JPH03164172A - Purification of urokinase precursor-like protein - Google Patents

Abstract

PURPOSE:To simply remove germ cell-derived protein impurities, etc., and obtain the high purity title precursor-like protein by dialyzing urokinase precursor-like aqueous solution obtained from the germ cell with water or buffer and then bringing the solution into contact with an anion exchanger. CONSTITUTION:An urokinase precursor-like protein (proUK)-containing aqueous solution, preferably produced by using Escherichia coli bacterium as a host and capable of expressing the activity is dialyzed using water such as distilled water or buffer having pH 4-7. After dialysis, anion exchanger (DEAE group) is treated with a strong acid or strong base in order to remove pyrodiene and the anion exchanger after the treatment is packed into a column and the above- mentioned dialyzed aqueous solution is passed through the above mentioned column and impurities are absorbed to carry out purification of the aimed proUK. Furthermore, the resultant aqueous solution may preferably be subjected to free-drying.

Description

Detailed Description of the Invention (Industrial Application Field) The present invention relates to a method for purifying a urokinase precursor-like protein (hereinafter referred to as proUK in this specification) produced in bacterial cells, and more specifically, the present invention relates to a method for purifying a urokinase precursor-like protein (hereinafter referred to as proUK) produced in bacterial cells. The present invention relates to a method for removing contaminant proteins derived from bacterial cells in an aqueous solution.

(Prior Art) proUX is a precursor of urokinase, an enzyme that activates plasminogen, which is a precursor of an enzyme that degrades blood clots formed in the blood, and converts it into plasmin.

Conventionally, urokinase obtained from human urine has been used for the treatment of blood clots, but when administered in large amounts, it may cause side effects, so recently attempts have been made to use proUK, which has fewer side effects.

Since prouk is rapidly converted into urokinase after being produced in vivo, it exists in only a trace amount under normal conditions. Therefore, the production of large quantities of proUK using genetic engineering techniques has been developed and is now at the stage of industrial production.

Regarding the genetic production of proUK, reference may be made to, for example, JP-A No. 59-51300.

(Problems with the Prior Art) According to the genetic engineering method, proUK, which naturally exists in trace amounts, can be produced in large quantities as described above. Since contamination with contaminant proteins cannot be avoided, the manufactured proU
A method for purifying K is required.

Conventionally, in order to purify foreign proteins produced using E. coli, for example, polymer gels with hydrophobic groups such as phenyl or butyl groups, polymer gels with iminodiacetic acid groups, etc. have been used ( Journa! or Bloc
hemlcal Chemistry, Volume 256, N
o. 8, pp. 3770-3775, 198l or J.
internal orBiochemical Chem
istry, Volume 256, No. l3S No. 7035-7
p. 041, 1981, etc.). However, these methods do not sufficiently remove contaminant proteins for purified proUK to be used as a medicine, and there is a demand for the development of alternative purification methods.

(Means for solving the problem) In view of the problem that conventional purification methods do not sufficiently remove contaminant proteins, the present inventors have conducted intensive research on a new purification method that can solve these problems. ,
The present invention has now been completed. That is, the present invention involves dialyzing an aqueous solution containing a urokinase precursor-like protein produced by bacterial cells against water or a buffer solution having a pH of 4 to 7, and then contacting the dialyzed solution with an anion exchanger. This is a characterized method for purifying urokinase precursor-like protein.

proUK as used herein refers to proUK that has the same primary structure as the natural urokinase precursor protein, as well as some amino acid residues in the protein as described in JP-A No. 62-143686. This includes proteins in which groups are substituted with other amino acid residues. Therefore, in this specification, pr
Describe as oUK.

In the present invention, the following operations are carried out on an aqueous solution containing proUK produced using a suitable bacterial cell, particularly E. coli, as a host using a genetic method. here,
This aqueous solution is not particularly limited as long as it contains proUK in a state where it can exhibit activity. When proUK is produced using Escherichia coli as the bacterial cell, pr
It is known that oUK is folded in a state in which it cannot express activity. Before carrying out the present invention, proUK in such a state may be treated by using a protein denaturant or an alkaline water-soluble relative as described in, for example, JP-A-59-161321 and JP-A-60-500893. It is sufficient to perform refolding (refolding) to a state in which activity can be expressed by a folding method or the like. In addition, if membrane fragments derived from bacterial cells are found in the aqueous solution containing proUK, these should be removed by a known method such as membrane separation method or gel filtration method prior to carrying out the present invention. It is best to remove it.

The aqueous solution as described above is first dialyzed against water or a buffer solution. The water may be one commonly used in the field of biochemistry, such as distilled water or ion-distilled water.

Moreover, the buffer solution is adjusted to pl+4 to pl{9, so that proUK will not be adsorbed to the anion exchanger in the next operation.

There are no particular restrictions on the buffering agent, and those commonly used in the field of biochemistry can be used without restriction.

Next, the post-dialysis solution obtained by the dialysis operation is brought into contact with an anion exchanger. This operation can be carried out in the form of so-called column chromatography, by introducing an anion exchanger into the solution or by filling a suitable column with an anion exchanger. If this operation is performed in the form of column chromatography, proUK can be obtained in the fraction that passes through when the dialysis solution is applied to the column.
This is preferable because it allows for faster purification.

In addition, in this case, contaminant proteins adsorbed on the anion exchanger can be quickly washed away, which is advantageous when the present invention is repeatedly carried out.

Any anion exchanger may be used as long as it supports an anion exchange group such as a DEAE group or a trimethylbenzylammonium group. For example, DEAE-Toyopearl
(manufactured by Tosoh Corporation, product name), DE52-Ce
Examples of anion exchangers include DOWEX 1 strongly basic anion (■ type) (manufactured by Dow Chemical Company). Through the above operations, the contaminant protein derived from the bacterial cells in the aqueous solution containing proUK produced by the bacterial cells is adsorbed to the anion exchanger, so when the present invention is carried out using the supernatant liquid or column chromatography. The pr purified to the pass-through fraction is
oUK fraction can be obtained.

By the way, when proUK is used as a medicine, it is necessary to remove a pyrogen called pyrogen in addition to contaminant proteins derived from E. coli, but the ion exchanger used in the present invention contains a trace amount of pyrogen. Contains. In terms of removing these pyrogienes, it is recommended to treat the anion exchanger containing DEAE groups etc. with a strong acid or strong alkali before use (for example, JP-A-55-92687, JP-A-61 -227782 etc.).

By the way, when the substance supporting the ion exchange group is a substance unstable to acid or alkali treatment, such as agarose, it is difficult to treat the carrier in advance. Therefore, in order to remove the pyrodiene from the ion exchanger in advance, an anion exchange group supported on a carrier that will not be denatured by the above-mentioned strong acid or strong alkali treatment is used. For example, it is preferable to use an anion exchanger using a hydrophilic vinyl polymer, acrylic polymer, styrene polymer, etc. as a carrier. Examples of commercially available materials include DEAE-Toyopearl (manufactured by Tosoh Corporation, trade name).

Carrying out the present invention under these conditions is also effective in terms of removing pyrogenic substances derived from bacterial cells.

Note that this publication does not impose any restrictions on the subsequent freeze-drying operation of the obtained proUK-containing aqueous wave to prepare a proUK specimen.

(Effects of the Invention) According to the present invention, contaminant proteins derived from bacterial cells in an aqueous solution containing protlK produced by bacterial cells are adsorbed to an anion exchanger, and as a result, protein free from contaminant proteins is obtained.
A roUK aqueous solution can be obtained.

Moreover, the operation of the present invention itself is extremely simple and can be carried out without requiring any special equipment.

The aqueous solution obtained by the purification method of the present invention has a lower amount of contaminant proteins than those obtained by conventional purification methods, and can be used as a variety of products. This makes it possible to provide materials suitable for use as such.

In addition, in the present invention, if an aqueous solution containing proUK after dialysis is brought into contact with an anion exchanger treated with a strong acid or a strong alkali, proU is produced by pyrodiene derived from the exchanger.
It is also possible to prevent K contamination, and as a result, a purified product with a lower amount of contaminant proteins and a lower pyrodiene content than proUK purified according to the conventional method can be obtained.

(Examples) Examples will be described below to explain the present invention in more detail, but the present invention is not limited to these Examples.

Example 1 Using a plasmid carrying DNA encoding proUK described in JP-A-62-143686.

After transforming E. coli (KY1436 strain) selected as a host. ProUK was produced by culturing in LB medium.

After culturing, the E. coli was crushed using a homogenizer,
After extracting the insoluble fraction, centrifugation was performed to remove bacterial body fragments and the like. Regarding proUK in the insoluble fraction,
After carrying out the refolding operation according to the method described in JP-A No. 59-161321, an additional 20 to 60
% concentration of ammonium sulfate precipitation? proUK was recovered as a precipitate and subjected to the following operation.

First, the proUK-containing aqueous solution obtained in the above procedure was applied to a hydrophobic chromatography column (manufactured by Tosoh Corporation, Butyl).
-Toyopearl) and metal affinity column (
After treatment with Tosoh Co., Ltd.'s Chelate T yopearl) and adjusting the absorbance to a specific activity of 70,000 units/280, the 5001 was added to 40 times the volume of 20mM phosphate buffer (pll7,5). was dialyzed twice as an external solution for a total of 8 hours.

Next, DEAE-T regenerated in advance with hydrochloric acid and caustic soda
Oyopear R1 (manufactured by Tosoh Corporation, trade name) was added to the above-mentioned post-dialysis solution, stirred for 2 hours, and then centrifuged (8000 rpm, 10 flllg).
) to precipitate the anion exchanger and obtain both supernatants.

The specific activity of proUK before and after addition of the anion exchanger was 61.55 million units before addition, whereas it was approximately 56 million units in the obtained supernatant fraction, and the recovery rate was approximately 91%. . Contaminant protein in the solution was 70 ng/proUK 1 mg before adding the anion exchanger, but after adding the anion exchanger, it was 10 ng/pr, which is the detection limit.
It had decreased to below oUK IB.

In addition, in this example, proUK activity 7]111
The determination is based on PyrG1yArg-pN using commercially available urokinase (manufactured by Midori Juji Co., Ltd.) of proUK activated to urokinase by the action of blasmin.
Determined by the hydrolysis activity of A (Pyr represents a pyroglutamyl group, Daiichi Chemical Co., Ltd., synthetic substrate S-2444) (Ilayashl, S, et al., TIIroIIIb
．． Res. 22, pp. 573-578, 1981).

The concentration of pro-LIK was determined based on absorbance at 280 ns. The amount of contaminant protein derived from E. coli was measured as in the reference example described later.

Example 2 A post-dialysis solution was obtained in the same manner as in Example 1. Next, D.E.A.
After regenerating E-Toyopear 1 with hydrochloric acid and caustic soda, it was packed into a 1.78×20 cm column.

The post-dialysis solution was applied to this column, and the fraction that passed through the column was obtained.

The total activity of proUK before and after column treatment was 6.
The amount was 5.23 million units, whereas the amount obtained in the pass-through fraction was approximately 65.89 million units, giving a recovery rate of approximately 101%. Contaminant protein in the solution was 70 ng before column treatment.
/proUKIB 1 mg, but after treatment, it decreased to below the detection limit of 10 ng/proUKIB.

Comparative Example 1 Contaminant protein gt (ECP; E.co
li. Removal of cell protein) A post-dialysis solution was obtained in the same manner as in Example 1. Add this solution to 50II in advance.
M ethylenediaminetetraacetic acid was added to an AF chelate Toyopearl force column (manufactured by Tosoh Corporation, 1.78 cm x 20 cm) that had been equilibrated with sodium chloride for 1 hour, and was adsorbed. 5
0IIIM Tris-HCl buffer (pll 7.5) was added to the column to wash and elute substances other than the adsorbed matter, and then
I! Further washing was performed with 1M Tris-HCl buffer (p116.O). Next 0. An acetate buffer (pH 5.0) containing 5 M sodium chloride was added to the column to elute proUK adsorbed on the column.

The total activity of proUK before and after column treatment was 26% before treatment.
460,000 units, whereas the pro obtained after treatment
In the UK elution fraction, it was 22.05 million units, and the recovery rate was 8.3 million units.
%Met.

The contaminant protein derived from E. coli was 70 n before column treatment.
g/proUK 1 mg, but after treatment it was 30 ng/proUK 1 mg, which was a larger amount of contaminant protein remaining than the detection limit (10 ng/proUK 1 mg).

Reference Example 1; Measurement of amount of contaminant protein In this example, contaminant protein (ECP; E.co
ll. The protein amount of Call Prote1n) was measured by enzyme immunoassay.

First, an antibody against ECP was prepared. Escherichia coli (KY1436 strain) similar to that used in Example 1, but not transformed with the plasmid used in Example 1, was cultured in the same medium as in Example 1. After culturing,
The insoluble fraction was extracted in the same manner as in Example 1, followed by a refolding operation and hydrophobic chromatography ram treatment.

5 With the above steps: J! Antibodies against ECP were obtained by immunizing rabbits with the J-prepared crude preparations.

For the quantification of ECP, fetal bovine serum, anti-ECP antibody (200 μl) prepared as described above, and the sample to be measured (
100 μl) were mixed, ECP derived from 03 Vacant strain was added at room temperature, and reacted at room temperature for 15 minutes.

Anti-rabbit IgG antibody insolubilized by bacterial cell walls was added to the above reaction solution, and enzyme-labeled ECP - anti-EPC antibody - anti-rabbit IgG antibody or ECP - anti-EPC antibody - anti-rabbit I
After incubating the immune complex consisting of gG antibodies, 0.9%
A sodium chloride solution was added and centrifuged to remove the liquid phase. Repeat this operation and collect the resulting precipitate,
Washed.

A phosphate buffer (pH 7) containing 4-methylumbelliferyl β-D-galactoside and bovine serum albumin was added to the solid phase obtained in the above procedure, and after being left at room temperature for 15 minutes, an alkaline substance was added. to bring the pH to 11,
The reaction was stopped.

After measuring the fluorescence intensity, standard E containing a known concentration of ECP
The ECPffi in the wipe was measured by comparing it with a calibration curve obtained by performing the same illll determination for the CP solution. The fluorescence intensity is fl at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm.
ll+ was determined.

Claims (1)

[Claims] (1) The aqueous solution containing the urokinase precursor-like protein produced by bacterial cells is dialyzed against water or a buffer solution with a pH of 4 to 7, and then the dialyzed solution is brought into contact with an anion exchanger. A method for purifying urokinase precursor-like protein.