JP2005060244A - Glutathione-immobilized magnetic fine particles and protein separation and purification method using the same - Google Patents

Abstract

To provide a glutathione-immobilized magnetic microparticle capable of rapidly recovering a target protein from a protein mixture and a method for separating and purifying a protein using the same.
A glutathione-immobilized magnetic fine particle in which magnetic fine particles and glutathione are immobilized via a stimulus-responsive polymer, the average particle diameter of which is 1-1000 nm. By fine particles.
[Selection figure] None

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to glutathione-immobilized magnetic microparticles and a method for separating and purifying a target protein using the same.
[0002]
[Prior art]
As a method for separating a target substance from a mixed solution, affinity chromatography using a column is widely performed. However, column chromatography takes time because the mixed solution is passed through a column packed with a packing material. When the target substance is a protein, the protein may be decomposed and the yield may be reduced.
On the other hand, there is known a method of adding particles to which a ligand is fixed to a mixed solution, adsorbing a target substance, collecting fine particles, and separating and collecting the target substance from the fine particles. Specifically, at least one selected from biotin and avidin is a temperature-responsive magnetic fine particle fixed to a magnetic fine particle via a polymer having a lower critical solution temperature (hereinafter abbreviated as “LCST”) and a magnet. A biological material separation method using magnetic force has been developed (see, for example, Patent Document 1). However, even if this method was used, the target protein could not be rapidly recovered from the cell disruption suspension of microorganisms.
[0003]
[Patent Document 1]
International Publication No. 02/16571 Pamphlet [0004]
[Problems to be solved by the present invention]
Accordingly, an object of the present invention is to provide glutathione-immobilized magnetic microparticles that enable rapid recovery of a target protein from a protein mixture and a protein separation and purification method using the same.
[0005]
[Means for solving the problems]
The present inventors have made extensive studies to solve the above problems. As a result, the inventors have found that the problems of the present invention can be solved by adopting the following configurations, and have completed the present invention based on this finding.
[0006]
The present invention has the following configuration.
(1) Glutathione-immobilized magnetic fine particles in which magnetic fine particles and glutathione are immobilized via a stimulus-responsive polymer, wherein the average particle size is 1-1000 nm. .
(2) The glutathione-immobilized magnetic fine particles according to (1), wherein the glutathione-immobilized magnetic fine particles have an average particle diameter of 1 to 200 nm.
(3) The glutathione-immobilized magnetic fine particles according to (1) or (2) above, wherein the stimuli-responsive polymer is a polymer exhibiting thermal responsiveness.
(4) Glutathione-fixed magnetic microparticles in which magnetic microparticles and glutathione are fixed via a polymer exhibiting thermal responsiveness, and using glutathione-fixed magnetic microparticles having an average particle diameter of 1 to 1000 nm A method for separating and purifying a target protein from a cell disruption suspension of microorganisms by the following procedure.
(1) A microorganism produces a fusion protein of a target protein and glutathione-S-transferase.
(2) Microorganisms are disrupted, a cell disruption suspension is prepared, and glutathione-immobilized magnetic microparticles are mixed therewith to produce a conjugate of the fusion protein and glutathione-immobilized magnetic microparticles.
(3) The temperature of the cell disruption suspension is set to the lower critical solution temperature or the upper critical solution temperature to aggregate the conjugate.
(4) Separate and collect the aggregated aggregates with a magnet.
(5) The protein is separated from the conjugate and purified.
(5) The method for separating and purifying a protein according to (4) above, wherein the average particle size of the glutathione-immobilized magnetic fine particles is 1 to 200 nm.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
In the glutathione-immobilized magnetic fine particles of the present invention, magnetic fine particles and glutathione are fixed via a stimulus-responsive polymer, and the average particle size is 1-1000 nm, preferably 1-200 nm.
[0008]
The magnetic fine particles used in the present invention are ferromagnetic materials that can be recovered by a magnet, and can be used without particular limitation as long as the average particle diameter is 0.9 or more and less than 1000 nm. In order to enhance the recognition of the target protein, the average particle size of the magnetic fine particles is preferably 0.9 or more and less than 200 nm. Examples of the magnetic fine particle material include magnetite particles, nickel oxide particles, ferrite particles, cobalt iron oxide, barium ferrite, carbon steel, tungsten steel, KS steel, rare earth cobalt magnet fine particles, and hematite. The method for preparing these magnetic fine particles is not particularly limited, and examples thereof include a method for preparing magnetite in which magnetite is made into double micelles using oleic acid and sodium dodecylbenzenesulfonate and dispersed in an aqueous solution. (Biocatalysis 1991, volume 5, pages 61-69).
Although the preferable magnetic force of the magnet used for this invention changes with magnetic force of the magnetic fine particle to be used, the neodymium magnet made from Magna can be mentioned as a specific example.
[0009]
In addition, in order to increase the recovery rate of the magnetic fine particles, it is necessary to fix the stimulus-responsive polymer on the surface of the magnetic fine particles. The stimulus-responsive polymer-immobilized magnetic fine particles can be obtained by a method of polymerizing a stimulus-responsive polymer in the presence of magnetic fine particles (Appl Microbiol Biotechnol (1994) 41: 99-105 or Journal of Fermentation and Bioengineering (described in J Ferment Bioeng (1997) 84; 337-341). The stimulus-responsive polymer may be any as long as it has the ability to agglomerate magnetic particles that are difficult to recover in a dispersed state to a size that can be recovered under specific conditions. Examples of the stimulus include heat, pH, salt concentration, light, and electricity. The stimulus-responsive polymer is preferably a polymer that exhibits thermal response that allows easy control of stimulation. Examples of the polymer exhibiting thermal responsiveness include poly-N-isopropylacrylamide exhibiting a lower critical solution temperature, and a copolymer of N acetylacrylamide and acrylamide exhibiting an upper critical solution temperature disclosed in JP-A-11-171928. is there.
[0010]
In the present invention, glutathione can be specifically bound to a target protein having glutathione-S-transferase (hereinafter abbreviated as “GST”) by immobilizing glutathione on magnetic fine particles. Such a GST-containing protein may be prepared by methods well known in the art. As a method for immobilizing glutathione on a stimulus-responsive polymer immobilized on a magnetic fine particle, as described in WO 01/09141, a polymerized ligand is used to polymerize the polymer. Copolymerization at the time of polymerization or at the time of polymerization, monomers having functional groups such as carboxylic acid, amino group, and epoxy group are copolymerized with other monomers, and the ligand is immobilized on the polymer according to methods well known in the art. Can be used.
[0011]
Using the glutathione-immobilized magnetic fine particles of the present invention, a target protein can be separated and purified from a microbial cell disruption suspension by the following procedure.
(1) A microorganism produces a fusion protein of a target protein and glutathione-S-transferase.
(2) Microorganisms are disrupted, a cell disruption suspension is prepared, and glutathione-immobilized magnetic microparticles are mixed therewith to produce a conjugate of the fusion protein and glutathione-immobilized magnetic microparticles.
(3) The temperature of the cell disruption suspension is set to the lower critical solution temperature or the upper critical solution temperature to aggregate the conjugate.
(4) Separate and collect the aggregated aggregates with a magnet.
(5) The protein is separated from the conjugate and purified.
For protein separation and purification, the average particle diameter of glutathione-immobilized magnetic fine particles is preferably 1 to 200 nm.
[0012]
【Example】
EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited at all by these.
In addition, the measuring method of the physical property in an Example and a comparative example, and the composition of the used material are as follows.
[0013]
SDS-PAGE: Electrophoresis was performed at an electric current of 25 mA using an electrophoresis apparatus equipped with a 10% by weight polyacrylamide gel made from acrylamide and bisacrylamide. The marker used was a colored molecular weight marker for protein electrophoresis manufactured by Amersham Biosciences. After electrophoresis, the protein in the gel was stained by immersing the polyacrylamide gel in 0.5% by weight Coomassie brilliant blue solution. In addition, when performing SDS-PAGE, a denaturation process is performed, but a target protein remove | deviates from a glutathione fixed magnetic microparticle by this process.
-Amount of purified GST: GST band was captured by a scanner, and the density was calculated by digitization.
-Particle diameter of glutathione-immobilized magnetic fine particles: The particle diameter was measured by ELS-8000SD type "light scattering photometer" manufactured by Otsuka Electronics Co., Ltd.
OD: Measurement was performed using a UV-1200 “spectrophotometer” manufactured by Shimadzu Corporation.
LB medium: Yeast extract (2.5 g), sodium chloride (2.5 g), and tryptone (bacto tryptone: 5 g) were suspended in distilled water (500 ml) and sterilized.
[0014]
Preparation of magnetic fine particles Ferrous sulfate heptahydrate (83.4 g), sodium nitrite (10.4 g) and distilled water (500 ml) were placed in a 1 L flask and stirred at 40 ° C. for 20 minutes.
Thereafter, concentrated ammonium (125 ml) was added, and the generated insoluble matter was collected by filtration and washed twice with distilled water to obtain magnetite. The obtained magnetite was put in a 1 L flask, distilled water (500 ml) was added, and the mixture was heated. The temperature of the solution was adjusted to 80 ° C., and then sodium oleate (7.5 g) was added. Stir for 20 minutes. Thereafter, the pH was adjusted to 5.5 with 1N hydrochloric acid, and the resulting insoluble matter was collected by filtration and washed twice with distilled water to obtain a magnetite having an oleic acid layer. This was placed in a 1 L flask, distilled water (500 ml) was added, and the mixture was heated to 70 ° C. Then, sodium dodecylbenzenesulfonate (7.5 g) was added and stirred overnight. Magnetic fine particles were obtained.
[0015]
Preparation of glutathione monomer Reduced glutathione hydrochloride (307 mg), 1,4 butanediol glycidyl ether (1010 mg), pH 8.0 0.5 M sodium phosphate buffer (500 μl) and 0.2 N sodium hydroxide solution (550 μl) ) Was adjusted with distilled water so that the total volume of the mixture was 5 ml, mixed, and reacted at room temperature for 1 hour. An equal amount of acetone was added to the reaction solution, the produced precipitate was separated, and an excess amount of 1,4 butanediol glycidyl ether was removed. The mixture of the precipitate (50 mg), dithiothreitol (15 mg), and pH 8.0 0.5 M sodium phosphate buffer (50 μl) was adjusted with distilled water to a total volume of 500 μl, mixed, and then mixed at room temperature. For 1 hour. Acetone (5 ml) was added to the reaction solution, the resulting precipitate was separated, suspended in 50 mM sodium phosphate buffer (500 μl), and glycidyl methacrylate (11.4 μl) was further added. After reacting by allowing to stand at room temperature for 1 hour, acetone (5 ml) was added and the precipitate was separated and purified by reverse phase column chromatography to obtain a polymerizable glutathione monomer.
[0016]
The glutathione-immobilized magnetic fine particles were prepared by the following method.
Distilled water was added to N-isopropylacrylamide (488 mg), the prepared glutathione monomer (5 μg), and the prepared magnetic fine particles (50 mg) to make a 25 ml solution. To this, ammonium persulfate (25 mg) and N, N, N ′, N′-tetraethylmethylenediamine (10 μl) were added, and the reaction was allowed to proceed with stirring for 6 hours under nitrogen substitution. The particle size of the obtained glutathione-immobilized magnetic fine particles was confirmed to be about 100 nm by a light scattering photometer. The particles had LCST at 29 ° C. and were completely dispersed in an aqueous solution lower than LCST, and recovery with a magnet was difficult. However, when the solution was LCST or more, the particles immediately aggregated and were easily recovered with a magnet. Was possible.
[0017]
Example 1
The fusion protein of GST (glutathione-S-transferase) and the target protein was purified by the following method.
E. coli Novablue strain (Novagen) transformed with plasmid pGEX6P-1 (Amersham Biosciences) was cultured overnight at 37 ° C. in LB medium (5 ml), then inoculated into LB medium (100 ml), The culture was performed at 37 ° C. until OD600 = 0.3. To this, isopropylthiogalactoside was added to a concentration of 50 μM to induce GST. Two hours later, the cells were collected by a centrifuge to disrupt the cells and obtain a cell disruption suspension containing GST. This cell disruption suspension (50 μl), 50 mM sodium phosphate buffer pH 7.0 (850 μl) and the prepared glutathione-immobilized magnetic microparticle solution (2.0 wt%, 100 μl) were mixed, and incubation was performed at room temperature for 30 minutes. After the separation, the temperature of the solution was set to 30 ° C. to aggregate the glutathione-immobilized magnetic fine particles. The aggregated glutathione-immobilized magnetic fine particles were collected with a magnet. Discard the supernatant of the solution, add 50 mM sodium phosphate buffer (1000 μl) to it, disperse the particles, then discard the supernatant of the solution again, add 50 mM sodium phosphate buffer (1000 μl) to it, After the washing step of dispersing the particles was repeated three times, the dispersed glutathione-immobilized magnetic fine particle solution (20 μl) was taken out and subjected to SDS-PAGE (FIG. 1).
The GST was purified in the same manner by changing the incubation time between 1 and 60 minutes. The obtained GST was subjected to SDS-PAGE. The obtained GST band was captured by a scanner, and the amount of purified GST was calculated.
[0018]
Example 2
The plasmid pGEXZZ in which the ZZ domain of protein A was inserted into the BamHI and XhoI sites in the multicloning site of pGEX6p-1 was similarly transformed into an E. coli Novablue strain to express the fusion protein. The fusion protein was purified according to the method of Example 1 except that it was used.
[0019]
From FIG. 1, it is clear that GST and the fusion protein of GST and ZZ domain were specifically purified by the prepared glutathione-immobilized magnetic microparticles.
[0020]
FIG. 1 shows the result of electrophoresis of the purified protein.
Lane 1 is a molecular weight marker (showing molecular weights 220,000, 97400, 66000, 46000, 30000, 21500, 14300 from the top), lane 2 is a cell disruption suspension of E. coli expressing GST, and lane 3 is adjusted. GST purified in Example 1 using glutathione-immobilized magnetic microparticles, lane 4 is a cell disruption suspension of Escherichia coli expressing a fusion protein of GST and ZZ in Example 2, and lane 5 is prepared. It is a fusion protein of GST and ZZ purified in Example 2 using glutathione-immobilized magnetic microparticles.
From these results, it was found that the expressed GST (molecular weight 25000) and GST-ZZ (molecular weight 35000) were purified to a single unit.
[0021]
Comparative Example 1
GST was purified in the same manner as in Example 1 except that 10 μl of GST Sepharose was used instead of glutathione-immobilized magnetic microparticles while changing the incubation time from 1 to 60 minutes. The obtained GST was subjected to SDS-PAGE.
The obtained GST band was captured by a scanner, and the amount of purified GST was calculated.
[0022]
The change with time of the amount of GST binding to each particle is shown in FIG.
From the result of FIG. 2, the glutathione-immobilized magnetic fine particles obtained in Example 1 can bind to the target protein in a shorter time than the glutathione sepharose (trade name) manufactured by Amersham Biosciences used in Comparative Example 1. I knew it was possible.
[0023]
【The invention's effect】
The glutathione magnetic fine particles of the present invention can rapidly recover a target protein from a protein mixture such as a cell disruption suspension.
[Brief description of the drawings]
FIG. 1 is an SDS-PAGE electrophoretic photograph of purified protein.
FIG. 2 is a graph showing a change over time in the amount of GST binding to each magnetic fine particle.

Claims (5)

A glutathione-immobilized magnetic fine particle in which magnetic fine particles and glutathione are fixed via a stimulus-responsive polymer, the average particle diameter of which is 1-1000 nm. The glutathione-immobilized magnetic fine particles according to claim 1, wherein the glutathione-immobilized magnetic fine particles have an average particle diameter of 1 to 200 nm. The glutathione-immobilized magnetic fine particles according to claim 1 or 2, wherein the stimulus-responsive polymer is a polymer exhibiting thermal responsiveness. Using glutathione-immobilized magnetic fine particles in which magnetic fine particles and glutathione are fixed via a polymer showing thermal responsiveness, the average particle diameter of which is 1 to 1000 nm, the following procedure is performed. To isolate and purify the protein of interest from the microbial cell suspension.
(1) A microorganism produces a fusion protein of a target protein and glutathione-S-transferase.
(2) Microorganisms are disrupted to prepare a cell disruption suspension, which is mixed with glutathione-immobilized magnetic microparticles to produce a conjugate of the fusion protein and glutathione-immobilized magnetic microparticles.
(3) The temperature of the cell disruption suspension is set to the lower critical solution temperature or the upper critical solution temperature to aggregate the conjugate.
(4) Separate and collect the aggregated aggregates with a magnet.
(5) The protein is separated from the conjugate and purified. The method for separating and purifying a protein according to claim 4, wherein the average particle diameter of the glutathione-immobilized magnetic fine particles is 1 to 200 nm.

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