JPS63245680A - Novel recombined plasmid pgif1 - Google Patents

Abstract

PURPOSE:To readily give a fused protein measurable enzymatic activity by making up a novel recombined plasmid pGIF1 including a gene coding fused protein from dehydrofolate reductase and somastatin to solubilize the fused protein. CONSTITUTION:The recombinant plasmid pGIF1 is stably copied in E. coli and gives the host E. coli, resistance to trimethoprim and ampicillin. In the plasmid, the gene giving trimethoprim resistance is recombined by modifying a part of the 3'-terminal of dihydrofolate reductase in Bacillus subtilis to code the fused protein with 4789 pairs of bases. The recombinant plasmid pGIF1 is introduced into E. coli C600 strain and the transformed strain is deposited as FERM P-9301.

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-T
A peptide consisting of a 14 amino acid sequence of hy-3er-Cys, hereinafter 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 polypeptide hormones produced in the gastrointestinal tract such as insulin and glucagon. G
IF is expected to be used as a therapeutic agent for dwarfism, diabetes, and the like.

The novel recombinant plasmid pGIF1 of the present invention has the DNA sequence shown in FIG. E, c containing this new recombinant plasmid pGIF1 and pGIF1
Oli C600 strain is suitable for fields such as microbial industry, fermentation industry, and pharmaceutical industry.

BACKGROUND OF THE INVENTION The technical background of the present invention is the so-called gene manipulation technology. Regarding the plasmid incorporating the GIF gene and its expression in E. coli, the results of Itakura et al.
ience+vol1.198. pp, 1056 1
063 (1977)). Generally, polypeptides with two molecules ff of 110,000 or less do not exist stably because they are degraded by proteases and the like even if they are produced in a host such as E or colj. This is thought to be due to the fact that it cannot maintain a stable conformation due to its small size (9 molecules). 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 involves chemically synthesizing the gene encoding GIF, fusing it with the β-galactosidase gene, and incorporating it into a multicopy plasmid.

The recombinant plasmid was introduced into E. coli and expressed as a fusion protein. fusion protein.

β-galactosidase and GIF are linked via a methionine residue. From this, GIF can be specifically cleaved and separated from the fusion protein by bromcyan treatment.

Problems However, in the case of the above known method, (1) E;
The fusion protein expressed in colj is insolubilized; (2) the activity of β-galactosidase itself is easy to measure, and during the separation and purification of the fusion protein;

It may be possible to use enzyme activity as an indicator.

In reality, separation and purification of the fusion protein has become a problem because it no longer has enzymatic activity due to fusion with GIF.

OBJECT OF THE INVENTION The object of the present invention is to solve the above problems, namely: HOGIF
The fusion protein that incorporates becomes insolubilized.

and (2) the lack of easily measurable enzymatic activity in the fusion protein, and to improve the GIF production technology.

GIF is a stable conformo'111 because it is small as a molecule.
1.1,1 Because I can't get the main yong* E, c o 1 i
Even if it is produced in a host of fj''5-J, it does not exist stably because it is degraded by protease etc. Therefore, when attempting to produce GIF using genetic engineering technology.

It is inevitable to create fusion genes and express them as fusion proteins. 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 so far,
Until now, fusion proteins that are soluble and have enzymatic activity have not been known.

As a result of intensive research, the present inventors found that the above problems could be solved by using the dihydrofolate reductase gene of Bacillus subtilis, and based on that knowledge, dihydrofolate reductase (hereinafter abbreviated as DHFR) The present invention was completed by constructing a recombinant plasmid pGIF1 incorporating a gene encoding a GIF fusion protein.

Structure of the Invention The present inventors have found that the DHFR of B.5ubtilis (1) consists of 168 amino acids, (2) the C- Even if the terminal amino acid sequence consisting of 6 amino acids is replaced with another amino acid sequence, D
There is no problem with HFR productivity and activity, +3)C
- It has been revealed that DHFR whose terminal amino acid sequence has been changed does not become insolubilized.

By utilizing this result and integrating the gene encoding GIF with its reading frame downstream of the EcoRI site of the B.5ubtilis DHFR gene, it is possible to synthesize a DHFR-GIF fusion protein that retains enzymatic activity. It is thought that this problem can be solved.

The recombinant plasmid pGIF1 of the present invention is (
1) Molecular design and chemical synthesis of DNA that encodes GIF.

and (2) chemically synthesized NA B, which was obtained as a result of the integration of the DHFR gene of 5ubtilis downstream of the EcoRI site, and is a completely new compound.

6DNA and Lri who kill GIF, below 1. The sequences shown were chemically synthesized by the phosphoramidite method and used by combining them, but the present invention is not limited by the two synthesis methods.

1.5'-AATTCTATGGCTGGTTGT-3
'2.5'-AAGAACTTCTTTTGGAAGA
CTTTCACTTCGTGTTAAG-3'3.5'-GATCCTTAACACCAAGTGAA
AGTCTTCCAAAA-3'4.5'-GAAGTTCTTAACAACCAGCCA
TAG-3' The plasmid pGIF1 of the present invention is 478
It is a novel recombinant plasmid, which has a size of 9 base pairs and can transform the host E.coli to be resistant to trimethoprim and ampinrin, and is determined by the base sequence shown in FIG. Plasmid pGIF1
are restriction enzymes EcoRI, BamHI.

Bgln, BstEIl, PstI, PvuI
I, Sat I cut one place each y A
a t TI+ CI a I + Hand■+H
Each is cut at two places by p a I.

FIG. 1 is a diagram showing the entire base sequence of pGIFl, and only one of the two mRNA sequences is shown. FIG. 2 is a diagram showing the base sequence of the portion encoding the DHFR-GIF fusion protein present in pGIFl and the amino acid sequence of the protein.

The recognition cleavage site of the restriction enzyme EcoRI is at bases 676 to 681 in Figure 1, and at base 4 in Figure 2.
It exists between bases 80 and 485. Restriction enzyme BamHI
The recognition cleavage site exists at bases 731 to 736 in FIG. The sequence consisting of 55 nucleotides in length obtained by this EcoRI and BamHI cleavage is the sequence introduced by the above synthetic NA.

The DHFR-GIF fusion protein is composed of 177 amino acids as shown in FIG.

The 162nd position from the amine end of the fusion protein is *
The amino acid sequence is the same as that of DHFR of B. 5ubtilis, and the sequence from positions 164 to 177 is the sequence of GIF. The 163rd amino acid is methio, *”)”l0J
ie t), and is a structure in which GIF can be excised by treating the fusion protein with bromcyanide. The molecular weight of the fusion protein is 20,380.

E. coli containing pGIFl is DHFR-GI
F fusion proteins can be made intracellularly. That is, E. coli containing pGIFl was cultured.

DHF was added to the supernatant obtained by collecting bacterial cells, disrupting them by ultrasonication, and centrifuging them at 20,000 revolutions per minute.
Almost all R-GIF fusion proteins exist. That is, the fusion protein is not insoluble but soluble in E,
It is produced in coi cells. Also.

From this supernatant, the fusion protein was purified using DHFR enzyme activity as a guideline, and was determined by enzyme immunoassay.
Reacts with antibodies against. That is, the DHFR-GIF fusion protein has DHFR enzyme activity, and can be separated and purified using this as an indicator. Therefore, the pGIF of the present invention
GI using genetic engineering technology by using l.
The above-mentioned problems related to F production can be solved.

The novel recombinant plasmid pGIF1 according to the present invention is p
The plasmid can be produced using BSFOLEKI (Patent Application No. 6l-234003) and chemically synthesized DNA according to the method described in Example 1, but the present invention is not limited by the method for producing the plasmid.

The plasmid pGIF1 of the present invention can be used in E, coli C
The E. coli C600 strain, which has been introduced into 600 strains and maintained in a stable state, and which contains the pGIFl of the present invention, has been deposited with the FEI as FERM P-9301.

Next, examples of the present invention will be shown.

Example 1 Creation of recombinant plasmid pGIF1 o, ooi+r@ plasmid pBSFOLEKI (
Patent application 6l-234003) with restriction enzyme EcoRI
After cleavage using BamHI and BamHI; separation by 196 agarose gel electrophoresis. Approximately 4.7 kilobase pairs of DNA
Take out the A fragment and put it in a dialysis chamber at 1d of 50mM.
Tris-HCI, pH 8.0 was added, sealed, and electroelution method + T, Ma
Niatis et al., Molecular CIoning
A Laboratory Manual + p.
164t Co1d Spring Harbor
Laboratory (1982), document 1).

DNA was collected from the gel, precipitated with ethanol, and the precipitate was dried under reduced pressure (referred to as DNA-1). This DNA sequence is 1-677 and 731-47 in Figure 1.
This is the 89th array. (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.

As a design guideline for this sequence, (1) pBsFOLEK
(2) the EcoRI cleavage site can be introduced into the 1 EcoRI and BamHI cleavage sites of DHFR;
(3) DHFR and GIF are fused via methionine by encoding the 60th to 162nd to match the open reading frames of the genes.

We considered creating a structure that allows GIF to be cut out using Bromsian processing.

1.5°-AATTCTATGGCTGGTTGT-3
'2.5''-AAGAACTTCTTTTGGAAGA
CTTTCACTTCGTGTTAAG-3'3.5'-GATCCTTAACACGAAGTGAA
AGTCTTCCAAAA-3゜4.5'-GAAGTTCTTAACAACCAGCCA
Four DNAs of TAG-3' were chemically synthesized according to the phosphoramidite method, purified, and phosphorylated at the 5°-end with polynucleotide kinase.
(containing approximately 0.0001 rl 1 g of DNA) was taken and annealed by incubating at 60°C (this is referred to as DNA-2).

Add DNA-1 to 0.05141 gauze reaction solution (10
mM Trjs-HCI, pH7, 4, 5mM Mg
C1z, 10mM dithiothreitol, 0.5m
After dissolving in MATP), add 0.05 units of DNA-2 and 0.5 units of T4-DNA ligase,
DNA ligation reaction was carried out at 7°C for 1 hour. This reaction.

Transformation method
n me t hoa' murmuring) What self literature 1゜pp, 250
) was introduced into E. coli strain C600. The treated bacterial cells were placed on a nutrient agar medium containing 50 rag/II sodium ampinrin and 1 omg/l trimethoprim (in 1 liter, Ig of glutose, 1 g of dipotassium phosphate, 5 g of polypeptone, and 15 g of agar). 30 colonies were obtained by plating the cells on an agar medium (containing agar medium) and culturing at 37°C for 24 hours. From these colonies, 8 colonies were randomly selected and added to 1.5-YT+Ap medium (5 g of Na in Ig).
Ct, 8 g of tryptone.

The bacterial cells were cultured at 37°C in a liquid medium containing 5 g of yeast extract and 50 rrIg of ampinlin sodium. Each culture solution was placed in an Eppendorf centrifuge tube and centrifuged at 12,000 rpm for 10 minutes to collect bacterial cells. Discard the supernatant and add to it 0.1-electrophoresis sample preparation solution (0.0625M Tris-HCt, pH 6).
, 8,296 sodium lauryl sulfate (abbreviated as SDS), 1096 glycerol, 2-mercaptoethanol (591), and 0.00196 promphenol blue. ) and suspend the bacterial cells.

The bacterial cells were dissolved by keeping this in boiling water for 5 minutes. This treated sample was subjected to 5DS-polyacrylamide electrophoresis (U, Lammli, Nat-ur).
e, vol, 227. pp, 680-685 (197
0)). pBsF as a standard sample
E. coli containing OLEKl was treated in the same way, and molecular weight marker samples include lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, egg albumin, and bovine serum albumin. The sample was run. As a result, in 6 out of 8 colonies, pBSFOLEKI
It was revealed that the DHFR-leucine enkephalin fusion protein produced by E and C01i, which contains E and C01, disappeared, and a new protein about 1000 daltons larger was produced. In addition, the amount of the newly obtained protein band is the same as that of the band of the DIIFR-leucine enkephalin fusion protein produced by E. coli containing pBsFOLEKl (:, 'As there is no difference in Ai, the efficiency of gene expression does not change. It was thought that one of these six cells was not properly selected and cultured in YT+Ap medium.
Tanaica and Weisblum's method (T,
Tanaka + B, Weisblumy J, Ba
cteriology, vol 121 + pp,
354 (1975)).

The obtained plasmid was treated with restriction enzymes EcoRI, B
amHI.

Bg l II t Bs t En, P s t L
Sat L Aat II+ C1a I tHi
When cleavage was attempted with ndIII and HpaI, 1. 1. 1. 1. 1. 1. 2. 2
．． 2. It was revealed that the plasmid was cleaved at the 2" site. The obtained plasmid was named pGIF'1.p
The entire base sequence of GIF-1 was determined according to the dideoxy method. As a result, the base sequence shown in FIG. 1 was revealed, and it was revealed that plasmid pGIF1 consists of 4789 base pairs.

Example 2 E, col containing recombinant plasmid pGIF1
Purification of DHFR-GIF fusion protein from strain 1C600.

Contains pGIF1, colt
After culturing C600 strain in 31 YT+Ap medium at 37°C overnight.

Bacterial cells were collected by centrifugation. Bacterial cells with a wet weight of about 14 g were obtained. The bacterial cells were mixed with 28ml of 1mM dithiothreitol (DTT) and 0.1mM ethylenediamine4.
After suspending the cells in 10 mM potassium phosphate buffer pH 7.0 containing disodium acetate (EDTA) and disrupting the cells by ultrasonic disruption, supernatant 30- was obtained by centrifugation at 20,000 rpm.

When the DHFR enzyme activity of the obtained supernatant was measured,
The value was 144 units/mJ (total activity: 4320 units, total protein: 1t1,200 mm, specific activity: 3.6 units/■ protein). supernatant.

DEAE-) Yopar 650M column ('250rr
mx1500 mm, about 75 ca), and O
Elution was performed by applying a KCI concentration gradient from M to 50mM. Approximately 5
Fractions were collected in ml portions and the enzyme activity of DHFR was measured.

Both fractions with enzyme activity were collected. An enzyme solution of 8211/ml was obtained (recovery activity 1830 units (42%)).

Amount of recovered protein: 20.2■, specific activity: 90.6-S nits/ff1g protein). This was concentrated to about 11 rLl using an Amicon ultrafiltration device, and this was
Fractionation was performed by W55 column chromatography.

Approximately 2.8 fractions were collected, the enzyme activity of DHFR was measured, and the fractions with the peak enzyme activity were collected (approximately 3.4
rnl, recovered activity 462 units (10.7%).

Amount of recovered protein: 1.9 [11 g, specific activity: 243 units/■ protein). 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 21,000, which was almost the same as the predicted molecular weight of 20,380 from the base sequence.

When the purified protein having DHFR activity was measured by enzyme immunoassay, it was revealed that the antigen-antibody reaction was competitively inhibited by GIF. Furthermore, when the carboxy-terminal sequence of the purified protein with DHFR activity was examined using the carboxypeptidase method, it was found that -Trp Lys-(Th
r, Phe, Thr)-3er- (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,
The content of thiol (SH) groups in the DHFR-GIF fusion protein was measured using 5,5°-dithiobis2-nitrobenzoic acid using the method of 4.2-4.6 residues/molecule. value was obtained. DHFR shown in Figure 2
The CyS content of -GIF was 5 residues/molecule, which almost matched this value. Further, the SH group content of DHFR (1-168) whose carboxy terminal side was not changed was similarly measured, and the result was a value of 2.4 to 2.7 residues/molecule. The above results were obtained from E.coli strain C600 containing two recombinant plasmids pGIFI.
F R≦G, = I' The F fusion protein is certainly GIF
It shows that it contains.

Effects of the Invention As mentioned above, the novel recombinant plasmid pGIF1 is
encodes the HFR-GIF fusion protein and p
E, coli C600 containing GIFl is DH
The FR-GIF fusion protein is produced in a soluble state.

Furthermore, the produced DHFR-GIF fusion protein
It exhibits FR enzyme activity and can be easily purified.

Because it has such properties, the novel recombinant plasmid pGIFI of the present invention and E. coli containing it.
C600 is useful for the production of DHFR-GIF fusion protein and the production of GIF using it.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the entire base sequence of pGIFl,
Only one DNA strand sequence of the Shizumoto strand DNA is described in the direction from the 5' end to the 3' end. The symbols in the figure represent nucleobases, and A represents adenine. C represents cytosine, G represents guanine, and T represents thymine. The numbers in the figure indicate the restriction enzyme H4ndlI among the two restriction enzyme C1aI cleavage recognition sites that exist in pGIFl [the ATCGA of the C1aI cleavage recognition site that is closer to the cleavage site].
The numbers are shown with the first 'A' of T as number 1. Figure 2 is a diagram showing the base sequence of the part encoding the DHFR-GIF fusion protein present in pGIFl and the amino acid sequence of the protein. The groups in the figure represent nucleobases and amino acids, A is adenine, C is cytosine, G
stands for guanine, T stands for thymine, and Ala stands for alanine.
rg is arginine + Asn is asparagine, Asp
represents aspartic acid, Cys represents cysteine, Gin represents glutamine, Glu represents glutamic acid, and G+y represents glycine. His stands for histidine, ■ce stands for isoleucine. L eu is leucine, Lys is lysine, Met is methionine + Phe is phenylalanine'; i:, P
r. is Proline, Set is Serine, Thr is Tre No. 37, Trpl;! h! Jp, 7 aa e T y
r Edf stands for + Vat stands for valine. The numbers in the diagram are. ATG encodes the first amino acid, methionine
The numbers are shown starting from codon "A" as number 1.

Claims (1)

[Claims] 1.E. It is stably replicated in the host E.coli. The gene conferring trimethoprim resistance encodes a dihydrofolate reductase-somatostatin fusion protein by partially modifying the 3' end of the Bacillus subtilis dihydrofolate reductase gene. The new recombinant plasmid pGIF1 has a size of 4789 base pairs and has the DNA sequence shown in FIG. 2. Novel recombinant plasmid p described in claim 1
E. containing GIF1. coli strain C600.