JPH01226900A - Method for purifying protein c - Google Patents

Abstract

PURPOSE:To efficiently purify the title compound for promoting anticoagulant fibrinolysis, etc., by treating a protein C-containing solution at a specific pH by an isoelectric precipitating method and subjecting the treatment product to gel filtration, treatment with immobilized sultate of polysaccharides and reversed phase chromatography. CONSTITUTION:A protein C-containing solution is treated under conditions of pH4-6 by an isoelectric precipitating method to recover a supernatant, which is then added to a cation exchanger under conditions of pH4-6, eluted under conditions of 0.1-2 M salt concentration, subsequently added to an anion exchanger under conditions of pH6-9 and eluted at a concentration gradient of 0.2-2 M salt concentration to afford a protein C-containing fraction. The resultant fraction is subjected to gel filtration using a carrier for the gel filtration having a fractionation molecular weight within the range of 1,000-1,000,000, added to immobilized sulfate of polysaccharides under conditions of pH5-7, eluted at a concentration gradient of 0-2 M salt concentration, then added to a carrier for reversed phase chromatography under conditions of pH1-2 and eluted at a concentration gradient of 10-80vol./vol.% organic solvent concentration to afford the aimed high-purity protein C.

Description

[Detailed description of the invention] [Industrial application field] The present invention relates to a method for purifying protein C.

[Prior art]

Protein C is a protein containing the amino acid γ-hydroxyglutamic acid (Gla), which is synthesized in the presence of vitamins, and has a chromatographic fraction number “C°” during the purification process from plasma. It was named after Protein C, which has a molecular weight of 62.000.
It is mainly synthesized in the liver as a single-chain glycoprotein, and exists mostly as two-chain molecules (L chain and H chain) in the circulating blood. Protein C is a Gla-containing coagulation factor factor
It is a serine protease proenzyme similar to factor

Thrombin, which is generated by the body's coagulation mechanism, forms a complex with thrombomodulin (thrombos + odulin), a specific receptor present on the vascular endothelial cell membrane, and loses its various catalytic functions as an original coagulation factor. A powerful activator of protein C. Protein C undergoes limited decomposition by this complex to become activated protein C (APC: activated protein C), which is a highly efficient coagulation fibrinolytic tJ4 that has both anticoagulant and fibrinolytic promoting effects. It becomes a node factor. Its mechanism of action is to selectively degrade and deactivate coenzymes F, Va, F, and VIIIa in the so-called tennase and prothrombinase complexes on the phospholipids on the surface of platelet membranes and endothelial cell membranes. Strongly inhibits blood coagulation reactions. APC also selectively degrades FAI (plasminogen activator inhibitor), a tPA inhibitor, and also promotes fibrinolysis.

Known methods for purifying protein C include precipitation adsorption with barium chloride, ammonium sulfate fractionation, treatment with anion exchangers, preparative electrophoresis, antibody columns for protein C, and antibody columns for contaminant proteins. @
(J, Biol, Chew, l.

355-363 (1976), 25iL191
4-1920 (1983).

J, Nara Mad Ass,, J5! , 44
8-454 (1984).

J, Biol, Chem, , 2j2J, 11
097-11105 (1986).

Thromb, l1aes+ostas,, ill
, 1-5 (1983)) However, the present invention reveals that the above methods do not provide satisfactory results in terms of purity, yield, and work efficiency, or are unsuitable for large-scale purification of proteins. It was discovered by these people.

[Problem to be solved by the invention]

Therefore, the present inventors investigated methods for efficiently purifying and isolating protein C, and found that isoelectric precipitation and reversed-phase chromatography were effective. discovered a method for purifying protein C, and completed the present invention.

[Means to solve the problem]

That is, the present invention is as follows.

Invention 1 Niprotin C-containing solution was adjusted to pH 4-4 by isoelectric precipitation method.
6. A method for purifying protein C, comprising the steps of treating under the conditions of 6 and collecting a supernatant.

Invention 2 Niprotin C-containing solution is added to a carrier for reverse phase chromatography at pH 1~
A method for purifying protein C, which comprises adding the protein C under the conditions of item 2 and eluting with an organic solvent concentration gradient from 10 v/v % to 80 v/v %.

Invention 3 A step of treating a Niprotin C-containing solution by (a) isoelectric precipitation under conditions of pH 4 to 6 and collecting the supernatant; (C) Addition to the anion exchanger under conditions of pH 6 to 9 followed by elution with a salt concentration gradient of 0.2 to 2M. (dl step of performing gel filtration using a carrier for gel filtration with a molecular weight cut-off range of 1,000 to 1,000,000, (e) adding to the immobilized sulfuric acid w under conditions of pH 5 to 7, and then adjusting the salt concentration to OM. Step of eluting with a concentration gradient up to 2M, (") After adding to the carrier for reverse phase chromatography under conditions of pH 1 to 2, organic solvent 4 times from 10 v/v % to 8 ° v / v
A method for purifying protein C, comprising the step of eluting with a concentration gradient of up to

[1] Starting material The protein C-containing solution used in the present invention is not particularly limited as long as it is derived from human plasma, and specifically, blood IF! Examples include a coagulation factor-containing fraction, a concentrated prothrombin complex fraction, and other protein C-containing fractions.

[2] Information production method (a) Isoelectric focusing method 5 methods Isoelectric focusing precipitation method is a method for purifying proteins that utilizes the fact that the solubility of proteins is minimized at the isoelectric point and a precipitate is formed. It is.

That is, in this step, the protein C-containing solution is adjusted to pH 4.
Treat under the conditions of ~6 and collect the supernatant.

The processing temperature at that time is 2-6°C, and the processing time is 6-6°C.
It is 24 hours.

After the treatment, the supernatant is collected by centrifugation at 5,000 to 20,000 g for 10 to 60 minutes.

(b) Treatment with a cation exchanger The protein C-containing solution obtained in (a) is added to the cation exchanger under conditions of pH 4 to 6.

As a cation exchanger, Bakerbond CBx or CM-Se with a carboxymethyl group as a ligand is used.
An example is pharose CL-6B.

Thereafter, a protein C-containing fraction eluted under open pH 1 salt concentration conditions of 0.1 to 2M is collected.

(C) Treatment with anion exchanger The protein C-containing solution obtained in (b) is added to the anion exchanger under conditions of pH 6 to 9.

As an anion exchanger, Mo for high performance liquid chromatography is used.
An example is no Q (QAE system).

After that, after washing under the conditions of the same p)l and a salt concentration of 0.2 to 0.3M, the same p)l was washed with a salt concentration of 0.2M to 2.0M.
Elution is performed with a concentration gradient up to 100%, and a protein C-containing fraction is collected.

((Sub) Protein C-containing solution obtained by gel filtration (C) was
000 to 1,000,000 to a carrier for gel filtration.

Examples of such carriers include 5apense 12 for high performance liquid chromatography, which is based on agarose.

Elution is performed at pH 6-B and salt concentration 0.1-2M, and protein C-containing fractions are collected.

(e) Treatment with immobilized sulfuric acid poly(Am) The protein C-containing solution obtained in (d) was treated with immobilized sulfuric acid poly(I) at pH 5 to 7. Add to class.

Examples of the immobilized sulfate polysaccharide include dextran sulfate and 5epharose CL4B whose ligand is heparin.

Thereafter, elution is performed with a concentration gradient from open pH 2 salt concentration OM to 2.0M, and a protein C-containing fraction is collected.

(f) A protein C-containing solution obtained by reverse phase chromatography tel is added to a carrier for reverse phase chromatography under conditions of pH 1 to 2.

Examples of the carrier for reversed phase chromatography include a reversed phase column using Cl as a ligand.

Thereafter, elution is performed with a concentration gradient of open-pH 1 organic solvent (e.g., acetonitrile, methanol, ethanol, propatool, dioxane, etc.) from 10 v/v% to 80 v/v%, and the protein C-containing fraction is collected. .

Note that after performing each step, ammonium sulfate fractionation, dialysis, etc. are performed as desired.

That is, the ammonium sulfate fraction contains a protein C-containing solution of 60 to 8
This is carried out by treating with ammonium sulfate at 0% saturation concentration and collecting the precipitated fraction.

In addition, dialysis is performed at 2-6°C under conditions of pH 4-7.
It will be held for 24 hours.

In the present invention, it is also possible to combine known purification steps.

Protein C obtained in this manner may be used as a reagent for biochemistry or pharmacology, and when used as a pharmaceutical, it may be sterilized, sterilized, or sterilized according to customary pharmaceutical manufacturing techniques. Lyophilization, formulation, etc. may be performed.

〔effect〕

According to the method of the present invention, protein C can be efficiently isolated and purified. Furthermore, the method of the present invention is simple to operate and can be applied to large-scale production. Therefore, it is extremely useful as a method for purifying protein C.

(Examples) In order to explain the present invention in more detail, Examples are given, but the present invention is not limited by these in any way.

(11 Purification method 1) Add 10 ml of distilled water to a vial of Crissin preparation and dissolve completely, then add 20 mM sodium acetate,
Dialysis was performed with a pH 5.0 buffer. After dialysis, io, oo
The supernatant fraction was collected by centrifugation at OC for 20 minutes.

2) The cation exchange chromatography supernatant fraction was added to Bakerbond CBx. 20mM sodium acetate, PH5.0 was used as the starting buffer, and 1M sodium chloride, 20mM sodium acetate, p) 15.0 was used as the ejection buffer. After adding the sample and washing with the starting buffer, switch to the elution buffer.
Pass fractions were collected.

3) Anion exchange chromatography dialysis retentate was purified using Mono Q HR60/10 (or Q
SepharoseHigh Performance
60/100). 20vM in starting buffer
) Lis-HCl, pH 7,15 was used as the elution buffer, and 1M sodium chloride, 20mM) Lis-HCl, p) 17.15 was used as the elution buffer. After adding the sample, the sample was washed with 30% (V/v) elution buffer/initiation buffer, and then linear gradient elution from 30% to 40% elution buffer/starting buffer was performed to collect the active fraction.

4) Gel filtration chromatography dialysis retentate with 5uperoSe 12 prep grade 6
Added at 0/600. The active fraction was collected using 20-M Tris-HCl, 500 mM sodium chloride, pH 7.2 buffer as i8 separation buffer.

5) Dextran sulfate chromatography The dialysis retentate was added to Sepharose CL4B with dextran sulfate as a ligand. 25mM in starting buffer
Imidazole, pH 6, was used as the elution buffer, 1 Sodium Chloride, 25++M Imidazole, pH 6.0. After addition of the sample, 0%→50% elution buffer/initiator buffer gradient elution was performed, and the active fraction was collected.

6) Reverse phase chromatography Both obtained fractions were applied to a reverse phase column using 08 as a ligand. Stock solution A (distilled water containing 0.1% tetrafluoroacetic acid) / B (800-acetonitrile was added to Afi,
1.000 aZ), and the starting bread was made by adding liquid A to 400II7 liquid B to make 1.00 aZ.
0 at (final concentration of acetonitrile 32%), elution buffer was 500aZ by adding solution A to solution B and making 1.00
0- (acetonitrile final concentration 40%) was used. After washing with the starting buffer, linear gradient elution was performed from 0% to 50% elution buffer (actual acetonitrile concentration was 32% to 36%). After the 0 fraction, the organic solvent in each fraction was dried under reduced pressure.

[2] Measurement 1) Ophthalony method 10μ of each sample and each antiserum were added to 1 or 2% agar gel prepared in a conventional manner. After leaving it at 4°C overnight and checking the sedimentation line, the gel was dehydrated with filter paper and washed with physiological saline, followed by dehydration and washing two more times.

After repeating the process, Coomassie staining was carried out in the usual manner◆2)
Immunoelectrophoresis method Prepare a 1.2% agar gel p) 1B, 6, μm 0.
06), after adding 10 parts of the sample, 2 m per 10 parts of the gel
The gel was run at 4°C for about 4 hours in A. After electrophoresis, 100μ of antiserum was added to each groove, left overnight at 4°C, and after confirming the sedimentation line, the gel was dehydrated with filter paper and washed with physiological saline, and the dehydration and washing process was repeated two more times. It was then stained with Coomassie in the usual manner.

3) Activity measurement method The sample was diluted with 20-tris-HCl, 100@M sodium chloride, pH 5 buffer and then subjected to measurement.

PROTACC (3 units/vial) is a 0 chromogenic substrate MCA diluted with distilled water.
-3112v, l vial (5, IN, 7.0
After completely dissolving the contents of the 85-μmole in dimethyl sulfoxide, 85-20mM Tris-HCl, p
) The 0 reaction stop solution diluted with 18.5 buffer was
A 20% (v/v) aqueous acetic acid solution was used. The measurement flow sheet is shown in Table 1.

Table 1 Protein C analysis method using Protac C Protac C sample 50st ■ Incubation at 30°C for 520 minutes Incubation at 30°C for 520 minutes The purification steps, activity recovery rate and degree of purification are shown in Table 2.

Table 2 Isoelectric precipitation 2000 36.9 1.020
100 196 cation exchange I mulberry 2400
18.0 808 95
318 Ri ■ Madograph (- Anion exchange 290 25.9
4.012 57 1.099 Chromatograph Egel filtration 67 28.0 13.195
43 3.342 chromatography reverse phase 350 0.32 2,001 3
4 44,342 Chromatography [4] Physicochemical properties of purified protein C 5O3-PA
As a result of molecular weight measurement by GE method, 5OS-P under non-reduction
In AGE, one band was observed at a molecular weight of 62,000,
Under reducing conditions, the molecular weight is 41,000 (Hllt) and 21
,000 (L chain), two bands were observed.

In immunoelectrophoresis, one clear sedimentation line was observed only at the α position, and anti-normal human serum, rabbit serum, anti-human PS,
No sedimentation lines were formed with rabbit serum F, II, F, IX, F, and X.

As a result of isoelectric focusing, protein C has a pl of 3.8~
It was recognized as a continuous band between 4.2 and 4.2.

From the above results, isolated protein C has a purity of 99%.
It is estimated that this is the above.

m-" Procedure Juho Seisho (spontaneous) 1. Indication of the case Patent Application No. 54159 of 1988 2. Name of the invention Process for purifying protein C 3. Person making the amendment Relationship to the case Name of the patent applicant (name ) Midori Juji Co., Ltd. 4, Agent 541 Address 4-56 Hirano-cho, Higashi-ku, Osaka (Basue Building) Ta (06) 227-1156 6. Contents of the amendment (1) "In the second line of page 15 of the specification" Correct "Protac C sample" to "Protin C sample".

(2) Protrocybin 2200 67.1 in Table 2, page 16 of the specification
927 100 98 complex concentration
Trocybin 2200 67.
1 927 100 98 complex concentrate
Correct to.

Claims (3)

[Claims] (1) A protein C-containing solution was subjected to isoelectric precipitation using p
A method for purifying protein C, comprising a step of treating under H4-6 conditions and collecting a supernatant. (2) Pour protein C-containing solution onto a carrier for reversed phase chromatography.
After adding under the conditions of H1-2, the organic solvent concentration was 10v/
A method for purifying protein C, comprising a step of eluting with a concentration gradient from v% to 80v/v%. (3) A step of treating a protein C-containing solution under conditions of pH 4 to 6 by (a) isoelectric focusing precipitation method and collecting the supernatant; (b) applying the solution to a cation exchanger under conditions of pH 4 to 6; (c) Adding to the anion exchanger under conditions of pH 6 to 9 and then eluting with a concentration gradient of salt concentration from 0.2 to 2M. (d) performing gel filtration using a carrier for gel filtration with a molecular weight cut-off range of 1,000 to 1,000,000; (e) adding to the immobilized sulfuric acid polysaccharide under conditions of pH 5 to 7, and then adding the salt concentration from 0M to (f) elution with a concentration gradient up to 2M, (f) adding to a carrier for reversed phase chromatography under conditions of pH 1 to 2, and then elution with an organic solvent concentration of 10v/v% to 80%;
A method for purifying protein C, comprising the steps of eluting with a concentration gradient up to v/v%.