JPS5838151B2 - Kallikrein purification method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for purifying kallikrein.

More specifically, the present invention relates to a method for simply purifying kallikrein to a high degree of purity by using an insoluble kallikrein antibody.

Kallikrein is a proteolytic enzyme that is widely distributed in living bodies such as animal urine, serum, saliva, sweat, tears, submandibular gland, pancreas, accessory sex glands, and kidneys.

In addition, kallikrein is a kinin-releasing enzyme that releases the active peptide kinin from kininogen in plasma.
It is important as a therapeutic agent for cardiovascular diseases and is widely used clinically.

Conventional methods for purifying kallikrein include ammonium sulfate salting out, solvent precipitation, ion exchange chromatography using cation and anion exchangers, electrophoresis, and affinity chromatography using inhibitors. However, these methods have the disadvantage that it is difficult to separate and remove kallikrein and hybrid proteins, lack specificity, and require considerable time and effort.

Recently, instead of such a purification method, a method for purifying kallikrein using an insoluble kinetic antibody is being applied.

However, since the purification method using insoluble recrein antibodies utilizes the specificity of the antigen-antibody reaction, the binding strength of the antigen-antibody complex is strong. I had no choice but to use

(Biochem. J. 189, 153-159.1
980 and JP-A-56-5095) As a result, these eluents change the structure of the protein, and deactivation of kallikrein during the elution process cannot be avoided, and these eluents cannot be used immediately after elution. It was necessary to remove the reagents used as

As a result of various studies on a method for purifying kallikrein using an insoluble kallikrein antibody, the present inventor found that by eluting the insoluble kallikrein antibody adsorbed substance with an alkaline aqueous solution, a method of purifying kallikrein with a recovery rate of approximately 95% and high purity and stability. We have found a way to obtain kallikrein eluate.

In the present invention, first, an insoluble kallikrein antibody is prepared by chemically covalently bonding an antibody obtained by immunizing an animal with kallikrein of a desired purity to an insoluble carrier.

As the insoluble carrier, an inert, sparse, and insoluble carrier such as agarose, glass beads, dextran, polyacrylamide, etc. can be used, and the carrier is chemically and covalently bonded to the antibody by a known method. Insoluble kallikrein antibodies can be prepared.

After treating the crude kallikrein solution with such an insoluble kallikrein antibody to specifically adsorb kallikrein, impurities are completely removed by washing, and then an alkaline aqueous solution, preferably 0. 2
By eluting kallikrein with an aqueous M Na 2 CO s solution, highly pure kallikrein can be obtained in high yield.

The crude kallikrein used in the present invention is urine of animals including humans,
It can be prepared by known methods from known kallikrein-containing raw materials such as blood and pancreas.

The present inventors found that kallikrein is unstable in solution on the acidic side, but relatively stable on the alkaline side, and utilized this property to establish the present purification method.

That is, the eluent used in the present invention, for example, 0. 2M
Na 2 CO s aqueous solution (pH 11.0-11
．． 5), it has been found that 97% or more of the activity remains even after treatment at 25°C for 3 hours.

Furthermore, the alkaline aqueous solution used in the present invention has no effect on the insoluble carrier-antibody binding, and can be reused semi-permanently by regenerating it with an appropriate buffer solution with a pH around 8. There is no change in rate and purity.

The eluent also has the advantage of being inexpensive and very easy to use and dispose of afterwards.

From the above points, it can be said that the method for purifying kallikrein using an insoluble kinetic antibody using an alkaline solution as an eluent is extremely superior to conventional methods.

Next, the present invention will be explained with reference to examples, but the present invention is not limited to these specific examples. Example 1 45 liters of adult urine is adjusted to pH 8.0 with IN sodium hydroxide.

This is filtered, and the filtrate is concentrated using an ultrafilter to a total volume of 1000 ml.

This concentrated solution was flown down a Sepharose 4B column (200 m) immobilized with anti-kallikrein antibody, and after adsorption (1 hour), 0.05
Wash thoroughly with MIJ acid buffer.

Next, elute with 0.2M sodium carbonate solution, desalt with Sephadex G-25, and freeze-dry.

As shown in Table 1, this treatment resulted in a specific activity of 104
The activity recovery rate was 94%.

Example 2 Partially purified human urinary kallikrein solution 500 liters 71 liters (0
．． 0.05M Tris-HCl buffer containing 15M sodium chloride (pH 8.0) was poured into a Sepharose 4B column (200m0) on which anti-kallikrein antibodies were immobilized, and after adsorption (about 30 minutes), 0.15M sodium chloride was added. including 0
．． After thorough washing with 05M Tris-HCl buffer pH 8.0,
．． Elute with 2M sodium carbonate solution, desalt with Sephadex G-25, and lyophilize.

As shown in Table 2, this treatment increased the specific activity by about 8 times, and the activity recovery rate was 97%.

Example 3 Partially purified kallikrein solution 2007rLl (0.07rLl)
1 0.05MIJ acid buffer I)H8.0 containing 5M sodium chloride was flowed down a column (200m) of glass beads immobilized with anti-kallikrein antibodies, and adsorption (approximately 1
After 0 min), phosphate buffer containing 0.15 M sodium chloride 1) H8. After thorough washing with O, elution is carried out with 0.2M sodium carbonate solution, which is desalted with Sephadex G-25 and freeze-dried.

As shown in Table 3, this treatment increased the specific activity by 7.5 times, and the activity recovery rate was 85%.

Example 4 Partially purified kallikrein solution 500ml (0.1
A 0.05 MIJ acid buffer containing 5 M sodium chloride (PH 8.0) was flowed down a Sepherose 4B column (200 mA) on which anti-kallikrein antibodies were immobilized, and adsorption (
After washing thoroughly with 0.05 MIJ acid buffer pH 8.0 containing 0.15 M sodium chloride,
1M glycine-sodium hydroxide buffer (pH 1 1
．． 5'), desalted with Sephadex G-25, and lyophilized.

As shown in Table 4, this treatment increased the specific activity by 7.3 times, and the activity recovery rate was 80%.

Example 5 100g of vancreatin was suspended in 1000'fILl of cold water, extracted with stirring for about 2 hours, centrifuged at 5000 rpm for 10 minutes, and the supernatant was collected. pH with IN sodium hydroxide
After adjusting the pH to 8.0, it was poured onto a Sepharose 4B column (200rrLl) on which antireactive recrein antibodies were immobilized, and after adsorption (about 50 minutes), phosphate buffer containing 0.15M sodium chloride, pH 8.0, was sufficient. After washing, it is eluted with 0.2M sodium carbonate solution, desalted with Sephadex G-25, and then freeze-dried.

As shown in Table 5, this treatment increased the specific activity by 3700 times, and the activity recovery rate was 96%.

Example 6 An extract obtained from 100 g of porcine pancreas bulk powder by the same procedure as in Example 5 was mixed with 0.0 g of porcine pancreas bulk powder. IN sodium hydroxide l- IJ um p
H8. After adjusting to 0, the antireactive recrein antibody was applied to a Sepharose 4B column (2007rt) to be adsorbed (about 50 minutes), and then eluted with 0.1 M glycine-sodium hydroxide buffer pH 11.5. After desalting with Sephadex G-25, it is freeze-dried.

As shown in Table 6, this treatment increased the specific activity by 3600 times and the activity recovery rate was 82%.

Example 7 Mince 500g of raw pig pancreas, add 1500ml of water,
Extract by autolysis.

This extract was filtered to obtain a filtrate, which was adjusted to pH 8.0 with 0.1N sodium hydroxide, and then allowed to flow through a column of glass beads immobilized with anti-kallikrein antibodies for adsorption (approximately 70 minutes). ), elute with 0.2M sodium carbonate, desalt with Sephadex G-25, and freeze-dry.

As shown in Table 7, this treatment increased the specific activity by 3500 times, and the activity recovery rate was 85%.

0 Example of regulation of anti-kallikrein antibody-immobilized Sepharose 4B GNBr-activated Sepharose 4B (Phar-m
acis Fine Chemicals) 10
Wash and swell 0g with 1mM HCl, 0.5
Coupled with 1.5 g of anti-kallikrein antibody in 0.1 M sodium bicarbonate buffer pH 8.3 containing M sodium chloride, stirred at room temperature for 2 hours, and unreacted active groups were removed with IM ethanolamine pH 8.0. Plotking (keep going for 2 hours).

Next, the blocking reagent is washed with the above sodium bicarbonate buffer to obtain Sepharose 4B on which the antireactive recrein antibody is immobilized.

0 Adjustment of anti-kallikrein antibody-immobilized glass beads After thoroughly washing 200 g of glass beads with a diameter of 2 mm,
Dry at 150°C for 24 hours.

Thereafter, 200,771 liters of a 2% r-aminoprobyltriethoxysilane-acetone solution was added, and the mixture was refluxed at 45°C for 24 hours.

Next, after washing the beads with water, add 200 yards of 1% glutaraldehyde.
and shake at 4°C for 24 hours.

Then add 50mM'J acid buffer pH 7. Wash with 0, 200 ml of the same buffer, anti-kallikrein antibody 140
Add (2) and shake at 4°C for 24 hours.

Then 2001 rLl of 50 mM ethanolamine dissolved in 0.1 M sodium carbonate is added and shaken at 4°C for 24 hours.

After thoroughly washing the beads with 50mM acetate buffer pH 5.0, 50% solution containing 0.9% sodium chloride, 0.05% sodium azide, 0.3% bovine serum albumin and 0.6% Triton X-405 was added. Store at 4°C in mM Tris-HCl buffer pH 5.0.

Pro-Phe-Arg-MCA activity measurement method Using prolyluphenylalanyl-arginine-4-methyl-coumaryl-7-amide as a substrate, the method of Kato et al.
ochem. 87.1127-1132.1980).

0. 0.0.2 ml containing 2 ml of 0.15 M sodium chloride.
Add 10 μl of the sample to 1 M Tris-HCl buffer pH 8.0, and after pre-incubation for 37°05 min,
Add MPro-Phe-Arg-MCA solution and perform enzyme reaction for 60 minutes.

0.1M acetate buffer pH 4. 2.5ml of 5! In addition, the reaction is stopped, and fluorescence is measured at an excitation wavelength of 380 nm and a fluorescence wavelength of 440 nm.

For kallikrein activity, the amount of enzyme that releases 1 μmol of 4-methyl-coumarin-7-amine per minute was defined as lunit.

Claims (1)

[Claims] 1. A method for purifying kallikrein, which comprises treating a crude kallikrein solution with an insoluble kallikrein antibody to specifically adsorb kallikrein, washing the adsorbed material, and then eluting kallikrein using an alkaline aqueous solution as an eluent.