JPH062060B2 - Purification method of porcine pancreatic kallikrein - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for purifying kallikrein, and particularly to a method for purifying porcine pancreatic kallikrein.

(Prior Art) Kallikrein is a proteolytic enzyme that specifically and rapidly releases physiologically active peptides and kinins from kininogen in plasma and tissues, and is present in body fluids and tissues of various animals. It is known. This kallikrein has a pharmacological action such as blood flow increase and blood pressure lowering action, and is already used as a cardiovascular drug, for example, as a therapeutic drug for hypertension, cerebrovascular disorder, arteriosclerosis, etc., and its demand is It tends to increase in recent years.

Kallikrein is also extracted and purified from human urine, but industrially it is usually produced by extracting and purifying from mammalian organs, especially porcine pancreas. As a conventional method for highly purifying kallikrein for use as a drug, ammonium sulfate salting out, solvent precipitation, ion exchange chromatography using a cation and anion exchanger, gel filtration, etc., or a method in which these are appropriately combined or expensive Affinity chromatography and the like using an inhibitor which is a reagent has been adopted.

(Problems to be Solved by the Invention or Objects of the Invention) As a result of intensive studies on the porcine pancreatic kallikrein, the present inventors have shown that the quality of kallikrein among contaminating proteins that cannot be separated by conventional purification methods is high. It was found that a protease that deteriorates was contained. That is, since the molecular weight and the isoelectric point are extremely close to those of kallikrein, they behave similarly to kallikrein when a conventional purification method is carried out, and at least two kinds of proteases which are clearly different from trypsin and chymotrypsin A are It was found to be present in the purified product of porcine pancreatic kallikrein. As a result of repeated studies on these proteases, although there are differences in the substrate specificity and the manner of action of the inhibitor, etc., it is a protease with a low specificity that has casein degrading ability and has a kallikrein inactivating action and a quinine degrading action. It turned out to be an enzyme.

Therefore, since such a protease exists,
It is presumed that the conventional porcine pancreatic kallikrein preparation has deteriorated the quality of preparation such as stability.

Therefore, the main object of the present invention is to be able to separate and remove the above-mentioned proteases that have been overlooked in conventional purification methods, and thus to provide a more stable method for purifying porcine pancreatic kallikrein. To provide.

A secondary object of the present invention is to provide a purification method for industrially producing high-purity porcine pancreatic licacrein of high purity in a large amount and at low cost in view of the fact that the demand for kallikrein is increasing as described above. Especially.

(Means and Actions for Solving Problem and Achieving Object) According to the present invention, a water-insoluble carrier —X—R (wherein X represents a —O—CH ₂ CHOHCH ₂ —O— group, R represents an aliphatic or aromatic hydrocarbon residue having 4 or more carbon atoms), and a solution containing a crude porcine pancreatic kallikrein is contacted with a hydrophobic group-bonded water-insoluble carrier to adsorb porcine pancreatic kallikrein And a step of separating the adsorbed porcine pancreatic kallikrein and contaminants by hydrophobic interaction chromatography, the method for purifying porcine pancreatic kallikrein, the above problem The point is solved and the above object is achieved.

In the case of an aliphatic hydrocarbon residue, examples of the hydrophobic group represented by R in the above formula include butyl, pentyl, hexyl, heptyl, octyl and nonyl groups, and in the case of an aromatic hydrocarbon residue. Phenyl, tolyl, naphthyl. Examples thereof include a phenanthryl group. The water-insoluble carrier is required to be inert and insoluble and has hydrophilicity. Examples of such a water-insoluble carrier include agarose (registered trademark Sepharose: Pharmacia, Fine, Chemicals, Sweden). , Dextran (registered trademark Sephadex: Pharmacia, Fine, Chemicals, Sweden), cellulose (registered trademark Celex: bio, Rad, USA), polyacrylic amide gel (registered trademark Bio-Ge)
l: Bio, Rad, USA) and the like can be used. The introduction of the bonding group represented by X in the above formula is carried out by the aliphatic alcohol (butanol, pentanol, hexanol, heptanol, octanol, etc.) or the phenol (having a hydrophobic group represented by R). Phenol, cresol, naphthol, etc.) is bound to a water-insoluble carrier as described above by the epichlorohydrin method (S. Hjertn et al., “Journal, Of, Chromatography” 101: 281, 1974). it can.

In addition, phenyl sepharose (R in the above formula is C ₆ H ₅ ) as an aryl group-bonding carrier, and octyl sepharose (R in the above formula is C ₈ C ₁₇ ) as an alkyl group-bonding carrier.
Are commercially available from Pharmacia, Fine, and Chemicals (Sweden), and they can be purchased and used as a hydrophobic group-bonded water-insoluble carrier.

The crude porcine pancreatic kallikrein used as a raw material in the method of the present invention can be partially purified with an ion exchange resin such as DEAE-Sepharose. Preparation of a solution of this partially purified porcine pancreatic kallikrein and equilibration of the hydrophobic group-bonded water-insoluble carrier are carried out by using a suitable buffer containing an electrolyte such as sodium chloride, potassium chloride or ammonium sulfate, preferably at a concentration of 1 M or more, preferably p
H6-8, particularly preferably pH 6.5-8, is used.

When the prepared crude, ie, partially purified porcine pancreatic kallikrein-containing solution described above is brought into contact with a hydrophobic group-bonded water-insoluble carrier, kallikrein and a contaminant protease to be removed are hydrophobic group-bonded water-insoluble. Adsorbed on the carrier. This adsorption mainly occurs due to the hydrophobic interaction between the hydrophobic group and the enzyme molecule. Since there is a difference in this hydrophobic interaction between kallikrein and the contaminant protease, the method of the present invention utilizes this difference to separate the two.

The hydrophobic interaction chromatography employed in the method of the present invention to perform this separation reduces the ionic strength (electrolyte concentration) of the solution after adsorbing kallikrein and contaminant proteases on the hydrophobic group-bound water-insoluble carrier. Alternatively, it can be carried out by increasing the hydrophobicity of the solution. In the former case, for example, a gradient elution method or a stepwise elution method is adopted, and in the latter case, for example, ethyl alcohol or ethylene glycol. It can be carried out by adopting an elution method of adding an organic solvent such as.

It should be noted that such hydrophobic interaction chromatography itself is known and is reported to be applied to the purification of human urinary kallikrein (Japanese Patent Laid-Open No. 57-203015), but when compared with human urine, pigs are used. The types of proteins in the pancreas are extremely diverse, so even if hydrophobic interaction chromatography can be used to purify human urinary kallikrein, it cannot be said that it can be applied to the purification of porcine pancreatic kallikrein. It should be noted that this is the case.

(Examples and Test Examples) Next, the present invention will be specifically described with reference to Examples and Test Examples. The characteristic values described in each example are measured by the following measuring methods.

a) Kallikrein activity H is a synthetic substrate specific for kallikrein using a standard of kalidinogenase manufactured by National Institute of Health as a standard.
It is measured using -P-valyl-L-leucyl-L-arginine-paranitroanilide (product code S-2266, manufactured by Mold, Sweden).

b) Protease activity Hammer Stencasein (E. Merck, West Germany)
The casein hydrolyzing activity is measured by using as a substrate, and the measured activity unit is expressed in Kunitz unit to be protease activity.

c) Protein amount It is indicated by the absorbance (A280 nm) at a wavelength of 280 nm or the amount of Lowry protein measured by the Lowry-Folin method.

Example 1 Crude porcine pancreatic calylein (partially purified using an ion exchange resin and having a total kallikrein activity of 620)
IU, having a kallikrein specific activity of 18 IU / A280, casein hydrolyzing activity of 1.82 U / A280)
-Prepare an equilibrated purified porcine pancreatic kallikrein-containing solution in NaCl-containing buffer solution (pH 8.0). On the other hand, 20m
The phenyl sepharose of Example 1 was packed in a column, the phenyl sepharose was equilibrated with the above buffer, and then the solution containing the above-mentioned crude porcine pancreatic kallikrein was passed through the column for adsorption treatment. Then, the kallikrein was separated by washing with the above-mentioned buffer solution and then performing gradient elution with a buffer containing 3M to OM NaCl. Purified porcine pancreatic kallikrein obtained by desalting and concentrating the eluate has a total kallikrein activity of 580 IU,
Recovery rate 93%, kallikrein specific activity 450 IU / A2
80 (purity increase 25 times), and casein hydrolyzing activity was 0.07 U / A280.

Example 2 Crude porcine pancreatic kallikrein (partially purified using an ion exchange resin and having a total kallikrein activity of 79)
00IU, kallikrein specific activity 33IU / A280, casein hydrolyzing activity 0.76U / A280) 2.5
It is prepared by equilibrating with a buffer solution containing M-NaCl (pH 8.0). On the other hand, 100 ml of phenyl sepharose was packed in the column, the phenyl sepharose was equilibrated with the above buffer, and then the above-mentioned crude porcine pancreatic kallikrein-containing liquid was passed through this column for adsorption treatment. It is then washed with the above buffer, then 1M-N
Kallikrein was separated by stepwise elution with aCl containing buffer. The purified porcine pancreatic kallikrein obtained by desalting and concentrating the eluate had a total kallikrein activity of 7,000 IU, a recovery rate of 88%, and a kallikrein specific activity of 470 IU / A280 (purity increase 14 times).
The casein hydrolyzing activity was 0.04 U / A280.

Example 3 Crude porcine pancreatic kallikrein (partially purified using an ion exchange resin. Total kallikrein activity 15
00IU, kallikrein specific activity 36IU / A280, casein hydrolyzing activity 0.89U / A280) 1M-
A crude swine pancreatic kallikrein-containing solution is prepared by equilibrating with a (NH ₄ ) ₂ SO ₄ -containing buffer solution (pH 6.5). On the other hand, 25 ml of octyl sepharose was packed in a column, the octyl sepharose was equilibrated with the above buffer, and then the above-mentioned crude porcine pancreatic kallikrein-containing liquid was passed through this column for adsorption treatment. Then, it was washed with the above buffer solution, and then 0.2M (NH
₄ ) Kallikrein was separated by stepwise elution with a buffer containing ₂ SO ₄ . Purified porcine pancreatic kallikrein obtained by desalting and concentrating the eluate had a total kallikrein activity of 1230 IU, a recovery rate of 82%, and a kallikrein specific activity of 520 IU / A280 (purity increase of 14 times).
And the casein hydrolyzing activity was 0.05 U / A280.

Example 4 Crude porcine pancreatic kallikrein (partially purified using an ion exchange resin and having a total kallikrein activity of 17
60 IU, kallikrein specific activity 30 I / A280, casein hydrolyzing activity / 108 U / A280) 1.5 M
- _{(NH 4)} was equilibrated with 2 SO ₄ containing buffer (pH 6.5) to prepare a crude porcine pancreatic kallikrein-containing solution. On the other hand, 25 m of octyl sepharose was packed in a column, the octyl sepharose was equilibrated with the above buffer, and then the above-mentioned crude porcine pancreatic kallikrein-containing liquid was passed through this column for adsorption treatment. After that, the kallikrein was separated by washing with the above buffer and eluting with a buffer containing 30% ethylene glycol and 1.5 M (NH ₄ ) ₂ SO ₄ . The purified porcine pancreatic kallikrein obtained by desalting and concentrating the eluate had a total kallikrein activity of 1510 IU / A280, a recovery rate of 86%, and a kallikrein specific activity of 450 IU / A280.
(The degree of increase in purity is 15 times), and the casein hydrolyzing activity is 0.
It was 07 U / A280.

Test Example 1 Porcine pancreatic properties prepared with a pH 7 buffer using the crude (partially purified) porcine pancreatic kallikrein used as a raw material in Examples 1 to 4 and the purified porcine pancreatic kallikrein obtained in Examples 1 to 4 Kallikrein solution was used as a control and test sample, and after passing through a sterilization filter, 37 ℃
Each sample was incubated under sealed conditions. Synthetic substrate HP-Valyl-L-leucyl-L-arginine-
The kallikrein activity of each sample was measured using para-nitroanilide (product code S-2266, manufactured by Kavi Ltd. of Sweden) at 0 days (ie, before incubation) and 0.25, 1 and 2 days after incubation, and 0 days were measured. The results of examining the residual rate (residual activity) when the activity of 100% was defined as shown in the graph of FIG.

From this graph, the kallikrein activity of the partially purified product as the raw material is gradually decreased, whereas the purified product obtained by the method of the present invention retains the initial activity even after 2 days from the incubation and is stable. It can be seen that the property is extremely high.

Test Example 2 Rabbits in good health were fasted for 2 days, then laparotomy was performed under anesthesia by subcutaneous injection of urethane 1.5 g / kg, and the mesenteric artery and vein under the control of the lower ileum 30-40 cm were cannulated. The following test substances (AE) were administered into the intestinal tract in which the both ends were ligated and the artificial nutrient solution (Krebs-Ringa-bicarbonate buffer solution (9% at 37 ° C containing 6% dextran) was administered.
5% O ₂ −5% CO ₂ )), and the perfusate reaching the venous side is sampled at regular time intervals to obtain a sample, which is the synthetic substrate Pro-Phe-Arg-MCA (prolyl-phenyl). The hydrolytic activity of alanyl-arginine-4-methylcoumaryl-7-amide, manufactured by Peptide Laboratories) was measured in the presence of SBTI (soybean trypsin inhibitor), and the obtained measurement values were converted into kallikrein international units. It was as shown in the table.

Test substance and dose: A (control): modified kallikrein 500 IU / animal L-lysine hydrochloride 0.5 mg L-arginine hydrochloride 0.5 mg / animal B: purified kallikrein (Example 1) 500 IU / animal L-lysine hydrochloride 0. 5 mg L-arginine hydrochloride 0.5 mg / animal C: purified kallikrein (Example 2) 500 IU / animal L-lysine hydrochloride 0.5 mg L-arginine hydrochloride 0.5 mg bovine serum albumin 2.0 mg / animal D: purified kallikrein (implementation) Example 3) 500 IU / animal Sodium chondroitin sulfate 1.0 mg / animal E: Purified kallikrein (Example 4) 500 IU / animal Sodium carboxymethylcellulose 1.0 mg / animal From the results shown in the above table, it is understood that the porcine pancreatic kallikrein purified by the method of the present invention has excellent intestinal absorbability. It was presumed that this increase in intestinal absorbability was due to no structural change in kallikrein during the purification process.

(Effects of the Invention) The method for purifying porcine pancreatic kallikrein according to the present invention can be carried out easily at low cost and therefore on an industrial scale,
In addition, it is an enzyme having a quinine-degrading activity that reduces kallikrein activity and can effectively remove a contaminating protease that has been overlooked in the conventional method for purifying porcine pancreatic kallikrein, resulting in the method of the present invention. The purified product is purified 11 to 25 times in purity as compared with the crude or partially purified product of the raw material, the casein hydrolyzing activity is reduced to about 1/20, and the stability is remarkably improved. Absorption is also improved.

[Brief description of drawings]

The figure shows that the porcine pancreatic kallikrein obtained by the method of the present invention and partially purified porcine pancreatic kallikrein, which is a raw material thereof, are incubated in a buffer solution, the kallikrein activity is measured over time, and the stability test results are expressed as residual activity. It is a graph which shows.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Junko Ueda 3546-1, Asanoda Kita-cho, Kitase-cho, Inabe-gun, Mie Sanwa Kitase Dormitory (72) Inventor Akihiro Morioka 3546-1 Asaita Asakita, Kitase-cho, Inabe-gun, Mie Kita Sanwa Dormitory (56) Reference JP-A-57-203015 Hoppe-Seyler's Z. P hysiol. Chem. (1981) P. 883-896

Claims (5)

[Claims] 1. A water-insoluble carrier —X—R (wherein X represents —O—CH ₂ CHOHCH ₂ —O— group, and R represents an aliphatic or aromatic hydrocarbon residue having 4 or more carbon atoms. The step of adsorbing porcine pancreatic kallikrein by contacting a solution containing crude porcine pancreatic kallikrein with a hydrophobic group-bonded water-insoluble carrier represented by And a step of separating by sexual interaction chromatography, the method for purifying porcine pancreatic kallikrein. 2. The method for purifying porcine pancreatic kallikrein according to claim 1, wherein a water-insoluble carrier bonded to a hydrophobic group in which R is octyl is used. 3. The hydrophobic group-bonded water-insoluble carrier is octyl sepharose.
Alternatively, the method for purifying porcine pancreatic kallikrein according to Item 2. 4. The method for purifying porcine pancreatic kallikrein according to claim 1, wherein a water-insoluble carrier bound to a hydrophobic group in which R is phenyl is used. 5. The hydrophobic group-bonded water-insoluble carrier is phenyl sepharose.
Alternatively, the method for purifying porcine pancreatic kallikrein according to Item 4.