KR20010085045A - Method of concentration and purification of protein applicable to wide range of protein - Google Patents

Abstract

PURPOSE: A concentration and purification method of proteins is provided, thereby being applicable to broad range of proteins. CONSTITUTION: The concentration and purification method of proteins comprises the steps of: binding proteins with resins, such as kaolinite, silica and silicate, and a buffer solution having pH 4 to 8 as a binding solution; removing unbound proteins or substances using the unbound protein removing solution; and isolating and eluting proteins from resins using an eluate, wherein the unbound protein removing solution having pH 4 to 8 contains 5 to 30% of alcohol, especially lower alcohol such as methanol, ethanol and propanol, the eluate having pH 7 to 10 contains 0.5 to 2% of SDS, and each kaolinite, silica and silicate has the diameter of 0.1 to 10 micrometer.

Description

Method of concentration and purification of protein applicable to wide range of protein}

The present invention relates to methods for concentrating and purifying proteins that can be applied to a wide range of proteins.

Protein concentration and purification methods are essential technologies in the fields of biotechnology, medicine, pharmacy and industry. Conventionally, methods for concentrating and purifying proteins have been developed, but different methods are used according to individual properties such as physical and chemical properties of proteins.

As a method widely used for concentrating proteins, a precipitation method using an organic solvent, a dialysis method, a low temperature freeze drying method, a gel chromatography method and a method using a micropermeable membrane are known.

Precipitation using organic solvents has the advantage that the process is simple and inexpensive. In this method, however, the protein may be denatured by the organic solvent, the precipitated protein may be insolubilized, and since the precipitation degree in the organic solvent is different for each protein, it is impossible to concentrate and recover almost all proteins simultaneously. .

Dialysis is a method of concentrating proteins using a concentration gradient of protein and salt. This method is simple, but it is time consuming and limited in the amount of protein that can be concentrated.

Low temperature freeze drying is a method of maintaining protein physiological activity at a low temperature and mechanically drying the protein. As the salt concentration increases with time, additionally, low salt treatment is required. Therefore, this method is not suitable as a method for concentrating a large amount of protein samples in a short time.

Gel chromatography using a column and a method using a micropermeable membrane are widely used to concentrate a small amount of protein sample, but have a disadvantage of being expensive.

On the other hand, widely used methods for purification of proteins include ion exchange method, hydrophobic bond separation method, size fractionation method and immuno-affinity separation method, and these methods are based on unique properties such as ionic and molecular weight of the protein. It is based on the principle of separation and purification.

The ion exchange method is a method of separating proteins according to electrical properties by attaching a negative or positive charge material to the filling beads.

The hydrophobic bond separation method is a method using the degree that the degree of hydrophobicity of the protein varies depending on the type of protein.

Size fractionation is a method of fractionating proteins by varying the diameter of the porous beads and using a difference in the rate of passage through the pores of the beads according to the size of the protein.

Immune affinity separation method is a method using the binding between the antigen and the antibody or ligand and the receptor, it is useful when there is an antibody or receptor that selectively binds to the protein to be isolated.

However, these conventional methods cannot be universal separation methods for all proteins because they are used according to the characteristics of the proteins. In particular, proteins that do not know exactly their characteristics must undergo repeated trial and error.

Therefore, research is being actively conducted on new materials that can be used as adsorption carriers for the concentration and purification of proteins applicable to a wide range of proteins. Representative examples thereof include silica and silicates, which are silicic anhydrides obtained in abundance from natural minerals. In the case of silica, it is used as a filler of an ion exchange column for separation and purification of various materials, and clay minerals such as kaolin and diatomaceous earth, which are amorphous silica, are widely used as filtration aids and fillers.

As such, there is a demand for the development of a carrier material for concentrating and purifying proteins applicable to a wide range of proteins, and the invention for the optimum concentration and purification conditions.

An object of the present invention is to provide a protein concentration and purification method that can be applied to a wide range of proteins regardless of physical and chemical properties.

In order to achieve the above object, the present invention is to select a carrier material capable of binding to a wide range of proteins, to provide the optimum binding conditions between the protein and the carrier, the optimum washing conditions of foreign matters and the optimal dissolution conditions of the protein.

1 is a diagram showing the results of comparing the protein binding capacity of kaolin, silica, silicate and hydrophobic polymers.

Figure 2 is a diagram showing the result of comparing the protein binding power of the heterogeneous mixture of kaolin, silica and silicate.

3 is a diagram showing the result of concentrating and purifying purified proteins of known molecular weight using the protein concentration and purification method of the present invention.

Figure 4 is a diagram showing the results of the concentration and purification of proteins from the cell extract of E. coli DH5α diluted using the protein concentration and purification method of the present invention.

5 shows a result of concentrating and purifying proteins present in human fibroblast culture medium using the protein concentration and purification method of the present invention, and b is concentrated using the protein concentration and purification method of the present invention. It is a figure which shows the result which detected the interferon beta in human fibroblast culture medium by the Western assay.

Figure 6 is a diagram showing the results of protein extraction from the liver of the mouse and electrophoresis analysis with polyacrylamide gel, using a protein concentration and purification method of the present invention to isolate and purify the protein of a specific size from the gel .

FIG. 7 shows protein extracts from recombinant E. coli strains producing GST fusion protein and subjected to electrophoresis analysis with polyacrylamide gel, followed by extraction and separation of GST fusion proteins of specific size from the gel using the protein concentration and purification method of the present invention. Shows the result of purification.

The present invention relates to methods for concentrating and purifying proteins that can be applied to a wide range of proteins.

Protein binding carriers used in the present invention are kaolin, silica, and silicates, which have excellent adsorption properties and ion exchange ability, and thus have adsorption to all kinds of proteins. Kaolin, silica and silicates used as protein binding carriers in the present invention preferably have a particle size in the range of 0.1-10 μm.

Specifically, the protein binding carrier of the present invention may use one material selected from kaolin, silica, and diatomaceous earth, or a heterogeneous mixture of two materials in a predetermined ratio. In the present invention, when two kinds of substances are mixed and used as the protein binding carrier, the mixing ratio of each component is preferably in the range of 1: 1-1: 5.

The protein binding carrier of the present invention is preferably used in the form of a colloidal liquid slurry. In preparing a carrier slurry of the present invention, a buffer in the range of pH 4-8 is used to prepare a final concentration of 10-500 mg / ml buffer by weight.

In the present invention, the process of concentrating and purifying the protein using the protein binding carrier consists of the following three steps.

1) binding the protein to the carrier

In the present invention, the binding of the protein to the carrier refers to the reversible adsorption or binding of the protein in a liquid aqueous solution in which the protein is dissolved by using all possible non-covalent methods such as ionic bond, hydrophobic bond, van der Waals bond and hydrogen bond. Means that.

These bonds can be optimally made at weakly acidic pH and low salt concentrations. In the case of ionic bonding, most of the proteins in the above conditions are positively charged, and thus can be ionically bonded to silicic acid having a negative charge. Therefore, in the present invention, the solution used for binding the protein and the carrier is preferably in the range of pH 4-8.

In the present invention, when the protein and the carrier are combined, the protein to be concentrated and purified is dissolved in purified water, buffer, and cell culture medium having a neutral, weakly acidic or weakly alkaline pH. However, when the protein to be concentrated and purified is dissolved in an alkaline buffer containing a surfactant, a weakly acidic buffer having a pH range of 4-7 is added to the protein solution so that the binding between the protein and the carrier is optimal. Allow the pH to range from 4-8.

2) washing the foreign matter not bound to the carrier

The washing of the foreign matter not bound to the carrier refers to a process of washing and removing substances remaining in the aqueous solution, that is, lipids, sugars, nucleic acids, salts, etc., after binding of the protein to the carrier, without binding to the carrier.

In the present invention, the washing solution used to wash the foreign matter uses a solution containing 5-30% alcohol in a buffer of pH 4-8 range.

As the alcohol that can be used in the present invention, it is preferable to use lower alcohols such as methanol, ethanol and propanol.

3) separating and eluting the protein from the carrier

After the foreign matter is removed, the protein bound to the carrier is separated and eluted under conditions that can lead to breakage of the non-covalent bond between the protein and the carrier.

In the present invention, it is preferable to use a solution containing 0.5-2% of sodium dodecyl sulfate (SDS) in a buffer of pH 7-10 as a solution for eluting the protein.

Since the protein concentration and purification method of the present invention can be applied to a wide range of proteins regardless of the physical and chemical properties of the protein, any protein can be obtained in high yield with a simple operation.

The protein concentration and purification method of the present invention is effective for a small amount of protein, but can be used for the separation of medically important proteins and the concentration and purification of industrial raw protein by expanding the application scale.

Hereinafter, the protein concentration and purification method according to the present invention will be described in detail by examples. However, the present invention is not limited to these examples.

Example 1 Comparison of Protein Adhesion of Kaolin, Silica, Silicate, and Hydrophobic Polymers

In order to find a substance showing excellent binding ability to a wide range of proteins among hydrophobic polymers used as a filler of kaolin, silica, silicate and column, the binding strength with the protein was compared using a variety of materials as a carrier.

Hydrophobicity such as natural silica and modified silica such as kaolin, silica, diatomaceous earth and bentonite, hydroxyapatite, BND cellulose, amberite XAD-2 and dowex resin The binding polymer was purchased from Sigma.

Silica, kaolin, diatomaceous earth, and bentonite were used having particle sizes of 0.1-10 μm, suitable for slurry production. Each powder was dissolved in strong acid, washed several times with 50% ethanol to remove suspended solids, and dissolved in purified water or acetate buffer. The final concentration of the slurry was used fixed at 100 mg / ㎖ in all experiments.

On the other hand, hydrophobic binding polymers such as hydroxyapatite, BND cellulose, Amberlite XAD-2, and dopex resin were used by dissolving them in purified water or acetate buffer at a weight ratio of 10% without pretreatment.

Since the protein used in the experiment should include various types, the whole protein extracted from E. coli DH5α was used directly. The solution used for protein extraction was a RIPA solution consisting of 50 mM Tris-HCl, pH 8.0, 1% NP-40, 150 mM sodium chloride, 5 mM EDTA, 1 mM PMSF. After quantifying the extracted protein at 10 mg / ㎖, diluted 1: 100 in a 100 mM, pH 5.0 acetate buffer solution, the carrier slurry prepared by the above process was added and reacted for 10 minutes at room temperature. 1 x Lamley sample consisting of 62.5 mM Tris-HCl, pH 6.8, 1% SDS, 0.005% bromophenol blue, 1% beta mercaptoethanol in a precipitate obtained by centrifugation for 30 seconds. After adding the buffer and boiling for 5 minutes, the supernatant obtained by centrifugation was analyzed on a 10% polyacrylamide gel. After electrophoresis, the gel was stained with a solution containing Coomassie Brilliant Blue R250 for 1 hour, followed by destaining with a solution containing methanol and acetic acid. As a control of the experiment, undiluted E. coli extracts were directly analyzed on the gel to compare the results, and the results are shown in FIG. 1.

According to FIG. 1, diatomaceous earth, kaolin and silica have adsorptive power to all kinds of proteins under the above conditions, and in particular, kaolin and silica exhibit very strong adsorptive power. Bentonite also has a binding ability to a protein, but has a problem of being difficult to handle, which is not suitable as a carrier to be used in the present invention. In addition, hydrophobic polymers such as hydroxyapatite, BND cellulose, amberlite XAD-2, dopex resin, and the like, have very poor binding to proteins.

Based on the results, it was confirmed that the effect on the protein binding when using a heterogeneous mixture of two kinds of substances showing a strong binding force to the protein. The binding conditions and electrophoresis conditions between the protein and the carrier were the same as those of the above method, and in order to analyze the separation state of the protein in more detail, the silver staining method, which is superior to the Coomassie blue staining method, was used and the results are shown in FIG. 2.

As shown in Figure 2, when using a mixture of kaolin and silica was excellent protein recovery than when using kaolin or silica alone. However, in the experimental group in which diatomaceous earth and kaolin were mixed, and the experimental group in which diatomaceous earth and silica were mixed, the recovery rate was much lower than that in the case of using kaolin or silica alone. In the experimental group in which hydroxyapatite was mixed with kaolin or silica, it was found that protein binding was not performed better than that treated with kaolin or silica alone.

To quantify the results obtained in FIGS. 1 and 2, the absorbance was measured at a wavelength of 595 nm using a protein quantitative solution containing Coomassie Brilliant G250. From this, the recovery of protein was calculated, and the recovery rate was calculated as a percentage of the total amount compared to the initial protein sample, and the results are shown in Table 1.

Comparison of protein recovery in protein enrichment experiments using kaolin, silica, silicate and hydrophobic polymers as carriers Used polymer Recovery rate (%) ± standard deviation Diatomaceous earth 22 ± 5 china clay 86 ± 8 Silica 88 ± 6 Bentonite 25 ± 2 Amberlite 2 ± 1 Dows Resin 0 BND resin 18 ± 5 Hydroxyapatite 4 ± 4 Diatomaceous Earth + Kaolin 85 ± 2 Diatomaceous Earth + Silica 84 ± 4 Kaolin + Silica 92 ± 3 Kaolin + Hydroxyapatite 35 ± 2 Silica + hydroxyapatite 42 ± 4

Example 2 Establishment of binding conditions, washing conditions and elution conditions in the concentration and purification of the protein of the present invention

In order to establish the optimum binding conditions between the protein and the carrier in the concentration and purification of the protein of the present invention, a 1: 1 (by volume) mixture of kaolin and silica showing the best protein binding capacity in Example 1 was used as a carrier. The degree of binding between the protein and the carrier was confirmed under various binding conditions. The buffer used for binding between the protein and the carrier was used solutions having a range of pH 4-10, the experiment was carried out in the same manner as in Example 1 and the results are shown in Table 2.

Comparison of Protein Recovery According to Binding Conditions between Protein and Carrier Composition of Binding Buffer Recovery Rate (%) ± Standard Deviation 100 mM Tris-HCl, pH7.0 82 ± 6 100 mM Tris-HCl, pH8.0 75 ± 5 100 mM Tris-HCl, pH9.0 50 ± 2 100 mM Tris-HCl, pH10.0 35 ± 7 100 mM Sodium acetate, pH4.0 84 ± 8 100 mM Sodium acetate, pH5.0 89 ± 6 100 mM Sodium acetate, pH6.0 87 ± 5 100 mM Phosphate buffer, pH7.0 79 ± 4 100 mM Sodium carbonate, pH7.0 81 ± 4 10 mM Sodium acetate, pH5.0 75 ± 5 50 mM Sodium acetate, pH5.0 84 ± 4 100 mM Sodium acetate, pH5.0 89 ± 6 200 mM Sodium acetate, pH5.0 87 ± 5

As shown in Table 2, regardless of the composition of the buffer solution was excellent in the binding strength between the protein and the carrier at a neutral near pH range of pH 4-8, it was found that the recovery of the protein is high. In particular, as the pH of the buffer approached the weak acidity, the recovery rate increased, but as the alkalinity decreased, the binding between the protein and the carrier was significantly lower. The concentration of acetate in the buffer had little effect on the recovery.

Therefore, in the protein concentration and purification method that can be applied to a wide range of proteins, it can be seen that the binding between the protein and the carrier is optimal when using a buffer solution in the pH range of 4-8.

On the other hand, in the protein concentration and purification method of the present invention, in order to establish the optimal washing conditions of foreign matter, the degree of protein purification under various washing conditions was confirmed. The solution used for washing the foreign matter was used an acetate solution containing ethanol at various concentrations. Based on the optimal binding conditions obtained in Table 2, 1: 1 mixture of kaolin and silica was added to the protein solution diluted in 100 mM acetate solution, and then bound at room temperature for 10 minutes. After washing the precipitate obtained by centrifugation twice with each washing solution, the recovery rate of the protein is measured and the results are shown in Table 3.

Comparison of Protein Recovery According to Washing Conditions of Foreign Matter Composition of Washing Buffer Recovery Rate (%) ± Standard Deviation 100 mM Sodium acetate, pH5.0 88 ± 6 100 mM Sodium acetate, pH5.0 + 10% ethanol 89 ± 4 100 mM Sodium acetate, pH5.0 + 20% ethanol 87 ± 4 100 mM Sodium acetate, pH5.0 + 30% ethanol 74 ± 8 100 mM Sodium acetate, pH5.0 + 50% ethanol 45 ± 7

As shown in Table 3, the wash solution diluted with ethanol in the range of 10-30% in 100 mM acetate buffer showed excellent protein recovery. However, when the ethanol concentration was increased to 50%, not only the foreign matter was removed but also the binding force between the protein and the carrier was affected, indicating that the protein recovery was significantly reduced.

Therefore, in protein concentration and purification methods that can be applied to a wide range of proteins, when a solution containing 5-30% alcohol in a buffer in the pH 4-8 range is used as the washing solution, the binding between the protein and the carrier is not affected. It can be seen that foreign matter is optimally cleaned without

On the other hand, in the protein concentration and purification method of the present invention, in order to establish the optimum elution conditions of the protein, the recovery rate of the protein was confirmed under various elution conditions. The optimal conditions obtained from the results of the above example were combined with the carrier and the washing process, the protein was eluted under various conditions and the protein recovery was measured and the results are shown in Table 4.

Comparison of Protein Recovery by Protein Elution Conditions Composition of Protein Elution Buffer Recovery Rate (%) ± Standard Deviation 50 mM Tris-HCl, pH8.0, boil 72 ± 6 50 mM Tris-HCl, pH8.0, Room temperature 12 ± 4 50 mM Tris-HCl, pH8.0 + 0.1% SDS, room temperature 34 ± 2 50 mM Tris-HCl, pH8.0 + 0.5% SDS, room temperature 66 ± 8 50 mM Tris-HCl, pH8.0 + 1% SDS, room temperature 88 ± 5 50 mM Tris-HCl, pH8.0 + 1% SDS + 5 mM EDTA, room temperature 89 ± 7 50 mM Tris-HCl, pH8.0 + 1% SDS + 0.1M NaCl, room temperature 82 ± 3 50 mM Tris-HCl, pH8.0 + 1% SDS + 0.5M NaCl, room temperature 72 ± 6 50 mM Tris-HCl, pH8.0 + 1% SDS + 1M NaCl, room temperature 56 ± 4 50 mM Tris-HCl, pH8.0 + 0.5M TMAC, room temperature 58 ± 2

As shown in Table 4, the SDS treatment increased the protein recovery rate in a concentration-dependent manner. However, treatment with TMAC or EDTA, a chelator of divalent ions, which increased salt concentration or affected hydrophobic binding, had little effect on protein elution.

Therefore, in the protein concentration and purification method that can be applied to a wide range of proteins, it can be seen that the protein is optimally eluted when a solution containing 0.5-2% SDS in a pH 7-10 buffer is used as the protein elution solution.

Example 3 Concentration and Purification of Purified Proteins of Known Molecular Weight Using the Protein Concentration and Purification Method of the Present Invention

In order to confirm that the protein concentration and purification method of the present invention is suitable for the concentration and purification of diluted proteins, experiments were conducted on purified proteins of known molecular weight.

Purified proteins used were phosphorylase B (98 KDa), BSA (bovine serum albumin, 66 KDa), egg albumin (egg albumin, 45 KDa), carbonic anhydrase (29 KDa) , Trypsin inhibitor (21 KDa), and lysozyme (14 KDa). Each protein was diluted in an acetate buffer of 100 mM, pH 5.0 at a concentration of 10 µg / ml, and 10 µl (100 mg / ml) of a 1: 1 mixture of kaolin and silica was added and reacted at room temperature for 10 minutes. The precipitate obtained by centrifugation was washed twice with 500 μl of 100 mM acetate buffer pH 5.0 containing 20% ethanol. To the washed precipitate was added 10 μl of a protein elution solution of 50 mM Tris-HCl, pH 8.0, 1% SDS, 5 mM EDTA, followed by reaction at room temperature for 10 minutes. Supernatants obtained by centrifugation were analyzed on 12% polyacrylamide gels. Purified protein was identified using a staining solution containing Coomassie Brilliant Blue R250, the results are shown in FIG.

As shown in FIG. 3, all proteins were purified with high recovery. Therefore, it can be seen that the protein concentration and purification method of the present invention can be effectively applied to a wide range of proteins regardless of the individual characteristics of the protein, such as molecular weight.

Example 4 Concentration and Purification of Proteins from Escherichia Coli DH5α Cell Extracts Using the Protein Concentration and Purification Methods of the Present Invention

In order to confirm that all proteins can be effectively concentrated and purified from cell extracts using the protein concentration and purification method of the present invention, experiments were performed on protein mixtures extracted from E. coli DH5α.

Cell extracts of DH5a were diluted 1: 100 in 100 mM acetate binding buffer, bound to the carrier in the same manner as in Example 3, washed and eluted. After polyacrylamide gel electrophoresis analysis, silver staining was used to increase the sensitivity in protein detection, and the results are shown in FIG. 4.

As shown in Figure 4, by the protein concentration and purification method of the present invention, the proteins in the cell extract were all purified with high recovery regardless of their molecular weight. In addition, it can be seen that the protein in the diluted cell extract was well concentrated at a high concentration through the protein concentration and purification method of the present invention.

Example 5 Concentration of Trace Proteins in Cell Culture Using Protein Concentration and Purification Method of the Present Invention

In order to confirm that the concentration and purification of trace proteins present in the cell culture is possible using the protein concentration and purification method of the present invention, the interferon beta was concentrated and purified from the culture solution of human fibroblasts secreting interferon beta. .

10 μl of a liquid carrier slurry was added to 1 ml of the culture medium of the cells, followed by reaction for 10 minutes. After washing and eluting in the same manner as in Example 3, the result was analyzed by polyacrylamide gel electrophoresis and the results are shown in FIG. 5A.

To confirm the presence of interferon beta in the purified protein solution, Western analysis was performed using an antibody against human interferon beta after polyacrylamide gel electrophoresis, and the results are shown in FIG. 5B.

As shown in Figure 5a, the protein concentration and purification method of the present invention was able to concentrate and purify all the proteins present in the culture at a high recovery rate. In addition, in Figure 5b it can be seen that through the protein concentration and purification method of the present invention, the interferon beta secreted from the cells into the culture medium was efficiently concentrated and purified.

Example 6 Protein Separation and Purification from Polyacrylamide Gel Using Protein Concentration and Purification Method of the Present Invention

To ensure that proteins can be efficiently isolated and purified from polyacrylamide gels using the protein concentration and purification methods of the present invention, whole proteins extracted from the liver of mice were electrophoresed on polyacrylamide gels. After electrophoresis, the gel was stained with a solution containing Coomassie Brilliant Blue R250 and destained with a solution containing methanol and acetic acid. After the desired protein was cut out from the gel stained with a razor, the gel pieces were crushed in a buffer of 0.1% SDS, 0.1 M sodium carbonate, pH 7.4 and reacted at 37 ° C. for 1 hour. The supernatant obtained by centrifugation was concentrated and purified in the same manner as in Example 3, and the results are shown in FIG.

To reconfirm the above results, the protein extract obtained from the recombinant E. coli strain producing GST (glutathione-S-tranferase) fusion protein was subjected to electrophoresis analysis using polyacrylamide gel, and then GST fusion proteins of various sizes were prepared as described above. Was extracted from the gel and purified. In order to confirm that the GST fusion protein was properly purified, Western analysis using an antibody against GST and the results are shown in FIG. 7.

As shown in Figures 6 and 7, it was confirmed that the protein concentration and purification method of the present invention can efficiently separate and purify various types of proteins present in the polyacrylamide gel.

Protein concentration and purification method of the present invention can be applied to all kinds of proteins irrespective of the physical and chemical properties of the protein, any protein has the effect of obtaining a pure protein in a high yield through a simple operation.

Since the method of concentrating and purifying the protein of the present invention can effectively concentrate and purify all proteins contained in the cell extract, it can be usefully used when obtaining proteins from tissue or culture cells for diagnostic purposes.

In the protein concentration and purification method of the present invention, even a small amount of protein secreted from the cell and diluted in the culture medium can be concentrated and purified in high yield. Significant advances can be expected in the isolation and production of industrial proteins.

The method for concentrating and purifying the protein of the present invention can effectively extract and purify the protein from the polyacrylamide gel, and thus, a breakthrough in the field of research related to the protein can be expected.

Claims (10)

A method for concentrating and purifying a wide range of proteins, comprising the steps of: 1) binding a protein to a carrier using kaolin, silica and silicate as a carrier, and using a buffer in the range of pH 4-8 as a binding solution, and 2) not binding to the carrier. Method for concentrating and purifying proteins, comprising three steps of removing foreign matter and 3) separating and eluting the protein from the carrier The method for concentrating and purifying proteins according to claim 1, wherein a solution containing 5-30% alcohol in a buffer in the range of pH 4-8 is used as a solution for removing foreign matter not bound to the carrier. The method for concentrating and purifying proteins according to claim 1, wherein a solution containing 0.5-2% SDS in a buffer in a pH range of 7 to 10 is used as a solution for separating and eluting the protein from the carrier. The method of claim 1, wherein the kaolin, silica and silicate have a particle size in the range of 0.1-10 μm. [Claim 2] The method for concentrating and purifying proteins according to claim 1, wherein a carrier for binding to a protein is used as a carrier selected from kaolin, silica, and diatomaceous earth, or a heterogeneous mixture of two substances mixed at a predetermined ratio. 6. The method for concentrating and purifying proteins according to claim 5, wherein in the heterogeneous mixture, the mixing ratio of each component is in the range of 1: 1-1: 5. The method of claim 1, wherein the carrier to be bound to the protein is in the form of a colloidal liquid slurry. 8. The method of claim 7, wherein the slurry for preparing the slurry is in the range of pH 4-8, and the final concentration of the slurry is 10-500 mg / ml of the buffer by weight. 3. The method of claim 2, wherein the alcohol is a lower alcohol of methanol, ethanol and propanol. The method of claim 1, wherein step 2) is performed by removing a foreign substance not bound to the carrier using a solution containing 5-30% of a lower alcohol of methanol, ethanol and propanol in a buffer ranging from pH 4-8. The step of separating and eluting the protein from the carrier using a solution containing 0.5-2% SDS in a buffer in the pH range of 7-10, characterized in that the concentration and purification of the protein