KR20100070171A - A nanoparticle for separating protein, method for preparing the same, and method for separating and purifying protein using the same - Google Patents

Abstract

PURPOSE: A nanoparticle for isolating proteins is provided to isolate and purify GST-tagged protein using a magnetic nanoparticle on which glutathione is conjugated. CONSTITUTION: A nanoparticle for isolating GST-tagged protein has a magnetic nanoparticle surface on which glutathione group is conjugated. The magnetic nanoparticle is conjugated with a silica layer. The glutathione group is conjugated with 2-pyridyl disulfide which is bound to the silica layer. A method for manufacturing the nanoparticle for isolating the GST tagged protein comprises: a step of coating the surface of magnetic nanoparticle with the silica layer; a step of binding 2-pyridyl disulfide group to the silica layer; and a step of binding glutathione group to 2-pyridyldisulfide group.

Description

Nanoparticles for protein separation, preparation method thereof and protein separation purification method using the same {A NANOPARTICLE FOR SEPARATING PROTEIN, METHOD FOR PREPARING THE SAME, AND METHOD FOR SEPARATING AND PURIFYING PROTEIN USING THE SAME}

The present invention relates to a nanoparticle for protein separation, a manufacturing method, and a method for separating and purifying protein using the same, and more particularly, to a method for separating and purifying GST tagging protein using magnetic nanoparticles having a glutathione group bonded to a surface thereof. According to the present invention, a specific protein can be isolated very simply and quickly, which can be usefully used for disease treatment research such as cancer.

Current biological research focuses on how to organize, store, and use the information that results from research. Biological information in the knowledge-information society is being produced in a larger quantity than in the past, and the information production speed is also very fast. In addition, this phenomenon is becoming more serious at the present time when the human genome sequence is completely decoded after the human genome project is in force.

This method of mass production of biological information is driven by DNA arrays, proteomics, combinatorial chemical libraries (CCL), high throughput screening (HTS) using robotics, and bioinformatics technology. There is already a great deal of information on biology and medicine. However, out of 100,000 human genes, only about 10,000 genes are known for their function, and the rest of the unknown parts of the biological system, including animals and plants, remain to be revealed in future studies. . As a field of functional genetics that studies the function of genes whose functions are unknown, proteomics is utilized as an effective technique for studying the function of genes or proteins.

Proteomics can quickly provide a large amount of information on changes in expression and modification or biochemical activity of biological, disease-related genes or proteins. Therefore, in order to produce and accumulate useful information based on this information acquisition ability, and to effectively utilize this information for biological and medical research and industrial use, this mass information production method is introduced and quickly settled in biological and medical research. Develop your skills.

This proteomics technology is being studied to overcome the limitations of the application range of the current analysis platform. In particular, techniques that make proteomics information more reliable by reducing the complexities of proteins of low levels present in cells and increasing the concentration of phosphopeptides and target peptides such as cysteine peptides in accordance with physical and chemical properties Required.

Recently, nanoparticles are widely used in the biotechnology field, for example, in vitro and in vitro experiments such as MRI, drug delivery, and cell epidemiological studies. In particular, magnetic nanoparticles are getting more and more attention in the separation and purification of specific cells and biological molecules due to the ease of separation. In addition, the surface modification of nanoparticles allows for the separation of specific species from the mixed solution, and offers significant advantages over the use of conventional micron-sized bead technology due to the much wider surface area, faster binding speed, and higher compatibility and selectivity. Have

Among them, superparamagnetic nanoparticles are mainly used in biomedical fields such as drug delivery or imaging of cells in the body such as MRI. Because supermagnetic nanoparticles are magnetic only when a magnetic field is applied, they can also be used to isolate and purify specific proteomes. Many efforts have been made to introduce specific ligands into nanoparticles for such applications. As an example, Hyeon et al. Successfully demonstrated the availability of Ni / NiO core / shell nanoparticles for the isolation and purification of His-tagging proteins.

The technology of separating specific proteins well from different protein mixtures plays a crucial role in increasing the amount of information within the limited scope of current proteomics applications. Currently, a general separation technique is a selective separation method of peptides using beads larger than a micron, which does not have the level of selectivity, efficiency, and rapidity required by high-sensitivity proteomics, and impurity as the process time increases. Problems such as a decrease in selectivity by the peptide and peptide loss occurring in the concentration and washing steps.

Meanwhile, Korean Patent Laid-Open No. 10-2004-0074807 discloses an invention related to magnetic nanoparticles. However, since the above technique suggests magnetic nanoparticles for separating any protein, it is not possible to selectively separate only specific peptides or proteins present in a low ratio among the most complex protein combinations, and further separation process is required for proteomics experiments. The disadvantage is that.

In addition, Korean Patent Publication No. 10-0762969 discloses a method for separating and purifying nucleic acids using functional silica coated magnetic nanoparticles. The surface of the nanoparticles includes amine groups and alkyl groups, which bind to any phosphate group and electrostatic attraction. Thereby, nucleic acids can be separated, but only specific nucleic acids or proteins cannot be separated, and since the bonds formed are electrostatic bonds by electrical action, there is a problem in that the bonds may be very weak due to changes in conditions such as solvents.

In addition, Korean Patent Publication No. 10-2007-0018501 proposes a method for separating and purifying DNA using silica-coated magnetic nanoparticles having an amino substituent, but it is also impossible to selectively separate specific peptides. There is a disadvantage that the bound peptide is lost again upon hydrophobic interaction of.

Therefore, none of the above prior arts suggests an alternative to the separation of specific peptides required by high sensitivity proteomics.

It is a first object of the present invention to provide magnetic nanoparticles which are capable of very selective and simple separation of a specific protein from a protein mixture.

The second object of the present invention is to provide a method for preparing the nanoparticles for protein separation.

The third object of the present invention is to provide a method for separating and purifying a specific protein using the nanoparticles.

In order to solve the first problem, the present invention provides a nanoparticle for separating GST tagging protein, characterized in that the glutathione group (glutathione) is bonded to the surface of the magnetic nanoparticles. At this time, the magnetic nanoparticles are preferably coated with a silica layer.

According to one embodiment of the invention, the glutathione group may be bonded to the 2-pyridyl disulfide group, wherein the 2-pyridyl disulfide group is generally bonded to the silica layer.

According to another embodiment of the present invention, the surface of the nanoparticles may further include a functional group for preventing the aggregation of the particles, the functional group for preventing the aggregation may include, for example, a phosphonate group.

According to another embodiment of the present invention, the magnetic nanoparticles may be selected from the group consisting of iron oxide, cobalt and nickel doped with one or more of iron oxide, manganese or zinc, the diameter of the nanoparticles is 25 to 35nm Is preferably.

In order to solve the second problem, coating the surface of the magnetic nanoparticles with a silica layer; Bonding a 2-pyridyldisulfide group to the silica layer; And it provides a method for producing nanoparticles for GST tagging protein separation comprising the step of bonding a glutathione group to the 2-pyridyl disulfide group.

According to an embodiment of the present invention, the coating of the silica layer may include dispersing the magnetic particles together with a dispersant in a solvent; Coating a surface of the magnetic particles with a silica layer by adding ammonia (NH ₃ ) and tetraethylorthosilicate (TEOS) to the solution in which the magnetic particles are dispersed; Precipitating the silica coated magnetic particles may include the step of separating from the solvent.

According to another embodiment of the present invention, the step of bonding the 2-pyridyl disulfide group to the silica layer comprises the steps of dispersing the nanoparticles in a solvent; Coupling 3-aminopropyl group to the silica layer of the nanoparticle by adding 3-aminopropyltriethoxysilane to the solvent in which the nanoparticles are dispersed; And adding N-succinimidyl 3- (2-pyridyldithio) propionate to the nanoparticle dispersion having the 3-aminopropyl group bound thereto.

According to another embodiment of the present invention, in the step of bonding the glutathione group to the 2-pyridyldisulfide group, the glutathione solution is mixed with the nanoparticles to which the 2-pyridyldisulfide group is bonded and then washed using a nanoparticle. And removing glutathione that is absorbed on the surface of the nanoparticles without forming disulfide bonds.

In addition, according to another embodiment of the present invention, the method for producing nanoparticles according to the present invention may further comprise the step of bonding a functional group to prevent the aggregation of particles to the silica layer, the function of preventing the aggregation of the particles Groups may include, for example, phosphonate groups. In this case, the step of bonding the phosphonate group may include dispersing the nanoparticles in a solvent; And adding 3- (trihydroxysilyl) propylmethylphosphonate to the solvent in which the nanoparticles are dispersed.

In order to solve the third problem, mixing a magnetic nanoparticle having a glutathione group and a protein mixture; Binding the GST tagging protein to the nanoparticles to bind the glutathione group in the protein mixture; And it provides a separation and purification method of GST tagging protein using magnetic nanoparticles comprising the step of separating the nanoparticles bound to the GST tagging protein from the protein mixture.

In the protein separation and purification method of the present invention, the step of separating the nanoparticles bound to a specific protein from the protein mixture may be performed using a magnetic material capable of moving the magnetic nanoparticles. In addition, after separating the nanoparticles bound to the GST tagging protein from the protein mixture, may further comprise the step of separating the GST tagging protein from the nanoparticles.

In addition, according to another embodiment of the present invention, the protein mixture may be a mixture of one or more proteins selected from enolase, yeast protein, human serum protein and GST tagging ubiquitin, using magnetic nanoparticles according to the present invention Thus, GST tagging proteins can be highly selectively separated and purified from these protein mixtures.

Magnetic nanoparticles according to the present invention is coupled to a functional group that selectively reacts with a specific protein on its surface, by using this can be isolated and purified very selectively and quickly through a simple process, the protein metabolism and It can be usefully used in research fields such as various disease treatments.

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples provide only reference information for illustrating the present invention, and are not to be construed as limiting the scope of the present invention.

Example 1 Preparation of Nanoparticles for Protein Separation

Example 1-1 Coating of Silica Layer

1 is a view showing a method for preparing a nanoparticle for protein separation according to an embodiment of the present invention. Referring to FIG. 1, first, a silica layer 110 is coated on Fe ₃ O ₄ magnetic particles 100 (step a).

In more detail, the method for preparing the nanoparticles was first dispersed in 90 mg of Fe ₃ O ₄ having a diameter of 12 nm at 50 ° C. for 1 hour by sonication in 120 mL of cyclohexane in the presence of oleic acid (2 mL; Aldrich, 90%). Solution A was prepared. In addition, dispersant Igepal CO-520 (24 g, Aldrich) was dissolved in cyclohexane (300 mL) using magnetic stirring in a 1-L Erlenmeyer flask, and solution A was added to the solution along with 300 mL of cyclohexane. Then, the mixture was stirred for 10 minutes. Then, 28-40% ammonia (aq), which serves as a catalyst in coating the silica layer, was mixed dropwise into the stirred mixture with a syringe, followed by stirring for 5 minutes. Subsequently, tetraethylorthosilicate (TEOS), which coats the surface of the magnetic particles using a syringe, is mixed in drops, and the surface of the magnetic particles is coated with a silica layer in the form of Si-O-Si. It was.

Then, the mixture was further stirred for 20 hours at room temperature, and 500 mL of methanol was added to obtain the nanoparticles in the form of a precipitate. After the precipitate of the nanoparticles was obtained, the supernatant was removed, and a precipitate was obtained by using a centrifuge at 3500 rpm. The precipitate was then purified by a series of processes called sonication in hexane (3 x 90 mL), precipitation with chloroform (30 mL) and centrifugation (3500 rpm), after which the purification process was repeated once more.

The precipitate obtained through the above process was Fe ₃ O ₄ , the core particles were nanoparticles coated with a silica layer on the core particles. However, since Igepal, which is a dispersant, is adsorbed on the surface of the nanoparticles, protein separation is not easy, and thus Igepal is removed from the nanoparticles.

2A and 2B are TEM images of nanoparticles before and after surface coating by TEOS. Referring to Figure 2a, it can be seen that the Fe ₃ O ₄ particles present in the nanoparticles present in the form of a sphere, referring to Figure 2b, the surface of the Fe ₃ O ₄ particles by the method according to the invention It can be seen that is coated with silica of a certain thickness.

Example 1-2: Nanoparticle Modification with 3- (trihydroxysilyl) propylmethylphosphonate (THPMP) and 3-aminopropyltriethoxysilane (APTS) (step b of FIG. 1)

In the above example, 150 mg of the nanoparticles obtained in powder form were mixed with methanol (50 mL) in a 250 mL round bottom flask and sonicated for 1 hour. 3-aminopropyltriethoxysilane (2.75 μmol / mg, 75 μL, Adrich, 'APTS') and 3- (trihydroxysilyl) propylmethylphosphonate (2.2 μmol / mg, 149.5 μL, Adrich, below ' THPMP ') was added to the flask and then mechanically stirred (350 rpm) using an impeller. Subsequently, the nanoparticles were treated by refluxing while heating at 80 ° C. for 7 hours, and the treated nanoparticles were washed with methanol (3 × 25 mL) and dried to obtain APTS and THPMP combined nanoparticles.

Example 1-3 Surface Treatment of Nanoparticles with N-Succinimidyl 3- (2-pyridyldithio) propionate (Step c of FIG. 1)

100 mg of nanoparticles treated with APTS and THPMP were dispersed in methanol (9.5 mL) by sonication for 1 hour, and N-succinimidyl 3- (2-pyridyldithio) propionate (0.2 μmol / mg, 6.2 mg, Calbiochem, hereinafter 'SPDP',) and 500 μL of DMSO solution were added, washed with methanol (3 × 25 mL) and dried. This prepared nanoparticles for protein separation in which the thiol-reactive 2-pyridyldisulfide was covalently bonded to the surface.

Example 1-4: Surface Treatment of Nanoparticles with Glutathione (Step d of FIG. 1)

The nanoparticles in which the thiol-reactive 2-pyridyldisulfide group covalently bonded to the surface were dispersed in the Tris-HCl solution. Glutathione (0.29 μmol / mg) consisting of three amino acids containing one cysteine was dissolved in 50 mM Tris and 10 mM EDTA (Tris-HCl buffer pH 7.5) solution and the pH was adjusted to 7.5. The glutathione solution was mixed with the nanoparticles prepared in the above step at room temperature for 2 hours. After mixing, the mixture was washed with water and 80% acetonitrile to remove glutathione adsorbed on the surface of the nanoparticles without forming disulfide bonds with the nanoparticles. Thereafter, the final concentration of the nanoparticles was dispersed in a Tris-HCl solution so that 0.5 mg / 20 μL. According to this procedure, nanoparticles having a functional group in which a 2-pyridyldisulfide group and S of cysteine in glutathione are disulfide bonds were obtained.

Experimental Example 1: Separation and purification experiment using standard N-terminal GST tagging ubiquitin

Ubiquitin protein with N-terminus tagged with GST was dissolved in Tris-HCl solution to a concentration of 1 μg / μL. GST tagging ubiquitin was purchased from calbiochem.

After mixing 3 μg of GST tagging ubiquitin and 0.5 m (20 μL) of the nanoparticles obtained in the above example at room temperature for 10 minutes, the nanoparticles were separated using a metal capable of reacting with the magnetic particles, and the supernatant was collected. The separated nanoparticles were washed with Tris-HCl buffer (20 μL) to separate and collect proteins remaining in the form of adsorption without binding to the nanoparticles, and then mixed with the supernatant. The supernatant and nanoparticles separated for LC / MS analysis were dissolved in 100 mM ammonium carbonate buffer and peptided with trypsin.

4 shows the results of LC / MS analysis according to the present experimental example. First, chromatogram A is a base peak chromatogram obtained by LC / MS analysis by trypsinizing 3 μg of standard N-terminal GST tagging ubiquitin at the same time as trypsinizing the supernatant and nanoparticles of the above experiment. 4B is a base peak chromatogram analyzed after trypsinization of the protein remaining in the supernatant. 4C is a base peak chromatogram analyzed after trypsinization of the protein separated by nanoparticles.

In B of FIG. 4, since no peptide peak of GST tagging ubiquitin was found, it can be seen that all of the GST tagging ubiquitin added was bound to the nanoparticles. 4C shows that the detection intensity of the peptide peak of GST tagging ubiquitin is the same level as that of chromatogram A. From these results, it was confirmed that all of the GST tagging ubiquitin added was bound to the nanoparticles.

In this experiment, GST tagging ubiquitin bound to nanoparticles was separated from nanoparticles for analysis by SDS-PAGE gel electrophoresis. 20 μL of a 50 mM glutathione 1 mM TCEP (pH 7.4) solution was added to the nanoparticles obtained through the above experimental procedure, followed by mixing for 4 hours to separate the solution. This process separates GST tagging ubiquitin from nanoparticles by adding an overwhelming amount of glutathione to the surface of nanoparticles to stop the enzyme-substrate reaction between glutathione and GST, and by reacting with glutathione with GST attached to nanoparticles. to be.

Lane 1 of FIG. 5 was analyzed by adding 3 μg of standard GST tagging ubiquitin, lane 2 was analyzed by adding supernatant obtained in this experiment, and lane 3 was added to the solution separated from nanoparticles using glutathione solution. will be. There is no protein band in lane 2, and the protein band shown in lane 3 is almost identical to the band shown in lane 1.

From the above results, it can be seen that the nanoparticles prepared according to the present invention bind to GST tagging ubiquitin very efficiently.

Experimental Example 2: Separation and Purification Experiments on a Standard N-Terminal GST Tagging Ubiquitin and Enolase Mixture

Yeast enolase (3 μg) was mixed with standard N-terminal GST tagging ubiquitin (3 μg) to make a mixture with a non-reactive protein with nanoparticles prepared according to the present invention to make a mixture. . Then, after mixing with the nanoparticles prepared according to the present invention in the same manner as in Experimental Example 1, after separating and collecting the protein that is not bound to the nanoparticle-bound protein, analysis using SDS-PAGE gel electrophoresis It was.

Lane 1 of FIG. 6 was analyzed by adding a mixture of standard GST tagging ubiquitin and enolase, lane 2 was analyzed by adding supernatant obtained in this experiment, and lane 3 was separated from nanoparticles using glutathione solution. The solution was removed. Since only the band of enolase is shown in lane 2, the band of enolase is not shown in lane 3, and only the band of GST tagging ubiquitin is shown, the nanoparticles prepared according to the present invention are selectively selected even if a nonspecific protein is present. It can be seen that only GST tagging ubiquitin can be isolated.

Experimental Example 3 Separation and Purification Experiments on a Mixture of Standard N-Terminal GST Tagging Ubiquitin and Yeast Protein

To apply nanoparticles prepared to more complex protein mixtures, yeast protein mixtures were prepared by mixing with standard GST tagging ubiquitin.

After the separation by the same method as in Experimental Example 1 above was analyzed by SDS-PAGE gel electrophoresis.

7 is analyzed by adding a mixture of yeast protein mixture and standard GST tagging ubiquitin, lane 2 is analyzed by adding the supernatant obtained in the experiment, and lane 3 is separated from the nanoparticles using glutathione solution The solution was removed. Lane 2 shows bands of complex yeast protein mixtures and lane 3 shows only bands of GST tagging ubiquitin with no protein bands due to other nonspecific reactions. Accordingly, it was confirmed that the nanoparticles prepared according to the present invention can selectively separate and purify only GST tagging ubiquitin among complex protein samples in which a large number of nonspecific proteins are present.

Experimental Example 4: Isolation and Purification of a Mixture of Standard N-Terminal GST Tagging Ubiquitin and Protein of Human Serum

Human serum protein mixtures were prepared by preparing a mixture with standard GST tagging ubiquitin as in Experiment 3 above. Thereafter, the resultant was separated through the same experimental procedure as in Experimental Example 1, and analyzed by SDS-PAGE gel electrophoresis.

Lane 1 of FIG. 8 was analyzed by adding a mixture of human serum protein mixture and standard GST tagging ubiquitin, lane 2 was analyzed by adding supernatant obtained in the experiment, and lane 3 was separated from nanoparticles using glutathione solution. Solution was added. As in Experiment 3, it was confirmed that the nanoparticles prepared by the above experiments were able to selectively separate only GST tagging ubiquitin, despite being a complex protein sample having many nonspecific proteins.

The above results show that the nanoparticles according to the present invention can effectively bind to GST tagging protein, and can effectively separate and purify GST tagging protein by not chemically binding to other proteins.

1 is a view showing a method for producing a nanoparticle for protein separation according to an embodiment of the present invention.

2A and 2B are TEM images of nanoparticles before and after surface coating by TEOS. Referring to Figure 2a, it can be seen that the Fe ₃ O ₄ particles present in the nanoparticles present in the form of a sphere, referring to Figure 2b, the surface of the Fe ₃ O ₄ particles by the method according to the invention It can be seen that the silver is coated with a certain thickness of silica.

3A is VMS data of Fe ₃ O ₄ , and FIG. 3B is VMS data of Fe ₃ O ₄ @SiO ₂ nanoparticles.

4 shows the results of LC / MS analysis according to the present experimental example. First, chromatogram A is a base peak chromatogram obtained by LC / MS analysis by trypsinizing 3 μg of standard N-terminal GST tagging ubiquitin at the same time as trypsinizing the supernatant and nanoparticles of the above experiment. 4B is a base peak chromatogram analyzed after trypsinization of the protein remaining in the supernatant. 4C is a base peak chromatogram analyzed after trypsinization of the protein separated by nanoparticles.

5 is an SDS-PAGE gel electrophoresis picture of a protein separated into nanoparticles according to the present invention when only GST tagging protein is present without nonspecific protein.

Figure 6 is a SDS-PAGE gel electrophoresis picture of the protein obtained by separating the GST tagging ubiquitin and enolase mixture into nanoparticles according to the present invention.

7 is a SDS-PAGE gel electrophoresis picture of a protein obtained by separating GST tagging ubiquitin and yeast protein mixture into nanoparticles according to the present invention.

8 is an SDS-PAGE gel electrophoresis picture of a protein obtained by separating a mixture of GST tagging ubiquitin and human serum protein into nanoparticles according to the present invention.

<Description of Major Symbols in Drawing>

100: magnetic particles

110: silica layer

Claims (24)

GST tagging protein separation nanoparticles characterized in that the glutathione group (glutathione) is bonded to the surface of the magnetic nanoparticles. The nanoparticle for separating GST tagging protein according to claim 1, wherein the magnetic nanoparticle is coated with a silica layer. The nanoparticle for separating GST tagging proteins according to claim 2, wherein the glutathione group is bonded to a 2-pyridyldisulfide group. The nanoparticle for separating GST tagging proteins according to claim 3, wherein the 2-pyridyldisulfide group is bonded to the silica layer. The nanoparticle for separating GST tagging protein according to claim 1, further comprising a functional group for preventing agglomeration of particles on the surface of the nanoparticle. The nanoparticle for separating GST tagging proteins according to claim 5, wherein the functional group for preventing aggregation includes a phosphonate group. The nanoparticle of claim 1, wherein the magnetic nanoparticle is selected from the group consisting of iron oxide, cobalt, and nickel doped with at least one of iron oxide, manganese, and zinc. According to claim 1, wherein the nanoparticles are nanoparticles for protein separation, characterized in that the diameter of 25 to 35nm. Coating the surface of the magnetic nanoparticles with a silica layer; Bonding a 2-pyridyldisulfide group to the silica layer; And Method for producing a nanoparticles for GST tagging protein separation comprising the step of bonding a glutathione group to the 2-pyridyl disulfide group. The method of claim 9, wherein the coating of the silica layer comprises: dispersing magnetic particles together with a dispersant in a solvent; Coating a surface of the magnetic particles with a silica layer by adding ammonia (NH ₃ ) and tetraethylorthosilicate (TEOS) to the solution in which the magnetic particles are dispersed; Precipitating the silica-coated magnetic particles, and then separating from the solvent. The method of claim 9, wherein the bonding of the 2-pyridyldisulfide group to the silica layer comprises dispersing nanoparticles in a solvent; Coupling 3-aminopropyl group to the silica layer of the nanoparticle by adding 3-aminopropyltriethoxysilane to the solvent in which the nanoparticles are dispersed; Preparation of nanoparticles for separating GST tagging protein, comprising adding N-succinimidyl 3- (2-pyridyldithio) propionate to the nanoparticle dispersion having 3-aminopropyl group bound thereto Way. The method of claim 9, wherein the bonding of the glutathione group to the 2-pyridyldisulfide group is performed by mixing a glutathione solution into the nanoparticles to which the 2-pyridyldisulfide group is bonded and then using a washing solution to form a nanoparticle without disulfide bonds. A method for producing nanoparticles for separating GST tagging proteins, comprising the step of removing glutathione that has been absorbed on the surface of the particles. 10. The method of claim 9, further comprising the step of binding a functional group to prevent the aggregation of particles to the silica layer. The method for preparing nanoparticles for separating GST tagging proteins according to claim 13, wherein the functional group for preventing aggregation of the particles comprises a phosphonate group. The method of claim 14, wherein the bonding of the phosphonate group comprises dispersing the nanoparticles in a solvent; And adding 3- (trihydroxysilyl) propylmethylphosphonate to the solvent in which the nanoparticles are dispersed. Mixing a magnetic nanoparticle having a glutathione group and a protein mixture; Binding the GST tagging protein to the nanoparticles to bind the glutathione group in the protein mixture; And separating the nanoparticles bound to the GST tagging protein from the protein mixture. 17. The method of claim 16, wherein separating the nanoparticles bound to the GST tagging protein from the protein mixture is performed using a magnetic material. The method of claim 16, further comprising, after separating the nanoparticles bound to the GST tagging protein from the protein mixture, separating the GST tagging protein from the nanoparticles. Isolation and purification method of GST tagging protein. 17. The method of claim 16, wherein the glutathione group is bonded to a 2-pyridyldisulfide group. The method of claim 16, wherein the magnetic nanoparticles further comprise a functional group for preventing aggregation of the particles. The method for separating and purifying GST tagging protein using magnetic nanoparticles according to claim 20, wherein the functional group for preventing aggregation of the particles comprises a phosphonate group. 17. The method of claim 16, wherein the magnetic nanoparticles are selected from the group consisting of iron oxide, cobalt, and nickel doped with at least one of iron oxide, manganese, and zinc. . 17. The method of claim 16, wherein the surface of the magnetic nanoparticles is coated with a silica layer. The method of claim 16, wherein the protein mixture is a mixture of one or more proteins selected from enolase, yeast protein, and human serum proteins, and GST tagging ubiquitin.

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