JPH0364110B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to somatostatin (Ala-Gly-Cys-Lys-Asn-
Phe−Phe−Trp−Lys−Thr−Phe−Thr−Ser−
A peptide consisting of the 14 amino acid sequence of Cys, hereinafter abbreviated as GIF. ) at the carboxy terminus - dihydrofolate reductase - somatostatin (hereinafter referred to as
It is abbreviated as DHFR-GIF. ) relates to a fusion protein and a method for producing the same. 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 dwarfism, diabetes, and the like.

The DHFR-GIF fusion protein of the present invention is a novel protein and has the amino acid sequence shown in FIG. This DHFR-GIF fusion protein is suitable for fields such as the pharmaceutical industry.

Prior Art The technical background of the present invention includes so-called genetic manipulation technology. A plasmid incorporating the gene encoding the DHFR-GIF fusion protein of the present invention has already been constructed by the present inventors (Japanese Patent Laid-Open No. 1983-1993-1).
245680), but it was completely unknown before then. In addition, plasmid pGIF1 that incorporates the gene encoding the DHFR-GIF fusion protein
was introduced into the E.coliC600 strain and kept in a stable state, and the E.coliC600 strain containing pGIF1 was transferred to the Microtech Institute.
It has been deposited as FERMP-9301.

Problems Since GIF is a polypeptide consisting of 14 amino acids, it is easy to chemically synthesize the gene encoding it, insert it into an appropriate expression vector, and express it in a host such as E. coli. Conceivable. However, short peptides etc.
Even if it is produced in a host such as E. coli, it will be degraded by proteases, so even if the efficiency of gene expression is improved, it will not be able to accumulate within the bacterial body. Therefore, using genetic engineering technology, GIF
When trying to produce a short polypeptide such as a protein, it is necessary to create a fusion gene and express it as a fusion protein.

Itakura et al. chemically synthesized the gene encoding GIF, fused it with the β-galactosidase gene, inserted it into a multicopy plasmid, introduced the recombinant plasmid into E. coli, and expressed it as a fusion protein in E. coli. (K. Itakura et al.
Science, vol.198, pp.1056 (1977)). In the fusion protein created by their method, β-galactosidase and GIF are bonded via a methionine residue, and GIF can be specifically cleaved and separated from the fusion protein by bromcyan treatment. . However, in the case of this method, (1) the fusion protein expressed in E. coli is insolubilized, and (2) β-galactosidase itself is easy to measure the activity of, so when separating and purifying the fusion protein, the enzyme It was thought that it would be possible to use activity as an indicator, but in reality, it has lost its enzymatic activity by fusion with GIF, which poses a problem in the separation and purification of the fusion protein.

The present inventors have already conducted research to solve the above problem, constructed a recombinant plasmid pGIF1 containing the DHFR-GIF fusion protein gene, and used this as an E.
They introduced this into a coli host and succeeded in stably expressing it (see Japanese Patent Application Laid-Open No. 63-245680). However, there have been no reports regarding DHFR-GIF 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-GIF fusion protein from E. coli C600 strain containing pGIF1. antigenic (i.e.
The present invention was completed by clarifying that the protein is a multifunctional protein that retains immunological activity.

Structure of the Invention The DHFR-GIF fusion protein of the present invention is composed of 177 amino acids as shown in FIG. The 162nd amino acid sequence from the amino terminus of the fusion protein is the same as the amino acid sequence of B. subtilis DHFR, and the 164th to 177th sequences are the GIF sequence. The 163rd amino acid is methionine (Met), which has a structure that allows GIF to be excised by treating the fusion protein with bromcyanide. The molecular weight of the fusion protein is 20380.

FIG. 1 also depicts the DNA sequence encoding the DHFR-GIF fusion protein. this
The amino acid sequence could be determined from the DNA sequence.

The DHFR of B. subtilis is composed of 168 amino acids, and the sequence from positions 1 to 162 is shown in Figure 1, and the sequence from positions 163 to 168 is Ser-Lys-Val- Gly-Gly-Phe. Normally, when the primary structure of a protein is changed, its physiological activity is often lost, but in the case of the present invention,
Even if the C-terminal sequence of B. subtilis DHFR is changed, no effect is observed on DHFR activity.

In addition, as shown in the Examples, the fusion protein purified from this supernatant using DHFR enzyme activity as a guideline was purified by enzyme immunoassay method.
Reacts with antibodies against GIF. That is, DHFR−
GIF fusion proteins react with GIF antibodies and have immunological properties of GIF.

The DHFR-GIF fusion protein with these characteristics is produced by E. coli containing the recombinant plasmid pGIF1.
It can be obtained from coli. That is, almost all of the DHFR-GIF fusion protein was found in the supernatant obtained by culturing E. coli containing pGIF1, collecting cells, sonicating them, and centrifuging them at 20,000 rpm for 1 hour. exists. That is, the fusion protein is produced in E. coli cells in a soluble rather than insoluble state. DHFR-GIF fusion protein is
E. coli C600 strain containing pGIF1 was grown in YT+Ap medium (5 g of NaCl, 8 g of tryptone, 5 g of
After culturing at 37°C until the end of the logarithmic growth phase or the normal phase,
Bacterial cells are collected and disrupted by ultrasonication 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. can.

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 investigated by measuring the change in absorbance at 340 nm over time. This measurement can be easily performed using a spectrophotometer.

Next, examples of the present invention will be shown.

Example 1 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 YT+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 20 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 ultrasonic disruption. 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 DEAF-Toyopearl.
It was adsorbed onto 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 20.2 mg, specific activity
90.6 units/mg protein). 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 enzyme activity of DHFR was measured, and the peak fraction of enzyme activity was collected (approximately 8.4 ml, recovered activity 462 units (10.7%), recovered protein amount 1.9 mg, specific activity 243 units/mg protein). The obtained enzyme protein
When analyzed by SDS electrophoresis, it was found to be homogeneous, and purified DHFR-GIF using lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase, glyceraldehyde-3-phosphate dehydrogenase, ovalbumin, and bovine serum albumin as molecular weight markers. The estimated molecular weight of the fusion protein was 21,000, which was almost the same as the predicted molecular weight of 20,380 from the base sequence.

When the purified DHFR-GIF protein was measured by enzyme immunoassay, it was revealed that it reacted with an antibody against GIF, and that this antigen-antibody reaction was competitively inhibited by chemically synthesized GIF.

Example 2 Amino acid sequence of DHFR-GIF fusion protein The present inventors have already revealed the entire base sequence of plasmid pGIF1 containing the DHFR-GIF fusion protein gene (see Japanese Patent Application Laid-Open No. 63-245680). The part encoding the DHFR-GIF fusion protein was extracted from the obtained base sequence and translated into amino acids using a 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-GIF fusion protein and the corresponding amino acid sequence. As a result, (1) the DHFR-GIF fusion protein is composed of 177 amino acids, and the 162nd amino acid from the amino terminal side of the fusion protein is B.
(2) The 164th to 177th sequences are the sequence of GIF; (2) the 163rd amino acid is Met; by
(3) The structure is such that it is possible to extract GIFs.
It was revealed that the molecular weight of the fusion protein was 20,380.

In addition, when we examined the carboxy-terminal sequence of purified protein with DHFR activity using the carboxypeptidase method, we found that -Trp-Lys-
It turned out to be (Thr, Phe, Thr)-Ser- (carboxy terminal). The carboxy-terminal amino acid of DHFR-GIF is Cys (cysteine),
Since this amino acid cannot be detected by the method we used, we further tested it using 5,5′-dithiobis2-nitrobenzoic acid using the method of Ellman.
When the content of thiol (SH) groups in the DHFR-GIF fusion protein was measured, a value of 4.2 to 4.6 residues/molecule was obtained. DHFR-GIF shown in Figure 1
The Cys content was 5 residues/molecule, which was almost in agreement with this value. Furthermore, the SH group content (3 residues/molecule) 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 indicate that the DHFR-GIF fusion protein obtained from the E. coli C600 strain containing the recombinant plasmid pGIF1 is
Indicates that it contains GIFs.

Effects of the invention As mentioned above, the DHFR-GIF fusion protein
It has DHFR activity and can be easily purified. This property makes it useful for GIF production.

[Brief explanation of drawings]

FIG. 1 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 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)

[Scope of Claims] 1. A dihydrofolate reductase-somatostatin fusion protein produced by E. coli strain C600 containing a recombinant plasmid pGIF1 having the DNA sequence shown in the following formula, and having the amino acid sequence shown in the following formula. [Table] [Table] [Table] [Table] [Table] [Table] 2. Culture E. coli C600 strain containing the recombinant plasmid pGIF1 having the DNA sequence shown below.
Using dihydrofolate reductase activity as a guideline, dihydrofolate reductase-somatostatin fusion protein is purified from a cell-free extract of cultured bacterial cells using ion exchange column chromatography and subsequent gel filtration column chromatography. A method for producing a dihydrofolate reductase-somatostatin fusion protein, characterized by: [Table] [Table] [Table] [Table]