JPH0371111B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to somatostatin (Ala-Gly-Gys-Lys-Asn-
Phe−Phe−Trp−Lys−Thr−Phe−Thy−Ser
- A peptide consisting of the 14 amino acid sequence of Cys,
Hereinafter, it will be abbreviated as GIF. ) is related to a new recombinant plasmid that enables the production of plasmids.

GIF is a type of hypothalamic peptide that suppresses the secretion of anterior pituitary hormones such as growth hormone, and many peptide hormones produced in the gastrointestinal tract such as insulin and glucagon. Because of this effect, GIF is expected to be used as a therapeutic agent for gigantism, diabetes, and the like. The novel recombinant plasmid pGIF1 of the present invention has the DNA sequence shown in FIG. This new recombinant plasmid pGIF1 and E.coli containing pGIF1
Strain C600 is suitable for fields such as microbial industry, fermentation industry, and pharmaceutical industry.

Prior Art The technical background of the present invention includes so-called genetic manipulation technology. Regarding the plasmid incorporating the GIF gene and its expression in E. coli, the results of Itakura et al. are known.
Science'vol.198, pp.1056-1063 (1977)). Generally, polypeptides with a molecular weight of 10,000 or less do not exist stably because they are degraded by proteases and the like even when produced in a host such as E. coli. this is,
It is thought that this is because the molecules are too small to form a stable conformation. Therefore, when attempting to produce a short polypeptide such as GIF using genetic engineering technology, it is necessary to create a fusion gene and express it as a fusion protein. Their method involved chemically synthesizing the gene encoding GIF, fusing it with the β-galactosidase gene, inserting it into a multicopy plasmid, introducing the recombinant plasmid into E. coli, and expressing it as a fusion protein. It is. The fusion protein combines β-galactosidase and GIF via a methionine residue. From this,
from the fusion protein by bromcyan treatment.
GIF can be specifically cleaved and separated.

Problems However, in the case of the above known method, (1)
The fusion protein expressed in E. coli is insoluble, (2) β-galactosidase itself is easy to measure its activity, and when separating and purifying the fusion protein,
Although it is thought that it is possible to perform this using enzymatic activity as an indicator, in reality, separation and purification of the fusion protein has become a problem because it has already lost its enzymatic activity by fusion with GIF.

Purpose of the invention The purpose of the present invention is to solve the above problems, namely (1)
The purpose of the present invention is to improve GIF production technology by solving the problems of insolubilization of a fusion protein incorporating GIF and (2) lack of easily measurable enzymatic activity in the fusion protein.

GIF is a small molecule and cannot maintain a stable conformation, so even if it is produced in a host such as E. coli, it is degraded by proteases and does not exist stably. Therefore, when attempting to produce GIF using genetic engineering technology, it is unavoidable to create a fusion gene and express it as a fusion protein. Therefore, in order to improve GIF production technology, it is considered important to consider proteins to be fused with GIF. However, although various studies have been conducted to date, no fusion protein has been known to be soluble and have enzymatic activity.

As a result of intensive research, the present inventors discovered that the above-mentioned problems could be solved by using the dihydrofolate reductase gene of Bacillus subtilis.
Abbreviated as DHFR. ) and GIF fusion protein integrated recombinant plasmid pGIF1
and completed the present invention.

Structure of the Invention The present inventors have discovered that DHFR of B. subtilis
(1) It is made up of 168 amino acids, (2)
Even if the amino acid sequence consisting of 6 amino acids on the C-terminal side encoded by the sequence downstream of the EcoRI site present in the gene is replaced with another amino acid sequence, there is no problem with the productivity and activity of DHFR;
(3) DHFR with a changed C-terminal amino acid sequence is
It is clear that it does not become insolubilized. Utilizing this result, the gene encoding GIF was inserted downstream of the EcoRI site of the B. subtilis DHFR gene with the same reading frame, thereby retaining the enzyme activity.
It is possible to synthesize DHFR-GIF fusion protein,
It is thought that the above-mentioned problems can be solved.

The recombinant plasmid pGIF1 of the present invention is produced by (1) molecular design and chemical synthesis of DNA encoding GIF;
(2) Chemically synthesized DNA of B. subtilis DHFR gene
It was obtained as a result of integration downstream of the EcoRI site, and is a completely new compound.

The DNA encoding GIF was chemically synthesized using the phosphoramidite method using the sequence shown below, and was used by combining them, but the present invention is not limited by the synthesis method.

1 5′−AATTCTATGGCTGGTTGT−3′ 2 5′−
AAGAACTTCTTTTGGAAGACTT
TCACTTCGTGTTAAG-3' 3 5'-
GATCCTTAACACCAAGTGAAAGT
CTTCCAAA-3' 4 5'-GAAGTTTCTTACAACCAGCCATAG
-3′ The plasmid pGIF1 of the present invention has a size of 4789 base pairs, can transform host E. coli to trimethoprim and ampicillin resistance, and has the base sequence shown in FIG. This is a new recombinant plasmid to be confirmed. plasmid
pGIF1 contains restriction enzymes EcoRI, BamHI, Bgl,
BstE, Pst, Pvu, Sal cut at one place each, Aat, Cla, Hind, Hpa
Each section is cut at two places.

FIG. 1 is a diagram showing the entire base sequence of pGIF1, showing only one sequence of the double-stranded DNA.
FIG. 2 is a diagram showing the base sequence of the portion encoding the DHFR-GIF fusion protein present in pGIF1 and the amino acid sequence of the protein. restriction enzyme
The EcoRI recognition cleavage site is shown in Figure 1.
At bases 676 to 681, in Figure 2, bases 480 to 681
It is present at base 485. The recognition cleavage site for the restriction enzyme BamHI is present at bases 731 to 736 in FIG. The 55 nucleotide long sequence obtained by this EcoRI and BamHI cleavage is the sequence introduced by the synthetic DNA.

The DHFR-GIF fusion protein is composed of 177 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 177th sequences are GIF sequences. The 163rd amino acid is methionine (Met), and by treating the fusion protein with bromcyanide,
It has a structure that allows you to extract GIFs. The molecular weight of the fusion protein is 20380. E. coli containing pGIF1 can make DHFR-GIF fusion protein intracellularly. That is, most of the DHFR-GIF fusion protein was found in the supernatant obtained by culturing E. coli containing pGIF1, collecting the bacterial cells, disrupting them by ultrasonication, and centrifuging them at 20,000 rpm for 1 hour. Everything exists. That is, the fusion protein is produced in E. coli cells in a soluble rather than insoluble state. Furthermore, the fusion protein purified from this supernatant using DHFR enzyme activity as a guide reacts with an antibody against GIF by enzyme immunoassay. That is, the DHFR-GIF fusion protein
It has enzymatic activity and can be used for separation and purification using this as an indicator. Therefore, by using pGIF1 of the present invention, the above-mentioned problems associated with GIF production using genetic engineering techniques can be solved.

Novel recombinant plasmid pGIF1 according to the present invention
can be constructed according to the method described in Example 1 using pBSFOLEK1 (Japanese Unexamined Patent Publication No. 63-87981) and chemically synthesized DNA, but the present invention is limited by the method of constructing the plasmid. It's not something you can do.

The plasmid pGIF1 of the present invention is introduced into E. coli C600 strain and maintained in a stable state, and the plasmid pGIF1 of the present invention is
E. coli C600 strain containing pGIF1 was sent to the Microtech Institute.
It has been deposited as FERM P-9301.

Next, examples of the present invention will be shown.

Example 1 Creation of recombinant plasmid pGIF1 0.001 mg of plasmid pBSFOEK1 (JP-A-63-
87981) using restriction enzymes EcoRI 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 HCl, pH 8.0, seal, and electroelution method (T. Maniatis et al., Molecular
Cloning A Loboratory Manual, p.164, Cold
DNA was collected 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 DNA sequence is from 1 to 1 in Figure 1.
These are the 677th and 731st to 4789th sequences. (In Figure 1, the arrangement of the circular structure is shown as a linear arrangement for convenience, so the 4789th base and the 1st base are connected next to each other.) The DNA that encodes GIF is as follows: A compound having the sequence shown in was chemically synthesized by the phosphoramidite method and used. The design guidelines for this array are (1)
It can be introduced into the EcoRI and BamHI cleavage sites of pBSFOLEK1, and (2) the EcoRI cleavage site is
From encrypting 160th to 162nd of DHFR,
Aligning the open reading frames of genes, (3) DHFR
We considered fusing and GIF via methionine to create a structure that allows GIF to be excised by bromcyan treatment.

1 5′−AATTCTATGGCTGGTTGT−3′ 2 5′−
AAGAACTTCTTTTGGAAGACTT
TCACTTCGTGTTAAG-3' 3 5'-
GATCCTTAACACGAAGTGAAAGT
CTTCCAAA-3' 4 5'-GAAGTTTCTTACAACCAGCCATAG
-3' four DNAs were chemically synthesized using the phosphoramidite method, purified, and then treated with polynucleotide kinase.
After phosphorylating the 5′-end, approximately 0.1 ml of each (approx.
(containing 0.0001 mg of DNA) was taken and annealed by incubating at 60°C (this is called DNA-2).

DNA-1 was mixed with 0.05ml of ligase reaction solution (10mM Tris-HCl, pH7.4, 5mM MgC12,
After dissolving in 10mM dithiothreitol, 0.5mM ATP), 0.05ml of DNA-2 and 0.5 units of T4-DNA ligase were added, and the mixture was incubated at 37°C for 1 hour.
A DNA ligation reaction was performed. This reaction was incorporated into the E. colC600 strain according to the transformation method (cited in Reference 1, pp. 250). The treated bacterial cells were transferred to a nutrient agar medium containing 50 mg of sodium ampicillin and 10 mg of trimethoprim (agar containing 1 g of glucose, 1 g of dipotassium phosphate, 5 g of polypeptone, and 15 g of agar). 30 colonies were obtained by coating the cells on a medium (medium) and culturing at 37°C for 24 hours. Select 8 colonies from these colonies and add 1.5 ml of YT+Ap medium (1.
Inside, 5g NaCl, 8g tryptone, 5g
Bacterial cells were cultured overnight at 37°C in a liquid medium containing yeast extract and 50 mg of ampicillin sodium. Each culture solution was placed in an Etzpendorf centrifuge tube and centrifuged at 12,000 rpm for 10 minutes to collect bacterial cells. Discard the supernatant and add 0.1 ml of electrophoresis sample preparation solution (0.0625M Tris-HCl, pH 6.8,
2% sodium lauryl sulfate (abbreviated as SDS),
Contains 10% glycerin, 5% 2-mercaptoethanol, and 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 (UKLammli, 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 6 out of 8 colonies,
It was revealed that the DHFR-leucine enkephalin fusion protein produced by E. coli containing pBSFOLEK1 disappeared, and a new protein approximately 1000 Daltons larger was produced. In addition, the amount of newly obtained protein band is
Since there was no significant difference in the amount of the band of the DHFR-leucine enkephalin fusion protein produced by E. coli containing pBSFOLEK1, it was thought that the efficiency of gene expression had not changed. Choose one out of these six and culture it in YT+Ap medium.
Tanaka and Weisblum's method (T. Tanaka, B.
Weisblum; J. Bacteriology, vol121, pp.354
(1975). The obtained plasmid was treated with restriction enzymes EcoRI, BamHI,
Bgl、BstE、Pst、Sal、Aat、Cla、
Trimming with Hind and Hpa resulted in
It became clear that the cuts were made at 1, 1, 1, 1, 1, 1, 2, 2, 2, and 2 locations, respectively. The resulting plasmid was named pGIF1. The entire base sequence of pGIF1 was determined according to the dideoxy method. the result,
The base sequence shown in Figure 1 was revealed, and it was revealed that plasmid pGIF1 consists of 4789 base pairs.

Example 2 E.coliC600 containing recombinant plasmid pGIF1
Purification of DHFR-GIF fusion protein from strains.

E. coli C600 strain containing plasmid pGIF1 was cultured in 3 YA+Ap medium at 37° C. overnight, and the bacterial cells were collected by centrifugation. Bacterial cells with a wet weight of approximately 14 g were obtained. The cells were suspended in 28 ml of 10 mM potassium phosphate buffer pH 7.0 containing 1 mM dithiothreitol (DTT) and 0.1 mM disodium ethylenediaminetetraacetate (EDTA), and the cells were disrupted by ultrasonication. 30 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 144 units/ml (total activity 4320 units, total protein amount
The specific activity was 3.6 units/mg protein). Transfer the supernatant to DEAE-Toyo Pearl.
It was adsorbed on a 650M column (250mm x 1500mm, approximately 75cm ³ ) and eluted using a KC1 concentration gradient from 0M to 50mM. Approximately 6 ml of fractions were collected, the enzyme activity of DHFR was measured, and the fractions having enzyme activity were collected. 82 ml of enzyme solution was obtained (recovered activity 1830 units (42%), amount of recovered protein 20.0 mg, specific activity
9.06 units/mg protein). This was concentrated to about 1 ml using an ultrafiltration device, and fractionated using Toyopearl HW55 column chromatography. Fractions of approximately 2.8 ml were collected, the enzyme activity of DHFR was measured, and the peak fraction of enzyme activity was collected (approximately 8.4 ml, collected activity 462 units (10.7%),
Amount of recovered protein: 1.9 mg, specific activity: 243 units/mg protein). When the obtained enzyme protein was analyzed by SDS electrophoresis, it was found to be homogeneous and contained lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glycerolaldehyde-3-phosphate dehydrogenase, egg albumin, and bovine serum albumin. The estimated molecular weight of purified dihydrofolate reductase as a molecular weight marker was 21,000, which was almost the same as the predicted molecular weight of 20,380 from the base sequence.

When the purified protein with DHFR activity was measured by enzyme immunoassay,
It has become clear that the antigen-antibody reaction is competitively inhibited by GIF. In addition, purified
When the carboxy-terminal sequence of a protein with DHFR activity was examined using the carboxypeptase method, it was found that -Trp-Lys-(Thr, Phe, Thr)
-Ser- (carboxy terminal). The carboxy-terminal amino acid of DHFR-GIF is
Cys (cysteine), but since this amino acid cannot be detected by the method used by the present inventors, we further performed DHFR-GIF using 5,5'-dithiobis2-nitrobenzoic acid using Ellman's method. The content of thiol (SH) groups in the fusion protein was determined to be 4.2 to 4.6 residues/molecule. The Cys content of DHFR-GIF shown in Figure 2 is
5 residues/molecule, which was almost in agreement with this value. Also, the carboxy terminal side is not changed.
The SH group content of DHFR (1-168) was similarly measured and was found to be 2.4 to 2.7 residues/molecule. The above results are based on the DHFR-GIF obtained from E. coli C600 strain containing the recombinant plasmid pGIF1.
This shows that the fusion protein does indeed contain GIF.

Effects of the invention As mentioned above, the recombinant plasmid pGIF1 is
The E. coli C600 strain that encodes the DHFR-GIF fusion protein and contains pGIF1 is DHFR-GIF.
Produce GIF fusion protein in soluble form. Furthermore, the produced DHFR-GIF fusion protein
It exhibits DHFR enzymatic activity and can be easily purified. Because of these properties, the novel recombinant plasmid pGIF1 of the present invention and E. coli C600 containing it are useful for the production of DHFR-GIF fusion protein and the production of GIF using it.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the entire base sequence of pGIF1, and only one DNA strand sequence of the double-stranded DNA is written in the direction of the 5' end. The symbols in the figure are
Nucleic acid bases are represented, with A representing adenine, C representing cytosine, G representing guanine, and T representing thymine.
The numbers in the figure indicate the restriction enzyme ClaI, which exists in two places in pGIF1.
The number of the ClaI cleavage recognition site that is closer to the restriction enzyme Hind cleavage site among the cleavage recognition sites is shown, counting from the first "A" of ATCGAT as number 1. FIG. 2 is a diagram showing the base sequence of the portion encoding the DHFR-GIF fusion protein present in pGIF1 and the amino acid sequence of the protein. The symbols in the figure are
Represents a nucleobase and an amino acid, A is adenine,
C is cytosine, G is guanine, T is thymine,
Ala is alanine, Agr 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, Lys is lysine, Met is methionine, Phe
is phenylalanine, Pro is proline, Ser
represents serine, Thr represents threonine, Trp represents tryptophan, Tyr represents tyrosine, and Val represents valine. The number in the diagram is “A” of the ATG codon that encodes the first amino acid, methionine.
It shows the number counted starting from 1.

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-somatostatin fusion protein created by partially modifying the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. The novel recombinant plasmid pGIF1 encodes , has a size of 4789 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-somatostatin fusion protein created by partially modifying the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. E. coli strain C600 containing the novel recombinant plasmid pGIF1 encoding , having a size of 4789 base pairs and having the DNA sequence shown below. [Table] [Table] [Table] [Table] [Table]