A method for rapid purification of proteins by adsorption and elution on a silica gel column

Abstract

The present invention discloses a method for rapidly purifying proteins by adsorption and elution on a silica gel column, which relates to the field of biotechnology. The method includes the following steps: S1, obtaining the target protein; S2, adding a protein binding buffer; S3, preparing a sol column; S4, adding a washing solution; S5, adding a protein elution solution; S6, detecting the recovery effect. The present invention purifies the target protein by utilizing the principle that the silica gel column adsorbs proteins in a certain buffer system. Protein samples often contain surfactants such as SDS. Using NaI + NaCl + β-mercaptoethanol as the protein binding buffer can relieve the inhibition of surfactant on the adsorption of proteins by silica gel. Selecting anhydrous ethanol as the protein washing solution can greatly reduce the protein loss caused by washing. Using a low-concentration SDS solution or a high-concentration urea and other protein elution agents as the elution buffer can effectively elute the protein from the silica gel column, and the operation steps only take 30-40 minutes.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a method for rapidly purifying proteins by adsorption and elution on a silica gel column. Background Art

[0002] Due to its excellent mechanical strength and easy surface modification, silica gel has become the most widely used chromatographic column packing. Silica gel columns have the characteristics of high column efficiency, good selectivity, and fast analysis speed, so they are widely used for separating polar small molecules. The commercialization degree of silica gel columns is relatively high and they are commonly used for the purification and recovery of nucleic acids. Nucleic acids are a class of biopolymers and are essential components of all known life forms. They are the most important substances among all biomolecules and are widely present in all animal and plant cells and microorganisms.

[0003] In the process of the method for rapidly purifying proteins by adsorption and elution on a silica gel column in the prior art, it is not convenient to purify the target protein and the buffer solution inhibits the adsorbed protein, resulting in easy loss of protein during the rinsing process, so that the protein cannot be effectively eluted from the silica gel column, reducing the operation effect and application scope. For this reason, we propose a method for rapidly purifying proteins by adsorption and elution on a silica gel column to solve the above problems. Summary of the Invention

[0004] In view of the deficiencies of the prior art, the present invention provides a method for rapidly purifying proteins by adsorption and elution on a silica gel column to solve the problems raised in the above background art.

[0005] To achieve the above object, the present invention provides the following technical solution: A method for rapidly purifying proteins by adsorption and elution on a silica gel column, comprising the following steps:

[0006] S1. Obtaining the target protein: Using 5-6% agarose gel to separate proteins. After separation, perform staining treatment and cut out the required target protein.

[0007] S2. Adding the protein binding buffer: Add the protein binding buffer to the stained target protein in step S1, mix, and perform a water bath at 70°C for 10 minutes to prepare the required sol solution for standby.

[0008] S3. Preparing the sol column: Transfer the sol solution after the water bath in step S2 into a silica gel column and centrifuge at a speed of 12000 rpm for 1 minute to obtain the required sol column.

[0009] S4. Adding the washing solution: Add the washing solution to the sol column prepared in step S3.

[0010] S5. Addition of protein eluent: Add 100 ul of protein eluent to the sol column prepared in step S3;

[0011] S6. Detection of recovery effect: Perform electrophoresis separation using SDS-PAGE and detect the protein recovery effect.

[0012] To further optimize this technical solution, the target protein cut in step S1 is stained using R-250 or KCL staining.

[0013] To further optimize this technical solution, the protein binding buffer in step S2 is a mixed solution of NaI + NaCl + β-mercaptoethanol or a binding solution of NaI, DEAB, and Tris-HCl.

[0014] To further optimize this technical solution, the DEAB is a nucleic acid binding buffer.

[0015] To further optimize this technical solution, the concentration of the NaI solution is ≥5 M, the concentration of the NaCl solution is ≥1 M, and the concentration of β-mercaptoethanol is 1%.

[0016] To further optimize this technical solution, the washing solution in step S4 is one of water, 70% ethanol solution, absolute ethanol, or methanol, preferably absolute ethanol, and the content of added absolute ethanol is 700 ul.

[0017] To further optimize this technical solution, the protein eluent in step S5 is one of water Tris-Cl buffer, low-concentration SDS solution, lysis solution, or high-concentration urea solution, preferably low-concentration SDS solution or high-concentration urea solution.

[0018] To further optimize this technical solution, the concentration of the low-concentration SDS solution is 0.2%.

[0019] Compared with the prior art, the present invention provides a method for rapidly purifying proteins by adsorption and elution through a silica gel column, having the following beneficial effects:

[0020] 1. For the method of rapidly purifying proteins by adsorption and elution through a silica gel column, the present invention utilizes the principle of adsorbing proteins by a silica gel column in a certain buffer system to purify the target protein. Protein samples often contain surfactants such as SDS. Using NaI + NaCl + β-mercaptoethanol as the protein binding buffer can relieve the inhibition of surfactant on silica gel adsorption of proteins. Selecting absolute ethanol as the protein washing solution can greatly reduce protein loss caused by washing. Using a low-concentration SDS solution or a high-concentration urea solution and other protein eluents as the elution buffer can effectively elute the protein from the silica gel column, and the operation steps only take 30 - 40 minutes.

[0021] 2. The method for rapid purification of proteins by adsorption and elution through a silica gel column. The preparation method of the present invention can be used for the purification of prokaryotically expressed proteins and the purification of high-abundance proteins in plant samples, enabling the preparation method to be used for protein concentration and also applicable to the purification of target proteins from complex protein samples, thus expanding the scope of application of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic flow chart of a method for rapid purification of proteins by adsorption and elution through a silica gel column proposed by the present invention;

[0023] Figure 2 is a schematic diagram comparing the flow-through components of protein samples using different protein binding buffers through a silica gel column in the present invention;

[0024] Figure 3 is a schematic diagram comparing the flow-through components after rinsing the proteins bound to the silica gel column with different rinsing solutions in the present invention;

[0025] Figure 4 is a schematic diagram showing the protein elution effect of different eluents in the present invention;

[0026] Figure 5 is a schematic diagram for the purification of prokaryotically expressed proteins in the present invention;

[0027] Figure 6 is a schematic diagram for the purification of high-abundance proteins in plant samples in the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0029] Example 1: Please refer to Figures 1 to 4 As shown, the present invention discloses a method for rapid purification of proteins by adsorption and elution through a silica gel column, including the following steps:

[0030] S1. Obtaining the target protein: Using 5% agarose gel to separate proteins, after separation, performing a staining treatment, using R-250 for staining, and cutting out the required target protein;

[0031] S2. Addition of protein binding buffer: Add protein binding buffer to the stained target protein in step S1, and mix. The protein binding buffer is a binding solution of NaI, DEAB (a nucleic acid binding buffer), and Tris-HCl. After water bath at 70 °C for 10 min, prepare the required sol solution for standby;

[0032] S3. Preparation of sol column: Transfer the sol solution after water bath in step S2 to a silica gel column, and centrifuge at a speed of 12000 rpm for 1 min to obtain the required sol column;

[0033] S4. Addition of washing solution: Add washing solution to the sol column prepared in step S3. The washing solution is 70% ethanol solution;

[0034] S5. Addition of protein elution solution: Add 100 ul of protein elution solution to the sol column prepared in step S3. The protein elution solution is water Tris-Cl buffer;

[0035] S6. Detection of recovery effect: Perform electrophoresis separation operation using SDS-PAGE and detect the protein recovery effect.

[0036] Example 2: Please refer to Figures 1 to 4 As shown, the present invention discloses a method for rapid purification of proteins by adsorption and elution through a silica gel column, including the following steps:

[0037] S1. Obtaining of target protein: Use 6% agarose gel to separate proteins. After separation, perform staining treatment, and cut out the required target protein, and perform staining treatment using KCL staining;

[0038] S2. Addition of protein binding buffer: Add protein binding buffer to the stained target protein in step S1. The protein binding buffer is a mixed solution of NaI + NaCl + β-mercaptoethanol. The concentration of the NaI solution ≥ 5M, the concentration of the NaCl solution ≥ 1M, and the concentration of β-mercaptoethanol is 1%. After mixing and water bath at 70 °C for 10 min, prepare the required sol solution for standby;

[0039] S3. Preparation of sol column: Transfer the sol solution after water bath in step S2 to a silica gel column, and centrifuge at a speed of 12000 rpm for 1 min to obtain the required sol column;

[0040] S4. Addition of washing solution: Add washing solution to the sol column prepared in step S3. The washing solution is absolute ethanol, and the added amount of absolute ethanol is 700 ul;

[0041] S5. Addition of protein eluent: Add 100 μl of protein eluent to the sol column prepared in step S3. The protein eluent is a low-concentration SDS solution or a high-concentration urea solution. The concentration of the low-concentration SDS solution is 0.2%.

[0042] S6. Detection of recovery effect: Perform electrophoresis separation using SDS-PAGE and detect the protein recovery effect.

[0043] Example 3: Please refer to Figures 1 to 4 As shown, the present invention discloses a method for rapidly purifying proteins by adsorption and elution through a silica gel column, including the following steps:

[0044] S1. Obtaining the target protein: Use 6% agarose gel to separate proteins. After separation, perform staining treatment, and cut out the required target protein, and use KCL staining for the staining treatment.

[0045] S2. Addition of protein binding buffer: Add protein binding buffer to the stained target protein in step S1. The protein binding buffer is a mixture of NaI + NaCl + β-mercaptoethanol. The concentration of the NaI solution is 5 M, the concentration of the NaCl solution is 1 M, and the concentration of β-mercaptoethanol is 1%. Mix and treat, and perform a water bath at 70 °C for 10 min to prepare the required sol solution for standby.

[0046] S3. Preparation of the sol column: Transfer the sol solution after the water bath in step S2 to a silica gel column and centrifuge at a speed of 12000 rpm for 1 min to obtain the required sol column.

[0047] S4. Addition of washing solution: Add a washing solution to the sol column prepared in step S3. The washing solution is methanol, and the added amount of methanol is 700 μl.

[0048] S5. Addition of protein eluent: Add 100 μl of protein eluent to the sol column prepared in step S3. The protein eluent is a lysis solution.

[0049] S6. Detection of recovery effect: Perform electrophoresis separation using SDS-PAGE and detect the protein recovery effect.

[0050] Research results of the present invention:

[0051] 1. Use NaI + NaCl + β-mercaptoethanol as the protein binding buffer.

[0052] Commercially available nucleic acid recovery silica columns are used as protein adsorption silica columns. The commercialization degree of silica columns is relatively high, and they are commonly used for the purification and recovery of nucleic acids. First, the effects of common high-concentration chaotropic salts used to dissolve agarose gels, namely guanidine isothiocyanate (GITC), guanidine hydrochloride (GHCl), and sodium iodide (NaI), as binding buffers during the protein adsorption process by silica columns were tested. The binding buffer was mixed with Escherichia coli protein samples, heated at 70 °C for 10 min, and then passed through the silica column by centrifugation at 12,000 rpm for 1 min. The flowing-through binding buffer was collected for SDS-PAGE electrophoresis separation to detect the binding-promoting effects of different chaotropic agents.

[0053] Under the condition that the protein sample does not contain SDS, using NaI, DEAB (a nucleic acid binding buffer), and Tris-HCl as binding buffers will not result in the flowing-through of proteins ( Figure 1 left), indicating that all three of these solutions have the ability to retain proteins on the silica column. However, when passing Escherichia coli protein samples containing 0.2% SDS or Escherichia coli proteins separated by SDS-agarose gel through the silica column, only NaI and DEAB have the ability to retain proteins on the silica column ( Figure 1 middle, right). Although DEAB has the ability to bind proteins to the silica column, subsequent experiments found that it is difficult to elute proteins from the silica column after using it. Therefore, a high-concentration NaI solution (final concentration ≥ 5 M) is a more suitable protein binding buffer. NaCl (final concentration ≥ 1 M) and β-mercaptoethanol (final concentration 1%) were added to the binding buffer, which were used to provide the ions required for binding and prevent oxidation, respectively, and could better promote the binding of proteins to the silica column and facilitate the subsequent elution of protein samples from the silica column.

[0054] Figure 2 : Comparison of the flowing-through components of protein samples using different protein binding buffers after passing through the silica column. CK, Escherichia coli protein sample for recovery; DEAB, commercially available nucleic acid binding buffer; GITC, 2.5 M guanidine isothiocyanate + 1 M NaCl + 1% β-mercaptoethanol; GHCl, 3 M guanidine hydrochloride + 1 M NaCl + 1% β-mercaptoethanol; NaI + NaCl, 5 M NaI + 1 M NaCl + 1% β-mercaptoethanol; Tris-HCl, 0.25 M Tris-HCl buffer at pH 6.8; -SDS, the recovered sample is an Escherichia coli protein solution without SDS; +SDS, the recovered sample is an Escherichia coli protein solution containing 0.2% SDS; +Gel, the purified sample is an Escherichia coli protein gel block separated by SDS-agarose gel, as shown in Figure 2 shown.

[0055] 2. Use ethanol as the protein washing solution;

[0056] After selecting NaI + NaCl + β-mercaptoethanol as the protein binding buffer, a solution for rinsing impurities on the silica gel column was then selected, and the rinsing effects of water, 70% ethanol solution, absolute ethanol, and methanol were tested respectively. After adding the rinsing solution, centrifuge at 12,000 rpm for 1 min, collect the rinsing solution after flow-through, and then obtain the protein that may exist in the flow-through solution through ultrafiltration for SDS-PAGE detection. It was found that when recovering protein from an E. coli protein solution without SDS, using 70% ethanol, absolute ethanol, or methanol as the rinsing solution hardly caused protein loss due to flow-through ( Figure 2 left), but when recovering protein from the protein melting gel sample separated by SDS-agarose gel, there was a small amount of protein in the flow-through solution after rinsing with 70% ethanol, ethanol, or methanol. Among them, when ethanol was used as the rinsing solution, only a trace amount of protein flowed through ( Figure 2 right), so ethanol was selected as the rinsing solution.

[0057] Figure 3 : Comparison of flow-through components after rinsing the protein bound to the silica gel column with different rinsing solutions.

[0058] CK, control for the E. coli protein sample used for recovery; H2O, water; 70% ethanol, 70% aqueous ethanol solution; ethanol, absolute ethanol; methanol, absolute methanol; -Gel, the purified sample is an E. coli protein solution; +Gel, the purified sample is an E. coli protein gel block separated by SDS-agarose gel, as shown in Figure 3 shown.

[0059] 3. Use protein solubilizing agents such as low-concentration SDS or high-concentration urea as protein elution solutions;

[0060] Select a protein solubilizing agent to elute the protein from the silica gel column. Some common protein solubilizing agents were tested, including water, Tris-Cl buffer, SDS solution, lysis solution (LB, 7M urea, 2M thiourea, 4% CHAPS, 2% DTT, pH 8.5), and urea solution (8M urea) as protein elution solutions. After adding the protein elution solution to the silica gel column, centrifuge at 12,000 rpm for 1 min, and collect the elution solution for SDS-PAGE detection. The results showed that different from nucleic acid elution, water solution and Tris-Cl buffer could not effectively elute the protein from the silica gel column, but the aqueous solution or Tris-Cl buffer containing 0.2% SDS could well elute the protein from the silica gel column. The common protein solubilizing agents LB and UA could also achieve good elution effects.

[0061] Figure 4 Protein elution effects of different elution solutions.

[0062] CK, E. coli protein sample control for recovery; H2O, water; H2O + SDS, aqueous solution of 0.2% SDS; Tris-HCl, 0.25 M pH 8.0 Tris-HCl buffer; Tris-HCl + SDS, 0.25 M pH 8.0 Tris-HCl buffer containing 0.2% SDS; LB, 7 M urea, 2 M thiourea, 4% CHAPS, 2% DTT, pH 8.5; LB + SDS, LB containing 0.2% SDS; UA, 6 M urea; UA + SDS, 6 M urea containing 0.2% SDS; CKG, direct electrophoresis without recovery of the E. coli protein agarose gel block dissolution solution; -Gel, the purified sample is an E. coli protein solution; +Gel, the purified sample is an E. coli protein gel block separated by SDS-agarose gel, see Figure 4 as shown:

[0063] The preparation method of the present invention can be used for protein concentration and is also applicable to purifying target proteins from complex protein samples.

[0064] 4. Purification of prokaryotic expressed proteins:

[0065] The availability of the protein recovery method was tested by recovering prokaryotic expressed proteins. E. coli proteins were separated by agarose gel and stained with cold KCl (final concentration 3 M) solution, and the high-abundance bands were stained white ( Figure 5 ). α-Galactosidase (81.8 kDa), α-amylase (49.7 kDa), and crf1 protein (28.8 kDa) induced and expressed in E. coli were all successfully recovered to obtain clear and high-purity target bands ( Figure 5 ).

[0066] Purification of E. coli expressed proteins: Figure 5 Left, after separation of E. coli proteins by agarose gel, stained with cold KCl (final concentration 3 M) solution; Figure 5 Right, SDS-PAGE of E. coli proteins and purified target proteins; M, 10 kDa - 170 kDa protein marker; 1, E. coli proteins expressing α-galactosidase (81.8 kDa); 1R, target protein band recovered from 1; 2, E. coli proteins expressing α-amylase (49.7 kDa); 2R, target protein band recovered from 2; 3, E. coli proteins expressing crf1 protein (28.8 kDa); 3R, target protein band recovered from 3, see Figure 5 as shown:

[0067] 5. The preparation method of the present invention can be used for purifying high-abundance proteins in plant samples.

[0068] Further test whether this protein purification method can be used for the recovery of plant proteins. The purification of the ~53 kDa RuBisCO protein in banana leaf proteins and the ~13.5 kDa REF138 protein in rubber tree latex proteins was tested, and clear and highly pure target bands were also obtained ( Figure 6 ). Purification of high-abundance proteins in plant tissues. M, 10 kDa - 170 kDa protein marker; 1, total protein of banana leaves; 1R, purified ~53 kDa RuBisCO protein from 1; 2, total protein of rubber tree latex; 2R, purified ~13.5 kDa REF138 protein from 2, as shown in Figure 6 .

[0069] Judgment criterion: Through the comparison of three examples and the research results, it is concluded that Example 2 has the best effect. Therefore, Example 2 is selected as the best example. Specific changes in quantity also fall within the scope protected by this technical solution.

[0070] Advantages of the present invention: This method for rapidly purifying proteins by silica column adsorption and elution. The present invention utilizes the principle of silica column adsorbing proteins in a certain buffer system to purify target proteins. Protein samples often contain surfactants such as SDS. Using NaI + NaCl + β-mercaptoethanol as the protein binding buffer can relieve the inhibition of surfactants on silica's adsorption of proteins. Selecting anhydrous ethanol as the protein rinsing solution can greatly reduce protein loss caused by rinsing. Using a low-concentration SDS solution or a high-concentration urea solution or other protein elution agents as the elution buffer can effectively elute proteins from the silica column, and the operation steps only take 30 - 40 minutes; The preparation method of the present invention can be used for the purification of prokaryotic expression proteins and can also be used for the purification of high-abundance proteins in plant samples, so that this preparation method can be used for protein concentration and is also applicable to purifying target proteins from complex protein samples, improving the scope of application of the present invention.

[0071] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for rapid purification of proteins by adsorption and elution on a silica gel column, characterized in that, It includes the following steps: S1. Obtaining the target protein: Use 5-6% agarose gel to separate proteins. After separation, perform staining and cut out the required target protein. S2. Adding the protein binding buffer: Add the protein binding buffer to the stained target protein in step S1, mix, and perform a water bath at 70°C for 10 minutes to prepare the required sol solution for standby. The protein binding buffer in step S2 is a mixed solution of NaI + NaCl + β-mercaptoethanol. The concentration of the NaI solution is 5M, the concentration of the NaCl solution is 1M, and the concentration of β-mercaptoethanol is 1%. S3. Preparing the sol column: Transfer the sol solution after the water bath in step S2 to a silica gel column and centrifuge at a speed of 12,000 rpm for 1 minute to obtain the required sol column. S4. Adding the washing solution: Add the washing solution to the sol column prepared in step S3. The washing solution is one of ethanol or methanol. S5. Adding the protein elution solution: Add 100 μL of the protein elution solution to the sol column prepared in step S4. The protein elution solution is an SDS solution or a urea solution. S6. Detecting the recovery effect: Use SDS-PAGE for electrophoresis separation and detect the protein recovery effect.

2. The method for rapidly purifying proteins by silica column adsorption and elution according to claim 1, characterized in that, The target protein cut out in step S1 is stained using R-250 or KCL staining.

3. A method for rapidly purifying proteins by adsorption and elution through a silica gel column according to claim 1, characterized in that, The ethanol in step S4 is a 70% ethanol solution or absolute ethanol, and the content of ethanol is 700 μL.

4. A method for rapidly purifying proteins by adsorption and elution through a silica gel column according to claim 1, characterized in that, When the protein elution solution in step S5 is a urea solution, the concentration of the urea solution is 8M.

5. A method for rapidly purifying proteins by adsorption and elution through a silica gel column according to claim 1, characterized in that When the protein elution solution in step S5 is an SDS solution, the concentration of the SDS solution is 0.2%.

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