JPH0364518B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention is directed 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. ) on the carboxy-terminal (hereinafter referred to as C-terminal) side of the enzyme (hereinafter referred to as DHFR)-somatostatin fusion protein (hereinafter referred to as DHFR-GIF).
and its separation and purification method. DHFR-GIF of the present invention has the amino acid sequence shown in FIG. 1 and is suitable for fields such as fermentation industry and pharmaceutical industry. Prior Art and Problems GIF is a type of hypothalamic peptide, and suppresses the secretion of many peptide hormones produced in the gastrointestinal tract, such as anterior pituitary hormones such as growth hormone, and insulin and glucagon. Because of this effect, GIF is expected to be used as a therapeutic agent for gigantism, diabetes, and the like. DHFR-GIF of the present invention is a protein with a structure in which GIF is fused to the C-terminal side of modified dihydrofolate reductase of Escherichia coli, and is a novel substance that has not been reported so far. As a substance similar to the DHFR-GIF of the present invention, the Bacillus subtilis DHFR C
There is a protein fused to GIF at the end. (JP Publication No. 63-258597). The technical background of the present invention is so-called genetic manipulation technology. The recombinant plasmid pTPGIF2 containing the gene encoding DHFR-GIF of the present invention has already been constructed by the present inventors. Furthermore, E. coli containing pTPGIF2 has been deposited with the Institute of Fine Technology as FERMBP-1577. However, the method for separating and purifying DHFR-GIF from E. coli containing pTPGIF2 was completely unknown. OBJECT OF THE INVENTION The object of the present invention is to develop a novel substance, DHFR-
The purpose of this project is to establish a method for separating and purifying GIF and to utilize DHFR-GIF. In addition, the present invention uses genetic engineering techniques to
This was done as part of the development of a method to mass produce GIFs. Structure of the invention 1 Structure of DHFR-GIF. DHFR-GIF of the present invention is composed of 175 amino acids, as shown in FIG.
The sequence from positions 1 to 159 counting from the amino terminus of DHFR-GIF is the wild type of E. coli.
This is a sequence in which one amino acid substitution has occurred in DHFR (Cys-152 (wild type) → Glu-152), and the sequence from positions 162 to 175 is GIF. The amino acid immediately preceding the GIF sequence is methionine (Met). This allows DHFR−
By applying Bromsian processing to GIF, GIF
It is a structure that can be specifically cut out.
The 160th amino acid is isoleucine (lle). This sequence was created to match the reading frame of the genetic code when creating a fusion protein gene. The molecular weight of DHFR-GIF is
It is 19891. Figure 1 shows how to encrypt DHFR-GIF.
It describes the DNA sequence at the same time. We were able to determine the amino acid sequence from this DNA sequence. DHFR-GIF has a structure in which the carboxy-terminal side of Escherichia coli DHFR is partially modified and further fused with GIF. Normally, when the primary structure of a protein is changed, it often loses its physiological activity, but in the case of DHFR-GIF of the present invention, DHFR
No effect on enzyme activity was observed. 2 DHFR- from E. coli containing pTPGIF2
Separation and purification of GIF. The separation and purification of DHFR-GIF of the present invention consists of the following steps: culture of bacterial cells, disruption of bacterial cells, DEAE-Toyopearl column treatment, mesotrixate-coupled affinity chromatography, and DEAE-Toyopearl column chromatography. There is. Culture of bacterial cells pTPGIF2 is pTP70-1 JP-A-63-46193
This is a recombinant plasmid obtained by inserting a chemically synthesized DNA that encodes GIF into the BamHI site of the plasmid (see Japanese Patent Publication No. 2003-111002), and had already been constructed by the present inventors. pTPGIF2 is introduced into Escherichia coli and stably exists, and the Escherichia coli containing pTPGIF2 has been deposited as FERMBP-1577 at the Institute of Fine Technology. Culture of E. coli containing pTPGIF2
YT+Ap medium (5g NaCl in medium 1,
Liquid medium containing 8 g tryptone, 5 g yeast extract and 50 mg ambicillin sodium. ) can be cultured. In addition to this, the culture medium includes ST+Ap medium (a liquid medium containing 2 g of glucose, 1 g of dipotassium phosphate, 5 g of polypeptone, 5 g of yeast extract, and 50 mg of ambicillin sodium in medium 1). Any medium can be used as long as it grows, but as far as I have investigated,
YT+Ap medium was optimal for DHFR production. E. coli containing pTPGIF2 is inoculated into a medium and cultured at 37°C until the late logarithmic growth phase or stationary phase. Depending on the culture temperature,
The amount of DHFR-GIF accumulated varied, and as far as we investigated, the higher the culture temperature, the greater the amount accumulated. The cultured bacterial cells are collected by centrifugation at 5000 rpm. From medium 1, bacterial cells with a wet weight of 2 to 5 g are obtained. Bacterial collection and subsequent operations are performed at a constant temperature (between 0 and 10°C, preferably 4°C) unless otherwise specified. Disruption of bacterial cells The cultured bacterial cells were suspended in buffer solution 1 (10 mM potassium phosphate buffer containing 0.1 mM sodium ethylenediaminetetraacetate, pH 7.0) at 3 times the wet weight, and then subjected to a French press. to crush the bacterial cells. 20,000 microbial cell disruption liquid
Spin and centrifuge for 1 hour to obtain supernatant (cell-free extract). DEAE-Toyopearl column treatment This operation is performed for the purpose of pretreatment for the next purification process. Add the same volume of 0.6M to the cell-free extract.
Add Buffer 1 containing KCl. This enzyme solution was applied to a DEAE Toyopearl column equilibrated with buffer 1 containing 0.3M KCl in advance.
Elute the enzyme with buffer 1 containing 0.3M KCl. Fractionate a certain amount of the eluate using a fraction collector. Measure the DHFR activity of the fractionated eluate, and collect the fractions containing enzyme activity. Mesotrixate-bound affinity chromatography The enzyme solution obtained by the above procedure is adsorbed onto a mesotrixate-bound Sepgarose affinity column equilibrated with buffer 1 in advance. After adsorption, buffer 1 containing 1M KCl
wash with Washing is done by washing the eluate from the column.
Measure the absorbance at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.1 or less. Elution of the enzyme is performed using a 10 mM potassium phosphate buffer containing 1 M KCl and 3 mM, pH 9.0, and the eluate is fractionated into fixed amounts using a fraction collector. About the fractionated eluate
Measure DHFR activity and collect fractions containing enzyme activity. The obtained enzyme solution was added to buffer solution 1.
Dialyze against 3 times. DEAE-Toyopearl column chromatography The dialyzed enzyme solution is adsorbed onto a DEAE-Toyopearl column equilibrated with buffer 1 in advance. After adsorption, buffer 1 containing 50mM KCl
wash with Washing is done by washing the extract from the column.
Measure the absorbance at 280 nm, and continue to flow the same buffer until the absorbance becomes 0.01 or less. Enzyme elution was performed using buffer 1 from 50mM to 0.4M.
A linear concentration gradient of KCl is used, and a fixed amount of the eluate is fractionated using a fraction collector. 280nm for fractionated eluate
Measure the absorbance and DHFR activity. Collect fractions with a constant value of enzyme activity/absorbance at 280 nm. With the above operations, the reproducibility is good and DHFR−
GIF can be electrophoretically purified to complete homogeneity. According to the present invention, purification of DHFR-GIF can be performed within one week including culturing. Next, examples of the present invention will be shown. Example Purification of DHFR-GIF A Amount of bacterial cells used: wet weight 12 g B Enzyme purification table (purification processes in the table are cell-free extract, DEAE-Toyopearl column treatment,
Represents mesotrixate-linked affinity chromatography, and DEAE-Toyopearl column chromatography.

[Table] When the obtained enzyme protein was analyzed by SDS electrophoresis, about 23,000 single protein bands were shown, indicating that the obtained enzyme preparation was homogeneous. When purified DHFRGIF was examined by enzyme immunoassay, it was shown to react with antibodies against GIF. In other words, it has been revealed that the purified protein has a structure immunologically equivalent to GIF. In order to clarify the amino acid sequence of the carboxy-terminal side of the purified protein, carboxypeptidase Y was applied to the purified protein at varying times, and the released amino acids were quantified (carboxypeptidase method). method for determining the carboxy-terminal amino acid sequence). As a result, −−Trp−Lys−Thr−Phe−
It was expected to be Thr-Ser- (carboxy terminal). The carboxy-terminal amino acid of DHFR-GIF is Cys (cysteine), but this amino acid cannot be detected by the above method. Ellman
When the content of thiol (SH group) in purified protein was measured using 5,5'-dithiobis2-nitrobenzoic acid (DTNB) using the method of value was obtained. When pTP70-1DHFR was similarly measured, the value was 0.8 to 1.0 residues/enzyme molecule. The above results indicate that the protein with DHFR activity obtained by purification from E. coli harboring pTPGIF2 is DHFR-GIF. Effects of the invention As mentioned above, from Escherichia coli having pTPGIF2,
Purification can be easily performed using DHFR activity as an indicator. The obtained DHFR-GIF not only has DHFR activity but also has the immunological properties of GIF. Furthermore, DHFR-GIF has a structure in which GIF can be excised by treating DHFR-GIF with Promcyan because the DHFR and GIF parts are bonded via a methionine residue. From this, DHFR-GIF can be used as a raw material for the structure of GIF, and is useful for producing GIF using it.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the base sequence of the portion encoding DHFR-GIF 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 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, le is isoleucine, Leu is leucine, Lys is lysine,
Met represents methionine, Phe represents phenylalanine, Pro represents 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 counted 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 Escherichia coli containing the plasmid pTPGIF2 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 Plasmids with the DNA sequences shown below
Cell-free extraction of dihydrofolate reductase-somatostatin fusion protein from the centrifuged supernatant obtained by culturing E. coli containing pTPGIF2 and disrupting the cultured cells, using dihydrofolate reductase activity as a guide. From liquid, ion exchange column treatment,
1. A method for separating and purifying a dihydrofolate reductase-somatostatin fusion protein, the method comprising purifying it using mesotrixate-coupled affinity column chromatography and anion exchange column chromatography. [Table] [Table] [Table] [Table]