Fusion protein capable of self-assembling by comprising purification tag and alpha-spiral tag, and method for purifying recombinant protein using same

Abstract

The present invention relates to a fusion polypeptide having any one of a His tag, a HAT tag and a HQ tag, and an alpha-helical peptide tag, the main amino acids of which are charged amino acids and hydrophobic amino acids, and a method for purifying a target protein by using the fusion polypeptide. Since the fusion polypeptide has the ability to induce the formation of a self-assembly by fusing with the target protein, the target protein purification method using the fusion polypeptide can separate and purify the target protein only by 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. 10-2022-0122186 filed on September 27, 2022, and incorporates all contents disclosed in the corresponding Korean patent application document as a part of this specification.

[0003] The present invention relates to a fusion protein comprising a purification tag and an alpha-helix tag; a composition for purifying a recombinant protein comprising the fusion protein; and a method for purifying a recombinant protein using the fusion protein. Background Art

[0004] 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 beads (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 is required after purification, 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.

[0005] 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.

[0006] 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

[0007] Technical issues

[0008] 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.

[0009] One embodiment of the present application provides an α-helical peptide tag that forms a self-assembly, in which the main amino acids are composed of charged amino acids and hydrophobic amino acids.

[0010] Another embodiment of the present application provides a fusion polypeptide, comprising a first polypeptide represented by an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 and a second polypeptide which is an α-helix tag, wherein the main amino acids in the α-helix peptide tag are composed of charged amino acids and hydrophobic amino acids.

[0011] 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.

[0012] 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.

[0013] 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.

[0014] Other embodiments of the present application provide a host cell transformed with an expression vector.

[0015] 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.

[0016] Other embodiments of the present application provide a composition for purifying protein, comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8.

[0017] Other embodiments of the present application provide a self-assembly comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3, and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO:4 to SEQ ID NO:8.

[0018] Technical Solution

[0019] 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.

[0020] In order to achieve the above-mentioned purpose of the present invention, the present invention provides an α-helical peptide tag that forms a self-assembly, wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids.

[0021] The charged amino acids of the α-helical peptide tag (in the α-helical peptide tag, the main amino acids are composed of charged and hydrophobic amino acids) that form the self-assembly body can be selected from the positively charged amino acids of lysine (Lys, K), arginine (Arg, R) and histidine (His, H), and the negatively charged amino acids of aspartic acid (Asp, D) and glutamic acid (Glu, E), and the hydrophobic amino acids can be selected from alanine (Ala, A), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), proline (Pro, P), phenylalanine (Phe, F), methionine (Met) and tryptophan (Trp, W), and are characterized in that the content of charged amino acids and hydrophobic amino acids in the α-helical tag excluding the hinge region or the connection site is 60% or more, 65% or more, 70% or more, preferably 75% or more.

[0022] The α-helical peptide tag can be any one selected from the group consisting of mIZ (modified isoleucine zipper), TZ1H, and artificially designed DLH, IAQ or EAH peptides based on the main amino acid characteristics of these peptides being charged amino acids and hydrophobic amino acids, including but not limited to these.

[0023] The α-helical peptide tag may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:4 to SEQ ID NO:8, and in one embodiment, may be represented by or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:4 to SEQ ID NO:8.

[0024] The α-helical peptide tag can be used in the form of a fusion protein fused to a purification tag such as a His tag, wherein the "α-helical peptide tag" or the "fusion protein fused to an α-helical peptide tag and a purification tag such as a His tag" has the ability to induce the formation of a self-assembly by fusing with a target protein.

[0025] In addition, the present invention provides a fusion polypeptide comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 and a second polypeptide which is an α-helix tag, wherein the main amino acids are composed of charged and hydrophobic amino acids.

[0026] 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, the HAT tag in its original form, or a modified HQ tag (or HQ6 tag) of the His tag may be, but is not limited thereto. The His tag, the HAT tag, and the HQ tag may be represented or composed of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

[0027] The second polypeptide may be a motif, domain or peptide capable of nucleation or formation of a polymer such as a dimer, etc. Specifically, the second polypeptide may be any one selected from the group consisting of mIZ (modified isoleucine zipper), TZ1H, and artificially designed DLH, IAQ or EAH peptides based on the main amino acid characteristics of these peptides including DLH, IAQ or EAH peptides as charged amino acids and hydrophobic amino acids, including but not limited to this. mIZ, TZ1H, DLH, IAQ or EAH peptides may be represented or composed of the amino acid sequences of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0028] The first polypeptide and the second polypeptide may be linked sequentially.

[0029] 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.

[0030] 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.

[0031] 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).

[0032] 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.

[0033] 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.

[0034] 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.

[0035] The hinge region may comprise the amino acid sequence of SEQ ID NO:33.

[0036] 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.

[0037] 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.

[0038] The polynucleotide comprises each polynucleotide encoding the first polypeptide and the second polypeptide. The polynucleotide encoding the first polypeptide may comprise or consist of a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO:40 to 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:47.

[0039] 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.

[0040] 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.

[0041] 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.).

[0042] In the present invention, expression refers to the production of a protein or a nucleic acid in a cell.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The content of the fusion polypeptide or target protein is as described above.

[0051] The method of purifying the target protein may further include extracting the fusion protein from the host cells after culturing.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] The method for purifying a target protein may further comprise selectively isolating the formed self-assembly.

[0065] Separation can be performed by centrifugation or filtration.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In addition, the present invention provides a composition for purifying a protein, comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8.

[0071] The contents of the first polypeptide and the second polypeptide are as described above.

[0072] In addition, the present invention provides a self-assembly comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8. The contents of the first polypeptide and the second polypeptide are as described above.

[0073] Beneficial Effects

[0074] The present invention relates to a fusion polypeptide, wherein one of a His tag, a HAT tag, and an HQ tag is fused with an α-helical peptide tag consisting of charged amino acids and hydrophobic amino acids as main amino acids; and a method for purifying a target protein using the fusion polypeptide, wherein the fusion polypeptide is fused with 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

[0075] 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.

[0076] 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.

[0077] 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.

[0078] Figure 2a This is a diagram showing the helical amino acid distribution of the mIZ peptide that forms an α-helical structure, analyzed using a wheel diagram.

[0079] Figure 2bThis is a diagram showing the helical amino acid distribution of the TZ1H peptide that forms an α-helical structure using a wheel diagram.

[0080] Figure 2c This is a diagram showing the helical amino acid distribution of the IAQ peptide that forms an α-helical structure using a wheel diagram.

[0081] Figure 2d This is a diagram showing the helical amino acid distribution of the DLH peptide that forms an α-helical structure using a wheel diagram.

[0082] Figure 2e This is a diagram showing the helical amino acid distribution of the EAH peptide that forms an α-helical structure using a wheel diagram.

[0083] Figure 3a This figure confirms whether the formation of macromolecular self-assembly is induced when treating with various cations by fusing the target protein GFP with the His tag alone as a purification tag.

[0084] Figure 3b This figure examines whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing the trimer-forming TZ1H tag alone to GFP as a purification tag.

[0085] Figure 3c This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing the His tag and TZ1H peptide tag to GFP as purification tags.

[0086] Figure 3d This figure examines whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing the trimer-forming mIZ tag alone to GFP as a purification tag.

[0087] Figure 3e This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing the His tag and mIZ peptide tag to GFP as purification tags.

[0088] Figure 3f This figure examines whether the formation of macromolecular self-assemblies is induced when the DLH tag is used alone as a purification tag and fused to GFP.

[0089] Figure 3g This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing the His tag and DLH peptide tag to GFP as purification tags.

[0090] Figure 3hThis figure examines whether the formation of macromolecular self-assemblies is induced when the IAQ tag is used alone as a purification tag and fused to GFP.

[0091] Figure 3i This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing the His tag and IAQ peptide tag as purification tags to GFP.

[0092] Figure 3j This figure examines whether the formation of macromolecular self-assemblies is induced when the EAH tag is used alone as a purification tag and fused to GFP.

[0093] Figure 3k This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing the His tag and EAH peptide tag to GFP as purification tags.

[0094] Figure 4a The His tag or TZ1H peptide tag was used as a purification tag and fused to GFP alone or both tags were fused to 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.

[0095] Figure 4b The His tag or mIZ peptide tag was used as a purification tag and fused to GFP alone or both tags were fused to 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.

[0096] Figure 4c The His tag or DLH peptide tag was used as a purification tag and fused to GFP alone or both tags were fused to 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.

[0097] Figure 4d The His tag or IAQ 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 cationic Ni 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0098] Figure 4e The His tag or EAH 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.

[0099] Figure 5a 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.

[0100] Figure 5b The TZ1H peptide tag alone was used as a purification tag 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.

[0101] Figure 5c The His tag and TZ1H peptide tag were fused to GFP as purification tags 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.

[0102] Figure 5d The mIZ 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.

[0103] Figure 5e The His tag and mIZ peptide tag were fused to GFP as purification tags 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.

[0104] Figure 5f The DLH 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.

[0105] Figure 5g The His tag and DLH peptide tag were fused to GFP as purification tags 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.

[0106] Figure 5hThe IAQ 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.

[0107] Figure 5i The His tag and IAQ peptide tag were fused to GFP as purification tags 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.

[0108] Figure 5j The EAH peptide tag alone was used as a purification tag 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.

[0109] Figure 5k The His tag and EAH peptide tag were fused to GFP as purification tags 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.

[0110] Figure 6a The combination of His-tag and TZ1H 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.

[0111] Figure 6b The combination of His-tag and mIZ-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.

[0112] Figure 6c The His-tag and DLH-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.

[0113] Figure 6dThe His-tag and IAQ-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.

[0114] Figure 6e The His-tag and EAH-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.

[0115] Figure 7a The His tag and TZ1H peptide tag were fused to GFP as purification tags to analyze the expression of GFP when treated with ammonium sulfate 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.

[0116] Figure 7b The His tag and mIZ peptide tag were fused to GFP as purification tags to analyze the expression of GFP when treated with ammonium sulfate 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.

[0117] Figure 7c The His tag and DLH peptide tag were combined as purification tags and fused to GFP to analyze the expression of cytokines when treated with ammonium sulfate and the cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0118] Figure 7d The His tag and IAQ peptide tag were combined as purification tags and fused to GFP to analyze the activity of the cytosolic proteins when treated with ammonium sulfate and the cationic Ni 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0119] Figure 7e The His tag and EAH peptide tag were combined as purification tags and fused to GFP to analyze the expression of GFP when treated with ammonium sulfate 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.

[0120] Figure 8aThe combination of His-tag and TZ1H peptide tag was fused to GFP as purification tag to analyze the expression of Ni in the presence of ammonium sulfate as salt, depending on the selected cation. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0121] Figure 8b The His-tag and mIZ-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 chosen cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0122] Figure 8c The combination of His-tag and DLH peptide tag was fused to GFP as purification tag to analyze the expression of Ni in the presence of ammonium sulfate as salt, depending on the selected cation. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0123] Figure 8d The combination of His-tag and IAQ-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 selected cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0124] Figure 8e The combination of His-tag and EAH 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 selected cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0125] Figure 9a The His-tag and TZ1H peptide-tag were combined as purification tags and fused to GFP 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.

[0126] Figure 9b The His-tag and mIZ-peptide-tag were combined as purification tags and fused to GFP 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.

[0127] Fig.9cThe His-tag and DLH-peptide-tag were combined as purification tags and fused to GFP 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.

[0128] Figure 9d The His-tag and IAQ-peptide-tag were combined as purification tags and fused to GFP 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.

[0129] Fig.9e The His-tag and EAH-peptide-tag were combined as purification tags and fused to GFP 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.

[0130] Fig.10a The combination of His-tag and TZ1H peptide tag was fused to GFP as purification tag to analyze the expression of Ni in the presence of ammonium sulfate as salt, depending on the selected cation. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0131] Fig.10b The His-tag and mIZ-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 chosen cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0132] Fig.10c The combination of His-tag and DLH peptide tag was fused to GFP as purification tag to analyze the expression of Ni in the presence of ammonium sulfate as salt, depending on the selected cation. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0133] Fig.10d The combination of His-tag and IAQ-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 selected cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0134] Fig.10eThe combination of His-tag and EAH 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 selected cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0135] Fig.11a 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.

[0136] Fig.11b The TZ1H peptide tag alone was used as a purification tag 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.

[0137] Fig.11c The HQ tag and TZ1H peptide tag were fused to GFP as purification tags 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.

[0138] Fig.11d The mIZ 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.

[0139] Fig.11e The HQ tag and mIZ peptide tag were fused to GFP as purification tags 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.

[0140] Fig.11f The HAT 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.

[0141] Figure 11g The HAT tag and mIZ peptide tag were fused to GFP as purification tags 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.

[0142] Fig.12a The combination of HQ tag and TZ1H peptide tag as a modified form of His tag was fused to GFP as a purification tag 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.

[0143] Figure 12b The results were analyzed by fusing a combination of HQ tag, a modified form of His tag, and mIZ peptide tag to GFP as purification tags 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.

[0144] Fig.12c The HAT tag in its original form was fused to GFP as a purification tag to analyze the selectivity 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.

[0145] Fig.12d The combination of a HAT tag as the original form of the His tag and an mIZ peptide tag was fused to GFP as a purification tag to analyze the selective activity of the cationic Ni in the presence of various concentrations of ammonium sulfate as the salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0146] Fig.13 When the His tag and each peptide tag combination are fused to GFP as a purification tag, and in the presence of ammonium sulfate as a salt, the selected cation Ni is used as a specific inducer. 2+ During the treatment, the yield and purity of each unit were analyzed by the degree of self-assembly formation.

[0147] Fig.14 When HAT tag alone was fused to GFP as purification tag, or HQ or HAT tag and each peptide tag were fused to GFP as purification tag in combination, and Ni was used as a specific inducer in the presence of ammonium sulfate as salt. 2+ During the treatment, the yield (recovery rate) and purity of each unit are analyzed by the degree of self-assembly formation.

[0148] Fig.15aThe His tag alone, or a combination of the His tag and the DLH peptide tag, the combination of the HQ tag and the mIZ peptide tag, or the combination of the HAT tag and the mIZ peptide tag were fused to GFP as purification tags to analyze the activity of the cationic 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.

[0149] Fig.15b The results were analyzed by fusing a combination of His tag and TZ1H peptide tag, a combination of His tag and mIZ peptide tag, a combination of His tag and IAQ peptide tag, a combination of His tag and EAH peptide tag, or HAT tag alone as purification tags to GFP to analyze the effect of cationic Ni on the expression of GFP 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.

[0150] Fig.15c The combination of HQ tag and TZ1H peptide tag was fused to GFP as purification tag to analyze the activity of 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.

[0151] Fig.16a It is a diagram showing the amino acid sequence information of a peptide tag constructed by substituting each amino acid of the TZ1H peptide tag based on the α-helix tag.

[0152] Fig.16b The analysis was performed when a peptide tag constructed by replacing each amino acid of the TZ1H peptide tag based on the α-helical tag was fused to GFP and the cationic Ni was selected in the presence of ammonium sulfate as the salt. 2+ Figure 3. Capacity of macromolecular self-assembly formation when treated with specific inducers.

[0153] Fig.16c This is a diagram analyzing the ability to form a self-assembly when a peptide tag constructed by substituting each amino acid of the TZ1H peptide tag based on the α-helix tag is fused to GFP and treated with ammonium sulfate as a salt under specific pH conditions in a buffer.

[0154] Fig.17a 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.

[0155] Fig.17b 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:mIZ:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0156] Fig.17c 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:TZ1H:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0157] Fig.17d is based on the selection of the cation Ni in the presence of ammonium sulfate as a salt. 2+ Figure 3 Analysis of fluorescence intensity per pixel of fluorescence images captured under conditions of treatment with specific inducers for His6:GFP, His6:TZ1H:GFP, TZ1H:GFP, His6:mIZ:GFP, and mIZ:GFP to quantitatively analyze the formation of macromolecular self-assemblies by fluorescence microscopy.

[0158] Fig.18a The His-tag and DLH-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 the applicability to recombinant proteins other than GFP.

[0159] Fig.18b The His-tag and IAQ-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 the applicability to recombinant proteins other than GFP.

[0160] Fig.18c The combination of His tag and mIZ peptide tag was fused to the light chain of the therapeutic antibody Herceptin 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 the applicability to recombinant proteins other than GFP.

[0161] Fig.18dThe His-tag and TZ1H 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 the applicability to recombinant proteins other than GFP.

[0162] Fig.18e The His-tag and TZ1H 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 the applicability to recombinant proteins other than GFP.

[0163] Fig.18f The His-tag and mIZ-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 the applicability to recombinant proteins other than GFP.

[0164] Figure 18g The His-tag and mIZ-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 the applicability to recombinant proteins other than GFP.

[0165] Figure 18h The His-tag and DLH-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 the applicability to recombinant proteins other than GFP. DETAILED DESCRIPTION

[0166] 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.

[0167] [Experimental Materials and Methods]

[0168] <Experimental Materials>

[0169] 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.

[0170] Example 1. Construction of a vector for expressing protein

[0171] 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.Subsequently, vectors pET 28a-His6:TZ1H:GFP, pET 28a-His6:TZ1H(IA / GG):GFP, pET 28a-His6:TZ1H(Q / G):GFP, pET 28a-His6:TZ1H(H / G):GFP, pET 28a-His6:TZ1H(H / K):GFP, pET 28a-His6:TZ1H(E / G):GFP, pET 28a-His6:TZ1H(K / G):GFP, pET 28a-His6:mIZ:GFP, pET 28a-His6:DLH:GFP, pET 28a-His6:IAQ:GFP and pET 28a-His6:EAH:GFP were constructed based on primer pairs F-Nco I, BamH I-His6:TZ1H / R-Kpn I-TZ1H; F-NcoI, BamH I-His6:TZ1H(IA / GG) / R-Kpn I-TZ1H(IA / GG); F-Nco I, BamH I-His6:TZ1H(Q / G) / R-Kpn I-TZ1H(Q / G); F-Nco I, BamH I-His6:TZ1H(H / G) / R-Kpn I-TZ1H(H / G); F-Nco I, BamHI-His6:TZ1H(H / K) / R-Kpn I-TZ1H(H / K); F-Nco I, BamH I-His6:TZ1H(E / G) / R-Kpn I-TZ1H(E / G); F-Nco I, BamH I-His6:TZ1H(K / G) / R-Kpn I-TZ1H(K / G); F-Nco I, BamH I-His6:mIZ / R-Kpn I-mIZ; F-Nco I, BamH I-His6:DLH / R-Kpn I-DLH; F-Nco I, BamH I-His6:IAQ / R-KpnI-IAQ; F-Nco I, BamH I-His6:EAH / R-Kpn I-EAH, respectively.In order to construct vectors other than the His6 tag, pET 28a-TZ1H:GFP, pET 28a-TZ1H:GFP, and pET 28a-TZ1H:GFP were constructed based on the primer combinations F-Nco I, BamH I-TZ1H / R-Kpn I-TZ1H; F-Nco I, BamH I-TZ1H(H / G) / R-Kpn I-TZ1H(H / G); F-Nco I, BamH I-TZ1H(K / G) / R-Kpn I-TZ1H(K / G); F-Nco I, BamH I-mIZ / R-Kpn I-mIZ; F-Nco I, BamH I-DLH / R-Kpn I-DLH; F-Nco I, BamH I-IAQ / R-Kpn I-IAQ; and F-Nco I, BamH I-EAH / R-Kpn I-EAH. 28a-TZ1H(H / G):GFP, pET 28a-TZ1H(K / G):GFP, pET 28a-mIZ:GFP, pET 28a-DLH:GFP, pET 28a-IAQ:GFP and pET 28a-EAH:GFP.

[0172] In addition, to test the feasibility of using the His6 tag pseudo sequence, pET 28a-HAT:GFP, pET 28a-HAT:mIZ:GFP, pET 28a-HQ:TZ1H:GFP and pET 28a-HQ:mIZ:GFP vectors were constructed based on the primer combinations of F-Nco I, BamH I-HAT / R-Kpn I-HAT; F-Nco I, BamH I-HAT:mIZ / R-Kpn I-mIZ; F-Nco I, BamH I-HQ:TZ1H / R-Kpn I-TZ1H; F-Nco I and BamH I-HQ:mIZ / R-Kpn I-mIZ, respectively. To evaluate the scalability of purification of various protein drugs, PCR was performed with primer combinations F-Bgl II-rhG-CSF / R-Hind III-rhG-CSF; F-Bgl II-INF-a2 / R-Hind III-INF-a2; F-Bgl II-HER LC / R-Hind III-HER LC, which were then cut with BglII / Hind III restriction enzymes and inserted into the GFP site of pET 28a-His6:DLH:GFP, pET 28a-His6:IAQ:GFP, pET 28a-His6:mIZ:GFP, and pET 28a-His6:TZ1H:GFP vectors to complete pET 28a-His6:DLH:rhG-CSF, pET 28a-His6:IAQ:INF-a2, and pET 28a-His6:mIZ:HER LC, His6:TZ1H:rhG-CSF, pET 28a-His6:TZ1H:INF-a2, pET 28a-His6:mIZ:INF-a2, pET 28a-His6:mIZ:rhG-CSF, pET 28a-His6:DLH:INF-a2 representative vectors. The specific sequence information of each tag and designed recombinant protein is shown in Tables 1 to 3 below.

[0173] [Table 1]

[0174] Amino acid sequence and nucleotide sequence information of the first purification tag

[0175]

[0176] [Table 2]

[0177] Amino acid sequence and nucleotide sequence information of α-helical tag (or second polypeptide tag)

[0178]

[0179]

[0180] [Table 3]

[0181] Amino acid sequence and nucleotide sequence information of each fusion polypeptide, target protein and hinge region.

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194] [Table 4]

[0195] Amino acid sequence and nucleotide sequence information of mutant clones based on major amino acid analysis of TZ1H

[0196]

[0197]

[0198] Example 2. Design of α-helical peptide tags and analysis of wheel maps

[0199] Based on the charged amino acids and hydrophobic amino acids that are the main amino acids constituting the α-helical peptide tags, the mIZ, TZ1H, IAQ, DLH, and EAH peptide tags were artificially designed to promote the formation of self-assemblies by adjusting the relative arrangement positions of the main amino acids in the three-dimensional structure of the α-helix through wheel diagram analysis using pepwheel (https: / / www.bioinformatics.nl / cgi-bin / emboss / pepwheel). The arrangement of the mIZ peptide tag allows the charged and hydrophobic main amino acids to be bidirectionally distributed in the three-dimensional structure of the α-helix, and the TZ1H peptide tag is arranged so that the main amino acids are evenly distributed in all directions. The IAQ peptide tag is arranged so that, in addition to the charged and hydrophobic main amino acids, polar amino acids partially form clusters in the three-dimensional structure of the α-helix. The DLH and EAH peptide tags are arranged so that clusters of main amino acid combinations are distributed in three directions on the α-helical three-dimensional structure. The results of the wheel diagram analysis of the relative positions of the main amino acids in these α-helical peptide tags are shown in Figure 2. Figure 2a to Figure 2e Displayed in.

[0200] Example 3. Induction and extraction of fusion protein expression

[0201] 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.

[0202] Example 4. Induction and optimization of macromolecular self-assembly formation

[0203] Example 4-1. Optimization of ammonium sulfate concentration as salt

[0204] To optimize the ammonium sulfate concentration, a concentration of 0.5 mM Ni 2+ The supernatant obtained after centrifugation was treated with ammonium sulfate in the range of 12 to 22% [w / v] at 2% intervals.

[0205] Example 4-2. Optimization of sodium chloride concentration as salt

[0206] In order to analyze the effect of the treatment concentration of sodium chloride as salt, the Ni 2+ deal with.

[0207] Example 4-3. Optimization of buffer

[0208] 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 mM 2+ The supernatant was obtained by treatment with ammonium sulfate followed by centrifugation under buffered conditions.

[0209] Example 4-4. Ni 2+ Concentration Optimization

[0210] In order to optimize Ni 2+ Concentration, with Ni in the range of 0 to 2 mM 2+ The supernatant obtained by centrifugation after ammonium sulfate treatment was processed.

[0211] Example 4-5. Optimization of buffer pH

[0212] To optimize the buffer pH, the supernatant obtained by ammonium sulfate treatment and centrifugation was used after adjusting the pH to a range of 5-10 using HCl and NaOH. 2+ The supernatant obtained by ammonium sulfate treatment and centrifugation under each pH condition was treated.

[0213] Example 4-6. Optimization of reaction time for self-assembly formation

[0214] In order to optimize the formation of macromolecular self-assemblies, the supernatant obtained after centrifugation after ammonium sulfate treatment was treated with 0.5 mM Ni 2+Treatment, measurements were taken at 10-min intervals from immediately after treatment to 60 min after treatment, and additional measurements were taken up to 120 min.

[0215] Example 5. Recovery of macromolecular self-assembly recombinant protein and conversion into monomers

[0216] The recombinant protein that forms the macromolecular self-assembly in Example 4 in a specific inducer or condition-dependent manner is recovered by methods such as centrifugation and filtration.

[0217] Specifically, in the purification method by centrifugation, centrifugation is performed at 3,000rpm or higher speed at 4 ° C for 10 minutes, and the macromolecular assembly recombinant protein in the form of a precipitate is obtained, and 5mM EDTA is added to the same buffer for the macromolecular assembly induction reaction to obtain the recombinant protein of the 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 the macromolecular self-assembly, the same buffer used in the macromolecular assembly induction reaction is used to wash the process. In addition, the macromolecular assembly is converted into a monomer or eluted with the same buffer to which a chelating agent such as EDTA is added. In order to visually confirm this, the filter is visually confirmed by irradiating the filter with a UV wavelength of 360nm UV.

[0218] Example 6. Confirmation of protein purification by SDS-PAGE

[0219] 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 universal SDS-PAGE method.

[0220] Example 7. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0221] In order to analyze the formation and approximate size of macromolecular self-assemblies, a fluorescence microscope was used, and to detect the GFP fluorescence signal, a filter with excitation: 488 nm and emission: 520 nm was used. In order to quantitatively analyze the extent of macromolecular self-assembly formation, the relative scale of self-assembly formation was compared by measuring the fluorescence intensity per pixel of the captured fluorescence images.

[0222] [Experimental results]

[0223] Experimental Example 1. Identification of an inducing agent suitable for inducing the formation of self-assembly of His-tag and α-helix-based unit peptide tag fusion protein

[0224] 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. As described in Examples 1, 2 and 3, the fusion proteins His6:TZ1H:GFP, His6:mIZ:GFP, His6:DLH:GFP, His6:IAQ:GFP and His6:EAH:GFP were constructed by combining the His tag as a purification tag and the TZ1H peptide tag or the mIZ peptide tag (an α-helix-based unit composed of charged amino acids and hydrophobic amino acids) as well as the DLH peptide tag, the IAQ peptide tag and the EAH peptide tag (artificially designed based on these characteristics). For the control group experiment, the His6:GFP fusion protein (in which the His tag alone was used as a purification tag), the TZ1H:GFP, mIZ:GFP, DLH:GFP, IAQ:GFP and EAH:GFP fusion proteins (in which each α-helix-based unit was fused separately) were constructed (see Figure 1b ). Then, the protein extract containing the fusion protein was treated with various cationic inducers to induce the formation of macromolecular self-assemblies. The protein that formed macromolecular self-assemblies induced by the cationic inducers 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. Figures 3a to 3k middle.

[0225] The results are as follows Figures 3a to 3k As shown in Figure , it was confirmed that the His6:TZ1H:GFP fusion protein was expressed by Ni 2+ 、Co 2+ , Cu 2+ 、Zn 2 + , Fe 2+ , Ba 2+ Inducing the formation of macromolecular self-assemblies, in particular, Ni 2+ 、Zn 2+ , Fe 2+ , Ba 2+ Shows excellent induction of self-assembled structure formation (see Figure 3e ). His6:mIZ:GFP fusion protein was expressed by Ni 2+ 、Co 2+ , Cu 2+ 、Ag 2+ 、Zn 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies, in particular, Ni 2+ , Cu 2+、Ag 2+ , Fe 2+ Shows excellent induction of self-assembled structure formation (see Figure 3e ). His6:DLH:GFP and His6:IAQ:GFP fusion proteins were expressed by Ni 2+ 、Co 2+ , Cu 2+ 、Zn 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 3h and Figure 3i ), and His6:EAH:GFP by Cu 2+ 、Zn 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 3k In contrast, when the control group used the His tag alone (His6:GFP) or the peptide tag alone (TZ1H:GFP, mIZ:GFP, DLH:GFP, IAQ:GFP, EAH:GFP), the cationic inducer Ni 2+ , Cu 2 + 、Zn 2+ , Fe 2+ When treated with Figure 3a , Figure 3b , Figure 3d , Figure 3f , Figure 3h and Figure 3j ). In summary, it was confirmed that the combination of the His tag and the peptide tag significantly increased the induction of macromolecular self-assembly formation by various cationic inducers, and it was confirmed that the type of cationic inducer that increased the induction of self-assembly formation differed depending on the type of α-helix-based unit peptide tag. The combination of the His tag and all peptide tags generally showed an effect on the inducer Ni 2+ The excellent reactivity of Ni 2+ Experiments were performed as specific inducers.

[0226] Experimental Example 2. When the cation Ni 2+ Confirmation of self-assembly formation when treated as an inducer

[0227] Since Ni 2+ Suitable as an inducing agent for the formation of self-assembly, so when Ni 2+ When processing, it is confirmed that the use of Ni 2+, the formation of self-assemblies of each of the His6:TZ1H:GFP, His6:mIZ:GFP, His6:DLH:GFP, His6:IAQ:GFP, and His6:EAH:GFP fusion proteins.

[0228] Specifically, for each protein extract obtained in Example 3 comprising His6:TZ1H:GFP, His6:mIZ:GFP, His6:DLH:GFP, His6:IAQ:GFP, His6:EAH:GFP fusion proteins, His6:GFP (wherein the His tag is fused alone), and TZ1H:GFP, mIZ:GFP, DLH:GFP, IAQ:GFP, EAH:GFP fusion proteins (wherein each α-helix-based unit is fused alone), the cationic inducer Ni 2+ The protein induced to form a macromolecular self-assembly was recovered by centrifugation and dissolved in the same buffer used during protein elution. The structure was confirmed by SDS-PAGE by adding SDS sample buffer and heating. Figures 4a to 4e middle.

[0229] The results are as follows Figures 4a to 4e As shown, it was confirmed that His6:TZ1H:GFP fusion protein, His6:mIZ:GFP fusion protein, His6:DLH:GFP fusion protein, His6:IAQ:GFP fusion protein and His6:EAH:GFP were expressed by Ni 2+ Induced the formation of macromolecular self-assemblies, while the His tag alone (His6:GFP) control group and the peptide tag alone (TZ1H:GFP, mIZ:GFP, DLH:GFP, IAQ:GFP, EAH:GFP) control group did not show any significant difference in the Ni 2+ The inducing agent treatment did not induce the formation of macromolecular self-assemblies (see Figures 4a to 4e ).

[0230] Experimental Example 3. Establishment of the Optimal Conditions for the Formation of Macromolecular Self-Assemblies

[0231] In order to optimize the purification conditions for high purity by maximizing the induction of macromolecular self-assembly formation and minimizing the nonselective removal and binding of proteins other than the target protein of the fusion protein with His tag and α-helix-based unit (mainly composed of charged amino acids and hydrophobic amino acids) as purification tags, 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.

[0232] Experimental Example 3-1. Optimization of ammonium sulfate concentration conditions as salt

[0233] The following experiment was performed to confirm the ammonium sulfate concentration conditions suitable for the formation of macromolecular self-assembly of fusion proteins of His tag and α-helix-based units (mainly composed of charged amino acids and hydrophobic amino acids) as purification tags.

[0234] Specifically, for each of the protein extracts obtained in Example 3, including His6:TZ1H:GFP, His6:mIZ:GFP, His6:DLH:GFP, His6:IAQ:GFP and His6:EAH:GFP fusion proteins, His6:GFP in which the His tag is fused alone, and TZ1H:GFP, mIZ:GFP, DLH:GFP, IAQ:GFP, EAH:GFP fusion proteins in which each α-helix-based unit is fused alone, the formation of a macromolecular self-assembly was induced by the method described in Example 4-1. The protein induced to form a macromolecular self-assembly was recovered by centrifugation and dissolved in the same buffer used during protein elution, and confirmed by SDS-PAGE by adding SDS sample buffer and heating, and the results showed that Figures 5a to 5k middle.

[0235] The results are as follows Figures 5a to 5k As shown, it was confirmed that the formation of macromolecular self-assembly was induced in the following range: 12-22% (w / v) for His6:TZ1H:GFP, 14-22% (w / v) for His6:mlZ:GFP, His6:DLH:GFP and His6:IAQ:GFP, and 16-22% (w / v) for His6:EAH:GFP. The optimal macromolecular self-assembly formation for each unit was analyzed, which was 16% (w / v) for His6:TZ1H:GFP, and 20% (w / v) for His6:DLH:GFP, His6:mlZ:GFP, His6:IAQ:GFP and His6:EAH:GFP.

[0236] Experimental Example 3-2. Optimization of the conditions for the concentration of sodium chloride as salt

[0237] The following experiment was performed to confirm the sodium chloride concentration conditions suitable for the formation of macromolecular self-assembly of the fusion protein based on the His tag and the α-helical unit (in which the main amino acids are composed of charged and hydrophobic amino acids) as purification tags.

[0238] Specifically, under the treatment conditions of NaCl in the concentration range of 0-3M as salt, after inducing self-assembly by the method described in Example 4-2, they were confirmed by SDS-PAGE. Figures 6a to 6e Displayed in.

[0239] The results are as follows Figures 6a to 6e As shown, analysis showed that His6:DLH:GFP and His6:EAH:GFP fusion proteins did not form macromolecular self-assemblies at all NaCl concentrations, and the formation of macromolecular self-assemblies was best when 100 mM NaCl was added to His6:mlZ:GFP and His6:IAQ:GFP and 300 mM NaCl was added to His6:TZ1H:GFP.

[0240] The results of Experimental Examples 3-1 and 3-2 confirmed that the induction of self-assembly was relatively excellent when ammonium sulfate was used as compared to sodium chloride, and therefore in subsequent experiments, ammonium sulfate was used as the salt for the experiments.

[0241] Experimental Example 3-3. Optimization of buffer conditions

[0242] In order to confirm the buffer suitable for the formation of macromolecular self-assembly by fusion proteins using His tag and α-helical unit (mainly composed of charged and hydrophobic amino acids) as purification tags, the following experiment was performed.

[0243] 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 pH7.4)), 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 all buffers. Then, ammonium sulfate was used as a salt treatment, and Ni was added at a concentration of 0.5 mM. 2+ The results of the analysis of the effects of inducing self-assembly by SDS-PAGE according to the type of buffer (e.g. HEPES, Tris, phosphate, etc.) are shown in Figures 7a to 7e Displayed in.

[0244] The results are as follows Figures 7a to 7eAs shown, it was confirmed that all fusion proteins except His6:TZ1H:GFP showed the best effect of inducing the formation of macromolecular self-assemblies in HEPES (pH 7.4) buffer solution, and when PB buffer (pH 7.4) was used, His6:TZ1H:GFP fusion protein showed the best effect of inducing the formation of macromolecular self-assemblies.

[0245] Experimental Example 3-4. Ni 2+ Concentration Optimization

[0246] In order to confirm that the Ni-based fusion protein is suitable for forming a macromolecular self-assembly based on His tag and α-helical unit (mainly composed of charged and hydrophobic amino acids) as a purification tag 2+ The following experiments were performed under the following concentration conditions.

[0247] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate as a salt by the method described in Example 3-4, and the inducer Ni 2+ The cations were treated at different concentrations ranging from 0 to 2 mM to induce self-assembly, and then the effect of inducing self-assembly was confirmed. Figures 8a to 8e Displayed in.

[0248] The results are as follows Figures 8a to 8e As shown, it was confirmed that after ammonium sulfate treatment, the cationic inducer Ni 2+ Upon treatment, His6:TZ1H:GFP, His6:mIZ:GFP, His6:DLH:GFP, His6:IAQ:GFP, and His6:EAH:GFP fusion proteins formed macromolecular self-assemblies of nearly all monomers present in the protein extract within 20 min.

[0249] Experimental Example 3-5. Optimization of buffer pH

[0250] In order to confirm the pH conditions of the buffer suitable for the formation of macromolecular self-assembly of fusion proteins using His tags and α-helical units (mainly composed of charged and hydrophobic amino acids) as purification tags, the following experiment was performed.

[0251] 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 Example 4-5, and then confirmed by SDS-PAGE. Figures 9a to 9e Displayed in.

[0252] The results are as follows Figures 9a to 9eAs shown, in the case of His6:TZ1H:GFP and His6:mlZ:GFP fusion proteins, the induction of the formation of macromolecular self-assemblies occurred normally at pH 7-pH 8, but the induction of the formation of macromolecular self-assemblies tended to decrease under more alkaline conditions. It was confirmed that in all other units, the induction of the formation of macromolecular self-assemblies occurred normally under alkaline conditions of pH 7-pH 10, and the precipitation of non-specific proteins occurred under acidic conditions of pH 5 or lower.

[0253] Example 3-6. Optimization of the induction time of the self-assembly formation of the His tag and the α-helical unit-based fusion protein

[0254] In order to confirm the induction time conditions suitable for the formation of macromolecular self-assembly by fusion proteins using His tags and α-helical units (mainly composed of charged and hydrophobic amino acids) as purification tags, the following experiment was performed.

[0255] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate as a salt, 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 4-6 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 10a to 10e Displayed in.

[0256] The results are as follows Figures 10a to 10e As shown in , it was confirmed that each fusion protein, in which a peptide tag whose main amino acid composition consists of charged amino acids and hydrophobic amino acids is bound to a His tag, formed a macromolecular self-assembly within 20 minutes.

[0257] Experimental Example 3-7. Confirmation of the ability of fusion proteins of HQ tag or HAT tag and α-helix-based tag to induce self-assembly formation

[0258] To confirm whether the ability to induce the formation of macromolecular self-assemblies is enhanced under specific inducer conditions, HAT tags (the original form of His tags) and modified HQ tags were fused with TZ1H peptide tags and mIZ peptide tags, either individually or in combination, and green fluorescent protein was selected as a model protein to construct each of the following HQ6:TZ1H:GF, HQ6:mIZ:GFP, HAT:GFP, and HAT:mIZ:GFP fusion proteins. 2+As a specific inducing agent, ammonium sulfate was used as salt to induce macromolecular self-assembly and recovered by centrifugation. Then, SDS sample buffer was added, heated, and analyzed by SDS-PAGE.

[0259] First, in order to confirm the ability of the concentration conditions of ammonium sulfate to induce the formation of self-assembly of HQ tag or HAT tag and α-helix tag-based fusion proteins, an experiment was performed in the same manner as in Example 4-2.

[0260] Results, such as Figures 11a to 11g As shown, HQ6:mIZ:GFP and HAT:mIZ:GFP induced macromolecular self-assembly formation in the range of 12-22% (w / v), HAT:GFP in the range of 16-22% (w / v), and HQ6:TZ1H:GFP in the range of 18-22% (w / v). The best macromolecular self-assembly formation for each unit was analyzed, and HQ6:mlZ:GFP, HAT:GFP, HAT:mlZ:GFP were 18% (w / v), and HQ6:TZ1H:GFP was 22% (w / v).

[0261] In order to optimize the reaction time of the self-assembly formation of the HQ tag or HAT tag and the α-helix-based tag fusion protein, experiments were performed using the HQ tag or HAT tag and the α-helix-based tag fusion protein in the same manner as in Examples 4-6, and the results are shown in Figures 12a to 12d middle.

[0262] The results are as follows Figures 12a to 12d As shown, it was confirmed that each of the HQ tag or HAT tag and α-helix-based tag fusion proteins formed a macromolecular self-assembly within 20 minutes.

[0263] Experimental Example 4. Confirmation of the recovery rate of recombinant protein induced to form macromolecular self-assembly

[0264] Experimental Example 4-1. Confirmation of Yield and Purity of Fusion Proteins of His-Tag and α-Helix-Based Tags

[0265] After inducing the formation of self-assembly of the fusion protein of the His tag and the α-helix tag under the optimized conditions for the formation of self-assembly confirmed in Experimental Example 3, they were recovered by centrifugation.

[0266] Specifically, the centrifugation method induces the formation of supramolecular 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 Image Lab program of Bio-Rad. 2+ After inducing self-assembly, the yield was calculated by the following formula (Formula 1).

[0267] [Formula 1]

[0268] Yield = intensity of recovered target protein band / (intensity of unrecovered target protein band + intensity of recovered target protein band) × 100

[0269] Purity was expressed as a percentage by measuring the ratio of the purified protein band to the total protein.

[0270] The results are as follows Fig.13 As shown, after treatment with ammonium sulfate, 0.5 mM of the cation inducer Ni 2+ When processed, the maximum yield and maximum purity of each fusion protein analyzed for the His tag and α-helix-based tag were 96% and 97% for His6:TZ1H:GFP, 98% and 97% for His6:mlZ:GFP, 96% and 99% for His6:DLH:GFP, 98% and 96% for His6:IAQ:GFP and 89% and 97% for His6:EAH:GFP, respectively.

[0271] Experimental Example 4-2. Confirmation of the yield and purity of fusion proteins of HQ tag, HAT tag and α-helix-based tag

[0272] After inducing self-assembly formation of the fusion protein of the HQ tag or HAT tag and the α-helix-based tag under the conditions optimized for self-assembly formation confirmed in the above Experimental Example 3, the protein was recovered in the same manner as in Experimental Example 4-1 to confirm the yield and purity.

[0273] The results are as follows Fig.14 As shown, after treatment with ammonium sulfate, 0.5 mM of the cation inducer Ni 2+ When processed, the maximum yield and maximum purity of each HQ-tag or HAT-tag and α-helix-based tag fusion protein analyzed were 62% and 96% for HQ6:TZ1H:GFP, 90% and 97% for HAT:GFP, 97% and 99% for HQ6:mIZ:GFP, and 96% and 99% for HAT:mIZ:GFP, respectively.

[0274] Based on the above results, it was confirmed that when used alone as a purification tag, the HAT tag showed an enhanced ability to form macromolecular self-assemblies compared to the His tag, and the combination of the HAT tag or HQ tag with the TZ1H peptide tag and the mIZ peptide tag showed similar abilities to form macromolecular self-assemblies compared to the His tag.

[0275] Experimental Example 4-3. Confirmation of purification of His-tag, HQ-tag, HAT-tag and α-helix-based tag fusion proteins using filters

[0276] The method using the filter is as follows: using a 0.2 syringe filter (Satorius, Goettingen, GEU), the recombinant protein that forms the macromolecular self-assembly with the optimal conditions and cationic inducing agent 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.

[0277] The results are as follows Figures 15a to 15c As shown, in the presence of ammonium sulfate and Ni 2+ 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 the filter does not show fluorescence even when irradiating UV. 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.

[0278] Experimental Example 4-4. Confirmation of the main amino acids for the formation of macromolecular self-assembly of fusion protein of His tag and α-helix tag

[0279] The following experiment was performed to identify the main amino acids important for forming a macromolecular self-assembly among the amino acids constituting a His tag as a purification tag and a fusion protein based on an α-helix unit in which the main amino acids are composed of charged amino acids and hydrophobic amino acids.

[0280] Specifically, by replacing the amino acids of the His6:TZ1H:GFP fusion protein, His6:TZ1H(IA / GG):GFP, His6:TZ1H(Q / G):GFP, His6:TZ1H(H / G):GFP, His6:TZ1H(H / K):GFP, His6:TZ1H(E / G):GFP and His6:TZ1H(K / G):GFP fusion proteins were obtained. In addition, by replacing the amino acids of the TZ1H:GFP fusion protein, TZ1H(H / G):GFP and TZ1H(K / G):GFP fusion proteins were obtained, and the respective amino acid sequences were in Fig.16a Then, by using Ni as a specific inducer 2+ At a concentration of 0.5 mM and in the absence of Ni 2+ The formation of macromolecular self-assemblies was induced by shifting the pH to 8 under each optimal buffer condition and in the presence of ammonium sulfate as salt, and recovered by centrifugation, heated in SDS sample buffer, and analyzed by SDS-PAGE.

[0281] The results are as follows Fig.16b As shown, in the presence of ammonium sulfate as salt and Ni as a specific inducer 2+ At a concentration of 0.5 mM, His6:TZ1H(IA / GG):GFP did not form macromolecular self-assemblies, compared to the 73% formation ability of His6:TZ1H:GFP, and the formation abilities of His6:TZ1H(Q / G):GFP, His6:TZ1H(H / G):GFP, His6:TZ1H(H / K):GFP, His6:TZ1H(E / G):GFP, and His6:TZ1H(K / G):GFP were reduced to 68%, 65%, 63%, 46%, and 63%, respectively. Fig.16c As shown, in the presence of ammonium sulfate as a salt, without Ni 2+ and the pH changed to 8, the formation ability of His6:TZ1H:GFP was 40%, while His6:TZ1H(IA / GG):GFP, His6:TZ1H(Q / G):GFP and His6:TZ1H(H / K):GFP did not form macromolecular self-assemblies, and the formation ability of His6:TZ1H(E / G):GFP increased to 61%, while the formation abilities of His6:TZ1H(H / G):GFP and His6:TZ1H(K / G):GFP decreased to 19% and 15%, respectively.

[0282] The above results confirmed that the specific inducer Ni in the presence of ammonium sulfate as a salt and 0.5 mM concentration 2+Under the conditions of , the composition of hydrophobic and charged residues within the tag is crucial for the formation of macromolecular self-assemblies, because the main amino acids are the key to the formation of macromolecular self-assemblies. 2+ In the absence of ATP and when pH was changed to 8, the composition of most amino acid residues within the tag was important for the formation of macromolecular self-assemblies, but glutamic acid (a negatively charged residue) had no significant effect on the formation of macromolecular self-assemblies.

[0283] Experimental Example 4-5. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0284] In order to recover the macromolecular self-assembly by filtering of the low-speed centrifugal precipitate or the filter of μm-level filtering, the assembly 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. His6:mIZ:GFP and His6:TZ1H:GFP were selected as representatives for analysis, and His6:GFP was used as a control group. As specific experimental conditions, the optimized purification conditions of Experimental Examples 3 and 4 were applied.

[0285] First, the formation of macromolecular assemblies was observed. In the case of His6:GFP, very small assemblies were partially observed, but in the case of His6:mIZ:GFP and His6:TZ1H:GFP, macromolecular self-assemblies were observed that formed as a whole ( Figures 17a to 17c ). These macromolecular self-assemblies are formed three-dimensionally, so it is difficult to measure the exact size, but by analyzing the size of the macromolecular self-assemblies based on a planar image of a focal plane, it can be confirmed that they are formed in the size of hundreds of μm to mm ( Figure 17b to Figure 17c ). 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.

[0286] In addition, in order to quantitatively analyze the extent of macromolecular assembly formation, the fluorescence intensity per unit area was measured and compared. Generally, during self-assembly formation, the number of proteins assembled per unit area increases with the increase in assembly size, and in this experiment, the fluorescence intensity was measured using fused GFP.

[0287] The results confirmed that the relative intensity of fluorescence of His6:mIZ:GFP and His6:TZ1H:GFP increased by at least tens to thousands of times compared with the control group, and when fresh protein was added, the fluorescence intensity of His6:mIZ:GFP and His6:TZ1H:GFP and the size of the macromolecular self-assemblies increased ( Fig.17d ).

[0288] Experimental Example 5. Confirmation of the feasibility of purifying each target protein

[0289] 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.

[0290] Results, such as Figures 18a to 18h As shown, it was confirmed that the fusion protein optimally formed a macromolecular self-assembly depending on the target protein, and the maximum yield and maximum purity of the target protein sought in the analysis of the present invention were: 92% and 98% for His6:DLH:rhG-CSF, 84% and 96% for His6:IAQ:INF-a2, 80% and 94% for His6:mIZ:HER LC, 93% and 99% for His6:TZ1H:rhG-CSF, 96% and 99% for His6:TZ1H:INF-a2, 80% and 99% for His6:mIZ:INF-a2, 84% and 99% for His6:mIZ:rhG-CSF, and 87% and 98% for His6:DLH:INF-a2. That is, 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 from the various units provided in the present invention.

Claims

1. A fusion polypeptide comprising a first polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 and a second polypeptide which is an α-helical tag, wherein the main amino acids in the α-helical tag are composed of charged amino acids and hydrophobic 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:4 to SEQ ID NO:

8.

3. The fusion polypeptide according to claim 1, wherein The first polypeptide and the second polypeptide are linked sequentially. A fusion protein comprising the fusion polypeptide according to claim 1 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 protein according to any one of claims 4 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 an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3, or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8.

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