Self-assembly-forming fusion protein comprising amorphous peptide tag with master amino acid comprising charged and hydrophobic amino

Abstract

The present invention relates to an amorphous peptide tag in which a main amino acid consists of a charged amino acid and a hydrophobic amino acid, a fusion protein in which the tag is fused with a target protein, and a method for purifying the target protein using the same. Since the peptide tag can induce the formation of a self-assembly when fused to the target protein, the method for purifying the target protein using the same can separate and purify the target protein using only 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. 10-2022-0122187 filed on September 27, 2022, and all the contents disclosed in the corresponding Korean patent application document are incorporated as part of this specification. The present invention relates to a fusion protein comprising an amorphous (disordered, unstructured) peptide tag, wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids; a composition for purifying a recombinant protein comprising the same; and a method for purifying a recombinant protein using the same. Background Art

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

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

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

[0006] Technical issues

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

[0008] An example of the present disclosure provides an amorphous tag that forms a self-assembly, wherein the main amino acids consist of charged amino acids and hydrophobic amino acids.

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

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

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

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

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

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

[0015] Other embodiments of the present application provide a composition for purifying a protein, comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5.

[0016] Other embodiments of the present application provide a self-assembly comprising a first polypeptide represented by SEQ ID NO: 1 and / or a second polypeptide represented by an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5.

[0017] Technical Solution

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

[0019] In order to achieve the above-mentioned object of the present invention, the present invention provides an amorphous peptide tag, wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids.

[0020] The charged amino acids of the amorphous peptide tag forming the self-assembly (wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids) 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, M) 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.

[0021] The amorphous peptide tag may be any one selected from the group consisting of a PSIAEH tag, a PSIAHEK tag, a PSIHEKIHEK tag, and a PSIA tag, and may be a peptide capable of nucleation or forming a multimer such as a dimer or the like.

[0022] The amorphous peptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:2 to SEQ ID NO:5.

[0023] The term "amorphous peptide tag" used in this specification may refer to a peptide comprising a disordered region that does not have a fixed conformation under normal conditions, such as a secondary or tertiary structure of an α-helix or β-helix, and the term "amorphous peptide tag" may be used interchangeably with "unstructured peptide tag" or "second polypeptide".

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

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

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

[0027] In addition, the present invention provides a fusion polypeptide comprising a first polypeptide having an amino acid sequence of SEQ ID NO: 1 and a second polypeptide having amorphous characteristics, wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids.

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

[0029] The second polypeptide may be a motif, domain or peptide capable of nucleation or forming a multimer such as a dimer, etc. Specifically, the second polypeptide may be any one selected from the group consisting of PSIAEH, PSIAHEK, PSIHEKIHEK and PSIA peptides, but is not limited thereto.

[0030] The second polypeptide may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5. The PSIAEH, PSIAHEK, PSIHEKIHEK and PSIA peptides may be represented by or consist of the following amino acids: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, respectively.

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

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

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

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

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

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

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

[0038] The hinge region may comprise, or consist of, the amino acid sequence of SEQ ID NO: 20.

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

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

[0041] The polynucleotide comprises each polynucleotide encoding the first polypeptide and the second polypeptide. The polynucleotide encoding the first polypeptide may comprise or consist of the nucleotide sequence of SEQ ID NO: 21, and the polynucleotide encoding the second polypeptide may comprise or consist of a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO: 22 to SEQ ID NO: 25.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0073] In addition, the present invention provides a composition for purifying a protein, comprising an amorphous peptide tag having an amorphous characteristic in which amino acids mainly consist of charged amino acids and hydrophobic amino acids.

[0074] The polypeptide tag may be any one selected from the group consisting of a PSIAEH tag, a PSIAHEK tag, a PSIHEKIHEK tag, and a PSIA tag, and may be a peptide capable of nucleation or forming a multimer such as a dimer.

[0075] The amorphous peptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5.

[0076] In addition, the present invention provides a self-assembly comprising an amorphous peptide tag having an amorphous characteristic in which major amino acids consist of charged amino acids and hydrophobic amino acids.

[0077] The amorphous polypeptide tag may be any one selected from the group consisting of a PSIAEH tag, a PSIAHEK tag, a PSIHEKIHEK tag, and a PSIA tag, and may be a peptide capable of nucleation or forming a multimer such as a dimer or the like.

[0078] The polypeptide tag may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 to SEQ ID NO: 5.

[0079] Beneficial Effects

[0080] The present invention relates to an amorphous polypeptide, wherein the main amino acids are composed of charged amino acids and hydrophobic amino acids; a fusion protein, wherein the tag is fused to a target protein; and a method for purifying a target protein using the fusion protein, and a single peptide or fusion protein induces the formation of a macromolecular self-assembly in a specific inducer or treatment condition-dependent manner, 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 the expensive beads or resin-filled purification columns mainly used in conventional affinity chromatography, and the expensive devices or equipment for running them. In addition, because the formation of the macromolecular self-assembly is induced by salt treatment of a protein extraction solution and specific inducers or conditions, 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 recombinant proteins 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

[0081] 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.

[0082] 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.

[0083] 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.

[0084] Figure 2a This figure confirms whether the formation of macromolecular self-assembly is induced when treated with various cations by fusing a single His tag as a purification tag to the target protein GFP.

[0085] Figure 2bThis is a diagram of confirming whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing the PSIAEH peptide tag alone as a purification tag to GFP. The PSIAEH peptide tag is artificially designed (de novo) to be able to nucleate or form multimers such as dimers.

[0086] Figure 2c This figure examines whether the formation of macromolecular self-assemblies is induced when treated with various cations by fusing a combination of a His tag and a PSIAEH peptide tag to GFP as a purification tag.

[0087] Figure 2d This figure confirms whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing the PSIAHEK peptide tag alone as a purification tag to GFP. The PSIAHEK peptide tag is artificially designed (de novo) to be able to nucleate or form multimers such as dimers.

[0088] Figure 2e This figure examines whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing a combination of a His tag and a PSIAHEK peptide tag to GFP as a purification tag.

[0089] Figure 2f This figure examines whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing a combination of a His tag and a PSIA peptide tag to GFP as a purification tag.

[0090] Figure 2g This figure confirms whether the formation of macromolecular self-assemblies is induced when various cations are treated by fusing the PSIHEKIHEK peptide tag alone as a purification tag to GFP. The PSIHEKIHEK peptide tag is artificially designed (de novo) to be able to nucleate or form multimers such as dimers.

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

[0092] Figure 3a The His tag or PSIAEH peptide tag was used as a purification tag alone and fused to GFP, or both tags were used as purification tags and fused to GFP, one of the model proteins of the target protein, to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0093] Figure 3bThe His tag or PSIAHEK peptide tag was used as a purification tag and fused to GFP alone or both tags were used as purification tags and 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.

[0094] Figure 3c The His tag or PSIHEKIHEK peptide tag was 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 3d The His tag or PSIAHEK peptide tag was used as a purification tag alone or in combination with GFP to confirm the expression of cationic Ni at different concentrations. 2+ A diagram showing whether the treatment induces the formation of macromolecular self-assemblies.

[0096] Figure 4a The His tag was used as a purification tag alone and fused to GFP to analyze the selection of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0097] Figure 4b The PSIAEH peptide tag was used alone as a purification tag and fused to GFP to analyze the activity of the cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0098] Figure 4c The combination of His-tag and PSIAEH peptide tag was fused to GFP as 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.

[0099] Figure 4d The PSIAHEK peptide tag was used alone 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.

[0100] Figure 4eThe combination of His-tag and PSIAHEK peptide tag was fused to GFP as 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.

[0101] Figure 4f The combination of His-tag and PSIA peptide tag was fused to GFP as 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.

[0102] Figure 4g The PSIHEKIHEK peptide tag was used alone 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.

[0103] Figure 4h The combination of His-tag and PSIHEKIHEK peptide tag was fused to GFP as 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.

[0104] Figure 5a The His-tag and PSIAEH-peptide-tag were combined as purification tags fused to GFP to analyze the selective activity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0105] Figure 5b The combination of His-tag and PSIAHEK 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.

[0106] Figure 5c The His-tag and PSIHEKIHEK peptide-tag were combined as purification tags and fused to GFP to analyze the selectivity of cationic Ni in the presence of various concentrations of sodium chloride as salt. 2+ A diagram showing whether it promotes the formation of macromolecular self-assemblies when treated as a specific inducer.

[0107] Figure 5dThe His-tag and PSIA-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.

[0108] Figure 6a The His tag and PSIAEH peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0109] Figure 6b The His tag and PSIAHEK peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0110] Figure 6c The His tag and PSIHEKIHEK peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0111] Figure 6d The His tag and PSIA peptide tag were combined as purification tags and fused to GFP to analyze the activity of the purified protein when ammonium sulfate was used as the salt and the cationic Ni was selected. 2+ Plot of the extent of formation of macromolecular self-assemblies upon treatment of various buffers as specific inducers.

[0112] Figure 7a The combination of His-tag and PSIAEH peptide tag was fused to GFP as purification tag to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0113] Figure 7b The analysis was performed by fusing a combination of a His-tag and a PSIAHEK peptide tag as purification tags to GFP in the presence of ammonium sulfate as salt, depending on the choice of the cation Ni 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0114] Figure 7cThe analysis was performed by fusing a combination of a His-tag and a PSIAHEK peptide tag as purification tags to GFP in the presence of ammonium sulfate as salt, depending on the choice of the cation Ni 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0115] Figure 7d The His-tag and PSIA-peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of formation of macromolecular self-assemblies as a function of treatment concentration of a specific inducer.

[0116] Figure 8a The His tag and PSIAEH peptide tag were combined with GFP as purification tags to analyze the activity of the purified protein when treated with ammonium sulfate as salt and with the cationic Ni under various pH conditions of the buffer. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0117] Figure 8b The His-tag and PSIAHEK 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.

[0118] Figure 8c The His tag and PSIHEKIHEK peptide tag were fused to GFP as purification tags to analyze the activity of the protein under various pH conditions of the buffer, treated with ammonium sulfate as salt, and selected cationic Ni. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0119] Figure 8d The His-tag and PSIA-peptide-tag were combined with GFP as purification tags to analyze the activity of the purified protein when treated with ammonium sulfate as salt and with the cationic Ni under various pH conditions of the buffer. 2+ as a graph of the extent of formation of macromolecular self-assemblies when not treated or treated with a specific inducer.

[0120] Figure 9a The combination of His-tag and PSIAEH peptide tag was fused to GFP as purification tag to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0121] Figure 9b The analysis was performed by fusing a combination of a His-tag and a PSIAHEK peptide tag as purification tags to GFP in the presence of ammonium sulfate as salt, depending on the choice of the cation Ni 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0122] Fig.9c The analysis was performed by fusing a combination of a His-tag and a PSIHEKIHEK peptide tag as a purification tag to GFP in the presence of ammonium sulfate as a salt, depending on the choice of the cation Ni 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0123] Figure 9d The His-tag and PSIA-peptide-tag were combined as purification tags fused to GFP to analyze the expression of cytokines in the presence of ammonium sulfate as salt, depending on the choice of cation Ni. 2+ Plot of the extent of macromolecular self-assembly formation as a function of treatment time with a specific inducing agent.

[0124] Fig.10 The His tag and a combination of each of the PSIAEH, PSIAHEK, PSIHEKIHEK, and PSIA peptide tags were fused to GFP as purification tags in the presence of ammonium sulfate as a salt, depending on the choice of the cation Ni. 2+ The yield (recovery rate) and purity of each unit were analyzed as the degree of formation of the macromolecular self-assembly as a function of the treatment time with a specific inducing agent.

[0125] Fig.11a The His tag alone as a purification tag was fused to GFP, or the combination of the His tag and the PSIAEH peptide tag was fused to GFP as a purification tag to analyze the activity of the cationic Ni in the presence of ammonium sulfate as a salt. 2+ Diagram showing the possibility of recovering recombinant protein using filter filtration and UV irradiation when treated with specific inducers.

[0126] Fig.11b The results were analyzed by fusing a combination of a His tag and a PSIAHEK peptide tag, a combination of a His tag and a PSIHEKIHEK peptide tag, or a combination of a His tag and a PSIA peptide tag 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.

[0127] Fig.11c The results were analyzed by fusing a combination of a His tag and a PSIAHEK peptide tag, a combination of a His tag and a PSIAHEK peptide tag, or a combination of a His tag and a PSIA peptide tag to GFP as a purification tag to analyze the effect of the cationic Ni on the expression of the purified protein 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.

[0128] Fig.12a In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images were obtained under conditions of treatment with specific inducers for His6:GFP in order to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0129] Figure 12b In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:PSIAEH:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0130] Fig.12c 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:PSIAHEK:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0131] Fig.12d In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:PSIHEKIHEK:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0132] Fig.12e In the presence of ammonium sulfate as a salt, the cation Ni is selected 2+ Images obtained under conditions of treatment with specific inducers for His6:PSIA:GFP to analyze the formation and size of macromolecular self-assemblies by fluorescence microscopy.

[0133] Fig.13a The combination of His-tag and PSIAHEK peptide tag was fused to TEV protease as purification tag to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0134] Fig.13b The His-tag and PSIAEH-peptide-tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0135] Fig.13c The His-tag and PSIHEKIHEK peptide-tag combination was fused to GM-CSF as purification tags to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0136] Fig.13d The His-tag and PSIHEKIHEK peptide-tag were fused to TEV protease as purification tags to analyze the selectivity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0137] Fig.13e The His-tag and PSIA-peptide-tag were fused to TEV protease as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP.

[0138] Fig.13f The His tag and PSIHEKIHEK peptide tag were fused to interferon α2 as purification tags to analyze the selective activity of cationic Ni in the presence of various concentrations of ammonium sulfate as salt. 2+ Figure 2 shows the extent of macromolecular self-assembly formation upon treatment with specific inducers and their applicability to recombinant proteins other than GFP. DETAILED DESCRIPTION

[0139] 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.

[0140] [Experimental Materials and Methods]

[0141] <Experimental Materials>

[0142] 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.

[0143] Example 1. Construction of a vector for expressing protein

[0144] 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 the 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 the pET 28a-GFP subvector. In addition, the basic vector pET28a-His6: GFP vector was completed by overlapping PCR using the F-Nco I, BamH I-His6 / R-Kpn I-His6 primers, then cut with restriction enzymes, and then inserted into the Nco I / Kpn I site of the pET 28a-GFP subvector. Afterwards, pET28a-His6:PSIAEH:GFP, pET 28a-His6:PSIAHEK:GFP, pET 28a-His6:PSIHEKIHEK:GFP, and pET28a-His6:PSIA:GFP vectors were produced based on the primer combinations F-Nco I, BamH I-His6:PSIAEH / R-Kpn I-PSIAEH; F-Nco I, BamH I-His6:PSIAHEK / R-Kpn I-PSIHEKIHEK; and F-Nco I, BamH I-His6:PSIA / R-Kpn I-PSIA, respectively. In order to construct vectors other than the His6 tag and use them as a control group, representative vectors of pET 28a-PSIAEH:GFP, pET 28a-PSIAHEK:GFP, and pET 28a-PSIHEKIHEK:GFP were produced based on the primer combinations F-Nco I, BamH I-PSIAEH / R-Kpn I-PSIAEH; F-Nco I, BamH I-PSIAHEK / R-Kpn I-PSIAHEK; F-Nco I, BamH I-PSIHEKIHEK / R-Kpn I-PSIHEKIHEK.To evaluate the purification scalability of various protein drugs other than GFP, PCR was performed with primer combinations of F-Bgl II-rhG-CSF / R-Hind III-rhG-CSF; F-Bgl II-INF-a2 / R-Hind III-INF-a2; and F-Bgl II-TEV protease / R-Hind III-TEV protease, which were then cut with Bgl II / Hind III restriction enzymes and inserted into the GFP site of pET 28a-His6:PSIAEH:GFP, pET 28a-His6:PSIAHEK:GFP, pET 28a-His6:PSIHEK:GFP, and pET 28a-His6:PSIA:GFP vectors to complete the purification. Construction of representative vectors of pET 28a-His6:PSIHEKIHEK:rhG-CSF, pET 28a-His6:PSIHEKIHEK:TEV protease, pET 28a-His6:PSIA:TEV protease, and pET 28a-His6:PSIHEKIHEK:INF-a2. The specific sequence information of each tag and designed recombinant protein is shown in Tables 1 to 3 below.

[0145] [Table 1]

[0146] Amino acid sequence and nucleic acid sequence information of the first polypeptide tag

[0147]

[0148] [Table 2]

[0149] Amino acid sequence and nucleic acid sequence information of amorphous polypeptide tag (or second polypeptide tag)

[0150]

[0151] [Table 3]

[0152] Amino acid sequence and nucleic acid sequence information of each fusion polypeptide, target protein and hinge region.

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160]

[0161]

[0162] Example 2. Induction and extraction of fusion protein expression

[0163] 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.

[0164] Example 3. Induction and optimization of macromolecular self-assembly formation

[0165] Example 3-1. Optimization of ammonium sulfate concentration as salt

[0166] 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.

[0167] Example 3-2. Optimization of sodium chloride concentration as salt

[0168] In order to analyze the effect of the treatment concentration of sodium chloride as salt, the Ni 2+ deal with.

[0169] Example 3-3. Optimization of buffer

[0170] 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.

[0171] Example 3-4. Ni 2+ Concentration Optimization

[0172] In order to optimize Ni 2+ Concentration, with Ni in the range of 0 to 2 mM 2+ The supernatant was obtained by centrifugation after treatment with ammonium sulfate.

[0173] Example 3-5. Optimization of buffer pH

[0174] To optimize the buffer pH, the supernatant obtained by treatment with ammonium sulfate and centrifugation was used by adjusting the pH to a range of 5-10 with HCl and NaOH. 2+ The supernatant obtained by ammonium sulfate treatment and centrifugation under each pH condition was treated.

[0175] Example 3-6. Optimization of self-assembly formation reaction time

[0176] 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 additionally up to 120 min.

[0177] Example 4. Recovery of macromolecular self-assembly recombinant protein and conversion into monomers

[0178] The recombinant protein that forms the macromolecular self-assembly in Example 3 in a specific inducer or condition-dependent manner is recovered by methods such as centrifugation and filtration.

[0179] 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.

[0180] Example 5. Confirmation of protein purified by SDS-PAGE

[0181] 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.

[0182] Example 6. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0183] In order to analyze the formation and approximate size of the macromolecular self-assembly, etc., a fluorescence microscope was used, and in order to detect the GFP fluorescence signal, a filter set of excitation: 488 nm, emission: 520 nm was used.

[0184] [Experimental results]

[0185] Experimental Example 1. Identification of an inducing agent suitable for inducing the self-assembly formation of a fusion protein of a His tag and an amorphous peptide tag

[0186] In order to confirm the inducibility of forming macromolecular self-assemblies in a manner dependent on a specific inducer or condition, GFP was selected as a model protein, and the His tag and the amorphous unit PSIAEH peptide tag, PSIAHEK peptide tag, PSIHEKIHEK peptide tag and PSIA peptide tag in which the main amino acids are composed of charged amino acids and hydrophobic amino acids were combined as purification tags by the method described in Examples 1 and 2 to prepare His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP and His6:PSIA:GFP fusion proteins. For control group experiments, His6:GFP fusion proteins in which the His tag was fused alone were prepared, as well as PSIAEH:GFP, PSIAHEK:GFP and PSIHEKIHEK:GFP fusion proteins in which each of the amorphous or unstructured basic units was fused alone as a purification tag were prepared (see Figure 1b ). Then, the protein extract containing the fusion protein was treated with various cationic 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. Figure 2a to Figure 2h middle.

[0187] The results are as follows Figure 2a to Figure 2h As shown in Figure , it was confirmed that the His6:PSIAEH:GFP fusion protein was expressed by Ni 2+ 、Co 2+ , Cu 2+ , Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2c ), and His6:PSIAHEK:GFP fusion protein was expressed by Ni 2+ 、Co 2 + , Cu 2+ 、Zn 2+ , Fe 2+ Excellent in inducing the formation of macromolecular self-assemblies (see Figure 2e ). His6:PSIHEKIHEK: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 (see Figure 2h ), and His6:PSIA:GFP fusion protein was expressed by Cu 2+ 、Zn 2+, Fe 2+ Inducing the formation of macromolecular self-assemblies (see Figure 2f ).

[0188] On the other hand, in the case of the control group using His tag alone (His6:GFP) or peptide tag alone (PSIAEH:GFP, PSIAHEK:GFP, and PSIHEKIHEK:GFP), when cationic inducers such as Ni 2+ , Cu 2+ 、Zn 2+ , Fe 2+ When treated with Figure 2a , Figure 2b , Figure 2d and Figure 2g ).

[0189] In summary, it was confirmed that the combination of His-tag and peptide-tag significantly increased the formation of induced macromolecular self-assemblies by various cationic inducers. 2+ The reactivity of Ni is generally excellent, and in subsequent experiments, Ni 2+ Experiments were performed as specific inducers.

[0190] Experimental Example 2. When the cation Ni 2+ Confirmation of self-assembly formation when treated as an inducer

[0191] Since Ni 2+ Suitable as an inducing agent for the formation of self-assembly, so when Ni 2+ When processing, it was confirmed that Ni 2+ , formation of self-assemblies of each of the His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP and His6:PSIA:GFP fusion proteins.

[0192] Specifically, for each protein extract obtained in Example 2 comprising His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP and His6:PSIA:GFP fusion proteins, His6:GFP (wherein the His tag is fused alone), PSIAEH:GFP, PSIAHEK:GFP and PSIHEKIHEK:GFP fusion proteins (fused alone with each amorphous tag), the cationic inducer Ni was treated at a concentration of 0-2 mM, respectively. 2+, to induce the formation of macromolecular self-assemblies. The protein induced to form macromolecular self-assemblies 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 the structure. Figures 3a to 3d middle.

[0193] Results, such as Figures 3a to 3d As shown in Figure 5, it was confirmed that the His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP, and His6:PSIA:GFP fusion proteins induced the formation of macromolecular self-assemblies, while it was confirmed that the His6:GFP (in which the His tag was fused alone), PSIAEH:GFP, PSIAHEK:GFP, and PSIHEKIHEK:GFP fusion proteins (in which each amorphous tag was fused alone) induced the formation of macromolecular self-assemblies in Ni 2+ The formation of macromolecular self-assembly is not induced in the case of inducer treatment (see Figures 3a to 3d ).

[0194] Experimental Example 3. Establishment of the Optimal Conditions for the Formation of Macromolecular Self-Assemblies

[0195] By removing non-selective proteins other than the target protein, maximizing the induction of macromolecular self-assemblies, optimizing high-purity purification conditions, and minimizing the binding of fusion proteins with His tags and amorphous peptide tags 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.

[0196] Experimental Example 3-1. Optimization of ammonium sulfate concentration conditions as salt

[0197] The following experiment was performed to confirm the ammonium sulfate concentration conditions suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (mainly composed of charged amino acids and hydrophobic amino acids) were fused as a purification tag.

[0198] Specifically, for each of the protein extracts obtained in Example 2 containing His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP and His6:PSIA:GFP fusion proteins, His6:GFP to which the His tag is fused alone, and PSIAEH:GFP, PSIAHEK:GFP and PSIHEKIHEK:GFP fusion proteins to which each peptide tag is fused alone, the formation of a macromolecular self-assembly was induced by the method described in Example 3-1. 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 Figure 4a to Figure 4h middle.

[0199] The results are as follows Figure 4a to Figure 4h As shown, it was confirmed that the formation of macromolecular self-assemblies was induced in the following ranges: 14-22% (w / v) for His6:PSIAEH:GFP fusion protein, 16-22% (w / v) for His6:PSIAHEK:GFP fusion protein, 12-22% (w / v) for His6:PSIHEKIHEK:GFP fusion protein, and 18-22% (w / v) for His6:PSIA fusion protein.

[0200] Experimental Example 3-2. Optimization of the conditions for the concentration of sodium chloride as salt

[0201] The following experiment was performed to confirm the sodium chloride concentration conditions suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (in which the main amino acids consist of charged amino acids and hydrophobic amino acids) were fused as a purification tag.

[0202] Specifically, under the treatment conditions of NaCl as a salt in the concentration range of 0-3M, after inducing self-assemblies by the method described in Example 3-2, they were confirmed by SDS-PAGE. Figure 5a to Figure 5d Displayed in.

[0203] The results are as follows Figure 5a to Figure 5d As shown, analysis showed that His6:PSIAEH:GFP and His6:PSIA: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 the His6:PSIAHEK:GFP fusion protein and 150 mM NaCl was added to the His6:PSIHEKIHEK:GFP fusion protein.

[0204] The results of Experimental Examples 3-1 and 3-2 confirmed that the induction of self-assembly was relatively superior when treated with ammonium sulfate compared to sodium chloride, and therefore in subsequent experiments, ammonium sulfate was used as the salt for the experiments.

[0205] Experimental Example 3-3. Optimization of buffer conditions

[0206] In order to confirm a buffer suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (in which the main amino acids are composed of charged amino acids and hydrophobic amino acids) are fused as a purification tag, the following experiment was performed.

[0207] Specifically, for each fusion protein extract, pH 7.4 HEPES buffer (HEPES; 50 mM HEPES buffer (pH 7.4)), pH 7.4 PBS buffer (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4 (pH 7.4)), pH 7.4 PB buffer (PB; 10 mM Na2HPO4, 1.8 mM KH2PO4 pH 7.4)), pH 7.4 Tris buffer (Tris; 50 mM Tris-HCl buffer (pH 7.4)) were used as each buffer, and 1% [v / v] Triton X-100 and 1X [v / v] protease inhibitor cocktail tablets (Roche) were added to 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 6a to 6d Displayed in.

[0208] The results are as follows Figures 6a to 6d As shown, when HEPES (pH 7.4) buffer was used, all fusion proteins showed the best effect of inducing the formation of macromolecular self-assemblies.

[0209] Experimental Example 3-4. Ni 2+ Concentration Optimization

[0210] In order to identify Ni suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (in which the main amino acids are composed of charged amino acids and hydrophobic amino acids) are fused as purification tags 2+ The following experiments were performed under the following concentration conditions.

[0211] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate as a salt, and the inducer Ni 2+The cations were treated at different concentrations ranging from 0 to 2 mM to induce self-assembly by the method described in Example 3-4, and then the effect of inducing self-assembly was confirmed. Figures 7a to 7d Displayed in.

[0212] The results are as follows Figures 7a to 7d As shown, it was confirmed that in the case of His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP, and His6:PSIA:GFP fusion proteins, after ammonium sulfate treatment, when the cationic inducer Ni was added at a concentration of 0.5 mM 2+ Upon treatment, nearly all monomers present in the protein extract formed macromolecular self-assemblies within 20 min.

[0213] Experimental Example 3-5. Optimization of buffer pH

[0214] The following experiment was performed to confirm the pH conditions of the buffer suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (in which the main amino acids consist of charged amino acids and hydrophobic amino acids) are fused as a purification tag.

[0215] Specifically, the supernatant obtained by centrifugation after ammonium sulfate treatment was adjusted to a pH range of 5-10 using HCl and NaOH, self-assembly was induced by the method described in Examples 3-5, and then confirmed by SDS-PAGE. Figures 8a to 8d Displayed in.

[0216] The results are as follows Figures 8a to 8d As shown, in the case of His6:PSIA:GFP fusion protein, the induction of macromolecular self-assembly formation occurs normally at pH 7-pH 8, but the induction of macromolecular self-assembly formation tends to decrease under more alkaline conditions. It was confirmed that in other fusion proteins, the induction of macromolecular self-assembly formation occurs normally under alkaline conditions of pH 7-pH 10, and the precipitation of non-specific proteins occurs under acidic conditions of pH 5 or lower.

[0217] Example 3-6. Optimization of the induction time of self-assembly formation of fusion protein of His tag and amorphous peptide tag

[0218] The following experiment was performed to confirm the induction time conditions suitable for forming a macromolecular self-assembly of a fusion protein in which a His tag and an amorphous peptide tag (in which the main amino acids consist of charged amino acids and hydrophobic amino acids) were fused as a purification tag.

[0219] Specifically, the protein supernatant containing each fusion protein was treated with ammonium sulfate and the induction agent Ni 2+The cations were treated at a concentration of 0.5 mM. 2+ After the cationic treatment, self-assemblies were induced by the method described in Examples 3-6 every 10 minutes for up to 60 minutes, and they were induced for a maximum of 120 minutes, and then the effect of inducing self-assembly was confirmed by SDS-PAGE. Figures 9a to 9d Displayed in.

[0220] The results are as follows Figures 9a to 9d As shown in , it was confirmed that each fusion protein (in which a His tag was combined with an amorphous basic unit in which the main amino acid composition consisted of charged amino acids and hydrophobic amino acids) formed a macromolecular self-assembly within 20 minutes.

[0221] Experimental Example 4. Confirmation of the recovery rate of recombinant protein induced to form macromolecular self-assembly

[0222] Experimental Example 4-1. Confirmation of Yield and Purity of Fusion Protein of His Tag and Amorphous Peptide Tag

[0223] After inducing the formation of self-assembly of the fusion protein of the His tag and the amorphous peptide tag under the optimized conditions for forming the self-assembly confirmed in Experimental Example 3, the self-assembly was recovered by centrifugation.

[0224] Specifically, the centrifugation method induces the formation of macromolecular self-assemblies under optimal conditions and cationic inducing agents, and then centrifuges at 3,000 rpm or higher for 10 minutes at 4°C to recover the recombinant protein in the form of precipitates, and treats them with EDTA to convert the multimers into monomers. The converted monomers were confirmed by SDS-PAGE, and the yield and purity were measured using the Image Lab program of Bio-Rad. 2+ After inducing self-assembly, the yield was calculated by the following formula (Formula 1).

[0225] [Formula 1]

[0226] Yield = intensity of recovered target protein band / (intensity of unrecovered target protein band + intensity of recovered target protein band) × 100

[0227] Purity was measured by measuring the ratio of purified protein band to total protein and expressed as a percentage.

[0228] The results are as follows Fig.10 As shown, after treatment with ammonium sulfate, 0.5 mM of the cation inducer Ni 2+During the treatment, the maximum yield and maximum purity of each fusion protein analyzed for the His tag and α-helix-based peptide tag were 83% and 99% for the His6:PSIAEH:GFP fusion protein, 98% and 98% for the His6:PSIAHEK:GFP fusion protein, 90% and 97% for the His6:PSIHEKIHEK:GFP fusion protein, and 83% and 97% for the His6:PSIA:GFP fusion protein.

[0229] Experimental Example 4-2. Confirmation of purification of His-tag and amorphous peptide-tag fusion protein using a filter

[0230] The method using the filter is as follows: using 0.2 and 5.0 μm syringe filters (Satorius, Goettingen, GEU), 10 μm syringe filters (Tisch, OH, USA), the recombinant protein that forms the macromolecular self-assembly with the optimal conditions and cationic inducers is filtered into the protein extract, and then the washing process is carried out using the same buffer as the macromolecular assembly induction reaction. In addition, the macromolecular assembly is converted into a monomer or eluted using the same buffer with the addition of a chelating agent such as EDTA. In order to visually confirm this, the filter is irradiated with a 360nm UV wavelength to visually confirm it.

[0231] The results are as follows Figures 11a to 11c 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.

[0232] Experimental Example 4-3. Confirmation of the formation of macromolecular self-assembly by fluorescence microscopy

[0233] In order to recover the macromolecular self-assembly by filtering of the low-speed centrifugal precipitate or the filter filtered by μm level, 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. In the analysis, His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP and His6:PSIA:GFP were used, 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.

[0234] First, since the formation of macromolecular assemblies was observed, in the case of His6:GFP, assemblies of very small size were partially observed, but in the case of His6:PSIAEH:GFP, His6:PSIAHEK:GFP, His6:PSIHEKIHEK:GFP, and His6:PSIA:GFP, the overall formation of macromolecular self-assemblies was observed. The formation of these macromolecular self-assemblies is three-dimensional, so it is difficult to measure the exact size, but by analyzing the size of the macromolecular self-assemblies based on a planar image on one focal plane, it was confirmed that they were formed in a size of several hundred μm to mm ( Figures 12a to 12e ). 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.

[0235] Experimental Example 5. Confirmation of the feasibility of purifying each target protein

[0236] The biopharmaceutical market size (including antibody new drugs, vaccines, protein new 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 for purifying recombinant proteins using the specific inducer or condition-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 and TEV protease, purification of various target proteins was carried out, and the purification method optimized in Experimental Examples 3 and 4 was used.

[0237] The results are as follows Figures 13a to 13fAs shown, the fusion proteins that optimally form macromolecular self-assemblies according to the target protein were analyzed, and the maximum yield and maximum purity of the target protein sought in the present invention were: 86% and 93% for His6:PSIAHEK:TEV protease, 95% and 95% for His6:PSIAEH:INF-a2, 83% and 98% for His6:PSIHEKIHEK:rhG-CSF, 80% and 98% for His6:PSIHEKIHEK:TEV protease, 74% and 95% for His6:PSIA:TEV protease, and 83% and 97% for His6:PSIHEKIHEK:INF-a2. In other words, it was confirmed that in the purification of many target proteins other than the target protein sought in the present invention, they can be purified with the best yield and purity by selecting them in the various units provided in the present invention.

Claims

1. A fusion polypeptide comprising a first polypeptide represented by the amino acid sequence of SEQ ID NO: 1 and a second polypeptide as a peptide tag, wherein the main amino acid components of the peptide tag are composed of charged amino acids and 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: 2 to SEQ ID NO:

5.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

5.

21. An amorphous peptide capable of forming a self-assembly, wherein: The main amino acids are composed of charged amino acids and hydrophobic amino acids.

22. The peptide according to claim 21, wherein The peptide is represented by an amino acid sequence selected from the group consisting of SEQ ID NO:2 to SEQ ID NO:5.

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