JPH0948817A - Affinity resin for purification of plasma kallikrein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject resin capable of easily separating and purifying a plasma kallikrein and easily producing a large amount of a plasma kallikrein having extremely high purity by using a specific compound acting as a specific and strong inhibitor on plasma kallikrein and fixing the compound as a ligand. SOLUTION: This affinity resin is composed of (A) a ligand consisting of N-(trans-4-aminomethylcyclohexylcarbonyl)-L-phenylalanine-4- carboxymethylanilide fixed to (B) a substrate (preferably a hydrophiliuc vinyl polymer having amino group and especially having an amino group content of 150-200μmol/ml-gel). The resin is produced preferably by reacting the component A having amino group with the amino-protected component B in the presence of a condensation agent and deprotecting the amino-protecting group.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an affinity resin used for separating and purifying plasma kallikrein. The present invention also relates to a method for easily separating and purifying high-purity plasma kallikrein from plasma protein using the affinity resin according to an affinity chromatography method.

[0002]

2. Description of the Related Art Enzymes that specifically release quinine from kininogen are widely present in various organs and body fluids such as urine and sweat, and are generally called kallikrein. Of these, kallikrein present in plasma (plasma kallikrein or plasma kallikrein) exists as a precursor, prekallikrein, and is activated by factor XII (Hageman factor) of the blood coagulation system.
(Nagasawa, S., et al., Biochem. Biophys. Res. Commu
ns., 32, pp.644-649). Plasma kallikrein plays an important role in blood coagulation, and acts in amplification of coagulation system and activation of endogenous fibrinolysis.

Separation and purification of plasma kallikrein from plasma is generally performed by activating the prekallikrein in plasma by casein precipitation or addition of 20% acetone, followed by ion exchange chromatography, gel filtration, or p-. Aminobenzamidine, soybean trypsin inhibitor, etc. are used as ligands such as affinity chromatography.
(1) Nobuo Aoki et al., "Coagulation / Fibrinolysis / Kinin", Chugai Medical Co., Ltd., 1979, Chapter 6: Kinin enzymes and proteins, pp.389-396; (2) Moriya Hiro et al. , "Kallikrein kinin", Kodansha Co., Ltd., 1982, Chapter 2: Plasma calilein quinine system, pp.42-46).

Although there are many reports on the purification method of human plasma kallikrein, the recovery rate of high-purity kallikrein is generally high because activated plasma kallikrein rapidly forms a complex with α ₂ -macroglobulin. The problem is that is very low. For example, it was reported that human plasma kallikrein was separated and purified by ethanol precipitation fractionation of human plasma, followed by isoelectric focusing and CM- and DEAE-cellulose chromatography (Col.
man, RW, et al., J. Clin. Invest., 48, pp.11-22,
1969). In the above method, the presence of three types of human plasma kallikrein was confirmed, and the most cationic plasma kallikrein (molecular weight 100,000, sedimentation coefficient S ₂₀ = 5.7) was found to be kallikrein.
I, the slightly acidic kallikreins of molecular weight 163,000 and 124,000 are kallikrein II and kallikrein, respectively.
It is called III.

On the other hand, as an inhibitor of plasma kallikrein,
For example, soybean trypsin inhibitor (SBTI), aprotinin (trasylol), diisopropylfluorophosphate (DFP), pancreatic trypsin inhibitor (BBTI) and the like are known. Recently, N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide [N- (trans-4-aminomethylcyclohexylc), which is a specific and potent inhibitor,
arbonyl) -L-phenylalanine-4-carboxymethylanilide] has been reported (Wanaka, K., et al., Chem. Pharm. B
ull., 40, pp.1814-1817, 1992). There is also a report that human plasma-derived kallikrein III is not inhibited by soybean trypsin inhibitor, pancreatic trypsin inhibitor, or aprotinin (Colman, RW, et al., J. Cl.
in. Invest., 48, pp. 11-22, 1969).

[0006]

An object of the present invention is to provide a method for easily separating and purifying plasma kallikrein from plasma by a chromatographic method. Another object of the present invention is to provide a resin for chromatography used in the above method and a method for producing the same.

[0007]

[Means for Solving the Problems] As a result of diligent efforts to solve the above problems, the present inventors have found that N- (trans-4-aminomethylcyclohexylcarbonyl) which is a specific and potent inhibitor of plasma kallikrein By using an affinity resin with -L-phenylalanine-4-carboxymethylanilide (PKSI-527) immobilized as a ligand, plasma kallikrein can be easily separated and purified according to the affinity chromatography method. It has been found that pure plasma kallikrein can be produced easily and in large quantities. The present invention has been completed based on the above findings.

That is, the present invention relates to the ligand N- (trans-4-aminomethylcyclohexylcarbonyl) -L-
It is intended to provide an affinity resin comprising a support on which phenylalanine-4-carboxymethylanilide is immobilized; and the above affinity resin used for separating and purifying plasma kallikrein by affinity chromatography. According to a preferred embodiment of these inventions, the above affinity resin in which the carboxyl group of the ligand and the amino group of the support are covalently bonded by condensation;
The above affinity resin in which the support is a hydrophilic vinyl polymer having an amino group; the amount of amino groups in the support is
The above affinity resin is 150 to 200 μmol / ml-gel; and the above affinity resin in which the plasma kallikrein is human plasma kallikrein.

According to another aspect of the present invention, a support having an amino group and an amino-protected N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide are prepared. There is provided a method for producing the above affinity resin, which comprises a step of reacting in the presence of a condensing agent; and a step of deprotecting an amino protecting group. According to a preferred embodiment of this invention, the amino group is Boc
The above-mentioned method of reacting a group-protected N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide; the above-mentioned method wherein the support is a hydrophilic vinyl polymer; There is provided the above method, which comprises the step of acetylating unreacted amino groups of the support.

According to yet another aspect of the present invention, there is provided a method for separating and purifying plasma kallikrein from plasma, which comprises the steps of: (a) converting plasma kallikrein contained in a plasma sample to N
-(Trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide is adsorbed on an affinity resin comprising a support, and (b) plasma adsorbed on the support. Eluting kallikrein; According to a preferred embodiment of the present invention, the above method wherein the affinity resin is washed with a substantially neutral washing solution in step (a) and then the step (b) is eluted; There is provided the above method of elution with an eluent; the above method of using a plasma sample that has been subjected to prekallikrein activation treatment; and the above method of using a plasma sample that has been subjected to a removal treatment of kallikrein inhibitor.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION The affinity resin provided by the present invention is N- (trans-4-aminomethylcyclohexylcarbonyl) -L- which is an inhibitor of plasma kallikrein.
It is characterized in that phenylalanine-4-carboxymethylanilide is immobilized as a ligand, and enables efficient and simple separation / purification of plasma kallikrein according to the method of affinity chromatography. In the present specification, separation / purification should be construed in the broadest sense, a step of separating low-purity plasma kallikrein from plasma that is a natural product, and purification of high-purity plasma kallikrein from separated low-purity plasma kallikrein It is a concept including a process and a process of separating and purifying high-purity plasma kallikrein from plasma in one step.

N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide used as a ligand can be prepared by the method described in the literature (C
hem.Pharm. Bull., 40, pp.1814-1817, 1992). Without wishing to be bound by any particular theory, one of the reasons why the compounds have an inhibitory effect on plasma kallikrein is that the amino-terminal part of the compound having an amino group and a carboxyl group (trans-4
-Aminomethylcyclohexyl moiety) and plasma kallikrein aspartic acid (Asp) have been suggested to interact with each other (see above). Therefore, when the compound is immobilized on the support as a ligand, it is preferable to react the carboxyl group in the compound with the reactive functional group in the support.

As the support for immobilizing the ligand (support for immobilizing the ligand), those generally used as the support for immobilizing the ligand in the affinity chromatography method can be used. . The type of the support is not particularly limited, and any one can be used as long as it can immobilize a sufficient amount of the above ligand by covalent bond and the immobilized ligand can substantially interact with plasma kallikrein. Preferably, immobilization of a ligand having a reactive functional group (eg, amino group, hydroxyl group, or thiol group) capable of reacting with a carboxyl group of the above compound used as a ligand to form an amide group, an ester group, a thioester group, etc. A support for can be used.

More specifically, as the support, for example, hydrophilic vinyl polymer, agarose, cellulose and the like can be used, and a support having an amino group introduced therein is used as a support for immobilizing a ligand. It can be preferably used. Various supports for immobilizing such a ligand are commercially available and can be easily obtained by selecting an appropriate type according to the purpose and conditions of separation / purification.

For example, AF-aminotoyopearl 6 commercially available from Tosoh Corporation as a hydrophilic vinyl polymer.
A support such as 50 is a support for immobilizing a ligand most preferably used in the present invention (TSK-GEL for Affinity C
hromatography, AF-Toyopearl 650, Technical Catalog, Tosoh Corporation). When a support for immobilizing a ligand having an amino group is used, the amount of the amino group in the support is, for example, 50 to 500 μmol / ml-gel, preferably 100 to 300 μm.
Mol / ml-gel, particularly preferably about 150 to 200 μmol / ml-gel can be used.

A method for reacting the ligand with a support for immobilizing the ligand to produce the affinity resin having the ligand immobilized thereon may be a method well known to those skilled in the art. As an example, the case of using AF-Aminotoyopearl 650 as a support for immobilizing a ligand is described. Generally, after washing the gel with water and a 0.5 M NaCl aqueous solution adjusted to pH 4.5-6.0, the ligand is Add 0.5 M NaCl solution in which is dissolved in the gel, add a condensing agent, and stir at room temperature for 24-48 hours. By this reaction, the carboxyl group of the above-mentioned ligand and AF-aminotoyopearl 650
React with the amino group to form an amide bond, and the ligand is immobilized on a support. After that, the affinity resin can be produced by washing the obtained gel with water, a 1 M NaCl aqueous solution, water and an appropriate buffer solution.

Examples of the condensing agent include N-ethyl-N '-(3
-Dimethylaminopropyl) carbodiimide hydrochloride (EDC) and the like can be preferably used. The amino group remaining in the affinity resin is preferably acetylated, but acetylation can be performed according to a known method (for example, O'Carra,
P., et al., FEBS Letters, 21, 281, 1972). Specifically, after washing the above gel with distilled water, add 0.2 M sodium acetate and acetic anhydride and incubate at 0 ° C for 30 minutes.Add acetic anhydride and incubate at room temperature to remove amino groups. Fully acetylated. After the reaction,
The gel is washed with distilled water, 0.1 N NaOH, distilled water and an appropriate buffer solution in this order.

The method for separating and purifying plasma kallikrein from plasma using the above-mentioned affinity resin may be generally carried out according to a method well known to those skilled in the art as a method of affinity chromatography. The type of plasma kallikrein that can be separated and purified by the method of the present invention is not particularly limited, and examples thereof include mammals including humans (for example, humans, chimpanzees, monkeys, pigs, guinea pigs, mice,
Plasma kallikrein contained in plasma derived from goat, sheep, horse, dog, cat, cow, killer whale, dolphin, etc.) can all be separated and purified by the method of the present invention. Further, for example, human plasma kallikrein is known to exist in subclasses (kallikreins I, II, and III), but any mixture of such plasma kallikrein subclasses and plasma kallikrein of a specific subclass are also included in the present invention. It can be manufactured by a separation / purification method.

Plasma used as a raw material is casein precipitate,
It is preferable to perform pretreatment by a method such as addition of 20% acetone, addition of kaolin, or alcohol fractionation to sufficiently activate prekallikrein in plasma in advance. Also,
It is desirable to remove plasma kallikrein inhibitor prior to separation / purification. For this purpose, for example, acid treatment can be carried out. In one example, citrated plasma is adjusted to pH 2.0, treated at 37 ° C for 15 minutes, and then adjusted to pH 7.4 with NaOH in ice (for these pretreatments, Sampaio, C., et. al., Arch. Bio
chem. Biophys., 165, pp.133-139, 1974).

In order to carry out separation / purification according to the method of the present invention, generally, the above affinity resin is suspended in an appropriate buffer solution, packed in a column, and equilibrated with the buffer solution. it can. Plasma treated with sodium citrate is acid-treated to remove plasma kallikrein inhibitor, and then kaolin is added to react at 37 ° C for about 20 minutes to activate prekallikrein and centrifuge the reaction mixture. The separated supernatant is separated and applied to the above column to adsorb plasma kallikrein to the affinity resin.

Subsequently, a neutral washing solution having a pH of about 7.0, preferably a neutral buffer solution (for example, 0.01 M phosphate buffer solution, pH 7.
After thoroughly washing the column with (0, etc.), a protein fraction containing plasma kallikrein using a weakly acidic buffer solution, preferably about pH 3.0 (eg 0.01 M phosphate buffer, pH 3.0, etc.) as the eluent. To elute. As the eluent, any one can be used as long as it has a property of decreasing the binding force or affinity between plasma kallikrein and the immobilized ligand, but generally, the type of affinity resin or the type of plasma sample PH of the eluent, salt concentration,
Alternatively, the urea concentration or the like should be appropriately selected.

Preferably, it is mildly acidic, eg pH 2.0-4.0.
Plasma kallikrein can be efficiently recovered from the affinity resin by using an eluent of a certain degree.
In addition, N- (trans-4-
It is also preferred to add aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide. The above-mentioned separation / purification step is generally performed at room temperature,
It can be carried out at a temperature of preferably 15 to 25 ° C, more preferably about 20 ° C. The detection of the protein fraction can be performed by measuring the ultraviolet absorption spectrum.

The method for separating / purifying plasma kallikrein of the present invention can be used in combination with any method conventionally used for separating / purifying plasma kallikrein.
For example, an extremely high-purity plasma kallikrein can be produced by purifying plasma kallikrein by the method of the present invention and then performing purification using Sephadex G-150 or the like.

[0024]

The present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited to the following examples. Example 1: Preparation of affinity resin of the present invention N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide (hereinafter referred to simply as "ligand" in Examples) (1 g)
Was dissolved in water, and triethylamine (0.7 ml) was added under ice cooling. (Boc) ₂ CO (0.48 g) was added to this solution under ice cooling.
Stir for 3-4 hours. After the solvent was distilled off, ethyl acetate and citric acid were added to adjust the pH to 4, and the mixture was washed with saturated saline and water. The ethyl acetate layer was dried over anhydrous Na ₂ SO ₄ and ethyl acetate was distilled off. The residue was crystallized from petroleum ether and collected by filtration to obtain a ligand in which the amino group was Boc.

AF-Amino Toyopearl 650 M (Tosoh Corporation), which is a support for immobilizing a ligand, was washed with 0.5 M NaCl solution (pH 4.5-6.0),
Boc-ized ligand as described above dissolved in dioxane (40 ml)
(0.4 g) was added, 1-ethyl- (3-dimethylaminopropyl) -carbodiimide hydrochloride (0.18 g) was further added, and the mixture was shaken and stirred at room temperature for 24-48 hours. After completion of the reaction, the support was washed with dioxane, water, 1M NaCl solution, and water in this order to obtain a support having a ligand whose amino group was protected by a Boc group. Then, the Boc group was removed with trifluoroacetic acid, and the residual amino group in the support was acetylated with 0.2M sodium acetate and acetic anhydride to obtain the affinity resin of the present invention.

Example 2: Binding of plasma kallikrein to affinity resin The affinity resin obtained in Example 1 was treated with 0.01 M phosphate buffer.
After washing with (pH 7.0, containing 0.15 M NaCl), centrifugation was performed, and the above buffer was added to the residue to prepare a suspension. To 0.4 ml of this suspension, add 2 mM S-2302 [HD-Pro-Phe-Arg-pN
A, 0.05 ml, mold (now Chromogeni
x); same as below) and 0.12 U / ml human plasma kallikrein (0.05 ml, mold) were added and heated at 37 ° C for 4 minutes, and then 50% acetic acid (0.05 ml) was added to react. After stopping, the amount of free paranitroaniline was measured by the absorbance at 405 nm.
As a control, AF-Aminotoyopearl 650 M without immobilized ligand was used. As a result, the affinity resin of the present invention inhibits plasma kallikrein in a concentration-dependent manner.
The concentration showing 0% inhibition was 2.0 mg / ml. AF-Aminotoyopearl 650 M used as a control showed no inhibitory activity against plasma kallikrein.

Example 3: Purification of human plasma kallikrein sample The affinity resin prepared in Example 1 was suspended in 0.01 M phosphate buffer (pH 7.0, containing 0.15 M NaCl) and packed in a column (diameter 1.6 cm x 2.1 cm). Then, the column was equilibrated with 0.01 M phosphate buffer (pH 7.0, containing 0.15 M NaCl) to prepare an affinity column.

Commercially available human plasma kallikrein (0.4 ml, mold, 0.24 U / ml) was adsorbed on the above column, and 0.01 M phosphate buffer (pH 7.0, containing 0.15 M NaCl) and 0.01 M were added.
Human plasma kallikrein was eluted at 20 ° C. using a phosphate buffer (pH 3.0, containing 0.15 M NaCl) as an eluent.
A 1 ml aliquot was collected at a flow rate of 0.20 ml / min, and protein quantification by the Lowry method and absorbance (UV 280 nm) and plasma kallikrein activity were measured.

As a result, plasma kallikrein was adsorbed on the column, and the protein having almost no activity was eluted first. After thorough washing with 0.01 M phosphate buffer (pH 7.0)
By elution with 0.01 M phosphate buffer (pH 3), a protein fraction having plasma kallikrein activity was obtained. The results are shown in FIG. The recovery rate of plasma kallikrein by this method was 61%, and the specific activity increased 14-fold (Table 1). As a control, the same procedure was performed using AF-aminotoyopearl 650 with no immobilized ligand, but plasma kallikrein adsorption was not observed.

[0030]

[Table 1] ───────────────────── Recovery rate etc. Before refining After refining ────────────────── ─── Total protein amount (mg) 0.790 0.035 Total activity (nmol / min) 67.6 41.1 Specific activity (nmol / min / mg) 85 1174 Collection yield (%) 100 61 Increase in specific activity (fold) 1 14 ──── ─────────────────

Plasma kallikrein activity can be measured using S-2302 (H
-D-Pro-Phe-Arg-pNA, final concentration 0.2 mM, mold)
The fraction was added to 0.01 M phosphate buffer (pH 7.0) to adjust the amount of free paranitroaniline per minute to 405 n.
The absorbance was measured at m 2. Incidentally, hydrochloric acid or sodium hydroxide was added to 0.01 M phosphate buffer to prepare solutions having various pHs, and the effect of pH on plasma kallikrein activity was examined.
The highest activity was exhibited at 9.0, but almost no activity was observed at pH 3.0.

Example 4: Separation and purification of plasma kallikrein from human plasma sample Sodium citrate was added to human plasma and pH was adjusted to 2. with hydrochloric acid.
The pH was adjusted to 0 and treated at 37 ° C for 15 minutes, and then adjusted to pH 7.4 with NaOH in ice to remove plasma kallikrein inhibitor. Then kaolin (Yamazen Pharmaceutical, final concentration
0.2 mg / ml) was added and reacted at 37 ° C. for 20 minutes to activate prekallikrein in plasma. The reaction solution was centrifuged (3.000 rpm, 10 min) and the resulting supernatant (2.5
(ml) was applied to the above column and plasma kallikrein was separated from the plasma sample at 20 ° C (flow rate 0.50 ml / min, preparative amount).
2.5 ml). As the eluent, 0.01 M phosphate buffer (pH 7.
0,0.15 M NaCl) and 0.01 M phosphate buffer (pH 3.
0, 0.15 M NaCl) was used for protein quantification and plasma kallikrein activity measurement according to the method of Example 3 above.

As a result, a protein having almost no plasma kallikrein activity was first eluted with 0.01 M phosphate buffer (pH 7.0) and then 0.01 M phosphate buffer (pH 3.0) was used as an eluent to obtain plasma kallikrein. An active protein fraction was obtained. The elution profile obtained was almost the same as in Example 3 above. The results are shown in FIG. The recovery rate of plasma kallikrein activity by this method was 74%, and the specific activity increased 34 times.
Purified human plasma kallikrein was obtained with almost the same purity as the commercially available human plasma kallikrein used in (Table 2).

[0034]

[Table 2] ───────────────────── Recovery plasma, etc. Activated plasma After purification ────────────────── ──── Total protein amount (mg) 170 4 Total activity (nmol / min) 520 386 Specific activity (nmol / min / mg) 3 102 Recovery rate (%) 100 74 Increase in specific activity (fold) 1 34 ─── ──────────────────

Example 5: Separation and purification of plasma kallikrein from human plasma sample After the reaction solution supernatant was applied to the column in the same manner as in Example 4, 0.01
After washing with M phosphate buffer (pH 7.0, containing 0.15 M NaCl), human plasma kallikrein was eluted with 0.05 M glycine buffer (pH 3.0, containing 0.15 M NaCl) as an eluent.
The results are shown in FIG. Protein fractions with plasma kallikrein activity were collected, applied to a Sephadex G-150 column, and eluted with 0.05 M glycine buffer (pH 3.0, containing 0.15 M NaCl) to have plasma kallikrein activity. The protein fraction was collected. The recovery rate by the above two-step purification was 70%, and the specific activity was increased by 109 times (Table 3).

[0036]

[Table 3] ─────────────────────────── Activation of recovery rate Affinity Sephadex Plasma After column After column ────── ───────────────────── Preparative amount (ml) 2.0 7.5-Total protein amount (mg) 160 11.3 1.1 Total activity (nmol / min) 780 725 545.6 Ratio Activity (nmol / min / mg) 4.9 64.3 529.1 Recovery rate (%) 100 93 70 Increase in specific activity (fold) 1 13 109 ─────────────────────── ─────

Example 6 Investigation of Substrate Specificity of Plasma Kallikrein Substrate specificity of human plasma kallikrein purified from human plasma sample in Example 4 and commercially available human plasma kallikrein (used in Example 3, mold, 0.24) Substrate specificity of (U / ml) was compared using specific synthases, and both showed similar substrate specificity. That is, the plasma kallikrein substrate was well decomposed, and the thrombin, glandular kallikrein, plasmin, urokinase, and coagulation factor Xa substrate were hardly decomposed [Table 4: In the table, the S2302 substrate degrading activity was converted to 100. Indicates the value).

[0038]

[Table 4] ──────────────────────────── Substrate Commercially purified product Example 4 purified product ΔO.D.405 / min ΔO .D.405 / min ──────────────────────────── S2302 (plasma kallikrein) 0.13 (100) 0.13 (100) S2238 (thrombin ) 0.026 (20) 0.033 (26) S2266 (glandular kallikrein) 0.0196 (15) 0.0170 (13) S2251 (plasmin) 0.004 (3) 0.004 (4) S2444 (urokinase) 0.0096 (7) 0.00825 (6) S2222 (coagulation) Factor Xa) 0.0032 (2) 0.0016 (1) ────────────────────────────

[0039]

EFFECT OF THE INVENTION By using the affinity resin of the present invention, plasma kallikrein can be separated efficiently and easily.
It can be purified. In addition, by using the above-mentioned affinity resin, high-purity plasma kallikrein can be easily produced from a plasma sample in a large amount.

[Brief description of drawings]

FIG. 1 is a diagram showing an elution profile of affinity chromatography when a human plasma kallikrein sample was purified according to the method of the present invention. The arrow in the figure indicates substitution with the eluent at pH 3.0.

FIG. 2 is a view showing an elution profile of affinity chromatography when separating and purifying human plasma kallikrein from a human plasma sample according to the method of the present invention. The arrow in the figure indicates substitution with the eluent at pH 3.0.

FIG. 3 is a view showing an elution profile of affinity chromatography when separating and purifying human plasma kallikrein from a human plasma sample according to the method of the present invention. The arrow in the figure indicates the eluent glycine buffer (G
lycine buff. pH 3.0).

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Yuko Tsuda 1440-1 Shimizu, Uozumi-cho, Akashi-shi, Hyogo (72) Inventor Keiko Wanaka 3-15-18 Asahigaoka, Tarumi-ku, Kobe-shi, Hyogo Kobe Project Committee Member (72) Inventor Mayako Yasuda 3-15-18 Asahigaoka, Tarumi-ku, Kobe-shi, Hyogo Prefecture Kobe Project Committee for Thrombosis Hemostasis Research Project

Claims (15)

[Claims] 1. A ligand, N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine.
-4-Affinity resin consisting of a support on which carboxymethylanilide is immobilized. 2. The affinity resin according to claim 1, which is used for separating and purifying plasma kallikrein by affinity chromatography. 3. The carboxyl group of the ligand and the amino group of the support are covalently bonded by condensation.
The affinity resin described in 1. 4. The affinity resin according to claim 3, wherein the support is a hydrophilic vinyl polymer having an amino group. 5. The amount of amino groups in the support is 150 to 200 μm.
The affinity resin according to any one of claims 1 to 4, which is mol / ml-gel. 6. The affinity resin according to any one of claims 1 to 5, wherein the plasma kallikrein is human plasma kallikrein. 7. A support having an amino group and N- (trans
4. Aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide with an amino-protected compound in the presence of a condensing agent; and a step of deprotecting the amino-protecting group. 7. The method for producing the affinity resin according to any one of 6 above. 8. The method according to claim 7, wherein N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine-4-carboxymethylanilide having an amino group protected with a Boc group is reacted. 9. The method according to claim 7, wherein the support is a hydrophilic vinyl polymer. 10. The method according to claim 7, further comprising a step of acetylating unreacted amino groups of the support. 11. A method for separating and purifying plasma kallikrein from plasma, comprising the steps of: (a) converting plasma kallikrein contained in a plasma sample to N- (trans-4-aminomethylcyclohexylcarbonyl) -L-phenylalanine; -4-A method of adsorbing carboxymethylanilide on an affinity resin composed of a support, and (b) eluting plasma kallikrein adsorbed on the support. 12. The method according to claim 10, wherein the elution in step (b) is carried out after washing the affinity resin with a substantially neutral washing solution in step (a). 13. The method according to claim 11 or 12, wherein elution is performed with a substantially weakly acidic eluent in step (b). 14. The method according to any one of claims 11 to 13, wherein a plasma sample subjected to activation treatment of prekallikrein is used. 15. The method according to claim 11, wherein a plasma sample that has been subjected to a treatment for removing kallikrein inhibitor is used.