JPH05207891A - Method for producing brain natriuretic peptide - Google Patents

Abstract

(57) [Summary] [Structure] X-Glu-BNP (X is interferon γ, Δ
Cys interferon gamma or interferon alpha 80A2
A leader sequence of 70-170 amino acids; a Glu-glutamic acid residue; and BNP is an amino acid sequence of brain natriuretic peptide), such as the N-terminal partial sequence of Glu, and the C-terminal of Glu such as V8 protease. A method for producing brain natriuretic peptide (BNP), which is cleaved by an enzyme that specifically cleaves a part. This fusion protein is BNP
It can be easily obtained by a genetic engineering technique using an expression vector containing a DNA encoding [Effect] BNP can be easily obtained by the above method. The obtained BNP can be suitably used as a vasodilator, particularly as a drug for heart failure, or as a reagent for various analyses.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention produces a brain natriuretic peptide (hereinafter abbreviated as BNP) as a fusion protein, which is then treated with an enzyme to isolate BNP.
Method for efficiently producing NP, DNA sequence encoding BNP used in the method, expression vector having the DNA sequence,
And a transformant having the expression vector.

[0002]

2. Description of the Related Art Atrial natriuretic peptide (atrial
Following the discovery of natriuretic peptide (ANP), brain natriuetic peptide (BNP) has been isolated and identified from porcine brain. Later, it became clear that BNP is a cardiac hormone, which is present in much higher concentrations than in the brain, for example in pigs, especially in the atria. Sudoh, T. Et al. Used a cDNA fragment encoding a porcine BNP precursor as a probe to screen a human cDNA library,
NA sequence was determined (Biochemical and Biophysical
Reserch Communications, Vol.159, No.3, 1427-1434,
1989; JP-A-2-231082). Since the DNA sequence of human BNP was clarified in this way, human BNP-related peptides were also chemically synthesized based on this (JP-A-2-237999).
After that, human BNP-32 consisting of 32 amino acid residues was isolated directly from the atrium (FEBS Letters, Vol.259, N.
o.2,341-345). In addition to this, BNP of rat, cow, etc. is known, and its structure is known to have a considerable race difference except for a certain common part.

As described above, the structure of BNP, particularly human BNP, has been clarified, but an example of expressing BNP by a genetic engineering method has not been known so far. If BNP can be mass-produced by genetic engineering, a vasodilator,
In particular, it is considered that it can be suitably used as a drug effective for treating heart failure or as a diagnostic agent for heart failure and the like.

[0004]

It is an object of the present invention to provide a method for producing BNP, particularly human BNP, by a genetic engineering technique.

Another object of the present invention is to provide a method for efficiently producing BNP by producing BNP as a fusion protein, treating it with an enzyme and isolating BNP.

Still another object of the present invention is to provide an expression vector having a DNA sequence encoding the above fusion protein and capable of efficiently expressing the information of the DNA sequence in E. coli, and the expression vector introduced into E. coli. To provide a transformant.

[0007] Still another object of the present invention is to provide a DNA sequence encoding BNP and comprising a codon which is frequently used in E. coli.

[0008]

The method for producing BNP of the present invention comprises the following formula (I): X-Glu-BNP (I) (wherein X is a leader sequence of 70 to 170 amino acids, Glu is a glutamic acid residue, BNP Represents the amino acid sequence of BNP); and cleaves the fusion protein with an enzyme capable of specifically cleaving the C-terminal portion of Glu to collect human brain natriuretic peptide. Include.

In a preferred embodiment, the BNP is
Harvested by a process comprising the steps of: solubilizing the fusion protein with a denaturant or detergent; diluting the resulting lysate to reduce the concentration of the denaturant or detergent, Adding the enzyme to a diluent to obtain a reaction solution containing a soluble intermediate in which the fusion protein is partially decomposed; desalting the reaction solution to contain the intermediate and the enzyme, and A step of obtaining a fraction containing no denaturant or a surfactant; a step of subsequently performing a reaction with an enzyme contained in the fraction to release BNP; and a step of isolating the BNP.

The expression vector of the present invention has a DNA sequence encoding the above fusion protein and efficiently expresses the above fusion protein in E. coli.

The transformant of the present invention can be obtained by introducing the above expression vector into E. coli.

The present invention includes the DNA sequence set forth in SEQ ID NO: 2 which encodes human brain natriuretic peptide.

In the fusion protein X-Glu-BNP (I) prepared by the method of the present invention, the leader sequence represented by X is selected as an amino acid sequence which gives a high expression level. When the X-encoding sequence is introduced and expressed in E. coli, which is a suitable host, the fusion protein
It is preferable that the sequence be 20% or more of the total protein produced, and that the fusion protein forms an inclusion body in the bacterial cells. Molecular weight is 10-20k Dalton
(70 to 170 amino acids) is suitable. X is, for example, interferon γ, ΔCys interferon γ,
Interferon α80A2, interferon σ, interleukin-1β, bovine growth hormone, β-galactosidase, human interleukin-8, Escherichia coli pyruvate kinase I, aminoglycoside 3'-phosphotransferase (APH), N-terminal part of these polypeptides And the like. In particular, sequences containing interferon γ, ΔCys interferon γ, and the N-terminal 90-140 amino acid portion of interferon α80A2 are preferable. By constructing an expression vector having a DNA sequence encoding a polypeptide in which these sequences are bound to BNP via a Glu residue and transforming E. coli with the expression vector, BNP can be efficiently transformed in E. coli. Moreover, it can be expressed as stable fusion protein granules.

The method for producing BNP of the present invention will be described below in the order of steps. The DNA recombination method in this step is, for example, molecular cloning (Cold Spring Harbor
Laboratory, New York, 1982).

(A) Design and chemical synthesis of DNA sequence encoding BNP The amino acid sequence of human BNP is shown in SEQ ID NO: 1. The DNA sequence encoding this human BNP has already been clarified by Sudoh, T. et al. (Supra) (SEQ ID NO: 1).
This DNA sequence may be used in the present invention, for example,
When Escherichia coli is selected as the host cell, BNP DNA that is used by giving priority to the codons that are frequently used in E. coli
Advantageously, an array is used. The present inventors designed and synthesized a human BNP DNA sequence having a codon frequently used in Escherichia coli. The DNA sequence is shown in SEQ ID NO: 2 and FIG.

Since the DNA sequence encoding this human BNP has a relatively short chain, it can be obtained, for example, by chemical synthesis. Such DNA is obtained by, for example, synthesizing 12 kinds of fragments (Fr1 to 12) shown in FIG. 1 using an automatic nucleic acid synthesizer and purifying by chromatography such as high performance liquid chromatography (HPLC). It can be synthesized by a conventional method in which a phosphate group is added and ligated with ligase. The ligated DNA is isolated by conventional methods such as phenol extraction, ethanol precipitation, agarose gel electrophoresis. The recovery of DNA fragments by agarose gel electrophoresis was performed by Nucl. Acids Res., 8 , 253-264, 19
According to the method described in 80, it can be performed by elution from an agarose gel.

A recombinant plasmid (expression vector) is constructed using the DNA sequence encoding BNP.

(B) Construction of Expression Vector TrppATΔCyshuIFNγ, which is an example of the expression vector of the present invention
The construction of / huBNPΔApTc ^r is described below. This expression vector, as shown in FIG. 3, contains the plasmid pUC119 / hBNP
Pst I- Xba I fragment (fragment containing the sequence encoding BNP) and plasmid TrppATΔCyshuIFNγ / gulucag
on (European Patent Application Publication No. 0381433) Pst I- Xba
It is constructed from the I fragment.

To construct this expression vector, first, as shown in FIG. 2, plasmid vector pUC119 (Takara Shuzo)
Is digested with Pst I and Xba I to give the larger fragment. A synthetic gene fragment of about 110 bp containing the BNP-encoding sequence shown in FIG. 1 is inserted into this to obtain a plasmid pUC119 / hBNP which is a plasmid for cloning.

Then, as shown in FIG.
pUC119 / hBNP is cut with Pst I and Xba I to give the shorter fragment.

Separately, TrppATΔCyshuIFNγ, a human interferon γ fusion glucagon expression plasmid
/ glucagon (European Patent Application Publication No. 0381433) Ps
Cleavage with t I and Xba I gives the larger fragment. By ligating the above fragment and using T4 DNA ligase, the expression vector TrppATΔCyshuINFγ / huBNPΔAp
Tc ^r is obtained. The base sequence of the DNA fragment inserted into this expression vector is, for example, Proc. Natl. Acad. Sci. USA,
74, 5463-5467, 1977, by examining the D
It is confirmed that the NA fragment correctly encodes the 32 amino acids of human BNP. This TrppATΔCyshuIFNγ / huBNPΔ
ApTc ^r is a human IFN gene under the control of the Trp gene promoter.
It has a gene sequence encoding the 4th Gln to the 135th Ser residue of γ, and a nucleotide sequence encoding the amino acid sequence of BNP via the nucleotide sequence encoding the Glu residue about 30 bases downstream thereof. Including. The sequence near the junction of the 5'ends of the BNP-encoding DNA of this expression vector is shown in the lower right of FIG.

In the above example, an expression vector having a sequence containing the N-terminal sequence of ΔCys interferon γ as X was prepared, but in addition to this, an expression vector having a partial sequence such as interferon γ and interferon α80A2 described above is prepared as appropriate. Can be done.

(D) Transformation and Expression of Host Cell Expression plasmid of the present invention, such as TrppATΔCyshuIFN
γ / huBNPΔApTc ^r is introduced into an appropriate host cell according to the method of molecular cloning (supra). Transformants are selected using tetracycline resistance as an index.
The transformant into which the expression plasmid was introduced was human BN
It produces a fusion protein of P and human IFNγ. Examples of host cells include Escherichia coli K12 strain C600, JM103, AG-1, and 931. Especially, K12 strain C600 is preferable, and by using this strain, a fusion protein containing human BNP can be efficiently produced. ..

The above expression vector, TrppATΔCyshuIFNγ / h
Transformants E. coli K12 strain C600 was transformed with UBNPderutaApTc ^r was TrppATderutaCyshuIFNganmafusedhuBNP / C600 nomenclature.

When the transformant of the present invention is cultured, human BN
P is expressed in the host cell as a fusion protein.

(E) Solubilization of Fusion Protein, Degradation by Enzyme, and Isolation / Purification of BNP In order to collect BNP from a fusion protein containing BNP (X-Glu-BNP), the fusion protein is used as a granular protein in a host. Since it is accumulated in cells, the granule protein is first taken out and solubilized.

To this end, for example, first, the culture solution is centrifuged to collect the bacterial cells according to a conventional method, and the bacterial cells are crushed to obtain a pellet. A buffer containing a denaturant or a surfactant is added to the pellet to solubilize it. Guanidine (guanidine hydrochloride), urea, etc. are used as the denaturing agent. The denaturant is used by adding it to the buffer in an amount sufficient to solubilize the pellet. Guanidine and urea can be used by adding them until saturation is reached, but usually 6-8M
It is preferable to use it at a concentration of. Tw as a surfactant
een-80 (trade name) is preferably used, usually 0.05-0.2%
Used in a concentration of. The pH of the buffer solution is preferably set in the vicinity of the optimum pH of the enzyme used in the step in consideration of the next enzyme treatment step. For example, when S. aureus- derived protease V8 (Sigma, No. P-8400) is used as the enzyme, the pH is preferably 7.8. Therefore, as the buffer solution, it is preferable to use a buffer solution having a buffer capacity near pH 7.8, for example, an ammonium hydrogen carbonate buffer solution is preferably used. When the enzyme used in the enzyme treatment contains a neutral enzyme or the like as an impurity, the buffer solution contains EDTA as an enzyme inhibitor for inhibiting the activity.
Etc. may be added.

The solubilized solution thus obtained contains a denaturant or a surfactant at a high concentration, and the enzyme may not work sufficiently if it is used as it is in the subsequent enzyme treatment step. Therefore, the solubilized solution is usually diluted with a diluent such as the above buffer solution containing no denaturant or surfactant. The dilution is performed to such an extent that the protein can be maintained in a solubilized state and the enzyme used for the enzyme treatment described later can sufficiently act.
For example, when guanidine or urea is used as a denaturant, the concentration is diluted to 4M or less, preferably about 2M. However, be careful that the protein concentration in the solubilized solution is 0.1 to 5 mg / ml, preferably about 0.5 mg / ml.

Next, BNP is separated from the X protein by enzymatic treatment. In the above solubilization solution, the target BNP is
It exists in a state of being fused with the X protein or its partially modified form via the Glu residue. Since there is no Glu residue in human BNP, BNP can be completely excised by treatment with an enzyme that specifically cleaves the C-terminal peptide bond of the Glu residue. As such an enzyme, the protease V8 derived from S. aureus (Sigma, No. P-8400) as described above is preferable, but not limited thereto.
Any enzyme that specifically cleaves only the peptide bond on the carboxyl side of the Glu residue can be used. For example, a V8-like protease derived from S. aureus ATCC No. 12600 strain or a V8-like protease derived from B. licheniformis can also be preferably used. The amount of enzyme used is 1/50 to 1 / 1,000 (w / w) with respect to the amount of protein in the above diluted solution.
It is preferably about 1/125 (w / w). An enzyme reaction solution obtained by adding such an enzyme to the above diluted solution is reacted at room temperature to 40 ° C for 30 minutes to 7 hours with gentle stirring. The reaction time varies depending on the amount of the enzyme to be added, but for example, when the enzyme is added in an amount of about 1/125 (w / w), it is preferable to react for about 3 hours. The enzymatic reaction is stopped by adding an acid such as hydrochloric acid or acetic acid to lower the pH to about 3. The final product, BN, is contained in the obtained enzyme reaction solution.
P, and intermediates that are smaller than X-Glu-BNP are included. It is considered that this intermediate is produced by cleaving the Glu residue in the X protein with an enzyme. This intermediate is soluble even when the modifier or surfactant in the reaction solution is removed. The cause of the formation of such an intermediate is that the enzyme in the above-mentioned enzyme reaction solution is temporarily denatured by a denaturant or a surfactant and its activity is significantly reduced; The low-molecular-weight degradation product generated by cleavage inhibits the enzymatic action; the Glu residue cleavage site in the fusion protein is less susceptible to the enzymatic action due to higher-order structure such as molecular folding. Being in a state;
In order to increase the yield of BNP, this intermediate may be further treated with the above enzyme and cleaved. To do so, denaturants or surfactants in the enzyme reaction solution, and protein X
It is effective to remove the low molecular weight degradation products generated by the cleavage of the Glu residue inside. To remove them, gel filtration, dialysis, reverse phase chromatography, etc. are effective. Especially, gel filtration is preferably used. To perform gel filtration, it is preferable to employ conditions under which the denaturant or surfactant and the degradation product of low molecular weight are removed, and at the same time, the enzyme and the intermediate are eluted in the same fraction. By collecting the fractions containing the intermediate and enzyme obtained by gel filtration and adjusting the pH to the optimum pH of the enzyme, it is thought that the enzyme will reactivate and act on the solubilized intermediate. Be done. BNP is released by reacting the obtained intermediate-containing fraction under the same conditions as in the enzyme reaction.

The BNP cleaved from the fusion protein in this way is subjected to various chromatography such as gel filtration chromatography, affinity chromatography, ion exchange chromatography, hydrophobic chromatography, reverse phase HPLC, etc., and centrifugal separation operation in a conventional manner. And the like can be combined appropriately for purification. According to the above method, Glu used
The amount of enzyme that cleaves residues is small and economical.

The present invention is not limited to the method for producing human BNP as a fusion protein, and a vector expressing the protein, a transformant, and a specific DNA sequence encoding BNP are included in the present invention. It

[0032]

The present invention will be described with reference to the following examples.

In the following experiments, the DNA recombination method was
All molecular cloning (Molecular Cloning,
Cold Spring Harbor Laboratory, 1982). All enzymes used were purchased from Takara Shuzo.

(A) Design and chemical synthesis of DNA sequence encoding BNP A DNA sequence encoding the amino acid sequence of human BNP was designed using the codons most frequently used in Escherichia coli in principle. This DNA sequence is the first amino acid Ser
To the 32nd amino acid His, which is the DNA sequence shown in FIG.

12 kinds of short chain fragments shown in FIG. 1 were chemically synthesized using an automatic nucleic acid synthesizer. After adding a phosphate group, these fragments were ligated with ligase to obtain a DNA sequence having a double-stranded structure shown in FIG. This DNA sequence contains GAA and Pst I encoding Glu at the 5'end.
It has a cleavage site, and one stop codon TAA and an Xba I cleavage site at the 3'end.

(B) Construction of Expression Vector TrppAThuIFNγ / huBNPΔApTc ^r , which is an expression vector capable of expressing human BNP in Escherichia coli, is prepared as described in (1) and (2) below.
It was constructed by ligating the two DNA fragments obtained in the above section. The outline of the construction of this expression vector is shown in FIG. 2 and FIG.
Shown in.

(1) Pst I- Xba I of plasmid pUC119 / hBNP
Fragment (fragment containing sequence encoding BNP): As shown in FIGS. 2 and 3, plasmid pUC119 / hBNP, which is a vector for cloning a target gene, was constructed using plasmid vector pUC119 (Takara Shuzo) as a prototype. First, the above-mentioned pUC119 was cleaved with Pst I and Xba I, and the synthetic gene of about 110 bp obtained in section A (DNA sequence shown in FIG. 1)
And was named pUC119 / hBNP.

Then, as shown in FIG. 3, plasmid pUC119
/ hBNP was digested with Pst I and Xba I to obtain the shorter fragment.

(2) Plasmid vector TrppATΔCyshuIFN
Pst I- Xba I fragment of γ / glucagon: As shown in FIG. 3, a human interferon γ fusion glucagon expression plasmid [TrppATΔCyshuIFNγ / glucagon (European Patent Publication No. 0381433)] was cleaved with Pst I and Xba I to give a large fragment. One fragment was obtained.

DNA fragments of these and (Nucl. Aci
ds. Res. 8253-264, 1980, which was isolated by agarose gel electrophoresis) as shown in FIG.
Construct an expression vector by ligation using DNA ligase,
This was designated as TrppATΔCyshuIFNγ / huBNPΔApTc ^r .
Then, the base sequence of the DNA fragment inserted into this expression plasmid was changed to Proc. Natl. Acad. Sci. USA, 74, 5463-5467, 1
It was confirmed by the method described in 977, that the DNA fragment correctly encodes 32 amino acids of human BNP.

This TrppATΔCyshuIFNγ / huBNPΔApTc ^r is T
The fourth G of human IFNγ under the control of the rp gene promoter
It has a gene sequence encoding from ln to the 135th Ser residue and has a base sequence encoding the amino acid sequence of BNP via a base sequence encoding a Glu residue about 30 bases downstream thereof.

(C) Transformation and Expression of Host Cell Expression plasmid TrppAThuIFNγ / huBNPΔApTc ^r constructed as described above using Escherichia coli K12 strain C600 as a host cell.
Transformation was carried out. Since the transformant into which the expression plasmid was introduced acquired resistance to tetracycline, it was cultured overnight at 37 ° C on a tetracycline-containing medium (LB plate medium containing 15 µg / ml tetracycline) to form colonies. I chose what I did. Then, the formed colonies were cultured in a modified M9 medium at 37 ° C for 24 hours, and the cultured cells were collected by centrifugation. Buffer cells A (8g / L NaCl, 0.2g / L KCl, 2.9g / L Na
_{2 HPO 4 · 12H 2 O,} 0.2g / L KH 2 PO 4, 5mM EDTA, 10mMβ- mercaptoethanol and 10mM benzamidine, pH 6.65)
The cells were suspended in water, stirred well, then centrifuged and washed. The washed cells were collected, suspended again in 100 mL of the above buffer solution A, lysozyme was added so that the final concentration would be 60 mg / g, and the cells were reacted at 37 ° C for 1 hour. Then, the reaction solution was subjected to ultrasonic treatment and centrifuged at 4 ° C. for 15 minutes (7000 rpm) to precipitate the granular fraction, and the pellet was recovered.

(D) Solubilization of fusion protein, degradation with V8 protease, and isolation / purification of BNP Pellet (1.65 g) containing the granular fraction obtained in (C) was added to 8M.
Buffer B containing guanidine hydrochloride (50 mM NH ₄ HCO ₃ , 20 mM E
DTA and 10 mM dithiothreitol (DTT), pH 7,8) 75 m
Buffer solution B containing no guanidine hydrochloride after being dissolved in L
(225 mL) was added for dilution. Here, the amount of protein was measured (150 mg; by Bio-Rad protein assay), and 1/125 amount of the protein amount was used for protease V8 (Sigma, No. p.
8400) 1,2 mg was added, and the mixture was reacted at room temperature for 3 hours. The target BNP is fused with IFNγ via the Glu residue, and since there is no Glu residue in the BNP, it is completely formed by the action of V8 protease (however, the reaction solution contains the reducing agent DTT). It is cut out as a reduced form. However, in this initial digestion, not all bonds on the carboxyl side of the Glu residue were cleaved, and BNP and human IFNγ were decomposed in the resulting reaction solution (preparation a). Peptides of various lengths are mixed. This authentic sample a was purified as follows. First, add 1M hydrochloric acid to the standard a and adjust the pH.
After adjusting to 2-3, reverse phase chromatography ( ₅₀ C ₁₈
Column, 30 × 200mm), 0.1% trifluoroacetic acid (T
It was sequentially eluted with 5%, 30% and 60% acetonitrile containing FA). As a result, the target BNP (reduced type) and BNP
All incomplete digests containing in the molecule were eluted in the 30% acetonitrile fraction. This means that each eluate was redigested with Protease V8 and then analyzed by reverse phase HPLC,
It was confirmed whether or not the BNP peak was detected. Obtained 3
The 0% acetonitrile fraction was freeze-dried to obtain about 30 mg of powder. Next, in the same manner as above, the 30% acetonitrile fraction (about 30 m
g of powder) was obtained. The 30% acetonitrile fractions obtained above were combined (about 60 mg), and this was mixed with 50 mM ammonium acetate (pH
7.8-8.0, 50mL) and dissolve it in protease V8
(0.16 mg) was added and the mixture was reacted at room temperature for 3 hours. Here, the reaction solution contains no reducing agent (DTT), so BNP itself is produced. The reaction mixture (preparation b) and standard human BNP-32 were each subjected to analytical reverse phase HPLC ( ₅ C ₁₈ column, 4.6 × 150 mm) and eluted with 0.1% TFA containing acetonitrile (however, the acetonitrile concentration was 30 0% to 60% in minutes
The peak at a retention time of 14.895 minutes coincided with the standard BNP (see the chart in FIG. 4). Therefore, the preparation b was prepared under the same conditions as the analytical HPLC (however, the concentration of acetonitrile was 0 to 36%), and the preparative HPLC ( ₅ C ₁₈ column, 20
× 150 mm). The obtained HPLC chart is shown in FIG. Fractions eluting at the position corresponding to the 14.895 min peak when using analytical HPLC were collected. This fraction (preparation sample c) was subjected to analytical HPLC and eluted with 0.1% TFA containing 19.2% acetonitrile (flow rate 1.13 ml / min) (the result is shown in FIG. 6), and the retention time was 7.215 minutes. The main component was detected. Therefore, under the same conditions, the preparation c was subjected to HPLC with a preparative column (see FIG. 7), and the main fraction (preparation d) eluted at the corresponding position was collected and used as a purified peptide (yield 2.1 mg ). The elution position of this standard d in reverse phase HPLC was completely the same as that of standard human BNP-32.

Next, amino acid analysis and amino acid sequence analysis were carried out by the following methods. The results are shown in Table 1 and Table 2, respectively.

Amino acid analysis method: The sample is hydrolyzed with 6M hydrochloric acid in a sealed tube in the presence of a small amount of phenol for 72 hours. This hydrolyzate is analyzed by Hitachi amino acid analyzer (Model 835).

Amino acid sequence analysis method: The sample was analyzed by Applied Bi
Analyze using the osystems Protein Sequencer (Model 477A).

[0047]

[Table 1]

[0048]

[Table 2]

Furthermore, the bioassay of the standard sample d was carried out by the following method.

Bioassay method: The rat thoracic aorta was spirally cut to obtain a strip having a width of 2 to 3 mm and a length of about 2 cm.
A sample piece is obtained by dividing the sample into 0.5 cm lengths, and the smooth muscle relaxing activity is measured as follows. This strip of sample is placed in Krebs sodium bicarbonate buffer and suspended vertically. Equilibrate at 37 ° C with a gas mixture of O ₂ 95% and CO ₂ 5%. First, contract in the same buffer containing 50 mM KCl. Then, various concentrations of the specimen (specimen d) are added to relax this sample piece. Finally, 0.
Papaverine hydrochloride is added to 1 mM, the relaxation rate of the induced sample piece is regarded as 100%, and the degree of relaxation by the sample is calculated.

FIG. 8 shows the measurement results of the standard d and the standard human BNP-32. The results when using human α-ANP are also shown for comparison.

[0052]

[Sequence list]

[0053]

[SEQ ID NO: 1] Sequence length: 96 Sequence type: Nucleic acid Number of strands: Double-strand Topology: Linear Sequence type: cDNA Origin organism name: Human sequence characteristics Characteristic symbol: CDS characteristics Method determined: S sequence AGC CCC AAG ATG GTG CAA GGG TCT GGC TGC TTT GGG AGG AAG ATG GAC 48 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 CGG ATC AGC TCC TCC AGT GGC CTG GGC TGC AAA GTG CTG AGG CGG CAT 96 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30

[0054]

[SEQ ID NO: 2] Sequence length: 96 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Synthetic DNA sequence TCT CCG AAG ATG GTT CAG GGT AGC GGG TGC TTT GGC CGC AAA ATG GAT 48 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 CGT ATC AGC TCC TCT TCC GGT CTG GGT TGC AAG GTA CTG CGT CGC CAC 96 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30

[Brief description of drawings]

FIG. 1 is a sequence diagram showing a synthetic fragment containing the DNA sequence of the present invention encoding human brain natriuretic peptide.

FIG. 2: Expression vector TrppATΔCyshuIFNγ / huB of the present invention
FIG. 6 is a schematic diagram showing the construction of plasmid pUC119 / hBNP used for the construction of NPΔApTc ^r .

FIG. 3: Expression vector TrppATΔCyshuIFNγ / huB of the present invention
FIG. 3 is a schematic diagram showing the construction of NPΔApTc ^r .

FIG. 4 is an analytical HPLC chart of a fraction containing human BNP obtained by degrading the fusion protein produced by the method of the present invention.

FIG. 5 is a preparative HPLC chart of the fraction containing human BNP obtained by degrading the fusion protein produced by the method of the present invention.

FIG. 6 is an analytical HPLC of a purified fraction containing human BNP obtained by degrading the fusion protein produced by the method of the present invention.
Is a chart of.

FIG. 7: Preparative HPLC of a purified fraction containing human BNP obtained by degrading the fusion protein produced by the method of the present invention
Is a chart of.

FIG. 8 is a graph showing the in vitro vasorelaxation effect in rats using BNP obtained by the method of the present invention.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12N 15/62 ZNA 15/70 C12P 21/02 C 8214-4B // A61K 37/02 ABR 8314- 4C 37/24 ABN 8314-4C (C12N 1/21 C12R 1:19) (C12P 21/02 C12R 1:19) (72) Inventor Ken Inoue 1296 Ikaidanicho, Nishi-ku, Kobe-shi, Hyogo (72) Inventor Kunio Watanabe 125-67 Sugitani, Hashimoto-cho, Otsu City, Shiga Prefecture

Claims (12)

[Claims] 1. Formula (I): X-Glu-BNP (I) (In the formula, X represents a leader sequence of 70 to 170 amino acids, Glu represents a glutamic acid residue, and BNP represents an amino acid sequence of brain natriuretic peptide. And a step of obtaining a fusion protein represented by the above); and a step of cleaving the fusion protein with an enzyme capable of specifically cleaving the C-terminal portion of Glu to collect a brain natriuretic peptide. Law. 2. The method according to claim 1, wherein the X is a sequence containing an N-terminal partial sequence of interferon γ, ΔCys interferon γ, or interferon α80A2. 3. The method according to claim 1, wherein the amino acid sequence of the brain natriuretic peptide has the amino acid sequence set forth in SEQ ID NO: 1. 4. A process comprising the step of culturing a transformant obtained by introducing an expression vector expressing the fusion protein represented by the formula (I) into Escherichia coli to produce the fusion protein. Claim 1 obtained by
The method for producing the brain natriuretic peptide according to 1. 5. The method according to claim 1, wherein the brain natriuretic peptide is collected by a process comprising the steps of: solubilizing the fusion protein with a denaturant or a surfactant; A step of diluting the solubilized solution to reduce the concentration of a denaturant or a surfactant; a step of adding the enzyme to the diluted solution to obtain a reaction solution containing a soluble intermediate in which the fusion protein is partially decomposed; Desalting the reaction solution to obtain a fraction containing the intermediate and the enzyme and containing no denaturant or surfactant;
A step of subsequently performing a reaction with an enzyme contained in the fraction to release a brain natriuretic peptide; and a step of isolating the brain natriuretic peptide. 6. The protease V8 derived from Staphylococcus aureus, wherein the enzyme is Staphylococcus aureus .
From Staphylococcus aureus ATCC 12600 strain
The method according to claim 1, which is a V8-like protease or a V8-like protease derived from Bacillus licheniformis . 7. A DNA sequence according to SEQ ID NO: 2 which encodes a brain natriuretic peptide. 8. Formula (I): X-Glu-BNP (I) (wherein, X is a leader sequence of 70 to 170 amino acids, Glu is a glutamic acid residue, and BNP is an amino acid sequence of brain natriuretic peptide. 4. An expression vector having a DNA sequence encoding the fusion protein shown in FIG. 9. The expression vector according to claim 8, wherein the amino acid sequence of the brain natriuretic peptide has the amino acid sequence set forth in SEQ ID NO: 1. 10. The expression vector according to claim 8, wherein the amino acid sequence of the brain natriuretic peptide has the DNA sequence set forth in SEQ ID NO: 2. 11. The expression vector according to claim 8, which is TrppATΔCyshuIFNγ / huBNPΔApTc ^r . 12. A transformant obtained by introducing the expression vector according to claim 8 into Escherichia coli.