Protein purification method

Abstract

The invention relates to the technical field of protein purification, in particular to a biological method for purifying fusion protein through two different affinity tags. Two affinity purification tags and a dissolution promoting tag are perfectly combined, so that soluble expression of recombinant protein is ensured, high-purity target protein can be obtained through a two-step mild and efficient affinity purification process, the function and activity of the protein are ensured, a complex'capture-medium purification-fine purification 'multi-step purification process is avoided, and the method is suitable for industrial production. The purification process is shortened, the purification time is shortened, the purification efficiency is improved, and the method can be used for production and purification of functional proteins.

Description

Technical Field

[0001] The present invention relates to the technical field of protein purification, and in particular to a biological method for purifying a fusion protein through two different affinity tags. Background Art

[0002] The Gram-negative bacterium Escherichia coli (E. coli) is currently the most commonly used host bacterium in life science laboratories. It has a clear genetic background, is easy to culture, and grows rapidly, and is widely used for purposes such as plasmid amplification and protein expression. The E. coli recombinant expression system also has disadvantages, for example: 1) When some exogenous proteins are expressed in E. coli, they will misfold and form inclusion bodies; 2) During the expression and purification process, the target protein may break and degrade, making it difficult to obtain high-purity recombinant proteins.

[0003] For the problem of easy formation of inclusion bodies, strategies such as low-temperature culture, molecular chaperone assistance, and adding solubilizing tags can be used. Common tags that promote the solubilization and expression of inclusion body proteins include GST, MBP, SUMO, Trx, etc. SUMO is a ubiquitin-like protein from Saccharomyces cerevisiae. It not only has a small molecular weight and solubilizing properties similar to MBP, but also has a specific protease (ULP protease) that can specifically cut it. The efficiency of this processing is much higher than that of tool proteases such as Factor Xa, Thrombin, TEV, and 3C, and the target protein after ULP protease cutting has no redundant amino acid residues. Therefore, the combination of His-tag tag and SUMO tag gradually becomes the latecomer, and the frequency of use tends to surpass other solubilizing tags.

[0004] As for the problem of recombinant protein degradation, it is generally overcome through a multi-step purification strategy, such as a combined purification method of affinity chromatography, ion exchange and exclusion chromatography, which is the classic chromatography process of "capture-intermediate purification-fine purification". The chromatography conditions of the ion exchange medium in this method require complex conditions to determine the appropriate elution salt concentration. In addition, the purification efficiency of fine exclusion chromatography is relatively low, and the sample load is only about 5% of the volume of the chromatography column. In addition, the sample will be diluted, and the chromatography time is too long, which will affect the protein activity.

[0005] The most commonly used affinity chromatography process is the histidine tag, because metal ions such as nickel, cobalt, copper, and zinc can form coordination bonds with histidine in proteins. Therefore, when constructing a recombinant protein expression vector, 6-10 histidine tags (His-tag) can be introduced at the N-terminus or C-terminus of the target protein, and then the metal ion chelating filler can be used to achieve one-step efficient purification. Since the histidine tag is highly hydrophilic, it usually does not cause significant interference to the folding of the target protein after introduction, and the elution method is imidazole 10mM, 25mM, 50mM, 100mM, 250mM concentration gradient elution, and the conditions are mild. Therefore, the chromatography purification method of metal ion chelating filler has developed into the most common affinity purification method in life science laboratories. The corresponding metal ion chelating filler has developed from the first generation Ni IDA Beads 6FF, the second generation Ni NTA Beads 6FF, to the third generation Ni Smart Beads 6FF. The main advantage of the third-generation affinity filler is its strong tolerance to reducing agents, EDTA, and acid-base column cleaning. Its marginal working conditions have been greatly expanded, and it has higher production efficiency in large-scale production applications, which can effectively reduce production costs. At the same time, its metal ion shedding is low, and there is no need to repeatedly re-hang nickel ions, which is more conducive to environmental protection. However, the current traditional 6-10 histidine tags have weak affinity with the third-generation nickel metal ion chelating filler, which is not conducive to increasing the imidazole concentration of the impurities during the purification process to improve the impurity washing effect.

[0006] Another commonly used affinity tag is Strep-tag II, which is an 8-residue peptide that can simulate biotin and bind to the substrate binding pocket of strepavidin. This system has excellent selectivity and relatively mild elution conditions. The streptavidin filler corresponding to Strep-tag II has also developed to the third generation. The second generation mainly improves the affinity of mutant Streptactin for the tag, but retains the high affinity binding to the original substrate Biotin. Therefore, during use, the Biotin in the environment will gradually block the binding site of the filler, causing the filler load to decrease. The third-generation Streptactin XT mutant further improves the binding to Twin Strep-tag II, reduces the binding to Biotin, and increases the service life of the filler. Through screening, we also obtained a third-generation mutant, STarmStreptactin, which has moderate affinity for biotin, good binding specificity with Strep-tag II, high loading capacity, and long service life. Using Biotin instead of desthiobiotin as a competitive substrate for eluting the target protein reduces the cost of use and simplifies the regeneration process of the filler. Summary of the invention

[0007] In order to overcome the shortcomings of the existing technology, in view of the complexity of the existing "capture-intermediate purification-fine purification" process, an improved histidine tag and Strep-tag II are used as dual purification tags, and the solubility-promoting effect of the SUMO tag is combined to ensure the soluble expression of the protein. The histidine tag can be removed by SUMO protease, and can be further purified by Strep-tag II to remove the cut tag and further purify the protein. The purification conditions of this method are mild, and there is no need for complex ion exchange chromatography. The process and risks are controllable, which is convenient and fast for protein purification.

[0008] In order to solve the above problems, the technical solution provided by the present invention is as follows:

[0009] A protein purification method, comprising the following steps:

[0010] (1) Producing a fusion protein with a tag; the tag is a prokaryotic expression vector comprising an N-terminal improved histidine tag, a SUMO solubility-promoting tag fused behind the histidine tag, and a Strep-tag II fused behind the multiple cloning site;

[0011] (2) The tagged fusion protein is affinity purified using Ni filler, and after loading, it is equilibrated, washed, and finally eluted using imidazole;

[0012] (3) using SUMO protease to remove the histidine tag and SUMO lytic tag;

[0013] (4) Strep-tag II affinity purification to obtain the purified protein.

[0014] The present invention attempts to solve the problem of protein soluble expression and high-purity purification through a combination of multiple purification tags and lytic tags, as well as an enzyme cleavage purification system, and specifically describes a method in one embodiment, which comprises the following steps:

[0015] (1) A prokaryotic expression vector was designed. In addition to the basic components of the vector, it contained an improved N-terminal histidine tag, a SUMO solubility tag fused after the histidine tag, and a Strep-tag II fused after the multiple cloning site. Based on the principle of recombinant protein expression and through a prokaryotic expression system, various fusion proteins with tags were produced;

[0016] (2) The expressed fusion protein can be affinity purified through the improved histidine tag and high-tolerance Ni filler. After loading, it is balanced, washed and finally eluted with imidazole. The improved histidine tag has stronger binding force on the high-tolerance Ni filler than the traditional histidine tag. The imidazole concentration of the washing is increased and can be washed in the range of 5-50mM. 250-500mM imidazole is used for elution, and 250mM imidazole is the best. The suitable pH is 6.0-8.0, and the optimal pH is 7.0;

[0017] (3) After the histidine tag is purified, although the molecular weight of the histidine tag is small enough and will not affect the structure of the protein, the SUMO soluble tag is large. SUMO protease can be used to remove the histidine tag and the SUMO soluble tag to facilitate the next step of purification.

[0018] (4) Since only the C-terminus of the fusion protein has Strep-tag II, the tag can be used to affinity purify the target protein after SUMO protease cleavage. In this process, the histidine tag and SUMO solubilizing tag after the previous enzymatic cleavage can be removed, and the added SUMO protease can be removed. At the same time, the impurity proteins that are non-specifically bound during the affinity process of the histidine tag can also be removed in this step, and finally a high-purity soluble target protein can be obtained.

[0019] The perfect combination of two affinity purification tags and one solubility-promoting tag ensures the soluble expression of the recombinant protein while obtaining a very high purity target protein through a two-step gentle and efficient affinity purification process, ensuring the function and activity of the protein and avoiding the use of a complex "capture-intermediate purification-fine purification" multi-step purification process. It shortens the exploration of the purification process, speeds up the purification time, and improves the purification efficiency. It can be used for the production and purification of functional proteins.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1) The modified histidine affinity tag has stronger binding force, so that higher concentrations of imidazole can be used to remove impurities during purification;

[0022] 2) A flexible and versatile recombinant expression system that can add the required exogenous protein sequences (structural proteins or active proteins of other species (such as eukaryotic organisms), such as histones, cell membrane proteins, etc.) to the multiple cloning site for protein expression and ensure solubility;

[0023] 3) SUMO protease cleavage system ensures that no amino acid sequence remains after the N-terminal tag of the fusion protein is removed, ensuring that the function of the fusion protein is not affected in any way;

[0024] 4) Strep-tag II affinity purification can remove multiple impurity proteins produced in the previous steps at one time and purify them under mild conditions to ensure protein activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is the SDS-PAGE electrophoresis diagram of RN157 and RN161 expressing the dual affinity tag protein in Example 2 before and after induction;

[0026] Figure 2 This is the elution profile of the conventional histidine tag (8XHis) in Example 3;

[0027] Figure 3 This is the elution profile of the optimized histidine tag (HHHRHHHRHH) in Example 3;

[0028] Figure 4 The SDS-PAGE electrophoresis diagrams of histidine tag and STarm Sterptactin MagaroseBeads combined with SUMO protease cleavage purification in Example 5, Example 6, and Example 7;

[0029] Figure 5 It is the vector map;

[0030] Figure 6 is the multiple cloning site sequence. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the embodiments, but is not limited thereto.

[0032] Example 1: Vector modification

[0033] The 6X histidine tag at the N-terminus of the vector was transformed into an improved histidine with the sequence of HHHRHHHRHH through gene synthesis, and the tag was fused with a SUMO solubility-promoting tag. The vector contains multiple cloning restriction sites such as BamHI, NcoI and XhoI, and the C-terminus is fused with Strep-tag II. Figure 5 , Figure 6 As shown:

[0034] Example 2: Construction of fusion protein expression plasmid

[0035] Using the double restriction enzyme method, the target protein gene (RN157 and RN 161 represent different proteins fused into the expression vector, in order to verify the expression effect of the vector) was recombined into the multiple cloning restriction site. At the same time, an unmodified vector (conventional histidine tag, sequence HHHHHH) was used, and the same target protein gene was also fused into it as a control, see Figure 1 .

[0036] Example 3: Ni Smart Beads 6FF affinity purification of fusion protein

[0037] Refer to the protein expressed in Example 2. After collecting the bacterial sludge by centrifugation, it was lysed with a hammer lysis solution to identify that the fusion proteins were all in a soluble state. The supernatant was directly purified with Ni Smart Beads 6FF, and the filler was loaded into a gravity column tube; 20 column volumes of 20mM NaH2PO4, 500mM NaCl, pH 8.0 equilibrium solution were used for equilibrium; the supernatant sample was added to the balanced NiSmart Beads 6FF, mixed and incubated at room temperature for 30 minutes for binding; after the incubation, it was continued to be rinsed with 20 column volumes of equilibrium solution; after the equilibrium was completed, 0-250mM imidazole linear gradient elution was performed, and samples were collected in segments to test the new histidine tag elution imidazole concentration and observe the effect of improving the binding force of the new histidine tag (measured by the imidazole concentration corresponding to the elution peak. The higher the imidazole concentration required for elution, the stronger the binding force of the histidine tag is, see Figure 2 , Figure 3 ).

[0038] Example 4: Expression of fusion protein

[0039] Select GFP protein, and fuse GFP protein into the modified expression vector according to the steps of Examples 1 and 2. After the constructed plasmid DNA is purified, 100ul competent cells are taken and placed on ice to melt. In the clean bench, add 50ng of plasmid DNA (the volume cannot exceed 5ul) to the competent cells, gently rotate to mix, and place in ice for 30min. Place the test tube in a 42℃ water bath and let it stand for exactly 90s without shaking. Quickly transfer the test tube to ice and bathe it in ice for 1-2min. In the clean bench, add 900ul LB culture medium to the test tube and seal it, then place it in a 37℃ constant temperature shaking incubator and culture it at 180rpm for 45min. In the clean bench, transfer 200ul of transformed competent cells to a solid LB plate containing antibiotics and spread it evenly. Place the plate upright at room temperature until the liquid is completely absorbed. Invert the plate and place it in a constant temperature incubator at 37℃ and culture it for 16h.

[0040] Pick a single colony on the LB plate and place it in LB medium containing the corresponding antibiotics, culture it at 37°C, 180rpm until OD600 equals 0.6, add 0.1mM IPTG and induce it at low temperature overnight at 16°C, 180rpm.

[0041] Example 5: Ni Smart Magarose Beads Affinity Purification of Fusion Protein

[0042] Protein expressed in reference Example 4. After centrifugation to collect bacterial sludge, it was lysed with a hammer lysis solution to identify that the fusion proteins were all in a soluble state. The supernatant was directly purified with Ni Smart Magarose Beads, and the filler was placed in a gravity column tube; 20 column volumes of 20mM NaH2PO4, 500mM NaCl, pH 8.0 equilibrium solution were used for equilibrium; the supernatant sample was added to the balanced NiSmart Magarose Beads, mixed and incubated at room temperature for 30 minutes to bind; after the incubation, the equilibrium solution was continued to be used for 20 column volumes for washing; after the equilibrium was completed, low imidazole concentration was used for washing (imidazole concentration range 0-150mM), and then high imidazole concentration was used for elution (imidazole concentration range 150-250mM), see Figure 4 Line 1-4 of the microplate reader is used to collect the washed magnetic beads.

[0043] Example 6: SUMO protease cleavage

[0044] Take the magnetic beads of Example 5, add 50-60ug Sumo Protease to 100ul of magnetic beads, and digest at 30℃ for 1h. After digestion, collect the flow-through sample as the target protein, and continue to rinse with 20mM NaH2PO4, 500mM NaCl, pH 8.0 equilibrium solution, collect the fraction containing the target protein, and merge it with the flow-through. Figure 4 Line 5.

[0045] Example 7: Strep-tag II affinity purification

[0046] To the combined sample in Example 6, STarm Sterptactin Magarose Beads were added, mixed and incubated at room temperature for 30 min for binding, washed with equilibration solution, and eluted with 5 mM biotin. Figure 4 Line6-8 was used to obtain a high-purity target protein, GFP protein, with a purity of >95% (SDS-PAGE purity) and a purity of 30-40% before purification (SDS-PAGE purity).

[0047] The above is only a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several modifications and improvements without departing from the inventive concept of the present invention, which all belong to the protection scope of the present invention.

[0048] Sequence Listing

[0049] SEQ ID NO: 1

[0050] HHHRHHHRHH

[0051] SEQ ID NO:2

[0052] MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMD

[0053] SLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG

[0054] SEQ ID NO:3

[0055] WSHPQFEKGGGSGGGSGGSSAWSHPQFEK

Claims

1. A protein purification method, characterized in that: The method comprises the following steps: (1) producing a fusion protein with a tag; the tag is a prokaryotic expression vector, comprising an N-terminal improved histidine tag, a SUMO soluble tag fused behind the histidine tag, a multiple cloning site fused behind the SUMO soluble tag, and a Strep-tag II fused behind the multiple cloning site; (2) The tagged fusion protein is affinity purified using Ni filler, and after loading, it is equilibrated, washed, and finally eluted using imidazole; (3) using SUMO protease to remove the histidine tag and SUMO lytic tag; (4) Strep-tag II affinity purification to obtain the purified protein.

2. The method according to claim 1, characterized in that: The multiple cloning site in step (1) includes XbaI, NcoI, NdeI, BamHI, and XhoI.

3. The method according to claim 1, characterized in that The sequence of the improved histidine tag in step (1) is SEQ ID NO: 1, the sequence of the SUMO lytic tag is SEQ ID NO: 2, and the sequence of the Strep-tag II is SEQ ID NO:

3.

4. The method according to claim 1, characterized in that The Ni filler in step (2) is Ni Smart Magarose Beads or Ni Smart Beads 6FF.

5. The method according to claim 1, characterized in that In the step (2), 250-500 mM imidazole is used for elution, and the pH is 6.0-8.

0.

6. The method according to claim 1, characterized in that The purified target proteins include histones, cell membrane proteins, GFP, RN157 or RN161.

7. The method according to claim 1, characterized in that The step (4) Strep-tag II affinity purification specifically includes: adding STarm Sterptactin Magarose Beads, mixing and incubating at room temperature for binding, washing with 20mM Tris-HCl, 150mM NaCl, 1mM EDTA, pH 8.0 equilibrium solution, and eluting with biotin to obtain the target protein.

8. The method according to claim 1, characterized in that The SUMO protease in step (3) is SumoProtease.

9. The method according to claim 7, characterized in that: The incubation time was 30 min, the biotin concentration was 5 mM, and the equilibrium solution was 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, pH 8.0.

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