JPH02299589A - Fused protein of dihydrofolic acid reductase and anti-allergic pentapeptide - Google Patents

Abstract

PURPOSE:To produce DHFR-DSDGK fused protein in a mass by transforming an E.coli with a recombinant plasmid containing a base sequence coding a DHFR-DSDGK fused protein. CONSTITUTION:An E.coli is transformed with a recombinant plasmid containing a base sequence coding dihydrofolic acid reductase-anti-allergic pentapeptide (DHFR-DSDGK) fused protein. The transformed E.coli is cultured and the objective DHFR-DSDGK fused protein is separated from the cultured bacterial cell. The separation of the DHFR-DSDGK fused protein is preferably carried out by purifying a cell-free extract of the cultured cell by successively treating the extract by an ion exchange column, a methotrexate-bonded affinity column chromatography and an anion exchange column chromatography.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to dihydrofolate reductase (hereinafter abbreviated as DIIPR) and an anti-allergic peptide aspartic acid (Asp)-serine (Ser)-aspartic acid. (A
sp)-Glycine (Gay)-Lysine (Lys) peptide (hereinafter referred to as DSDGK)
It is abbreviated as ), Escherichia coli containing the plasmid, dihydrofolate reductase-antiallergic pentapeptide fusion protein (hereinafter abbreviated as DHPR-DSDGK), DI (FR -Production method of DSDGK and DSD
This relates to the manufacturing method of GK. The present invention can be effectively utilized in fields such as fermentation industry 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 preventive or therapeutic agent for allergic rhinitis, allergic dermatitis, bronchial asthma, and other allergic diseases caused by chemical mediators.

The technical background of the present invention is so-called genetic manipulation technology. So far, nothing is known about a plasmid incorporating a gene encoding DSDGK and its expression in E. coli.

Generally, polypeptides with a molecular weight of 10,000 or less do not exist stably because they are degraded by proteases and the like even when produced in a host such as E. coli. This is thought to be because the molecules are too small to adopt a stable conformation.

Therefore, when attempting to produce a short polypeptide such as DSDGK using genetic engineering technology, it is necessary to create a fusion gene and express it as a fusion protein. For example, Itakura et al. reported the synthesis of somatostatin, which consists of 14 amino acids, using genetic manipulation. Their method involves chemically synthesizing the gene encoding somatostatin, fusing it with the β-galactosidase gene, integrating it into a multicopy plasmid, introducing the recombinant plasmid into E. coli, and translating the carboxy-terminal side of β-galactosidase into is expressed as a fusion protein in which somatostatin is fused via methionine (K, It
Akura et al, +5science, vol.
198+pl), 1056 (1977)) are considered to be obstacles in the isolation and purification of the product due to the insolubility of the fusion protein and the lack of easily measurable enzymatic activity in the fusion protein.

[Problem to be solved by the invention]

The object of the present invention is to synthesize DSDGK by genetic engineering technology, and for this purpose, it is an attempt to create an effective plasmid incorporating a gene encoding DSDGK, and in particular, following the example of somatostatin mentioned above. Since there are problems with expressing DSDGK and β-galactosidase as a fusion protein, we first attempted to develop a recombinant plasmid containing a gene that expresses a fusion protein that is soluble and has easily measurable enzymatic activity. It is something. By using a bacterial cell containing such a recombinant plasmid, it is possible to produce a fusion protein containing DSDGK, and by using easily measurable enzyme activity, the fusion protein can be easily purified. .

(Means for Solving the Problems) As a result of intensive research, the present inventors found that the problem described in item 1 can be solved by using the E. coli dihydrofolate reductase gene, and based on that knowledge,
The present invention was completed by creating a recombinant plasmid incorporating a gene encoding a fusion protein of DSDGK and DSDGK.

That is, the present invention provides: (1) A recombinant plasmid containing a base sequence encoding a dihydrofolate reductase-antiallergic pentapeptide (D) IFR-DSDGK) fusion.

(2) The above (2) wherein the antiallergic bentapetide portion of the fusion protein consists of the amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine.
1) Recombinant plasmid described.

(3) The above (1) in which the dihydrofolate reductase-antiallergic pentapeptide (DHF, R-DSDGK) fusion protein has the following amino acid sequence:
Recombinant plasmids as described.

Met Ice Ser Leu lie Ala A
la Leu Ala ValAsp Arg V
al Ice Gay Met Glu A
sn Ala MetPro Trp Asn
Leu Pro Aha Asp Leu
Ala TrpPhe Lys Arg
Asn Thr Leu Asn Lys
Pro Valle Met Gly Ar
g Old s Thr Trp Glu Ser
IceGly Arg Pro Leu
Pro Gly Arg Lys Asn
l1e11e Leu Ser Ser Gi
n Pro Gly Thr Asp As
pArg Vat Thr Trp Val Lys
Serνal Asp GluAla Ile A
la Aha Cys Gay Asp V
al Pro Glulle Metνal Il
e Gly Gly Gly Arg Val Tyr
Glu Gln Phe Leu Pro
Lys Ala Gln Lys LeuTy
r Leu Thr 1lis Ile A
sp Ala Glu Val GluGly
Asp Thr Old s Phe Pro
Asp Tyr Glu Pr.

8sp Asp Trp Glu Ser
Val Phe Ser Glu PheHi
s Asp Ala Asp Ala Gl
n Asn Ser 1lis 5erTyr
Glu Phe Glu Ile Leu
Glu Arg ArB IleArg A
sp Ser Asp Gly Lys(4)
The recombinant plasmid according to (1) above, wherein the base sequence encoding a fusion protein of dihydrofolate reductase-antiallergic pentapeptide (DHFR-DSDGK) is the following base sequence.

GAT GACTGG GAA TCG GT
ATTCAGCGAA TTC (5) The recombinant plasmid described in (1) above, which is stably replicated in E. coli, confers trimethoprim resistance and ampicillin resistance to the host E. coli, and has a size of 4204 base pairs. Replacement plasmid.

(6) The recombinant plasmid according to (1) above, wherein the recombinant plasmid is the recombinant plasmid pBBK2MA having the base sequence shown in FIG.

(7) E. coli transformed with the recombinant plasmid described in (1) above.

(8) Dihydrofolate reductase-antiallergic bentabepti I” (DIIFR-D) having the following amino acid sequence:
SDGK) fusion protein.

Met Ile Ser Leu Ile
Ala Aha Leu Ala Vat 8s
To ArB Val lie Gly Me
t Glu Asn Aha MetPro
Trp Asn Leu Pro Ala
Asp Leu Ala TrpPhe L
ys Arg Asn Thr Leu A
sn Lys Pro Valle Met
Gly Arg Old s Thr Trp
Glu Ser TleGly Arg P
ro Leu Pro Gly Arg L
ys Asn l1e11e Leu Ser
Ser Gin Pro Gly Thr
Asp Asp8r Val Thr
Trp Val Lys Ser Val
spG] u to Ia Ile Ala Ala
Cys Gly Asp Val Pro
Glulle Met Val Ile G
ly Gly Gly Arg Val T
yrGlu Gin Phe Leu Pro
1. ys Ala Gln Lys 1.
cuTyr Leu Thr old s Ile
Asp Ala Glu Val GluG
ay Asp Thr l1is Phe Pro A
sp Tyr GIu Pr.

Asp Asp Trp Glu Ser Val P
he Ser Glu P11eH4s Asp
Ala Asp ΔIa Gin Asn
Ser 1lis 5erTyr Glu P
he Glu Ile Leu Glu A
rg Arg IleArg Asp Ser
Asp Gly Lys (9) The Escherichia coli described in (7) above is cultured, and the cultured cells are treated with dihydrofolate reductase-antiallergic pentapeptide (DHPR-DSD).
GK) A method for producing a dihydrofolate reductase-antiallergic pentapeptide fusion protein, which comprises collecting the fusion protein.

00) Dihydrofolate reductase-antiallergic pentapeptide (DHF) l-DSDGK) fusion protein is collected from a cell-free extract of cultured bacterial cells by ion exchange column treatment, mesotrixate-coupled affinity column chromatography, and The manufacturing method described in (9) above, which includes a step of purifying using anion exchange column chromatography sequentially.

(11) Dihydrofolate reductase-antiallergic pentapeptide (DIIFR-DSDGK) described in (8) above
1. A method for producing an anti-allergic pentapeptide comprising an amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine, which comprises allowing arginyl endopeptidase to act on a fusion protein.

It is related to.

The details of the present invention are as follows: (1) Creation of novel recombinant plasmid (2) Transformed E. coli (3) DHPR-DS
In order of the manufacturing process of DGK fusion protein (4) Obtained DHPR-DSDGK fusion protein (5) D, 5D
The manufacturing of GK will be explained.

(1) Creation of new recombinant plasmid i) Preparation of DNA encoding DSDGK 1,
5'-GATCCGCGATTCAGATGGC88A
T88-3'2, 5'-to CG to ATTTG (
:CATCTGAATCGCG-3' DNA consisting of two 25 nucleotides was chemically synthesized and heated at 60°C.
Both DNAs were annealed by incubation with the following double-stranded DNA.

5'-GATCICGCIGATTCAGATGGCA
AAITAAl -3'3'-IGCGjCTA
The site of the double-stranded DNA that binds to the AGTCTACCGTTTlATTIGCGC-5' cleavage site is clearly indicated. )i
i) Insertion of the DNA encoding DSDGK into the vector plasmid As a vector for expressing the DNA encoding DSDGK, the recombinant plasmid pLEK1 (Japanese Patent Application No. 79681/1983), which has already been prepared by the present inventors, is used. ) was used. pLEK 1 is stably retained in E. coli, and the E. coli containing pLEK 1 was sent to FERM at FIKEN.
It has been deposited as BP-1818. pLEK 1 can be isolated and purified from E. coli containing pLEKl according to a conventional plasmid isolation method and used.

The DNA fragment obtained by cutting the purified p[, EK 1 with Sekiguchi and Mlu I was combined with the double-stranded DNA of i) above and T4-
Ligate using DNA ligase to create the new plasmid pBB
Obtain K2MA.

iii) Base sequence of new plasmid pBBK2MA Figure 1 shows the entire base sequence of pBBK2MA of the present invention. The figure shows the DNA sequence of one of the double-stranded circular DNAs at the beginning of 5'-ATCGΔT-3', the cleavage recognition site of the restriction enzyme Ridan I, which is the only one present in the plasmid.
8" as number 1, and written in the direction from the 5' end to the 3' end. pBBK2MA of the present invention is a novel recombinant plasmid. pBBK2MA has a size of 4204 base pairs, Trolimethoprim and ampicillin resistance can be imparted to host E. coli. pBBK 2 MA consists of 4204 base pairs, and pLIEKl (consists of 4207 base pairs)
The larger DNA fragment (consisting of 4179 base pairs) obtained by SHI and Mlu I digestion of
A 25 base pair chemically synthesized N containing the GK-coating sequence
It has a structure in which A is combined. In Figure 1, 533
The sequence from position 557 to position 557 is a sequence derived from chemically synthesized NA. The other sequences are pLEK1-derived sequences. By the way, the entire nucleotide sequence of pLEK 1 has already been revealed by the present inventors (Japanese Patent Application No. 1986-7).
9681), 53 of the base sequences shown in Figure 1
This structure has the sequence from position 3 to position 557 replaced with the 28 base pair DNA sequence shown below.

5'-GATCCGTATGTACGGTGGTTTC
CTGT/IA-3' The sequence of the chemically synthesized DNA includes a recognition cleavage site for restriction enzyme I, 5'-ACGCGT-
3' (array from 557th to 562nd in Figure 1)
, is included.

Since there is a Mlu 1 site in the pLEK 1-derived part, foreign DNA is introduced in a specific direction, and DHFR
A fusion gene with the if trochanter can be easily created.

The sequence from 57th to 554th in Figure 1 is llH
DIIFR-DSDGK, in which DSDGK is bound to the F1 lupoxy terminal side via arginine, is encoded.

Upstream of the sequence encoding DIIPR-DSDGK is D.
HPR-DSDG) [Sequences exist that allow efficient gene expression (Japanese Patent Laid-Open No. 63-267276).

In other words, the sequence from the 43rd to the 50th is called the SD sequence, and is useful for efficient translation.
The second position, 190190, is a consensus transcription promoter and contributes to efficient transcription.

From this, pBBK2MA produces a large amount of DHFR-DSDGK when introduced into E. coli. D made
HP R-DSDGK is in a soluble state within the bacterial body,
It accumulates about 20% of the bacterial protein. Trimethoprim is an inhibitor of this DI (PR) and DHPR-
By accumulating a large amount of DSDGK, pBBK
E. coli with 2MA becomes trimethoprim resistant. Furthermore, pBBK2MA has a gene derived from pLEK1 that confers resistance to ampicillin. From this, E. coli into which pBBK2MA has been introduced also exhibits resistance to ampicillin. pBBK2MA is introduced into E. coli and kept stable.

pBBK2MA having such features can be constructed according to Example 1, but the present invention is not limited by the method for constructing the recombinant plasmid.

(2) Transformed E. coli pBBK2MA prepared in (1) above is incorporated into E. coli according to a conventional method. This transformed E. coli exhibits resistance to trimethoprim and ampicillin. pB
E. coli containing BK2M8 is
)ll? As a result of efficient expression of the R-DSDGK gene, a large amount of DIlPR-DSDGK is accumulated in the bacterial body in a soluble state. E. coli containing pBBK2MA was transformed into YT
+Ap medium (in 1 ml of medium, 5 g of NaCl, 8 g of 1
- Ribton, 5g yeast extract, and LOOmg
using a liquid medium containing ampicillin sodium).
When cultured at 37°C to stationary phase, DI (F
R-DSDGK 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 sonication method, and then separated into supernatant and precipitate using centrifugation, almost all of DHPR-DSDGK is recovered in the supernatant. Escherichia coli containing pBBK2MA
hiacol i) IIBIOI/pBBK 2 M
A is FEI??l to FEI?
BP-2389).

(3) Manufacturing process of DIIPR-DSDGK fusion protein The manufacturing process of DIIPR-DSDGK of the present invention includes i.
) Culture of bacterial cells, 11) Crushing of bacterial cells, 1ii) DE meter-Toyovar column treatment, iv) Mesotrixate (MTX)
) binding affinity chromatography, and v) D
(Go to E8-) It is made up of each step of Yopal Lamb chromatography.

Culture of Escherichia coli containing pBBK2MA is carried out using YT+AP.
It can be cultured in a medium (liquid medium containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract and 50 mg of ampicillin sodium in medium Il). In addition to this medium, ST+Ap medium (medium II
! , a liquid medium containing 2 g of glucose, 1 g of disodium phosphate, 5 g of polypeptone, 5 g of yeast extract and 50 n+g of sodium ampicillin.

) can be used as long as it allows the bacterial cells to grow, but as far as we have investigated, DHPR-
YT1Ap medium was optimal for the production of DSDGK.

E. coli containing pBBK2MA was inoculated into the medium, and 3
Culture at 7°C until late logarithmic growth phase or stationary phase. The amount of DIIPR-DSDGK 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 bacterial cells were rotated at 5,000 revolutions/
Collect by centrifugation for minutes. Medium 1! From this, bacterial cells with a wet weight of 2 to 4 g can be obtained.

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

ii) Buffer the bacterial cells obtained by crushing and culturing the bacterial cells to 3 times the wet weight. ff1
l (10mM potassium phosphate buffer containing 0.1mM sodium ethylenediaminetetraacetate (EDTA), p
H7,0) and crush the bacterial cells using a French press. The cell suspension is centrifuged at 5,000 rpm for 10 minutes to obtain a supernatant. Furthermore, the supernatant was added to 35,000
Ultracentrifugation is performed at rotation/min for 1 hour to obtain a supernatant (this supernatant is hereinafter referred to as cell-free extract).

1ii) DEAE-)Jovar column treatment This operation is performed for the purpose of pretreatment for the next purification step. The cell-free extract was applied to a DEAE-) Jovar force ram pre-equilibrated with Buffer 1 containing 0.1M KCI and
Wash the column with buffer 1 containing CI. Enzyme elution is 0
．． Buffer 1 containing 3M KCI is used. Fractionate a certain amount of the eluate using a fraction collector.

The DHPR activity of the fractionated eluate is measured, and both fractions containing enzyme activity are collected.

iv) MTX binding affinity chromatography The enzyme solution obtained by the above operation is adsorbed onto an MTX binding 5 epharose affinity column equilibrated with buffer 1 in advance. After adsorption, wash with buffer 2 containing IM KCI (10mM potassium phosphate buffer containing 0.1mM EDTA, pH 8.5). For washing, measure the absorbance of the eluate from the column at 280 nm, and if the absorbance is 0.
．． Continue to flow the same buffer solution until it becomes 1 or less. Elution of the enzyme is performed using Buffer 2 containing IM MCI and 3mM folic acid, and a fixed amount of the eluate is fractionated using a fraction collector. DHPR on the fractionated eluate
Measure the activity and collect both fractions containing enzyme activity. The obtained enzyme solution is dialyzed against buffer solution 1 three times. At this stage, DHFR-DSDGK with a purity of 90% or more is obtained.

V) DEAE-1-Yopard Lamb chromatography Dialyzed enzyme solution was equilibrated with buffer solution 1 in advance.
Adsorb onto EAE-Toyopearl column. After adsorption, 0.
Wash with buffer 1 containing 1M MCI. For washing, measure the absorbance of the eluate from the column at 280 nm, and if the absorbance is 0.
．． Continue to flow the same buffer solution until it becomes below 0.01. Enzyme elution was performed using buffer 1 with MCI of 0.1M to 0.3M.
A linear concentration gradient is used to fractionate a fixed amount of the eluate using a fraction collector. The absorbance at 280 nm and DHPR activity of the fractionated eluate are measured. Both values of enzyme activity/absorbance at 280 nm are collected.

Through the above operations, DHPR-DSDGH can be highly purified and homogenized with good reproducibility.

According to the present invention, purification of DI (PR-DSDGK) can be performed within one week including culturing, and the recovery rate is 20%.
With the above steps, a uniform enzyme preparation can be obtained.

DHPR enzyme activity was measured using a reaction solution (0.05mM dihydrofolic acid, 0.06mM NADPH, 12mM 2-mercaptoethanol, 50rnM phosphate buffer (pH 7).
.

One unit of enzyme reacts at 1 unit per minute under the above reaction conditions.
It is defined as the amount of enzyme required to reduce micromoles of dihydrofolate. This measurement can be easily performed using a spectrophotometer.

(4) Obtained D) IFI? -1) SDGK fusion protein Figure 2 shows the DNA sequence of the portion encoding DIIPR-DSDGK and the amino acid sequence of the protein made from it. DIIPR-DSDGK is 16
It is a novel protein consisting of 6 amino acids. The sequence from ■ to position 160 counting from the amino terminal side is p
1. This is the amino acid sequence of D II FR produced by E K1, and the sequence from positions 162 to 166 is DSDGK. The amino acid immediately preceding the sequence of DSDGK is arginine (Arg).

As a result, by treating DHPR-DSDGK with arginyl endopeptidase enzyme, DSDGK
can be cut out. The molecular weight of DIII'R-DSDGK is 18.796.

Note that DHPR-DSDGK has DHPR enzyme activity despite having a structure in which DSDGK is bound to the carboxy terminal side of DHPR. Therefore, when E. coli produces a large amount of DI (PR-DSDGK), DI
It becomes resistant to trimethoprim, an IFR inhibitor and antibacterial agent.

(5) Manufacture of DSDGK D) IFI in (4) above? The amino acid immediately before the DSDGK sequence in the -DSDGK fusion protein is arginine (Arg), and this D)IFR-DSDG fusion protein is enzymatically treated with arginyl endopeptidase to obtain DSIIGK.

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

(Example 1) The DNA used to encode the 5DGK created into pBBK2M is 1, 5'.
-GATCCGCGATTCAGATGGCAAATA
A-3'2, 5'-CGCGTTATTTGC,C
DNA consisting of two 25 nucleotides ATCTGAATCGCG-3' was synthesized using a chemical synthesizer (manufactured by Applied Biosystems, 380B) according to the phosphoramidite method.
), and after purification using an oligonucleotide purification cartridge (manufactured by Applied Biosystems), the DNA
Take approximately 0.02 ml of the DNA (containing approximately 0.1 Mg of DNA) and incubate it at 60°C to anneal both DNAs to form the following double-stranded DNA.
Get A 5'-GATCCGCGATTCAGATGGC
888cho88 -3'3'Hehehe Hehe GC
To GCT 88 GT, TACCGTTTATTGCGC
-5' (hereinafter referred to as DNAI). Approximately 1 μg of purified pL[EKl was transferred to Ram1ll (
After cutting with 20 units of Mlu I (manufactured by Zenshuzo Co., Ltd.) and 20 units of Mlu I (manufactured by Zenshuzo Co., Ltd.), they were separated by 0.85% agarose gel electrophoresis. A DNA fragment of approximately 4.2 kilobase pairs was excised and the DNA was recovered from the gel (D
(referred to as NA2). The operation of cutting DNA by the method of ``Molecular Cloning'' and ``Molecular Cloning''
A Laboratory Manual” (T, Man
iatis, E., F., Frltsch, J., Sambr.
ook.

eds, cold Spring Harbor La
boratory (1982), hereinafter referred to as Document 1. ) according to the method described. DNA 2 to 4
5μ! Reaction solution for glue gauze (10mM Tris-HC
I pH 7, 4, 5mM MgCl2, 10mM dithiothreitol, 5mM
A1 was added, and 1 unit of 74-DNA ligase (manufactured by Zenshuzo Co., Ltd.) was added thereto to carry out a DNA ligation reaction at room temperature for 2 hours. This reaction product was transformed using a transformation method (tra
Escherichia coli (E, coli 1 (BIOI)
Competent cells (manufactured by Zenshuzo Co., Ltd.) were incorporated.

The treated bacterial cells were treated with loomg/ffi ambisilanna 1-lium and 5mg/i! Nutrient agar medium containing trimethoprim (in medium 1!, 2 g glucose, 1 g dipotassium phosphate, 5 g yeast extract,
Contains 5g polypeptone and 15g agar. ) and cultured at 37°C for 24 hours.
We were able to obtain several colonies.

These colonies were transferred to 2 ml of YT+Ap medium (Culture 1
! 5g of NaC inside! , 8g tryptone, 5g yeast extract, and 100mg ampicillin sodium. ), the bacterial cells were cultured at 37°C for 1 night. Transfer each culture solution to an Eppendorf centrifuge tube and incubate for 12
．． The cells were centrifuged at 000 rpm for 10 minutes and collected as a precipitate. To this, 0.1 mfl of electrophoresis sample preparation solution (0,0709M Tris-HCI,
Sodium lauryl sulfate (S
DS), 11% glycerin, 5% 2-mercaptoethanol, and 0.045% bromophenol blue) were added to suspend the bacterial cells, and this was kept in boiling water for 5 minutes. , lysed the bacterial cells. The sample after this processing is 5DS
- Polyacrylamide electrophoresis (U., Laemm)
li; Nature, vol, 227. p680(1
970)). D as a standard sample
11 FR and lactalbumin (molecular weight 14,200) as a molecular weight marker, trypsin inhibitor (
molecular weight 20.100), trypsinogen (molecular weight 24
．． 000), carbonic anhydrase (molecular weight 29
,000), glyceraldehyde-3-phosphate dehydrogenase (molecular weight 136,000), egg albumin (molecular weight 45.000), and live serum albumin (molecular weight 66.000).
000) containing samples (DALTON MARK■-
LTM (manufactured by Sigma) was run on a 10 to 20% polyacrylamide concentration gradient gel (manufactured by Daiichi Chemical Co., Ltd.). As a result, all three colonies were pLEK
The hand of DHPR-leucine enkephalin fusion protein 1 disappears, and DHPI? It has been revealed that a new protein that is approximately 500 Daltons larger than other proteins is being produced. Select one or two appropriately from these three and culture them in YT+AP medium.
eisblum;J, Bacteri-ology v
Plasmids were prepared according to J.D. ol, 12L p, 354 (1975)). The obtained plasmid is pBBK
2 Named MA. 2MA to pBI3, pLEK
It should have a structure in which the sequence between the part 1 and the part 1 is synthesized and replaced with NA. For synthetic NA,
Sequence recognized by restriction enzyme 11, 5'-ACGC
Since it contains GT-3', pBBK with Mlul
When I tried to disconnect 2MA, it was indeed disconnected. It was also obtained by cleavage of pBBK 2MA with L-R1 (manufactured by Zenshuzo Co., Ltd.) (sequences 471-476 in Fig. 1) and 5aII (manufactured by Zenshuzo Co., Ltd.) (sequences 563-568 in Fig. 1). The dideoxy method using M13 phage (J, Mess.
ingHMethods in Bnzymology
, vol. 1OL, p. 20 (1983)).
The base sequence was determined in the direction from EcoRI to 5a11.

As a result, the sequence from 471st to 568th of the entire base sequence of pBBK 2 MA shown in FIG. 1 was confirmed.

In addition, approximately 4.2 kilobase pairs of DNA obtained by Bam old-3all cleavage of pBBK2HA is
T, uniform 11.1° anus 1 ■ 1. Sarashi I, Hayo■, Rika■
As a result of a restriction enzyme cleavage experiment using , SuaT (manufactured by Zenshuzo Co., Ltd.) and Aatll (manufactured by Toyobo Co., Ltd.), pL
IEK 1 double aim old - gong (approximately 4 pieces obtained by 1 cutting)
．． It was shown to be exactly identical to that of 2 kilobase pairs.

From the above results, it was determined that the entire base sequence of p138K 2 MA was the sequence shown in FIG.

(Example 2) DIIPI produced by Escherichia coli containing pBBK 2 MA
The amino acid sequence of I) IIPR-DSI) GK produced by E. coli containing I-DSDGKpBBK 2 M8 is D
It can be predicted from the sequence of the HPR-DSDGK gene. The array from 57th to 554th in Figure 1 is D
Since IIPR-DSDGK is encoded, the amino acid sequence was estimated using a triplet code table. As a result, the amino acid sequence shown in FIG. 2 was obtained. pBBK
2 From E. coli containing MA, D liver 17-DSDG
K was separated and purified, and the purified protein was used to confirm the following.

Purification IA of DIIFR-DSDGK A. Amount of bacterial cells used: Wet weight 13 g. B. Enzyme purification step: Shown in Table 1 below. (■ in the table
indicates cell-free extract, ■ indicates DEAE-TOYOPEAR column (Toyopearl ion exchanger, DEAE-TOYOPEAR
L650 manufactured by Tosoh Corporation) processing, ■ is manufactured by Shiso Corporation 8g rotor
se (MTX-agarose, manufactured by Sigma) affinity chromatography, and ■ DEAE-1-Yobal column (Toyopearl ion exchanger, DEAR-TO)
YOPEARL650 (manufactured by Tosoh Corporation)) Chromatogushiso Corporation (manufactured by Chromatogushiso Corporation) For enzyme purification, the cell-free extract of
This was carried out by subjecting it to the purification step (2). ) Table 1 ■ 47 731 6,167 100 ■ 70 44
4 5.388 87.4■ 30 38 1,47
7 24.0D HP R enzyme activity was determined by reaction solution (0,0
5mM dihydrofolic acid, 0.06mM NADPII
, 12mM 2-mercaptoethanol, 50mM phosphate buffer (pH 7.0)) were placed in a 1mρ cuette, an enzyme solution was added thereto, and the temperature was determined by measuring the change in absorbance at 340nm over time. One unit of enzyme was defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute under the reaction conditions described in section 1.

When the obtained enzyme protein was analyzed by SDS electrophoresis (method described in the above example), approximately 21,50
No single protein hand was shown, indicating that the enzyme preparation obtained was homogeneous.

(Example 3) Purified DIIFR-DSDGK solution (2.2 mg/m
Add 1 ml of 1 mg/ml arginyl endopeptidase (manufactured by Zenshuzo Co., Ltd.) solution to 5 ml of IM Tris-HCl (p) (8,0) 1 ml,
Incubated overnight at 37°C. After the reaction, it was concentrated to about 1 ml using a centrifugal freeze dryer. This 0.1
mfl using high performance liquid chromatography equipment (Hitachi 655
type) using a reversed-phase column (Waters Nova Pak
Cl8). ? A 0.1% trifluoroacetic acid aqueous solution was used for elution, and fractions with an elution time of 2 to 3 minutes were collected. The column was regenerated by flowing acetonitrile containing 0.1% trifluoroacetic acid.

Separation was repeated under the same conditions, and the collected fractions were dried in a centrifugal freeze dryer and dissolved by adding 1 mR of purified water. This was separated again using a gel filtration column (Asahi Back G5-320H) using a high performance liquid chromatography device. Elution was performed using 0.05M ammonium acetate buffer pH 6,7 at a flow rate of 0.1 mR/min. The antiallergenic pentapeptide DSDGK is approximately 6
It was eluted at the 0 minute position. When the DSDGK fractions were collected, 45 μg of peptide was recovered.

[Effect of the invention] As described above, the novel recombinant plasmid pBBK of the present invention
2 MA encodes DHPR-DSDGK, and E. coli harboring pBBK 2 old encodes DIIPR-DSDGK.
DSDGK is accumulated and produced in large quantities in a soluble state. Furthermore, the generated DHPR-DSDGK is DIIFIII
It retains enzymatic activity and can be easily purified. By following the purification method of the present invention, DIIFII-
DSDGH can be purified quickly and efficiently.

Further, the DHFI'1-DSDGK obtained in this way is then converted to D 11 P by an appropriate enzyme or chemical treatment.
After cleavage between R and DSDGK, D S 1) G K can be isolated and purified by a combination of known separation techniques such as high performance liquid chromatography, gel filtration, and electrophoresis. The purified ItsDGK is useful as a therapeutic agent for allergic diseases. Therefore, the present invention has provided an extremely advantageous means for producing 5DGK, which is useful as a therapeutic agent for allergic diseases, by genetic engineering means.

[Brief explanation of drawings]

FIG. 1 shows the entire base sequence of pBBK2M, in which only one DNA 8M sequence of the double-stranded DNA is written in the direction from the 5' end to the 3' end. 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 number 1, starting from the first groove of the restriction enzyme history cleavage recognition site, 5'-8TCGAT-3', which exists in pBBK2MA. is the Kano FR-DSD present in pBBK2MA
FIG. 2 is a diagram showing the base sequence of the portion encoding GK and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A is adenine, C is cytosine, G is guanine, T is thymine, Gly is glycine, Al
a stands for alanine, Val stands for valine, Leu stands for leucine, Ile stands for isoleucine, Set stands for serine, T
hr is threonine, Cys is cysteine, Met is methionine, Asp is aspartic acid, 8sn is asparagine, Glu is glutamic acid, Gin is glutamine, Arg is arginine, Lys is lysine,
His stands for histidine, Phe stands for phenylalanine,
Tyr is tyrosine, Trp is tryptophan, Pr
o indicates proline. The numbers in the figure indicate the numbers counted starting from methionine, which is the amino terminal amino acid of the protein. FIG. 3 shows a high performance liquid chromatogram on a gel filtration column of a fraction obtained by treating DHPR-DSDGK with arginyl endopeptidase and collecting it on a reverse phase column. The horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units. The arrow in the figure indicates the position where the antiallergic pentapeptide DSDGK was eluted.

Claims (11)

[Claims] (1) A recombinant plasmid containing a base sequence encoding a dihydrofolate reductase-antiallergic pentapeptide fusion protein. (2) The recombinant plasmid according to claim (1), wherein the antiallergic pentapetide portion of the fusion protein consists of the amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine. (3) The recombinant plasmid according to claim (1), wherein the dihydrofolate reductase-antiallergic pentapeptide fusion protein has the following amino acid sequence. [There is a gene sequence] (4) The recombinant plasmid according to claim (1), wherein the base sequence encoding the dihydrofolate reductase-antiallergic pentapeptide fusion protein is the following base sequence. [There is a gene sequence] (5) The recombinant plasmid according to claim (1), 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 4204 base pairs. . (6) The recombinant plasmid according to claim (1), wherein the recombinant plasmid is a recombinant plasmid pBBK2MA having the base sequence shown in FIG. (7) Escherichia coli transformed with the recombinant plasmid according to claim (1). (8) A dihydrofolate reductase-antiallergic pentapeptide fusion protein having the following amino acid sequence. [There is a gene sequence] (9) A dihydrofolate reductase-antiallergic pentapeptide characterized by culturing the Escherichia coli according to claim (7) and collecting the dihydrofolate reductase-antiallergic pentapeptide fusion protein from the cultured cells. Method for producing fusion proteins. (10) The step of collecting dihydrofolate reductase-antiallergic pentapeptide fusion protein involves ion exchange column treatment, mesotrixate-coupled affinity column chromatography, and anion exchange column chromatography from a cell-free extract of cultured bacteria. The manufacturing method according to claim 9, which includes the steps of sequentially using and purifying. (11) Aspartic acid-serine-aspartic acid-glycine characterized in that arginyl endopeptidase is made to act on the dihydrofolate reductase-antiallergic pentapeptide fusion protein according to claim (8).
A method for producing an anti-allergic pentapeptide consisting of a lysine amino acid sequence.