JPS63258597A - Dihydrofolic acid reductase-somatostatin fused protein - Google Patents

Abstract

PURPOSE:To enable purification of the titled protein from a culture liquid, by passing a cell-free extract liquid of cultured microbial cells through an ion exchange column and gel-filtration column. CONSTITUTION:Microbial cells are collected from cultured product of E.coli C600 (FERM P-9301) containing recombinant plasmid pGIF1 and are disintegrated by ultrasonic radiation. The disintegrated product is subjected to centrifugal separation and the obtained supernatant liquid is purified by ion exchange chromatography and subsequent gel-filtration chromatography. The titled protein having amino acid sequence of formula, i.e. fused protein of dihydrofolic acid reductase and somatostatin is produced by the process.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to somatostatin (Ala-Gly Cys-Lys-Asn-P), which is a growth hormone secretion inhibitor.
he-Phe Trp Lys-Thr-Phe-
A peptide consisting of the 14 amino acid sequence of Thr-3er Cys, hereinafter abbreviated as GIF. ) at the carboxy terminus of dihydrofolate reductase-somatostatin (hereinafter abbreviated as DHFR-GIF) fusion protein. GIF is a type of hypothalamic peptide and 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 it has such an 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.

BACKGROUND OF THE INVENTION The technical background of the present invention is so-called genetic manipulation technology. A plasmid incorporating a gene encoding the DHFR-GIF fusion protein of the present invention has already been constructed by the present inventors (patent pending), but was previously unknown. In addition, the plasmid pGIF1 incorporating the gene encoding the DHFR-GIF fusion protein was introduced into the eE. coli C600 strain and kept in a stable state, and the E. coli C60 strain containing pGIFl was
The 0th strain has been deposited with the Microtech Institute as FERM P-9JO1.

Problem Since GIF is a polypeptide consisting of 14 amino acids, the gene encoding it has to be chemically synthesized (l,
It is thought that it can be easily integrated into an appropriate expression vector and expressed in a host such as E. coli. However, even if short peptides are produced in a host such as E. coli, they will be degraded by proteases or the like, so even if the efficiency of gene expression is improved, they cannot be accumulated within the cells. 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.

Itagane et al. chemically synthesized the gene encoding GIF, fused it with the β-galactosidase gene, and incorporated it into a multicopy plasmid.9 The recombinant plasmid was injected into E. coli.
was introduced into E. coli as a fusion protein (K, I, Takura et al., 5ci).
ence.

vol, 198, pp, 1056 (1977
) ). The fusion protein made by their method is
- 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, with this method.

(11E, the fusion protein expressed in coli is insolubilized; (2) the activity of β-galactosidase itself is easy to measure, and is used during separation and purification of the fusion protein;

It was thought that it would be possible to use enzyme activity as an indicator.

In fact, it has become a problem in the separation and purification of the fusion protein because it has lost its enzymatic activity by fusion with G IIFj','.

The present inventors have already conducted research to solve the above problem, constructed a recombinant plasmid pGIF1 containing the DHFR-GIF fusion protein gene, introduced it into an E. coli host, and succeeded in stably expressing it. (Patent pending) 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 extensive research and established a method for separating and purifying DHFR-GIF fusion protein from E. coli C600 strain containing pGIFl, and also demonstrated that the obtained fusion protein exhibits DHFR activity and GIF activity. The present invention was completed by revealing that the protein is a multifunctional protein that retains antigenicity (ie, immunological activity).

Structure of the Invention 1 The DHFR-G'IF fusion protein of the present invention is composed of 177 amino acids as shown in FIG. + B from the amino terminal side of the fusion protein to position 162
It is the same as the amino acid sequence of DHFR of p. 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 20.380.

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

B. The DHFR of 5ubtilis 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 5er-Lys-Val- Gly-Gty-P
It is he. Normally, when the primary structure of a protein is changed, it often loses its physiological activity.1. Yes, but.

In the case of the present invention, even if the C-terminal sequence of DHFR of B. 5ubtilis is changed, no effect on DHFR activity is observed.

Also from this supernatant, as shown in the Examples.

The fusion protein purified using DHFR enzyme activity as a guide reacts with an antibody against GIF using an enzyme immunoassay method. That is, the DHFR-GIF fusion protein is
It reacts with GIF antibodies and has immunological properties of GIF.

The DHFR-GIF fusion protein with these characteristics is an E
, can be obtained from colt. Namely.

E. coli containing pGIFl was cultured, and the bacterial cells were collected.

Almost all of the DHFR-GIF fusion protein is present in the supernatant obtained by sonication and centrifugation at 20,000 rpm. That is, the fusion protein is produced in E. coli cells in a soluble rather than insoluble state. DHFR-GIF fusion protein =
E. coli C600 strain containing pGIFl was transformed into YT
+A p, 5 g Nact in medium (il).

8g tryptone, 5g yeast extract, and 50r
I! After culturing at 37°C until the end of the logarithmic growth phase or the normal phase using a liquid medium containing ampicillin sodium (g), the cells were collected, disrupted by ultrasonication, and the centrifuged supernatant (cell-free extract) was collected. This supernatant can be highly purified by ion exchange column chromatography followed by gel filtration column chromatography.

DHFR enzyme activity was determined by 0.05 mM dihydrofolate;
0.06mM NADPH (reduced nicotinamide dinucleotide phosphate)), 12mM 2-mercaptoethanol, 50mM potassium phosphate buffer (
pH 7.0) was placed in a 1- cuvette.

An enzyme solution is added to this, and dihydrofolic acid and NADPH, which decrease with the enzymatic reaction at 30°C, can be investigated by measuring the change in absorbance at 340 nm over time.

This measurement can be easily performed using a 9 spectrophotometer.

Next, examples of the present invention will be shown.

Example 1 E. coliC containing recombinant plasmid pGIF1
Purification of DHFR-GIF fusion protein from strain 600.

E, cot i C containing plasmid pGIF1
After culturing 600 strains in 31 YT+Ap medium at 37°C overnight, the bacterial cells were collected by centrifugation. 14 g of bacterial cells were obtained in terms of wet weight. The bacterial cells were mixed with 20d of 10mM dithiothreitol (DTT) and 0.1mM disodium ethylenediaminetetraacetate (EDTA). I
Suspend in potassium phosphate buffer pH 7.0.

After crushing the cells by ultrasonic crushing, the cells were crushed at 20,000 revolutions/
A supernatant of 30 min was obtained by centrifugation for a rotation time of 30 min. When the DHFR enzyme activity of the obtained supernatant was measured, it was found that 144
Units/f (total activity: 4320 units, total protein amount: 1200 units, specific activity: 3.6 units)/mg protein). Transfer the supernatant to DEAE-Toyo Pearl 650
M column (250 mm, x 1500 mm, approx. 75
01115) and eluted using a 50mM KCI concentration gradient from OM. Fractions of approximately 6 d each were collected, the enzyme activity of DHFR was measured, and both fractions having enzyme activity were collected. An enzyme solution of 82d was obtained (recovery activity 183
0 units) (4296), recovered protein ff120.2
mgt specific activity 90.6 units/mg protein). This was concentrated to about 1d using an Amicon ultrafiltration device,
This was fractionated by Toyopearl HW55 column chromatography. Collect fractions of approximately 2.8 ml each.

The enzyme activity of DHFR was measured, and the peak fraction of enzyme activity was collected (approximately 3.4 m/, collected activity 462 units (10
, 796), total recovered protein 1.9rf1g, specific activity 2
43 units/■ protein). The obtained enzyme protein was analyzed by SDS electrophoresis.

homogeneous, lactalbumin, trypsin inhibitor, trypsinogen, carbonic anhydrase,
The molecular weight of the purified DHFR-GIF fusion protein was estimated to be 21,000 using glyceraldehyde-3-phosphate dehydrogenase, ovalbumin, and bovine serum albumin as molecular weight markers, which was approximately the same as the predicted molecular weight of 20,380 from the base sequence. Met.

When the purified DHFR-GIF protein was measured by enzyme immunoassay, it reacted with an antibody against GIF, and this antigen-antibody reaction was also caused by chemically synthesized GI
It has become clear that this is competitively inhibited by F.

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 (patent pending). From the obtained base sequence, DHFR-
The part encoding the GIF fusion protein was extracted and translated into amino acids using a triplet codon table.

As a result, the results shown in FIG. 1 were obtained. Figure 1 shows D
DNA encoding the HFR-GIF fusion protein
The sequence and its corresponding amino acid sequence are shown. As a result, the fllDHFR-G IF fusion protein is composed of 177 amino acids, and the 7
From the mino terminal side to the 162nd.

B, The sequence is the same as the sequence from 1 to 162 of DHFR of G. 5ubtilis, and the sequence from 164 to 177 is G.
IF sequence (2N63rd amino acid is M
et, and that the structure allows GIF to be excised by treating the fusion protein with bromcyanide, and (3) that the molecular weight of the fusion protein is 20.380.

Furthermore, when the carboxy-terminal sequence of the purified protein with DHFR activity was examined using the carboxypeptidase method, + -Trp-Lys -(Thr,
Phe, Thr) 5er- (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 detected 5,5'-dithiobis2- by using Ellman's method. DHFR-GIF using nitrobenzoic acid
When the content of thiol (SH) groups in the fusion protein was measured, a value of 4.2 to 4.6 residues/molecule was obtained.

The 'Cya content of DHFR-GIE- shown in Figure 1 is 5 residues/molecule.

This value almost matched. In addition, the SH group content of DHFR (1-168) whose carboxy terminal side has not been changed (
3 residues/molecule) were similarly measured and the results were 2.4 to 2.
The value was 7 residues/molecule.

The above results demonstrate that DHFR-GIF obtained from the E. coccine strain C600 containing the two recombinant plasmids pGIF1.
This shows that the fusion protein does indeed contain GIF.

Effects of the Invention As mentioned above, the DHFR-GIF fusion protein
It has FR activity and can be easily purified. Since it has such properties, it is useful for producing GIF.

[Brief explanation of drawings]

FIG. 1 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 symbols in the figure represent nucleobases and amino acids, where A'' is adenine, C is cytosine, G is guanine, T is thymine, Ala is alanine, Arg is arginine, Asn is asparagine, and A
sp is aspartic acid *Cys is cysteine, Gi
n stands for glutamine, Glu stands for glutamic acid, Gt
y is glycine. His stands for histidine and Ile stands for iturocine. Leu is leucine, I, ys is lysine, Met is methionine, Phe is phenylalanine,
Pro is habrolin, Set is serine, Thr is threonine. T rp stands for tryptophan and Tyr stands for tyrosine. Vat indicates valine. The numbers in the diagram indicate the ATG codon that encodes the first amino acid, methionine.
The numbers are shown with A'' as number 1.

Claims (1)

[Claims] 1. E. coli containing recombinant plasmid pGIF1. col
A dihydrofolate reductase-somatostatin fusion protein produced by iC600 and having the amino acid sequence shown in FIG. 2. E. coli containing recombinant plasmid pGIF1. col
iC600 was cultured, and using dihydrofolate reductase activity as a guide, dihydrofolate reductase-somatostatin fusion protein was extracted from a cell-free extract of the cultured cells using ion exchange column chromatography and subsequent gel filtration column chromatography. 1. A method for producing, separating and purifying a dihydrofolate reductase-somatostatin fusion protein, the method comprising: purifying a dihydrofolate reductase-somatostatin fusion protein.