JPS6032717A - Purification of kallikrein - Google Patents

Abstract

PURPOSE:To purify large amount of kallikrein easily, in high purity and yield, by passing crude kallikrein solution in a weakly basic anion exchange resin column to effect the absorption of kallikrein, and eluting kallikrein with a mixture of buffer solution and an organic solvent miscible with water. CONSTITUTION:Drude kallikrein solution is passed through a weakly basic anion exchange resin (e.g. acrylamide resin such as Diaion WA-10) to effect the absorption of kallikrein. The absorbed kallikrein is desorbed with a mixture of an organic solvent miscible with water (e.g. methanol, ethanol, etc.) and a buffer solution (e.g. buffer solution of acetate, phosphate, borate, etc.) to obtain purified kallikrein. The crude kallikrein can be prepared by fractionating and precipitating the extract of pig pancreas e.g. with an organic solvent, and dissolving the produced precipitate of kallikrein in water or a buffer solution. The eluent mixture is preferably composed of 10-30% orgnic solvent and 0.2-0.5M buffer solution of 4.5-7pH.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for purifying kallikrein, and more particularly, to a method for purifying kallikrein present in the pancreas of mammals easily and in large amounts, with high 5 rit and high yield.

Kallikrein is a type of proteolytic enzyme produced in various tissues of mammals, and is also known as a type of kinin-releasing enzyme. Free kinin has a peripheral vasodilating effect, and kallikrein is therefore used for the treatment of cardiovascular diseases. Since kallikrein is usually extracted from mammalian organs, the crude extract contains many foreign proteins, and in order to use this extract as a pharmaceutical, it is necessary to perform further purification.

Conventional methods for purifying kallikrein include ammonium sulfate salting-out method, solvent precipitation method (% 1987-102495), and ion exchange chromatography (JP-A-53-62893).

No. 54-2270, No. 54-14647), electrophoresis (Japanese Patent Application Laid-open No. 49-41583), inhibitors, adsorbents,
Affinity chromatography using antibodies, etc.
JP-A-54-ti2312, Sho-56-117791, Sho-57-50924, Sho-57-146581, Sho-58-5191, Sho-58-55043), etc. are known. However, these methods were insufficient in separating kallikrein and hybrid proteins, and the operations were complicated.
It has drawbacks such as problems with the stability of the carrier, and has not yet been satisfactory for industrially mass-producing high-purity kallikrein at low cost.

The present inventor further developed a method for purifying kallikrein that is simple and industrially advantageous (after conducting intensive research, it was found that in order to elute kallikrein adsorbed to a weakly basic anion exchange resin, The present invention was completed based on the discovery that kallikrein and hybrid proteins can be separated more effectively by using a mixture with a mild solution.

Therefore, the present invention is characterized in that after a crude kallikrein solution is passed through a weakly basic anion exchange resin column to adsorb kallikrein, kallikrein is eluted with a mixture of a water-miscible organic solvent and a buffer solution. The present invention provides a method for purifying kallikrein.

To carry out the method of the present invention, first, a crude kallikrein solution is passed through a weakly basic anion exchange resin to adsorb kallikrein. 1fL of crude kallikrein solution, which is the raw material
is obtained by fractionating and precipitating a pig pancreas extract or the like using a known organic solvent precipitation method, and dissolving the resulting kallikrein precipitate in water or a buffer solution, and the pH of the solution is 45 to 45. It is preferable to set the value to around 7, especially around 5, to allow adsorption. The weakly basic anion exchange resin used in the present invention may be of any type, but it is preferably a carrier such as polystyrene, acrylic acid amide, metaphenylene diamine, etc., and a primary amine, secondary amine, sb is a resin with a tertiary amine exchange group, examples of which include acrylic acid amide resins such as Diaion WA-10 and WA-11;
Acrylic resins such as Amberlite IRA-68, Diaion WA-20, WA-30, Amberlite I
Styrenic resins such as RA-45, IRA-47, Dowex3, Amberlite I R-4B, Wofati
The effect of oiy-y enylenediamine-based trees such as tM is overshadowed.

The adsorbed kallikrein is then eluted with a mixture of a water-miscible organic solvent and a buffer. Organic solvents miscible with water include methanol, ethanol, acetone,
Examples include isopropyl alcohol, dioxane, tetrahydrofuran, acetonitrile, and propylene glycol, and these are preferably used in an amount of 10 to 30q6 in the total eluate.

As the tfC buffer, acetate, phosphate, borate, citrate and other buffers are preferred. Prior to elution of adsorbed kallikrein, resin'i 0.05 to 0°15
It is preferable to wash with a buffer solution of M (pH 4,5 to 7), and the elution of kallikrein is carried out at a pH of 0.2 to α
It is preferable to use a mixture with a 5M buffer solution (pH 4,5 to 7).

The present invention will be described in detail below with reference to Test Examples and Examples. In addition, in the examples, purification was performed using a column method using a concentrated buffer gradient method, but the present invention is not limited to this, and the present invention can also be carried out using a patch method or a method in which elution is performed using a buffer solution of a certain performance. I can do it.

Reference example 1. (Conventional method) 1 oor of porcine pancreas powder was suspended in 0.251 sodium chloride aqueous solution 1iIA, adjusted to pH 7.5 with aqueous ammonia, and subjected to fractional sedimentation with 40-55% acetone. The resulting precipitate was dissolved in 400 gd of water, the pH was adjusted to 4.5 with 6N vinegar, and the resulting precipitate was removed by centrifugation to obtain a kallikrein-containing solution.

This solution was equilibrated with 0.05 M acetate buffer (pH 5,0) and mixed with Diamond 1-on WA-11 (manufactured by Mitsubishi Kasei Corporation) 400.
kallikrein was adsorbed onto the resin. Next, after washing the column with 0.1 M vinegar aim buffer (pH 5,0), α15M-Q, 5M acetate buffer (1) H5,0
) (4t-4t) concentration gradient method to elute sicicalifrein. (Flow rate: 3 td/br, 50 t
d/tube) The collected kallikrein active fractions were concentrated by ultra-V filtration, desalted by adding acetone, and lyophilized to obtain purified kallikrein 527q (total strength: 43780 KU). This elution pattern is shown in FIG.

Reference example 2. (Standard method) Porcine pancreas extract 20f was dissolved in 200-ml water, adjusted to pi (7,5) with aqueous ammonia, and precipitated with acetone 55-ml. After dissolving the obtained precipitate in 2001-d water, 6N acetic acid The pH was adjusted to 4.5, and the resulting precipitate was removed by centrifugation to obtain a kallikrein-containing solution.This solution was treated in exactly the same manner as in Test Example 1, except that Diaion WA-11 was changed to WA-10, to obtain purified kallikrein 409. ■(Comprehensive 49120KU)
I got it. This elution pattern is shown in FIG.

Example 1 100 vol of porcine pancreas xi powder was suspended in 1 t of 0.2-eu sodium chloride aqueous solution, adjusted to pH 7.5 with aqueous ammonia, and subjected to fractional sedimentation with 40-55% acetone. After dissolving the resulting precipitate in 400 d of water, 6N6#! The resulting precipitate was removed by centrifugation to obtain a kallikrein-containing solution. This solution was passed through a column of Diaion WA-11400- equilibrated with 0.05 M acetate buffer (pH 5,0), and kallikrein was adsorbed onto the resin. Next, after washing the column with 0.1M acetate buffer (pH 5,0),
-) 0.15 M including L and f - Q, 5 MJ! rlli
#ii buffer solution (pH 5,0) C4l-4t) no 1jiI
υ kallikrein was eluted by the K gradient method. (Flow rate: 3
00at/br, 50w! /tube) The collected kallikrein active fractions were subjected to concentrated MI by ultrafiltration, desalted by adding acetone, and lyophilized to obtain f##kallikrein 368~ (overall 43,000 KU) t-. This elution pattern is shown in FIG. In comparison with Reference Example 1 (Figure 1) (7), the separation of kallikrein and hybrid protein was excellent.

Example 2 Purified kallikrein 387W (general quality 44500
KU) was obtained. This elution pattern is shown in Figure @4. Compared to Reference Example 1 (Fig. 1), the separation of kallikrein and hybrid protein is excellent.

Example 1 20 ff of porcine pancreas extract was dissolved in 200 ml of water, adjusted to pH 7.5 with aqueous ammonia, and precipitated with 55 ml of acetone. The nine precipitates obtained were dissolved in 200 m of water, and then purified with 6N acetic acid.
The resulting precipitate was removed by centrifugation to obtain a kallikrein-containing solution. This solution'Q0.05M acetate buffer (p
Diaion WA-1o, 40 equilibrated with H5,0)
0- column to adsorb kallikrein onto the resin.

Next, after washing the column with 1M acetate buffer (pH 5.0), 0.15M-0.5M containing 20% gold
1): Cicarifurin was eluted by a concentration gradient method using an acid buffer (pH 5,0) (4t-4t). (Flow rate: 30
After concentrating the collected active fractions of kallikrein (0-/br, 50 m/tube) by ultrafiltration, desalting by adding acetone and freeze-drying the purified kallikrein 258 (overall 53200
KU) was obtained. This elution pattern is shown in FIG. Compared to Reference Example 2 (Fig. 2), the separation of kallikrein and hybrid protein was excellent.

Example 4 Purified kallikrein 2519 (general quality 4
68tiOKU) was obtained. This elution pattern was compared with Reference Example 2 (FIG. 2) shown in FIG. 6, and the separation of kallikrein and hybrid protein was excellent.

Example 5 Table 1 shows the specific activity, recovery rate, trypsin activity and chymotrypsin activity of each purified kallikrein obtained in Reference Example 1.2 and Examples 1 to 4.

Although there was no difference in the recovery rate of kallikrein, the specific activity of kallikrein and the removal rate of trypsin and chymotrypsin were better when eluted with a buffer containing an organic solvent.

In the present invention, the following activity measurement method was used.

(1) Method for determining the physiological activity of kallikrein [The Journal of Biochemistry (J, Blochem) Vol. 58, p. 201, 1
956] K followed.

(2) Kallikrein esderase activity G, W, 8h
Methods such as wer t [Biochemistry, Engineering, Bait 7
i') ly -7 near 7 (Biocht=m
et Bicphya Acta) Vol. 16, p. 570, 1955] (using benzoyl-L-flea arginine ethyl ester as a substrate, the reaction was carried out at 25° C. for 15 minutes).

(a) Trypsin activity Takami's method (Biochemistry, Vol. 41, p. 777, 1969)
(N-benzoyl-DL-arginine,
(P-nitroanili Ftl was used and reacted at 37°C for 15 minutes).

(4) Chymotrypsin activity The method of Sasaki et al.
Anal, Hlochem) Volume 86, Page 159, 1
978 (N.benzoyl-L-tyrosyl-P-nitroanilide was used as a substrate and the reaction was carried out at 37°C for 300 minutes).

Margin below (11)・ (12)

[Brief explanation of drawings]

FIG. 1 and FIG. 2 are drawings showing the elution patterns of kallikrein in Reference Examples 1 and 2 (comparative method), respectively. Figures 3 to 6 show Example 1, respectively. or 4 (
2 is a drawing showing the elution pattern of kallikrein in the method of the present invention. Applicant Nisnis Pharmaceutical Co., Ltd. (1 Figure 1 Kallikrein activity fraction number Figure 2 Kallikrein activity A280 nm EU/ml (----'I (e-o) Fraction number Figure 3 Fraction number Figure 4 Kallikrein Active fraction number Figure 5 Fraction blue number Figure 6 Nratanyo/Kou number

Claims (1)

[Scope of Claims] L A crude kallikrein solution is passed through a weakly basic anion exchange resin column to adsorb kallikrein, and then kallikrein is eluted with a mixture of a water-miscible organic solvent and a buffer solution. A method for purifying kallikrein, which is characterized by: 2 The organic solvent miscible with water is methanol, ethanol, etc.
J and is selected from acetone, isopropyl alcohol, dioxane, tetrahydrofuran, acetonitrile or propylene glycol.
The method for purifying kallikrein according to item 1.