JPH0364118B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a blood peptide Bradykinin (Arg-Pro-Gly-Phe-Ser-
A peptide consisting of a nine amino acid sequence of Pro-Phe-Arg, hereinafter abbreviated as BK. ).

BK is a type of blood peptide and has a blood pressure lowering effect (vasodilatory effect). Because of this effect, BK is expected to be used as a therapeutic agent for hypertension and the like. The BK production method of the present invention is suitable for fields such as medicine and pharmaceutical industry.

BACKGROUND ART BK is a type of blood peptide and is known to have effects such as blood pressure lowering effect (vasodilatory effect), intestinal constriction effect, and vascular permeability effect.
BK is Arg−Pro−Gly−Phe−Ser−Pro−Phe
It has been revealed that it is composed of the nine amino acid sequence of -Arg. BK itself is an active peptide, and Kallidin with Lys attached to the N-terminus or Met-Lys-bradykinin with Met-Lys attached are known to have lower activity than BK. It is being BK is a short peptide, already known as Boissonnas (Boissonnas
et al., Helv.Chim.Acta, vol.43, pl349 (1960))
Chemical synthesis is carried out by.

The present invention was carried out as part of the development of a new synthesis method for BK. The technical background of the present invention is as follows:
There is so-called genetic manipulation. B. subtilis dihydrofolate reductase (hereinafter abbreviated as DHFR) and BK
The present inventors have already constructed a plasmid incorporating a gene that encodes a fusion protein with DHFR-BK (hereinafter abbreviated as DHFR-BK) (Japanese Patent Application Laid-Open No. 63
-245679) was completely unknown before then. In addition, the plasmid pBLAK1, which incorporates a gene encoding DHFR-BK, was developed by E. coli.
introduced into C600 strain and kept in a stable state,
Contains pBLAK1. The E. coli C600 strain has been deposited with the Institute of Fine Technology as FERM P-9300. Furthermore, from E. coli C600 strain containing pBLAK1,
Although the present inventors had already conducted the separation of DHFR-BK (Japanese Unexamined Patent Publication No. 1-38099), it was completely unknown before then.

Problems Since BK is a peptide consisting of nine amino acids, it is possible to chemically synthesize the corresponding gene, create a recombinant plasmid that expresses the synthesized gene, and express it in a host such as E. coli. considered to be a thing. However, short peptides and the like are rapidly degraded by proteolytic enzymes present in Escherichia coli and the like, so even if gene expression efficiency is improved, the target peptide cannot be accumulated within the host cell body. At this time, in many cases, it has been considered that the fusion protein becomes insolubilized and the fusion protein does not have easily measurable enzymatic activity, which pose obstacles to the isolation and purification of the product. The present inventors have already conducted research to solve this problem and constructed a recombinant plasmid pBLAK1 containing the DHFR-BK gene.
We introduced this into an E. coli host and succeeded in stably expressing it (Japanese Patent Application Laid-Open No. 1982-245679), and we are now separating and purifying DHFR-BK from the bacterial cells (Japanese Patent Application Laid-Open No. 1-38099). Public bulletin). However, DHFR−BK
The separation of BK from the plant was completely unknown.

Purpose of the invention The present inventors have developed an E.coli strain containing pBLAK1.
We conducted intensive research with the aim of efficiently producing BK by using the C600 strain, and first,
DHFR from E. coli C600 strain containing pBLAK1
-BK was separated, the obtained DHFR-BK was then treated with bromocyanide, and the present invention was completed by showing that BK could be easily separated and purified by high performance liquid chromatography.

Structure of the Invention DHFR-BK of the present invention is composed of 172 amino acids as shown in FIG. The 162nd position from the amino terminus of the fusion protein is B.
It is identical to DHFR (consisting of 168 amino acids) of S. subtilis, and the sequence from positions 164 to 172 is the BK sequence. The 163rd amino acid is Met, and by treating the fusion protein with bromcyanide,
The structure allows BK to be cut out. DHFR
-The molecular weight of BK is 19801. In Figure 1,
The DNA sequence encoding DHFR-BK is also described.

The DHFR-BK fusion protein is produced in soluble form in E. coli cells containing the recombinant plasmid pBLAK1. E.coli containing pBLAK1
In the supernatant obtained by culturing, collecting the bacterial cells, crushing the bacterial cells, and centrifuging at 20,000 rpm,
Almost all of DHFR-BK is present.

DHFR-BK transforms E.coli containing pBLAK1 into
YT + Ap medium (in medium 1, 5 g NaCl, 8
g Bactotryptone, 5g yeast extract,
After culturing at 37°C until the end of the logarithmic growth phase or the stationary phase using a liquid medium containing 50 mg of ampicillin sodium, the cells are collected, disrupted, and centrifuged to obtain a supernatant. The supernatant can be highly purified by ion exchange column chromatography followed by gel filtration column chromatography. During column chromatography, it is necessary to determine in which fraction the target DHFR-BK is present, but since the target DHFR-BK has DHFR enzyme activity, it is necessary to measure the activity. The fraction in which the fusion protein is present can be determined by

DHFR enzyme activity was determined by 0.05mM dihydrofolate;
0.06mM NADPH (reduced nicotinamide dinucleotide phosphate), 12mM 2-mercaptoethanol, 50mM phosphate buffer (PH7.0)
This can be done by taking a 1 ml cuvette, adding an enzyme solution to it, and measuring the time change in absorbance at 340 nm to see how dihydrofolic acid and NADPH decrease as a result of the enzymatic reaction at 30°C. This measurement can be easily performed using a spectrophotometer. DHFR-BK can be uniformly purified through the above purification process.

The purified DHFR-BK was freeze-dried, and 1
Add 70% formic acid to give a concentration of 10 mg protein/ml and dissolve. Add about 20 times the amount of protein in crystalline bromide, seal tightly, and allow to react at room temperature for 24 hours. Lyophilize the reaction solution to remove excess reagent.
Dissolve the dried sample in 70% formic acid to give 1 to 10 mg protein/ml. The dissolved sample was transferred to a high-performance liquid chromatography device (Shimadzu LC-6A, inertsil-
It can be eluted and separated using a concentration gradient of 15% to 50% acetonitrile in 0.1% trifluoroacetic acid using a 0DS column). The eluate is
This can be done by measuring absorbance at 220 nm. FIG. 2 shows a high performance liquid chromatogram of a DHFR-BK sample treated with bromocyanide. When commercially available chemically synthesized BK was separated under the same conditions, a single peak eluted 15 minutes after sample injection, and DHFR-
When BK is added to a BK sample, only the height of the peak increases 15 minutes after sample injection. By separating the peak eluate that appears 15 minutes after sample injection,
BK can be separated.

In the example of the present invention, bacterial cells with a wet weight of approximately 12 g were obtained from 3 liters of culture medium, and this bacterial cell (calculated approximately
15.6mgkDHFR-BK, containing approximately 0.84mg BK. ), 5.7 mg of DHFR-BK (yield: 36.5%, calculated to contain approximately 0.31 mg of BK) can be obtained by purification.
Approximately 0.20 mg of BK (yield: 24%) was obtained by separating and purifying it using high-performance liquid chromatographies.

Next, examples of the present invention will be shown.

Example E. coli C600 strain containing plasmid pBLAK1 was cultured overnight at 37°C in 3 liters of YT+Ap medium, and the cells were collected by centrifugation. Bacterial cells with a wet weight of about 12 g were obtained. Bacterial cells were treated with 0.1mM ethylenediamine 4
After suspending the bacterial cells in 10 mM potassium phosphate buffer (PH7.0) (hereinafter referred to as buffer 1) containing disodium acetate (EDTA) and crushing them using a French press, the cells were washed at 20,000 rpm. 35 ml of supernatant was obtained by centrifugation for 1 hour. In the obtained supernatant
DHFR enzyme activity was measured and found to be 90 units/ml (total activity 3105 units, total protein 902 mg, specific activity 3.5 units/mg protein).
DHFR enzyme activity was determined using the reaction solution (0.05mM dihydrofolic acid, 0.06mM NADPH, 12mM 2-mercaptoethanol, 50mM phosphate buffer (PH).
7.0)) was carried out by taking a 1 ml cuvette, adding the enzyme solution thereto, and measuring the change in absorbance at 340 nm over time. One unit of enzyme was defined as the amount of enzyme required to reduce 1 micromole of dihydrofolate per minute under the above reaction conditions. The obtained supernatant was added to DEAE-Toyopearl 650M.
Adsorb it onto a column (250mm x 1500mm, approx. 75cm ³ ),
Elution was performed with buffer 1 containing 50mM KCl. Approximately 6 ml of each fraction was collected, the enzyme activity of DHFR was measured, and the fractions having enzyme activity were collected. 65 ml of enzyme solution was obtained (recovered activity 1897 units (60.2
%), recovered protein 25 mg, specific activity 75.9 units/mg protein). This was concentrated to about 1 ml using an Amicon ultrafiltration device, and fractionated using Toyopearl HW55 column chromatography. Collect approximately 2.8 ml of fractions each,
DHFR enzyme activity was measured and the peak fractions of enzyme activity were collected. 11.2 ml of enzyme solution was obtained (recovered activity 1151 units (36.5%), recovered protein 5.7
mg, specific activity 202 units/mg protein). The obtained purified protein was freeze-dried, and about 2 ml of it was
After dissolving 50 mg of bromocyanide in 70% formic acid and dissolving it, the mixture was reacted at 37°C with shaking for 24 hours. After the reaction, 20 ml of water was added and then freeze-dried. The specimen obtained by freeze-drying was dried at 5.5
ml of 30% acetic acid. Take 0.1 ml of it, and transfer it to a high-performance liquid chromatography system (Shimadzu LC).
-6A) and was separated using an lnertsil-0DS 5 μm column. Elution was performed by applying a concentration gradient of acetonitrile (15% to 50%) in 0.1% trifluoroacetic acid. From 0 to 2 minutes, 15% acetonitrile was used, and from 2 minutes to 32 minutes, a linear concentration gradient of 15% to 50% acetonitrile was applied. Between 32 and 35 minutes, a 50% concentration was used, and thereafter a 15% acetonitrile concentration was used. The elution was determined by measuring the absorbance at 220 nm. FIG. 2 shows the elution pattern under the above conditions. A in FIG. 2 shows a high performance liquid chromatogram of the obtained sample. Moreover, B in FIG. 2 shows the result of separating commercially available BK under the same conditions. C in Figure 2 indicates that the obtained sample has 5μ
The results of separation after adding g of BK are shown.
BK was eluted at about 15 minutes under the above conditions (Figure B). When the sample obtained by bromcyan treatment is separated, there is a product eluting at the approximately 15 minute position (Figure A), and in the sample with BK added, only the peak at this position becomes high. (C
figure). This indicates that BK is contained in the bromic cyanide-treated specimen, and that it is eluted in about 15 minutes under the above conditions. Furthermore, by comparing the peak heights, it was revealed that approximately 3.7 μg of BK was contained in 0.1 ml of the standard sample. Therefore, approximately 203 μg of BK is contained in 5.5 ml of the specimen obtained by bromcyan treatment. This sample was separated in 0.1 ml portions under the same conditions, and the peaks eluted after about 15 minutes were collected. A portion of the sample was taken and analyzed for amino acids. As a result, the amino acid composition ratio was Arg:Gly:
The values of Phe:Pro:Ser=1.8:1.0:1.6:2.7:0.8 were obtained, which almost matched the values of the amino acid composition of BK. Furthermore, no other amino acids were detected.

Effects of the invention The present invention uses DHFR-BK protein.
Although the method for producing BK has been described, the present invention provides a method for producing BK using genetic recombination technology, which is beneficial for the use of BK.

[Brief explanation of drawings]

Figure 1 shows DHFR-BK present in pBLAK1.
FIG. 2 is a diagram showing the base sequence of a portion that encrypts a protein 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, Ala is alanine, Arg is arginine, Asn is asparagine, Asp is aspartic acid, Cys is cysteine, Gln is glutamine, Glu is glutamic acid, Gly is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lsy is lysine,
Met is methionine, Phe is phenylalanine,
Pro is proline, Ser is serine, Thr is threonine, Trp is tryptophan, Tyr is tyrosine,
Val indicates valine. The symbols in the figure indicate numbers starting from "A" of the ATG codon that encodes the first amino acid, methionine. Figure 2 shows DHFR-BK sample treated with bromide (Figure A), commercially available BK (5 μg)
(Figure B) and bromcyane-treated DHFR-
A high-performance liquid chromatogram of a sample (Figure C) in which 5 μg of BK was added to BK is shown. The horizontal axis represents time in minutes, and the vertical axis represents absorbance at 220 nm in arbitrary units.

Claims (1)

[Claims] 1 E.coli containing recombinant plasmid pBLAK1
1. A method for producing bradykinin, which comprises separating and purifying a dihydrofolate reductase-bradykinin fusion protein produced by strain C600, decomposing this by a bromcyanic degradation method, and then separating and purifying bradykinin.