Fusion protein comprising peptide tag having main amino acid consisting of charged and polar amino acids and forming self-assembly, and method for purifying recombinant protein using same

Abstract

The present invention relates to: a fusion polypeptide in which a His tag is fused with a peptide tag having a major amino acid consisting of a charged amino acid and a polar amino acid; a fusion protein wherein the fusion polypeptide is fused to a target protein; and a target protein purification method using the same. Since the peptide tag is fused to the target protein and thus has the ability to induce the formation of a self-assembly, the target protein purification method using the same can be used for separating and purifying the target protein only using a very simple method such as centrifugation or filtration.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0122190 filed on September 27, 2022, and all the contents disclosed in the corresponding Korean patent application document are incorporated as part of this specification. The present invention relates to a fusion protein comprising a peptide tag, wherein the main amino acids consist of charged amino acids and polar amino acids; a composition for purifying a recombinant protein comprising the same; and a method for purifying a recombinant protein using the same. Background Art

[0003] Typically, His tag (or His6 tag) or HAT tag (native histidine affinity tag) is widely used together with MBP (maltose binding protein) tag and GST (glutathione S-transferase) tag for purification of recombinant proteins. The general method of purifying recombinant proteins using His tag is to purify them by the following method: fuse the His tag to the recombinant protein so that it can bind to Ni 2+ -NTA beads (or resin) or Co 2+ -CMA bead binding (where nickel ions or cobalt ions are immobilized by affinity chromatography), and then the recombinant protein bound to the beads is eluted by excess imidazole or low pH treatment. In other words, the recombinant protein fused to the His tag is eluted using the following principle: imidazole, which has a structure similar to the amino acid histidine, competes with the His tag and binds to Ni 2+ -NTA beads or Co 2+ -CMA beads (Kerpe K.2003). As such, the most widely used method for purifying recombinant proteins including protein drugs is adsorption (affinity) chromatography using affinity tags or using protein A having binding affinity to antibodies. However, such purification methods have various problems, such that they should be subjected to a time-consuming, complicated multi-step purification process, and the purification purity is slightly reduced due to contamination by proteins having non-specific binding ability to the beads, and a process for removing large-sized tags after purification is required, and it is difficult to use the recovered beads to produce other types of recombinant proteins, etc. These problems serve as the main factors limiting the scale-up of the purification step and increasing the production cost.

[0004] On the other hand, many studies have been conducted on motifs, domains or peptides that can nucleate or form multimers such as dimers. It has been reported that the formation of Ni-dependent ligands can be achieved by artificially inducing histidine residues in isoleucine zipper peptides that form helical structures. 2+ 、Zn 2+ , Cu 2+ In addition, the TZ1H peptide, which is known to have a helical structure, is also known to be a trimer of metal ions.2+ Or pH-dependent structural transformation to form a trimer. In addition, it has been reported that ferritin, an iron storage protein found in various species, has the ability to bind iron ions, and 24 ferritin proteins are combined to form a ferritin complex. In addition, α-helical peptide 18A and β-strand peptide ELK16 are known. Amphipathic surfactant-like peptide L6KD and hydrophobic peptide GFIL8, etc. form inclusion bodies in cells. In addition, ELP (elastin-like polypeptide) has been reported to undergo protein aggregation, and studies have been conducted on applying this property to recombinant protein purification, and it has been reported that calmodulin and M13 peptide specifically bind in a calcium ion-dependent manner, and the binding ability between Atox1 and WD4 depends on Zn 2+ , Cu 2+ , Pt 2+ , as well as FRB (FKBP12-rapamycin binding protein) and FKBP (FK506 binding protein) specifically bind in the presence of rapamycin.

[0005] Against the background of the above-mentioned technology or the characteristics of the motif, domain or peptide of a specific protein, the inventors have completed the present invention, which can solve the problems of conventional methods for purifying recombinant proteins, and induce the formation of macromolecular (supramolecular) assemblies through specific inducers or conditional treatments, while high-purity recombinant proteins can be selectively purified by simple methods (such as centrifugation or filtration), which is a completely different concept from conventional methods. Summary of the invention

[0006] Technical issues

[0007] The technical problem to be solved by the present invention is to solve various problems in conventional recombinant protein methods (i.e., purification processes), such as the use of expensive beads or resins, the need for expensive purification devices or equipment, the consumption of a large amount of time, and the multi-step complex process, etc., and involves a method of inducing the formation of a tag macromolecular self-assembly fused to a recombinant protein by a specific inducer or condition-dependent manner, and separating and purifying the target recombinant protein only by simple methods (e.g., centrifugation, filtration, etc.). Thus, a competitive next-generation method for purifying recombinant proteins is developed, which does not require expensive beads or resins, and can purify recombinant proteins very easily, quickly, simply, with high purity, high efficiency, and low cost.

[0008] One embodiment of the present application provides a peptide tag, wherein the main amino acids forming the self-assembly are composed of charged amino acids and polar amino acids.

[0009] Another embodiment of the present application provides a fusion polypeptide comprising a first polypeptide represented by SEQ ID NO: 1 and a second polypeptide as a peptide tag, wherein the main amino acids in the peptide tag are composed of charged amino acids and polar amino acids.

[0010] Other embodiments of the present application provide a fusion protein, comprising a fusion polypeptide and a target protein, wherein the fusion polypeptide comprises a first polypeptide and a second polypeptide.

[0011] Other embodiments of the present application provide a polynucleotide encoding a fusion polypeptide comprising a first polypeptide and a second polypeptide, or a fusion protein comprising a fusion polypeptide and a target protein.

[0012] Other embodiments of the present application provide an expression vector comprising a polynucleotide encoding a fusion polypeptide comprising a first polypeptide and a second polypeptide or a fusion protein comprising a fusion polypeptide and a target protein.

[0013] Other embodiments of the present application provide a host cell transformed with an expression vector.

[0014] Other embodiments of the present application provide a method for purifying a target protein, comprising culturing cells containing a polynucleotide in a culture medium, wherein the polynucleotide encodes a fusion polypeptide containing a first polypeptide and a second polypeptide or a fusion protein containing the fusion polypeptide and the target protein.

[0015] Other embodiments of the present application provide a composition for purifying a protein, comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0016] Other embodiments of the present application provide a self-assembly comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0017] Technical Solution

[0018] Each description and embodiment disclosed in this application can also be applied to each other description and embodiment. In other words, all combinations of the elements disclosed in this application belong to the scope of this application. In addition, the scope of this application cannot be considered to be limited by the specific description described below. In addition, those skilled in the art can identify or confirm many equivalents of the specific aspects of the application disclosed in this application using only ordinary experiments. In addition, these equivalents are intended to be included in this application.

[0019] In order to achieve the above-mentioned object of the present invention, the present invention provides a peptide tag, characterized in that the main amino acids are composed of charged amino acids and polar amino acids.

[0020] The main amino acids forming the self-assembly are composed of charged amino acids and polar amino acids, and the charged amino acids of the peptide tag can be selected from the positively charged amino acids of lysine (Lys, K), arginine (Arg, R) and histidine (His, H), the negatively charged amino acids of aspartic acid (Asp, D) and glutamic acid (Glu, E), and the polar amino acids can be selected from serine (Ser, S), threonine (Thr, T), cysteine ​​(Cys, C), asparagine (Asn, N), glutamine (Gln, Q) and tyrosine (Tyr, Y), and is characterized in that the content of charged amino acids and polar amino acids in the peptide tag except the hinge region or the connection site is 55% or more, 60% or more, 65% or more, preferably 70% or more.

[0021] The peptide tag can be selected from the group consisting of a P(SE)2 tag, a P(SE)4 tag, a P(SE)2IA tag, a PSEIAH tag, a PS(E)6 tag, a PS(E) 10 Label, PS(D) 10 Any one of the group consisting of a tag, an Ectp1 tag and a SHD tag, and may be a peptide capable of nucleation or formation of a multimer such as a dimer or the like.

[0022] The peptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0023] The term “peptide tag in which the main amino acids consist of charged amino acids and polar amino acids” used in the present specification may refer to a peptide tag in which polar amino acids are arranged at both ends of a charged amino acid patch, or a peptide tag in which a mixed composition of polar amino acids and charged amino acids is repeated, and the term “peptide tag in which the main amino acids consist of charged amino acids and polar amino acids” may be used interchangeably with “second polypeptide”.

[0024] In addition, the present invention provides a peptide tag or a fusion protein (in which the peptide tag and the target protein are fused), a polynucleotide encoding the same, a vector expressing the same, and a host cell transformed with the expression vector.

[0025] In addition, the present invention provides a method for purifying a target protein, comprising culturing a cell comprising a polynucleotide encoding a peptide tag or a recombinant fusion protein in a culture medium, wherein the peptide tag and the target protein are fused.

[0026] In addition, the present invention provides a composition for purifying a protein, comprising a peptide tag or a fusion protein comprising a peptide tag and a target protein.

[0027] In addition, the present invention provides a fusion polypeptide comprising a first polypeptide having an amino acid sequence of SEQ ID NO: 1 and a second polypeptide characterized in that main amino acids consist of charged amino acids and polar amino acids.

[0028] The first polypeptide may be a purification tag. Specifically, the first polypeptide may be a polypeptide that can bind Ni 2+ or Co 2+ The His tag may include, but is not limited to, a His tag of SEQ ID NO: 1, a HAT tag of its original form, or a modified HQ tag (or HQ6 tag) of the His tag. The His tag may include or consist of the amino acid sequence of SEQ ID NO: 1.

[0029] The second polypeptide may be a motif, domain or peptide capable of nucleation or forming a multimer such as a dimer. Specifically, the second polypeptide may be selected from P(SE)2, P(SE)4, P(SE)2IA, PSEIAH, PS(E)6, PS(E) 10 、PS(D) 10 , EctP1 and SDH peptide, but not limited thereto.

[0030] The second polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10. P(SE)2, P(SE)4, P(SE)2IA, PSEIAH, PS(E)6, PS(E) 10 、PS(D) 10 , SHD and EctP1 peptides can be represented by or composed of the following amino acids: SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively.

[0031] The first polypeptide and the second polypeptide may be linked sequentially.

[0032] In addition, the present invention provides a recombinant fusion protein comprising a fusion polypeptide and a target protein, wherein the fusion polypeptide comprises a first polypeptide and a second polypeptide.

[0033] The fusion polypeptide comprising the first polypeptide and the second polypeptide can be inserted not only into the C-terminus of the target protein, but also into the N-terminus or any position in the protein, as long as it does not substantially affect the function of the protein. In this article, substantially not affecting the function of the protein means that the activity of the protein before the fusion polypeptide fusion is maintained at 80% or more, preferably 95% or more.

[0034] In the present invention, "recombinant protein" refers to a polymer of amino acids (peptides, oligopeptides, polypeptides or proteins) comprising at least 2 parts, wherein each part comprises a separate function. At least one first part of the recombinant protein comprises at least one fusion polypeptide comprising a first polypeptide and a second polypeptide of the present invention, and at least one second part of the recombinant protein comprises at least one target protein (or peptide).

[0035] In the present invention, "target protein" refers to any protein for the purpose of production or purification, including peptides, and can be used interchangeably with the term "target protein" in this specification.

[0036] As target proteins, green fluorescent protein (GFP), granulocyte macrophage colony stimulating factor (CSF), interferon α2 (INF-α2), TEV protease and therapeutic antibody Herceptin light chain are included, but are not limited thereto. The term "target protein" refers to a protein produced by the biotechnology method according to the present invention, is not particularly limited to any one, and preferably includes proteins that can be used for medical, industrial, diagnostic and experimental purposes, etc.

[0037] The fusion protein may also be a cleavage site of a protease between a fusion polypeptide comprising the first polypeptide and the second polypeptide and a target protein, a hinge region of an immunoglobulin, a target protein, or all of them.

[0038] The hinge region may comprise, or consist of, the amino acid sequence of SEQ ID NO:41.

[0039] The fusion protein may contain a cleavage site between the fusion polypeptide and the target protein that is commonly used for protein purification, such as a TEV protease cleavage site or a Ni 2+ The SNAC tag is cut by the same method to remove the fusion polypeptide containing the first polypeptide and the second polypeptide in the recombinant fusion protein.

[0040] In addition, the present invention provides a fusion polypeptide comprising a first polypeptide and a second polypeptide, or a polynucleotide encoding a fusion protein comprising a fusion polypeptide and a target protein.

[0041] The polynucleotide comprises each polynucleotide encoding the first polypeptide and the second polypeptide. The polynucleotide encoding the first polypeptide may comprise or consist of the nucleotide sequence of SEQ ID NO:42, and the polynucleotide encoding the second polypeptide may comprise or consist of a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO:43 to SEQ ID NO:51.

[0042] The nucleotide sequences in the sequence listing described in the present specification are described in the direction from the 5' end to the 3' end even if there is no separate description.

[0043] In addition, the present invention provides a recombinant expression vector, comprising a fusion polypeptide comprising a first polypeptide and a second polypeptide, or a polynucleotide encoding a fusion protein comprising the fusion polypeptide and a target protein.

[0044] In the present invention, recombinant can be used interchangeably with genetic manipulation, which refers to the production of genes of a type that does not exist naturally by using molecular cloning techniques (eg, modifying, cutting, connecting genes, etc.).

[0045] In the present invention, expression refers to the production of a protein or a nucleic acid in a cell.

[0046] In the present invention, a recombinant expression vector is a vector capable of expressing a target protein or nucleic acid (RNA) in a suitable host cell, and refers to a gene construct comprising key regulatory factors operably linked to express a polynucleotide (gene) insert.

[0047] In this specification, "operably linked" means that the nucleic acid expression regulatory sequence and the nucleic acid sequence encoding the target protein or RNA are in functional connection to exert a general function, and they are connected so that the gene can be expressed through the expression regulatory sequence. The expression regulatory sequence refers to a DNA sequence that controls the expression of a polynucleotide sequence that is operably linked in a specific host cell. The regulatory sequence includes a promoter for transcription, any operator sequence that regulates transcription, a sequence encoding an appropriate mRNA ribosome binding site, a sequence that regulates transcription and translation, a start codon, a stop codon, a polyadenylation signal, and an enhancer, etc.

[0048] The type of the recombinant expression vector of the present invention is not particularly limited, as long as it is a vector commonly used in the cloning field, and examples include plasmid vectors, cosmid vectors, phage vectors and viral vectors, but are not limited thereto. Plasmids include plasmids derived from Escherichia coli (E. coli) (pBR322, pBR325, pUC118 and pUC119, pET-22 (+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5) and plasmids derived from yeast (pPICZ, YEp13, YEp24 and YCp50), etc., and as viruses, animal viruses such as retroviruses, adenoviruses or vaccinia viruses, insect viruses such as baculoviruses, etc. can be used, and preferably pET-28a vectors can be used.

[0049] In addition, the present invention provides a host cell transformed with an expression vector, wherein the expression vector comprises a polynucleotide encoding a fusion polypeptide or a fusion protein, wherein the fusion polypeptide comprises a first polypeptide and a second polypeptide, and the fusion protein comprises the fusion polypeptide and a target protein.

[0050] The type of host cell according to the present invention is not particularly limited, as long as it can be used for expressing the polynucleotide contained in the recombinant expression vector of the present invention. The cell (host cell) transformed with the recombinant expression vector according to the present invention can be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cells), an animal cell (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells), an insect cell or a hybridoma derived therefrom, preferably, can be Escherichia coli, but is not limited thereto.

[0051] The recombinant expression vector according to the present invention can be introduced into cells to transform by methods known in the art (such as but not limited to transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene guns, and known methods for nucleic acid flow into cells) to produce antibodies or fragments thereof. Cells transformed with the recombinant expression vector according to the present invention can overexpress or mass-produce the fusion protein of the present invention.

[0052] In addition, the present invention provides a method for purifying a target protein, comprising culturing a host cell transformed with an expression vector in a culture medium, wherein the expression vector comprises a polynucleotide encoding a fusion polypeptide or a fusion protein, wherein the fusion polypeptide comprises a first polypeptide and a second polypeptide, and the fusion protein comprises the fusion polypeptide and the target protein.

[0053] The content of the fusion polypeptide or target protein is as described above.

[0054] The method of purifying the target protein may further include extracting the fusion protein from the host cells after culturing.

[0055] The buffer solution used to extract the fusion protein may be a buffer solution generally (usually) used for extracting proteins, such as HEPES, Tris or phosphate, and preferably it may be a HEPES buffer solution, but is not limited thereto.

[0056] When extracting the fusion protein, a surfactant such as Trion X-100 may be added to improve the efficiency of cell lysis, and a protease inhibitor cocktail in which various protease inhibitors are mixed may be added to inhibit proteolysis.

[0057] The method for purifying a target protein may also include inducing the formation of a self-assembly of a fusion protein obtained from a host cell, wherein the host cell is transformed with an expression vector comprising a polynucleotide encoding a fusion polypeptide or a fusion protein, wherein the fusion polypeptide comprises a first polypeptide and a second polypeptide, and the fusion protein comprises the fusion polypeptide and the target protein.

[0058] Inducing the formation of self-assembly can also include processing under specific conditions to promote the formation of self-assembly. The specific conditions for inducing the formation of self-assembly can be physical-chemical conditions, such as the type of inducing agent, the concentration of inducing agent, the type of salt, the concentration of salt, pH conditions, the type of buffer solution, temperature, reaction time, etc.

[0059] Inducing the formation of the self-assembly may include adding an inducing agent to promote the formation of the self-assembly. According to the addition of the inducing agent, the formation of the macromolecular self-assembly can be promoted.

[0060] The inducer can be a cationic substance, specifically, can be selected from cations such as Ni 2+ 、Co 2+ 、Zn 2+ , Cu 2+ 、Ag 2+ , Fe 2+ and Ba 2+ Any one or more of the group consisting of, and the type of the appropriate inducing agent for inducing the formation of the self-assembly may differ depending on the combination of the first polypeptide and the second polypeptide.

[0061] The inducing agent may be 0.1-2.0 mM, 0.1-1.5 mM, 0.1-1.0 mM, 0.1-0.7 mM, 0.3-2.0 mM, 0.3-1.5 mM, 0.3-1.0 mM, 0.3-0.7 mM, 0.4-2.0 mM, 0.4-1.5 mM, 0.4-1.0 mM, 0.4-0.7 mM, 0.4-0.6 mM or 0.5 mM, but is not limited thereto.

[0062] Inducing the formation of the self-assembly may include adding salt. The salt may be a salt commonly used for protein precipitation, such as ammonium sulfate or sodium chloride, etc., and may preferably be ammonium sulfate, but is not limited thereto.

[0063] Ammonium sulfate may be 5-30% (w / v), 5-27% (w / v), 5-25% (w / v), 5-23% (w / v), 7-30% (w / v), 7-27% (w / v), 7-25% (w / v), 7-23% (w / v), 10-30% (w / v), 10-27% (w / v), 10-25% (w / v), 10-23% (w / v), 12-30% (w / v), 12-27% (w / v), 12-25% (w / v), 12-23% (w / v) or 12-22% (w / v), but is not limited thereto.

[0064] The buffer solution inducing the formation of the self-assembly may be a buffer solution generally used for extracting proteins, such as HEPES, Tris or phosphate, and preferably it may be a HEPES buffer solution, but is not limited thereto.

[0065] Inducing the formation of the self-assembly may be performed at pH 6 to pH 10, and may be performed at pH 7 to pH 10, but is not limited thereto.

[0066] Inducing the formation of the self-assembly may be performed immediately after treatment with the inducing agent for 10 minutes, 10 minutes or longer, 15 minutes or longer, 20 minutes or longer, 10 minutes to 120 minutes, 15 minutes to 120 minutes, 20 minutes to 120 minutes, but is not limited thereto.

[0067] The method for purifying a target protein may further comprise selectively isolating the formed self-assembly.

[0068] Separation can be performed by centrifugation or filtration.

[0069] The fusion protein comprising a fusion polypeptide (comprising a first polypeptide and a second polypeptide) and a target protein forms a macromolecular self-assembly to increase size and density. Therefore, centrifugation or filtration can be used as a separation method. Centrifugation can use density differences to precipitate them, and filtration can filter out molecules larger than a specific size.

[0070] In order to minimize contamination by non-specific proteins that may remain after separation by centrifugation or filtration, the recombinant protein fraction may also be washed with a buffer solution.

[0071] In addition, the present invention is a method for reversibly converting a recombinant protein purified by treatment with a specific inducer and / or conditional treatment to induce the formation of a macromolecular self-assembly into a monomer, and may also include treatment with a chelating agent such as EDTA or EGTA that can strongly bind to a specific cationic inducer.

[0072] The method for purifying the target protein may further include cleaving the TEV protease cleavage site or the SNAC tag contained in the fusion protein, and the cleavage may be performed by TEV protease or Ni 2+ Processing to be carried out.

[0073] In addition, the present invention provides a composition for purifying a protein, comprising a peptide tag having a characteristic that main amino acids consist of charged amino acids and polar amino acids.

[0074] The polypeptide tag can be selected from P(SE)2, P(SE)4, P(SE)2IA, PSEIAH, PS(E)6, PS(E) 10 、PS(D) 10 , EctP1 and SHD peptide tags, and may be a peptide capable of nucleation or formation of a multimer such as a dimer.

[0075] The polypeptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0076] In addition, the present invention provides a self-assembly comprising a peptide tag having a characteristic that main amino acids consist of charged amino acids and polar amino acids.

[0077] The polypeptide tag can be selected from P(SE)2, P(SE)4, P(SE)2IA, PSEIAH, PS(E)6, PS(E) 10 、PS(D) 10 , EctP1 and SHD peptide tags, and may be a peptide capable of nucleation or formation of a multimer such as a dimer.

[0078] The polypeptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0079] In addition, the present invention provides a composition for purifying a protein, comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0080] In addition, the present invention provides a self-assembly comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 10.

[0081] The contents of the first polypeptide and the second polypeptide are as described above.

[0082] Beneficial Effects

[0083] The present invention relates to a fusion polypeptide in which a His tag and a peptide tag composed of charged amino acids and polar amino acids are fused; and a method for purifying a target protein using the fusion polypeptide, wherein the fusion polypeptide is fused to the target protein and induces the formation of a macromolecular self-assembly in a manner dependent on a specific inducer or treatment condition, so that the recombinant protein can be separated and purified only by a very simple method (such as centrifugation or filtration), without the need to use a purification column filled with expensive beads or resins mainly used in conventional affinity chromatography, and an expensive device or equipment for running it. In addition, because the formation of the macromolecular self-assembly is induced by salt treatment of a protein extraction solution and a specific inducer or condition treatment, the present invention has the advantages of a very simple and easy treatment process, a very short time required for purification, and the ability to purify the recombinant protein with high efficiency and high purity. In addition, the present invention can easily convert the purified recombinant protein into a water-soluble monomer by chelating agent treatment, and if necessary, these tags can be easily removed by inducing the re-formation of the self-assembly after cutting the tag fused to the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1a is a conceptual diagram showing the induction of macromolecular self-assembly formation by specific inducers acting on fused purification tags and self-assembly tags.

[0085] Figure 1b It is a schematic diagram showing the configuration of a vector for expressing a target protein, the vector comprising a gene region encoding a His6 tag and a self-assembly body tag, and comprising a gene region encoding the target protein.

[0086] Figure 1c The figure shows a method for selectively purifying a recombinant protein by a method such as centrifugation or filtration after inducing the formation of a macromolecular self-assembly by a specific inducing agent or treating under specific conditions.

[0087] Figure 2a This figure confirms whether the formation of macromolecular self-assembly is induced when treated with various cations by fusing a single His tag as a purification tag to the target protein GFP.

[0088] Figure 2bThis is a diagram to confirm whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing a P(SE)2 peptide tag designed de novo to enable nucleation or formation of multimers such as dimers.

[0089] Figure 2c This figure examines whether the formation of macromolecular self-assemblies is induced by treatment with various cations by fusing a combination of a His tag and a P(SE)2 peptide tag to GFP as a purification tag.

[0090] Figure 2d This is a diagram to confirm whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing a combination of a His tag and a P(SE)2IA peptide tag designed from scratch to enable nucleation or formation of multimers such as dimers as purification tags to GFP.

[0091] Figure 2e This figure examines whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing a combination of a His tag and a PSEIAH peptide tag to GFP as a purification tag.

[0092] Figure 2f This is a diagram to confirm whether the formation of macromolecular self-assembly is induced when treated with various cations by fusion of a de novo designed P(SE)4 peptide tag capable of nucleation or formation of multimers such as dimers as a purification tag with GFP.

[0093] Figure 2g This is a diagram to confirm whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing a combination of a His tag and a P(SE)4 peptide tag designed from scratch to enable nucleation or formation of multimers such as dimers as purification tags to GFP.

[0094] Figure 2h This is a diagram in which a P(SE)6 peptide tag designed de novo to be capable of nucleation or formation of multimers such as dimers was fused to GFP alone as a purification tag to confirm whether the formation of macromolecular self-assemblies was induced when treated with various cations.

[0095] Figure 2i This figure examines whether the formation of macromolecular self-assemblies is induced by treatment with various cations by fusing a combination of a His tag and a PS(E)6 peptide tag to GFP as a purification tag.

[0096] Figure 2j It is a PS(E) designed from scratch to be able to nucleate or form multimers such as dimers by adding a His tag. 10 A combination of peptide tags was used as purification tags fused to GFP to confirm whether the formation of macromolecular self-assemblies was induced when treated with various cations.

[0097] Figure 2k It is a PS(D) designed from scratch to be able to nucleate or form multimers such as dimers by adding a His tag 10 A combination of peptide tags was used as purification tags fused to GFP to confirm whether the formation of macromolecular self-assemblies was induced when treated with various cations.

[0098] Figure 2l This figure examines whether the formation of macromolecular self-assemblies is induced when the EctP1 tag is used alone as a purification tag and fused to GFP.

[0099] Figure 2m This figure examines whether the formation of macromolecular self-assemblies is induced by treatment with various cations by fusing a combination of a His tag and an EctP1 peptide tag to GFP as a purification tag.

[0100] Figure 2n The SHD peptide tag designed from scratch so that the Pol2[H2(-)]3Pol2 sequence (Pol: refers to polar amino acids, (-): refers to negatively charged amino acids) is repeated, thereby enabling nucleation or formation of polymers such as dimers, etc., and is fused to GFP as a purification tag to confirm whether the formation of macromolecular self-assemblies is induced when treated with various cations.

[0101] Figure 2o This figure examines whether the formation of macromolecular self-assemblies is induced by treatment with various cations by fusing a combination of a His tag and an SHD peptide tag to GFP as a purification tag.

[0102] Figure 3a The His tag or P(SE)2 peptide tag was used as a purification tag alone and fused to GFP (the GFP was one of the model proteins used as the target protein) to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0103] Figure 3b The His tag or P(SE)2 peptide tag alone was used as a purification tag and fused to GFP, or the combination of the His tag and the P(SE)2IA peptide tag was used as a purification tag and fused to GFP to confirm the effect of different concentrations of cationic Ni on the expression of GFP. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0104] Figure 3cThe His tag or P(SE)2 peptide tag alone was used as a purification tag and fused to GFP, or the combination of the His tag and the PSEIAH peptide tag was used as a purification tag and fused to GFP to confirm the effect of different concentrations of cationic Ni on the expression of GFP. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0105] Figure 3d The His tag or P(SE)4 peptide tag was used as a purification tag alone or in combination with GFP to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0106] Figure 3e The His tag or P(SE)6 peptide tag was used as a purification tag and fused to GFP alone or in combination with GFP to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0107] Figure 3f The His tag or P(SE)6 peptide tag alone as a purification tag was fused to GFP or the His tag and P(SE)6 peptide tag were fused to GFP. 10 The combination of peptide tags as purification tags fused to GFP was used to confirm that the expression of cationic Ni at different concentrations was 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0108] Figure 3g The His tag or P(SE)6 peptide tag alone as a purification tag was fused to GFP or the His tag and P(SE)6 peptide tag were fused to GFP. 10 The combination of peptide tags as purification tags fused to GFP was used to confirm that the expression of cationic Ni at different concentrations was 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0109] Figure 3h The His tag or EctP1 peptide tag was used as a purification tag alone and fused to GFP, or the combination of these two tags was used as a purification tag and fused to GFP to confirm that the expression of cationic Ni at different concentrations was 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0110] Figure 3i The His tag or SHD peptide tag was fused to GFP as a purification tag alone or a combination of the two tags as a purification tag to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0111] Figure 4a The His tag was used as a purification tag alone and fused to GFP to analyze the selection of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0112] Figure 4b The P(SE)2 peptide tag was used alone as a purification tag and fused to GFP to analyze the selective cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0113] Figure 4c The His-tag and P(SE)2 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0114] Figure 4d The His-tag and P(SE)2IA peptide-tag were combined as purification tags fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0115] Figure 4e The His-tag and PSEIAH peptide-tag were combined as purification tags fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0116] Figure 4f The P(SE)4 peptide tag was used alone as a purification tag and fused to GFP to analyze the activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0117] Figure 4g The His-tag and P(SE)4 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0118] Figure 4h The P(SE)6 peptide tag was used alone as a purification tag and fused to GFP to analyze the activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0119] Figure 4i The His-tag and P(SE)6 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0120] Figure 4j The His tag and PS(E) 10 A combination of peptide tags as purification tags fused to GFP were used to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0121] Figure 4k The His tag and PS(D) 10 A combination of peptide tags as purification tags fused to GFP were used to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0122] Figure 4l The EctP1 peptide tag alone was used as a purification tag and fused to GFP to analyze the selective cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0123] Figure 4m The His-tag and EctP1 peptide-tag were combined as purification tags fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0124] Figure 4n The SHD peptide tag was used alone as a purification tag and fused to GFP to analyze the activity of the cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0125] Figure 4o The His-tag and SHD-peptide-tag were combined as purification tags fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0126] Figure 5a The His-tag and P(SE)2 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0127] Figure 5b The His-tag and P(SE)2IA peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0128] Figure 5c The combination of His-tag and PSEIAH peptide tag was fused to GFP as purification tag to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0129] Figure 5d The His-tag and P(SE)4 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0130] Figure 5e The His-tag and PS(E)6 peptide-tag were combined as purification tags and fused to GFP to analyze the selectivity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0131] Figure 5f The His tag and PS(E) 10 The combination of peptide tags as purification tags fused to GFP was used to analyze the selective cationic Ni in the presence of various concentrations of NaCl as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0132] Figure 5g The His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was used to analyze the selective cationic Ni in the presence of various concentrations of NaCl as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0133] Figure 5h The EctP1 peptide tag was used alone as a purification tag and fused to GFP to analyze the selective cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0134] Figure 5i The His-tag and EctP1 peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0135] Figure 5j The SHD peptide tag was used alone as a purification tag and fused to GFP to analyze the selective cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0136] Figure 5k The His-tag and SHD-peptide-tag were combined as purification tags and fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0137] Figure 6a The His tag and P(SE)2 peptide tag were fused to GFP as purification tags to analyze the activity of Ni in the presence of ammonium sulfate as salt. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0138] Figure 6b The His tag and P(SE)2IA peptide tag were combined as purification tags and fused to GFP to analyze the activity of Ni in the presence of ammonium sulfate as the salt. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0139] Figure 6cThe His tag and PSEIAH peptide tag were combined as purification tags and fused to GFP to analyze the activity of Ni in the presence of ammonium sulfate as the salt. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0140] Figure 6d The His tag and P(SE)4 peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0141] Figure 6e The His tag and PS(E)6 peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0142] Figure 6f The His tag and PS(E) 10 The combination of peptide tags as purification tags fused to GFP was used to analyze the expression of proteins in ammonium sulfate as salt and the selected cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0143] Figure 6g By combining the His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was used to analyze the expression of proteins in ammonium sulfate as salt and the selected cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0144] Figure 6h The EctP1 peptide tag was used as a purification tag and fused to GFP to analyze the expression of GFP in the presence of ammonium sulfate and the selected cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0145] Figure 6i The His tag and EctP1 peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0146] Figure 6jThe SHD peptide tag was fused to GFP as a purification tag to analyze the expression of GFP in ammonium sulfate as a salt and the selected cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0147] Figure 6k The His tag and SHD peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0148] Figure 7a The His-tag and P(SE)2 peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0149] Figure 7b The His-tag and P(SE)2IA peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0150] Figure 7c The combination of His-tag and PSEIAH peptide tag was fused to GFP as purification tag to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0151] Figure 7d The His-tag and P(SE)4 peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0152] Figure 7e The combination of His-tag and PS(E)6 peptide tag was fused to GFP as purification tag to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0153] Figure 7f The His tag and PS(E) 10The combination of peptide tags as purification tags fused to GFP was analyzed in the presence of ammonium sulfate as salt, depending on the choice of cation Ni 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0154] Figure 7g By combining the His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was analyzed in the presence of ammonium sulfate as salt, depending on the choice of cation Ni 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0155] Figure 7h The EctP1 peptide tag alone was used as a purification tag fused to GFP to analyze the expression of GFP in the presence of ammonium sulfate as a salt, depending on the choice of cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0156] Figure 7i The His-tag and EctP1 peptide-tag combination was fused to GFP as purification tags to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0157] Figure 7j The SHD peptide tag was used alone as a purification tag fused to GFP to analyze the presence of ammonium sulfate as a salt, depending on the choice of cation Ni 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0158] Figure 7k The His-tag and SHD-peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0159] Figure 8a The His tag and P(SE)2 peptide tag were fused to GFP as purification tags to analyze the activity of the GFP under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0160] Figure 8bThe His tag and P(SE)2IA peptide tag were combined with GFP as purification tags to analyze the activity of the purified protein when treated with ammonium sulfate as salt and with the cationic Ni under various pH conditions of the buffer. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0161] Figure 8c The His tag and PSEIAH peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0162] Figure 8d The His tag and P(SE)4 peptide tag were fused to GFP as purification tags to analyze the activity of the GFP under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0163] Figure 8e The His-tag and PS(E)6 peptide-tag were fused to GFP as purification tags to analyze the activity of the GFP-DNA under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0164] Figure 8f The His tag and PS(E) 10 The combination of peptide tags as purification tags fused to GFP was analyzed when treated with ammonium sulfate as salt and selected cationic Ni under various pH conditions of the buffer. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0165] Figure 8g The His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was analyzed when treated with ammonium sulfate as salt and selected cationic Ni under various pH conditions of the buffer. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0166] Figure 8h The EctP1 peptide tag was used alone as a purification tag and fused to GFP to analyze the activity of the GFP under various pH conditions of the buffer, treated with ammonium sulfate as a salt, and selected cationic Ni2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0167] Figure 8i The His-tag and EctP1 peptide-tag combination was fused to GFP as purification tags to analyze the activity of the GFP under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0168] Figure 8j The SHD peptide tag was used alone as a purification tag and fused to GFP to analyze the expression of GFP under various pH conditions of the buffer, treated with ammonium sulfate as a salt, and selected cationic Ni 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0169] Figure 8k The His-tag and SHD-peptide-tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0170] Figure 9a The His-tag and P(SE)2 peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0171] Figure 9b The His-tag and P(SE)2IA peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0172] Fig.9c The combination of His-tag and PSEIAH peptide tag was fused to GFP as purification tag to analyze the expression of GFP in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0173] Figure 9dThe His-tag and P(SE)4 peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0174] Fig.9e The combination of His-tag and PS(E)6 peptide tag was fused to GFP as purification tag to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cationic Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0175] Figure 9f The His tag and PS(E) 10 The combination of peptide tags as purification tags fused to GFP was analyzed in the presence of ammonium sulfate as salt, depending on the choice of cation Ni 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0176] Figure 9g By combining the His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was analyzed in the presence of ammonium sulfate as salt, depending on the choice of cation Ni 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0177] Figure 9h The EctP1 peptide tag alone was used as a purification tag fused to GFP to analyze the expression of GFP in the presence of ammonium sulfate as a salt, depending on the choice of cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0178] Figure 9i The His-tag and EctP1 peptide-tag combination was fused to GFP as purification tags to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0179] Figure 9j The SHD peptide tag was used alone as a purification tag fused to GFP to analyze the presence of ammonium sulfate as a salt, depending on the choice of cation Ni 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0180] Figure 9kThe His-tag and SHD-peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0181] Fig.10 The His tag is combined with P(SE)2, P(SE)4, P(SE)2IA, PSEIAH, PS(E)6, PS(E) 10 、PS(D) 10 Each of the combinations of EctP1 and SHD peptide tags, or EctP1 and SHD peptide tags alone were fused to GFP as purification tags, in the presence of ammonium sulfate as the salt, by a process depending on the choice of cation Ni. 2+ The yield (recovery rate) and purity of each unit are analyzed as the degree of formation of the macromolecular self-assembly as a function of the treatment time with a specific inducing agent.

[0182] Fig.11a The His tag alone, the SHD tag alone, the combination of the His tag and the PSEIAH peptide tag, or the combination of the His tag and the SHD peptide tag were fused to GFP as purification tags to analyze the activity of the cationic Ni in the presence of ammonium sulfate as salt. 2+ Diagram showing the possibility of recovering recombinant protein using filter filtration and UV irradiation when treated with specific inducers.

[0183] Fig.11b The His tag and P(SE)2 peptide tag combination or the His tag and EctP1 peptide tag combination were fused to GFP as purification tags to analyze the activity of Ni in the presence of ammonium sulfate. 2+ Diagram showing the possibility of recovering recombinant protein using filter filtration and UV irradiation when treated with specific inducers.

[0184] Fig.11c The His tag and P(SE)2IA peptide tag combination, the His tag and P(SE)4 peptide tag combination, the His tag and PS(E)6 peptide tag combination, the His tag and PS(E) 10 Combination of peptide tag or His tag and PS(D) 10 The combination of peptide tags as purification tags fused to GFP was used to analyze the activity of Ni in the presence of ammonium sulfate as the salt. 2+ Diagram showing the possibility of recovering recombinant protein using filter filtration and UV irradiation when treated with specific inducers.

[0185] Fig.11d The EctP1 tag was used as a purification tag and fused to GFP to analyze the effect of ammonium sulfate as a salt when the cationic Ni 2+ Diagram showing the possibility of recovering recombinant protein using filter filtration and UV irradiation when treated with specific inducers.

[0186] Fig.11e The method comprises combining a His tag alone, an EctP1 tag alone, an SHD tag alone, a combination of a His tag and a P(SE)2 peptide tag, a combination of a His tag and a P(SE)2IA peptide tag, a combination of a His tag and a PSEIAH peptide tag, a combination of a His tag and a P(SE)4 peptide tag, a combination of a His tag and a PS(E)6 peptide tag, a combination of a His tag and a PS(E) 10 Combination of peptide tags, His tags and PS(D) 10 The combination of peptide tags, the combination of His tag and EctP1 peptide tag, or the combination of His tag and SHD peptide tag were fused to GFP as purification tags to analyze the effect of Ni on the expression of Ni in the presence of ammonium sulfate as salt. 2+ A graph showing the possibility of recovering recombinant proteins by filtration using filters with pore sizes increasing to 5.0 μm and 10 μm and by UV irradiation when treated with specific inducers.

[0187] Fig.12a In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0188] Figure 12b In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:P(SE)2:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0189] Fig.12c In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with His6:P(SE)2IA:GFP as a specific inducer to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0190] Fig.12d In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+Images obtained under conditions of treatment with specific inducers for His6:PSEIAH:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0191] Fig.12e In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:P(SE)4:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0192] Fig.12f In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:PS(E)6:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0193] Figure 12g In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ As a specific inducer for His6:PS(E) 10 : Images obtained under GFP treatment conditions to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0194] Figure 12h In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ As a specific inducer for His6:PS(D) 10 : Images obtained under GFP treatment conditions to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0195] Fig.12i In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for EctP1:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0196] Fig.12j In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ The formation and size of macromolecular self-assemblies were analyzed by fluorescence microscopy under the condition of His6:EctP1:GFP treatment as a specific inducer to obtain images.

[0197] Figure 12k In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+The images were obtained by analyzing the formation and size of macromolecular self-assemblies under the conditions of SHD:GFP treatment with specific inducers.

[0198] Figure 12l In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:SHD:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0199] Fig.13a The His tag and P(SE)2 peptide tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0200] Fig.13b The His tag and P(SE)2 peptide tag were fused to TEV protease as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0201] Fig.13c The His tag and P(SE)2 peptide tag were fused as purification tags to the light chain of the therapeutic antibody Herceptin to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0202] Fig.13d The His tag and P(SE)4 peptide tag were fused to TEV protease as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0203] Fig.13e The His tag and P(SE)4 peptide tag were fused as purification tags to the light chain of the therapeutic antibody Herceptin to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0204] Fig.13f The His tag and P(SE)2IA peptide tag were combined as purification tags to fusion with granulocyte macrophage colony stimulating factor to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0205] Figure 13g The His-tag and PSEIAH-peptide-tag were combined as purification tags to fusion with GM-CSF to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0206] Fig.13h The His tag and EctP1 peptide tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0207] Fig.13i The His-tag and EctP1 peptide-tag were fused to TEV protease as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0208] Fig.13j The His-tag and SHD-peptide-tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0209] Figure 13k The SHD peptide tag alone was used as a purification tag and fused to granulocyte macrophage colony stimulating factor to analyze the selective cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0210] Figure 13l The SHD peptide tag was fused to interferon α2 as a purification tag to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0211] Figure 13m The SHD peptide tag was fused to TEV protease as a purification tag to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0212] Fig.13n The His tag and P(SE)4 peptide tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP. DETAILED DESCRIPTION

[0213] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below. In other words, it should be understood that all applications and changes, equivalents and substitutes included in the concept and technology of the present invention are included.

[0214] [Experimental Materials and Methods]

[0215] <Experimental Materials>

[0216] Ammonium sulfate ((NH4)2SO 4、 CAT NO. A4915) and nickel chloride (NiCl2, CAT NO. 339350) were purchased from Sigma Aldrich (St. Louis, MO, USA) as ACS reagent grade and 98% or higher reagents, EDTA disodium salt dihydrate (C 10 H 14 N2Na2O8·2H2O, CAT NO. 0105) was purchased from VWR Life Science (Radnor, PA, USA) as biotechnology grade and used.

[0217] Example 1. Construction of a vector for expressing protein

[0218] In order to establish a model system for constructing a recombinant protein purification system using GFP in the bead-free self-assembly induction method, the GFP gene was amplified using F-Kpn I-hinge region-Bgl II-TEV cleavage site: GFP / R-Xho I, Hind III: GFP primers, cut with restriction enzymes, and then inserted into the Kpn I / Xho I site of the pET 28a vector to construct a pET 28a-GFP subvector. In addition, overlapping PCR was performed using F-Nco I, BamH I-His6 / R-Kpn I-His6 primers, followed by cutting with restriction enzymes and then inserting into the Nco I / Kpn I site of the pET 28a-GFP subvector to complete the basic vector pET 28a-His6: GFP vector. Afterwards, the primer combinations F-Nco I, BamH I-His6:P(SE)2 / R-Kpn IP(SE)2; F-Nco I, BamH I-His6:P(SE)4 / R-Kpn IP(SE)4; F-Nco I, BamH I-His6:P(SE)2IA / R-Kpn IP(SE)2IA; F-Nco I, BamH I-His6:PSEIAH / R-Kpn I-PSEIAH; F-Nco I, BamH I-His6:PS(E)6 / R-Kpn I-PS(E)6; F-Nco I, BamH I-His6:PS(E)6 10 / R-Kpn I-PS(E) 10 ;F-Nco I, BamH I-His6:PS(D) 10 / R-Kpn I-PS(D) 10 ; F-Nco I, BamH I-His6: EctP1 / R-Kpn I-EctP1; F-Nco I, BamH I-His6: SHD / R-Kpn I-SHD, producing pET 28a-His6: P(SE)2: GFP, pET 28a-His6:P(SE)2IA:GFP, pET28a-His6:P(SE)4:GFP, pET 28a-His6:PSEIAH:GFP, pET 28a-His6:PS(E)6:GFP, pET 28a-His6:PS(E) 10 :GFP, pET 28a-His6:PS(D) 10:GFP, pET 28a-His6:EctP1:GFP, pET 28a-His6:SHD:GFP vectors. In order to construct vectors other than the His6 tag and use them as controls, representative vectors of pET 28a-P(SE)2:GFP, pET 28a-P(SE)4:GFP, pET 28a-PS(E)6:GFP, pET 28a-EctP1:GFP, and pET 28a-SHD:GFP were produced based on the primer combinations F-NcoI, BamH IP(SE)2 / R-Kpn IP(SE)2; F-Nco I, BamH IP(SE)4 / R-Kpn IP(SE)4; F-Nco I, BamH I-PS(E)6 / R-Kpn I-PS(E)6; F-Nco I, BamH I-EctP1 / R-Kpn I-EctP1; F-Nco I, BamHI-SHD / R-Kpn I-SHD.To evaluate the purification scalability of various protein drugs other than GFP, PCR was performed with primer combinations of F-Bgl II-rhG-CSF / R-Hind III-rhG-CSF; F-Bgl II-INF-a2 / R-Hind III-INF-a2; F-Bgl II-TEV protease / R-Hind III-TEV protease; and F-Bgl II-HER LC / R-Hind III-HER LC, which were then cut with Bgl II / Hind III restriction enzymes and inserted into pET 28a-His6:P(SE)2:GFP, pET 28a-His6:P(SE)4:GFP, pET 28a-His6:P(SE)2IA:GFP, pET 28a-His6:PSEIAH:GFP, pET 28a-His6:EctP1:GFP, and pET 28a-His6:SHD:GFP, the GFP site of the pET28a-SHD:GFP vector, and finally completed pET 28a-His6:P(SE)2:INF-a2, pET 28a-His6:P(SE)2:TEV protease, pET 28a-His6:P(SE)2:HER LC, pET 28a-His6:P(SE)4:TEV protease, pET28a-His6:P(SE)4:HER LC, pET 28a-His6:P(SE)2IA:rhG-CSF, pET 28a-His6:PSEIAH:rhG-CSF, pET 28a-His6:EctP1:INF-a2, pET 28a-His6:EctP1:TEV protease, pET Construction of representative vectors of 28a-His6:SHD:INF-a2, pET 28a-SHD:rhG-CSF, pET 28a-SHD:INF-a2, pET 28a-SHD:TEV protease and pET 28a-His6:P(SE)4:INF-a2. The specific sequence information of each tag and designed recombinant protein is shown in Tables 1 to 3 below.

[0219] [Table 1]

[0220] Amino acid sequence and nucleic acid sequence information of the first polypeptide tag

[0221] Tag Name Amino acid sequence (N→C) SEQ ID NO: Nucleic acid sequence (5'→3') SEQ ID NO: <![CDATA[His6]]> HHHHHH 1 CATCACCATCACCACCAT 42

[0222] [Table 2]

[0223] Amino acid sequence and nucleic acid sequence information of a polypeptide tag (or a second polypeptide tag) whose main amino acids are composed of charged amino acids and polar amino acids

[0224]

[0225]

[0226] [Table 3]

[0227] Amino acid sequence and nucleic acid sequence information of each fusion polypeptide, target protein and hinge region.

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246] Example 2. Induction and extraction of fusion protein expression

[0247] The vector for expressing the fusion protein produced in Example 1 was transformed into BL21 (DE3) Escherichia coli and cultured in LB medium (containing kanamycin 50 μg / ml) at 37°C. In order to induce overexpression of the protein, the OD 600 When the value was 0.6, 1.0 mM IPTG (isopropyl-β) was added, and it was cultured at 18°C ​​for 18 hours. The cultured E. coli was centrifuged at 4,000 rpm for 15 minutes at 4°C to obtain a precipitate. In order to extract the overexpressed fusion protein, it was sonicated with HEPES buffer (HEPES; containing 50 mM HEPES buffer (pH 7.5), Triton X-100, protease inhibitor cocktail (Roche, Mannheim, GEU)), and then centrifuged at 13,000 rpm for 15 minutes at 4°C to obtain a supernatant containing the fusion protein.

[0248] Example 3. Induction and optimization of macromolecular self-assembly formation

[0249] Example 3-1. Optimization of ammonium sulfate concentration as salt

[0250] To optimize the ammonium sulfate concentration, a concentration of 0.5 mM Ni 2+ The treatment was carried out by treating the supernatant obtained by centrifugation with ammonium sulfate in the range of 12-22% [w / v] at 2% intervals.

[0251] Example 3-2. Optimization of sodium chloride concentration as salt

[0252] In order to analyze the effect of the treatment concentration of sodium chloride as salt, the Ni 2+ deal with.

[0253] Example 3-3. Optimization of buffer

[0254] To optimize the buffer, pH 7.4 HEPES buffer (HEPES; 50 mM HEPES buffer (pH 7.4)), pH 7.4 PBS buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.4)), pH 7.4 PB buffer (PB; 10 mM Na2HPO4, 1.8 mM KH2PO4 pH7.4)), pH 7.4 Tris buffer (Tris; 50 mM Tris-HCl buffer (pH7.4)) were used, and 1% [v / v] TritonX-100 and 1X [v / v] protease inhibitor cocktail tablets (Roche) were added to all buffers and used. Ni was added at a concentration of 0.5 mM2+ The supernatant was obtained by treatment with ammonium sulfate followed by centrifugation under buffered conditions.

[0255] Example 3-4. Ni 2+ Concentration Optimization

[0256] In order to optimize Ni 2+ Concentration, with Ni in the range of 0 to 2 mM 2+ The supernatant was obtained by centrifugation after treatment with ammonium sulfate.

[0257] Example 3-5. Optimization of buffer pH

[0258] To optimize the buffer pH, the supernatant obtained by treatment with ammonium sulfate and centrifugation was used by adjusting the pH to a range of 5-10 with HCl and NaOH. 2+ The supernatant obtained by ammonium sulfate treatment and centrifugation under each pH condition was treated.

[0259] Example 3-6. Optimization of self-assembly formation reaction time

[0260] To optimize the formation of macromolecular self-assemblies, 0.5 mM Ni was added to the supernatant obtained by centrifugation after ammonium sulfate treatment. 2+ Treatment was performed, and measurements were taken at 10-minute intervals from immediately after treatment to 60 minutes after treatment, and further measurements were taken up to 120 minutes.

[0261] Example 4. Recovery of macromolecular self-assembly recombinant protein and conversion into monomers

[0262] The recombinant protein that forms the macromolecular self-assembly in Example 3 in a specific inducer or condition-dependent manner is recovered by methods such as centrifugation and filtration.

[0263] Specifically, in the purification method by centrifugation, centrifugation is carried out at 4 ℃ for 10 minutes at 3000rpm or higher speed, and the macromolecular assembly recombinant protein in the form of precipitate is obtained, and 5mM EDTA is added to the same buffer for macromolecular assembly induction reaction to obtain the recombinant protein of monomer. In the filtering method, 0.2 and 5.0 μm syringe filters (Satorius, Goettingen, GEU) and 10 μm syringe filters (Tisch, OH, USA) are used, and after filtering the recombinant protein of macromolecular self-assembly body, the same buffer used in the macromolecular assembly induction reaction is used to carry out washing process. In addition, the macromolecular assembly is converted into a monomer or eluted with the same buffer solution adding a chelating agent such as EDTA. In order to visually confirm this, this is visually confirmed by irradiating the filter with the UV wavelength of 360nm UV.

[0264] Example 5. Confirmation of protein purified by SDS-PAGE

[0265] A certain amount of monomeric recombinant protein recovered by centrifugation and filtration was taken, SDS sample buffer (glycerol, 2M Tris-HCl (pH 6.8), 30% SDS, 2-mercaptoethanol) was added, and they were heated for analysis, and the macromolecular self-assembly induced optimization condition search, purification yield and purity of the purified protein were evaluated by the general SDS-PAGE method.

[0266] Example 6. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0267] In order to analyze the formation and approximate size of the macromolecular self-assembly, a fluorescence microscope was used, and in order to detect the GFP fluorescence signal, a filter set of excitation: 488 nm, emission: 520 nm was used.

[0268] [Experimental results]

[0269] Experimental Example 1. Identification of an inducing agent suitable for inducing the self-assembly formation of a peptide tag fusion protein in which the main amino acids are composed of charged amino acids and polar amino acids

[0270] In order to confirm the inducible ability to form macromolecular self-assemblies in a specific inducer or condition-dependent manner, GFP was selected as a model protein, and His tags and P(SE)2 peptide tags, in which the main amino acids are composed of charged amino acids and polar amino acids, P(SE)4 peptide tags, P(SE)2IA peptide tags, PSEIAH peptide tags, PS(E)6 peptide tags, PS(E) 10 Peptide tag, PS(D) 10 The peptide tag, EctP1 peptide tag and SHD peptide tag were combined as purification tags to prepare His6-:P(SE)2:GFP, His6:P(SE)4:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 :GFP, His6:EctP1:GFP and His6:SHD:GFP fusion proteins. For control experiments, His6:GFP fusion proteins in which the His tag was fused alone, and P(SE)2:GFP, P(SE)4:GFP, PS(E)6:GFP, EctP1:GFP and SHD:GFP fusion proteins in which each peptide tag was fused alone as a purification tag were prepared (see Figure 1b). Then, the protein extract containing the fusion protein was treated with various cationic inducing agents to induce the formation of macromolecular self-assemblies. The protein induced to form macromolecular self-assemblies by the cationic inducing agents was recovered by centrifugation and dissolved in the same buffer used during protein elution. SDS-PAGE was performed by adding SDS sample buffer and heating to confirm that the macromolecular self-assemblies were formed. Figure 2a to Figure 2o middle.

[0271] Results, such as Figure 2a to Figure 2o As shown, it was confirmed that the His6:P(SE)2:GFP fusion protein was expressed by Cu 2+ 、Zn 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2c ), and His6:P(SE)2IA:GFP fusion protein was expressed by Cu 2+ 、Ag 2+ 、Zn 2 + , Fe 2+ Excellent in inducing the formation of macromolecular self-assemblies (see Figure 2d ). His6:PSEIAH:GFP fusion protein was expressed by Ni 2+ 、Ag 2+ , Cu 2+ 、Zn 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2e ), and His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP and His6:PS(D) 10 GFP through Cu 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2g , Figure 2i , Figure 2j and Figure 2k ). It was confirmed that the EctP1:GFP fusion protein and His6:EctP1:GFP fusion protein were expressed by Ni 2+ 、Co 2+ , Cu 2+ 、Zn 2+ , Fe 2+ Induce the formation of macromolecular self-assembly (see Figures 21 and Figure 2m ), SHD:GFP fusion protein and His6:SHD:GFP fusion protein were expressed by Ni 2+ 、Co 2+ , Cu 2+ 、Ag 2+ 、Zn2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2n and Figure 2o ).

[0272] On the other hand, in the case of the control group using His tag alone (His6:GFP) or peptide tag alone (P(SE)2:GFP, P(SE)4:GFP, PS(E)6:GFP), when cationic inducers such as Ni 2+ , Cu 2+ 、Zn 2+ , Fe 2+ When treated with Figure 2a , Figure 2b , Figure 2f and Figure 2h ).

[0273] In summary, it was confirmed that the combination of the His tag and the peptide tag significantly induced the formation of macromolecular self-assemblies by various cationic inducers, and that the SHD peptide tag was excellent in inducing the formation of macromolecular self-assemblies by various cationic inducers not only when combined with the His tag but also when used alone. The combination of the His tag and all the peptide tags and the SHD peptide tag alone had no significant effect on the inducing agent Ni. 2+ The reactivity of Ni 2+ Used as a specific inducer for experiments.

[0274] Experimental Example 2. When the cation Ni 2+ Confirmation of self-assembly formation when treated as an inducer

[0275] Since Ni 2+ is suitable as an inducer to form self-assembly, so this method was used to confirm that when Ni 2+ Formation of self-assemblies of each fusion protein upon treatment.

[0276] Specifically, for His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10Protein extracts of His6:GFP, His6:EctP1:GFP and His6:SHD:GFP fusion proteins, His6:GFP (where His tag was fused alone), P(SE)2:GFP, P(SE)4:GFP, PS(E)6:GFP, EctP1:GFP and SHD:GFP fusion proteins (where a peptide tag whose main amino acids consist of charged amino acids and polar amino acids was fused), and cationic inducer Ni 2+ The protein induced by the macromolecular self-assembly was recovered by centrifugation, dissolved in the same buffer used during protein elution, and confirmed by SDS-PAGE by adding SDS sample buffer and heating. The results are shown in Figures 3a to 3i middle.

[0277] Therefore, if Figures 3a to 3i As shown in the figure, His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP and His6:PS(D) 10 :GFP fusion protein induced the formation of macromolecular self-assembly, and confirmed that Ni 2+ In the case of inducer treatment, the His tag alone (His6:GFP) control group did not induce the formation of macromolecular self-assemblies, and it was confirmed that the control group using P(SE)2:GFP, P(SE)4:GFP, and PS(E)6:GFP fusion proteins to which each peptide tag was fused alone did not induce the formation of macromolecular self-assemblies in the presence of Ni 2+ In the case of inducer treatment, except for P(SE)2:GFP, all other proteins weakly induced the formation of macromolecular self-assemblies (see Figure 3a to Figure 3g In addition, His6:EctP1:GFP fusion protein was expressed by Ni 2+ Induced the formation of macromolecular self-assemblies, and confirmed that the control group using the EctP1:GFP fusion protein in which the EctP1 peptide tag was fused alone was 2+ In the case of the inducer treatment, the formation of macromolecular self-assemblies was relatively weakly induced alone, and in the case of the SHD peptide tag, it was confirmed that not only the His6:SHD:GFP fusion protein fused in combination with His but also the SHD:GFP fusion protein fused with the peptide alone were induced by Ni. 2+ Inducing the formation of macromolecular self-assemblies (see Figure 3h and Figure 3i ).

[0278] Experimental Example 3. Establishment of the Optimal Conditions for the Formation of Macromolecular Self-Assemblies

[0279] In order to maximize the induction of macromolecular self-assembly and the optimization of high-purity purification conditions by removing nonselective proteins other than the target protein, and to minimize the binding of the His tag to the fusion protein in which the main amino acids consist of charged amino acids and polar amino acids as the purification tag, various conditions such as buffer, salt treatment, Ni 2+ Effects of treatment concentration, pH change, treatment time, etc. on the formation of induced self-assembly.

[0280] Experimental Example 3-1. Optimization of ammonium sulfate concentration conditions as salt

[0281] The following experiment was performed to confirm the ammonium sulfate concentration conditions suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag is fused with a peptide tag in which the main amino acids are composed of charged amino acids and polar amino acids as a purification tag.

[0282] Specifically, for the His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 Each protein extract of P(SE)2:GFP, P(SE)4:GFP, PS(E)6:GFP, EctP1:GFP and SHD:GFP fusion proteins, His6:GFP in which the His tag is fused alone, and P(SE)2:GFP, P(SE)4:GFP, PS(E)6:GFP, EctP1:GFP and SHD:GFP fusion proteins in which each peptide tag is fused alone, was induced to form a macromolecular self-assembly by the method described in Example 3-1. The protein induced to form a macromolecular self-assembly was recovered by centrifugation, dissolved in the same buffer used during protein elution, and confirmed by SDS-PAGE by adding SDS sample buffer and heating. The results are shown in Figure 4a to Figure 4o middle.

[0283] The results are as follows Figure 4a to Figure 4oAs shown, it was confirmed that the formation of macromolecular self-assemblies was induced in the following ranges: 12-22% (w / v) for SHD:GFP fusion protein, 14-22% (w / v) for His6:EctP1:GFP fusion protein and His6:SHD:GFP fusion protein, 16-22% (w / v) for His6:PSEIAH:GFP fusion protein, and 16-22% (w / v) for His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PS(E) 10 :GFP and His6:PS(D) 10 The concentration of His6:PS(E)6:GFP fusion proteins was 18-22% (w / v), and that of His6:P(SE)4:GFP and His6:PS(E)6:GFP fusion proteins was 20-22% (w / v).

[0284] Experimental Example 3-2. Optimization of the conditions for the concentration of sodium chloride as salt

[0285] The following experiment was performed to confirm the sodium chloride concentration conditions suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and a peptide tag in which the main amino acids are composed of charged amino acids and polar amino acids are fused as purification tags.

[0286] Specifically, under the treatment conditions of NaCl as a salt in the concentration range of 0-3M, after inducing self-assembly by the method described in Example 3-2, it was confirmed by SDS-PAGE that the result was Figures 5a to 5k Shown in.

[0287] Results, such as Figures 5a to 5k As shown, His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 :GFP and His6:SHD:GFP fusion proteins did not form macromolecular self-assemblies at all NaCl concentrations, and when 200mM NaCl was added for His6:EctP1:GFP fusion protein and 250mM NaCl was added for SHD:GFP fusion protein, macromolecular self-assembly formation was the best. The results of Experimental Examples 3-1 and 3-2 confirmed that the induction of self-assemblies was relatively excellent when treated with ammonium sulfate compared to sodium chloride, so in subsequent experiments, ammonium sulfate was used as the salt for the experiment.

[0288] Experimental Example 3-3. Optimization of buffer conditions

[0289] In order to confirm a buffer suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and a peptide tag in which the main amino acids consist of charged amino acids and polar amino acids are fused as a purification tag, the following experiment was performed.

[0290] Specifically, for each fusion protein extract, pH 7.4 HEPES buffer (HEPES; 50 mM HEPES buffer (pH 7.4)), pH 7.4 PBS buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.4)), pH 7.4 PB buffer (PB; 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4)), pH 7.4 Tris buffer (Tris; 50 mM Tris-HCl buffer (pH 7.4)) were used as each buffer, and 1% [v / v] Triton X-100 and 1X [v / v] protease inhibitor cocktail tablets (Roche) were added to each buffer. Then, ammonium sulfate was used as a salt treatment, and Ni was added at a concentration of 0.5 mM. 2+ The results of SDS-PAGE analysis of the effects of buffer types (such as HEPES, Tris, phosphate, etc.) on the induction of self-assembly are shown in Figures 6a to 6k Displayed in.

[0291] The results are as follows Figures 6a to 6k As shown in the results, it was confirmed that in the case of His6:P(SE)2IA:GFP and His6:PSEIAH:GFP fusion proteins, the formation of macromolecular self-assemblies was excellent in all buffers including HEPES (pH 7.4), and in the case of His6:P(SE)2:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 In the case of His6:EctP1:GFP, SHD:GFP and His6:SHD:GFP fusion proteins, the effect of inducing the formation of macromolecular self-assembly was the best when HEPES (pH 7, 4) buffer was used.

[0292] Experimental Example 3-4. Ni 2+ Concentration Optimization

[0293] In order to confirm that Ni is suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and a peptide tag in which the main amino acids are composed of charged amino acids and polar amino acids are fused as purification tags, 2+ The following experiments were performed under the following concentration conditions.

[0294] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate as salt and the inducing agent Ni 2+ The cations were treated at different concentrations ranging from 0 to 2 mM to induce self-assembly by the method described in Example 3-4, and then the effect of inducing self-assembly was confirmed. Figures 7a to 7k Displayed in.

[0295] The results are as follows Figures 7a to 7k As shown, it was confirmed that His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 In the case of His6:EctP1:GFP, SHD:GFP and His6:SHD:GFP fusion proteins, after ammonium sulfate treatment, when the cationic inducer Ni 2+ Upon treatment, nearly all monomers present in the protein extract formed macromolecular self-assemblies within 20 min.

[0296] Experimental Example 3-5. Optimization of buffer pH

[0297] In order to confirm the pH conditions of the buffer suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and a peptide tag in which the main amino acids consist of charged amino acids and polar amino acids are fused as a purification tag, the following experiment was performed.

[0298] Specifically, the supernatant obtained by centrifugation after ammonium sulfate treatment was adjusted to a pH range of 5-10 using HCl and NaOH, self-assembly was induced by the method described in Examples 3-5, and then confirmed by SDS-PAGE. Figures 8a to 8k Displayed in.

[0299] The results are as follows Figures 8a to 8k As shown, in His6:P(SE)2:GFP, His6:PSEIAH:GFP and His6:PS(D) 10 In the case of GFP fusion protein, the induction of the formation of macromolecular self-assembly occurred normally at pH 6-pH 10. It was confirmed that in all other units, the induction of the formation of macromolecular self-assembly occurred normally under alkaline conditions of pH 7-pH 10, while under acidic conditions of pH 5 or lower, precipitation of nonspecific proteins occurred.

[0300] Experimental Example 3-6. Optimization of the induction time for self-assembly formation of a fusion protein of a His tag and a peptide tag in which the main amino acids are composed of charged amino acids and polar amino acids

[0301] In order to confirm the induction time conditions suitable for the formation of a macromolecular self-assembly of a fusion protein in which a His tag and a peptide tag in which the main amino acids consist of charged amino acids and polar amino acids are fused as a purification tag, the following experiment was performed.

[0302] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate and the induction agent Ni 2+ The cations were treated at a concentration of 0.5 mM. 2+ After the cationic treatment, self-assemblies were induced by the method described in Examples 3-6 every 10 minutes for up to 60 minutes, and they were induced for a maximum of 120 minutes, and then the effect of inducing self-assembly was confirmed by SDS-PAGE. Figures 9a to 9k Displayed in.

[0303] The results are as follows Figures 9a to 9k As shown, it was confirmed that each fusion protein (in which a peptide tag whose main amino acids are composed of charged amino acids and polar amino acids is bound to a His tag) formed a macromolecular self-assembly within 20 minutes.

[0304] Experimental Example 4. Confirmation of the recovery rate of recombinant protein induced to form macromolecular self-assembly

[0305] Experimental Example 4-1. Confirmation of Yield and Purity of Fusion Proteins of His Tag and Peptide Tag in Which the Main Amino Acids Are Consistent with Charged Amino Acids and Polar Amino Acids

[0306] After inducing the formation of self-assembly of a fusion protein of a His tag and a peptide tag in which the main amino acids consist of charged amino acids and polar amino acids under the optimized conditions for forming self-assembly confirmed in Experimental Example 3, they were recovered by a centrifugation method.

[0307] Specifically, the centrifugation method induces the formation of macromolecular self-assemblies under optimal conditions and cationic inducing agents, and then centrifuges at 3,000 rpm or higher for 10 minutes at 4°C to recover the recombinant protein in the form of precipitates, and treats them with EDTA to convert the multimers into monomers. The converted monomers were confirmed by SDS-PAGE, and the yield and purity were measured using the ImageLab program of Bio-Rad. Ammonium sulfate and Ni were used. 2+ After inducing self-assembly, the yield was calculated by the following formula (Formula 1).

[0308] [Formula 1]

[0309] Yield = intensity of recovered target protein band / (intensity of unrecovered target protein band + intensity of recovered target protein band) × 100

[0310] Purity was measured by measuring the ratio of purified protein band to total protein and expressed as a percentage.

[0311] The results are as follows Fig.10 As shown, after treatment with ammonium sulfate, 0.5 mM of the cation inducer Ni 2+ When treated, the maximum yield and maximum purity of each fusion protein of the His tag and the peptide tag in which the main amino acids consist of charged amino acids and polar amino acids were analyzed as follows: 92% and 97% in the His6:P(SE)2:GFP fusion protein, 75% and 92% in the His6:P(SE)2IA:GFP fusion protein, 91% and 95% in the His6:PSEIAH:GFP fusion protein, 81% and 97% in the His6:P(SE)4:GFP fusion protein, 77% and 98% in the His6:PS(E)6:GFP fusion protein, and 91% and 95% in the His6:PS(E)4:GFP fusion protein. 10 : 75%, 98% in GFP fusion protein, His6:PS(D) 10 In the fusion protein of 1:1, 2:1, 3:2, 5:1, 7:0, 9:1, 7:1, 9:2 ...1, 9:2, 9:1, 9:1, 9:1, 9:1, 9:1, 9:1, 9:1, 9:1, 9:1

[0312] Experimental Example 4-2. Confirmation of purification of fusion protein of His tag and peptide tag in which the main amino acids are composed of charged amino acids and polar amino acids using a filter

[0313] The method using the filter is as follows: using 0.2 and 5.0 μm syringe filters (Satorius, Goettingen, GEU), 10 μm syringe filters (Tisch, OH, USA), the recombinant protein of the macromolecular self-assembly formed with the optimal conditions and cationic inducers is filtered into the protein extract, and then the washing process is carried out using the same buffer as the macromolecular assembly induction reaction. In addition, the macromolecular assembly is converted into a monomer or eluted using the same buffer with the addition of a chelating agent such as EDTA. In order to visually confirm this, the filter is irradiated with a 360nm UV wavelength to visually confirm it.

[0314] The results are as follows Figures 11a to 11e As shown, in the presence of ammonium sulfate and Ni2+ In the filter of the treated protein solution, fluorescence can be observed by irradiating UV because the macromolecular self-assembly is filtered through the filter. Thereafter, when the buffer to which EDTA is added passes through the filter, the recombinant protein polymer is converted into a monomer by EDTA and eluted, so even if UV is irradiated, the filter does not show fluorescence. Like this, the conversion from polymer to monomer by EDTA is reversible, and the reaction that occurs within seconds is very fast. In addition, the process of removing the label after purification can also be easily removed under specific inducers or conditions. Therefore, by using the purification method of the present invention, recombinant proteins can be easily, conveniently and quickly recovered with devices and materials generally available to anyone, and it is expected that they can be applied to the process of producing recombinant proteins to carry out large-scale production effortlessly in the future.

[0315] Experimental Example 4-3. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0316] In order to recover the macromolecular self-assemblies by low-speed centrifugation precipitation or filtration through a μm-grade filter, the assemblies should be formed to a size larger than that of bacteria. In order to visually analyze the formation and size of these macromolecular self-assemblies, they were observed under a fluorescence microscope. In the analysis, His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10 :GFP, EctP1:GFP, His6:EctP1:GFP, SHD:GFP and His6:SHD:GFP, His6:GFP was used as a control group. As specific experimental conditions, the optimized purification conditions of Experimental Examples 3 and 4 were applied.

[0317] First, since the formation of macromolecular assemblies was observed, in the case of His6:GFP, assemblies of very small size were partially observed, but in the case of His6:P(SE)2:GFP, His6:P(SE)2IA:GFP, His6:PSEIAH:GFP, His6:P(SE)4:GFP, His6:PS(E)6:GFP, His6:PS(E) 10 :GFP、His6:PS(D) 10In the case of :GFP, EctP1:GFP, His6:EctP1:GFP, SHD:GFP, and His6:SHD:GFP, the overall formation of macromolecular self-assemblies was observed. Since these macromolecular self-assemblies are formed in three dimensions, it is difficult to measure the exact size, but by analyzing the size of the macromolecular self-assemblies based on a planar image of one focal plane, it can be confirmed that they are formed in the size of hundreds of μm to mm ( Figures 12a to 12l ). Such a size is a huge size reaching several to ten times the general size of bacteria, and is estimated to be equal to or larger than the average size of plant cells classified as large-sized among eukaryotic cells.

[0318] Experimental Example 5. Confirmation of the feasibility of purifying each target protein

[0319] The biopharmaceutical market size (including new antibody drugs, vaccines, new protein drugs, etc.) is growing rapidly, and if the recombinant proteins including these biopharmaceuticals are easily and quickly purified to high purity, it is possible to ensure price competitiveness in the drug market. In other words, it is very important to expand versatility by applying the purification method to various target proteins. Therefore, it is confirmed that the method of purifying recombinant proteins using the specific inducer or conditionally dependent self-assembly induction method of the present invention can be applied to various target proteins, such as pharmaceutical proteins, enzymes and antibodies. As target proteins, granulocyte colony stimulating factor, interferon α2, TEV protease and therapeutic antibody Herceptin light chain, various target proteins were purified, and the purification method optimized in Experimental Examples 3 and 4 was used.

[0320] The results are as follows Figures 13a to 13nAs shown, the fusion proteins that best form macromolecular self-assemblies according to the target protein are analyzed, and the maximum yield and maximum purity of the target protein sought in the present invention are: 61% and 98% for His6:P(SE)2:INF-a2, 69% and 97% for His6:P(SE)2:TEV protease, 66% and 92% for His6:P(SE)2:HER LC, 83% and 97% for His6:P(SE)4:TEV protease, and 83% and 97% for His6:P(SE)4:HER LC was 64%, 96%, for His6:P(SE)2IA:rhG-CSF was 71%, 99%, for His6:PSEIAH:rhG-CSF was 92%, 98%, for His6:EctP1:INF-a2 was 92%, 97%, for His6:EctP1:TEV protease was 88%, 97%, for His6:SHD:INF-a2 was 96%, 98%, for SHD:rhG-CSF was 95%, 98%, for SHD:INF-a2 was 87%, 96%, and for SHD:TEV protease was 97%, 97%. In other words, it was confirmed that in the purification of many target proteins other than the target protein sought by the present invention, they can be purified with optimal yield and purity by selecting them among the various units provided in the present invention.

Claims

1. A fusion polypeptide comprising a first polypeptide represented by the amino acid sequence of SEQ ID NO: 1, and a second polypeptide as a peptide tag, wherein the main amino acid components of the peptide tag are composed of charged amino acids and polar amino acids.

2. The fusion polypeptide according to claim 1, wherein The second polypeptide is represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

9.

3. The fusion polypeptide according to claim 1, wherein The first polypeptide and the second polypeptide are linked sequentially.

4. A fusion protein comprising the fusion polypeptide according to claim 1 or the single polypeptide according to claim 2, and a target protein.

5. The fusion protein according to claim 4, wherein The target protein is linked to the N-terminus or C-terminus of the fusion polypeptide. The fusion protein according to claim 5 , further comprising a cleavage site of a protease, a hinge region of an immunoglobulin, or both between the fusion polypeptide and the target protein.

7. A polynucleotide encoding the fusion polypeptide according to any one of claims 1 to 6. An expression vector comprising the polynucleotide according to claim 7 .

9. A host cell transformed with the expression vector according to claim 8.

10. A method for purifying a target protein, comprising culturing cells comprising a polynucleotide encoding the fusion polypeptide according to any one of claims 4 to 6. 11 . The method for purifying a target protein according to claim 10 , further comprising, after the culturing, inducing the formation of a self-assembly of the fusion protein produced in the cells.

12. The method for purifying a target protein according to claim 11, wherein: Inducing the formation of the self-assembly includes adding an inducing agent.

13. The method for purifying a target protein according to claim 12, wherein: The inducer is a cation.

14. The method for purifying a target protein according to claim 13, wherein: The inducing agent has a concentration of 0.1 mM-2.0 mM.

15. The method for purifying a target protein according to claim 11, wherein: Inducing the formation of self-assemblies includes adding a salt including ammonium sulfate.

16. The method for purifying a target protein according to claim 15, wherein: The ammonium sulfate has a concentration of 10%-25% (w / v).

17. The method for purifying a target protein according to claim 11, wherein: The induction of self-assembly formation was carried out under the conditions of pH 6-pH10. The method for purifying a target protein according to claim 11 , further comprising selectively separating the formed self-assembly.

19. The method for purifying a target protein according to claim 18, wherein: The separation is performed by centrifugation or filtration.

20. A composition for purifying a protein, comprising a first polypeptide represented by the amino acid sequence of SEQ ID NO: 1, or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO:

9.

21. A method for purifying a target protein, comprising culturing a cell comprising a polynucleotide in a culture medium, wherein the polynucleotide encodes a fusion protein, in which a fusion polypeptide is fused to a target protein, wherein the fusion polypeptide comprises a first polypeptide represented by an amino acid sequence of SEQ ID NO: 1 and a second polypeptide which is an EctP1 peptide tag, wherein the main amino acid components of the EctP1 peptide tag consist of polar amino acids and charged amino acids.

22. The method for purifying a target protein according to claim 1, wherein: The second polypeptide is represented by SEQ ID NO:

10.

23. A peptide capable of forming a self-assembly, wherein: The main amino acids are composed of charged amino acids and polar amino acids.

24. The peptide according to claim 23, wherein The peptide is represented by an amino acid sequence selected from the group consisting of SEQ ID NO:2 to SEQ ID NO:9.

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