JPH0354560B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a blood peptide, Bradykinin (Arg-Pro-Pro-Gly-Phe-
A peptide consisting of the nine amino acid sequence Ser-Pro-Phe-Arg, hereinafter abbreviated as BK. ) at the carboxy terminal side (hereinafter abbreviated as DHFR-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. of the present invention
DHFR-BK is a completely new protein having the amino acid sequence shown in FIG. 1, and is suitable for use in fields such as medicine and the 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−Pro−Gly−Phe−Ser−
It has been revealed that it is composed of nine L-amino acids of Pro-Phe-Arg. BK itself is an active peptide, with Lys at the N-terminus.
Kallidin with , also Met−Lys
-Bradykinin is known to have lower activity than BK. BK is a short peptide and has already been described by Boissonnas et al.
Chemical synthesis was carried out by Helv. Chim. Acta, vol 43, pp. 1349 (1960).

This study was carried out as part of the development of a new synthesis method for BK. The technical background of the present invention is so-called genetic manipulation technology. B.subtilis
A plasmid incorporating a gene encoding a fusion protein of DHFR and BK was previously constructed by the present inventors in Japanese Patent Application Laid-open No. 63-245679, but was completely unknown prior to that. Also, DHFR−
The recombinant plasmid pBLAK1 containing the gene encoding BK was introduced into E. coli strain C600 and kept stable, and the E. coli containing pBLAK1
Strain C600 has been deposited at the FERMP-9300.

Problems Since BK is a peptide consisting of nine amino acids, we chemically synthesized the corresponding gene and created a recombinant plasmid to express it.
It is thought that it can be expressed in a host such as E. coli. However, short peptides etc.
Since it is rapidly degraded by proteolytic enzymes present in E. coli and the like, it cannot be accumulated in the host bacterial body even if the efficiency of gene expression is improved. Therefore, when attempting to produce a short polypeptide such as BK using genetic engineering technology, it is necessary to create a fusion gene and express it as a fusion protein. 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 problems in the isolation and purification of the product. The present inventors have already conducted research to solve this problem, constructed a recombinant plasmid pBLAK1 containing the DHFR-BK fusion protein gene, introduced it into an E. coli host, and succeeded in stably expressing it. (Japanese Unexamined Patent Publication No. 63-245679). However, there are no reports regarding DHFR-BK fusion protein, and it is a completely new compound.

Purpose of the Invention The present inventors conducted intensive research and established a method for separating and purifying DHFR-BK fusion protein from E. coli C600 strain containing pBLAK1. It has been revealed that the present invention is a multifunctional protein that retains the antigenicity (i.e., immunological activity) of the protein, leading to the completion of the present invention.

Structure of the Invention The DHFR-BK fusion protein of the present invention is composed of 172 amino acids as shown in FIG. The 162nd amino acid sequence from the amino terminus of the fusion protein is the same as the 1-162nd amino acid sequence of B. subtilis DHFR (consisting of 168 amino acids), and the 164th-172nd sequence is the BK sequence. be.
The 163rd amino acid is Met, which has a structure that allows BK to be excised by treating the fusion protein with bromcyanide. The molecular weight of the fusion protein is 19801.

FIG. 1 also depicts the DNA sequence encoding the DHFR-BK fusion protein. this
We were able to determine the amino acid sequence from the DNA sequence.

DHFR of B. subtilis is composed of 168 amino acids, and the sequence from positions 163 to 168 shown in Figure 1 is Ser-Lys-Val-Gly-Gly.
-Phe, and has a completely different sequence from the DHFR-BK fusion protein. Normally, when the primary structure of a protein is changed, it often loses its physiological activity, but in the case of the present invention, the DHFR of B. subtilis
Even if the carboxy-terminal sequence of DHFR
No effect on activity was observed.

The DHFR-BK fusion protein of the present invention specifically reacts with antibodies against BK to form an immune complex. That is, as shown in the examples, DHFR
- After separating the immune complex precipitate obtained by reacting the BK fusion protein with an antibody against BK made in a rabbit and reacting the resulting immune complex with an antibody against rabbit immunoglobulin made in a goat, When the precipitate was solubilized and DHFR activity was measured, DHFR activity was present. Furthermore, the amount of DHFR activity was competitively reduced by coexisting BK in the reaction solution of DHFR-BK fusion protein and BK antibody. On the other hand, B. subtilis
When DHFR and BK antibody are reacted, DHFR activity cannot be detected during immune complex precipitation. From this, it became clear that the DHFR-BK fusion protein has immunological properties of BK.
From the above, the DHFR-BK fusion protein is
It was shown to have properties of both DHFR and BK.

The DHFR-BK fusion protein with these characteristics contains the recombinant plasmid pBLAK1.
It is produced in a soluble state in E. coli cells.
That is, E. coli containing pBLAK1 was cultured,
DHFR-BK was collected in the supernatant obtained by collecting bacterial cells, sonicating them, and centrifuging them at 20,000 rpm.
Almost all of the fusion proteins are present. That is,
The fusion protein is not insoluble but soluble in E.
It is produced in coli cells.

The DHFR-BK fusion protein was prepared by injecting E. coli containing pBLAK1 into YT+Ap medium (5 g in medium 1).
After culturing at 37°C until the end of the logarithmic growth phase or the stationary phase using a liquid medium containing NaC1, 8 g of Bactotributon, 5 g of yeast extract, and 50 mg of ampicillin sodium, the cells were collected and cultured. is sonicated to obtain a centrifuged supernatant (cell-free extract), and this supernatant can be highly purified by ion exchange column chromatography and subsequent gel filtration column chromatography. During column chromatography, it is necessary to fractionate the eluate and determine in which fraction the desired DHFR-BK fusion protein is present. In the present invention, since the target DHFR-BK fusion protein has DHFR enzyme activity, the fraction in which the fusion protein is present can be determined by measuring this activity.

DHFR enzyme activity was determined by 0.05mM dihydrofolate;
Add 0.06mM NADPH (reduced nicotinamide dinucleotide phosphate), 12mM 2-mercaptoethanol, and 50mM potassium phosphate buffer (PH7.0) to a 1ml cuvette, add the enzyme solution, and incubate at 30°C. Dihydrofolate and NADPH, which decrease with the enzymatic reaction, can be measured by measuring the change in absorbance at 340 nm over time.
This measurement can be easily performed using a spectrophotometer.

In the examples, DEAE-Toyopearl was used as a carrier for ion exchange column chromatography.
650M gel and Toyopearl HW55 gel are used as carriers for gel filtration column chromatography, but the feature of the purification method of the present invention is that the DHFR-BK fusion protein can be purified by measuring the DHFR enzyme activity. The purpose of the present invention is to monitor elution, etc. during the process, and the present invention is not limited by the carrier used.

Next, examples of the present invention will be shown.

Example 1 E. coli containing recombinant plasmid pBLAK1
Purification of DHFR-BK fusion protein from C600 strain E. coli C600 strain containing plasmid pBLAK1 was cultured overnight at 37°C in YT + Ap medium of 3.
Bacterial cells were collected by centrifugation. Bacterial cells with a wet weight of approximately 12 g were obtained. The bacterial cells were suspended in 10mM potassium phosphate buffer PH7.0 (Buffer 1) containing 0.1mM disodium ethylenediaminetetraacetate (EDTA),
After disrupting the cells by ultrasonic disruption, the cells were centrifuged at 20,000 rpm for 1 hour to obtain 35 ml of supernatant. When the DHFR enzyme activity of the obtained supernatant was measured, it was found to be 90 units/ml (total activity 3150 units, total protein 902 mg, specific activity 3.5).
units/mg). DHFR enzyme activity was measured using DHFR reaction solution (0.05mM dihydrofolic acid, 0.06mM
NADPH, 12mM 2-mercaptoethanol,
50mM potassium phosphate buffer (PH7.0)
The reaction was carried out at 30° C. in a spectrophotometer, and the change in absorbance at 340 nm over time was measured.
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 centrifugation supernatant was transferred to a DEAE-Toyopearl 650M column (250mm _x
1500 mm, approximately 75 cm ³ ), and eluted with buffer 1 containing 50 mM CK1. Approximately 6 ml of fractions were 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%), recovered protein 25
mg, specific activity 75.9 units/mg). This was concentrated to about 1 ml using an Amicon ultrafiltration device, and fractionated using Toyopearl HW55 column chromatography. Collect approximately 2.8ml of fractions,
The enzymatic activity of DHFR was measured, and the peak fraction of enzymatic activity was collected. 11.2 ml of enzyme solution was obtained (recovered activity 1151 units (36%), recovered protein 5.7 mg,
specific activity 202 units/mg). When the obtained enzyme protein was analyzed by SDS electrophoresis, it was found to be homogeneous, including lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, egg albumin, and bovine serum albumin. As a marker, the molecular weight of purified dihydrofolate reductase was estimated to be 20,000, which was almost the same as the predicted molecular weight of 19,801 from the base sequence.

Example 2 Immunoassay of DHFR-BK fusion protein Commercially available rabbit anti-BK antibody (Biosys, manufactured by SA)
0.1 ml of sodium phosphate buffer containing 0.15 M NaCl, 0.1% bovine serum albumin, and 0.1% sodium azide, pH 7.0 (referred to as BSAPE).
0.1 ml and DHFR− diluted 1000 times with BSAPB.
0.1 ml of BK (10.3 mm units, 50.1 ng DHFR)
- BK) were mixed and reacted at 30° for 1 hour.
Next, add rabbit serum diluted 300 times with BSAPB to 0.1
ml and 0.1 ml of goat anti-rabbit gamma globulin diluted 300 times with BSAPB and further incubated at 30℃ for 1 hour.
Allowed time to react. The resulting precipitate was collected by centrifugation at 12,000 rpm, and the precipitate was further washed three times with BASPB. The resulting precipitate was solubilized by adding 0.02 ml of 0.005 N hydrochloric acid, and further 0.2 ml of 0.5 M potassium phosphate buffer containing 10 mM dithiothreitol, pH 7.0 was added. As a control, (1) 0.1 mg
(2) DHFR−BK instead of
using B. subtilis DHFR, (3) rabbit anti-
One without BK antibody and (4) one without goat anti-rabbit gamma globulin were prepared in the same manner, and the DHFR enzyme activity was measured. As a result, no significant DHFR activity was observed in samples (2) to (4) prepared as controls;
In the complete system, an enzyme activity of approximately 0.5 to 2% of the added DHFR-BK was observed, and it was also used as a control (1)
In the sample, about one tenth of that (added DHFR−
Enzyme activity of approximately 0.05-0.2% of BK was observed.

This result shows that the DHFR-BK fusion protein
This shows that chemically synthesized BK reacts with antibodies against BK and competitively inhibits the antigen-antibody reaction.

Example 3 Amino acid sequence of DHFR-BK fusion protein The present inventors have already revealed the entire nucleotide sequence of the recombinant plasmid pBLAK1 containing the DHFR-BK fusion protein gene (Japanese Patent Application Laid-Open No. 1983-1999).
245679). From the obtained base sequence, DHFR-
They extracted the part encoding the BK fusion protein and translated it into amino acids using the triplet codon table. As a result, the results shown in FIG. 1 were obtained.
FIG. 1 shows the DNA sequence of the portion encoding the DHFR-BK fusion protein and the corresponding amino acid sequence. As a result, (1) the DHFR-BK fusion protein is composed of 172 amino acids;
From the amino terminal side of the fusion protein to position 162,
It is identical to the amino acid sequence of DHFR of B. subtilis,
The 164th to 172nd examples are BK arrays.

(2) It became clear that the 163rd amino acid is Met, and that it has a structure that allows BK to be excised by treating the fusion protein with bromcyanide, and (3) that the molecular weight of the fusion protein is 19801. Ta.

In addition, when we examined the carboxy-terminal amino acid sequence of the purified protein with DHFR activity using the carboxypeptidase method, we found that -Phe
-Ser-Pro-Phe-Arg (carboxy terminal). This sequence completely matches the carboxy-terminal sequence of BK. The above results indicate that the DHFR-BK fusion protein does indeed contain BK.

Effects of the invention As mentioned above, the DHFR-BK fusion protein
It exhibits DHFR enzymatic activity and can be easily purified. It is useful for producing BK using it.

[Brief explanation of drawings]

Figure 1 shows DHFR-BK present in pBLAK1.
FIG. 2 is a diagram showing the base sequence of a portion encoding a fusion 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, A1a is alanine, Arg is arginine,
Asn represents asparagine, Asp represents aspartic acid, Cys represents cysteine, Gln represents glutamine,
Gln is glutamine, Glu is glutamic acid, Gly
is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine,
Met represents methionine, Phe represents phenylalanine, Pro represents proline, Ser represents serine, Thr represents threonine, Trp represents tryptophan, Tyr represents tyrosine, and Val represents valine. The numbers in the figure indicate the numbers starting from "A" of the ATG codon that encodes the first amino acid, methionine.

Claims (1)

[Claims] 1. E. coli containing recombinant plasmid pBLAK1
A dihydrofolate reductase-bradykinin fusion protein produced by strain C600 and having the amino acid sequence shown below. [Table] [Table] 2 E.coli containing recombinant plasmid pBLAK1
C600 strain was cultured, and from the cell-free extract of the cultured bacterial cells,
Dihydrofolate reductase, characterized in that it is purified using ion exchange column chromatography and subsequent gel filtration column chromatography using dihydrofolate reductase activity as a guide.
Production separation and purification method of bradykinin fusion protein.