem could be solved by using the E. coli dihydrofolate reductase gene, and based on that knowledge, the DHPR and DS
The present invention was completed by constructing a recombinant plasmid incorporating a gene encoding a fusion protein (It) consisting of a DGK multimer.

That is, the present invention resides in a recombinant plasmid containing a base sequence encoding a fusion protein (II) of dihydrofolate reductase antiallergic pentapeptide multimer.

The antiallergic pentapeptide portion consists of the amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine.

Dihydrofolate reductase in the above recombinant plasmid -
In the antiallergic pentapeptide multimer fusion protein (II), the fusion protein (II) in which the antiallergic pentapeptide is a dimer has the following amino acid sequence.

Met Ile Ser Leu Ile
Ala Ala Leu Ala ValAs
p Arg Val Ile Gly Me
t Glu Asn Ala MetPro
Trp Asn Leu Pro Ala Asp L
eu Ala TrpPhe Lys Arg Asn
Thr Leu Asn Lys Pro Vall
ie Met Gly Arg His Thr Tr
p Glu Ser l1eGly Arg Pro
Leu Pro Gly Arg Lys Asn 1
ie11e Leu Ser Ser Gin Pro
Gly Thr Asp AspArg Val T
hr Trp Val Lys Ser Val As
p GluAla Ile Ala Ala Cys
Gly Asp Val Pro Glulle Me
t Val lie Gly Gly Gly Arg
Val TyrGlu Gin Phe Leu P
ro Lys Ala Gin Lys Leu
Tyr Leu Thr His Ile Asp A
la Glu Val GluGly Asp Thr
) Iis Phe Pro Asp Tyr Glu
Pr.

Asp Asp Trp Glu Ser Val
Phe Ser Glu Phe old Asp A
la Asp Ala Gin Asn S
er His 5erTyr Glu, Phe
Glu lie Leu Glu Arg Arg
l1eLeu Met Asp Ser Asp G
ly Lys Asp Ser AspGly Ly
s (wherein represents an amino acid that mediates dihydrofolate reductase and antiallergic pentapeptide) Examples of the base sequence encoding the fusion protein (II) in which the antiallergic pentapeptide shown above is a dimer are as follows: Examples include the base sequence of

ATG ATCAGT CTG ATT GCG GC
G TTA GCG GTAGAT CGCGTT
ATCGGCATG GAA AACGCCAT
GCCG TGG AACCTG CCT GCCGA
T CTCGCCTGGTTT AAA CGCA
ACACCCTTA AAT AAA CCCGT
GATT ATG GGCCGCCAT ACCTGG
GAA TCA ATCGGT CGT CCG T
TG CCA GGA CGCAA AAT ATT
ATCCTCAGCAGT GAAGCCGGT AC
G GACGATCGCGTA ACG TGG
GTG AAG TCG GTG GAT
GAAGCCATCGCG GCG TGT
GGT GACGTA CCA GAAATCA
TG GTG ATT GGCGGCGGT
CGCGTT TATGAA CAG TTCT
TG CCA AAA GCG CAA A
AA CTGTAT CTG ACG CAT
ATCGACGCA GAA GTG GA
AGGCGACACCCAT TTCCCG GA
T TACGAG CCGGAT GACTGG
GAA TCG GTA TTCAGCGA
A TTCCAC GAT GCT GAT G
CG CAG AACTCT CACAGCTA
T GAG TTCGAA ATT CTG
GAG CGG CGG ATCCTG A
TG GAT TCA GAT GGCAAA
GAT TCT GACGGT AAA (in the formula, - indicates a base sequence encoding an amino acid that mediates dihydrofolate reductase and antiallergic pentapeptide) Furthermore, dihydrofolate reductase-antiallergic pentapeptide in the above recombinant plasmid The multimeric fusion protein (II) in which the antiallergic pentapeptide is a trimer has the following amino acid sequence.

Met Ile Ser Leu lie
Ala Ala Leu Ala Val eights
To Arg Vat lie Gly Me
t Glu Asn Ala MetPro
Trp Asn Leu Pro Ala Asp
Leu Ala Trp Phe Lys Arg
Asn Thr Leu Asn Lys Pro
Valle Met Gly Arg His
Thr Trp Glu Ser l1eGly
Arg Pro Leu Pro Gly Arg L
ys Asn Tlelie Leu Ser S
er Gin Pro Gly Thr Asp A
spArg Val Thr Trp Val L
ys Ser Val Asp GluAla I
le Ala Ala Cys Gly Asp
Val Pro Glulle Met Val
Ile Gly Gly Gly Arg Vat
TyrGlu Gin Phe Leu Pro Ly
s Ala Gin Lys LeuTyr Leu
Thr His lie Asp Ala Glu
Val GluGly Asp Thr His Ph
e Pro Asp Tyr Glu Pr.

^sp Asp Trp Glu Ser Val
Phe Ser Glu PheFlis Asp A
la Asp Ala Gln Asn Ser Hi
s 5erTyr Glu Phe Glu lie
Leu Glu Arg Arg l1eLeu
Met Asp Ser Asp Gly Lys
Asp Ser AspGly Lys Asp S
er Asp Gly Lys (wherein has the same meaning as above) Examples of the base sequence encoding the fusion protein (II), which is the antiallergic pentapeptide trimer, include the following base sequence.

ATCATCAGT CTG ATT GCG GCG
TTA GCG GTAGAT CGCGTT AT
CGGCATG GAA AACGCCATGCCG
TGG AACCTG CCT GCCGAT CTC
GCCTGGTTT AAA CGCAACACCTT
A AAT AAA CCCGTGATT ATG G
GCCGCCAT ACCTGG GAA TCA A
TCGGT CGT CCG TTG CCA GGA
CGCAAA AAT ATTATCCTCAGCA
GT CAA CCG GGT ACG GACGAT
CGCGT8 ACCTGG GTG AAG
TCG GTG GAT GAAGCCATCG
CG GCG TGT GGT GACGTA CCA
GAAATCATG GTG ATT GGCGGC
GGT CGCGTT TATGAA CAG TTC
TTG CCA AAA GCG CAA AAA C
TGTAT CTG ACG CAT ATCGACG
CA GAA GTG GAAGGCGACACCCA
T TTCCCG GAT TACGAG CCGGA
T GACTGG GAA TCG GTA TTCA
GCGAA TTCCACGAT GCT GAT CG CAG AACTCT CACAGCTAT G
AG TTCGAA ATT CTG GAG CGG
CGG ATCCTG ATG GAT TC
A GAT GGCAAA GAT TCT
GACGGT AAA GACTCG GACGGG
AAA (in the formula, - has the same meaning as above) Furthermore, the recombinant plasmid containing the nucleotide sequence encoding the fusion protein award (Il[), which is a dimer of the above-mentioned antiallergic pentapeptide, contains Escherichia coli. This includes those that are stably replicated in E. coli, confer trimethoprim resistance and ampicillin resistance to the host E. coli, and have a size of 4658 base pairs.

Furthermore, the recombinant plasmid containing the nucleotide sequence encoding the fusion protein (II), which is a trimer of the above-mentioned antiallergenic pentapeptide, is stably replicated in E. coli and confers trimethoprim resistance and ampicillin resistance to the host E. coli. and has a size of 4673 base pairs.

The recombinant plasmid mentioned above includes the recombinant plasmid pBBK80M having the base sequence shown in FIG.

Furthermore, the above-mentioned recombinant plasmid includes a recombinant plasmid pBBK97M having the base sequence shown in FIG.

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

Furthermore, the present invention resides in a dihydrofolate reductase-antiallergic pentapeptide dimer fusion protein (II) having the following amino acid sequence.

Met lie Ser Leu Ile Ala A
la Leu Ala Val^sp Arg V
al lie Gly Met Glu A
sn Ala MetPro Trp Asn L
eu Pro Ala Asp Leu Aha Tr
pPhe Lys Arg Asn Thr Leu
Asn Lys Pro Valle Met Gl
y Arg old s Thr Trp Glu Ser l
1eGly Arg Pro Leu Pro Gly
Arg Lys Asn 1ie11e Leu S
er Ser Gin Pro Gly Thr As
p AspArg Val Thr Trp Val
Lys Ser Val Asp GluAla Il
e Ala Ala Cys Gly Aspνal
Pro Glulle Met Val Ile Gl
y Gly Gly Arg Vat TyrGlu
Gln Phe Leu Pro Lys Ala G
in Lys LeuTyr Leu Thr His
Ile Asp Ala Glu Val GluG
ly Asp Tltr His Phe Pro A
sp Tyr Glu Pr.

Asp Asp Trp Glu Ser Val P
he Ser Glu PheHis Asp Ala
Asp Ala Gin Asn Ser His
5erTyr Glu Phe Glu Ile Le
u Glu Arg Arg l1eLeu Met
Asp Ser Asp Gly Lys Asp S
er AspGly Lys (wherein has the same meaning as above) Furthermore, the present invention provides a dihydrofolate reductase having the following amino acid sequence.
Antiallergic pentapeptide trimer fusion protein (II).

Met Ile Ser Leu Ile Ala A
la Leu Ala Val^sp Arg Val
Ile Gly Met Glu Asn Ala
MetPro Trp Asn Leu Pro Al
a Asp Leu Aha TrpPhe Lys
Arg Asn Thr Leu Asn Lys P
ro Valle Met Gly Arg His
Thr Trp Glu Ser l1eGly A
rg Pro Leu Pro Gly Arg Ly
s Asn l1e11e Leu Ser Ser
Gln Pro Gly Thr Asp AspAr
g Val Thr Trp Val Lys Ser
Val Asp GluAla Ile Ala A
la Cys Gly Asp Val Pro Gl
uTie Met Val Ile Gly Gly
Gly Arg Val Tyr Glu Gin Ph
e Leu Pro Lys Ala Gin Lys
LeuTyr Leu Thr His lie A
sp Ala Glu Val GluGly Asp
Thr His Phe Pro Asp Tyr
Glu Pr.

Asp Asp Trp Glu Ser Vat P
he Ser Glu PheHis Asp Ala
Asp Ala Gin Asn Ser His
5erTyr Glu Phe Glu Ile Le
u Glu Arg Arg Ile□ Asp
Ser Asp Gly Lys Asp
Ser AspGly Lys Asp Ser A
sp Gly Lys (in the formula, - has the same meaning as above) Furthermore, the present invention involves culturing the above-mentioned transformed Escherichia coli, and producing the above-mentioned dihydrofolate reductase-antiallergic pentapeptide trimer from the cultured cells. or dihydrofolate reductase-antiallergic pentapeptide trimer fusion protein (II), characterized in that the fusion protein (II) of dihydrofolate reductase-antiallergic pentapeptide trimer is obtained. II) in the manufacturing method. The step of collecting the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (I[) includes streptomycin treatment, ammonium sulfate precipitation, and mesotrixate-linked affinity chromatography from a cell-free extract of cultured bacterial cells. The method includes a step of purifying using sequentially the following.

Furthermore, the present invention provides aspartic acid-serine-aspartic acid-glycine- The objective is to collect an anti-allergic pentapeptide consisting of the amino acid sequence of lysine.

Examples of the enzyme used in the enzyme treatment include trypsin.

The details of the present invention are as follows: (1) Production of a novel recombinant plasmid, (2) Transformed E. coli, (3) DHFR-D
Production process of SDGK multimer fusion protein (II),
(4) The obtained DHFR-DSDGK multimer fusion protein (II), (5) DSDGK multimer manufacturing process, and (6) DSDGK manufacturing process will be explained in this order.

(1) Preparation of new recombinant plasmid i) Preparation of DNA encoding DSDGK multimer D
The preparation of DNA encoding the SDGK multimer consists of -heavy chain D
After each NA is chemically synthesized, both columns are annealed. If the target double-stranded DNA is long, it may be prepared by dividing it into several parts along the way.

The DNA encoding the DSDGK multimer has sticky ends on both ends to be ligated to the vector, and between them, from the side closest to the IFR coding region, D) at least one component for cleaving the bond between the IFR and the DSDGK multimer. This structure includes a base sequence encoding 1 amino acid, a base sequence encoding a DSDGK multimer, and a base sequence instructing termination of translation.

The sticky end is the nucleotide sequence of the cut surface generated by the action of a restriction enzyme, and the plasmid pJIEK2 (Unexamined Japanese Patent Publication No.
252289), a BamHI cut is made at the end near the DHFR coding region.
It is convenient to use a cut surface by Xhol at the opposite end.

The amino acid interposed between DFIFR and the DSDGK multimer consists of methionine or multiple amino acids containing methionine.

The degree of polymerization of the DSDGK multimer is not particularly limited, but is preferably from a dimer to a decamer. In addition, a dimer and a trimer were illustrated as specific examples described later.

ii) Insertion of DNA encoding DSDGK multimer into vector plasmid A vector capable of expressing DFIFR is used to express DNA encoding DSDGX multimer. An example of this is the recombinant plasmid pMEK2 (Japanese Unexamined Patent Publication No. 1-25228
(Refer to Publication No. 9). pMEK2 is stably retained in Escherichia coli, and Escherichia coli containing pMEK2 was sent to FIKEN.
It has been deposited as ERM BP-1816. pME
K2 can be isolated and purified from Escherichia coli containing pMEK2 according to a conventional plasmid isolation method and used. Purified pMEK2 was purified with BamHI and
The double strand D of i) above is added to the DNA fragment obtained by cutting with o l.
The NAs are ligated using 74-DNA ligase to obtain a novel recombinant plasmid that allows expression of the desired fusion protein (II) in E. coli cells.

ii) Base sequence of new plasmid Figure 1 shows the new recombinant plasmid pB, which is an example of the present invention.
B shows the entire 80M base sequence. double stranded circular DN
Only the base sequence of one DNA strand of A that encodes genetic information is processed by the restriction enzyme C1, which is the only one present in the plasmid.
The first "A" of the recognition cleavage site of a I, 5'-ATCGAT-3', is counted as number 1 and is described in the direction from the 5' end to the 3' end. ρBBK8DM is 465
It has a size of 8 base pairs and can confer trimethoprim and ampicillin resistance to the host E. coli.

pBBK8DM is the larger DNA fragment (consisting of 4614 base pairs) obtained by BamHI and Xho I digestion of pMEK2 (consisting of 4640 base pairs).

) and the nucleotide sequence encoding the DSDGK dimer.
It has a structure in which chemically synthesized base pairs and NA are bound together. In FIG. 1, the sequence from position 533 to position 576 is derived from chemically synthesized NA. By the way, pMEK
The entire nucleotide sequence of No. 2 has already been revealed by the present inventors (see Japanese Unexamined Patent Publication No. 1-252289), and the sequence from 533rd to 576th of the nucleotide sequences shown in Figure 1 is as follows: This is a structure that has been replaced with the 26 base pair sequence shown in .

5'-GATCCGTTACGGTGGTTTCAT
The terminal sequence of GTAAC-3' chemically synthesized double-stranded DNA includes a sticky end generated upon cleavage with the restriction enzyme Bam)tl, and 5'-GATC-3' (53 in Figure 1).
Array from 3rd to 536th. ), and restriction enzyme X
Sticky end generated upon cleavage by hoI, 3'-AG
CT-5' (a sequence complementary to the sequence from 577th to 580th in FIG. 1). pMEK
Since there is a sticky end site generated by BamHI and Xho I cleavage in the portion derived from D.2, a fusion gene with the D) IFR gene can be easily created by introducing heterologous DNA in a determined direction.

The array from 57th to 572nd in Figure 1 is DHF
DSDGK dimer has leucine-
DHPR bound via the methionine amino acid sequence
It encodes a DSDGK dimer fusion protein (II).

D) IFR-DSDGK dimer fusion protein (It
) upstream of the sequence encoding D) IFR-DSDG
There is a sequence that allows efficient expression of the K dimer fusion protein (II) gene (Japanese Patent Application Laid-Open No. 63-267276
No. Specification).

That is, the sequence from position 43 to position 50 is called the SD sequence and contributes to efficient translation, and position from position 4616 to 4644 is a consensus transcription promoter and contributes to efficient transcription. Therefore pBB
When K80M is introduced into E. coli, a large amount of D) IF
A fusion protein (rl) of R-DSDGK dimer is made. The produced D) IFR-DSD (J dimer fusion protein award (II) accumulates in a soluble state within the bacterial body, accounting for about 20% of the bacterial protein.

Although trimethoprim is an inhibitor of this DIIFR, E. coli harboring pBBK80M has DHPR-DSD.
Since the GK dimer fusion protein (It) accumulates in large quantities within the bacterial cells, it becomes resistant to trimethoprim.

FIG. 2 shows the entire base sequence of the novel recombinant plasmid pBBK9TM, which is another example of the present invention.

Similarly to Fig. 1, only the base sequence of one DMA strand of the double-stranded circular DNA encoding genetic information is cut into the 5'-ATCGAT- pBBK9TM is 4673 base pairs in size, and is written in the direction from the 5' end to the 3'' end, counting the first "A" of 3' as number 1.
BamHl of MEK2 (consisting of 4640 base pairs)
and Xho ■ Larger DNA obtained by cutting
fragment (consisting of 4614 base pairs) and a 59 base pair chemically synthesized N containing the base sequence that trimerizes the DSDGK trineid.
It has a structure in which A is combined. In Figure 2, 533
The sequence from position 591 to position 591 is a sequence derived from chemically synthesized NA.

The array from 57th to 587th in Figure 2 is DHF
D) IF in which DSDGK tricameral is linked to the carboxyl terminal side of R via a trimeric syn-methionine amino acid sequence
It encodes the R-DSDGK trimeric fusion protein (II).

pBBK97M has similar characteristics to pBBK8DM except that it produces a large amount of leaf FR-DSDGK trimer fusion protein (I[) when introduced into E. coli.

pBBK80M and p8897M can be produced according to Example 1 and Example 2, respectively, but the present invention is not limited by the method for producing recombinant plasmids.

(2) Transformed E. coli The recombinant plasmid prepared in (1) above is incorporated into E. coli according to a conventional method. The transformed E. coli exhibits resistance to trimethoprim and ampicillin. E. coli containing this plasmid has DHPR on the plasmid.
-As a result of efficient expression of the DSDGK multimer fusion protein (II) gene, a large amount of DFIFR-DSD (J multimer fusion protein (II)) is accumulated in the bacterial body in a soluble state. E. coli YT
+Ap medium (liquid medium containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 100 μl of ampicillin sodium in medium IL).
D) IFR- that accumulates when cultured at °C to stationary phase
The DSDGK multimer fusion protein (II) accounts for about 20% of the bacterial protein. If cultured bacterial cells are suspended in an appropriate buffer such as phosphate buffer, disrupted using the French press method or ultrasonic disruption method, and then separated into supernatant and precipitate using centrifugation, most All DHFI'1
-DSDGK multimeric fusion protein (I[) is recovered in the supernatant. Of this new recombinant plasmid, pB
BK80? Escherichia coli containing I
No. 2 (FERM BP2392) and pBBK
Escherichia coli containing 9TM was produced by Kaikoken Joyori No. 2393 (
FERM BP-2393).

(3) Production process of DHP R-D SDG K multimer fusion protein (II) The method consists of the following steps: 11) disruption of the bacterial cells, 1ii) streptomycin treatment, iv) ammonium sulfate precipitation, and ①) mesotrixate (MTX)-binding affinity chromatography.

i) Culture of bacterial cells E. coli containing the plasmid of the present invention is cultured using YT1Ap medium (a liquid containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 50 μm of ampicillin sodium in medium IL). culture medium.). In addition to this medium, ST+Ap medium (medium I
In L, 2g glucose, 1g disodium phosphate, 5g polypeptone, 5g yeast extract and 50 g
■Liquid medium containing ampicillin sodium. )Such,
Although any medium can be used as long as it allows bacterial cells to grow, YT+Ap medium is preferable for the production of D) IFR-DSDGK multimer fusion protein (I[).

E. coli containing the plasmid of the present invention is inoculated into a medium and cultured at 37°C until the late logarithmic growth phase or stationary phase. The amount of DHFR-DSDG multimeric fusion protein (II) accumulated in the bacterial cells varied depending on the culture temperature, and as far as we investigated, the higher the culture temperature, the greater the amount accumulated.

The cultured cells are collected by centrifugation at 4.700×g. Bacterial cells with a wet weight of 2 to 4 g are obtained from the medium IL.

Bacterial collection and subsequent operations are preferably carried out at low temperatures (between 0 and 10°C, preferably 4°C) unless otherwise specified.

ii) The bacterial cells obtained by disrupting the bacterial cells were added to the buffer solution [0.1 mM disodium ethylenediaminetetraacetate (EDTA-Na2) and 14IIIM2 at a ratio of 10 parts per culture solution IL.
The cells are suspended in 10 mM potassium phosphate buffer (pt(7,0)) containing mercaptoethanol and disrupted using a French press or an ultrasonic disruptor.

The cell suspension is centrifuged at 27,000 x g for 20 minutes to obtain a supernatant (hereinafter referred to as cell-free extract).

ij) Slowly add 1.5% (W/V) streptomycin sulfate to the streptomycin-treated cell-free extract while stirring with a stirrer. After stirring for 20 minutes, the streptomycin-treated solution was centrifuged at 27,0 OOX g for 20 minutes to obtain a supernatant.

iv) Ammonium sulfate precipitation 0.8 to 0 per volume of supernatant obtained by the above procedure
゜Slowly add 9 volumes of saturated ammonium sulfate solution while stirring with a stirrer. After room temperature for 20 minutes, the ammonium sulfate precipitated solution was centrifuged at 27,0 OOX g for 20 minutes to obtain a supernatant.

v) MTX binding affinity chromatography The supernatant obtained by the above procedure was added to a 25-MTX binding agarose gel (manufactured by Sigma) equilibrated with buffer 1 in advance.
Add. After allowing it to adsorb to the gel for 10 minutes, the gel is packed into a column. The column was mixed with buffer solution 1, buffer solution 1 containing IMKCI, buffer solution 2 containing IMKCI [0.1mM disodium ethylenediaminetetraacetate (EDTA・Na
g), 10m containing 14mM 2-mercaptoethanol
M potassium phosphate buffer (pif 8.5). ] Wash in this order. Elution of DHFR-DSDG multimeric fusion protein (II) was performed using a solution of buffer 1 containing 1M MCI and 3mM folic acid adjusted to pH 9.3 with 2N NaOH, and the eluate was Fractionate a certain amount using a fraction collector. D for the fractionated eluate
HPR activity is measured and both fractions containing enzyme activity are collected. The resulting solution is dialyzed against buffer 1. At this stage, D) IFR-DSDGK multimer fusion protein (II) with a purity of 90% or more is obtained.

By the above operations, the DI(FR-DSDGK multimer fusion protein (II)) can be purified with good reproducibility.

According to the present invention, purification of the DHPR-DSDGK multimer fusion protein (II) can be performed within 3 days including culturing, and a homogeneous protein preparation can be obtained with a recovery rate of about 80%. .

Measurement of DHPR enzyme activity was performed using a reaction solution [0.05n+M dihydrofolic acid, 0.06mM NADPFI, 12mM
50 mM potassium phosphate buffer (pH 7.0) containing 2-mercaptoethanol. ] 1 relative is placed in a cuvette, a sample is added to the cuvette, and the time change in absorbance at 340 nm is measured at 25°C. Enzyme activity is expressed in units, and 1 unit is defined as the amount of enzyme required to convert 1 micromole of dihydrofolate into ternary molecules per minute under the above reaction conditions.

This measurement can be easily performed using a spectrophotometer.

(4) Obtained DHFR-DSDGK multimer fusion protein (II) Figure 3 shows an example of the DHPR-DSDGK multimer fusion protein (II) contained in pBBK80M.
I (PR-DSD (J dimer fusion protein (II)
This shows the DNA sequence of the coding part and the amino acid sequence of the protein produced from it. D) IFR-D
SDGK dimer fusion protein (II) is a novel protein consisting of 172 amino acids. The sequence from positions 1 to 159 counting from the amino terminus has a single amino acid substitution in E. coli wild-type DHPR [C
ys-152(wild)→G1u-152] sequence, and the sequence from the 163rd to the 172nd is the sequence of the DSDGK dimer fusion protein (II). Isoleucine at position 160 (lie) and leucine at position 161 (L
eu) is pMEK2 Bamt (DSD (,
This sequence was created to align the reading frame of genetic information when introducing a gene encoding a dimer into a protein.

The 162nd amino acid, that is, the amino acid immediately before the DSDGK dimer sequence, is methionine (Met). Therefore, a fusion protein of DHFR-DSDGK dimer (
II) by Bromsian processing, DSDIJ
It is possible to produce two types of Nisei bodies. D) IFR-DSDG
The molecular weight of K dimer fusion protein (II) is 19,
It is 387.

In addition, FIG. 4 shows another example of the DHPR-DSDGK multimer fusion protein (II) contained in pBBK97H.
The DNA sequence of the portion encoding t) and the amino acid sequence of the protein produced therefrom are shown.

DHFR-DSDGK trimer fusion protein (II)
is a novel protein consisting of 177 amino acids.

The sequence from positions 1 to 172 counting from the amino terminal side is the same as the above DHPR-DSDGK dimer fusion protein (II), but D) IFR-DSDGK trimer fusion protein (II) is DI (PR-DSDGK
It has a structure in which another DSDGK is bound to the carboxyl terminal side of the dimeric fusion protein (II).

DHFR-DSDGK trimeric fusion protein '1 (II
) is 19,889.

In addition, DHFR-DSDGK dimer fusion protein (
II) and DHFR-DSDGK trimer fusion protein (II) have a structure in which the DSDGK di-form and the di-SDGK trimer are bound to the carboxy terminal side of DHFR, respectively. Has enzymatic activity. Therefore, when E. coli produces a large amount of Kano FR-DSDGK dimer fusion protein (II) and Kano FR-DSDGK trimer fusion protein (II), D) Becomes tolerant.

(5) Manufacturing process of DSDGK multimer D)IFl? in (4) above? - In the fusion protein (It) of the DSDGX multimer, the amino acid immediately before the DSDGK multimer is methionine (Net). So this DH
When PR-DSDGK multimer fusion protein (II) is chemically treated with brom cyanide, DSDGII:multimer is excised from DHFR-DSDGK multimer fusion protein f(II). The chemically treated decomposition product is purified by HPLC to obtain a DSDGK multimer.

(6) Production process of DSDGK The DSDGK multimer obtained in (5) above is treated with trypsin or with lysyl endopeptidase to produce DSDGK.
Get SDGK. Also, DI (FR-DS) obtained in (4)
DSDGK can also be obtained directly without going through the DSDGK multimer by treating the DIJ multimer fusion protein (II) with trypsin and purifying the decomposition product using 1iPLc.

〔Example] Next, the present invention will be explained in detail with reference to examples.

(Example 1) Preparation of pBBK80M The DNA encoding the DSDGK dimer was 1,
5'-GATCCTGATGGATTCAGATGGC
AAAGATTCTGACGGTAAATAAC-3 2,5'-TCGAGTTATTTACCGTCAGA
ATCTTTGCCATCTGAATCCATCAG-
DNA consisting of two 44 nucleotides at the 3' end was synthesized using a chemical synthesizer (manufactured by Millisien, 750) according to the phosphoramidide method.
After chemical synthesis using oligonucleotide purification cartridge (manufactured by Applied Biosystems),
Take about 0.02 d of DNA (contains about 0.1 gg of DNA) and add it to the reaction solution for caination (
50mM Tris-HCI pH7,8,10mM
gC1z, 5mM dithiothreitol, 0.5mM A
TP) at 180 μm and T4 polynucleotide kinase (
Add 1μm of Takara Shuzo Co., Ltd. 10 units/μm) to 37
The 5' end was phosphorylated by incubation for 30 minutes at 20°C. This was incubated at 80° C. for one time and then gradually cooled to anneal both DNAs to obtain the following double-stranded DNA (hereinafter referred to as DNAI).

1, 5'-GATCCTGATGGATTCAGAT
GGCAAAGATTCTGA2.3'-GACTAC
CTAAGTCTACCGTTTCTAAGACTCG
GTAAATAAC-3 GCCATTTATTGAGCT-5' purified approximately 1μ
g of pMEK2 was cleaved with 20 units of BamHI- (manufactured by Takara Shuzo Co., Ltd.) and 20 units of xhol (manufactured by Takara Shuzo Co., Ltd.), and then separated by 0.85% agarose gel electrophoresis. A gel containing a DNA fragment of approximately 4.6 kilobase pairs was cut out, and DNA was recovered from the gel (hereinafter referred to as D
Call it NA2. ).

The operation of DNA cleavage with Ban+Hr and
laboratory Manual” [T.

Maniatis, E.F., Fr1tsch+J.
Sambrook eds, coldSpring
Harbor Laboratory (1982)
It was carried out according to the method described in . Total 20μ
20μm ligase reaction solution (10ff
1M Tris-HCI pH7,4,5mM MgCl
2.10mM dithiothreitol, 5mM ATP),
Add 10 μl of DNAI and add 1 μm of T4-DN to this.
A ligase (Takara Shuzo Co., Ltd., 300 units/μl) was added, and the DNA ligation reaction was carried out at room temperature for 19 hours. This reaction product was transformed using a transformation method (transform+atio).
n method, described in Maniatis et al., supra. ), Escherichia coli (Takara Shuzo E, coli I
(BIOI competent cells). The treated cells were transferred to a nutrient agar medium containing 100 μ/l of ampicillin sodium and 5 μ/l of trimethoprim (
Medium LL contains 2 g glucose, 1 g dipotassium phosphate, 5 g yeast extract, 5 g voripeptone, and 15 g agar. ) and incubate at 37°C for 24 hours.
By culturing for several hours, several dozen colonies could be obtained. Six of these colonies were assigned a YT+ of 2-
The cells were cultured at 37° C. in AP medium (medium IL contains 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 100 μl of ampicillin sodium). Transfer the culture solution to each Eppentorf centrifuge tube (1,
5 m volume) and placed in an Eppendorf centrifuge (54 m volume).
14S type) at 12,000 rpm for 10 minutes, and the bacterial cells were collected as a precipitate. To this, 0.1
Sample preparation solution for electrophoresis [0,0709? T of I
ris41C1pl (6, 8, 2% (W/V) sodium lauryl sulfate (SDS), 11% (V/V) (7
) Contains glycerin, 5% (V/V) (7) 2-mercaptoethanol, and 0.045% (W/V) (7) Bromophenol blue. ] to suspend the bacterial cells,
This was kept in boiling water for 5 minutes to dissolve the bacterial cells. This treated sample was subjected to 5DS-polyacrylamide gel electrophoresis [U., Laemmli; Nature;
vol, 227. p680 (1970), ]. Lactalbumin (molecular weight 14,200) and trypsin inhibitor (molecular weight 14,200) were used as molecular weight markers.
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 live serum albumin (molecular weight 66
．． 000) mixture (DALTON MA [manufactured by Sigma]
Electrophoresis was carried out using a concentration gradient gel (manufactured by Daiichi Kagaku Co., Ltd.) in which the acrylamide concentration ranged from 10 to 20%. The results revealed that proteins of the estimated size were produced in three of the six colonies. This colony was cultured in YT+Ap medium, and T
anaka and -eisblum's method [T, Tanak
a, B, Weisblum; J, Bacteriol
ogy vol, 121. p, 354 (1975)
The plasmid was prepared according to [ , ]. The obtained plasmid was named pBBK80M. pBBW8IIM
should have a structure in which the sequence between pMEI(2's Bam) I and Xho 1 sites is synthesized and replaced with NA. Since the synthetic NA contains a sequence, 5'-CTCGAG-3', which is recognized and cleaved by the restriction enzyme Xho I, when we tried to cleave pBBK8DM with Xho I, it was indeed cleaved. In addition, approximately 400 nucleotides of DNA obtained by cleaving pBBK8DM with EcoRT (sequence 471-476 in Figure 1) and 5all (sequence 875-880 in Figure 1)
For A, the dideoxy method using M13 phage [J
, Messing;Methods in Enzym
ology vol, 10L p20 (1983). 1, the base sequence was determined in the direction from EcoRI to 5alI. As a result, the sequence from positions 471 to 875 of the entire base sequence of pBBK8D)1 shown in FIG. 1 was confirmed.

In addition, approximately 4.6 kilobase pairs of DNA obtained by BamH1-Xho I digestion of pBBK8DM was
RI rPstl, HindIn, Hpal, P
Vull, 8g per day 1. C1aX (manufactured by Takara Shuzo Co., Ltd.)
As a result of a restriction enzyme cleavage experiment using AatII and AatII (manufactured by Toyobo Co., Ltd.), the Bam)I-X of pMEK2
It was shown to be exactly identical to the approximately 4.6 kilobase pairs obtained by ho I cleavage.

Based on the above results, the entire base sequence of pBBK8DM was determined as shown in FIG.

(Example 2) D) IFR-D produced by E. coli containing pBBX8DM
SDGK mer fusion protein (II) DHFli'- produced by E. coli containing pBBK8DM
The amino acid sequence of the DSDGK dimer fusion protein (II) can be predicted from the base sequence of the DHFR-DSDfJ dimer fusion protein (II) gene.

The array from 57th to 572nd in Figure 1 is DHFR.
Since it encodes the -DSDGK dimer fusion protein (II), the amino acid sequence was estimated using the triblet code table. As a result, the amino acid sequence shown in FIG. 3 was obtained. Then, using E. coli containing pBBK8DM, D) lFI? -DS
The fusion protein (II) merging with DII; was isolated and purified and identified.

DHFR-DSDGK dimer fusion protein (II)
Purification A, amount of bacterial cells used: wet weight 13.3 g/3 L
Culture solution B, enzyme purification process: Shown in Table 1 below (in the table, ▪ indicates cell-free extract, ▪ indicates streptomycin treatment, ▪ indicates ammonium sulfate precipitation, and ▪ indicates mesotrixade-linked affinity chromatography.

(Margin below overlapping) Table 1 Clear view Liquid volume Recovered enzyme) Recovered enzyme
Yield process (ml) Quality (mg)
Activity (unit) (%) ■ 32
Li2O390100■ 30 960
340 87■ 54 630
280 72DHFR enzyme activity was measured using the reaction solution [
0.05mM dihydrofolic acid, 0.06+aM NAD
PI (, 12mM 2-mercaptoethanol, 50mM
M potassium phosphate buffer (pH 7,0)] Place 1 d in a cuvette, add the sample to it, and incubate at 25°C for 34 hours.
This was done by measuring the change in absorbance at 0 nm over time. Enzyme activity is expressed in units, and 1 unit is defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute under the above reaction conditions.

The obtained DIIFR-DSDGK dimer fusion protein (II) was analyzed by 5DS-polyacrylamide gel electrophoresis (method described in Example 1).
Approximately 22,000 single protein bands were shown, and the resulting DHFR-DSDGK dimer fusion protein (
II) The standard was shown to be homogeneous.

(Example 3) Purification and separation of DSDGK dimer from D)lFI'1-I) SDG dimer fusion protein (II) Purification and homogenization of 61d DHFRDS obtained in Example 2
The solution of DGK dimer fusion protein (II) was concentrated using an ultrafiltration membrane (Amicon YM5 diameter 25 cylinder) and dialyzed against buffer 1 to obtain a 5.5d DHPR-DSDGK dimer fusion protein. (II) Dark! One liquid was obtained. This is 26
．． It contained DI (PR-DSDGK dimer fusion protein (IT)) at a concentration of 8 μ/d.
SDGK dimer fusion protein (II) concentrate 100
ul to 0. Bromcyan (50 mg) dissolved in IN hydrochloric acid
/d) 180 μm, 0, IN hydrochloric acid 720 μm was added and stirred overnight at room temperature.

After the reaction, a small amount of water was added and then freeze-dried.

The obtained sample was dissolved in 500 μl of 0.1% trifluoroacetic acid. Take 300 μm of it and use a high performance liquid chromatography device (Hitachi 655 model) to
DS-5 column (Yamamura Chemical Co., Ltd. diameter 4.6 x 250 mm)
It was separated by Elution was performed by applying a concentration gradient of acetonitrile (0% to 10%) in 0.1% trifluoroacetic acid. From 0 to 5 minutes, use 0% acetonitrile, and from 5 to 35 minutes, use 0% to 10% acetonitrile.
A linear concentration gradient of acetonitrile was applied. the result,
An elution curve as shown in FIG. 5 was obtained. The peak fraction approximately 19 minutes after sample injection was separated, and a small amount of water was added to the separated eluate and lyophilized to remove the solvent to obtain a peptide.

After acid hydrolysis of the obtained peptide, 1/25th thereof was used for amino acid analysis. As a result, aspartic acid was 8.
8n mole, 4.4n mole each of serine, glycine, and lysine were detected. The amino acid composition was consistent with that of the DSDGK dimer. From these results, it is possible to recover the DSDGK dimer at a yield of about 66% by treating the purified homogenized DHPR-DSDGK dimer fusion protein (II) with brom cyanide treatment and then separating it using high performance liquid chromatography. It became clear. The yield of purification of DHPR-DSDGK dimer fusion protein (II,) was about 57%, and DI
(FR-O5[lG dimeric fusion protein (II)
The isolation yield of DSDGK from cell-free extracts was calculated to be about 37%, since the yield of isolation of DSDGK dimer from cell-free extracts was about 66%.

(Example 4) Separation of DSDGK from separated and purified D) IFR-DSDGK dimer fusion protein (II) 20 μm concentrated solution of D) IFR-DSDGK dimer fusion protein (II) used in Example 3 into an Ebbendorf centrifuge tube (1,5-volume) and add 80 μl of acetone to it.
added. Then, it was placed in a -20°C freezer for 30 minutes, and DHPR-DSD (J dimer fusion protein (II)
) was precipitated. This was heated at 12,000 rpm for 2 hours using an Eppendorf centrifuge (model 54145).
Centrifuge for 1 minute, remove the supernatant, add 10 μm 1M Tris-HCl buffer (p) 18.0), 90 μm purified water,
Add 0 μm TPCK trypsin (manufactured by Sigma) and
Incubated overnight at 7°C. After the reaction, 10 μl of this was taken and separated using a YMC-005-5 column (manufactured by Yamamura Chemical Co., Ltd., diameter 4.6 x 250 mm) using a high performance liquid chromatography device (Hitachi 655 model). Elution was performed under the same conditions as in Example 3. As a result, a peak of DSDGK was confirmed approximately 9 minutes after sample injection.

(Example 5) Creation of pBBK9TM The DNA for coating the 5DGK triketa is 1,
5'-GATCCTGATGGATTCAGATGG
CAAAGATTCTGACGGTAAAGACTCG
GACGGGAAAATAAC-32,5-TCGAGT
TATTTCCCGTCCGAGTCTTTACCGT
C8GAATCTTTGCCATCTGAATCCAT
The DNA of CAG-3' was chemically synthesized using a chemical synthesizer (Model 7500, manufactured by Millisien) according to the phosphoramidide method, and purified using an oligonucleotide purification cartridge (manufactured by Applied Biosystems).
1 d (containing about 0.1 μg of DNA) and add reaction solution for caination (50mM Tris) to this.
-HCI pH 7,8, 10mM MgCh, 5mM dithiothreitol, 0.5m-TP) at 180 μl.
l! :T4 polynucleotide kinase (manufactured by Zenshuzo Co., Ltd. 1
The 5' end was phosphorylated by adding 1 μl of 0 units/μl) and incubating at 37°C for 30 minutes. After incubating this at 80°C once, it was gradually cooled to anneal both DNAs.
The following double-stranded DNA was obtained (hereinafter referred to as DNA3).

1,5'-GATCCTGATGGATTCAGATG
GCAAAGATTCTGACGG2.3'-GACT
ACCTAAGTCTAAGACT
GCCTAAAGACTCGGACGGGAAATAA
C-3ATTTCTGAGCCTGCCCTTTATT
Add 20μl of DNA to GAGCT-510μm DNA3
AI, 20 μl of ligase reaction solution, 1 μl of T4-,
DNA ligase (both described in Example 1) was added to carry out a DNA ligation reaction at room temperature for 19 hours. Then, according to the method described in Example 1, E. coli was transformed, the transformant was cultured, and the cultured cells were subjected to 5DS-polyacrylamide gel electrophoresis.

As a result, production of protein of the size estimated for two colonies was observed. Therefore, one of these
colonies were cultured in YT+Ap medium, and the Tana
Plasmids were prepared according to the method of K. ka and Weisblum. The resulting plasmid was named pBBK97M. pBBK9T should have a structure in which the sequence between the Bam1 (I and ho1 sites of pMEK2) is replaced with synthesized NA.
o Sequence recognized and cleaved by I, 5'-CTCGAG-3
', so pBBK9TM with Xho I
When I tried to disconnect it, it was indeed disconnected. Also, pB
BK9T? The dideoxy method using M13 phage was performed on approximately 400 nucleotides of DNA obtained by digestion of 1 with EcoRI (sequence 471-476 in Figure 2) and Sal I (sequence 890-895 in Figure 2). Accordingly, the base sequence was determined in the direction from EcoRI to SalI.

As a result, the sequence from position 471 to position 890 of the entire base sequence of pBBK9TM shown in FIG. 2 was confirmed.

In addition, approximately 4.6 kilobase pairs of DNA obtained by Ban+HI-Xho I digestion of pBBK9TM is
coRI, Pstl, HindnI, Flpa
As a result of a restriction enzyme cleavage experiment using p. BamH of IEK2
It was shown to be exactly identical to the approximately 4.6 kilobase pairs obtained by IXho I cleavage.

From the above results, the entire base sequence of pBBK97M could be determined as shown in FIG.

(Example 6) D) IFR-D produced by E. coli containing pBBK97M
DHPR-DS produced by E. coli containing SDGK trimer fusion protein (II) pBBK9T-
The amino acid sequence of the DGK trimeric trimeric protein (II) is the DHFR-DSDGK trimeric fusion protein (I).
I) Can be predicted from the base sequence of the gene. Second
The array from 57th to 587th in the diagram is DHFR-D
Since it encodes SDGK trimer fusion protein (II), the amino acid sequence was estimated using a triplet code table. As a result, the amino acid sequence shown in FIG. 4 was obtained. Using Escherichia coli containing pBBK97M, the DHPR-DSDGK trimeric protein (II) was isolated and purified and identified as follows.

DHFR-DSD (J trimer fusion protein (II)
Purification A, amount of bacterial cells used: wet weight 13.1 g/3 L Culture solution B, enzyme purification step: shown in Table 2 below. (■ in the table represents cell-free extract, ■ streptomycin treatment, ■ ammonium sulfate precipitation, and ■ mesotrixate bond affinity chromatography. For enzyme purification, the cell-free extract in ■ is sequentially subjected to the purification steps of ■, ■, and ■. ) Table 2 Purification Liquid volume Recovered 9 proteins Recovered enzyme Yield process (IRl) Quality (-g)
Activity (Enift) (%) ■ 43
940 330■
40 670
300 ■ 49 580
170 ■ 95
152 130 DI (The method for measuring PR enzyme activity and the definition of activity were in accordance with Example 2.

When the obtained DHFR-DSDGK trimer fusion protein (II) was analyzed by 5DS-polyacrylamide gel electrophoresis (method described in Example 1), about 22,000 single protein bands were detected. The resulting DHPR-DSDGK trimeric protein (U
) The specimen was shown to be homogeneous.

(Example 7) Separation of DSDGK trimer from separated and purified DHFR-DSDGK trimer fusion protein (II) 95! obtained in Example 6! The purified Kano PR-DSDGK trimer fusion protein (II) solution of d was concentrated using an ultrafiltration membrane (Amicon YM5 diameter 25n+m), and the solution was concentrated for 6.5 d.
DHPR-DSDGK trimeric fusion protein (II
) A concentrated solution was obtained. This is DH at a concentration of 22.8■/-
It contained a FRDSDGK trimeric fusion protein (II).

This DI (PR-DSDGK trimer fusion protein (
II) 0.0 μl to 100 μl of concentrated solution. Bromsyanide (50 mg/d) dissolved in IN formic acid 180 u 1.0.
720 u 1 IN formic acid was added and stirred overnight at room temperature.

After the reaction, a small amount of water was added and then freeze-dried. The obtained sample was dissolved in 500 μl of 0.1% trifluoroacetic acid. Take 40 μl of it and use a high performance liquid chromatography device (Hitachi 655 model) to transfer YMC-005.
-5 column (manufactured by Yamamura Chemical Co., Ltd., diameter 4.6 x 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. A peak fraction approximately 31 minutes after sample injection was separated, and a small amount of water was added to the separated eluate and lyophilized to remove the solvent to obtain a peptide.

After acid hydrolysis of the obtained peptide, a quarter of it was used for amino acid analysis. As a result, aspartic acid was 7.8
n mole, serine, glycine, and lysine were detected in an amount of 3.9 n mole each. The amino acid composition is
It matched that of the trimer to DSDG. From this result, it was found that the DSDGK dimer could be recovered with a yield of about 56% by treating the purified DHPR-DSDGK trimer protein with brom cyanide and then separating it using high performance liquid chromatography.

DHPR-DSDGK trimeric fusion protein (I[)
The yield of purification of DHPR-DSDGK is about 40%.
Since the yield of separation of DSDGK trimer from trimeric fusion protein (II) is approximately 56%, we calculate that the yield of DSDGK trimer from cell-free extract is approximately 22%. be done.

(Example 8) Separation of DSDGK from separated and purified D) IFR-DSDGK trimer fusion protein (II) 10 μl of Di(PR-DSDGK trimeric protein (II) concentrate) used in Example 7 into an Eppendorf centrifuge tube (1,5I, 11 volumes) and add 40% acetone to it.
μl was added. Then, it was placed in a -20°C freezer for 30 minutes to precipitate the trimeric fusion protein (II) in D) IFR-DSDG.

This is an Eppendorf centrifuge (model 5414S).
The supernatant was removed by centrifugation at 12,000 rpm for 2 minutes, and 20 μl of 8M urea was added to the precipitate to dissolve it. Additionally 80 μl of 0.1M) Lis-HCl buffer (
p118.0), 10 μl of 0.5% trypsin (Di
fc.

Co., Ltd.) was added and incubated overnight at 37°C.

After the reaction, take 20 μl of the reaction and transfer it to YMC-ODS using a high performance liquid chromatography device (Hitachi 655 model).
5 column (manufactured by Yamamura Chemical Co., Ltd., diameter 4.6 x 250 mm). Elution was performed under the same conditions as in Example 3. the result,
A DSDGH peak was observed approximately 9 minutes after sample injection.

[Effect of the invention] As mentioned above, the novel recombinant plasmid of the present invention has DH
Since it encodes the PR-DSDGK multimer fusion protein (II), E. coli carrying this plasmid encodes the DHFR-DSDGK multimer fusion protein (II).
is produced and accumulated in large quantities in a soluble state. Furthermore, the produced DHFR-DSDGK multimer fusion protein (I
I) retains DHFR enzyme activity and can be easily purified. By following the purification method of the present invention, DI (PR-DSDGK multimer fusion protein (I
I) can be purified quickly and efficiently.

Alternatively, the fusion protein (II) of the DHPR-DSDGK multimer thus obtained can be directly treated with trypsin or other suitable enzyme, or the DSDIJ multimer can be cut out by chemical cleavage such as bromcyanide, and then trypsin can be applied. DSDGK can be isolated and purified by high performance liquid chromatography. Purified DSDG is useful as a therapeutic agent for allergic diseases. Therefore, the present invention provides an advantageous means for producing 5DGK, which is useful as a therapeutic agent for allergic diseases, by genetic engineering technology.

[Brief explanation of the drawing]

Figures 1 and 2 show pBBK8DM and pB, respectively.
It is a diagram showing the entire base sequence of BK9TM, and is a double-stranded DNA.
Only the base sequence of one DNA strand of A that encodes genetic information is written in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleobases, A represents adenine,
C represents cytosine, G represents guanine, and T represents thymine. Numbers in the figure are pBBK8DM and pBBK9TM
The first "A" of the recognition cleavage site of the restriction enzyme C1a I, 5'-ATCGAT-3', which exists at one location in each of the figures, is numbered as number 1. Figures 3 and 4 show pBBK8DM and pB, respectively.
FIG. 2 is a diagram showing the base sequence of the portion encoding the DHPR-DSDGK dimer fusion protein (II) and the DHPR-DSDGK trimer fusion protein (II) present in BK9TM, and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A for adenine, C for cytosine, G for guanine, T for thymine,
cty is glycine, Ala is alanine, Val is valine, Leu is 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, Gin is glutamine, ^rg is arginine, Lys is lysine, His is histidine, Ph
e is phenylalanine, Tyr is tyrosine, Trp
indicates tryptophan, and Pro indicates proline. The numbers in the figure indicate the numbers counted starting from methionine, which is the amino terminal amino acid of fusion protein (II). Figures 5 and 6 show DHFR-DSDGK dimer fusion proteins (II) and D) IFR-DSD, respectively.
This figure shows a chromatogram obtained when GK trimer fusion protein (II) was treated with brom cyanide and separated using a high-performance liquid chromatography device using a reversed phase column. The horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units. The arrows in the figure indicate the positions where the DSDGK dimer and DSDGK trimer are eluted, respectively.

Claims (1)

[Scope of Claims] 1. A recombinant plasmid containing a base sequence encoding a dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (II). 2. The recombinant plasmid according to claim 1, wherein the antiallergic pentapeptide portion consists of the amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine. 3. In the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (II), a fusion protein in which the antiallergic pentapeptide is a dimer (
Claim 1 wherein II) has the following amino acid sequence:
Recombinant plasmids as described. [There is a gene sequence. ] [There is a gene sequence. ] (In the formula, _ represents an amino acid that mediates dihydrofolate reductase and antiallergic pentapeptide.) 4. The base sequence encoding the fusion protein (II) in which the antiallergic pentapeptide is a dimer is as follows. The recombinant plasmid according to claim 3, which has the base sequence of [There is a gene sequence. ] [There is a gene sequence. ] (In the formula, _ represents a base sequence encoding an amino acid that mediates dihydrofolate reductase and antiallergic pentapeptide) 5. Dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (II) , a fusion protein in which the antiallergic pentapeptide is a trimer (
Claim 1 wherein II) has the following amino acid sequence:
Recombinant plasmids as described. [There is a gene sequence. ] [There is a gene sequence. ] (In the formula, _ has the same meaning as above.) 6. The set according to claim 5, wherein the base sequence encoding the fusion protein (II) in which the antiallergic pentapeptide is a trimer is the following base sequence. Replacement plasmid. [There is a gene sequence. ] [There is a gene sequence. ] (In the formula, _ has the same meaning as above) 7. The recombinant plasmid is stably replicated in E. coli, confers trimethoprim resistance and ampicillin resistance to the host E. coli, and has a size of 4658 base pairs. The recombinant plasmid according to claim 3, which is a recombinant plasmid. 8. The recombinant plasmid according to claim 5, wherein the recombinant plasmid is stably replicated in E. coli, confers trimethoprim resistance and ampicillin resistance to the host E. coli, and has a size of 4673 base material. 9. The recombinant plasmid according to claim 3, wherein the recombinant plasmid is pBBK8DM having the base sequence shown in FIG. 10. The recombinant plasmid according to claim 5, which is a recombinant plasmid pBBK9TM having the base sequence shown in FIG. 11. E. coli transformed with the recombinant plasmid according to claims 1 to 10. 12. Dihydrofolate reductase-antiallergic pentapeptide dimer fusion protein (II) having the following amino acid sequence. [There is a gene sequence. ] [There is a gene sequence. (In the formula, _ has the same meaning as above.) 13. Dihydrofolate reductase-antiallergic pentapeptide trimer fusion protein (II) having the following amino acid sequence. [There is a gene sequence. ] [There is a gene sequence. ] (In the formula, _ has the same meaning as above.) 14. Cultivating the Escherichia coli according to claim 11, and collecting the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (II) from the cultured bacteria. A method for producing a fusion protein (II) of dihydrofolate reductase-antiallergic pentapeptide multimer, characterized in that: 15. The step of collecting the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (II) consists of streptomycin treatment, streptomycin treatment,
15. The manufacturing method according to claim 14, which comprises purifying using ammonium sulfate precipitation and mesotrixate bond affinity chromatography sequentially. 16. Dihydrofolate reductase-antiallergic pentapeptide dimer fusion protein (II) or dihydrofolate reductase-antiallergic pentapeptide trimer fusion protein (II) according to claim 12 or 13. Aspartic acid-serine, which is characterized in that an anti-allergic pentapeptide consisting of an amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine is obtained by enzymatic treatment, chemical treatment, or a combination of these treatment methods. -Aspartic acid-Glycine-
A method for producing an anti-allergic pentapeptide consisting of a lysine amino acid sequence. 17. The production method according to claim 16, wherein the enzyme is trypsin.