Renaturation-assisting tag for purifying inclusion body protein, and gene-derived product thereof and use thereof

Abstract

The present application belongs to the technical field of protein purification. Provided in the present application are a renaturation-assisting tag for purifying an inclusion body protein, and a gene-derived product thereof and the use thereof. Provided in the present application is a renaturation-assisting tag P67, comprising at least one of the amino acid sequences as set forth in SEQ ID NO: 1 to SEQ ID NO: 3 and mutants thereof, wherein the mutant has a mutation at position 33 of the amino acid sequence from lysine to glutamic acid or proline. The renaturation-assisting tag P67 according to the present application is fused and expressed with a target protein in a prokaryote, enabling successful renaturation of a recombinantly expressed inclusion body (insoluble inclusion body) and conferring a biological activity thereon. The renaturation-assisting tag P67 establishes a foundation for subsequent protein functional research and a translational use.

Description

A complexing tag for purifying inclusion body proteins, its gene-derived products, and applications.

[0001] This application claims priority to Chinese Patent Application No. 202411867353.1, filed on December 18, 2024, entitled "A recombinant tag for purifying inclusion body proteins and its gene-derived products and applications", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application belongs to the field of protein purification technology, specifically relating to a recombinant tag for purifying inclusion body proteins, its gene-derived products, and applications. Background Technology

[0003] Proteins are the primary executors of life functions, and the successful purification of biologically active proteins is crucial for subsequent protein function research and translational applications. Currently, many proteins have been successfully purified using gene recombination and prokaryotic expression purification systems. The biggest obstacle to successful protein purification is the formation of inclusion bodies (IBs) in recombinant proteins. IB proteins are insoluble during the initial induced expression stage in host bacteria. During subsequent extraction and purification, the addition of denaturing agents (urea and guanidine hydrochloride) causes the IB protein to become soluble. However, the denatured IB must undergo a slow refolding process to remove the denaturing agent, allowing the IB protein to refold correctly and regain its solubility and biological activity. Therefore, the refolding process is particularly important for the successful purification of IB proteins. Based on the ease of refolding, IB proteins can be divided into two categories: refolding-easy inclusion bodies (REIBs) and refolding-resistant inclusion bodies (RRIBs). Among these, the formation of aggregates and precipitation during the refolding process of RRIBs is the main reason for the failure of inclusion body protein purification. Although some studies have found that adding small molecule compounds and altering the physical environment (pressure and pH) can improve the IB refolding process, the results have been minimal. Summary of the Invention

[0004] In view of this, the purpose of this application is to provide a refolding tag that enables denatured inclusion body proteins to refold, thereby achieving purification.

[0005] This application provides a recombinant tag P67, comprising at least one of the following: a PCC tag, a PC tag, an ACCA tag, and a mutant of the PCC tag, PC tag, or ACCA tag; the amino acid sequences of the PCC tag, PC tag, and ACCA tag are shown in SEQ ID NO:1 to SEQ ID NO:3; the mutant of the PCC tag, PC tag, or ACCA tag is based on the amino acid sequence of the PCC tag, PC tag, or ACCA tag, where the lysine at position 33 is mutated to glutamic acid or proline.

[0006] This application provides a gene encoding the recombinant tag P67, wherein the nucleotide sequence of the gene is at least one of the following shown in SEQ ID NO:4 to SEQ ID NO:8.

[0007] This application provides a prokaryotic protein expression tool plasmid comprising a nucleic acid molecule expressing the helper tag P67 or the encoding gene.

[0008] Preferably, the pET41a vector is used as the backbone vector, wherein the GST tag is replaced by the coding gene.

[0009] Preferably, it also includes HRV-3C protease recognition cleavage site or TEV protease recognition site.

[0010] This application provides a prokaryotic recombinant vector for expressing a target protein, which uses the prokaryotic protein expression tool plasmid as a backbone vector and also includes the target protein gene;

[0011] The cloning site of the target protein gene is between the NcoI and XhoI multiple cloning sites or between the BamHI and XhoI multiple cloning sites.

[0012] This application provides a fusion protein, which is obtained by fusing the fusion tag P67 and the target protein.

[0013] This application provides a fusion gene, which is obtained by tandemly connecting the coding gene and the target gene.

[0014] This application provides the application of the assisted recombinant tag P67, the encoding gene, the prokaryotic protein expression tool plasmid, the prokaryotic recombinant vector, the fusion protein, or the fusion gene in inclusion body purification.

[0015] This application provides a method for purifying inclusion body proteins, comprising the following steps:

[0016] The target protein gene was cloned into the prokaryotic protein expression tool plasmid to obtain a recombinant vector;

[0017] The recombinant vector was transformed into competent cells of a prokaryotic expression system, and inclusion body proteins were isolated after culture and induction culture of the target protein.

[0018] The inclusion body protein was denatured and dissolved to obtain denatured and dissolved inclusion body protein;

[0019] The denatured and dissolved inclusion body protein was refolded by dialysis to obtain purified inclusion body protein.

[0020] This application provides a refolding aid P67, with an amino acid sequence of at least one shown in SEQ ID NO:1 to SEQ ID NO:3, wherein the 33rd amino acid is glutamic acid or proline. This application fuses the refolding aid P67 with a target protein in a prokaryotic expression system, resulting in a fusion protein that is an inclusion body protein carrying P67. The inclusion body protein carrying P67 is denatured, dissolved, and then refolded in a urea gradient concentration refolding solution. More than 68% of the inclusion body protein regains its solubility, achieving purification. In contrast, inclusion bodies without P67 are partially or completely lost in PBS dialysis solution, indicating refolding failure. Therefore, the refolding aid P67 can effectively improve the refolding of inclusion bodies (difficult-to-refold inclusion bodies), laying the foundation for subsequent protein function research and application transformation. Furthermore, the refolding aid P67 is currently the smallest molecular weight refolding aid disclosed, which is beneficial for the expression of fusion proteins and is not limited by carrier capacity. Furthermore, the fusion expression of the complexing tag P67 with the target protein allows the resulting fusion protein to be readily recognized by biotin ligases in host cells (eukaryotes) or bacteria (prokaryotes), covalently linking biotin to the lysine side chain of the MKM sequence in the complexing tag P67, thus forming a biotinylated modification. The biotinylated fusion protein can then be purified using the highly specific and stable biotin-avidin system under denaturing conditions. Therefore, the complexing tag P67 provided in this application can be widely used in the purification of inclusion body proteins. Attached Figure Description

[0021] Figure 1 shows the origin and spatial structure analysis of the P67 tag. AE represents a conserved domain shared by five eukaryotic carboxylases, capable of biotinylation modification; the structure within the black dashed circle represents the spatial structure of the P67 tag. F represents P67, consisting of eight antiparallel β-sheets distributed on either side of the central MKM sequence. Note: Lysine K in MKM is the biotinylation site. The β-sheets are formed by the sequences with gray backgrounds accompanied by eight arrows. ACCA: Acetyl-CoA carboxylase A; ACCB: Acetyl-CoA carboxylase B; PC: Pyruvate carboxylase; PCC: Propionyl-CoA carboxylase; MCC: Methylcrotonyl-CoA carboxylase.

[0022] Figure 2 shows the structural diagrams of the tool plasmid and control plasmid used to express fusion proteins with and without the P67 tag, respectively. A. Full sequence diagram of the tool plasmid pET-41a-P67-MCS; B. Schematic diagram of the tool plasmid used to express the P67-tagged fusion protein at the T7 promoter and multiple cloning site; C. Schematic diagram of the structure of the vector constructed using the control plasmid pET-41a-MCS expressing the P67-tagged fusion protein; D. Schematic diagram of the sequence structure of the HRV-3C protease cleavage site and the MCS multiple cloning site.

[0023] Figure 3 shows the results of SDS-PAGE combined with Coomassie blue staining to observe the induction and lysis of inclusion body protein Gal-12; where A is the result of IPTG-induced expression in host bacterial culture with P67 tag; and B is the result of IPTG-induced expression in host bacterial culture without P67 tag.

[0024] Figure 4 shows the dialysis refolding results of inclusion body protein Gal-12 detected by Coomassie blue staining. A. Dialysis refolding results of Gal-12 protein with the P67 tag; B. Dialysis refolding results of Gal-12 protein without the P67 tag; C. Quantitative curves of the gray values ​​of the two target protein bands in the above dialysis refolding results, where the gray curve is the refolding curve of Gal-12 protein with the P67 tag; and the black curve is the refolding curve of Gal-12 protein without the P67 tag.

[0025] Figure 5 shows the results of induction and lysis of inclusion body protein Int-2 detected by Coomassie blue staining. In Figure A, the expression results of host bacterial culture with P67 tag induced by IPTG are shown, and in Figure B, the expression results of host bacterial culture without P67 tag induced by IPTG are shown.

[0026] Figure 6 shows the dialysis refolding results of inclusion body protein Int-2 observed by SDS-PAGE combined with Coomassie blue staining. A. Dialysis refolding results of Int-2 protein with the P67 tag; B. Dialysis refolding results of Int-2 protein without the P67 tag; C. Quantitative curves of the gray values ​​of the two target protein bands in the above dialysis refolding results, with the gray curve representing the refolding curve of Int-2 protein with the P67 tag and the black curve representing the refolding curve of Int-2 protein without the P67 tag.

[0027] Figure 7 shows the results of SDS-PAGE combined with Coomassie blue staining to observe the induction and lysis of inclusion body protein Gal-8S; where A is the result of IPTG-induced expression in host bacterial culture with P67 tag; and B is the result of IPTG-induced expression in host bacterial culture without P67 tag.

[0028] Figure 8. Dialysis refolding results of inclusion body protein Gal-8S observed by SDS-PAGE combined with Coomassie blue staining; where A. Dialysis refolding results of Gal-8S protein with P67 tag; B. Dialysis refolding results of Gal-8S protein without P67 tag; C. Quantitative curves of gray values ​​of the two target protein bands in the above dialysis refolding results, where the gray curve is the refolding curve of Gal-8S protein with P67 tag; the black curve is the refolding curve of Gal-8S protein without P67 tag;

[0029] Figure 9 shows the tag removal results of the fused inclusion body protein after refolding, observed by SDS-PAGE combined with Coomassie blue staining; where A is a schematic diagram of the removal of the N-terminal extra tag of the fusion protein using HRV 3C protease; B and D are the purification results of the three fusion proteins after digestion with 3C protease, with black arrows indicating tagged fusion proteins and gray arrows indicating the target protein after tag removal; E is the supernatant protein Int-2 solution after 3C digestion flowing through a nickel column (used to remove the 3C protease carrying the His tag and the N-terminal extra tag), and the effluent (Ni F lane) is collected to obtain the final target protein Int-2; black arrows indicate tagged fusion proteins; gray arrows indicate the target protein after tag removal; 3C protease MW: 22KD; N-terminal tag MW: 13.4KD;

[0030] Figure 10 shows the functional detection results of the fusion inclusion body protein after successful refolding. Column 1 is the control column, where no active protein was added, and the chicken blood cells agglutinated at the bottom, resulting in a clear solution. Columns 2-10 are the test columns.

[0031] Figure 11 shows the detection results of purifying refractory inclusion body proteins using P67 tags from other eukaryotic carboxylases; A. Detection results of P67 tags from 5 eukaryotic carboxylases; B. Quantitative results from electrophoresis, with black arrows indicating tagged fusion proteins.

[0032] Figure 12 shows a comparison of the purification results of refractory inclusion body proteins using different tags; where A represents the purification results of inclusion body proteins with different tags; B represents the quantitative results of electrophoresis; and black arrows indicate tagged fusion proteins.

[0033] Figure 13 shows the functional verification results of the P67 tag after the "MKM" was mutated into "MEM" and "MPM", where A. primary structure of the P67 tag and its mutant tags; B. DNA sequencing results; E. purification results of P67 tags with "MKM", "MEM" and "MPM"; F. quantitative results of electrophoresis.

[0034] Figure 14 shows the results of purification of the non-reducible inclusion body protein expressed by this system using a streptavidin chromatography column;

[0035] Figure 15 shows the results of soluble protein purification using streptavidin chromatography and nickel chromatography (Ni) columns, respectively. Detailed Implementation

[0036] This application provides a recombinant tag P67, comprising at least one of the following: a PCC tag, a PC tag, an ACCA tag, and a mutant of the PCC tag, PC tag, or ACCA tag. The amino acid sequence of the PCC tag is as shown in SEQ ID NO:1.

[0037] (LRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGTVKSVHCQAGDTVGEGDLLVELE); the amino acid sequence of the PC tag is as shown in SEQ ID NO:2 (IGAPMPGKVIDIKVVAGAKVAKGQPLCVLSAMKMETVVTSPMEGTVRKVHVTKDMTLEGDDLILEIE); the amino acid sequence of the ACCA tag is as shown in SEQ ID NO:3 (MRSPSAGKLIQYIVEDGGHVFAGQCYAEIEVMKMVMTLTAVESGCIHYVKRPGAALDPGCVLAKMQL), and the mutant of the PCC tag, PC tag or ACCA tag is based on the amino acid sequence of the PCC tag, PC tag and ACCA tag, with the lysine at position 33 mutated to glutamic acid or proline.

[0038] In this application, the synergistic tag P67 is obtained by cutting out a conserved domain that can undergo biotinylation modification from the sequence of a eukaryotic carboxylase. P67 consists of eight antiparallel β-sheets distributed on both sides of the middle MKM sequence. The lysine K in MKM is the biotinylation site, as shown in Figure 1. This application screened inclusion body proteins for refolding ability of eukaryotic carboxylases, including acetyl-CoA carboxylase A (ACCA), acetyl-CoA carboxylase B (ACCB), pyruvate carboxylase (PC), propionyl-CoA carboxylase (PCC), and methylcrotonyl-CoA carboxylase (MCC). The results showed that tag P67 derived from ACCA, PC, and PCC has similar refolding function, while tag P67 derived from ACCB (LRSPSAGKLTQYTVEDGGHVEAGSSYAEMEVMKMIMTLNVQERGRVKYIKRPGAVLEAGCVVARLEL, SEQ ID) NO:13) and MCC-derived label P67 (PLAPMTGTIEKVFVKAGDKVKAGDSLMVMIAMKMEHTIKSPKDGTVKKVFYREGAQANRHTPLVEFE, SEQ ID NO:14) do not have good rehabilitative function.

[0039] In this application, the eukaryotic carboxylase sequence contains the MKM sequence, which is easily recognized by biotin ligases of host cells (eukaryotes) or bacteria (prokaryotes). Biotin is covalently linked to the lysine side chain of the MKM sequence in the recoverable tag P67, forming a biotinylated modification. The biotinylated fusion protein can be purified under denaturing conditions using the highly specific and stable biotin-avidin system. To verify whether the biotinylation of the lysine (K) in the MKM sequence affects the recoverable function of the recoverable tag P67, this application mutated "MKM" in tag P67 to "MEM" and "MPM." The resulting tag P67 still exhibited good recoverable function, indicating that the aforementioned amino acid mutation does not affect the recoverable effect of tag P67.

[0040] This application provides a gene encoding the p67 auxiliary tag.

[0041] In this application, the gene encoding ACCA's P67 is:

[0042] The gene encoding P67 in PC:

[0043] The gene encoding P67 in PCC:

[0044] The gene encoding P67 MEM:

[0045] Ctgcgttccccgatgcccggagtggtggtggccgtctctgtcaagcctggagacgcggtagcagaaggtcaagaaatttgtgtgattgaagccatggaaatg cagaatagtatgacagctgggaaaactggcacggtgaaatctgtgcactgtcaagctggagacacagttggagaaggggatctgctcgtggagctggaa(SEQ ID NO:7), where the underline is the mutation site;

[0046] P67 MPM encoding gene:

[0047] Ctgcgttccccgatgcccggagtggtggtggccgtctctgtcaagcctggagacgcggtagcagaaggtcaagaaatttgtgtgattgaagccatgcctatgcagaatagtatgacagctgggaaaactggcacggtgaaatctgtgcactgtcaagctggagacacagttggagaaggggatctgctcgtggagctggaa(SEQ ID NO:8), where the underlined sites are mutation sites;

[0048] The gene encoding P67 in ACCB:

[0049] The gene encoding P67 in MCC:

[0050] In this embodiment, a reversibility test for inflammation using a control tag was also conducted, wherein the amino acid sequence of the Avi tag is GLNDIFEAQKIEWHE (SEQ ID NO:11). The amino acid sequence of the Trx tag is SDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQ NPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA (SEQ ID NO:12).

[0051] This application provides a prokaryotic protein expression tool plasmid containing the encoding gene of the synergistic tag P67 or the encoding gene itself.

[0052] In this application, the coding gene is preferably obtained by sequence optimization based on the codon preference of Escherichia coli.

[0053] In this application, the prokaryotic protein expression tool plasmid preferably uses the pET41a vector as the backbone vector, wherein the GST tag is replaced by the coding gene. The cloning site of the coding gene is preferably between the NdeI and SpeI restriction enzyme sites. The method for constructing the prokaryotic protein expression tool plasmid preferably involves connecting one end of the coding gene of the synergistic tag P67 to a sticky end of an NdeI restriction endonuclease (SEQ ID NO:15, TATTGTCGGATGGTTCAA) and the other end to a sticky end of a SpeI restriction endonuclease (SEQ ID NO:16, CTAGTTGAACCATCCGACA). The resulting coding gene of the synergistic tag P67 with restriction enzyme sites and the backbone vector are digested with NdeI and SpeI restriction endonucleases, respectively. The products are then ligated to obtain the prokaryotic protein expression tool plasmid, denoted as pET41a-P67-MCS.

[0054] In this application, the prokaryotic protein expression tool plasmid preferably further includes an HRV-3C protease recognition cleavage site or a TEV protease recognition site. The HRV-3C protease recognition cleavage site is LEVLFQ↑GP (SEQ ID NO:17), and the TEV protease recognition site is ENLYFQ↑S (SEQ ID NO:18), where ↑ represents the protease cleavage site. The pET41a vector originally carried thrombin and enterokinase recognition sites. However, due to the low specificity of thrombin (recognition site LVPR↑GS, SEQ ID NO:19) and enterokinase (recognition site DDDD↑K, SEQ ID NO:20), which recognize 6 and 5 consecutive amino acid conserved sequences respectively, non-specific cleavage of the target protein is easily caused when using these enzymes to remove the tag later. This application replaces the HRV-3C protease recognition cleavage site or the TEV protease recognition site, improving the specificity of the cleavage and helping to avoid non-specific cleavage of the target protein. The entire genome synthesis for the aforementioned vector modifications was outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0055] This application provides a prokaryotic recombinant vector for expressing a target protein, which uses the prokaryotic protein expression tool plasmid as a backbone vector and also includes the target protein gene; the cloning site of the target protein gene is between the NcoI and XhoI multiple cloning sites or between the BamHI and XhoI multiple cloning sites.

[0056] In this application, the method for constructing the prokaryotic recombinant vector preferably involves double digesting the target protein gene with BamHI and XhoI multiple cloning sites at both ends and the prokaryotic protein expression tool plasmid with BamHI and XhoI restriction endonucleases, respectively, and then ligating the digestion products to obtain the prokaryotic recombinant vector.

[0057] In this application, the target protein gene includes at least one of the following proteins or truncated versions of the gene: Int-2 protein (SEQ ID NO:21), Gal-8S protein (SEQ ID NO:22), and Gal-12 protein (SEQ ID NO:23). The amino acid sequence of the truncated version of the Gal-12 protein is shown in SEQ ID NO:24.

[0058] In one embodiment of this application, the inclusion body protein obtained by recombination expression in Escherichia coli using a prokaryotic recombinant vector was successfully refolded by the action of the refolding tag P67, while the inclusion body protein without the refolding tag P67 was completely lost in PBS dialysis solution, indicating that the refolding failed.

[0059] In one embodiment of this application, in order to further verify that the successful refolding of the target protein is affected by the refolding tag P67, this application uses the 3C protease cleavage site to remove the tag sequence of the target protein, and finds that the untagged target protein is partially or completely lost during the refolding process. This indicates that the P67 tag is crucial for maintaining the soluble state of the target protein.

[0060] In another embodiment of this application, to verify the activity of the successfully renatured target protein, three target protein activity assays were conducted. The results showed that P67-Int-2 and P67-Gal-8S possessed biological activity and could induce chicken blood agglutination, while P67-Gal-12 did not exhibit biological activity. This application further recombinantly expressed the truncated form of P67-Gal-12 (the N-terminal sequence of Gal-12) to obtain inclusion body protein, which was then successfully purified through denaturation and renaturation to obtain the biologically active target protein. This indicates that the C-terminal structure of Gal-12 may hinder protein function.

[0061] This application provides a fusion protein, which is obtained by fusing the fusion tag P67 and the target protein.

[0062] This application provides a fusion gene, which is obtained by tandemly connecting the coding gene and the target gene.

[0063] This application provides the application of the assisted recombinant tag P67, the encoding gene, the prokaryotic protein expression tool plasmid, the prokaryotic recombinant vector, the fusion protein, or the fusion gene in inclusion body purification.

[0064] This application provides a method for purifying inclusion body proteins, comprising the following steps:

[0065] The target protein gene was cloned into the prokaryotic protein expression tool plasmid to obtain a recombinant vector;

[0066] The recombinant vector was transformed into competent cells of a prokaryotic expression system, and inclusion body proteins were isolated after culture and induction of the target protein.

[0067] The inclusion body protein was denatured and dissolved to obtain denatured and dissolved inclusion body protein;

[0068] The denatured and dissolved inclusion body protein was refolded by dialysis to obtain purified inclusion body protein.

[0069] This application clones the target protein gene into the prokaryotic protein expression tool plasmid to obtain a recombinant vector.

[0070] In this application, the cloning method is the same as the method for constructing a prokaryotic recombinant vector expressing the target protein in the above-mentioned technical solutions, and will not be described in detail here.

[0071] After obtaining the recombinant vector, this application transforms the recombinant vector into competent cells of a prokaryotic expression system, and after culture and induction of the target protein, isolates inclusion body proteins.

[0072] This application does not impose any particular limitation on the transformation method, and transformation schemes well known in the art are adopted. In the embodiments of this application, the transformation is performed by heat shock. The prokaryotic expression system is preferably *Escherichia coli*. The competent *E. coli* cells can be BL21(DE3)PLysS (Weidi Bio, #EC1003). The culture is preferably performed by inoculating the bacterial strain in LB medium and culturing at 37°C and 225 rpm. The culture is preferably expanded to the logarithmic growth phase. The conditions for the expansion culture are the same as above and will not be repeated here. The induction agent is preferably IPTG. The induction concentration of IPTG is preferably 1 mM. The induction time is preferably 3-5 h, and can be 4 h.

[0073] After obtaining the inclusion body protein, this application denatures and dissolves the inclusion body protein to obtain denatured and dissolved inclusion body protein.

[0074] In this application, the denaturation and dissolution method preferably involves sequentially placing the inclusion body protein in buffer A, buffer B, and buffer C to separate the precipitate, then mixing the precipitate with buffer D to dissolve it, collecting the supernatant to obtain the denatured and dissolved inclusion body protein. Buffer A is preferably an aqueous solution comprising the following amounts: 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 100 mM NaCl, and 1% Triton X-100 (v / v). Buffer B is preferably an aqueous solution comprising the following amounts: 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 100 mM NaCl, 1% Triton X-100 (v / v), and 2 M urea. Buffer C is preferably an aqueous solution comprising the following amounts: 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 100 mM NaCl, 1% Triton X-100 (v / v), and 2 M guanidine hydrochloride. The buffer solution D is preferably an aqueous solution comprising the following contents: 50 mM Tris-HCl (pH 8.5), 1 mM EDTA, 100 mM NaCl, 10 mM DTT, 2 mM sodium deoxycholate, and 8 M urea.

[0075] After obtaining the denatured and dissolved inclusion body protein, this application performs dialysis refolding on the denatured and dissolved inclusion body protein to obtain purified inclusion body protein.

[0076] In this application, the dialysis refolding solution is an aqueous solution containing a gradient concentration of urea, preferably comprising the following components: 50 mM Tris-HCl (pH 8.5), 100 mM NaCl, gradient concentrations of urea, 1% glycine, 5% glycerol, 0.2% PEG (Mr3550), 1 mM oxidized glutathione, and 1 mM reduced glutathione. The gradient concentrations of urea are preferably 6 M, 4 M, 2 M, and 1 M. The denatured and dissolved inclusion body proteins are dialyzed sequentially according to the urea concentration from high to low. The dialysis time is 6 hours, and the dialysis temperature is preferably 4°C. The molecular weight cutoff of the dialysis bag is selected according to the size of the target protein.

[0077] In this application, after dialysis refolding, the assay preferably further includes protein content determination and activity determination. The protein content determination method preferably employs Coomassie blue staining. The activity determination method preferably employs a chicken blood agglutination test.

[0078] The following detailed description, in conjunction with embodiments, illustrates a recombinant tag for purifying inclusion body proteins, its gene-derived products, and applications provided in this application. However, these descriptions should not be construed as limiting the scope of protection of this application.

[0079] Example 1

[0080] A method for constructing plasmids for prokaryotic protein expression.

[0081] The GST tag on the pET41a vector (Novagen, #71071) was replaced with a P67 tag (LRSPMPGVVVAVSVKPGDAVAEGQEICVIEAMKMQNSMTAGKTGTVKSVHCQAGDTVG EGDLLVELE, SEQ ID NO:1), and the enterokinase site was modified to an HRV-3C protease cleavage site (LEVLFQ↑GP, SEQ ID NO:17, where ↑ indicates the cleavage site), resulting in the prokaryotic protein expression tool plasmid pET-41a-P67-MCS (SEQ ID NO:31). The entire gene synthesis for the above vector modification was outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0082] The multiple cloning site MCS on the tool plasmid pET-41a-P67-MCS is used for the insertion of the target protein coding sequence to form an expression vector; the HRV-3C protease cleavage site upstream of the MCS is used for the removal of the N-terminal redundant tag (AB in Figure 2).

[0083] This embodiment also constructed a control plasmid (pET-41a-MCS, SEQ ID NO:32). The construction method differs from that of the tool plasmid pET-41a-P67-MCS only in that it does not contain the cloning P67 tag sequence (C in Figure 2). The whole genome synthesis of the control plasmid was outsourced to Sangon Biotech (Shanghai) Co., Ltd.

[0084] Example 2

[0085] To verify the function of the P67 tag, this embodiment constructed six vectors for expressing three human RRIBs, including three expression vectors with the P67 tag and three control vectors without the P67 tag. Specific information on the target protein and vectors is shown in Table 1.

[0086] Table 1. Prokaryotic expression recombinant vectors and control plasmids used

[0087] The construction method includes the following steps:

[0088] 1. Primer sequence

[0089] The primers used to amplify the Intelectin-2 gene are as follows:

[0090] Int-2 BamHI FP: AATCGGGGATCCACCTGTGCGTTTAGCTTTA (SEQ ID NO: 25);

[0091] Int-2 XhoI RP: GTGGTGCTCGAGTTAACGGTAAAACAGCAGAACT (SEQ ID NO: 26);

[0092] The primers used to amplify the Galectin-12 gene are as follows:

[0093] Gal-12 NcoI FP:CCCAGTCCCATGGAGTCAACCGTCTGGCGGTC (SEQ ID NO: 27);

[0094] Gal-12 EcoRI RP: GCCTGTACAGAATTCTTAGCTATGAACGCAATACAGC (SEQ ID NO: 28);

[0095] The primers used to amplify the Galectin-8S gene are as follows:

[0096] Gal-8S BamHI FP: AATCGGGGATCCATGATGCTGAGCCTGAAC (SEQ ID NO: 29);

[0097] Gal-8S XhoI RP: GTGGTGCTCGAGTTACCAACTACGAACTTCCAG (SEQ ID NO: 30).

[0098] Using the ORF of the target protein encoding gene as a template, PCR amplification was performed using the primers containing the above-mentioned restriction sites to obtain the Int-2, Gal-12 and Gal-8S gene sequences. The PCR amplification system was 50 μL (including 25 μL of 2×PCR buffer, 1 μL each of 10 μM forward and reverse primers, 1 μL of template, and water added to make up to 50 μL). The PCR amplification program was 95℃ for 4 min, (95℃ for 10 s, 65℃ for 30 s, 72℃ for 1 min) for 35 cycles, and 72℃ for 5 min to obtain the PCR product.

[0099] The PCR product and the tool plasmid pET-41a-P67-MCS or the control plasmid pET-41a-MCS were double-digested with BamHI (NEB, #R3136) and XhoI (NEB, #R0146) restriction endonucleases in a 50 μL reaction volume (including 5 μL of 10× digestion buffer, 1 μL of each restriction endonuclease, 10 μg of template plasmid, and water to a final volume of 50 μL). The reaction program was 37 °C for 1 hour to obtain the PCR product with sticky ends and the linear tool plasmid pET-41a-P67-MCS or the control plasmid. A 10 μL ligation reaction volume (NEB, #M0202; including 1 μL of 10× digestion buffer, 1 μL of T4 DNA ligase, 1 μg of template plasmid, 0.5 μg of PCR digestion product, and water to a final volume of 10 μL) was prepared and ligated at room temperature for 2 hours to obtain the ligation product.

[0100] The ligation product was transformed into competent *E. coli* cells BL21(DE3)PLysS (Weidi Bio, #EC1003) using a heat shock method and cultured overnight at 37°C. Colony PCR identification was performed using the primers described above, with the following reaction program: 95°C for 4 min, (95°C for 10 s, 65°C for 30 s, 72°C for 1 min) for 35 cycles, followed by 72°C for 5 min. Agarose gel electrophoresis was performed on the amplified products; the expected band size indicated successful transformation. Plasmid DNA sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd., and no mutations were found.

[0101] Example 3

[0102] Recombinant expression and purification of target protein

[0103] 1. Recombinant expression methods for target proteins

[0104] Recombinant bacteria that were successfully transformed from the three target genes in Example 2 were selected and inoculated into 2 ml of LB liquid medium containing the corresponding antibiotics for activation and culture overnight. The temperature of the constant temperature shaker was set to 37°C and the rotation speed was 225 rpm.

[0105] Add 0.6 ml of overnight host bacteria to each Erlenmeyer flask containing 50 ml of LB medium (antibiotic-free) (5 times the volume of the culture medium), and incubate at 37°C on a shaker until OD (out of control) is reached. 600 It is 0.6, approximately 3 hours.

[0106] Add IPTG to the culture flask to achieve a final IPTG concentration of 1 mM, and incubate at 37°C in a shaker for 4 h to induce protein expression.

[0107] 2. Denaturation and lysis of host bacterial and inclusion body proteins

[0108] Centrifuge 5000g of the induced bacterial culture for 10 min, discard the supernatant, and retain the bacterial precipitate.

[0109] Resuspend the bacterial pellet in buffer A (5 ml of buffer A for every 100 ml of centrifuged LB culture), sonicate the bacterial suspension (45% power, 2 s on, 2 s off, 10 min on sonication), and centrifuge at 10,000 g for 10 min. Wash the inclusion body pellet collected by centrifugation once with PBS buffer (using a syringe to aspirate repeatedly), centrifuge, discard the supernatant, and collect the pellet.

[0110] Resuspend the precipitate in buffer B (by repeatedly aspirating the precipitate with a syringe), and then sonicate for 5 min under the same conditions; after sonication, centrifuge at 10,000 g for 10 min, discard the supernatant, and collect the inclusion body precipitate.

[0111] Sonicate with buffer C for 3 min to wash the inclusion body precipitate (repeatedly aspirate the precipitate with a syringe); centrifuge at 10,000g for 10 min to collect the precipitate.

[0112] Dissolve the inclusion bodies with buffer D (repeatedly aspirate the precipitate with a syringe) to allow the inclusion bodies to dissolve slowly. After incubation at room temperature for 30 minutes, centrifuge at 10,000g for 10 minutes. The supernatant is the denatured and dissolved inclusion body protein.

[0113] The concentration of dissolved inclusion body protein was determined using the BCA protein quantification kit; the protein concentration was then adjusted to 0.1–1.0 mg / ml.

[0114] 3. Dialysis refolding of inclusion body proteins

[0115] The prepared protein solution was placed into a dialysis bag and then placed in a beaker containing refolding buffer (containing 6M urea) (with a volume of at least 500 times the dialysis volume). Dialysis was performed slowly at 4°C for 6 hours.

[0116] Transfer the dialysis bag to refolding buffer (containing 4M urea) and slowly dialyze at 4°C for 6 hours.

[0117] Transfer the dialysis bag to refolding buffer (containing 2M urea) and dialyze slowly at 4°C for 6 hours.

[0118] Transfer the dialysis bag to refolding buffer (containing 1M urea) and dialyze slowly at 4°C for 6 hours.

[0119] Finally, place the dialysis bag in PBS and dialyze slowly overnight at 4°C.

[0120] Protein purity was determined by SDS-PAGE combined with Coomassie blue staining.

[0121] The solutions required for the induction, denaturation, dissolution, and dialysis refolding of inclusion body proteins in the above experiments are as follows:

[0122] PBS: 137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 1.8mM KH2PO4;

[0123] Buffer A: 50mM Tris-HCl (pH 8.5), 1mM EDTA, 100mM NaCl, 1% Triton X-100;

[0124] Buffer B: 50mM Tris-HCl (pH 8.5), 1mM EDTA, 100mM NaCl, 1% Triton X-100, 2M urea;

[0125] Buffer C: 50mM Tris-HCl (pH 8.5), 1mM EDTA, 100mM NaCl, 1% Triton X-100, 2M guanidine hydrochloride;

[0126] Buffer D: 50mM Tris-HCl (pH 8.5), 1mM EDTA, 100mM NaCl, 10mM DTT, 2mM sodium deoxycholate, 8M urea;

[0127] Refolding buffer: 50mM Tris-HCl (pH 8.5), 100mM NaCl, containing 6M, 4M, 2M, and 1M urea, 1% glycine, 5% glycerol, 0.2% PEG (Mr3550), 1mM oxidized glutathione, and 1mM reduced glutathione.

[0128] result

[0129] This embodiment conducted RRIB purification experiments on three target proteins. The results showed that Gal-12, Int-2, and Gal-8S proteins were all expressed in the form of inclusion bodies, as shown in Figure 3. The target protein Gal-12 only appeared in the precipitate after lysing the bacterial cells using buffer A. Both the P67-tagged (Figure 3A) and non-P67-tagged (Figure 3B) Gal-12 fusion proteins were completely dissolved in Buffer D containing 8M urea denaturant and appeared in the supernatant. During dialysis, as the urea concentration was gradually reduced from 8M to a completely urea-free PBS solution, 76% of the P67-tagged Gal-12 protein regained its solubility, indicating successful renaturation (Figure 4A). The non-P67-tagged Gal-12 protein was completely lost in the PBS dialysis buffer, indicating failed renaturation (Figure 4B).

[0130] Figure 5 shows that the Int-2 protein is expressed as inclusion bodies. The Int-2 protein only appeared in the precipitate after cell lysis using buffer A. Both the P67-tagged (Figure 5A) and non-P67-tagged (Figure 5B) Int-2 fusion proteins were completely dissolved in the supernatant using Buffer D containing 8M urea denaturant. During dialysis, as the urea concentration was gradually decreased from 8M to a completely urea-free PBS solution, 68% of the P67-tagged Int-2 protein regained its solubility, indicating successful renaturation (Figure 6A). The non-P67-tagged Int-2 protein was completely lost in the PBS dialysis buffer, indicating failed renaturation (Figure 6B).

[0131] Figure 7 shows that the Gal-8S protein is also expressed in the form of inclusion bodies. The Gal-8S protein only appears in the precipitate after cell lysis using buffer A. Both the P67-tagged (Figure 7A) and non-P67-tagged (Figure 7B) Gal-8S fusion proteins can be completely dissolved in Buffer D containing 8M urea and appear in the supernatant. During dialysis, as the urea concentration was gradually decreased from 8M to a completely urea-free PBS solution, 91% of the P67-tagged Gal-8S protein regained its solubility, indicating successful renaturation (Figure 8A). Only 26% of the non-P67-tagged Gal-8S protein regained its solubility (Figure 8B).

[0132] Example 4

[0133] Reverse verification of the effect of the p67 tag on the refolding of the target protein

[0134] Construct recombinant vectors expressing three target proteins (Gal-12, Int-2, and Gal-8S) according to Figure 2C. For specific steps, please refer to the recombinant vector construction method in Example 2.

[0135] Three fusion proteins were successfully prepared after refolding according to the purification method in Example 3. The P67 tag was then removed by 3C protease (Takara, #7360). The specific reaction conditions were as follows: 500 μL (including 50 μL of 10X digestion buffer, 10 μL of 3C protease, 100 μg of protein to be digested, and water added to make up to 500 μL), and the reaction program was 4°C for 16 hours.

[0136] After enzymatic digestion, the detagged proteins were purified using a nickel ion chromatography column (Beyotime, #P2233) to remove the 3C protease and P67 tag. Specifically, 500 μL of the digested protein solution was passed through a 100 μL nickel column at room temperature, and the eluent was collected, yielding three purified detagged target proteins. Electrophoresis analysis of these samples showed that the successfully renatured inclusion body proteins were fusion proteins carrying the P67 tag. However, after removing the excess N-terminal tag using the 3C protease cleavage site, the purified detagged target protein Gal-12 (Figure 9, B) was completely lost in the supernatant; while Gal-8S (Figure 9, C) and Int-2 (Figure 9, D) still had some residue in the supernatant, but most of the target proteins precipitated. This indicates that the P67 tag is crucial for maintaining the soluble state of the target protein, and some inclusion body proteins must carry the P67 tag to remain soluble.

[0137] The supernatant protein Int-2 solution after 3C digestion was passed through a nickel column (used to remove the His-tagged 3C protease and the N-terminal redundant tag P67), and the effluent was collected. Electrophoresis of the effluent showed that the final target protein with approximately 90% purity appeared in the Ni F lane (Figure 9, E). This indicates that the system can successfully renature and remove the tag from the difficult-to-renature inclusion body protein, ultimately yielding the target protein.

[0138] Example 5

[0139] The activity of successfully refolded inclusion body proteins was detected using a hemagglutination assay.

[0140] In a 96-well V-plate, column 1 was used as the negative control, and columns 2 through 10 were used as the experimental group. 75 μL of buffer containing 150 mM NaCl and 10 mM Tris (pH 7.5) was added to wells 1 through 10. 100 μg / mL of the target protein was added to the rightmost well. This 75 μL of liquid was removed and mixed with the liquid from the previous well. The mixture was then gradually diluted from right to left in a 1:1 ratio to the next well, after which the 75 μL of liquid was discarded. 25 μL of 2% (V / V) chicken erythrocyte suspension was added to each well and mixed well. The plate was incubated on ice for 1 hour, and the aggregation of chicken erythrocytes was observed by photographing. The minimum concentration required for complete erythrocyte aggregation induced by the target protein was recorded.

[0141] The results showed that P67-Int-2 and P67-Gal-8S were biologically active and could induce chicken blood agglutination (Figure 10). P67-Gal-12 did not show any biological activity, which is speculated to be because the C-terminal structure may prevent the protein from functioning.

[0142] The C-terminal structure of Gal-12 was removed, and the gene sequence encoding the N-terminal polypeptide (SEQ ID NO:24) expressing only Gal-12 (SEQ ID NO:33) was cloned into a tool plasmid, as detailed in Example 3. The P67-tagged Gal-12 truncated variant (P67-Gal-12 N-ter) was isolated and its activity was tested according to the recombinant expression and purification methods described in Example 3.

[0143] The results showed that the truncated protein also belongs to RRIB, and it could be successfully purified using this system and possessed biological activity (Figure 10).

[0144] Example 6

[0145] Compare the purification of the refractory inclusion body protein Int-2 using the P67 tag of other carboxylases.

[0146] In this embodiment, the p67 sequences of carboxylases (ACCA, ACCB, MCC, PC) from different eukaryotic sources were used to construct tool plasmids according to the method in Example 1. The resulting tool plasmids were cloned into the Int-2 gene to obtain a prokaryotic expression recombinant vector. The recombinant protein was expressed and purified according to the method in Example 3.

[0147] The results showed that the P67 tags from ACCA and PC had similar restorative functions as those from PCC; while the P67 tags from ACCB and MCC did not have restorative functions, and all Int-2 was lost during the PBS dialysate stage after dialysis (Figure 11).

[0148] Comparative Example 1

[0149] The recombinant proteins were expressed in E. coli by fusing them with the P67 tag, Avi tag, and Trx tag, respectively, and the recombinant proteins were purified according to the method in Example 3.

[0150] The results are shown in Figure 12 and Table 2.

[0151] Table 2 Results of purification of irreducible inclusion body proteins using different labels

[0152] The results showed that the Trx tag had some recovery-aiding function, but its effect was lower than that of the P67 tag; similarly, the Avi tag had no recovery-aiding function, and all Int-2 after dialysis was lost in the PBS dialysis solution stage.

[0153] Example 7

[0154] To avoid the potential impact of biotinylation modification on the P67 tag (occurring on the lysine side chain in MKM) on the use of the target protein, this embodiment verifies that the lysine in "MKM" of the P67 tag is mutated to proline and glutamic acid, and the purification function of the mutated tag P67 is tested according to the method in Example 3.

[0155] The results showed that mutating MKM to MEM and MPM in P67 did not affect the tag’s reproducibility function (Figure 13).

[0156] Example 8

[0157] The p67-tagged intractable inclusion bodies (purified under denaturing conditions) and soluble proteins (purified under non-denaturing conditions) were purified using a streptavidin chromatography system, respectively.

[0158] This embodiment first confirmed that the target protein carrying the P67 tag in this system can undergo biotinylation modification within the host bacteria. Host bacteria carrying the recombinant plasmid pET-41a-P67-Gal-12 N-ter were induced to express the protein by IPTG for 4 hours, and the bacterial culture was collected. The whole-cell lysate was analyzed by SDS-PAGE combined with Coomassie blue staining. Simultaneously, the IPTG-induced bacterial culture was centrifuged, and Buffer D containing 8M urea was added directly to lyse the cells (2 mL Buffer D per 50 mL of culture medium). The lysate was passed through a streptavidin chromatography column (200 μL column volume), and the column was washed with 10 column volumes of Buffer D. 20 μL of stationary phase beads was aspirated, and the bound proteins on the beads were detected by electrophoresis. The target protein was visible bound to the column, as indicated by the black arrow (bead lane A in Figure 14). The remaining beads were removed, placed in a dialysis bag, and dialyzed (according to the method in Example 3). HRV 3C protease was added to the dialyzed column for in-column digestion (4°C for 16 hours). Finally, the column was eluted with 3 column volumes of PBS. Electrophoresis and staining of the eluent revealed successfully renatured and tagged target proteins (gray arrows) in the eluent (Elution lane A in Figure 14). This demonstrates that the poorly renatured inclusion body protein induced by this system can be purified using a streptavidin-labeled column under deformable conditions. In this example, streptavidin was labeled with IRDye 680 and Western blot analysis was performed on the above samples. A strong streptavidin binding signal was observed at the location of the fusion protein (Figure 14B). This demonstrates that the P67 tag of this application can be modified with biotin.

[0159] The coding sequence of the soluble protein Galectin-8 was inserted between the BamHI and XhoI sites using the cloning method described in Example 2, forming the recombinant vector pET-41a-P67-Gal-8. This recombinant vector pET-41a-P67-Gal-8 was transformed into competent host cells, cultured, and then induced to express by IPTG. The recombinant protein was isolated and detected. The blue-stained band of the target protein appeared at the expected molecular weight position (Figure 15), with the black arrow indicating the Gal-8 fusion protein tagged with P67. Following the procedures in Example 3, the host cells were lysed with Buffer A and centrifuged. The supernatant and precipitate were then analyzed by SDS-PAGE and Coomassie blue staining. The target protein appeared in both the supernatant (Sup; this portion is the soluble protein) and the inclusion body precipitate (Pellet, Pel). Soluble proteins in the supernatant were purified using streptavidin and nickel (Ni) columns, respectively. Specifically, 5 mL of supernatant was passed through a 200 μL stationary phase column, followed by washing the column with 15 mL Buffer A. 20 μL of the stationary phase (Beads) was then added to electrophoresis loading buffer for SDS-PAGE blue staining. Results confirmed that the target protein was bound to the stationary phase (Beads lane). After digestion of the stationary phase with 3C protease (200 μL of 3C protease digestion solution containing 1 IU was added to the column phase, and digestion was performed at 4°C for 16 hours), the eluent was eluted with 1 mL PBS. Electrophoresis and staining of the eluent revealed a blue-stained band of Galectin-8 (gray arrow) with the tag removed in the Elution lane (Figure 15). Using this system, 0.4 mg of purified protein was obtained per liter of LB medium containing host bacteria, a yield comparable to that of the nickel column (see rightmost lane). As can be seen, soluble proteins induced by the recombinant vector provided in this application can be purified using a Streptavidin chromatography column under non-denaturing conditions. The results of the above examples demonstrate that the biotinylated P67 tag, which can be recognized by host cells and generates a biotinylated tag, can be used to purify both soluble and poorly soluble recombinant target proteins using a conventional streptavidin chromatography column.

[0160] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A reproducible label P67, characterized in that, Including at least one of the following: PCC tag, PC tag, ACCA tag, mutant of said PCC tag, mutant of PC tag and mutant of ACCA tag; The amino acid sequences of the PCC tag, PC tag, and ACCA tag are shown in SEQ ID NO:1 to SEQ ID NO:3; The mutant of the PCC tag, the mutant of the PC tag, or the mutant of the ACCA tag is an amino acid sequence obtained by mutating the 33rd lysine in the amino acid sequence of the PCC tag, the PC tag, or the ACCA tag to glutamic acid or proline.

2. A gene encoding the synergistic tag P67 as described in claim 1, characterized in that, The nucleotide sequence of the encoded gene is at least one of the following shown in SEQ ID NO:4 to SEQ ID NO:

8.

3. A prokaryotic protein expression tool plasmid, characterized in that, It comprises a nucleic acid molecule expressing the synergistic tag P67 of claim 1 or the encoding gene of claim 2.

4. The prokaryotic protein expression tool plasmid according to claim 3, characterized in that, The pET41a vector is used as the backbone vector, in which the GST tag is replaced with the coding gene.

5. The prokaryotic recombinant tool plasmid according to claim 4, characterized in that, It also includes HRV-3C protease recognition cleavage sites or TEV protease recognition sites.

6. A prokaryotic recombinant vector for expressing a target protein, characterized in that, It uses the prokaryotic protein expression tool plasmid as described in any one of claims 3 to 5 as a backbone vector, and also includes the target protein gene; The cloning site of the target protein gene is between the NcoI and XhoI multiple cloning sites or between the BamHI and XhoI multiple cloning sites.

7. A fusion protein, characterized in that, It is obtained by fusing the recombinant tag P67 as described in claim 1 with the target protein.

8. A fusion gene, characterized in that, It is obtained by tandemly connecting the coding gene and the target gene as described in claim 2.

9. The fusion gene according to claim 8, characterized in that, The target gene includes at least one of the following proteins or truncated genes: Int-2 protein, Gal-8S protein, and Gal-12 protein; The nucleotide sequence of the Int-2 protein gene is shown in SEQ ID NO:21; The nucleotide sequence of the gene for the Gal-8S protein is shown in SEQ ID NO:22; The nucleotide sequence of the gene for the Gal-12 protein is shown in SEQ ID NO:23; The amino acid sequence of the truncated form of the Gal-12 protein is shown in SEQ ID NO:

24.

10. The application of the recombinant tag P67 of claim 1, the encoding gene of claim 2, the prokaryotic protein expression tool plasmid of any one of claims 3 to 5, the prokaryotic recombinant vector of claim 6, the fusion protein of claim 7, or the fusion gene of claim 8 in inclusion body purification.

11. A method for purifying inclusion body proteins, characterized in that, Includes the following steps: The target protein gene is cloned into the prokaryotic protein expression tool plasmid described in any one of claims 3 to 5 to obtain a recombinant vector; The recombinant vector was transformed into competent cells of a prokaryotic expression system, and inclusion body proteins were isolated after culture and induction culture of the target protein. The inclusion body protein was denatured and dissolved to obtain denatured and dissolved inclusion body protein; The denatured and dissolved inclusion body protein was dialyzed and refolded to obtain purified refolded protein.

12. The purification method according to claim 11, characterized in that, The denaturation and dissolution method is as follows: the inclusion body protein is placed in buffer A, buffer B and buffer C in sequence to separate the precipitate, and then the precipitate is mixed with buffer D to dissolve it. The supernatant is collected to obtain the denatured and dissolved inclusion body protein. The buffer solution A is an aqueous solution comprising the following components: 50 mM pH 8.5 Tris-HCl, 1 mM EDTA, 100 mM NaCl, and 1% Triton X-100 (volume percentage). The buffer solution B is an aqueous solution comprising the following components: 50 mM pH 8.5 Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100 (volume percentage), and 2 M urea. The buffer solution C is an aqueous solution comprising the following components: 50 mM pH 8.5 Tris-HCl, 1 mM EDTA, 100 mM NaCl, 1% Triton X-100 (volume percentage), and 2 M guanidine hydrochloride. The buffer solution D is an aqueous solution comprising the following components: 50 mM pH 8.5 Tris-HCl, 1 mM EDTA, 100 mM NaCl, 10 mM DTT, 2 mM sodium deoxycholate, and 8 M urea.

13. The purification method according to claim 11, characterized in that, The dialysis refolding solution is an aqueous solution containing a gradient concentration of urea, comprising the following components: 50 mM pH 8.5 Tris-HCl, 100 mM NaCl, gradient concentrations of urea, 1% glycine by mass, 5% glycerol by volume, 0.2% PEG, 1 mM oxidized glutathione, and 1 mM reduced glutathione. The gradient concentrations of the urea were 6M, 4M, 2M and 1M.

14. The purification method according to claim 13, characterized in that, The dialysis refolding process involves dialyzing the denatured and dissolved inclusion body proteins sequentially according to the urea concentration, from highest to lowest.

15. The purification method according to claim 14, characterized in that, The dialysis time is 6 hours; the dialysis temperature is 4°C.

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