JPS5820592B2 - kallikreinoseihouhou - 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 easily purifying kallikrein obtained from mammalian organs to high purity in large quantities.

Kallikrein is a type of proteolytic enzyme produced in various parts of animal bodies, and is also a type of kinin-releasing enzyme that specifically and promptly releases the active peptide kinin from kininogen in plasma.

Kallikrein has physiological activities such as peripheral vasodilation and is said to be effective in the treatment of circulatory system diseases. has been carried out.

However, the demand for kallikrein as a medicine has been increasing steadily in recent years, and there is currently a strong desire to produce high-purity kallikrein in large quantities at low cost.

In general, since kallikrein is sourced from animal organs, there are a large number of coexisting foreign proteins, making separation of these extremely difficult and extremely difficult to obtain highly pure kallikrein.

Conventionally, in order to remove foreign proteins from kallikrein, a combination of complicated operations such as ammonium sulfate salting out, solvent precipitation, ion exchange chromatography, gel filtration, etc. was required, or foreign proteins were removed using inhibitors, which are expensive reagents. A method of specifically adsorbing and removing proteins had to be used.

However, none of these methods can be said to have sufficient industrial efficiency to produce kallikrein at low cost and in large quantities.

The present inventor conducted intensive research in search of a method for purifying kallikrein, which is difficult to purify, with easy operation and high purification efficiency, and as a result, the inventors succeeded in specifically adsorbing kallikrein using insoluble concanavalin A. The present invention has been completed by successfully separating kallikrein from kallikrein-like heterologous proteins at low cost and by allowing the foreign proteins to remain unadsorbed. It is something.

The present invention is characterized by bringing a kallikrein-containing solution into contact with insoluble concanavalin A to adsorb kallikrein, and then flowing down with a saline solution containing about 10% of glucose or/and mannose to elute highly purified kallikrein. This is a method for purifying kallikrein.

The first feature of the present invention is that kallikrein containing a foreign protein is brought into contact with insoluble concanavalin A to specifically adsorb kallikrein.

This adsorption does not adsorb foreign proteins, so if it is washed away with saline, only kallikrein will remain in the insoluble concanavalin A.

A second feature of the present invention is that kallikrein specifically adsorbed to insoluble concanavalin A is eluted with a saline solution containing about 10 parts of glucose or/and mannose.

By this treatment, most of kallikrein is eluted from insoluble concanavalin A, and kallikrein of extremely high purity can be obtained.

Concanavalin A used in the present invention is, for example, Biochimica et Biophidica acta (BIOCHIMICA).
It can be easily extracted and purified from the seeds of rapeseed by the method described in AET BIOPHYSICAACTA) Vol. 147, pp. 262-271 (1967).

To make concanavalin A insoluble, concanavalin A can be transferred to an insoluble carrier, such as an inert, sparse, insoluble, and hydrophilic insoluble carrier, such as agarose (registered trademark Sepharose: Pharmacia Pharmaceutical Co., Ltd.).
Fine Chemical Co., Ltd.), Dextran (registered trademark Sephadex: Pharmacia Fine Chemical Co., Ltd.)
Concanavalin A can be chemically covalently bonded to cellulose powder or the like, or it can be made insolubilized by a known method such as polymerization and gelation of concanavalin A using a polyaldehyde such as glutaraldehyde.

The insoluble concanavalin A obtained here has a specific affinity for kallikrein, so it is either added to the kallikrein-containing material and stirred, or the insoluble concanavalin A is packed in a column and the kallikrein-containing material is passed through the column. After specifically adsorbing kallikrein, it is possible to wash away foreign proteins by washing with a saline solution.

Kallikrein can be specifically eluted with a saline solution containing about 10 g of glucose or/and mannose on the adsorbed kallikrein from which foreign proteins have been washed away.

The salt solution can be selected from a fairly wide range of concentrations, but preferably about 0.5 to 1.5M, particularly preferably IM.

Insoluble concanavalin A from which kallikrein has been eluted can be used immediately by simply washing with a washing buffer, and is also highly workable, as it does not require regeneration after use.

Purification of kallikrein in the present invention is extremely simple;
Moreover, the obtained kallikrein has an amazingly high purity, and is extremely useful for preparations of kallikrein.

Next, test examples of the present invention will be shown.

Test Example Crude kallikrein solution obtained by hot water extraction from pig pancreas extract (pancreatin) was treated with (1) insoluble concanavalin A obtained in the same manner as in Example 1 (method of the present invention), (2) ) Diethyl aminoethyl (DEAE-
) treated with cellulose (known method according to Japanese Patent Publication No. 37-11697), and adsorbed using the same method as in Example 1 for (1), and the same method as in Example 1 for (2), The kallikrein obtained by purification by the method of Example 1 was subjected to isoelectric point fractionation, and the degree of purification was compared and examined.

The fractionation conditions were Ampholine (manufactured by LKB) with a pH range of 3.5 to 10, final concentration 1%, and initial voltage 7.
00V, 12mA, final voltage 700V, 1.5m
A. The energization time was set to 42 hours, and the temperature during energization was set to 5°C.

After energization, 21 rll fractions were collected, and the -, casein decomposition activity, N-benzoyl arginine ethyl ester (BAEE) decomposition activity, and absorbance at 280 mμ were measured for each fraction.

The results are as shown in Figures 1 and 2.
Kallikrein activity indicated by BAEE degrading activity is (1
) and (2), the isoelectric point (hereinafter referred to as pI) is 3.
．． 9 to 4.1.

Since there is a correlation between this BAEE-degrading activity and the physiological activity of kallikrein to release kinin, the BAEE-degrading activity was expressed as kallikrein activity.

On the other hand, high-purity kallikrein does not show any degrading activity towards casein, but low-purity kallikrein exhibits casein-degrading activity if it contains coexisting proteases. It is very likely that kallikrein contains kininase, which breaks down the active peptide kinin released by the action of kallikrein into inactive substances.

As shown in FIG. 2, in the known method (2), pI 4.5, pI 4.9, pI 5.3
A strong peak of caseinolytic activity was observed in , and furthermore, the protein absorbance measurement at 280 m
I 4.5, pI 4゜9, pH 5.3, pI
There are many different proteins that have isoelectric points at pH 5.8, pH 6.8, and pI 8.1.

In this way, in the known method, many heterologous proteins are involved along with strong casein-degrading activity, whereas in the method (1) of the present invention, as shown in FIG.
Compared to the figure, the number of peaks and the peak values in absorbance measurement at 280 mμ are very small, and the values are low.Also, almost no casein-degrading activity is observed, and kininase has also been removed.

Therefore, kallikrein purified by the method of the present invention contains almost no heterologous protein similar to kallikrein compared to kallikrein purified by a known method.

In this way, foreign proteins similar to kallikrein, such as trypsin, chymotrypsin, and kininase, which are present when extracting and purifying kallikrein derived from animal organs, can be removed efficiently and economically in one step.

The activity measurement method used in this test example and examples is as follows.

1. Kallikrein Physiological Activity 9. Method of Kallikrein et al. [The Journal of Biochemistry (J, Biohem, ) vol. 58.

201 (1956)].

2 Casein degrading activity Kunitz's method [The Journal
of General Physiology (J.

Gen, Physiol) Volume 30, Page 291 (19
1947)].

3 BAEE degrading activity Trautschold (I, Trautschold)
[Hoppe-8e
yIers Z, Physiul, Chem, )
Volume 325.

48 (1961)].

Next, examples of the present invention will be shown.

Example 1 50 liters of defatted pork pancreas powder was suspended in 500 rnl of water, extracted with hot water at 48°C for 1 hour, and then fractionated with acetone. The resulting precipitate was dissolved in water and dialyzed to obtain a kallikrein-containing solution. Ta.

On the other hand, Sepharose 4B was activated with bromic cyanide, concanavalin A was covalently bonded to it to obtain concanavalin A-Sepharose, 100% of this was packed in a column, and the above kallikrein-containing solution was passed through to convert kallikrein to concanavalin A-Sepharose. It was adsorbed to Sepharose.

After washing the column with 1M saline solution, kallikrein was eluted from the adsorbent with 1M saline solution containing 10% glucose.

The eluate was dialyzed against water and then freeze-dried to obtain purified kallikrein 85.

When the physiological activity of purified kallikrein was measured, it was 25k.
u/1nIi! Met.

Example 2 Concanavalin A100f is dissolved in 0.1M phosphate buffer, 80rrll of 25% glutaraldehyde is added thereto, stirred under water cooling for 1 hour, and gelled concanavalin A is obtained by centrifugation.

This gelled concanavalin A was packed into a column, and a kallikrein-containing solution obtained from 300 vol of defatted pork pancreas powder in the same manner as in Example 1 was passed through the column.

After washing the column with a 1M salt solution, kallikrein in the adsorbent was eluted with a 1M salt solution containing 10% glucose.

The eluate was then dialyzed against water and freeze-dried.
Kallikrein 12 was obtained.

The physiological activity of this kallikrein was 30ku/7INi.

[Brief explanation of drawings]

FIG. 1 shows the isoelectric point fractionation pattern of kallikrein purified by the method of the present invention, and FIG.
This figure shows the isoelectric point fractionation pattern of kallikrein purified by the EAE-cellulose method.

Claims (1)

[Claims] 1 Purification of kallikrein characterized by contacting a kallikrein-containing solution with insoluble concanavalin A to adsorb kallikrein, and then flowing down with a saline solution containing about 10% glucose or/and mannose to elute highly pure kallikrein. Method.