KR101135054B1 - A nanoparticle for separating protein, method for preparing the same, and method for separating and purifying protein using the same - Google Patents

Abstract

The present invention relates to glutathione S transferase tagging (GST tagging) protein separation nanoparticles, a method for preparing the protein, and a protein separation purification method using the same. More specifically, glutathione S transfer using magnetic nanoparticles having a glutathione group bonded to a surface thereof It relates to a method for separating and purifying GST tagging proteins. 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.

Magnetic Nanoparticles, Peptides, Cysteine

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 a protein using the same, and more specifically, to isolate a glutathione S transferase tagging (GST tagging) protein using magnetic nanoparticles having a glutathione group bonded to a surface thereof. It relates to a method of purification. 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 isolating and purifying histidine 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 glutathione S transferase tagging (GST tagging) protein nanoparticles 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 a nanoparticles for glutathione S transferase tagging (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 a glutathione S transferase tagging protein (GST tagging) protein that binds the glutathione group in the protein mixture with the nanoparticles; And separating the nanoparticles bound to the glutathione S transferase tagging protein from the protein mixture, and separating and purifying the glutathione S transferase tagging protein using magnetic nanoparticles. .

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. The method may further include separating the glutathione S transferase tagging protein from the protein mixture after separating the nanoparticles bound to the glutathione S transferase tagging protein from the protein mixture. It may be.

According to another embodiment of the present invention, the protein mixture may be a mixture of glutathione S transferase tagging (GST tagging) ubiquitin and at least one protein selected from enolase, yeast protein, and human serum protein, and the present invention. Using magnetic nanoparticles according to the present invention, glutathione S transferase tagging (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 preparation method of the nanoparticles, first, 90 mg of Fe ₃ O ₄ having a diameter of 12 nm in a cyclohexane 120mL in the presence of oleic acid (2mL; purchased from Aldrich, 90%) for 50 hours by sonication Dispersion was performed to prepare Solution A. In addition, dispersant Igepal CO-520 (24 g, Aldrich) was dissolved in cyclohexane (300 mL) in a 1-L Erlenmeyer flask using magnetic stirring, and the solution was added to the solution together with 300 mL of cyclohexane. A was added and 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, a dispersant, is adsorbed on the surface of the nanoparticles, Igepal was removed from the nanoparticles because it is not easy to separate proteins.

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.

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 with heating at 80 ° C. for 7 hours, and the treated nanoparticles were washed with methanol (3 × 25 mL) and dried to form 3-aminopropyltriethoxysilane (APTS) and 3- ( Nanoparticles bound to trihydroxysilyl) propylmethylphosphonate (THPMP) were obtained.

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

100 mg of nanoparticles surface-treated with 3-aminopropyltriethoxysilane (APTS) and 3- (trihydroxysilyl) propylmethylphosphonate (THPMP) were dispersed in methanol (9.5 mL) by sonication for 1 hour. , N-succinimidyl 3- (2-pyridyldithio) propionate (0.2 μmol / mg, 6.2 mg, purchased from Calbiochem, hereinafter 'SPDP',) and 500 μL of dimethyl sulfoxide (DMSO) solution was added, then 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 having the thiol-reactive 2-pyridyldisulfide group covalently bonded to the surface prepared in the above step were dispersed in a Tris-HCl solution. Glutathione (0.29 μmol / mg) consisting of three amino acids containing one cysteine was added to 50 mM Tris and 10 mM ethylenediaminetetraacetic acid (EDTA) (Tris-HCl buffer pH 7.5). After dissolution 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 tris-hydrochloric acid (Tris-HCl) solution so as to be 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 glutathione S transferase tagging (GST tagging) ubiquitin

Ubiquitin protein with N-terminus Tagged with GST was dissolved in Tris-HCl solution to a concentration of 1 μg / μL. Glutathione S transferase tagging (GST tagging) ubiquitin was purchased from Calbiochem.

After mixing 3 μg of glutathione S transferase 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 magnetic particles. , 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 glutathione S transferase tagging (GST tagging) ubiquitin at the same time as trypsinizing the supernatant and nanoparticles of the above experiment. to be. 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.

4B, since no peptide peak of glutathione S transferase tagging (GST tagging) ubiquitin was found, it can be seen that all of the applied glutathione S transferase tagging (GST tagging) ubiquitin binds to nanoparticles. 4C shows that the detection intensity of the peptide peak of glutathione S transferase tagging (GST tagging) ubiquitin is the same as that of chromatogram A. From this result, the glutathione S transferase tagging (GST tagging) ubiquitin was added. It was confirmed that all of them were combined with the nanoparticles.

In the present experiment, glutathione S transferase tagging (GST tagging) ubiquitin bound to nanoparticles was separated from nanoparticles for analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 20 μL of a 50 mM glutathione 1 mM tris (2-carboxyethyl) phosphine (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. It adds an overwhelming amount of glutathione to the surface of the nanoparticles to stop the enzyme-substrate reaction between glutathione and GST, and reacts the glutathione with GST attached to the nanoparticles to the glutathione S transferase tagging (GST tagging). ) It is the process of separating ubiquitin.

Lane 1 of FIG. 5 was analyzed by adding 3 μg of standard glutathione S transferase tagging (GST tagging) ubiquitin, lane 2 was analyzed by adding supernatant obtained in this experiment, and lane 3 was analyzed using glutathione solution. The solution separated from the particles was added. No protein bands were found in lane 2, and the protein bands shown in lane 3 were almost identical to the bands shown in lane 1.

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

Experimental Example 2 Separation and Purification Experiments on Standard N-Terminal Glutathione S Transferase Tagging (GST Tagging) Ubiquitin and Enolase Mixed Solution

Yeast enolase (3 μg) was added to the standard N-terminal glutathione S transferase tagging (GST tagging) ubiquitin (3 μg) to make a mixture with a non-reactive protein with the nanoparticles prepared according to the present invention. ) To make a mixed solution. 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 nanoparticles combined with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used for analysis.

6 is analyzed by adding a mixture of standard glutathione S transferase tagging (GST tagging) ubiquitin and enolase, lane 2 is analyzed by adding the supernatant obtained in this experiment, and lane 3 is glutathione The solution separated from the nanoparticles using a solution was added. Only the band of enolase is shown in lane 2, the band of enolase is not visible in lane 3, and only the band of glutathione S transferase tagging (GST tagging) ubiquitin is shown. It can be seen that even in the presence of non-specific proteins, only glutathione S transferase tagging (GST tagging) ubiquitin can be isolated.

Experimental Example 3: Isolation and Purification of a Mixture of Standard N-Terminal Glutathione S Transferase Tagging (GST Tagging) Ubiquitin and Yeast Protein

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

After the separation through the same method as in Experimental Example 1 above was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

7 is analyzed by adding a mixture of yeast protein mixture and standard glutathione S transferase tagging (GST tagging) ubiquitin, lane 2 is analyzed by adding the supernatant obtained in the experiment, and lane 3 is glutathione solution Was added to the solution separated from the nanoparticles. Lane 2 shows bands of a complex yeast protein mixture and lane 3 shows only a band of glutathione S transferase tagging (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 glutathione S transferase tagging (GST tagging) ubiquitin even among complex protein samples in which many nonspecific proteins are present.

Experimental Example 4 Isolation and Purification of a Mixture of Standard N-Terminal Glutathione S Transferase Tagging (GST Tagging) Ubiquitin and Protein of Human Serum

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

Lane 1 of FIG. 8 was analyzed by adding a mixture of human serum protein mixture and standard glutathione S transferase tagging (GST tagging) ubiquitin, lane 2 was analyzed by adding supernatant obtained in the experiment, and lane 3 was glutathione solution. Was added to the solution separated from the nanoparticles. As in Experimental Example 3 above, the nanoparticles prepared in the above experiments were found to be able to selectively separate only Glutathione S transferase tagging (GST tagging) ubiquitin, despite being complex protein samples containing a large number of nonspecific proteins.

The above results indicate that the nanoparticles effectively bind glutathione S transferase tagging protein (GST tagging) protein, and do not chemically bond with other proteins, thereby effectively glutathione S transferase tagging protein (GST tagging) protein. Shows that it can be isolated and purified.

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 tetraethylorthosilicate (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 glutathione S transferase tagging (GST tagging) ubiquitin at the same time as trypsinizing the supernatant and nanoparticles of the above experiment. to be. 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 a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph of a protein separated into nanoparticles according to the present invention when only glutathione S transferase tagging protein is present without the presence of a nonspecific protein. to be.

6 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph of a protein obtained by separating a glutathione S transferase tagging ubiquitin and enolase mixture into nanoparticles according to the present invention.

7 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph of a protein obtained by separating a glutathione S transferase tagging ubiquitin and yeast protein mixture into nanoparticles according to the present invention.

8 is a sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) photograph of a protein obtained by separating a mixture of glutathione S transferase tagging ubiquitin and human serum proteins into nanoparticles according to the present invention.

<Description of Major Symbols in Drawing>

100: magnetic particles

110: silica layer

Claims (24)

Glutathione S transferase tagging (GST tagging) protein separation nanoparticles, characterized in that the glutathione group (glutathione) is attached through the binding of the 2-pyridyl disulfide group attached to the surface of the magnetic nanoparticles. The nanoparticles of claim 1, wherein the magnetic nanoparticles are coated with a silica layer. delete According to claim 2, The 2-pyridyl disulfide group Glutathione S transferase tagging (GST tagging) nanoparticles for protein separation, characterized in that bonded to the silica layer. According to claim 1, Glutathione S transferase tagging (GST tagging) nanoparticles for protein separation characterized in that it further comprises a functional group to prevent the aggregation of particles on the surface of the nanoparticles. The nanoparticle for separating glutathione S transferase tagging (GST tagging) protein according to claim 5, wherein the functional group for preventing aggregation includes a phosphonate group. The method of claim 1, wherein the magnetic nanoparticles for the separation of glutathione S transferase tagging (GST tagging) protein, characterized in that selected from the group consisting of iron oxide, cobalt and nickel doped with one or more of iron oxide, manganese or zinc. Nanoparticles. 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 A method of preparing nanoparticles for glutathione S transferase tagging (GST tagging) protein separation, comprising bonding a glutathione group to the 2-pyridyldisulfide 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; Method for producing a nanoparticles for the separation of glutathione S transferase tagging (GST tagging) protein, characterized in that it comprises the step of precipitating the silica-coated magnetic particles separated 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; Glutathione S transferase tagging (GST tagging), comprising adding N-succinimidyl 3- (2-pyridyldithio) propionate to the nanoparticle dispersion having 3-aminopropyl group bound thereto. Method for producing nanoparticles for protein separation. 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 a nanoparticle for separating glutathione S transferase tagging (GST tagging) protein, comprising the step of removing glutathione adsorbed on the surface of the particle. 10. The method of claim 9, further comprising the step of binding a functional group that prevents the aggregation of the particles to the silica layer, characterized in that the method for producing a nanoparticles for the separation of glutathione S transferase tagging (GST tagging) protein. The method of claim 13, wherein the functional group that prevents the aggregation of the particles comprises a phosphonate group. 15. A method of preparing nanoparticles for separating GST tagging protein, wherein the glutathione S transferase tagging protein is separated. 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 a glutathione S transferase tagging protein (GST tagging) protein that binds the glutathione group in the protein mixture with the nanoparticles; And separating the nanoparticles bound to the glutathione S transferase tagging protein from the protein mixture, and separating and purifying the glutathione S transferase tagging protein using magnetic nanoparticles. 17. The glutathione S transfer using magnetic nanoparticles of claim 16, wherein the step of separating the nanoparticles bound to the glutathione S transferase tagging protein from the protein mixture is performed using a magnetic material. Method for the isolation and purification of GST tagging proteins. 17. The method of claim 16, wherein after separating the nanoparticles bound to the glutathione S transferase tagging protein from the protein mixture, the glutathione S transferase tagging protein is separated from the nanoparticles. Method for separation and purification of glutathione S transferase tagging (GST tagging) protein using magnetic nanoparticles, characterized in that it further comprises a step. 17. The method of claim 16, wherein the glutathione group is bonded to a 2-pyridyldisulfide group. 17. The method of claim 16, wherein the glutathione S transferase tagging protein is purified using magnetic nanoparticles. 17. The method of claim 16, wherein the magnetic nanoparticles further comprise a functional group to prevent aggregation of the particles. The method for separating and purifying glutathione S transferase tagging (GST tagging) proteins using magnetic nanoparticles according to claim 20, wherein the functional group for preventing aggregation of the particles comprises a phosphonate group. The glutathione S transferase tagging (GST) using magnetic nanoparticles 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. tagging) method for the isolation and purification of proteins. The method of claim 16, wherein the surface of the magnetic nanoparticles is coated with a silica layer. 17. The method of claim 17, wherein the glutathione S transferase tagging protein is purified using magnetic nanoparticles. The glutathione S using magnetic nanoparticles of claim 16, wherein the protein mixture is a mixture of at least one protein selected from enolase, yeast protein, and human serum protein, and glutathione S transferase tagging ubiquitin. Method for the isolation and purification of transferase tagging protein.

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