JPH0364111B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to leucine enkephalin (Tyr-Gly-Gly-Phe-
The present invention relates to a novel recombinant plasmid capable of producing a pentapeptide consisting of the five amino acid sequence of Leu. Novel recombinant plasmid of the present invention
pBSFOLEK1 is the DNA shown in Figure 1.
It has an array. Industrial applications of this new recombinant plasmid include microbial industry, fermentation industry,
Suitable for fields such as the pharmaceutical industry.

Prior Art Leucine enkephalin is known as an endogenous peptide that exhibits morphine-like analgesic action, and is an interesting polypeptide that is expected to be used as a non-addictive analgesic or anesthetic. The technical background of the present invention is so-called genetic manipulation technology.
As a plasmid incorporating leucine enkephalin, there is a plasmid already developed by the present inventors.
pLEG1 (Japanese Unexamined Patent Publication No. 61-260885) is known. pLEG1 has the characteristic that the gene sequence encoding leucine enkephalin can be excised and utilized by cutting with the restriction enzyme EcoRI.

Problems However, pLEG1 incorporates the E. coli dihydrofolate reductase-leucine enkephalin fusion protein gene, and although it has excellent gene expression efficiency such as promoters, pLEG1 cannot be used. When they introduced it into E.coli and attempted to express it, it was found that the amount produced was far short of the expected amount. Furthermore, the fusion protein lost dihydrofolate reductase activity. For this reason, immunochemical methods using antibodies are the only method for detecting fusion proteins made with E. coli, and since this method is complicated, there are issues that need to be improved in terms of separation and purification. There is.

OBJECTS OF THE INVENTION The present inventor has conducted extensive research in order to solve the above-mentioned problems, and has clearly analyzed the base sequence of the dihydrofolate reductase gene of B. subtilis.
As a result, (1) dihydrofolate reductase of B. subtilis is composed 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, the productivity of dihydrofolate reductase and It was revealed that there was no relationship with activity. Utilizing this result, the target peptide gene can be integrated downstream of the EcoRI site of the B. subtilis dihydrofolate reductase gene with the open reading frame of the gene, and a dihydrofolate reductase-target peptide fusion protein that retains enzymatic activity can be synthesized. It is possible. Due to this,
It is thought that production of gene products such as peptides, which have not conventionally been stably produced in the host, and their separation and purification will become extremely easy. From the above, it was suggested that the above-mentioned problems could be solved by molecular design and chemical synthesis of DNA encoding leucine-enkephalin, and this was carried out downstream of the EcoRI site of the dihydrofolate reductase gene of B. subtilis. A new recombinant plasmid, pBSFOLEK1, was created, which was integrated into the plasmid pBSFOLEK1, and the invention was completed.

Structure of the Invention The plasmid pBSFOLEK1 of the present invention has a size of 4762 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 pBSFOLEK1 contains restriction enzymes EcoRI, BamHI,
By BglII, BstEII, PstI, PvuII, SalI,
AatII, ClaI, HindIII,
Each is cleaved at two positions by HpaI.

FIG. 1 shows the entire base sequence of pBSFOLEK1, showing only one sequence of the double-stranded DNA. FIG. 2 is a diagram showing the base sequence of the portion encoding the dihydrofolate reductase-leucine enkephalin fusion protein present in pBSFOLEK1 and the amino acid sequence of the protein. The recognition cleavage site of the restriction enzyme EcoRI is shown in Figure 1.
At bases 675 to 681, in Figure 2, bases 480 to 681
It is present at base 485.

The dihydrofolate reductase-leucine enkephalin fusion protein is 168
It is composed of amino acids. The amino acid sequence from the amino terminal to position 162 of the fusion protein is identical to the amino acid sequence of B. subtilis dihydrofolate reductase,
The sequence from positions 164 to 168 is that of leucine enkephalin. The 163rd amino acid is methionine (Met), which has a structure that allows leucine enkephalin to be excised by treating the fusion protein with bromcyan. The molecular weight of the fusion protein is 19296.

E. coli containing pBSFOLEK1 can make dihydrofolate reductase-leucine enkephalin fusion protein intracellularly. That is,
E. coli containing pBSFOLEK1 was cultured, bacterial cells were collected, and the supernatant obtained by disrupting the cells contained dihydrofolate reductase-leucine enkephalin fusion protein. The dihydrofolate reductase-leucine enkephalin fusion protein can be purified based on its activity. Furthermore, the purified dihydrofolate reductase-leucine enkephalin fusion protein
It can react with antibodies against leucine enkephalin.

Plasmid pBSFOLEK1 is pBSDHFR1 (Japanese Unexamined Patent Publication No. 63-28394), pBR322 (JGSutcliffe,
(1978) Cold Spring Harbor Symp.Quant.
Biol., vol. 43, p. 77) and chemically synthesized DNA according to the method described in Example 1, but the present invention is limited by the method of plasmid construction. isn't it.

The plasmid pBSFOLEK1 of the present invention is an E.
coliC600 strain and kept in a stable state.
E. coli C600 strain containing pBSFOLEK1 was sent to the Microtech Institute.
It has been deposited as FERM P-8969.

Next, examples of the present invention will be shown.

Example 1 Creation of pBSFOLEK1 0.001 mg of plasmid pBSDHFR1 was digested with restriction enzymes.
After cleavage using EcoRI and PstI, separation was performed by 1% alogase gel electrophoresis. 2 large and small
A DNA fragment was obtained. Cut out a small piece and place it in a dialysis tube with 1 ml of 50mM Tris-HCl, PH.
Add 8.0, seal, and electroelution
Law, T. Maniatis et al., Molecular CloningA
Loboratory Manual, p.164, Cold Spring
According to Harbor Laboratory (1982), Reference 1),
DNA was collected from the gel, precipitated with ethanol, and the precipitate was dried under reduced pressure (referred to as DNA-1).

Next, add 0.001 mg of plasmid pBR322 to the restriction enzyme
After cleavage using BamHI and PstI, separation was performed by 1% agarose gel electrophoresis. 2 large and small
A DNA fragment was obtained. Cut out the large pieces and place in a dialysis tube with 1 ml of 50mM Tris-HCl, PH.
8.0 and seal, collect DNA from the gel by electroelution method, precipitate the DNA with ethanol,
The precipitate was dried under reduced pressure (referred to as DNA-2).

As the DNA encoding leucine enkephalin, 1 5'-
AATTCTATGTACGGTGGTTTCCTGTA
AG-3' 2 5'-
GATCCTTACAGGAAACCACCGTACATA
G-3' DNA consisting of two 28 nucleotides was chemically synthesized according to the phosphoramidite method, purified, and the 5'-terminus was phosphorylated with polynucleotide kinase. (containing DNA-3) and annealed by incubating at 60°C
).

DNA-1 and DNA-2 were mixed with 0.05ml of ligase reaction solution (10mM Tris-HCl, PH7.4, 5mM
MMgCl ₂ , 10mM dithiothreitol, 0.5m
After dissolving and mixing in MATP), 0.001ml of DNA
-3 and 5.0 units of T4-DNA ligase were added, and the DNA ligation reaction was carried out at 37°C for 1 hour. This reaction product was transformed using the transformation method (reference 1, pp. 250).
It was incorporated into E. coli C600 strain according to the following. The treated bacterial cells were placed on a nutrient agar medium containing 50 mg/ampicillin sodium and 10 mg/trimethoprim (1 g glucose, 1 g dipotassium phosphate, 5 g yeast extract, 5 g polypeptone, By coating on an agar medium containing 15g of agar and culturing at 37℃ for 24 hours,
We were able to obtain 105 colonies. Select one appropriately from these colonies, culture the bacterial cells,
Tanaka and Weisblum's method (T. Tanaka, B.
Weisblum; J. Bacteriology, vol121, pp.354
(1975)). The obtained plasmid was treated with restriction enzymes EcoRI, BamHAI,
BglII, BstEII, PstI, PvuII, SalI, AatII,
When cleavage was attempted with ClaI, HindIII, and HpaI, the results were 1, 1, 1, 1, 1, 1, 1, 2, 2, respectively.
It became clear that it would be cut in two or two places. The resulting plasmid was named pBSFOLEK1.
The entire base sequence of pBSFOLEK1 was determined according to the dideoxy method. As a result, the nucleotide sequence shown in Figure 1 was revealed, and the plasmid pBSFOLEK1 was
It was revealed that it consists of 4762 base pairs.

Example 2 E.coliC600 containing plasmid pBSFOLEK1
Purification of dihydrofolate reductase-leucine enkephalin fusion protein from strains.

E.coliC600 containing plasmid pBSFOLEK1
After culturing the strain overnight at 37 °C in a nutrient medium containing 50 mg of ampicillin sodium (liquid medium containing 5 g of NaCl, 8 g of bactopeptone, 5 g of yeast extract in 1, pH 7.4). , the bacterial cells were collected by centrifugation. Bacterial cells with a wet weight of about 13 g were obtained. The bacterial cells were dissolved in 10mM containing 20ml of 0.1mM dithiothreitol (DTT) and 0.1mM disodium ethylenediaminetetraacetate (EDTA).
After suspending the cells in potassium phosphate buffer PH7.0 and disrupting them by ultrasonic disruption, the cells were incubated at 20,000 rpm for 1
25 ml of supernatant was obtained by centrifugation for hr. The dihydrofolate reductase activity of the obtained supernatant was measured and found to be 144 units/ml. The supernatant,
DEAE-Toyopearl 650M column (25cmx150cm,
(approximately 75 cm ³ ) and eluted using a KCl concentration gradient from 0 to 100 mM. Fractions of approximately 1 ml were collected, dihydrofolate reductase activity was measured, and fractions having enzyme activity were collected. 25 ml of enzyme solution was obtained. This was processed using an Amicon ultrafilter for approximately 1
ml, and this was fractionated by Toyopearl HW55 column chromatography. Fractions of approximately 1 ml were collected, dihydrofolate reductase activity was measured, and fractions with peak enzyme activity were collected (approx.
3.7ml, 48mg). When the obtained enzyme protein was analyzed by SDS electrophoresis, it was found to be homogeneous, and the molecular weight of lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, egg albumin, and bovine serum albumin was determined. The estimated molecular weight of purified dihydrofolate reductase as a marker was 19,000, which was consistent with the predicted molecular weight of 19,296 from the base sequence.

When purified dihydrofolate reductase was measured by enzyme immunoassay, it was revealed that it reacted with an antibody against leucine enkephalin, and the antigen-antibody reaction was competitively inhibited by chemically synthesized leucine enkephalin. It became.
In addition, approximately 1.2mg of purified dihydrofolate reductase
in 0.5 ml of 70% formic acid at 37°C with bromine cyanide at 24°C.
After processing for an hour, freeze-dry this and add 0.2ml of 70%
When dissolved in formic acid and analyzed by high performance liquid chromatography, approximately 0.013 mg of leucine enkephalin was detected. This result is
This shows that dihydrofolate reductase of E. coli C600 strain carrying pBSFOLEK1 exists as a fusion protein with leucine enkephalin.

[Brief explanation of drawings]

Figure 1 shows the entire base sequence of pBSFOLEK1, with only one DNA strand sequence of the double-stranded DNA described in the direction from the 5' end to the 3' 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 number of the ClaI cleavage recognition site that is closer to the restriction enzyme HindIII cleavage recognition site among the two restriction enzyme ClaI cleavage recognition sites that exist in pBSFOLEK1, counting the first “A” of ATCGAT as number 1. There is. Figure 2 shows
FIG. 2 is a diagram showing the base sequence of the portion encoding the DHFR-leucine enkephalin fusion protein present in pBSFOLEK1 and the amino acid sequence of the protein. The symbols in the figure represent nucleobases and amino acids, A for adenine, C for cytosine, G for guanine, T for thymine, Ala for alanine, Arg for arginine, Asn for asparagine, and Asp for 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 phenyl. Alanine Pro
represents proline, Ser represents serine, Thr represents threonine, Trp represents tryptophan, Tyr represents tyrosine, and Val represents valine. The number in the diagram encodes the first amino acid, methionine.
The numbers are shown starting from the ATG codon "A" as number 1.

Claims (1)

[Claims] 1. Stably replicates in E.coli and is a host
It is possible to confer trimethoprim resistance and ampicillin resistance to E. coli, and the gene that confers trimethoprim resistance has been partially modified at the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. A novel recombinant plasmid pBSFOLEK1 encoding the Allin fusion protein, having a size of 4762 base pairs and having the DNA sequence shown below. [Table] [Table] [Table] [Table] [Table] 2 Stably replicates in E.coli and is a host
It is possible to confer trimethoprim resistance and ampicillin resistance to E. coli, and the gene that confers trimethoprim resistance has been partially modified at the 3' end of the dihydrofolate reductase gene of Bacillus subtilis. The E.
coliC600 strain. [Table] [Table] [Table] [Table] [Table]