JPH0333315B2 - - Google Patents

Description

[Detailed description of the invention]

Industrial Application Field The present invention provides a recombinant plasmid pBSFOL14-1 incorporating the Bacillus subtilis dihydrofolate reductase gene.
Bacillus subtilis dihydrofolate reductase (hereinafter abbreviated as DHFR) produced by Escherichia coli containing
DHFR− with a peptide fused to the carboxy end
The present invention relates to a separation and purification method for purifying fusion proteins of leucine enkephalin, DHFR-bradykinin, and DHFR-somatostatin. DHFR is known as a target of various antibacterial agents, and some drugs that specifically inhibit this enzyme are used as chemotherapeutic agents. For these reasons, DHFR is indispensable for research and development of chemotherapeutic agents. In addition, it is possible to perform a specific reduction reaction of folic acid compounds using this enzymatic reaction, and that it is a coenzyme.
It can also be used to detect and quantify NADPH.
On the other hand, DHFR-leucine enkephalin,
DHFR-bradykinin and DHFR-somatostatin are fusion proteins that have the properties of a physiologically active peptide fused to the carboxy terminal side in addition to DHFR activity, and are proteins that are expected to have various uses. Therefore, the present invention is suitable for the fields of enzyme production, pharmaceutical production, clinical testing, and enzyme reactors. Conventional techniques and problems There are no reports regarding a method for purifying DHFR from Bacillus subtilis. The carboxy-terminal sequence of Bacillus subtilis DHFR was modified and a peptide was fused to the carboxy-terminus.
DHFR-leucine enkephalin (patent application 1982)
-249260), DHFR-bradykinin (patent application 1982)
The present inventors have described the purification of fusion proteins of DHFR-somatostatin (Japanese Patent Application No. 62-092881). Purification of fused DHFR, in which a peptide is fused to the carboxy-terminal side of DHFR, is carried out by sonicating the cultured bacterial cells and then centrifuging them. Separation was performed using anion exchange column chromatography, followed by gel filtration column chromatography such as Toyopearl HW55. However, in this method, since many nucleic acids etc. are present in the cell-free extract, the initial
When using anion exchange column chromatography such as DEAE-Toyopearl, problems such as the need for a large column capacity,
Problems such as the elution peak of DHFR protein broadened and the volume of recovered enzyme solution increased.
Furthermore, it has become clear that it is often difficult to obtain a completely homogeneous enzyme preparation with good reproducibility in the above two-step purification process. Purpose of the Invention The present inventors discovered that there are no reports on purification methods for Bacillus subtilis DHFR, and that various peptides were fused to the carboxy-terminal side of Bacillus subtilis DHFR.
Creating a DHFR fusion protein;
In addition, in view of the fact that there are some problems in the purification method of such fusion proteins, we have conducted intensive research on the purification method of Bacillus subtilis DHFR and its fusion protein, and obtained completely homogeneous enzyme preparations with good reproducibility. We have developed a method that allows this, and completed the present invention. Structure of the Invention The present invention comprises 1. Culture of bacterial cells, 2. Disintegration of bacterial cells, 3. Streptomycin sulfuric acid treatment, 4. Ammonium sulfate fractionation, 5. Butyl Toyopearl column chromatography, 6. Toyopearl HW55 column chromatography. Yi, 7.
DEAE - Consists of the process of Toyopearl column chromatography. 1 Culture of bacterial cells pBSFOL14-1 is derived from Bacillus subtilis strain 168.
A plasmid containing the DHFR gene,
This was already constructed by the present inventors.
pBSFOL14-1 is introduced into E. coli and stably exists, and E. coli containing pBSFOL14-1 is
It has been deposited with the Institute of Fine Technology as FERM P-9627. Also, pBSFOLEK1, pBLAK1, and
Escherichia coli containing pGIF1 was sent to the FIKEN.
FERM P-8969, FERM P-9300, and
It has been deposited as FERM P-9301. All of these E. coli cultures were YT+Ap
It can be cultured in a medium (liquid medium containing 5 g of NaCl, 8 g of tryptone, 5 g of yeast extract, and 50 mg of ampicillin sodium in 1 liter of medium). In addition to this as a medium,
ST + Ap medium (liquid medium containing 2 g glucose, 1 g dipotassium phosphate, 5 g polypeptone, 5 g yeast extract, and 50 mg ampicillin sodium in 1 liter of medium), etc.
Any medium that allows bacterial growth can be used, but as far as we have investigated, DHFR
The YT+Ap medium was optimal for the production of . These Escherichia coli are inoculated into a medium and cultured at 37°C until the late logarithmic growth phase or stationary phase. The cultured bacterial cells are collected by centrifugation at 5000 rpm. From 1 liter of culture medium, 2 to 5 g of wet cells can be obtained. Bacterial collection and subsequent operations are performed at low temperatures (between 0 and 10°C, preferably 4°C) unless otherwise specified. 2 Disruption of bacterial cells Cultured bacterial cells were mixed with 3 times the wet weight of buffer 1 (10 mM potassium phosphate buffer containing 0.1 mM sodium ethylenediaminetetraacetate, PH
7.0) and crush the bacterial cells using a French press. Centrifuge the cell suspension at 20,000 rpm for 1 hour to obtain a supernatant (cell-free extract). 3 Streptomycin sulfuric acid treatment A treatment performed to remove nucleic acids mixed in the cell-free extract. Add streptomycin sulfate to the cell-free extract to a concentration of 1.9% (weight/volume) and stir (20 minutes). Thereafter, centrifugation is performed at 20,000 rpm for 1 hour to obtain a supernatant (streptomycin sulfate treatment solution). The concentration of streptomycin sulfate used is effective between 1.0 and 2.2%, but in the examples a concentration of 1.9% is used. 4 Ammonium sulfate fraction Gradually add the same volume of saturated ammonium sulfate solution as the streptomycin sulfuric acid treatment solution while stirring. After further stirring for 20 minutes, centrifugation is performed at 20,000 rpm for 1 hour to obtain a supernatant (ammonium sulfate treated supernatant). The volume of saturated ammonium sulfate added is effective between 0.7 and 1.2 volumes of streptomycin sulfate treatment solution. 5 Butyl Toyopearl Column Chromatography The ammonium sulfate-treated supernatant is adsorbed onto a butyl Toyopearl column equilibrated in advance with Buffer 1 containing 2M KCl. After adsorption, wash with buffer 1 containing 2M KCl. Washing is done by washing the eluate from the column.
Measure at 280 nm and continue using the same buffer until the absorbance is below 0.1. Enzyme elution is 0.9M
The eluate is fractionated into fixed amounts using a fraction collector. Measure the DHFR activity of the fractionated eluate, and collect the fractions containing enzyme activity.
The obtained enzyme solution was passed through a concentration device using an ultrafiltration membrane (Amicon, Ultrafiltration Cell).
Concentrate to approximately 1 ml using Diaflo Ultrafiltration membrane YM5). 6 Toyopearl HW55 column chromatography Apply the concentrated enzyme solution to a Toyopearl HW55 column (gel filtration column chromatography) equilibrated with buffer 1 in advance and elute with the same buffer. 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. 7 DEAE-Toyopearl column chromatography The enzyme solution obtained by Toyopearl HW55 column chromatography was added to buffer solution 1 in advance.
Adsorb onto a DEAE-Toyopearl column equilibrated with After adsorption, wash with buffer 1. For washing, measure the eluate from the column at 280 nm and check the absorbance.
Continue to flow the same buffer until the concentration is below 0.01. Elution of the enzyme is performed using Buffer 1 containing 50 mM KCl, and a fixed amount of the eluate is fractionated using a fraction collector. Measure the absorbance at 280 nm and DHFR activity of the fractionated eluate. Collect fractions with a constant value of enzyme activity/absorbance at 280 nm. By the above procedure, Bacillus subtilis can be isolated with good reproducibility.
DHFR and its fusion proteins can be electrophoretically purified to complete homogeneity. In addition to the Toyopearl column carrier used in the present invention, dextran gel-based carriers are known, but Bacillus subtilis DHFR and its fusion protein are thought to be strongly adsorbed to dextran-based carriers, and such carriers are not recommended. The recovery rate of the enzyme during purification was significantly reduced. According to the present invention, purification of Bacillus subtilis DHFR and its fusion proteins can be performed within 3 to 4 days including culturing. Next, examples of the present invention will be shown. Example 1 From E. coli containing pBSFOL14-1
Purification of DHFR A Amount of bacterial cells used: Wet weight 12g B Enzyme purification table (The purification process in the table is cell-free extract, streptomycin sulfuric acid treatment solution,
It represents ammonium sulfate-treated supernatant, butyl Toyopearl column chromatography, Toyopearl HW55 column chromatography, and DEAE-Toyopearl column chromatography (hereinafter, the same applies to Examples 2, 3, and 4).

[Table] Example 2 DHFR from E. coli containing pBSFOLEK1
-Purification of leucine-enkephalin fusion protein A Amount of bacterial cells used: wet weight 13g B Enzyme purification table

[Table] Example 3 Purification of DHFR-bradykinin fusion protein from E. coli containing pBLAK1 A Amount of bacterial cells used: wet weight 12 g B Enzyme purification table

[Table] Example 4 Purification of DHFR-somatostatin fusion protein from E. coli containing pGIF1 A Amount of bacterial cells used: wet weight 10 g B Enzyme purification table

[Table] Effects of the Invention The present invention enables the production of Bacillus subtilis, which is expected to be used in various ways as described in the section on industrial application fields.
It has become possible to supply DHFR and its fusion proteins in high purity quality.

Claims (1)

[Claims] 1 Recombinant plasmid pBSFOL14-1 incorporating the Bacillus subtilis dihydrofolate reductase gene,
In the separation and purification of a fusion protein of Bacillus subtilis dihydrofolate reductase or dihydrofolate reductase and leucine enkephalin, bradykinin or somatostatin produced by Escherichia coli containing either pBSFOLEK1, pBLAK1 or pGIF1,
PBSFOL14-1, pBSFOLEK1, pBLAK1 or
Cultured E. coli cells containing either pGIF1 were disrupted and the supernatant obtained by centrifugation was treated with streptomycin sulfate, ammonium sulfate fractionation, hydrophobic column chromatography, gel filtration chromatography,
Separation and purification of a fusion protein of Bacillus subtilis dihydrofolate reductase or dihydrofolate reductase and lotosine enkephalin, bradykinin or somatostatin, which is highly purified by anion exchange chromatography. Law.