A method for purifying proteins and its use

Abstract

The application provides a protein purification method and application thereof. The method comprises the following steps: (1) constructing a nucleotide fragment encoding AeBlueM-protease recognition site-target protein, and cloning the nucleotide fragment into a basic vector at a multiple cloning site by using a homologous recombination method to obtain a carrier; (2) constructing a nucleotide fragment encoding AeBlueM-Im7, and cloning the nucleotide fragment into the basic vector at the multiple cloning site by using the homologous recombination method to obtain the carrier; (3) constructing a nucleotide fragment encoding CL7-protease recognition site-target protein, and cloning the nucleotide fragment into the basic vector at the multiple cloning site by using the homologous recombination method to obtain the carrier. The purification method is not dependent on traditional resins and chromatography columns, is simple to operate, is easy to scale up, and is low in cost.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a protein purification method and its application. Background Technology

[0002] Proteins play a vital role in scientific research, medicine, and industrial production. For in vitro analysis or applications, target proteins must be isolated from other cellular components; this process is called protein purification. Protein purification typically begins with cell lysis. Lysis releases intracellular substances. Centrifugation separates soluble proteins from various insoluble impurities, and various methods are then used to obtain proteins with high purity while retaining their activity. Currently, affinity chromatography is a widely used protein purification method. It is a separation and purification technique based on specific interactions between biomolecules. Its principle is to selectively adsorb target molecules (such as proteins) by utilizing the specific recognition between the target molecule and ligands in the stationary phase.

[0003] Traditional affinity chromatography requires the preparation of affinity resins and chromatography columns, which are expensive and not suitable for high-throughput, large-scale purification.

[0004] In recent years, researchers have focused on developing aggregation tags for protein separation and purification without relying on resins and chromatography columns. These tags can be divided into two categories: in vivo induced aggregation tags and in vitro induced aggregation tags. In vivo induced aggregation tags cause proteins to aggregate during expression. After cell lysis and centrifugation, the target protein remains in the precipitate, existing in either an active or inactive form. Active proteins can be released from the precipitate through protease cleavage and integrin self-cleavage. Inactive proteins require denaturation and refolding of the precipitate to obtain the active protein. However, denaturation and refolding are complex, have low success rates, and are costly; proteins expressed in their active form are more advantageous for downstream operations. However, these methods are only suitable for peptides and relatively stable proteins, and not for most proteins that are inherently prone to aggregation and precipitation, nor for membrane proteins that need to be expressed on membranes. In vitro induced aggregation tags purify proteins without denaturation and refolding, and the protein exists in its active form, making them suitable for proteins with more demanding requirements. Typical in vitro induced aggregation tags include elastin peptides (ELPs).

[0005] ELP-tagged fusion proteins are expressed in soluble form in vivo. Above the phase transition temperature, the fusion proteins aggregate and can be separated by centrifugation; lowering the temperature allows the fusion proteins to redissolve. At this point, the ELP tag can be removed by protease cleavage or integrity cleavage. After another phase transition to remove the ELP, a high-purity target protein (>90%) can be obtained. However, this method requires the target protein to be at a relatively high temperature (>37°C), which is detrimental to some heat-sensitive proteins. This method also requires multiple heating and cooling processes, making the procedure complex. ELP sequences have high repetition, making cloning difficult. The temperature required for inducing precipitation of ELP fusion proteins also needs to be determined individually for each protein. ELP fusion proteins have also been reported to potentially cause slowed cell growth and decreased protein expression levels.

[0006] Therefore, it is necessary to provide a protein purification method that is simple to operate, easy to scale up, and low in cost, and does not rely on traditional resins and chromatography columns. Summary of the Invention

[0007] This invention utilizes AeBlueM, a mutant of AeBlue, a colored protein derived from sea anemones. Its amino acid sequence is shown in SEQ ID NO.1: SEQ ID NO.1 MASLVKKDMCIKMTMEGTVNGHHFKCVGEGEGKPFEGTQVEKIRITEGGPLPFAYDILAPCCMYGSKTFIKHVSGIPDYFKESFPEGFTWERTQIFEDGGYLTIHQDTSLQGNN FIFKVNVIGANFPANGPVMQKKTAGWEPCVEMLYPRDGVLCGQSLMALKCTDGNHLTSHLRTTYRSRKPSNAVNMPEFHFGDHRIEILKAEQGKFYEQYESAVARYSEGMDELYK This invention discovered that the addition of imidazole to AeBlueM protein induces precipitation under freezing conditions. After removal of the inducing factor, AeBlueM cannot be redissolved in the buffer, indicating that this process is irreversible. Furthermore, the precipitate exhibits green fluorescence, suggesting that most of its three-dimensional structure is preserved after precipitation. AeBlueM expression levels are also high in *E. coli* (at least 120 mg of AeBlueM protein can be purified per liter of shake-flask culture). Based on these properties of AeBlueM, this invention utilizes it as a tag for protein purification, purifying proteins through induced precipitation.

[0008] The first objective of this invention is to provide the application of AeBlueM in protein purification, wherein the amino acid sequence of AeBlueM is shown in SEQ ID NO.1.

[0009] A second object of the present invention is to provide the application of a vector comprising AeBlueM in protein purification, wherein the vector comprising AeBlueM is selected from (1) or (2): (1) Direct carrier: A nucleotide fragment encoding the AeBlueM protease recognition site-target protein was constructed and cloned into the multiple cloning site of the basic vector using homologous recombination to obtain the direct vector; SEQ ID NO.2 Atggcttcactggttaaaaaagacatgtgcatcaaaatgacgatggaaggaacagtaaacggtcaccatttcaagtgtgtaggagaaggcgaaggcaaaccatttgaagggacccaggtggaaaagatacgcatcactgaaggtgggcccttaccatttgcgtatgatatt ttggccccttgttgcatgtatggcagtaaaaccttcattaagcatgtgtcgggtattccggattactttaaggagtcttttcctgagggctttacctgggaaagaacacaaatcttcgaggatggcggctatctcaccataacaccaggacacgagccttcagggtaataatt ttattttcaaagttaatgtcatcggtgccaacttccctgcaaacggtcccgtgatgcagaaaaaacagctggatgggaaccgtgcgttgagatgctttatccgcgggacggcgtcctgtgtggtcagagcctgatggccctgaaatgcactgatggcaatcatctgacgtc ccacctgcgcactacctatcgttctcgcaagccatccaatgcagttaacatgccggaatttcattttggggatcatcgcattgagattttgaaagctgaacaaggtaaattttatgaacaatacgagtcagcggtggcccgttacAgtgagGGCATGGATGAACTCTACAAA (2) Indirect carriers, including carrier A and carrier B: (2-1) Construct a nucleotide fragment encoding AeBlueM-Im7 and clone it into the multiple cloning site of the base vector using homologous recombination to obtain the vector A.

[0010] (2-2) Construct a nucleotide fragment encoding the target protein-protease recognition site-CL7, and clone it into the multiple cloning site of the basic vector using homologous recombination to obtain the vector B; Furthermore, the base carrier is pET28b or pPICZA.

[0011] In a particular embodiment, (2-1) cloned on pET28b; (2-2) cloned on pET28b or pPICZA.

[0012] Furthermore, the protease recognition site is selected from the recognition sites of HRV3C protease, TEV protease, thrombin, and enterokinase.

[0013] Furthermore, (1) the nucleotide sequence of the AeBlueM is shown in SEQ ID NO.2.

[0014] Furthermore, (2-1) the nucleotide sequence encoding AeBlueM-Im7 is shown in SEQ ID NO.3, and (2-2) the nucleotide sequence of CL7 is shown in SEQ ID NO.4.

[0015] SEQ ID NO.3 AtggcttcactggttaaaaaagacatgtgcatcaaaatgacgatggaaggaacagtaaacggtcaccatttcaagtgtgtaggagaaggcgaaggcaaaccatttgaagggacccaggtggaaaagatacgcatcactgaaggtgggcccttaccatttgcgtatgatattttggccccttgttgcatgtatggcagtaaaaccttcattaagcatgtgtcgggtattccggattactttaaggagtcttttcctgagggctttacctgggaaagaacacaaatcttcgaggatggcggctatctcaccatacaccaggacacgagccttcagggtaataattttattttcaaagttaatgtcatcggtgccaacttccctgcaaacggtcccgtgatgcagaaaaaaacagctggatgggaaccgtgcgttgagatgctttatccgcgggacggcgtcctgtgtggtcagagcctgatggccctgaaatgcactgatggcaatcatctgacgtcccacctgcgcactacctatcgttctcgcaagccatccaatgcagttaacatgccggaatttcattttggggatcatcgcattgagattttgaaagctgaacaaggtaaattttatgaacaatacgagtcagcggtggcccgttacAgtgagGGCATGGATGAACTCTACAAATCGAATTCTggtggctctagtattagtgattacacagaggctgagtttgttcaacttcttaaggaaattgaaaaagagaatgttgctgcaactgatgatgtgttagatgtgttactcgaacacttcgtaaaaattactgagcatccagatggaacggatctgatctattatcctagtgataatagagacgatagccccgaagggattgtcaaggaaattaaagaatggcgagctgctaacggtaagccaggatttaaacagggc SEQ ID NO.4 agcaaaagcaatgaaccgggtaaggcaaccggtgaaggtaagccggttaataacaaatggctgaacaatgccggtaaagatctgggtagtccggttccggatcgtattgcaaataaactgcgtgataaagaattcgagagcttcgatgattttcgtgaaaccttttgggaagaagttagcaaagatcctgaac tgagcaaacagtttagccgcaataacaatgatcgtatgaaagttggtaaagcaccgaaaacacgtacccaggatgttagcggtaaacgtacctcatttgaactgaatcatcagaaaccgattgaacagaatggtggcgtttatgatatggataacattagcgttgttaccccgaaacgcaacattgatattgaa Furthermore, when the vector is (1), the application is as follows: the vector is induced to express to obtain the AeBlueM-protease recognition site-target protein fusion protein, imidazole is added, mixed, and fully frozen. After thawing, the supernatant is discarded by centrifugation, the precipitate is washed and resuspended, protease is added for digestion, and then the precipitate is discarded by centrifugation. The supernatant contains the purified target protein.

[0016] Furthermore, the protease is selected from HRV3C protease, TEV protease, thrombin, and enterokinase. Furthermore, when the vector is (2), the application is as follows: the vector (2-1) is induced to express to obtain the AeBlueM-Im7 fusion protein, imidazole is added, mixed, and fully frozen. After thawing, the supernatant is discarded by centrifugation, and the precipitate is washed and resuspended to obtain protein resin; the vector (2-2) is induced to express to obtain the CL7-protease recognition site-target protein fusion protein, protein resin is added for incubation, the supernatant is discarded by centrifugation, the precipitate is washed and resuspended, protease is added for digestion, the precipitate is discarded by centrifugation, and the supernatant is the purified target protein.

[0017] When the above-mentioned vector is (1), the protein purification method is the direct method: Specifically, the target protein is fused with AeBlueM, with a protease (such as HRV3C protease) recognition site inserted in the middle. The AeBlueM portion can be precipitated by imidazole and low temperature induction, but the target protein remains soluble. Because the precipitation is irreversible, after washing, the target protein can be released from the precipitate by protease cleavage, and it retains high purity.

[0018] When the above-mentioned carrier is (2), the protein purification method is an indirect method: For proteins that cannot tolerate imidazole and low temperatures, as well as membrane proteins or non-monomer proteins, protein precipitation can be used as a resin for purification. AeBlueM-Im7 fusion proteins can be obtained by fusing AeBlueM with Im7, and CL7-tagged fusion proteins can be obtained by fusing the target protein with CL7; this is because Im7 and CL7 have a very high affinity (Kd≈10). -14 ~10 -17 By binding with CL7, the precipitated AeBlueM-Im7 can very effectively capture CL7-tagged target proteins. Introducing a protease cleavage site between the target protein and CL7 allows the target protein-CL7 fusion protein to be released from the resin via protease cleavage after capture and washing, resulting in a highly pure target protein.

[0019] The technical solution of this invention has the following beneficial effects: 1. This invention uses the colorimetric protein AeBlueM, which can be expressed at high levels soluble in cells, as an aggregation tag; 2. Aggregated tags can be induced to form irreversible aggregates and precipitates under specific conditions; 3. When expressed in fusion with the target protein, the way the aggregation tag is induced to precipitate does not affect the activity and solubility of the target protein; 4. The target protein can be separated from the precipitated tag by protease cleavage or intima-peptide self-cleavage; 5. When the target protein is affected by factors that induce precipitation, or when the target protein cannot be directly fused with the aggregation tag for expression, an interaction protein pair can be used to fuse the aggregation tag and the target protein with two interacting proteins respectively. Then, through the aggregation tag-induced precipitation and the interaction protein pair, the target protein is anchored to the precipitation formed by the aggregation-induced tag.

[0020] The protein purified using this invention, as analyzed by SDS-PAGE, exhibits high yield and purity of the released target protein, while maintaining its activity. This novel protein purification method eliminates the need for traditional resin preparation, is simple and efficient, easily scalable, low-cost, and more environmentally friendly, being a biodegradable system. Attached Figure Description

[0021] Figure 1. Structure of the AeBlueM-HRV3Cs-mCherry-pET28b expression plasmid. Figure 2 Here is the structure diagram of the AeBlueM-Im7-pET28b expression plasmid. Figure 3Structure diagram of the MBP-HRV3Cs-CL7-pET28b expression plasmid Figure 4 The structure diagram of the TrVKORL-HRV3Cs-CL7-pPICZA expression plasmid.

[0022] Figure 5 Purification flowchart.

[0023] Figure 6 Direct purification of MCherry, wherein: (a) Lane 1, cell lysate supernatant containing AeBlueM-HRV3Cs-mCherry; Lane 2, protein precipitate after freezing, centrifugation, and washing; Lane 3, protein supernatant after freezing and centrifugation purification; Lane 4, purified mCherry protein. M, protein molecular weight standard. An asterisk (*) indicates the location of the target protein mCherry. (b) Molecular sieve chromatography profile of mCherry purified by direct method; (c) Comparison of fluorescence spectra of mCherry purified by direct method and mCherry purified by nickel column.

[0024] Figure 7 Indirect purification of MBP: Lane 1, AeBlue-Im7 particles frozen with imidazole; Lane 2, cell lysate supernatant containing MBP-CL7; Lane 3, AeBlue-Im7 particle and MBP-CL7 protein complex; Lane 4, purified MBP protein; M, protein molecular weight standard.

[0025] Figure 8 Indirect purification of superfolded green fluorescent protein-bound vitamin K epoxide oxidoreductase-like protein (TrVKORL): Lane 1, AeBlue-Im7 particles; Lane 2, TrVKORL-CL7 cellular fraction dissolved in DDM; Lane 3, AeBlue-Im7 particles and TrVKORL-CL7 complex; Lane 4, purified TrVKORL; M, protein molecular weight standard. An asterisk (*) indicates the location of the target protein. Detailed Implementation

[0026] The present invention will be further explained below with reference to the embodiments, but the embodiments do not limit the present invention in any way.

[0027] Example 1 Construction of expression plasmid (1) Construction of expression plasmids by direct method Using mCherry as the target protein for purification, HRV3C protease was used as an example. DNA encoding AeBlueM, the HRV3C protease recognition site, and mCherry was used to obtain DNA encoding the AeBlueM-HRV3Cs-mCherry fusion protein via overlap extension PCR, the nucleotide sequence of which is shown in SEQ ID NO. 5. Then, this DNA was cloned into the multiple cloning site of the pET28b vector using homologous recombination to obtain the AeBlueM-HRV3Cs-mCherry-pET28b expression plasmid, as shown below. Figure 1 As shown. The primer sequences used are shown in SEQ ID NO.6-11, and the steps are as follows: a. Perform PCR reactions as shown in Table 1 to obtain fragment 1 encoding the AeBlueM and HRV3C protease recognition sites: Table 1 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.7 (10 μM) 0.5 AeBlueM template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. Target fragment 1 was determined by agarose gel electrophoresis.

[0028] PCR reactions were performed as shown in Table 2 to obtain fragment 2 encoding the HRV3C protease recognition site and mCherry: Table 2 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.8 (10 μM) 0.5 Primer SEQ ID NO.9 (10 μM) 0.5 mCherry template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. Target fragment 2 was determined by agarose gel electrophoresis.

[0029] PCR was performed as shown in Table 3 to obtain DNA fragment 3 encoding the AeBlueM-HRV3Cs-mCherry fusion protein: Table 3 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.9 (10 μM) 0.5 Segment 1 0.5 Segment 2 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 3, encoding the AeBlueM-HRV3Cs-mCherry fusion protein, was identified by agarose gel electrophoresis.

[0030] The PCR reaction was performed as shown in Table 4 to obtain plasmid backbone fragment 4 of the pET28b vector: Table 4 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.10 (10 μM) 0.5 Primer SEQ ID NO.11 (10 μM) 0.5 pET28b template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 3 minutes and 30 seconds, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 4 was identified by agarose gel electrophoresis. b. Take 3 μL of fragment 3 and 3 μL of fragment 4, and mix well. Add 2 μL to 40 μL of DH5α competent cells and incubate on ice for 30 minutes. Then heat shock at 42°C for 45 seconds and place on ice for 2 minutes. Add 200 μL of LB medium and incubate at 37°C for 45 minutes. Spread the bacterial culture evenly on LB plates containing kanamycin and incubate at 37°C for 14-16 hours.

[0031] c. Select 1-5 single clones, resuspend them in 20 μL of ddH2O, and perform colony PCR as shown in Table 5: Table 5 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.9 (10 μM) 0.5 Monoclonal colony suspension 1 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, 18 cycles; 72°C for 5 minutes; store at 16°C. Positive single clones with PCR products of the same size as fragment 3 were identified by agarose gel electrophoresis.

[0032] d. Inoculate the identified positive monoclonal antibodies into 4 ml of LB liquid medium and incubate overnight at 37°C. Extract the plasmid and sequence it to confirm the correct plasmid, thus obtaining the AeBlueM-HRV3Cs-mCherry-pET28b expression plasmid. Figure 1 As shown.

[0033] SEQ ID NO.5 SEQ ID NO.6 ctttaagaaggagatataccAtggcttcactggttaaaaaag SEQ ID NO.7 GGACCTTGAAACAAAACTTCCAAAGAATTCGATTTGTAGAGTTCATCCATGCCctc SEQ ID NO.8 GGAAGTTTTGTTTCAAGGTCCACTGGGATCCGTGAGCAAGGGCGAGGAGG SEQ ID NO.9 ggtggtggtggtgctcgagCTTGTACAGCTCGTCCATG SEQ ID NO.10 CTCGAGCACCACCACCACCACCACCACTGAG SEQ ID NO.11 CATGGTATATCTCCTTCTTAAAGTTAAAC (2) Construction of expression plasmids by indirect method Taking the purification of vitamin K epoxide oxidoreductase-like protein TrVKORL, which is constrained by maltose-binding protein MBP and superfolded green fluorescent protein, as examples, as target proteins, respectively.

[0034] (A) DNA encoding AeBlueM and Im7 was fused by overlap extension PCR to obtain DNA encoding AeBlueM-Im7, the nucleotide sequence of which is shown in SEQ ID NO.3; then, it was cloned into the multiple cloning site of the pET28b vector using homologous recombination to obtain the AeBlueM-Im7-pET28b expression plasmid. Figure 2 As shown. The primer sequences used are shown in SEQ ID NO.6, SEQ ID NO.10-11, NO.12-14, and the steps are as follows: a. Perform PCR reactions as shown in Table 6 to obtain fragment 5 encoding AeBlueM: Table 6 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.12 (10 μM) 0.5 AeBlueM template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 5 was determined by agarose gel electrophoresis.

[0035] The PCR reaction was performed as shown in Table 7 to obtain fragment 6 encoding Im7: Table 7 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.13 (10 μM) 0.5 Primer SEQ ID NO.14 (10 μM) 0.5 Im7 template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. Target fragment 6 was determined by agarose gel electrophoresis.

[0036] PCR was performed as shown in Table 8 to obtain DNA fragment 7 encoding the AeBlueM-Im7 fusion protein: Table 8 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.14 (10 μM) 0.5 Segment 5 0.5 Segment 6 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 7 was determined by agarose gel electrophoresis.

[0037] b. Take 3 μL of fragment 7 and 3 μL of fragment 4, and mix well. Add 2 μL to 40 μL of DH5α competent cells and incubate on ice for 30 minutes. Then heat shock at 42°C for 45 seconds and place on ice for 2 minutes. Add 200 μL of LB medium and incubate at 37°C for 45 minutes. Spread the bacterial culture evenly on LB agar plates containing kanamycin and incubate at 37°C for 14-16 hours.

[0038] c. Select 1-5 single clones, resuspend them in 20 μL of ddH2O, and perform colony PCR as shown in Table 8: Table 8 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.6 (10 μM) 0.5 Primer SEQ ID NO.14 (10 μM) 0.5 Monoclonal colony suspension 1 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, 18 cycles; 72°C for 5 minutes; store at 16°C. Positive single clones with PCR products of the same size as fragment 7 were identified by agarose gel electrophoresis.

[0039] d. Inoculate the identified positive monoclonal antibodies into 4 ml of LB liquid medium and incubate overnight at 37°C. Extract the plasmid, and sequence it to confirm the correct plasmid, thus obtaining the AeBlueM-Im7-pET28b expression plasmid. Figure 2 As shown.

[0040] SEQ ID NO.12 agagccaccAGAATTCGATTTGTAGAGTTCATCCATGCCctc SEQ ID NO.13 CAAATCGAATTCTggtggctctagtattagtgattacacag SEQ ID NO.14 ggtggtggtggtgctcgaggccctgtttaaatcctggc (B1) The DNA encoding the target protein MBP, the HRV3C protease recognition site, and CL7 was fused by overlap extension PCR to obtain DNA encoding MBP-HRV3Cs-CL7, the nucleotide sequence of which is shown in SEQ ID NO.15; then, it was cloned into the multiple cloning site of the pET28b vector using homologous recombination to obtain the MBP-HRV3cs-CL7-pET28b expression plasmid. Figure 3 As shown. The primer sequences used are shown in SEQ ID NO.10-11, NO.16-19, and the steps are as follows: a. Perform PCR reactions as shown in Table 9 to obtain fragment 8 encoding the MBP and HRV3C protease recognition sites: Table 9 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.16 (10 μM) 0.5 Primer SEQ ID NO.17 (10 μM) 0.5 MBP template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 8 was determined by agarose gel electrophoresis.

[0041] PCR reactions were performed as shown in Table 10 to obtain fragment 9 encoding the HRV3C protease recognition site and CL7: Table 10 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.18 (10 μM) 0.5 Primer SEQ ID NO.19 (10 μM) 0.5 CL7 template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 9 was determined by agarose gel electrophoresis.

[0042] PCR was performed as shown in Table 11 to obtain DNA fragment 10 encoding the MBP-HRV3Cs-CL7 fusion protein: Table 11 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.16 (10 μM) 0.5 Primer SEQ ID NO.19 (10 μM) 0.5 Segment 8 0.5 Segment 9 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 10 was determined by agarose gel electrophoresis.

[0043] b. Take 3 μL of fragment 10 and 3 μL of fragment 4, and mix well. Add 2 μL to 40 μL of DH5α competent cells and incubate on ice for 30 minutes. Then heat shock at 42°C for 45 seconds and place on ice for 2 minutes. Add 200 μL of LB medium and incubate at 37°C for 45 minutes. Spread the bacterial culture evenly on LB agar plates containing kanamycin and incubate at 37°C for 14-16 hours.

[0044] c. Select 1-5 single clones, resuspend them in 20 μL of ddH2O, and perform colony PCR as shown in Table 5: Table 12 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.16 (10 μM) 0.5 Primer SEQ ID NO.19 (10 μM) 0.5 Monoclonal colony suspension 1 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, 18 cycles; 72°C for 5 minutes; store at 16°C. Positive single clones with PCR products of the same size as fragment 10 were identified by agarose gel electrophoresis.

[0045] d. Inoculate the identified positive monoclonal antibodies into 4 ml of LB liquid medium and incubate overnight at 37°C. Extract the plasmid and sequence it to confirm the correct plasmid, namely the MBP-HRV3Cs-CL7 expression plasmid. Figure 3 As shown.

[0046] SEQ ID NO.15 SEQ ID NO.16 GTTTAACTTTAAGAAGGAGAGCAGCTATGCAGCTTAAAATCGAAGAAGGTAAACTG SEQ ID NO.17 GTTGGACCTTGAAACAAAACTTCCAAAGAATTCGAAGTCTGCGCGTCTTTCAG SEQ ID NO.18 GGAAGTTTTGTTTCAAGGTCCAACTGCTGCCGCCGCTagcaaaagcaatgaacc SEQ ID NO.19 GGTGGTGGTGGTGCTCGAGttcaatatcaatgttgcgtttcggggtaacaacgctaat (B2) The DNA encoding the target protein hyperfolded green fluorescent protein constrained vitamin K epoxide oxidoreductase-like protein (TrVKORL), the HRV3C protease recognition site, and CL7 was fused by overlap extension PCR to obtain DNA encoding TrVKORL-HRV3Cs-CL7, the nucleotide sequence of which is shown in SEQ ID NO.20; then, it was cloned into the pPICZA vector by homologous recombination to obtain the TrVKORL-HRV3Ccs-CL7-pPICZA expression plasmid. Figure 4 As shown. The primer sequences used are shown in SEQ ID NO.18, NO. 21-25, and the steps are as follows: a. Perform PCR reactions as shown in Table 13 to obtain fragment 11 encoding the TrVKORL and HRV3C protease recognition sites: Table 13 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.21 (10 μM) 0.5 Primer SEQ ID NO.22 (10 μM) 0.5 TrVKORL template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 11 was determined by agarose gel electrophoresis.

[0047] PCR reactions were performed as shown in Table 14 to obtain fragment 12 encoding the HRV3C protease recognition site and CL7: Table 14 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.18 (10 μM) 0.5 Primer SEQ ID NO.23 (10 μM) 0.5 CL7 template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. Target fragment 12 was obtained by agarose gel electrophoresis.

[0048] PCR was performed as shown in Table 15 to obtain DNA fragment 13 encoding the TrVKORL-HRV3Cs-CL7 fusion protein: Table 15 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.21 (10 μM) 0.5 Primer SEQ ID NO.23 (10 μM) 0.5 Segment 11 0.5 Segment 12 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 13 was determined by agarose gel electrophoresis.

[0049] The PCR reaction was performed as shown in Table 16 to obtain plasmid backbone fragment 14 of the pPICZA vector: Table 16 Components Volume (μL) <![CDATA[ddH2O]]> 8.5 2X Phanta Max Master Mix 10 Primer SEQ ID NO.24 (10 μM) 0.5 Primer SEQ ID NO.25 (10 μM) 0.5 pET28b template DNA (plasmid, 20 ng / μL) 0.5 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 3 minutes and 30 seconds, repeated 18 times; 72°C for 5 minutes; stored at 16°C. The target fragment 14 was determined by agarose gel electrophoresis.

[0050] b. Take 3 μL of fragment 13 and 3 μL of fragment 14, and mix well. Add 2 μL to 40 μL of DH5α competent cells and incubate on ice for 30 minutes. Then heat shock at 42°C for 45 seconds and place on ice for 2 minutes. Add 200 μL of LB medium and incubate at 37°C for 45 minutes. Spread the bacterial culture evenly on LB agar plates containing Zeocin and incubate at 37°C for 14-16 hours.

[0051] c. Select 1-5 single clones, resuspend them in 20 μL of ddH2O, and perform colony PCR as shown in Table 17: Table 17 Components Volume (μL) <![CDATA[ddH2O]]> 8.0 2X Phanta Max Master Mix 10 Primer SEQ ID NO.21 (10 μM) 0.5 Primer SEQ ID NO.23 (10 μM) 0.5 Monoclonal colony suspension 1 Reaction procedure: 95°C pre-denaturation for 3 minutes; 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 1 minute, 18 cycles; 72°C for 5 minutes; store at 16°C. Positive single clones with PCR products of the same size as fragment 13 were identified by agarose gel electrophoresis.

[0052] d. Inoculate the identified positive monoclonal antibodies into 4 ml LB liquid medium containing Zeocin antibiotic and incubate overnight at 37°C. Extract the plasmid and sequence it to confirm the correct plasmid, namely the TrVKORL-HRV3Cs-CL7-pPICZA expression plasmid. Figure 4 As shown.

[0053] SEQ ID NO.20 SEQ ID NO.21 GCTAGCCTCGAGCCACCATGAGTAAAGGAGAAGAACTTTTC SEQ ID NO.22 GGACCTTGAAACAAAACTTCCAAAGAATTCGATTTGTAGAGTTCATCCATGCC SEQ ID NO.23 GGTGATGGTGATGGTGacCGAGttcaatatcaatgttgcg SEQ ID NO.24 GGTCACCATCACCATCACCACCATCACCATCACTAATAGTC SEQ ID NO.25 CATGGTGGCTCGAGGCTAGCTTCG Example 2: Direct purification of mCherry The amino acid sequence of MCherry is shown in SEQ ID NO.26.

[0054] SEQ ID NO.26 MQLSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFNWERVMNFEDGGVVTVTQDSS LQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVDIKLDILSHNEDYTIVEQYERAEGRHSTGGMDELYK The plasmid AeBlueM-HRV3Cs-mCherry-pET28b encoding the fusion protein AeBlueM-HRV3Cs-mCherry constructed in Example 1 (1) is used (e.g.) Figure 1The cells (as shown) were transformed into BL21(DE3) chemocompetent cells and cultured overnight at 37°C on agar plates containing 50 µg / mL kanamycin. The next day, the cells were collected from the plates and inoculated into 500 mL of LB medium containing kanamycin, and cultured at 37°C until the OD600 reached 0.6. The temperature was then adjusted to 25°C, and 0.4 mM IPTG was added to induce protein expression, and the cells were cultured overnight. On the third day, the cells were collected by centrifugation at 3904 xg for 15 minutes. The collected cells were resuspended in 50 mL of lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl), and the cells were lysed by sonication. Cell debris or undiluted cells were then removed by centrifugation at 30,000 xg for 30 minutes at 8°C, yielding a supernatant containing the target protein mCherry.

[0055] Add imidazole to the supernatant to a concentration of 250 mM, mix well, and incubate at -25°C until completely frozen. After complete thawing, centrifuge at 1000 x g for 5 minutes; the target protein should be in the precipitate. Wash the precipitate three times with 10 mL of lysis buffer each time. Then resuspend in 6 mL of lysis buffer, add HRV3C protease, and digest at 4°C to release the target protein mCherry. Detect complete digestion using SDS-PAGE. After complete digestion, centrifuge at 3000 x g for 20 minutes and collect the supernatant to obtain the target protein mCherry. The purification process is as follows: Figure 2 As shown on the left.

[0056] SDS-PAGE results showed that the purity of mCherry reached 81%. Figure 6 As shown in (a); the purified mCherry exhibits a single peak on the molecular surface, indicating high purity, as shown in (a). Figure 6 As shown in (b).

[0057] Example 3: Spectroscopic determination of mCherry The concentration of mCherry protein purified by the direct method was adjusted to 10 μM. mCherry purified by immobilized metal ion chromatography was used as a control. The excitation and emission wavelengths were measured using a Shimadzu RF-6000 fluorescence spectrophotometer.

[0058] The results showed that the excitation and emission spectra of mCherry purified by the direct method and that purified by nickel column were consistent, indicating that the protein purified by this method does not affect the fluorescence properties of mCherry. Figure 6 As shown in (c).

[0059] Example 4: Preparation of protein resin in indirect purification method The plasmid AeBlue-Im7-pET28b encoding the fusion protein AeBlue-Im7 constructed in Example 1(2)A was used (as shown in Example 1(2)A). Figure 2 The cells (as shown) were transformed into BL21(DE3) chemocompetent cells and cultured overnight at 37°C on agar plates containing 50 µg / ml kanamycin. The next day, the cells were collected from the plates and inoculated into 500 mL of LB medium containing kanamycin, and cultured at 37°C until the OD600 reached 0.6. The temperature was adjusted to 25°C, 0.4 mM IPTG was added to induce protein expression, and the culture continued overnight. On the third day, the cells were collected by centrifugation at 3904 xg for 15 minutes. The cells containing AeBlue-Im7 were resuspended in 50 mL of lysis buffer, and the cells were lysed by sonication. Cell debris or undiluted cells were then removed by centrifugation at 30,000 xg for 30 minutes at 8°C, yielding a supernatant containing the AeBlue-Im7 fusion protein.

[0060] Add imidazole to the supernatant to a concentration of 250 mM, mix well, dispense into 10 mL / tubes, and place at -25°C until completely frozen to obtain AeBlue-Im7 protein resin, which can be stored at -80°C for later use.

[0061] Example 5: Indirect purification of maltose-binding protein (MBP) The amino acid sequence of MBP is shown in SEQ ID NO.27.

[0062] SEQ ID NO.27 KIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGV DNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQT The plasmid MBP-HRV3Cs-CL7-pET28b encoding the fusion protein MBP-HRV3Cs-CL7 constructed in Example 1(2)B1 was used (e.g., MBP-HRV3Cs-CL7-pET28b). Figure 3 The cells (as shown) were transformed into BL21(DE3) chemocompetent cells and cultured overnight at 37°C on agar plates containing 50 µg / mL kanamycin. The next day, the cells were collected from the plates and inoculated into 500 mL of LB medium containing kanamycin, and cultured at 37°C until the OD600 reached approximately 0.6. The temperature was then adjusted to 25°C, and 0.4 mM IPTG was added to induce protein expression, followed by overnight culture. On the third day, the cells were collected by centrifugation at 3904 xg for 15 minutes. The collected cells were resuspended in 50 mL of lysis buffer (20 mM Triss, pH 8.0, 150 mM NaCl), and the cells were lysed by sonication. Cell debris or undiluted cells were then removed by centrifugation at 30,000 xg for 30 minutes at 8°C, yielding a supernatant containing the target protein fusion protein.

[0063] Take one frozen sample of AeBlue-Im7 prepared in Example 4, thaw it, and add it to the supernatant above. After incubating at 4°C for 30 minutes, collect the precipitate by centrifugation at 1000 xg for 20 minutes. Wash the precipitate three times with lysis buffer, 10 mL each time. Then resuspend it in 6 mL of lysis buffer, add protease, and digest at 4°C to release the target protein MBP. Detect the completeness of digestion using SDS-PAGE. After complete digestion, centrifuge at 3000 xg for 20 minutes and collect the supernatant to obtain the target protein MBP. The purification process is as follows: Figure 2 As shown on the right. SDS-PAGE results show that the purity of MBP reached 86%, as... Figure 7 As shown.

[0064] Example 6 Purification of superfolded green fluorescent protein-bound vitamin K epoxide oxidoreductase-like protein (TrVKORL) The amino acid sequence of TrVKORL is shown in SEQ ID NO.28. SEQ ID NO.28 MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLRVSTPRWERIARVLVCLLGILLSLYAFHVEREHARDPSYKALCDVSSSISCSKVFGSR WGRGFGLLGSIFGNDSALNQPNSVYGIVFYAFQLLLGMTVSAMAALILMTTSIMSVVGSLYLGYILYFVLKDLCVICVTTYALNFILFVLNYKRLVYLNEAW KQKLQAKQDNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITHGMDELYK The plasmid TrVKORL-HRV3Cs-CL7-pPICZA (e.g., constructed in Example 1(2)B2) encoding TrVKORL-HRV3Cs-CL7) was used to construct Example 1(2)B2. Figure 4 (As shown) The cells were linearized by PmeI restriction enzyme digestion and then electroporated into freshly prepared P. pastoris competent cells. Single colonies with the brightest fluorescence, indicating the highest expression level of the target protein, were selected by fluorescence-detected molecular sieve chromatography. These single colonies were then inoculated into 2 mL of BMG medium (1% ammonium sulfate, 0.4 μg / mL biotin, 1.2% glycerol, 0.34% amino acid-free yeast nitrogen basal source, 100 mM potassium phosphate, pH 6.0) and cultured at 30°C until the OD600 exceeded 20. The medium was then changed to BMM (1% ammonium sulfate, 0.4 μg / mL biotin, 0.34% amino acid-free yeast nitrogen basal source, 200 mM potassium phosphate, pH 6.0, 0.7% methanol). The temperature was adjusted to 25°C, and cultured for 1-3 days, with 5 mL of methanol added per liter of culture every 24 hours to induce protein expression. The yeast cells were then collected at 3000 xg for 15 minutes. The yeast cells were resuspended in 40 mL of lysis buffer, and 5 mL of glass beads (0.1–0.2 µm) were added. The mixture was then homogenized using a cryogenic grinder. After homogenization, the glass beads were removed, and 1.2 g of detergent DDM was added. The mixture was incubated at 4 °C for 3 hours to extract the target membrane protein. Then, the mixture was centrifuged at 30,000 x g for 1 hour at 8 °C, and the supernatant was collected to obtain the target membrane protein TrVKORL.

[0065] Take one frozen sample of AeBlue-Im7 prepared in Example 4, thaw it, and add it to the supernatant above. After incubating at 4°C for 30 minutes, collect the precipitate by centrifugation at 3000 xg for 20 minutes. Wash the precipitate three times with lysis buffer, 10 mL each time. Then resuspend it in 6 mL of lysis buffer, add protease, and digest at 4°C to release the target protein. Detect the completeness of digestion using SDS-PAGE. After complete digestion, centrifuge at 3000 xg for 20 minutes and collect the supernatant to obtain the sfGFP-terminated vitamin K epoxide reductase TrVKORL. The purification procedure is as follows: Figure 2 As shown on the right.

[0066] SDS-PAGE results showed that the purity of TrVKORL reached 84%, such as Figure 8 As shown.

[0067] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of the mutant AeBlueM of AeBlue in protein purification, characterized in that, The amino acid sequence of AeBlueM is shown in SEQ ID NO.

1.

2. The application of vectors including AeBlueM in protein purification, characterized in that, The carrier comprising AeBlueM is selected from the following (1) or (2): (1) Direct carrier: A nucleotide fragment encoding the AeBlueM protease recognition site-target protein was constructed and cloned into the multiple cloning site of the base vector using homologous recombination to obtain the vector. (2) Indirect carriers, including carrier A and carrier B: (2-1) Construct a nucleotide fragment encoding AeBlueM-Im7 and clone it into the multiple cloning site of the basic vector using homologous recombination to obtain vector A; (2-2) Construct a nucleotide fragment encoding the CL7-protease recognition site-target protein and clone it into the multiple cloning site of the base vector using homologous recombination to obtain vector B.

3. The application according to claim 2, characterized in that, (1) The nucleotide sequence of AeBlueM is shown in SEQ ID NO.

2.

4. The application according to claim 2, characterized in that, (2-1) The nucleotide sequence encoding AeBlueM-Im7 is shown in SEQ ID NO.3, and (2-2) The nucleotide sequence of CL7 is shown in SEQ ID NO.

4.

5. The application according to claim 2, characterized in that, The underlying carrier is pET28b or pPICZA.

6. The application according to claim 2, characterized in that, The protease recognition sites are selected from the recognition sites of HRV3C protease, TEV protease, thrombin, and enterokinase.

7. The application according to claim 2, characterized in that, When the vector is (1), the application is as follows: the vector is induced to express to obtain the fusion protein of AeBlueM-protease recognition site-target protein, imidazole is added, mixed, and frozen completely. After thawing, the supernatant is discarded by centrifugation, the precipitate is washed and resuspended, protease is added for digestion, and then the precipitate is discarded by centrifugation. The supernatant is the purified target protein.

8. The application according to claim 2, characterized in that, When the vector is (2), the application is as follows: the vector (2-1) is induced to express to obtain the AeBlueM-Im7 fusion protein, imidazole is added, mixed, and frozen completely. After thawing, the supernatant is discarded by centrifugation, and the precipitate is washed and resuspended to obtain protein resin; the vector (2-2) is induced to express to obtain the CL7-protease recognition site-target protein fusion protein, protein resin is added for incubation, the supernatant is discarded by centrifugation, the precipitate is washed and resuspended, protease is added for digestion, the precipitate is discarded by centrifugation, and the supernatant is the purified target protein.

9. The application according to claim 7 or 8, characterized in that, The protease is selected from HRV3C protease, TEV protease, thrombin, and enterokinase.

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