JPH0371110B2 - - 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. ) is related to a new recombinant plasmid that enables the production of plasmids.

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 novel recombinant plasmid pBLAK1 of the present invention has the DNA sequence shown in FIG. Novel recombinant plasmid pBLAK1 and E.coliC600 containing pBLAK1
The strain is suitable for use in 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 contraction effect, and vascular permeability effect.
BK is Arg−Pro−Pro−Gly−Phe−Ser−Pro
-Phe-Arg has been revealed to be composed of nine L-amino acids. BK itself is an active peptide, and Kallidin, which has Lys at the N-terminus, and Met-Lys-Bradykinin, which has Met-Lys, are also known to have activity, although it is lower than BK. . BK is a short peptide and has already been described by Boissonnas et al., Helv.Chim.Acta, vol 43,
Chemical synthesis has been carried out by (pp. 1349 (1960)).

The present invention was carried out as part of the development of a new method for synthesizing BK. The technical background of the present invention is so-called genetic manipulation technology. A plasmid incorporating the gene encoding BK and its E.
So far, nothing is known about its expression in coli.

Problem Generally, polypeptides with a molecular weight of 10,000 or less are E.
Even when produced in a host such as coli, it does not exist stably because it is degraded by proteases. This is thought to be because the molecules are too small to form a stable conformation.
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. For example, Itakura et al. reported the synthesis of somatostatin, which consists of 14 amino acids, using genetic engineering. Their method involves chemically synthesizing the gene encoding somatostatin, fusing it with the β-galactosidase gene, integrating it into a multicopy plasmid, and translating the recombinant plasmid into E.
coli and expressed as a fusion protein in which somatostatin is fused to the carboxy-terminal side of β-galactosidase via methionine (K. Itakura et al., Science, vol. 198, pp. 1056
(1977)), but there are considered to be obstacles in the isolation and purification of the product due to the insolubility of the fusion protein and the lack of easily measurable enzymatic activity in the fusion protein.

Purpose of the Invention The present invention was carried out as part of the development of a new method for synthesizing BK.The present invention was carried out as part of the development of a new method for synthesizing BK. - Since there are problems with expressing a fusion protein with galactosidase, we aimed to develop a recombinant plasmid containing a gene that expresses a fusion protein that is soluble and has an easily measurable enzymatic activity. By using a bacterial cell containing such a recombinant plasmid, it is possible to produce a fusion protein containing BK, and by using easily measurable enzyme activity, the fusion protein can be easily purified. It is possible that

As a result of intensive research, the present inventors discovered that the above objective could be achieved by using the dihydrofolate reductase gene of Bacillus subtilis.
It is abbreviated as ) and BK fusion protein was created, and the present invention was completed by creating a recombinant plasmid pBLAK1 that incorporates a gene encoding a fusion protein of BK.

Structure of the Invention The present inventors have discovered that DHFR of B. subtilis
(1) It is made up of 168 amino acids, (2)
There is no problem with the productivity and activity of DHFR even if the amino acid sequence consisting of 6 amino acids on the carboxy-terminal side encoded by the sequence downstream of the EcoRI site present in the gene is replaced with another amino acid sequence. , (3) It has been revealed that DHFR with altered carboxy-terminal amino acid sequence does not become insolubilized. By using this result, we can identify the gene encoding BK with the reading frame B.sub.
-Integrated downstream of the EcoRI site of the tilis DHFR gene,
It is possible to synthesize a DHFR-BK fusion protein that retains enzymatic activity.

The recombinant plasmid pBLAK1 of the present invention is (1) BK
(2) molecular design and chemical synthesis of DNA encoding the B. subtilis DHFR gene;
This is the result of integration downstream of the EcoRI site.

(1) Molecular design and chemical synthesis of DNA encoding BK.

The DNA that encodes BK is shown below.
Two sequences consisting of 40 nucleotides are chemically synthesized by the phosphoramidite method and used by linking them together.

1 5'-
AATTCTATGCGCCCACCGGGTTT
CTCACCGTTCCGCTAG-3' 2 5'-
GATCCTTACCGGAACGGTGAGAA
ACCCGGTGGGCGCATAG-3′ EcoRI site (5′-
The sequence of GAATTC-3' (cleaved between GA), its upstream nucleotide sequence, and the amino acid sequence encoded by it are as shown below, 5'...GAA.AAA.AAG.AAT.TCT.... 3′Glu.Lys.
Lys.Asn.Ser. can be introduced into the EccRI cleavage site, and in order to determine the direction of introduction, the downstream side is the BamHI cleavage site (5′-
A sequence that can be introduced into the GGATCC-3' sequence (cleaved between GG) was selected. Furthermore, the fusion between BK and DHFR is mediated by methionine (Met), and the structure allows BK to be excised by treating the fusion protein with bromcyanide. Above array 1
is the sequence encoding BK, and 2 is its complementary strand sequence. By introducing the above array 1,
on the carboxy-terminal side of DHFR as shown below.
A sequence in which BK encodes a fusion protein via Met is obtained.

5′−GAAAAAAAGAATTCTATGCGCCCA…
GluLysLysAsnSerMetArgProCCGGGT
TTCTCACCGTTCCGCTAAG-3'...
ProGlyPheSerProPheArg (2) Chemically synthesized DNA of B. subtilis DHFR gene
Incorporation downstream of the EcoRI site.

Rows 1 and 2 are complementary to each other and have an EcoRI cleavage sequence at one end and a BamHI cleavage sequence at the other end. The present inventors have already developed a recombinant plasmid pBSFOLEK1 containing the gene for a fusion protein in which leucine enkephalin is fused to the carboxy terminal side of DHFR.
Publication pBSFOLEK1 contains a sequence encoding leucine enkephalin, an EcoRI site and
Each has one BamHI site. Therefore, the DNA that encodes the chemically synthesized BK is
A recombinant plasmid capable of expressing the DHFR-BK fusion protein can be created by introducing it into the EcoRI-BamHI site of pBSFOLEK1.

The plasmid pBLAK1 of the present invention has a size of 4774 base pairs, can transform host E. coli to trimethoprim and ampicillin resistance, and is determined by the base sequence shown in Figure 1. This is a new recombinant plasmid. Plasmid pBLAK1 contains restriction enzymes EcoRI, BamHI, Bgl
, BstE, Pst, Pvn, Sai,
Aat, Cla, Hind,
Each is cut at two places by Hpa.

FIG. 1 shows the entire base sequence of pBLAK1, showing only one sequence of the double-stranded DNA. Figure 2 shows DHFR present in pBLAK1.
- It is a diagram showing the base sequence of the portion encoding the BK fusion protein and the amino acid sequence of the protein. The recognition cleavage site for the restriction enzyme EcoRI is present at bases 676 to 681, and in FIG. 2, bases 480 to 485. The recognition cleavage site for the restriction enzyme BamHI is present at bases 715 to 721 in FIG. The sequence consisting of 40 nucleotides obtained by this EcoRI and BamHI cleavage is the sequence introduced by the above synthetic DNA.

The DHFR-BK fusion protein 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 the amino acid sequence of DHFR of S. subtilis,
The 164th to 172nd sequences are the BK sequences. 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
It is 19801.

E. coli containing pBLAK1 can produce DHFR-BK fusion protein intracellularly. Specifically, E.coli containing pBLAK1 was cultured, the bacterial cells were collected, the cells were sonicated, and the resulting supernatant was centrifuged at 20,000 rpm for 1 hour.DHFR-BK
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. Furthermore, it was revealed that the protein purified from this supernatant using DHFR enzyme activity as a guideline reacts with antibodies against BK. That is, the DHFR-BK fusion protein is
It has DHFR enzyme activity, and separation and purification can be performed using this as an indicator. Therefore, the present invention
By using pBLAK1, it became possible to produce BK using genetic engineering technology.

Novel recombinant plasmid according to the present invention
pBLAK1 was produced using pBSFOLEK1 JP-A-63-87981 and chemically synthesized DNA in Example 1.
However, the present invention is not limited by the method of plasmid construction.

The plasmid pBLAK1 of the present invention can be used in E. coli C600
The E. coli C600 strain containing pBLAK1 was introduced into the FERMP-
It has been deposited as 9300.

Next, examples of the present invention will be shown.

Example 1 Creation of recombinant plasmid pBLAK1 0.001 mg of plasmid pBSFOLEK1 (JP-A-63-
87981) using the restriction enzymes EcoIR and BamHI, and then separated by 1% agarose gel electrophoresis. Cut out a DNA fragment of approximately 4.7 kilobase pairs and place it in a dialysis tube with 1 ml of 50mM Tris-
Add HC1, pH 8.0, seal, and electroelution method (T. Maniatis et al., Molecular
Cloning A Loboratory Manual, p.164, Cold
DNA was recovered from the gel using Spring Harbor Laboratory (1982), reference 1), and then extracted with ethanol.
After precipitating the DNA, the precipitate was dried under reduced pressure (DNA
-1). This array is 77th and 716th in Figure 1.
This is the ~4774th array. (In Figure 1, the arrangement of the cyclic structure is shown as a linear arrangement for convenience, so
The 4774th base and the 1st base are connected next to each other. ) The DNA encoding BK was chemically synthesized using the phosphoramidite method using the sequence shown below.

1 5'-
AATTCTATGCGCCCACCGGGTTT
CTCACCGTTCCGCTAG-3' 2 5'-
GATCCTTAGCGGAACGGTGAGAA
ACCCGGTGGGCGCATAG-3' These two DNAs were chemically synthesized according to the phosphoramidite method, and after purification, the 5'-ends were phosphorylated with polynucleotide kinase, and each DNA was approximately 0.1
Take ml (contains about 0.0001mg of DNA),
This was annealed by incubating at 60°C (this was called DNA-2).

DNA-1 was mixed with 0.05ml of ligase reaction solution (10mM Tris-HC1, pH7.4, 5mM MgC12,
After dissolving (10mM dithiothreitol, 0.5mM ATP), add 0.05ml of DNA-2 and 0.5 units of
Add T4-DNA ligase and heat at 37℃ for 1 hour.
A DNA ligation reaction was performed. This reactant,
It was introduced into E. coli C600 strain according to the transformation method (reference 1, pp. 250). The treated bacterial cells were transferred to a nutrient agar medium containing 50 mg/ampicillin sodium and 10 mg/trimethoprim (1 g of golcose, 1 g of dipotassium phosphate, 5 g of yeast extract, 5 g of polypeptone, and 15 g of trimethoprim). Approximately 50 colonies were obtained by spreading the cells onto an agar medium (containing agar) and culturing at 37°C for 24 hours. Select eight colonies randomly from these colonies,
1.5ml of YT+Ap medium (5g of NaCl,
8 g trypne, 5 g yeast extract, and 50 g
in liquid medium containing mg ampicillin sodium).
The bacterial cells were cultured overnight at 37°C. Transfer each culture solution to an Etzpendorf centrifuge tube and spin at 12,000 rpm.
The cells were centrifuged for 10 minutes and the bacterial cells were collected. Discard the supernatant and
This was supplemented with 0.1 ml of electrophoresis sample preparation solution (0.0625 M Tris-HC1, pH 6.8, 2% sodium lauryl sulfate (abbreviated as SDS), 10% glycerin, 5% 2-mercaptoethanol, and
Contains 0.001% Bromophenol Blue. ) was added to suspend the bacterial cells, which were then kept in boiling water for 5 minutes to dissolve the bacterial cells. This treated sample was subjected to SDS-polyacrylamide electrophoresis (UK Lammli, Nature, vol.227, pp.680-
685 (1970). E.coli containing pBSFOLEK1 was treated in the same way as a standard sample, and lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, ovalbumin, and bovine serum were used as molecular weight marker samples. Samples containing albumin were run. As a result, in three of the eight colonies, the DHFR-leucine enkephalin fusion protein produced by E. coli containing pBSFOLEK1 disappeared, and a new protein containing approximately
It has become clear that proteins 1000 daltons larger are produced. In addition, the amount of newly obtained protein bands was determined from E. coli containing pBSFOLEK1.
Since there was no significant difference in the amount of the band from the DHFR-leucine enkephalin fusion protein produced by the method, it was thought that the efficiency of gene expression had not changed. Choose one from these three and use it as YT+
The method of Tanaka and Weisblum (T. Ta−naka, B. Weisblum; J. Bacteriology,
vol 121, pp. 354 (1975)). The obtained plasmid is digested with restriction enzymes.
EcoRI, BamHI, Bgl, BstE, PstI, Pvn
, SalI, Aat, ClaI, Hind. When cleavage was attempted with HpaI, 1, 1, 1, 1,
It became clear that it would be cut in 1, 1, 1, 2, 2, 2, 2 places. The obtained plasmid
It was named pBLAK1. The entire base sequence of pBLAK1 is
Determined according to the dideoxy method. As a result, the first
The nucleotide sequence shown in the figure was revealed, and the plasmid
It was revealed that pBLAK1 consists of 4774 base pairs.

Example 2 E.coli containing recombinant plasmid pBLAK1
Purification of DHFR-BK fusion protein from C600 strain.

E. coli C600 strain containing plasmid pBLAK1 was cultured in 3 YT+Ap medium at 37° C. overnight, and the 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 pH 7.0 (buffer 1) containing 0.1mM disodium ethylenediaminetetraacetic acid (EDTA)WO,
After disrupting the cells by ultrasonic disruption, 35 ml of supernatant was obtained by centrifugation at 20,000 rpm for 1 hour. 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 902mg, 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))
Transfer to a 1 ml cuvette and add the enzyme solution to it.
The reaction was carried out at 30°C in a spectrophotometer, and the change in absorbance at 340 nm was measured 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 centrifuged supernatant was transferred to a DEAE-Toyopearl 650M column (250 mm
x1500mm, approximately 75cm ³ ) and eluted with buffer 1 containing 50mM KC1. 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). Concentrate this to approximately 1 ml using an Amicon ultrafiltration device,
This was fractionated by Toyopearl HW55 column chromatography. Approximately 2.8 ml of fractions were collected, the enzyme activity of DHFR was measured, and the fractions with peak enzyme activity were collected. 11.2 ml of enzyme solution was obtained (recovered activity 1151 units, 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.

When the purified protein with DHFR activity was measured by immunoassay, it reacted with the antibody against BK, and the chemically synthesized BK caused the antigen-
It became clear that the antibody response was competitively inhibited. In addition, when we investigated the amino acid sequence on the carboxy-terminal side of the purified protein with DHFR activity using the carboxypeptidase method, we found that -
Phe−Ser−Pro−Phe−Arg (carboxy terminal)
It turned out to be. This sequence completely matches the carboxy-terminal sequence of BK. The above results indicate that the DHFR-BK fusion protein is indeed
It shows that it contains BK.

Effects of the invention As mentioned above, the novel recombinant plasmid
pBLAK1 produces the DHFR-BK fusion protein in a soluble state. In addition, the produced DHFR−
BK fusion protein exhibits DHFR enzymatic activity and can be easily purified. Because it has such properties, the novel recombinant plasmid of the present invention
pBLAK1 and E.coli containing it are DHFR-
Production of BK fusion protein and its use
Beneficial for BK production.

[Brief explanation of drawings]

Figure 1 shows the entire base sequence of pBLAK1, with only one DNA strand sequence of the double-stranded DNA written in the direction of the 5' end. The symbols in the figure represent nucleic acid bases; A represents adenine, C represents cytosine, G represents guanine, and T represents thymine. The numbers in the figure indicate the restriction enzyme Hind of the two restriction enzyme ClaI cleavage recognition sites that exist in pBLAK1.
of the ClaI cleavage recognition site closer to the cleavage site,
It shows the number counted from the first "A" of ATCGAT as number 1. Figure 2 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, Ala is alanine, Arg is arginine,
Asn represents asparagine, Asp represents aspartic acid, Cys represents cysteine, Gln represents glutamine,
Glu is glutamic acid, Gly is glycine, His is histidine, Ile is isoleucine, Leu is leucine, Lys is lysine, Met is methionine,
Phe stands for phenylalanine, Pro stands for 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. Stably replicates in E.coli and is a host
The gene that confers trimethoprim resistance can confer trimethoprim resistance and ampicillin resistance to E. coli, and the gene that confers trimethoprim resistance is a dihydrofolate reductase-bradykinin fusion protein produced by partially modifying the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. The novel recombinant plasmid pBLAK1 encodes , has a size of 4774 base pairs and has the DNA sequence shown below. [Table] [Table] [Table] [Table] [Table] 2 Stably replicates in E.coli and is a host
The gene that confers trimethoprim resistance can confer trimethoprim resistance and ampicillin resistance to E. coli, and the gene that confers trimethoprim resistance is a dihydrofolate reductase-bradykinin fusion protein produced by partially modifying the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. E. coli strain C600 containing the novel recombinant plasmid pBLAK1 encoding , having a size of 4774 base pairs and having the DNA sequence shown below. [Table] [Table] [Table] [Table] [Table]