JPS6038113B2 - Kallikrein purification method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for efficiently removing impurities from an aqueous solution containing kallikrein, such as an extract of a mammalian hawk or a crude kallikrein solution, and purifying kallikrein to a high degree of purity.

Kallikrein is known to exist in the viscera, submandibular gland, pig urine, and human urine of mammals such as cows, pigs, horses, and whales.There are various methods for purifying kallikrein. is proposed.

For example, a method in which crude kallikrein is adsorbed and separated on weakly basic anion exchange cellulose, and a method in which kallikrein precipitate, which is precipitated by adding lead or zinc salt to an aqueous solution containing kallikrein, is adsorbed and separated on weakly basic anion exchange cellulose, etc. There is a method in which crude kallikrein obtained by adding a protein precipitate salt to a glandular tissue extract is adsorbed and separated on a giant reticular weakly basic anionic sardine resin and purified. However, these methods have drawbacks such as the physical robustness of the ion exchanger and its susceptibility to microbial contamination, and when using a giant reticulated strongly basic anion exchange resin, elution of the target product is slow and incomplete. The drawback is that elution and separation require a large amount of solvent and a considerable amount of time.

A purification method using a support having a molecular binding effect is also known, but with this method it is difficult to remove impure proteins having a molecular weight comparable to that of kallikrein contained in a crude kallikrein solution. Further, even if the purification is carried out according to the conventionally known fractional precipitation method or isoelectric point fractionation method, it requires a lot of time and labor, and the loss of the sample is large, so that it cannot be said to be an industrially suitable method. In view of the above-mentioned current situation, the present invention has been made for the purpose of providing a method for purifying kallikrein simply and quickly with high purity.
Synthesis that exhibits a behavior in which the elution time becomes longer as the temperature rises in liquid chromatography using water as the scale liquid and polyethylene glycol as the solute, and that contains both a non-dissociable hydrophilic group and a hydrophobic group in its molecule. A method for purifying kallikrein is characterized in that it is carried out by liquid chromatography using a granular filler made of a polymer as a stationary phase and a buffer having a pH of 4 to 9 as an eluent. The filler used in the present invention is a granular material made of a synthetic polymer, and when the granular synthetic polymer is used as a filler, it is a high-speed liquid using polyethylene glycol as the sample and water as the eluent. This behavior shows that as the column temperature increases in chromatography, the sample elution time increases.

For example, if the column temperature is kept at room temperature (2000) and
The time taken to leave the port will be longer if it is kept at 50q0 than if it is kept at 0oo. As described above, the packing material used as the stationary phase in the present invention has two characteristics: water can be used as an eluent, and the elution time of the sample increases as the column temperature increases. Therefore, in the present invention, it has been found that a filler having such characteristics is effective for purifying kallikrein, and has therefore been developed. In order to have the above characteristics, the synthetic polymer constituting the filler must contain non-dissociable hydrophilic groups such as hydroxyl group, amide group, ethylene oxide group,
It is required that both propylene oxide groups and the like and hydrophobic groups such as alkyl groups, phenyl groups, alkylene groups, etc. are included. In order to produce the filler of the present invention, a known water suspension polymerization method can be suitably employed, and a monomer having the non-dissociable hydrophilic group and the hydrophobic group in the molecule is subjected to water suspension polymerization. A filler can be obtained by forming polymer particles.

Suitable examples of the above monomers include ethylene glycol dimethacrylate, tetramethylolmethane triacrylate, and ethylene glycol diacrylate.
It can also be produced by aqueous suspension polymerization of a monomer mixture in which the monomer having a non-dissociable hydrophilic group and the monomer having a hydrophobic group are mixed in an appropriate ratio. Preferred examples of the former monomer include tetraethylene glycol diacrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, acrylamide, etc., and preferred examples of the latter monomer include styrene, divinylbenzene, vinyl acetate, and acrylic acid. Examples include methyl and methyl methacrylate. By selecting one or more monomers from each group and mixing them, a polymerization catalyst and a suspension stabilizer are added and water suspension is carried out to form a monomer with a diameter of 5 to 200 mm. Micron polymer particles can be obtained. In any of the above polymerizations, if an organic solvent is added that dissolves the monomers to be polymerized but does not dissolve the polymer, the resulting spherical polymer becomes porous and the surface area of the filler decreases. is effective for increasing separation efficiency. In addition, as the requirements for a packing material for high performance liquid chromatography, it is required to have excellent mechanical strength, low swelling properties, etc.
For this reason, it is desirable that the filler is spherical, and furthermore, it is desirable that it be crosslinked. Therefore, the polymerizable monomer used to produce the filler used in the present invention preferably contains a monomer having a polyfunctional group capable of forming a crosslinked structure. The polymer particles obtained by water suspension polymerization are dried by heating or the like.

Then, by dispersing the dry particles in water, it is determined whether or not they get wet with water. Particles that do not leak cannot be used in high performance liquid chromatography using water as an eluent, and therefore cannot be used as the packing material of the present invention. Next, the polymer particles obtained by removing fine particles and coarse particles were packed into a liquid chromatography column, ion-exchanged water was used as an eluent, and polyethylene glycol with a molecular weight of 400 (PEG 400, average degree of polymerization was 8) was used as a sample. 4-15
The polyethylene glycol is separated using a polyethylene glycol containing each component with a degree of polymerization of In order to employ it as a filler in the present invention, it is necessary to measure the chromatogram at room temperature and at 50°C, and to obtain a result that the scale time at 50°C is longer than at room temperature. In the present invention, a packing material having the above-mentioned specific properties is used as a stationary phase, and the pH is 4.
Purification of kallikrein is carried out by liquid chromatography using a buffer of ~9 as an eluent.

Further, the temperature at that time is preferably a temperature suitable for using water as an eluent, usually in the temperature range of 0 to 85:00. Further, as the buffer in the present invention, phosphate buffer, acetate buffer, citrate buffer, etc. are suitably used. To purify kallikrein according to the present invention, a packing material having the above-mentioned specific properties is used as a stationary phase,
In liquid chromatography using the above-mentioned buffer as an eluent, kallikrein is adsorbed onto a packing material at approximately pH 5.0 to 6.0, and the pH is increased by a pH gradient method or pH switching, and kallikrein is absorbed below the increased pH. This can be done by desorbing and fractionating the thereby separated kallikrein. Further, by using a high performance liquid chromatography device, kallikrein can be purified in a short time of 30 minutes to 1 hour. The method for purifying kallikrein of the present invention is as described above, in which a packing material having particular properties is used as a stationary phase, and the pH is 4 to 9.
Since this buffer solution is used as an eluent, it is possible to purify kallikrein more easily and in a shorter time than with conventional methods, and with high precision.

The present invention will be explained below based on examples. Example
1. Into a 2-piece collapsible flask equipped with a condenser, a thermometer, and a dropping funnel, 400 g of a 4% by weight aqueous polyvinyl alcohol solution, 100 g of tetramethylolmethane triacrylate, and 1. penzoyl peroxide were added.
A mixed liquid consisting of the gunwale was supplied.

Next, the temperature was raised to 8,000 ℃ while stirring at a speed of 40 pm, reaction was carried out for 1 hour, and the mixture was cooled. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a spherical polymer having a particle size of 5 to 20 microns. Particles of 8 to 12 microns obtained by removing fine particles and coarse particles were dispersed in 800 ml of ion-exchanged water and transferred to a stainless steel column (diameter 7.9 bars, length 50 arcs) using a high-pressure constant flow pump. 1. Add ion exchange water. Filling was carried out by ejecting at a speed of 100 m/min. The obtained packed column was connected to a high-performance liquid chromatograph (trade name: Shimadzu DuPont High-Performance Liquid Chromatograph Model 830), and the column temperature was maintained at room temperature, 360, 50 qo, and 70 qo, respectively, and polyethylene glycol (PEG400) with a molecular weight of 400 was added. , manufactured by Wako Pure Chemical Industries, Ltd.) was used as a sample and chromatographic analysis was performed using ion-exchanged water as the eluent. In the obtained chromatograms at each temperature, the dissolution time of each component became longer as the temperature increased. Next, 1% of crude kallikrein (concentration IKU/9) that had been autolyzed and dried in advance
Inject M/Vol aqueous solution 100 and add phosphate buffer (
monopotassium dihydrogen phosphate - disodium monohydrogen phosphate)
A gradient was performed by using molten liquid as a molten liquid and gradually increasing the pH from 5.9 to 8.5. When the fractionated components at pH 8.0 and above in FIG. 1 were separated, desalted, and freeze-dried, 11 KU/9 of kallikrein was obtained.

Example 2 Using the filler prepared according to Example 1, DEAE
- 10% wt/vol aqueous solution 1 of crude kallikrein (3. chick U/fence) from which a small amount of impurity protein was removed with cellulose
Inject 0.00ml and use phosphate buffer as a molten liquid and adjust the pH to 6.
A gradient was performed from 0 to 9.2 increasing gradually.

The fractionated components after the dotted line A shown in Figure 2 were removed, and the remaining fractionated components were separated, desalted, and freeze-dried.1
5. We obtained 9 kallikreins of chick U/.

[Brief explanation of the drawing]

FIGS. 1 and 2 show liquid chromatograms of Examples 1 and 2, respectively. The vertical axis shows the absorbance at 28 mm, and the vertical axis shows the elution time (minutes). O'Kyou O2Ko

Claims (1)

[Claims] 1. Purification of kallikrein shows that in liquid chromatography using water as an eluent and polyethylene glycol as a solute, the elution time becomes longer as the temperature rises, and the molecule contains non-pyretic hydrophilic and hydrophobic groups. kallikrein, characterized in that it is carried out by liquid chromatography using a granular packing material made of a synthetic polymer containing both as a stationary phase and a buffer solution having a pH of 4 to 9 as an eluent. Purification method.