JP3193998B2 - Fusion protein of dihydrofolate reductase-antiallergic pentapeptide multimer - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to dihydrofolate reductase (hereinafter abbreviated as DHFR) and aspartic acid (Asp) -serine (Ser) -aspartic acid (Asp) -glycine ( Gly) -Lysine (Lys)
Consisting of five amino acid sequences (hereinafter referred to as DSDGK
Abbreviated. )) A novel recombinant vector capable of producing a large amount of the fusion protein (III) containing the multimer, Escherichia coli transformed with the recombinant vector, DHFR-DSDGK multimeric fusion protein (III), DHFR-DSDGK For the production of fusion protein (III), the production of DSDGK multimers and
It relates to the manufacturing method of DSDGK. INDUSTRIAL APPLICATION This invention is utilized effectively in fields, such as a fermentation industry and a pharmaceutical industry.

[Prior art] DSDGK is a kind of anti-allergic peptide, and is used as a preventive and therapeutic drug for allergic rhinitis, allergic dermatitis, bronchial asthma and other allergic diseases caused by chemical mediators. It is an interesting bioactive peptide expected to be used (Japanese Unexamined Patent Publication 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 (Japanese Patent Application Laid-Open No. 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, with the aim of increasing the production efficiency of DHFR, Iwakura and colleagues reported that the rrnB gene, which is known as an efficient
A recombinant plasmid pTP104-4 was prepared using P70-1 (Japanese Patent Application 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 a large amount of a fusion protein containing DSDGK by a genetic engineering technique. Therefore, a nucleotide sequence encoding a fusion protein containing a multimer of DSDGK is required. The purpose of the present invention is to prepare an effective vector into which is incorporated. This vector must satisfy the following requirements.

(I) It can be produced as a fusion protein containing a large number of DSDGKs repeatedly.

(Ii) Expression of the fusion protein gene in large amounts.

(Iii) The fusion protein to be produced is easily purified.

(Iv) DSDGK can be easily separated from the fusion protein.

[Means for Solving the Problems] As a result of intensive studies, the present inventors have completed the present invention by using the dihydrofolate reductase gene of Escherichia coli.

That is, the present invention relates to a set comprising a base sequence encoding a (dihydrofolate reductase)-(aspartate-serine-aspartate-glycine-lysine) n fusion protein (n is 11 to 15) having the following amino acid sequence: In the replacement vector.

Furthermore, the recombinant vector contains a fusion protein (II
A recombinant vector wherein I) has the following amino acid sequence is included.

Furthermore, the recombinant vector contains a fusion protein (II
A recombinant vector in which the nucleotide sequence encoding I) has the following nucleotide sequence is included.

Further, the above-mentioned recombinant vector includes a recombinant plasmid that is stably replicated in Escherichia coli and confers ampicillin resistance to the host Escherichia coli.

Further, the above-mentioned recombinant vector includes a recombinant plasmid pD314TX having a structure in which a base sequence of 30 bases between the restriction enzyme BamHI and HindIII cleavage sites of the plasmid pUC118 is replaced with the following base sequence.

Furthermore, the present invention relates to Escherichia coli transformed with the above recombinant vector.

The present invention also relates to a dihydrofolate reductase-antiallergic pentapeptide 15-mer fusion protein having the following amino acid sequence:

The present invention further comprises culturing the transformed Escherichia coli,
Production of a dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (III), comprising collecting a dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (III) from the culture It is also about the law.

The method for producing the dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (III) includes the following:
In some cases, the step of collecting the fusion protein includes a step of sequentially purifying the cell-free extract of the cultured cells using a streptomycin treatment and a mesotrixate binding affinity chromatography.

Furthermore, the present invention provides an aspartic acid-serine-aspartic acid-glycine-lysine by treating a fusion protein (III) of a dihydrofolate reductase-antiallergic pentapeptide multimer with an enzyme, a chemical treatment or a combination of these treatments. The present invention also relates to 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 the amino acid sequence of

Examples of the enzyme used for the above enzyme treatment include trypsin or lysyl endopeptidase.

In the present invention, the “dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (II
“I)” refers to a fusion protein of DHFR and a DSDGK multimer (11-mer or more). This fusion protein may be a protein having an extra peptide added to its carboxyl terminus. Further, an amino acid or an oligopeptide may be interposed between DHFR and the DSDGK multimer. Examples include arginine, lysine, leucine-methionine and isoleucine-glutamic acid-glycine-arginine [factor recognition sequence]. Therefore, the fusion protein of the present invention includes a fusion protein containing each of the above amino acids or oligopeptides.
The degree of polymerization of the multimer of the antiallergic pentapeptide is at least 11 mer, and the more it is within the range that does not reduce the productivity of the protein by the microorganism, the more desirable.

The nucleotide sequence encoding this fusion protein includes a region encoding DHFR and a region encoding DSDGK multimer, and even if a nucleotide sequence encoding an intervening amino acid or oligopeptide exists between these regions. Good.

A recombinant vector containing a nucleotide sequence encoding a fusion protein has a D sequence that repeatedly encodes many DSDGKs on the same chain.
It can be prepared by an ordinary method using an NA chain and a vector capable of expressing DHFR. In order to increase the expression level of the fusion protein, a commercially available terminator nucleotide sequence may be inserted immediately after the nucleotide sequence encoding the fusion protein. As an example of a vector capable of expressing DHFR, there is a recombinant plasmid pBBK10MM (described in Japanese Patent Application No. 2-123201) already prepared by Iwakura et al. pBBK10MM is stably retained in E. coli,
Escherichia coli containing BK10MM has been deposited with the Japan Institute of Fine Arts and Sciences as FERM BP-2394. pBBK10MM can be separated and purified from Escherichia coli containing pBBK10MM in accordance with a usual method for separating a plasmid.

The fusion protein is obtained by culturing Escherichia coli containing the above-mentioned recombinant plasmid in a large amount, disrupting the cells, and purifying from the cell-free extract. Microbial cells can be disrupted by ultrasonic waves or French press. Purification of the fusion protein from the cell-free extract can be performed by sequentially using streptomycin treatment and methotrexate (hereinafter abbreviated as MTX) binding affinity chromatography. MT
In X-binding affinity chromatography, a fraction containing the fusion protein is obtained using the enzyme activity of DHFR as an index.

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. 1 ml is placed in a cuvette, a sample is added to the cuvette, and the time change of the absorbance at 340 nM at 25 ° C. is measured. 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 is defined as one unit. This measurement can be performed using a spectrophotometer.

DSDGK degrades the purified fusion protein, and decomposes the degraded product by high performance fluid chromatography (hereinafter abbreviated as HPLC).
And can be obtained by fractionation. As a method for decomposing the fusion protein, there are methods of enzymatic treatment, chemical treatment, or a combination thereof. Examples of enzymes used in this enzyme treatment include trypsin and lysyl endopeptidase.

The DNA containing the nucleotide sequence encoding the repeat of the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein of the present invention was prepared by the following procedures (a) to (f).

(A) a single-stranded DNA consisting of two or more repeating units, each having a base sequence encoding a peptide as a repeating unit;
And (b) keeping the temperature and cooling to generate a double-stranded DNA in which both DNA strands are offset by at least one repeat unit or more, and (c) deoxyadenosine 5 ' -Triphosphate (dATP),
Deoxycytidine 5'-triphosphate (dCTP), deoxyguanosine 5'-triphosphate (dGTP), and deoxythymidine 5'-triphosphate (dTTP) (hereinafter abbreviated as four types of bases)
Is reacted in the presence of DNA polymerase to synthesize a complementary strand of the single-stranded DNA region into a double-stranded DNA, and (d) heating to convert the single-stranded DNA into a single-stranded DNA complementary thereto. (E) Perform the operations (b) to (d) at least once, and (f) Perform the operations (b) and (c).

This is described below with reference to FIG. In FIG. 3, (1) and (2) indicate that “a base sequence encoding a peptide is a repeating unit,
The single-stranded DNA composed as described above and the single-stranded DNA complementary thereto are shown. P and Q are each a nucleotide sequence encoding a peptide, and P 'and Q' are complementary sequences of P and Q, respectively.

The two single-stranded DNAs of (1) and (2) are kept warm and cooled, that is, heated to 70 to 100 ° C., and then slowly cooled to form a combination of regions related to the formation of complementary strands in the single-stranded DNA. Due to the difference, multiple types of double-stranded DNA are generated. That is, each single-stranded DNA is blunt-ended
In addition to the double-stranded DNAs complementary to each other, single-stranded DNAs are shifted and complemented to generate double-stranded DNAs whose ends are not blunt. One example is the double-stranded DNA of (3). Here, double-stranded DNA that is offset and complementary and whose ends are not blunt is used.

When four types of bases are reacted with the double-stranded DNA of (3) in the presence of a DNA polymerase such as Klenow fragment or Taq polymerase, the complementary strand of the single-stranded DNA region at the terminal portion of the double-stranded DNA is formed. It is synthesized to form the double-stranded DNA (4). The double-stranded DNA in (4) is longer than the double-stranded DNA in which the two single-stranded DNAs in (1) and (2) are complemented so that the ends are blunt, as shown by (L). The DNA strand is just extended. Since P and Q, which are the nucleotide sequences encoding the peptide, are present in the elongated DNA chain, the number of nucleotide sequences encoding the peptide contained in the DNA chain increases with the extension of the DNA chain. It is.

When this double-stranded DNA of (4) is heated, this double-stranded DNA
Dissociates into two single-stranded DNAs of (5) and (6).

When the two single-stranded DNAs of (5) and (6) are kept warm and cooled, the two single-stranded DNAs of (5) and (6) are added to the original double-stranded DNA of (4). The DNA shifts and complements, producing double-stranded DNA with blunt ends. One example is the double-stranded DNA of (7). Four bases are added to the double-stranded DNA of (7).
When the reaction is carried out in the presence of DNA polymerase, the complementary strand of the single-stranded DNA region at the end of the double-stranded DNA is synthesized (8).
Of double-stranded DNA is produced. The double-stranded DNA of (8) is longer than the double-stranded DNA of (4) by the length indicated by (M).

When the double-stranded DNA of (8) is heated, this DNA strand is dissociated into two single-stranded DNAs of (9) and (10).

The two single-stranded DNAs of (5) and (6) above are
When at least one cycle of cooling, reaction of the four bases in the presence of DNA polymerase and heating is performed at least once, and then the mixture is kept warm and cooled, and the four bases are reacted in the presence of the DNA polymerase. Double-stranded DN
A is obtained. One example is the double-stranded DNA of (11). Since P and Q, which are the nucleotide sequences encoding the peptide, are present in the extended portion of the DNA chain, the number of nucleotide sequences encoding the peptide contained in the DNA chain also increases with the extension of the DNA chain. To increase.

EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples.

Example 1 Preparation of Recombinant Vector Containing DNA Strand Having Repeatedly Many Nucleotide Sequences Encoding Antiallergic Pentapeptide (1) Nucleotide Sequence Encoding Peptide as Repeating Unit (Three Antiallergic Pentapeptides in Series) Preparation of Single-Stranded DNA Consisting of Encoding Base Sequence) Two oligonucleotides consisting of 45 bases shown in the following 1 and 2 were synthesized by a chemical synthesizer (Milligen, Model 7500) according to the phosphoramidite method.

After drying under reduced pressure, 10 mM Tris (hydroxymethyl) at pH 8.0 containing 1 mM disodium ethylenediaminetetraacetate.
Aminomethane (hereinafter abbreviated as Tris) -dissolved in hydrochloric acid buffer (hereinafter referred to as TE solution). 260n of this solution
The absorbance at m was measured and the oligonucleotide concentration was measured assuming an absorbance of 1.0 at 20 μg / ml. 0.2 pH of 8.0 containing 2 mM disodium ethylenediaminetetraacetate and 0.089 M boric acid.
16% polyacrylamide gel containing 7M urea (weight ratio of acrylamide and N, N'-methylenebisacrylamide is 19: 1) using a 089M tris-boric acid solution (hereinafter referred to as a TBE solution) as a buffer solution Use 70-100 μg
Was electrophoresed. After excising a gel containing oligonucleotides of the desired size, Smith's diffusion elution method [Smith, HO; Methods in Enzymolog
y, vol. 65, p. 371 (1980)], each oligonucleotide was dissolved in 30 μl of TE solution, and the single-stranded DN was dissolved.
Solution A was used.

(2) Preparation of a single-stranded DNA consisting of two or more repeating units and a single-stranded DNA complementary thereto, and a mixture thereof. Equal amounts of the two purified single-stranded DNAs are mixed, and each is mixed with T4 polynucleotide kinase. The 5 'end of the single-stranded DNA was phosphorylated. After inactivating the enzyme by heating at 70 ° C for 10 minutes,
It was cooled naturally to room temperature. This was ligated, and the single-stranded DNA was ligated by a phosphodiester bond. This was precipitated with ethanol to recover double-stranded DNA. The recovered double-stranded DNA was subjected to polyacrylamide gel electrophoresis and confirmed to be a double-stranded DNA having a chain length of two or more repeating units. The procedures of phosphorylation, ligation, ethanol precipitation and polyacrylamide gel electrophoresis are described in Molecular Cloning: A Laboratory Manual.
Manual) [T. Maniatis, EFFritsch, J. Sambrook, eds.,
Cold Spring Harbor Laboratory (1982) Hereinafter, the method of Maniatis et al. ].

(3) Generation of double-stranded DNA in which both DNA strands are complementary by being shifted by at least one repetition unit by heat retention and cooling Four incubators at 90 ° C, 75 ° C, 50 ° C and 37 ° C were prepared. 7 μl each of the single-stranded DNA solution obtained in (1) above
Starting from, the double-stranded DNA that has undergone the above operation (2) is converted to TE
After dissolving in 45 μl of the solution, the mixture was heated at 90 ° C. for 2 minutes to dissociate into two single-stranded DNAs. Subsequently, the mixture was sequentially cooled at 75 ° C. for 5 minutes, at 50 ° C. for 5 minutes, and at 37 ° C. for 5 minutes to obtain a double-stranded DNA complementary to each other with a shift of at least one repeat unit or more.

(4) Synthesis of complementary strand of single-stranded DNA region by reacting four kinds of bases in the presence of DNA polymerase A 10-fold concentration of synthesis buffer [dATP 5mM] was added to the obtained double-stranded DNA. , DCTP 5 mM, dGTP
5 mM, 5 mM dTTP, 10 mM ATP, 50 mM magnesium chloride and 2 mM dithiothreitol in 100 mM Tris-HCl buffer at pH 7.4] and 2 units of Klenow fragment (manufactured by Takara Shuzo). , And incubated for 20 minutes, and the single-stranded DNA present in the double-stranded DNA obtained in (3) above
The complementary strand of the region was synthesized to obtain double-stranded DNA.

(5) Heated single-stranded DNA and its complementary single-stranded D
Dissociation into NA The reaction solution (4) was heated at 90 ° C for 2 minutes, and the double-stranded DNA obtained in (4) was dissociated into a single-stranded DNA and a single-stranded DNA complementary thereto.

(6) Preparation of DNA chain having a large number of repeated base sequences encoding DSDGK The cycle consisting of the above (3) to (5) was repeated 15 times. The Klenow fragment was added at each cycle, and 1/10 volume of a 10-fold concentration of synthesis buffer was added every 5 cycles. Thereafter, the above operations (3) and (4) were performed.

(7) Incorporation of the obtained DNA chain into a plasmid vector The reaction solution of the above (6) was heated at 70 ° C for 10 minutes to inactivate the enzyme. This was digested with the restriction enzyme Hinc II, and treated with alkaline phosphatase.
Ligation with 19 (Takara Shuzo) was performed to obtain a ligation reaction product.

(8) Acquisition of Transformant and Analysis of Recombinant Vector According to the E. coli transformation method (the method of Maniatis et al.), The ligation reaction product obtained in (7) above was used to transform the ligation reaction product into E. coli MV.
It was incorporated into 1184 strain (a host bacterium of plasmid pUC118 manufactured by Takara Shuzo) to obtain a transformant. One of the obtained transformants was cultured to obtain a large amount of recombinant plasmid. Acquisition of large amounts of recombinant plasmids followed the method of Maniatis et al.

The resulting recombinant plasmid was named p1-9, digested with restriction enzymes EcoRI and HindIII, and the TBE solution was used as a buffer.
8% polyacrylamide gel (acrylamide and N, N '
-The weight ratio of methylene bisacrylamide was 29: 1. ), About 30 as shown in FIG.
A hand of 0 base pairs could be detected. In the figure, S indicates the injection position of the sample, and each dotted line and number indicate the position and size (unit is base pairs) of each molecular weight marker migrated in parallel. Arrows indicate the direction of electrophoresis.

The DNA sequence of the band of about 300 base pairs was determined by the dideoxy method of Sanger et al. [Sanger. F., Nicklen, S. & Couls
on, AR: Proc. Natl. Acad. Sci. USA 74, p-5463-5467 (1
977)], and it was confirmed that a base sequence having three base sequences encoding the amino acid sequence of aspartic acid-serine-aspartic acid-glycine-lysine was repeated five times. However, the last base was deleted. FIG. 5 shows the nucleotide sequence of the recombinant plasmid p1-9 inserted into pUC119. In the figure,
The numbers are counted from the cleavage site of pUC119 by the restriction enzyme Hinc II, and the number closer to the recognition cleavage site of the restriction enzyme Hind III is numbered 1. In the figure, symbols indicate nucleic acid bases, A indicates adenine, C indicates cytosine, G indicates guanine, and T indicates thymine.

p1-9 has a structure in which a base sequence repeatedly encoding an anti-allergic pentapeptide is inserted into pUC119 in the direction opposite to the direction of the lac promoter on pUC119. The base sequence shown in FIG. 5 is the base sequence described from the direction of the lac promoter, and the base sequence in the direction from the last base to the first base is complementary to the base sequence repeatedly encoding the anti-allergic pentapeptide. Base sequence.

Of the base sequences shown in FIG. 5, the first to eleventh base sequences encode this pentapeptide due to the deletion of bases when a DNA encoding the anti-allergic pentapeptide is repeatedly produced. The missing nucleotide sequence. In addition, E. coli containing p1-9 was
No. 11802 (FERM P-11802).

Example 2 Preparation of Recombinant Vector Having Nucleotide Sequence Encoding Dihydrofolate Reductase-Antiallergic Pentapeptide Multimeric Fusion Protein (III) Recombinant vectors were prepared by the following procedures (1) to (5). did. FIG. 6 shows the construction of this recombinant vector. In the figure, open arrows indicate the promoter and operator regions of the lactose operon, and solid arrows indicate DS.
DGK shows a region of nucleotide sequence encoding the multimers, arrows containing diagonal lines in represents a region of nucleotide sequence encoding the DHFR, Ap ^r represents the ampicillin resistance gene region,
r indicates a region having the base sequence of the universal translation terminator, dGCTTAATTAATTAAGC indicates the base sequence, and dCCTCCAGG indicates the base sequence of the linker of the restriction enzyme XhoI. Kb is an abbreviation for kilobase pairs.

(1) Plasmid p1-9 was replaced with restriction enzymes EcoR I and Hind I
After digestion with II, the digest was subjected to 8% polyacrylamide gel electrophoresis to separate a DNA fragment of 0.29 kilobase pairs (hereinafter abbreviated as kb).

(2) Plasmid pUC118 (Takara Shuzo) was replaced with restriction enzyme Ec
Digested with oR I and Hind III, and 0.29 kb DNA of the above (1)
The fragments were mixed and a ligation reaction was obtained using DNA ligase. In the same manner as in Example 1 (8), the ligation reaction product was incorporated into Escherichia coli MV1184 strain to obtain a transformant, and one of the transformants was cultured in large quantities to obtain a recombinant plasmid.

(3) The resulting recombinant plasmid was named p1-9RV, which was digested with the restriction enzyme PstI, and the resulting linear plasmid DNA was blunt-ended with Klenow fragment. Linker containing stop codon (Pharmacia universal translation terminator, dGCTTAATTA
ATTAAGC) was phosphorylated with T4 polynucleotide kinase, mixed with this linear plasmid DNA, and a ligation reaction was obtained using DNA ligase. Example 1
This ligation reaction was performed in the same manner as in (8).
Light transformants were obtained by incorporation into the MV1184 strain, and one strain of the light transformants was further cultured in large quantities to obtain a recombinant plasmid.

(4) The obtained recombinant plasmid was named p14T5. This recombinant plasmid contained three of the above linkers. This was digested with the restriction enzyme Hind III, and the ends of the resulting linear plasmid DNA were blunt-ended with Klenow fragment. And Xho I linker (Takara Shuzo, dCCTCGA
GG) was phosphorylated at the 5 'end with T4 polynucleotide kinase, mixed with this linear plasmid DNA, and a ligation reaction was obtained using DNA ligase. Example 1 (8)
This ligation reaction was performed in the same manner as in
A light transformant was obtained by incorporation into a strain, and one strain of the light transformant was further cultured in large scale to obtain a recombinant plasmid. The resulting recombinant plasmid was named p14TX.

(5) After the plasmid pBBK10MM was digested with the restriction enzyme BamHI, the ends of the resulting linear plasmid DNA were blunt-ended with Klenow fragment. This linear plasmid DNA was digested with restriction enzyme BglII, and the digest was subjected to 0.8% agarose gel electrophoresis to separate a 0.52 kb DNA fragment. p14TX was digested with restriction enzymes BamHI and HincII, mixed with the above 0.52 kb DNA fragment, and used as a ligation reaction product using DNA ligase.

(6) The ligation reaction product was incorporated into Escherichia coli MV1184 in the same manner as in Example 1 (8). 100 mg / l ampicillin sodium and 5 mg
nutrient agar medium containing 1 / l of trimethoprim and 1 mM of isopropyl-β-D-thiogalactopyranoside (containing 1 g of medium containing 5 g of sodium chloride, 5 g of yeast extract, 10 g of bactotripton and 15 g of agar) , Adjusted to pH 7.0 with NaOH), and cultured at 37 ° C for 24 hours to obtain a transformant. Plasmids were prepared from dozens of the obtained transformants according to the method of Maniatis et al. These plasmids were digested with restriction enzymes, and the desired recombinant plasmid was selected based on the cleavage pattern. The resulting recombinant plasmid was named pD314TX. FIG. 1 shows that the restriction enzymes BamHI and Hind of plasmid pUC118 among the entire nucleotide sequence of pD314TX are shown in FIG.
The base sequence replaced with the 30 base sequence between the III cleavage sites is shown. In the figure, symbols indicate nucleic acid bases, A indicates adenine, C indicates cytosine, G indicates guanine, and T indicates thymine. In the figure, the numbers are the numbers counted from the cleavage site of pUC118 by the restriction enzyme BamHI, and the number closer to the recognition cleavage site of the restriction enzyme EcoRI is number 1.
In addition, E. coli containing pD314TX was obtained from
No. (FERM P-11803).

Example 3 Purification of DHFR-DSDGK multimeric fusion protein (III) from Escherichia coli containing pD314TX A transformant containing pD314TX was transformed into a YT medium containing 100 μg / ml ampicillin (8 g of tryptone, 1 g of yeast, PH 7.0 liquid medium containing 5 g of extract and 5 g of NaCl.)
After culturing at 37 ° C. for several hours, 1 mM isopropyl-β-D-thiogalactopyranoside was added, followed by shaking culture at 37 ° C. until the stationary phase. The cultured cells were centrifuged at 4,700 × g and collected. Collection and subsequent operations were performed at low temperature (0 to 10 ° C, preferably 4 ° C).

Purification of the fusion protein from the cultured cells was performed according to the following procedures (1) to (3).

(1) Disruption of bacterial cells Cells were crushed at a rate of 10 ml per 1 L of culture solution in buffer 1 [0.1 m
M ethylenediamine tetraacetate disodium (EDTA.Na ₂ ) and 10 mM potassium phosphate buffer (pH 7.0) containing 14 mM 2-mercaptoethanol], and the cells were disrupted using an ultrasonic disrupter.

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

(2) Streptomycin treatment While stirring the cell-free extract with a stirrer, 1.5 (W / V)
Of streptomycin sulfate was slowly added. After stirring for 20 minutes, the solution treated with streptomycin is 27,000 × g
, And centrifuged for 20 minutes to obtain a supernatant.

(3) MTX-Binding Affinity Chromatography To the supernatant obtained by the above operation, 50 ml of MTX-bound agarose gel (manufactured by Sigma) preliminarily equilibrated with Buffer 1 was added. After allowing to stand for 10 minutes to adsorb to the gel, the gel was packed into a column. The column was washed with buffer 1 and buffer 1 containing 1M KCl in that order. For elution of the fusion protein, see 4.
Buffer 1 containing 5 mM folic acid was applied to a solution adjusted to pH 9.3 with 2N NaOH, and the eluate was fractionated by a fixed amount using a fraction collector. About fractionated eluate
DHFR activity was measured, and fractions containing enzyme activity were collected.
The resulting solution was dialyzed against buffer 1.

Table 1 summarizes the above purification steps and the yield in each step. For purification, 8.46 g of wet cells obtained from 3 L of a culture solution of Escherichia coli containing the recombinant plasmid pD314TX was used. Table 1 shows cell-free extract, streptomycin treatment, and MTX binding affinity chromatography.

Example 4 Obtained DHFR-DSDGK Multimer Fusion Protein (III) Obtained DHFR-DSDGK Multimer Fusion Protein (III)
Was subjected to SDS-polyacrylamide gel electrophoresis [UKLaemm
li; Nature, vol. 227, p. 680 (1970)]. Lactalbumin (molecular weight 1
4,200), trypsin inhibitor (molecular weight 20,100),
Trypsinogen (molecular weight 24,000), carbonic anhydrase (molecular weight 29,000), glyceraldehyde-3
-Phosphate dehydrogenase (MW 36,000), egg albumin (MW 45,000) and bovine serum albumin (MW 6
6,000) (Sigma, DALT ON MARK VII-
L) and electrophoresed on a concentration gradient gel (Daiichi Kagaku) having an acrylamide concentration of 10 to 20%. as a result,
A single protein band with a molecular weight of about 32,000 is shown,
The obtained DHFR-DSDGK multimer fusion protein (III) preparation was shown to be homogeneous.

The amino acid sequence of the DHFR-DSDGK multimeric fusion protein (III) can be deduced from the nucleotide sequence encoding it. FIG. 2 shows DHFR-DS contained in pD314TX as an example of a fusion protein (III) of DHFR-DSDGK multimer.
The nucleotide sequence encoding the fusion protein of DGK15 mer and the amino acid sequence of the protein produced therefrom are shown. In the figure, symbols 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 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 is histidine, Phe is phenylalanine, and Tyr is tyrosine. ,
Trp indicates tryptophan and Pro indicates proline.
In the figure, the numbers indicate the numbers obtained by counting methionine, which is the amino terminal amino acid of the fusion protein, as number 1. The fusion protein of DHFR-DSDGK15 mer is a novel protein consisting of 241 amino acids. The sequence from positions 1 to 159 counted from the amino terminal side is the wild-type D of E. coli.
One amino acid substitution occurred in HFR [Cys-152 (wil
d) → Glu-152] sequence, and the 161st to 235th positions are the amino acid sequence of the DSDGK15 mer.

The sequence from the 236th position to the 241st position is a sequence generated due to deletion of the base in the portion encoding the DSDGK multimer in the process of producing pD314TX. 160th isoleucine (Ile)
Is a sequence generated to match the reading frame of genetic information when pBBK10MM is prepared. The molecular weight of the fusion protein of DHFR-DSDGK15 mer calculated from the molecular weight of amino acids is 34,207.

Example 5 D from fusion protein of DHFR-DSDGK15 mer separated and purified
Separation of SDGK 72.2 μl of the fusion protein concentrate (protein concentration: 7.9 μg / μl) of the DHFR-DSDGK15 mer obtained in the example was placed in an Eppendorf centrifuge tube (1.5 ml), and 288.8 μl of acetone was added thereto. Then, the mixture was placed in a freezer at -20 ° C for 30 minutes to precipitate a fusion protein of DHFR-DSDGK15 mer. This is centrifuged by Eppendorf (5414S
Centrifugation at 12,000 rpm for 10 minutes to remove the supernatant, and 10 μl of 1M Tris-HCl buffer (pH 8.
0), 37 μl purified water, 23 μl 8M Urea and 30 μl TPC
K-trypsin (Sigma) was added, and the mixture was incubated at 37 ° C overnight. After the reaction, take 10 μl of this and use a high-performance liquid chromatography system (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 by applying a gradient of acetonitrile (0% to 10%) in 0.1% trifluoroacetic acid. From 0 to 5 minutes, 0% acetonitrile was used, and from 5 minutes to 35 minutes, a linear gradient of 0% to 10% acetonitrile was applied. As a result, an elution curve as shown in FIG. 7 was obtained. In the drawing, the horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units. Arrows in the figure indicate positions where DSDGK was eluted. The peak about 7 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.
The amount of K was determined to be 3.6 μg. From this, the recovery rate when DSDGK is obtained by trypsinizing the fusion protein of DHFR-DSDGK15 mer is calculated to be about 81%.

[Effect of the Invention] As described above, the novel recombinant plasmid of the present invention is DH
E. coli harboring this plasmid encodes the fusion protein (III) of FR-DSDGK multimer.
Produces and accumulates a large amount of DGK multimeric fusion protein (III) in a soluble state. Furthermore, the produced DHFR-DSDGK multimeric fusion protein (III) retains DHFR enzyme activity and can be easily purified. According to the purification method of the present invention, the fusion protein (III) of the DHFR-DSDGK multimer can be purified quickly and efficiently. Further, it is possible to isolate and purify DSDGK by subjecting the thus obtained DHFR-DSDGK multimeric fusion protein (III) to enzyme treatment or chemical treatment or fractionation by high performance liquid chromatography using them in combination. it can. 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 in large quantities by genetic engineering techniques.

[Brief description of the drawings]

FIG. 1 shows the nucleotide sequence of the recombinant plasmid pD314TX.
FIG. 3 is a view showing a nucleotide sequence substituted with a nucleotide sequence of 30 nucleotides between cleavage sites of restriction enzymes BamHI and HindIII of UC118. FIG. 2 shows the nucleotide sequence encoding the fusion protein of DHFR-DSDGK15 mer present in pD314TX and the amino acid sequence of this fusion protein. FIG. 3 is a scheme schematically showing a method for producing a DNA chain that repeatedly encodes a large number of peptides on the same chain. FIG. 4 shows an electrophoretogram obtained by decomposing a digest obtained by digesting the recombinant plasmid p1-9 with restriction enzymes EcoR I and Hind III by polyacrylamide gel electrophoresis. FIG. 5 shows the nucleotide sequence of the recombinant plasmid p1-9.
FIG. 3 is a view showing a nucleotide sequence inserted into pUC119. FIG. 6 is a construction diagram showing the procedure for preparing plasmid pD314TX using plasmid p1-9 as the first plasmid. FIG. 7 shows that DHFR-DSDGK15 mer is treated with trypsin,
FIG. 3 shows a chromatogram obtained by separation with an HPLC apparatus using a reversed-phase column.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI // (C12P 21/02 C12R 1:19) (72) Inventor Yoshio Tanaka Examiner 5-722, Matsushiro 5-chome, Matsushiro, Tsukuba-shi, Ibaraki Pref. Emiko Sakazaki (56) References JP-A-1-316398 (JP, A) JP-A-1-149992 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00 C07K 7/06 C07K 19/00 C12N 9/04 C12P 21/02 BIOSIS (DIALOG) CA (STN) GenBank / EMBL / DDBJ (GENETYX) REGISTRY (STN) WPI (DIALOG)

Claims (11)

(57) [Claims] 1. A fusion protein of (dihydrofolate reductase)-(aspartic acid-serine-aspartic acid-glycine-lysine) n having the following amino acid sequence (n is 11
To 15). 2. The recombinant vector according to claim 1, wherein the fusion protein comprises a 15-mer of (aspartic acid-serine-aspartic acid-glycine-lysine) and has the following amino acid sequence. 3. The recombinant vector according to claim 2, wherein the nucleotide sequence encoding the fusion protein is the following nucleotide sequence. 4. The recombinant vector according to claim 1, wherein the recombinant vector is a recombinant plasmid that is stably replicated in Escherichia coli and confers trimethoprim resistance and ampicillin resistance to the host Escherichia coli. 5. The recombinant vector according to claim 3, wherein the recombinant vector is a recombinant plasmid pD314TX having a structure in which a base sequence of 30 bases between the restriction enzyme BamH I and Hind III cleavage sites of plasmid pUC118 is replaced by the following base sequence. Recombinant vector. 6. Escherichia coli transformed with the recombinant vector according to any one of claims 1 to 5. 7. A fusion protein of dihydrofolate reductase-antiallergic pentapeptide 15-mer having the following amino acid sequence: 8. A dihydrofolate reductase comprising culturing the Escherichia coli according to claim 6, and collecting a dihydrofolate reductase-antiallergic pentapeptide multimeric fusion protein (III) from the culture. A method for producing a fusion protein (III) of an antiallergic pentapeptide multimer. 9. The step of collecting the dihydrofolate reductase-antiallergic pentapeptide multimer fusion protein (III) is carried out by sequentially using a streptomycin treatment and a mesotrixate-bound affinity chromatography from a cell-free extract of cultured cells. 9. The production method according to claim 8, comprising a purification step. 10. The dihydrofolate reductase according to claim 7,
Enzymatic treatment of the fusion protein of the anti-allergic pentapeptide 15-mer produces aspartic acid-serine-
Collecting an anti-allergic pentapeptide consisting of an amino acid sequence of aspartic acid-glycine-lysine, aspartic acid-serine-aspartic acid-
A method for producing an anti-allergic pentapeptide comprising a glycine-lysine amino acid sequence. 11. The method according to claim 10, wherein the enzyme is trypsin or lysyl endopeptase.