CSQ-tagged protein expression and purification method

Abstract

The present invention relates to methods for expressing, solubilizing, and purifying proteins using a CSQ tag (Calsequestrin tag), and provides a recombinant expression vector containing a CSQ tag and a target protein, a host cell transformed with the recombinant expression vector, and a method for expressing and purifying a target protein using the CSQ tag. The present invention is expected to reduce the costs of protein materials and pharmaceuticals by increasing the expression of proteins commonly used in pharmaceuticals and cosmetics using the CSQ tag and making them easier to separate with calcium.

Description

[Technical Field]

[0001] The present invention relates to methods for expressing, solubilizing, and purifying proteins using a CSQ tag (Calsequestrin tag), and provides a fusion protein containing a CSQ tag and a target protein, a nucleic acid containing a nucleotide sequence encoding the fusion protein, an expression vector containing the nucleic acid, cells transformed with the expression vector, and a method for expressing, solubilizing, and purifying a target protein using the CSQ tag. The present invention is expected to reduce the costs of protein materials and pharmaceuticals by improving the expression and solubilization of proteins commonly used in pharmaceuticals and cosmetics using the CSQ tag, making them easier to separate with calcium. [Background technology]

[0002] In recent years, advances in genetic engineering and biology have led to numerous attempts to mass-produce or obtain specific proteins for use in various industries and disease treatments. Therefore, efforts have focused on the development of protein preparation, mass production, and purification technologies to obtain desired proteins. Often, target proteins needed by humans can be produced by culturing cells transformed with an expression vector and expressing the desired protein. In some cases, such proteins can be expressed in eukaryotic or prokaryotic cells, and in particular, in transformed plants or animals. For example, attempts have been made to express the target protein in a transformed animal that secretes milk and obtain it through the milk of the transformed animal. In this case, the target protein can be isolated and purified from the cell culture or milk.

[0003] When expressing a protein in animals, plants, or microorganisms that lack a separate secretory method for extracting the target protein, it is necessary to first extract the protein from storage organs or cells. Isolating the target protein from such transformed cells is not easy. Therefore, to facilitate isolation, a method of modifying the target protein to a form containing a tag rather than its native form has been widely used. The use of tags for purification is one of the most efficient protein purification techniques. The tags used in this method are broadly classified into peptide tags and protein tags. Peptide tags consist of short amino acids, typically his-tags (histidine tags), with hexahistidine tags (His6-tags) being particularly popular. Histidine peptides have a specific chemical affinity for nickel, and fusion proteins containing such tags can be highly purified using a nickel-containing column. Protein tags contain protein domains that bind to specific components, utilizing their characteristics. A typical example is the GST tag (Glutathione S-Transferase tag), which can be highly purified using a column immobilized with glutathione, a substrate of GST.

[0004] Therefore, the inventors conducted extensive research to develop a novel tag that would facilitate the acquisition of target proteins. As a result, they confirmed that by fusing a CSQ (Calsequestrin) tag to a target protein, the target protein can be easily expressed, solubilized, and purified, thereby completing the present invention. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Republic of Korea Patent Publication No. 10-2014-0026781 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a fusion protein comprising a CSQ-tag and a protein of interest.

[0007] Another object of the present invention is to provide a nucleic acid comprising a nucleotide sequence encoding the fusion protein, and an expression vector comprising the nucleic acid. A schematic diagram of the recombinant expression vector of the present invention is shown in Figure 1.

[0008] It is still another object of the present invention to provide a cell transformed with the expression vector.

[0009] A further object of the present invention is to provide a method for expressing and purifying a target protein using a CSQ tag. An example of the method for expressing and purifying a target protein according to the present invention is shown in FIG. [Means for solving the problem]

[0010] The present invention relates to a CSQ tag for improving the expression and water solubility of a target protein, and a fusion protein containing the target protein.

[0011] The CSQ tag can be encoded by amino acids consisting of SEQ ID NO:1 or SEQ ID NO:2.

[0012] The CSQ tag is represented by SEQ ID NO: 3 Alternatively, it may be encoded by a nucleotide sequence consisting of SEQ ID NO:4.

[0013] The CSQ tag and the target protein can be fused via a hydrolase-digestible peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8.

[0014] The target protein is characterized by being selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. For example, the target protein may be interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α and -β, TGF-α, -β), Brain-Derived Neutrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (PIGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor 1 and 2 (FGF-1, -2), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (Luteinizing Hormone-Releasing Hormone)Hormone, Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone, Oxytocin, Thymosin alpha-1, Corticotropin-Releasing Factor, Calcitonin, Bivalirudin, Vasopressin, Phospholipase-Activating Protein (PLAP), Insulin, Tumor Necrosis Factor (TNF), Follicle-Stimulating Hormone, Thyroid-Stimulating Hormone, Antidiuretic Hormone, Pigmenting Hormone, Parathyroid Hormone, Luteinizing Hormone, Calcitonin Gene-Related Peptide The enzyme may be selected from the group consisting of growth hormone releasing peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

[0015] The target protein can be encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-19.

[0016] The present invention also relates to a nucleic acid comprising a nucleotide sequence encoding said fusion protein.

[0017] The present invention also relates to an expression vector containing the nucleic acid.

[0018] The present invention also relates to a cell transformed with the expression vector.

[0019] The cell can be Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium, Serratia marcescens, Pseudomonas species, yeast, insect cells, CHO cell lines (Chinese Hamster Ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, MDCK cell lines, or plant cells.

[0020] The present invention further comprises the steps of: producing an expression vector containing a nucleic acid encoding the fusion protein; B) transfecting a host cell with the expression vector to obtain a transformant; C) expressing a fusion protein containing a CSQ tag and a target protein from the transformant; D) precipitating the fusion protein containing the CSQ tag and the target protein from the expressed transformant using calcium; and E) separating the target protein from the fusion protein using a hydrolase; The present invention relates to a method for expressing and purifying a target protein using a CSQ tag, including: [Effects of the Invention]

[0021] According to the method of the present invention for expressing, solubilizing, and purifying a target protein using the CSQ tag, the high expression and high water solubility of CSQ make it possible to express proteins that are difficult to express or prone to aggregation in aqueous solution.Furthermore, since the protein precipitates with calcium, it can be easily separated and purified without using a column, which is expected to reduce the costs of protein materials and pharmaceuticals. [Brief explanation of the drawings]

[0022] [Figure 1] FIG. 1 is a schematic diagram showing a recombinant expression vector according to the present invention. [Figure 2]FIG. 1 is a schematic diagram showing a method for expressing and purifying a target protein according to the present invention. [Figure 3] FIG. 1 shows the results of cloning an EDB target protein gene into a CSQ1TEV or CSQ1Thrombin vector in Example 1. [Figure 4] This figure compares the results of purifying the CSQ1TEV-EDB and CSQ1Thrombin-EDB proteins with the results of purifying EDB using commercially available His-tag and GST fusion proteins in Example 1. In the case of the EDB14 protein, which is expressed using a His-tag or GST-tag, the target protein is not present in the supernatant but is mostly aggregated as a pellet and insolubilized. In the case of the EDB14, 21, and 26 proteins using the CSQ tag, some of the protein aggregates as a pellet, but approximately 30 to 50% or more of the protein is present in the supernatant (S) as a water-soluble protein, confirming that the target protein can be eluted. [Figure 5] This figure shows the results of cleaving the CSQ1TEV-EDB protein with TEV protease in Example 1. After separating the CSQ-tag and EDB protein with TEV protease, calcium treatment was performed to precipitate and remove the separated CSQ-tag, confirming that the target protein, EDB, could be easily separated. [Figure 6] FIG. 1 shows the results of cloning a target EGF protein gene into the CSQ1TEV and CSQ1Thrombin vectors in Example 2. [Figure 7] This figure shows the results of purifying CSQ1Thrombin-EGF and CSQ1TEV-EGF proteins in Example 2. It is known that EGF expressed in bacteria is normally aggregated and 100% is present in the pellet, but when the CSQ tag is used, 30-40% can be made water-soluble. [Figure 8] FIG. 1 shows the results of cleaving CSQ1Thrombin-EGF protein with thrombin protease in Example 2. [Figure 9]FIG. 1 shows the results of purification of CSQ1TEV-KGF1 protein in Example 3. [Figure 10] FIG. 1 shows the results of cleaving the CSQ1TEV-KGF1 protein with TEV protease in Example 3. [Figure 11] FIG. 1 shows the results of purification of CSQ1TEV-VEGF protein in Example 3. [Figure 12] FIG. 1 shows the results of cleaving CSQ1TEV-VEGF protein with TEV protease in Example 3. [Figure 13] FIG. 1 shows the results of purification of CSQ1TEV-FGF2 protein in Example 3. [Figure 14] FIG. 1 shows the results of cleaving CSQ1TEV-FGF2 protein with TEV protease in Example 3. [Figure 15] FIG. 1 shows the results of cloning BMP2 and TGFβ target protein genes into the TEVCSQ1 or ThrombinCSQ1 vector in Example 4. [Figure 16] FIG. 1 shows the results of purifying BMP2-TEVCSQ1 and BMP2-ThrombinCSQ1 proteins in Example 4. [Figure 17] FIG. 1 shows the results of cleaving BMP2-TEVCSQ1 or BMP2-ThrombinCSQ1 protein with TEV protease or thrombin protease in Example 4. [Figure 18] FIG. 1 shows the results of purifying TGFβ-TEVCSQ1 and TGFβ-ThrombinCSQ1 proteins in Example 4. [Figure 19] FIG. 1 shows the results of cleaving TGFβ-TEVCSQ1 or TGFβ-ThrombinCSQ1 protein with TEV protease or thrombin protease in Example 4. [Figure 20] FIG. 1 shows the results of purification of HRP-TEVCSQ1 protein in Example 4. [Figure 21]FIG. 1 shows the results of cleaving HRP-TEVCSQ1 protein with TEV protease in Example 4. [Figure 22] FIG. 1 shows the results of purification of the GLP1-TEVCSQ1 protein in Example 4. [Figure 23] FIG. 1 shows the results of cleaving the GLP1-TEVCSQ1 protein with TEV protease in Example 4. [Figure 24] FIG. 10 shows the results of cloning the target protein genes BMP2 and KGF1 into the CSQ2TEV or CSQ2Thrombin vector in Example 5. [Figure 25] FIG. 10 shows the results of purification of CSQ2TEV-BMP2 and CSQ2Thrombin-BMP2 proteins in Example 5. [Figure 26] FIG. 10 shows the results of cleaving CSQ2TEV-BMP2 or CSQ2Thrombin-BMP2 protein with TEV protease or thrombin protease in Example 5. [Figure 27] FIG. 1 shows the results of purification of CSQ2TEV-KGF1 and CSQ2Thrombin-KGF1 proteins in Example 5. [Figure 28] FIG. 1 shows the results of cleaving CSQ2TEV-KGF1 or CSQ2Thrombin-KGF1 protein with TEV protease or thrombin protease in Example 5. [Figure 29] FIG. 1 shows the results of purifying HRP-TEVCSQ2 and HRP-Thrombin-CSQ2 proteins in Example 6. [Figure 30] FIG. 1 shows the results of cleaving HRP-TEVCSQ2 protein with TEV protease in Example 6. [Figure 31] FIG. 1 shows the results of purification of GLP1-TEVCSQ2 protein in Example 6. [Figure 32] FIG. 1 shows the results of cleaving the GLP1-TEVCSQ2 protein with TEV protease in Example 6. [Figure 33] FIG. 10 shows the results of purification of CSQ1-Ssp Dna-EGF protein in Example 7. [Figure 34] FIG. 10 shows the results of cleaving CSQ1-Ssp Dna-EGF protein with dithiothreitol (DTT) in Example 7. [Figure 35] FIG. 10 shows the results of purification of CSQ2-Ssp Dna-KGF1 protein in Example 7. [Figure 36] FIG. 10 shows the results of cleaving CSQ2-Ssp Dna-KGF1 protein with dithiothreitol (DTT) in Example 7. [Figure 37] FIG. 10 shows the results of purification of EGF-GyrA-CSQ1 protein in Example 8. [Figure 38] FIG. 10 shows the results of cleaving the EGF-GyrA-CSQ1 protein using 20 mM Na-HEPES (pH 6.5) buffer in Example 8. [Figure 39] FIG. 10 shows the results of purification of BMP2-GyrA-CSQ2 protein in Example 8. [Figure 40] FIG. 10 shows the results of cleavage using 20 mM Na-HEPES (pH 6.5) buffer in Example 8. BEST MODE FOR CARRYING OUT THE INVENTION

[0023] As used herein, the term "calsequestrin (CSQ)" refers to a calcium-binding protein in the sarcoplasmic reticulum (SAR). Even when the calcium concentration is higher in the SAR than in the cytoplasm, it binds calcium after muscle contraction, allowing calcium ions to be stored in the cisternae of the SAR. A single SAR molecule can bind multiple calcium ions (e.g., 40-50 calcium-binding sites per SAR molecule), providing excellent calcium storage capacity. Calsequestrin exists as a monomer when calcium is scarce, but when calcium levels increase, it converts into a multimer and grows in size.

[0024] As used herein, the term "target protein" refers to any protein that needs to be obtained in high purity or in large quantities for a specific purpose, including, without limitation, native proteins, mutant proteins, or novel recombinant proteins. The target protein may be a protein that requires high purity or large quantities for industrial, medical, or academic reasons, preferably a recombinant protein for pharmaceutical or research use, and more preferably a protein selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. More preferably, the target protein may be either or both of the light and heavy chains of an antibody, and most preferably the light chain variable region (VL) or heavy chain variable region (VH) of an antibody.

[0025] As used herein, the term "hydrolase-degraded peptide" can be included between the CSQ tag and the target protein, which can be cleaved by a hydrolase to separate the CSQ tag from the target protein.

[0026] As used herein, the term "vector" refers to a nucleic acid construct capable of expressing a protein of interest in a suitable host cell, comprising the necessary regulatory elements operably linked to a nucleic acid insert for expression. The vector may be constructed by manipulating a plasmid commonly used in the art, such as a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vectors (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19), a phage (e.g., λgt4λB, λ-Charon, λΔzl, and M13), or a virus (e.g., CMV, SV40, etc.).

[0027] As used herein, the term "transformation" refers to the artificial genetic change that occurs when exogenous DNA is introduced into a host cell, either as a chromosomal factor or as a replicable DNA integrated into the chromosome. Transformation methods include, but are not limited to, the CaCl2 precipitation method, the Hanahan method, which uses a reducing agent called DMSO (dimethyl sulfoxide) to enhance the efficiency of the CaCl2 method, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicon carbide fibers, Agrobacterium-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and desiccation- and inhibition-mediated transformation.

[0028] As used herein, the term "host cell" refers to a cell that parasitizes other microorganisms or genes and provides nutrients to the host cell, and that, when transformed with a vector, exerts various genetic or molecular effects within the host cell. Host cells capable of stably and continuously cloning and expressing the vectors of the present invention in prokaryotic cells include, without limitation, host cells known in the art, including strains of the genus Bacillus, such as E. coli Rosetta, E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus subtilis, and Bacillus thuringiensis, as well as Enterobacteriaceae and strains thereof, such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. Furthermore, when the vector of the present invention is transformed into eukaryotic cells, yeast (Saccharomyce cerevisiae), insect cells, human cells (e.g., CHO cell lines (Chinese Hamster Ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, and MDCK cell lines), plant cells, etc. can be used as host cells.

[0029] According to a first embodiment, The present invention provides a fusion protein comprising a CSQ tag and a protein of interest.

[0030] In the fusion protein according to the present invention, the CSQ tag comprises CSQ1 or CSQ2.

[0031] In the fusion protein according to the present invention, the CSQ1 and CSQ2 are encoded by the amino acids of SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The amino acids of SEQ ID NO: 1 can be encoded by the nucleotide sequence of SEQ ID NO: 3, and the amino acids of SEQ ID NO: 2 can be encoded by the nucleotide sequence of SEQ ID NO: 4.

[0032] The fusion protein according to the present invention is characterized in that the CSQ tag and the target protein are fused via a peptide that is hydrolyzed by a hydrolase and consists of amino acids selected from the group consisting of SEQ ID NOs: 5 to 8.

[0033] In the fusion protein according to the present invention, the target protein is selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. For example, the target protein may be interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α and -β, TGF-α, -β), Brain-Derived Neutrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (PIGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor 1 and 2 (FGF-1, -2), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (Luteinizing Hormone)Hormone-Releasing Hormone, Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone, Oxytocin, Thymosin alpha-1, Corticotropin-Releasing Factor, Calcitonin, Bivalirudin, Vasopressin, Phospholipase-Activating Protein (PLAP), Insulin, Tumor Necrosis Factor (TNF), Follicle-Stimulating Hormone, Thyroid-Stimulating Hormone, Antidiuretic Hormone, Pigmenting Hormone, Parathyroid Hormone, Luteinizing Hormone, Calcitonin Gene-Related Peptide The enzyme may be selected from the group consisting of growth hormone releasing peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

[0034] In the fusion protein according to the present invention, the target protein is characterized in that it is encoded by amino acids selected from the group consisting of SEQ ID NOs: 9 to 19.

[0035] According to a second embodiment, The present invention provides a nucleic acid comprising a nucleotide sequence encoding a fusion protein comprising a CSQ tag and a protein of interest, and an expression vector comprising said nucleic acid.

[0036] In the nucleic acid or expression vector according to the present invention, the CSQ tag is encoded by amino acids of SEQ ID NO: 1 or SEQ ID NO: 2. The amino acids of SEQ ID NO: 1 can be encoded by the nucleotide sequence of SEQ ID NO: 3, and the amino acids of SEQ ID NO: 2 can be encoded by the nucleotide sequence of SEQ ID NO: 4.

[0037] In the nucleic acid or expression vector according to the present invention, the CSQ tag and the target protein are fused via a peptide that can be decomposed by a hydrolase and that consists of amino acids selected from the group consisting of SEQ ID NOs: 5 to 8.

[0038] In the nucleic acid or expression vector according to the present invention, the target protein is selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. For example, the target protein may be interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α and -β, TGF-α, -β), Brain-Derived Neutrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (PIGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor 1 and 2 (FGF-1, -2), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (Luteinizing Hormone)Hormone-Releasing Hormone, Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone, Oxytocin, Thymosin alpha-1, Corticotropin-Releasing Factor, Calcitonin, Bivalirudin, Vasopressin, Phospholipase-Activating Protein (PLAP), Insulin, Tumor Necrosis Factor (TNF), Follicle-Stimulating Hormone, Thyroid-Stimulating Hormone, Antidiuretic Hormone, Pigmenting Hormone, Parathyroid Hormone, Luteinizing Hormone, Calcitonin Gene-Related Peptide The enzyme may be selected from the group consisting of growth hormone releasing peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

[0039] In the nucleic acid or expression vector according to the present invention, the target protein is characterized in that it is encoded by an amino acid selected from the group consisting of SEQ ID NOs: 9 to 19.

[0040] According to a third embodiment, The present invention provides a cell transformed with an expression vector containing a fusion protein comprising a CSQ tag and a protein of interest.

[0041] In the transformed cell according to the present invention, the CSQ tag is characterized in that it is encoded by amino acids consisting of SEQ ID NO: 1 or SEQ ID NO: 2. The amino acids of SEQ ID NO: 1 can be encoded by the nucleotide sequence of SEQ ID NO: 3, and the amino acids of SEQ ID NO: 2 can be encoded by the nucleotide sequence of SEQ ID NO: 4.

[0042] In the transformed cell of the present invention, the CSQ tag and the target protein are fused via a peptide that can be degraded by a hydrolase and is composed of amino acids selected from the group consisting of SEQ ID NOs: 5 to 8.

[0043] In the transformed cell of the present invention, the target protein is selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. For example, the target protein may be interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α and -β, TGF-α, -β), Brain-Derived Neutrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (PIGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor 1 and 2 (FGF-1, -2), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (Luteinizing Hormone)Hormone-Releasing Hormone, Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone, Oxytocin, Thymosin alpha-1, Corticotropin-Releasing Factor, Calcitonin, Bivalirudin, Vasopressin, Phospholipase-Activating Protein (PLAP), Insulin, Tumor Necrosis Factor (TNF), Follicle-Stimulating Hormone, Thyroid-Stimulating Hormone, Antidiuretic Hormone, Pigmenting Hormone, Parathyroid Hormone, Luteinizing Hormone, Calcitonin Gene-Related Peptide The enzyme may be selected from the group consisting of growth hormone releasing peptide (CGPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

[0044] In the transformed cell according to the present invention, the target protein is characterized in that it is encoded by an amino acid selected from the group consisting of SEQ ID NOs: 9 to 19.

[0045] The transformed cell of the present invention is characterized in that the cell is Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium, Serratia marcescens, Pseudomonas species, yeast, insect cells, CHO cell line (Chinese Hamster Ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, MDCK cell line, or plant cell.

[0046] According to a fourth embodiment, The present invention provides a method for expressing and purifying a target protein using a CSQ tag, the method comprising: A) preparing an expression vector containing a nucleic acid encoding a fusion protein containing a CSQ tag and a protein of interest; B) transfecting a host cell with the expression vector to obtain a transformant; C) expressing a fusion protein containing a CSQ tag and a target protein from the transformant; D) precipitating the fusion protein containing the CSQ tag and the target protein from the expressed transformant using calcium; and E) separating the target protein from the fusion protein using a hydrolase.

[0047] In the method for expressing and purifying a target protein according to the present invention, the CSQ tag is encoded by amino acids represented by SEQ ID NO: 1 or SEQ ID NO: 2. The amino acids represented by SEQ ID NO: 1 can be encoded by the nucleotide sequence represented by SEQ ID NO: 3, and the amino acids represented by SEQ ID NO: 2 can be encoded by the nucleotide sequence represented by SEQ ID NO: 4.

[0048] The method for expressing and purifying a target protein according to the present invention is characterized in that the CSQ tag and the target protein are fused via a peptide that can be degraded by a hydrolase and is composed of amino acids selected from the group consisting of SEQ ID NOs: 5 to 8.

[0049] In the method for expressing and purifying a target protein according to the present invention, the target protein is selected from the group consisting of high molecular weight proteins, glycoproteins, cytokines, growth factors, blood products, vaccines, hormones, enzymes, and antibodies. For example, the target protein may be interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor-α and -β, TGF-α, -β), Brain-Derived Neutrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (PIGF), Hepatocyte Growth Factor (HGF), Fibroblast Growth Factor 1 and 2 (FGF-1, -2), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (luteinizing hormone receptor agonist).Hormone-Releasing Hormone, Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone, Oxytocin, Thymosin alpha-1, Corticotropin-Releasing Factor, Calcitonin, Bivalirudin, Vasopressin, Phospholipase-Activating Protein (PLAP), Insulin, Tumor Necrosis Factor (TNF), Follicle-Stimulating Hormone, Thyroid-Stimulating Hormone, Antidiuretic Hormone, Pigmenting Hormone, Parathyroid Hormone, Luteinizing Hormone, Calcitonin Gene-Related Peptide The enzyme may be selected from the group consisting of growth hormone releasing peptide (GHPR), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

[0050] In the method for expressing and purifying a target protein according to the present invention, the target protein is encoded by an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 19.

[0051] Various examples are presented below to help understand the invention. The examples described below are merely provided to make the invention easier to understand, and the scope of protection of the present invention is not limited by these examples. [Example]

[0052] Example 1: Separation of EDB using CSQ-tags 1-1. EDB cloning into CSQ1TEV or CSQ1Thrombin vector To clone EDB into the CSQ1TEV or CSQ1Thrombin vector, the following oligonucleotides, EDB-F1 and EDB-B1, were synthesized (Bioneer, Daejeon, South Korea). For comparison, fusion proteins of EDB with the known His tag or GST tag were prepared.

[0053] 5'-AATGGATCCGAGGTGCCCCAACTCACTGAC-3' (SEQ ID NO: 20)

[0054] 5'-ATTCTCGAGTTACGTTTGTTGTGTCAGTGTAGTAGG-3' (SEQ ID NO: 21)

[0055] For amplification, 20 pmol of EDB-F1, 20 pmol of EDB-B1, 4 μl of PCR PreMix (Elpisbio, Daejeon, South Korea), and 10 μg of template were mixed and distilled water was added to a total volume of 20 μl. This mixture was subjected to PCR (95°C for 5 minutes, 30 cycles: 95°C for 30 seconds, 42°C for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes). After amplification, the EDB14 (SEQ ID NO: 9), EDB21 (SEQ ID NO: 10), and EDB26 (SEQ ID NO: 11) genes were obtained by purification (PCR purification kit, GeneAll, Seoul, South Korea). To insert the EDB genes into the CSQ1TEV or CSQ1Thrombin vector, the CSQ1TEV or CSQ1Thrombin vector and insert DNA were digested with restriction enzymes. Approximately 1 μg of insert DNA was reacted with BamHI (New England Biolabs, Ipswich) and XhoI (NEB, Ipswich) for one day, and then purified using a PCR purification kit. Approximately 40 μg of CSQ1TEV or CSQ1Thrombin vector was added to CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich), reacted for 3 hours, and then purified using a PCR purification kit. The insert DNA was ligated to the CSQTEV or CSQ1Thrombin vector using T4 DNA ligase (Bioneer, Daejeon, South Korea) at room temperature for 3 hours (Figure 3).

[0056] The CSQ1TEV or CSQ1Thrombin vector and EDB insert were then ligated and transformed with DNA. Competent DH5α cells were thawed on ice and mixed with 2 μl of the ligation solution in 100 μl of competent cells, followed by incubation for 30 minutes. Next, the cells were heat-shocked at 42°C for 1 minute, and then 200 μl of SOC medium was added. After incubation at 37°C for 30 minutes, the cells were plated. Six colonies were randomly selected from the plated colonies, subjected to PCR, and the inserts were confirmed by DNA electrophoresis. The cloned colonies were sequenced by Bioneer for confirmation.

[0057] As a comparative example, fusion proteins of EDB with a His tag or GST tag were cloned into the pBT7-N-His and pBT7-N-GST vectors provided by Bioneer in the same manner as described above.

[0058] Purification of CSQ1TEV-EDB, CSQ1Thrombin-EDB, His-EDB, and GST-EDB All DNA sequences confirmed by Bioneer were transformed into BL21 cells and plated on agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. They were then mixed with glycerol at a 1:1 ratio to create a 1 ml stock, which was stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 20 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. The stock was then transferred to 400 ml of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.7 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 18°C ​​with 200 rpm for one day. The supernatant was centrifuged at 4,000 rpm for 20 minutes at 4°C, and the entire supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the supernatant was removed and completely dissolved. 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and centrifuged at 13,000 rpm for 1 hour at 4°C. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for 1 hour. The supernatant was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain CSQ1TEV-EDB and CSQ1Thrombin-EDB proteins (Figure 4).

[0059] His-EDB was expressed and purified as follows. EDB14 was cloned into the pBT7-N-His vector and transformed into BL21 cells, which were then plated on agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 220 rpm. They were then mixed with glycerol at a 1:1 ratio to make a 1 ml stock and stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 20 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 220 rpm. They were then transferred to 400 ml of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.7 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the cells were cultured at 37°C for one day at 220 rpm. The cells were centrifuged at 4000 x g for 10 minutes at 4°C, and the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the cells were removed and completely dissolved. Then, 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of isopropanol, and 1 ml of this solution was added to the lysed E. coli.

[0060] E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm for 1 hour at 4°C. Ni-NTA affinity resin (Elposbio, Daejeon, South Korea) was washed with distilled water and lysis buffer, and the supernatant was allowed to bind to the resin. After washing with 600 ml of washing buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 20 mM imidazole), the N-terminal His-tagged EDB14 protein was eluted and isolated using elution buffer (50 mM sodium phosphate (pH 8.0), 300 mM NaCl, and 250 mM imidazole) (Figure 4).

[0061] GST-EDB was expressed and purified as follows. EDB14 was cloned into the pBT7-N-GST vector and transformed into BL21 cells, which were then plated on agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 220 rpm. They were then mixed with glycerol at a 1:1 ratio to make a 1 ml stock and stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 20 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 220 rpm. They were then transferred to 400 ml of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.7 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the cells were cultured at 37°C for one day at 220 rpm. The cells were centrifuged at 4000 x g for 10 minutes at 4°C, and the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% NP-40). After storage at -80°C for one day, the cells were removed and completely dissolved. Then, 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of isopropanol, and 1 ml of this solution was added to the lysed E. coli.

[0062] E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm for 1 hour at 4°C. After washing the GST·Bind agarose resin (Elposbio, Daejeon, South Korea) with distilled water and lysis buffer, the supernatant was allowed to bind to the resin. After washing with 600 ml of washing buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, and 0.1 mM EDTA), the N-terminal GST-tagged EDB14 protein was eluted and isolated using elution buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1 mM EDTA, and 10 mM reduced (free) glutathione) (Figure 4).

[0063] As a result, it was confirmed that the EDB14 protein was not expressed in the supernatant when His-tag or GST-tag was used, but when CSQ-tag was used, it was water-soluble and could be expressed in the supernatant.

[0064] 1-3. Isolation of EDB using TEV protease To separate EDB from the CSQ1TEV-EDB protein, 5 μl of TEV protease (Genscript, USA) was added and incubated at 30°C for one day, followed by SDS-PAGE (Figure 5). The results confirmed that the CSQ-tag and EDB fusion proteins could be easily separated using TEV protease after calcium precipitation.

[0065] Example 2: Separation of EGF using CSQ-tag 2-1. Cloning of EGF into CSQ1TEV and CSQ1Thrombin vectors To clone EGF into the CSQ1TEV and CSQ1Thrombin vectors, the following two oligonucleotides, EGF-F1 and EGF-B1, were synthesized (Bioneer, Daejeon, Republic of Korea).

[0066] 5'-AATGGATCCAACTCTGATAGCGAATGCCCG-3' (SEQ ID NO: 22)

[0067] 5'-ATTCTCGAGTTA ACGCAGTTCCCACCATTT-3' (SEQ ID NO: 23)

[0068] To amplify the EGF gene, 20 pmol of EGF-F1, 20 pmol of EGF-B1, 4 μl of PCR PreMix (Elpisbio, Daejeon, South Korea), and 10 μg of template were mixed and distilled water was added to a total volume of 20 μl. This mixture was subjected to PCR (95°C for 5 minutes, 30 cycles: 95°C for 30 seconds, 42°C for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes). After amplification, the EGF gene (SEQ ID NO: 12) was purified (PCR purification kit, GeneAll, Seoul, South Korea). To insert the EGF gene into the CSQ1TEV and CSQ1Thrombin vectors, the vector and insert DNA were digested with restriction enzymes. Approximately 1 μg of insert DNA was reacted with BamHI (New England Biolabs, NEB, Ipswich) and XhoI (NEB, Ipswich) for one day, and then purified DNA was obtained using a PCR purification kit. Approximately 40 μg of CSQ1TEV and CSQ1Thrombin vectors were added to CIAP (Calf Intestinal Alkaline Phosphatase) (NEB, Ipswich), reacted for 3 hours, and then purified using a PCR purification kit. The insert DNA was ligated to the vector using T4 DNA ligase (Bioneer, Daejeon, South Korea) at room temperature for 3 hours (Figure 6).

[0069] The CSQ1TEV or CSQ1Thrombin vector was then ligated with the EGF insert and transformed with DNA. Competent DH5α cells were thawed on ice and mixed with 2 μl of the ligation solution in 100 μl of competent cells, followed by incubation for 30 minutes. The cells were then heat-shocked at 42°C for 1 minute, added with 200 μl of SOC medium, and incubated at 37°C for 30 minutes before plating. Six colonies were randomly selected from the resulting plate and subjected to PCR. The inserts were then confirmed by DNA electrophoresis. The cloned colonies were then sequenced by Bioneer.

[0070] 2-2. Purification of CSQ1Thrombin-EGF or CSQ1TEV-EGF The sequence-confirmed DNA was transformed into BL21 cells and plated on agar plates containing ampicillin. The colony grown on the agar plate was inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. It was then mixed with glycerol at a 1:1 ratio to create a 1 ml stock, which was stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 20 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. It was then transferred to 400 ml of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.7 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 18°C ​​with 200 rpm for one day. The mixture was centrifuged at 4,000 rpm at 4°C for 20 minutes, and the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the mixture was removed and completely dissolved. Then, 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and centrifuged at 13,000 rpm at 4°C for one hour. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for one hour. The mixture was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain CSQ1-Thrombin-EGF and CSQ1-TEV-EGF proteins (Figure 7).

[0071] 2-3. Isolation of EGF using thrombin protease To separate EGF from the CSQ1Thrombin-EGF protein, 5 μl of thrombin protease was added, and the mixture was incubated at 22°C for one day, after which it was confirmed by SDS-PAGE (Figure 8). EGF It was confirmed that the fusion protein was easily separated using thrombin protease.

[0072] Example 3: Separation of KGF1, VEGF, and FGF2 using CSQ-tag 3-1. Purification of CSQ1TEV-KGF1, CSQ1TEV-VEGF, and CSQ1TEV-FGF2 The genes for KGF1 (SEQ ID NO: 13), VEGF (SEQ ID NO: 14), and FGF2 (SEQ ID NO: 15) were synthesized at Bioneer, cloned into CSQ1TEV, transformed into BL21 cells, and plated on agar medium containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. They were then mixed with glycerol at a 1:1 ratio to prepare 1 ml of stock and stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. The stock was then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the culture was incubated at 17°C for one day at 200 rpm. After centrifugation at 4,000 rpm for 20 minutes at 4°C, the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the culture was removed and completely dissolved. 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm for one hour at 4°C. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for one hour. The culture was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain CSQ1TEV-KGF1, CSQ1TEV-VEGF, and CSQ1TEV-FGF2 proteins (FIGS. 9, 11, and 13).

[0073] 3-2. Separation of KGF1, VEGF, and FGF2 using TEV protease To separate KGF1 from the CSQ1TEV-KGF1 protein (hereafter, experiments were performed in the same manner for CSQ1TEV-VEGF and CSQ1TEV-FGF proteins), 5 μl of TEV protease (Genscript, USA) was added, incubated at 30°C for one day, and then analyzed by SDS-PAGE (Figures 10, 12, and 14). The results confirmed that the CSQ-tag and KGF1 fusion protein, the CSQ-tag and VEGF protein, and the CSQ-tag and FGF protein were all easily separated using TEV protease.

[0074] Example 4: Separation of BMP2, TGFβ, HRP, and GLP1 using CSQ-tag 4-1. Cloning of BMP2 and TGFβ into TEVCSQ1 or ThrombinCSQ1 vectors To clone BMP2 and TGFβ into the TEVCSQ1 or ThrombinCSQ1 vector, the following oligonucleotides, Amplify-F1 and Amplify-B1, were synthesized (Bioneer, Daejeon, Republic of Korea).

[0075] 5'-CAGCAAGACAGCGATGGATCC-3' (SEQ ID NO: 24)

[0076] 5'-CGACTTACAGGTGATCTCGAG-3' (SEQ ID NO: 25)

[0077] To amplify the BMP2 gene, 20 pmol of Amplify-F1, 20 pmol of Amplify-B1, PCR PreMix (Bioneer, Daejeon, South Korea), and 10 μg of template were mixed and distilled water was added to a total volume of 20 μl. This mixture was subjected to PCR (95°C for 5 minutes, 30 cycles: 95°C for 30 seconds, 47°C for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes). After amplification, the BMP2 gene was purified (PCR purification kit, GeneAll, Seoul, South Korea) to obtain the BMP2 (SEQ ID NO: 16) and TGFβ (SEQ ID NO: 17) genes. To insert the BMP2 gene (hereinafter, the TGFβ gene was also inserted in the same manner as for BMP2) into the TEVCSQ1 or ThrombinCSQ1 vector, the TEVCSQ1 or ThrombinCSQ1 vector and the insert DNA were digested with restriction enzymes. Approximately 1 μg of insert DNA was incubated with BamHI (New England Biolabs, Ipswich) for one day and then with XhoI (NEB, Ipswich) for two days. Purified DNA was then obtained using a PCR purification kit. Approximately 3 μg of TEVCSQ or ThrombinCSQ vector was incubated with CIAP (Calf Intestinal Alkaline Phosphatase) (Takara, Japan) for 3 hours and then purified using a PCR purification kit. The insert DNA was ligated to the TEVCSQ or ThrombinCSQ vector using T4 DNA ligase (Bioneer, Daejeon, South Korea) at room temperature for 3 hours (Figure 15).

[0078] Subsequently, DNA prepared by ligation of the TEVCSQ1 or ThrombinCSQ1 vector with the BMP2 insert was transformed. Competent DH5α cells were thawed on ice and mixed with 5 μl of the ligation solution in 100 μl of competent cells, followed by incubation for 30 minutes. The cells were then heat-shocked at 42°C for 30 seconds, and 200 μl of SOC medium was added. After incubation at 37°C for 40 minutes, the cells were plated. Colonies obtained from the plated cells were randomly selected, and the cloned colonies were sequenced and confirmed by BioNear. The HRP (SEQ ID NO: 18) and GLP1 (SEQ ID NO: 19) genes were also synthesized by BioNear and cloned into TEVCSQ1 in the same manner as above.

[0079] 4-2. Purification of BMP2-TEVCSQ1 or BMP2-ThrombinCSQ1, TGFβ-TEVCSQ1 or TGFβ-ThrombinCSQ1, HRP-TEVCSQ1, and GLP1-TEVCSQ1 The BMP2-TEVCSQ1, BMP2-ThrombinCSQ1, TGFβ-TEVCSQ1, TGFβ-ThrombinCSQ1, HRP-TEVCSQ1, and GLP-TEVCSQ1 vectors, sequence-confirmed by Bioneer, were transformed into BL21 cells and plated onto agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. Afterwards, the colonies were mixed with glycerol at a 1:1 ratio to make 1 ml stocks, which were then stored in a -80°C deep freezer. 100 μl of the stock was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. The cells were then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 200 rpm at 17°C for one day. The cells were centrifuged at 4000 rpm for 20 minutes at 4°C, and the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the cells were removed and completely dissolved. 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of the solution was added to the lysed E. coli. E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm at 4°C for 1 hour. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for 1 hour. The supernatant was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain BMP2-TEVCSQ1 and BMP2-ThrombinCSQ1 proteins (Figures 16, 18, 20, and 22).

[0080] 4-3. Separation of BMP2, TGFβ, HRP, and GLP1 using TEV protease To separate BMP2 from the BMP2-TEVCSQ1 protein (hereinafter, experiments for TGFβ-TEVCSQ1, HRP-TEVCSQ1, and GLP1-TEVCSQ1 proteins were performed in the same manner as for BMP2-TEVCSQ1), 2 μl of TEV protease (Genscript, USA) was added, and the mixture was incubated at 30°C for one day. After incubation, the mixture was analyzed by SDS-PAGE (Figures 17, 19, 21, and 23). The results confirmed that the CSQ-tag and BMP2 fusion proteins were easily separated using TEV protease.

[0081] 4-4. Separation of BMP2 and TGFβ using thrombin protease To separate BMP2 from the BMP2-ThrombinCSQ1 protein (hereinafter, the experiment was performed in the same manner as for BMP2-TEVCSQ1 for the TGFβ-TEVCSQ1 protein), 5 μl of thrombin protease was added, and the mixture was incubated at 22°C for one day, followed by SDS-PAGE (Figures 17 and 19). The results confirmed that the CSQ-tag and BMP2 fusion proteins could be easily separated using thrombin protease.

[0082] Example 5: Separation of BMP2 and KGF1 using CSQ-tag 5-1. Cloning of BMP2 and KGF1 into CSQ2TEV or CSQ2Thrombin vectors To clone BMP2 and KGF1 into the CSQ2TEV or CSQ2Thrombin vector, synthesized oligonucleotides Amplify-F1 and Amplify-B1 (Bioneer, Daejeon, South Korea) were used. For amplification, 20 pmol of Amplify-F1, 20 pmol of Amplify-B1, PCR PreMix (Bioneer, Daejeon, South Korea), and 10 μg of template were mixed and distilled water was added to a total volume of 20 μl. This mixture was subjected to PCR (95°C for 5 minutes, 30 cycles: 95°C for 30 seconds, 47°C for 30 seconds, 72°C for 45 seconds, and 72°C for 5 minutes). After amplification, the BMP2 (SEQ ID NO: 16) and KGF1 (SEQ ID NO: 13) genes were obtained by purification (PCR purification kit, GeneAll, Seoul, South Korea). To insert the BMP2 gene (KGF1 gene, hereafter, the same procedure as for BMP2) into the CSQ2TEV or CSQ2Thrombin vector, the CSQ2TEV or CSQ2Thrombin vector and insert DNA were digested with restriction enzymes. Approximately 1 μg of insert DNA was incubated with BamHI (New England Biolabs, NEB, Ipswich) for one day and then with XhoI (NEB, Ipswich) for two days. Purified DNA was then obtained using a PCR purification kit. Approximately 3 μg of the CSQ2TEV or CSQ2Thrombin vector was ligated with CIAP (Calf Intestinal Alkaline Phosphatase) (Takara, Japan), incubated for 3 hours, and then purified using the PCR purification kit. The insert DNA was ligated to the CSQ2TEV or CSQ2Thrombin vector using T4 DNA ligase (Bioneer, Daejeon, South Korea) at room temperature for 3 hours (Figure 24).

[0083] The CSQ2TEV or CSQ2Thrombin vector was then ligated with the BMP2 insert and transformed with DNA. Competent DH5α cells were thawed on ice and mixed with 5 μl of the ligation solution (100 μl of competent cells) for 30 minutes. The cells were then heat-shocked at 42°C for 30 seconds, added with 200 μl of SOC medium, and incubated at 37°C for 40 minutes before plating. Colonies were randomly selected, and the cloned colonies were sequenced by Bioneer for confirmation.

[0084] 5-2. Purification of CSQ2TEV-BMP2 or CSQ2Thrombin-BMP2, and CSQ2TEV-KGF1 or CSQ2Thrombin-KGF1 All DNA sequences confirmed by Bioneer were transformed into BL21 cells and plated on agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. They were then mixed with glycerol at a 1:1 ratio to create a 1 ml stock, which was stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured at 37°C with mixing at 200 rpm for one day. The stock was then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured at 17°C with mixing at 200 rpm for one day. The supernatant was centrifuged at 4,000 rpm for 20 minutes at 4°C, and the entire supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the supernatant was removed and completely dissolved. 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and centrifuged at 13,000 rpm for 1 hour at 4°C. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for 1 hour. The supernatant was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain CSQ2TEV-BMP2 and CSQ2Thrombin-BMP2 proteins (Figures 25 and 27).

[0085] 5-3. Isolation of BMP2 and KGF using TEV protease To separate BMP2 from the CSQ2TEV-BMP2 protein (hereafter, the same experiment will be performed for the CSQ2TEV-KGF1 protein), 2 μl of TEV protease (Genscript, USA) was added, and the mixture was incubated at 30°C for one day. After incubation, the mixture was analyzed by SDS-PAGE (Figures 26 and 28). The results confirmed that the CSQ-tag and BMP2 fusion proteins could be easily separated using TEV protease.

[0086] 5-4. Isolation of BMP2 and KGF using thrombin protease To separate BMP2 from the CSQ2Thrombin-BMP2 protein (hereinafter, the same experiment was performed for the CSQ2Thrombin-KGF1 protein), 5 μl of thrombin protease was added, and the mixture was incubated at 22°C for one day, after which the mixture was analyzed by SDS-PAGE (Figures 26 and 28). The results confirmed that the CSQ-tag and BMP2 fusion proteins could be easily separated using thrombin protease.

[0087] Example 6: Separation of HRP and GLP1 using CSQ-tag 6-1. Purification of HRP-TEVCSQ2 or HRP-ThrombinCSQ2, and GLP1-TEVCSQ2 The HRP (SEQ ID NO: 18) and GLP1 (SEQ ID NO: 19) genes were synthesized at BioNear, cloned into the TEVCSQ2 and ThrombinCSQ2 vectors, transformed into BL21 cells, and plated on agar plates containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. They were then mixed with glycerol at a 1:1 ratio to prepare a 1 ml stock solution, which was then stored in a deep freezer at -80°C. 100 μl of the stock solution was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. The solution was then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the culture was incubated at 17°C for one day at 200 rpm. After centrifugation at 4,000 rpm for 20 minutes at 4°C, the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 nM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the culture was removed and completely dissolved. 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm for one hour at 4°C. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for one hour. The culture was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain HRP-TEVCSQ2, HRP-ThrombinCSQ2, and GLP1-TEVCSQ2 proteins (FIGS. 29 and 31).

[0088] 6-2. Separation of HRP and GLP1 using TEV protease To separate HRP from the HRP-TEVCSQ2 protein (hereinafter, the same experiment was performed for the GLP1-TEVCSQ2 protein), 2 μl of TEV protease (Genscript, USA) was added, and the mixture was incubated at 30°C for 1 day. After incubation, the mixture was analyzed by SDS-PAGE (Figures 30 and 32). The results confirmed that the CSQ-tag and HRP fusion protein, as well as the CSQ-tag and GLP1 fusion protein, were easily separated using TEV protease.

[0089] Example 7: Separation of EGF and KGF1 using CSQ-tag 7-1. Purification of CSQ1-Ssp DNA-EGF and CSQ2-Ssp DNA-KGF1 The EGF (SEQ ID NO: 12) and KGF1 (SEQ ID NO: 13) genes were synthesized by BioNear, cloned into the CSQ1-SspDNA and CSQ2-SspDNA vectors, transformed into BL21 cells, and plated on agar medium containing ampicillin. The colonies grown on the agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. They were then mixed with glycerol at a 1:1 ratio to prepare a 1 ml stock solution, which was then stored in a deep freezer at -80°C. 100 μl of the stock solution was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. The solution was then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and the culture was incubated at 17°C for one day at 200 rpm. After centrifugation at 4,000 rpm for 20 minutes at 4°C, the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the culture was removed and completely dissolved. Then, 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of this solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and then centrifuged at 13,000 rpm for one hour at 4°C. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for one hour. The culture was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain CSQ1-Ssp DNA-EGF and CSQ2-Ssp DNA-KGF1 proteins (FIGS. 33 and 35).

[0090] 7-2. Separation of EGF and KGF1 using pH To separate EGF from the CSQ1-Ssp DNA-EGF protein (hereinafter, the same experiment was performed for the CSQ2-Ssp DNA-KGF1 protein), the mixture was incubated at room temperature for one day in 20 mM HEPES pH 6.5, 500 mM NaCl buffer, and then analyzed by SDS-PAGE (Figures 34 and 36). The results confirmed that the CSQ-tag and EGF fusion proteins were easily separated using pH.

[0091] Example 8: Separation of EGF and BMP2 using CSQ-tag 8-1. Purification of EGF-GyrA-CSQ1 and BMP2-GyrA-CSQ2 The genes for EGF (sequence number 12) and BMP2 (sequence number 16) were synthesized by BioNear, cloned into the GyrA-CSQ1 and GyrA-CSQ2 vectors, transformed into BL21 cells, and then plated on agar medium containing ampicillin.

[0092] The colonies grown on agar plates were inoculated into 5 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. They were then mixed with glycerol at a 1:1 ratio to create 1 ml of stock, which was then stored in a deep freezer at -80°C. 100 μl of the stock was inoculated into 30 ml of LB medium containing ampicillin (50 μg / ml) and cultured for one day at 37°C with mixing at 200 rpm. The stock was then transferred to 1 L of LB medium containing ampicillin (50 μg / ml) and cultured until an OD of 0.6 was reached. Next, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added and cultured for one day at 17°C with mixing at 200 rpm. The mixture was centrifuged at 4,000 rpm at 4°C for 20 minutes, and the supernatant, excluding the precipitated cells, was removed and suspended in lysis buffer (20 mM Tris (pH 8.0), 300 mM NaCl, and 10 mM imidazole). After storage at -80°C for one day, the mixture was removed and completely dissolved. Then, 10 mg of PMSF (phenyl methane sulfonyl fluoride) was dissolved in 1 ml of DMSO, and 1 ml of the solution was added to the lysed E. coli. The E. coli was lysed using a sonicator and centrifuged at 13,000 rpm at 4°C for one hour. 20 mM CaCl2 was added to the supernatant and incubated at 4°C for one hour. The mixture was then centrifuged at 5,000 rpm for 30 minutes. The supernatant was removed, and the precipitate was suspended in EDTA to obtain EGF-GyrA-CSQ1 and BMP2-GyrA-CSQ2 proteins (Figures 37 and 39).

[0093] 8-2. Separation of EGF and BMP2 using dithiothreitol (DTT) To separate EGF from the EGF-GyrA-CSQ1 protein (the same experiment will be performed for the BMP2-GyrA-CSQ2 protein below), the mixture was incubated at room temperature for one day in 20 mM HEPES pH 8.5, 500 mM NaCl, and 40 mM DTT buffer, and then analyzed by SDS-PAGE (Figures 38 and 40). The results confirmed that the CSQ-tag and EGF fusion proteins could be easily separated using DTT.

[0094] Although the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention should be defined by the appended claims and their equivalents.

Claims

1. A composition for purifying a target protein, comprising an expression vector containing a nucleic acid encoding a fusion protein, wherein the fusion protein comprises: CSQ tag, a target protein, and a peptide that can be degraded by a hydrolase, which is bound between the CSQ tag and the target protein; wherein the target protein is separated from the fusion protein using a hydrolase after precipitating the fusion protein using calcium.

2. The composition for purifying a target protein according to claim 1, wherein the CSQ tag comprises an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO:

2.

3. The composition for purifying a target protein according to claim 1, wherein the CSQ tag is encoded by a nucleotide sequence consisting of SEQ ID NO: 3 or SEQ ID NO:

4.

4. A composition for purifying a target protein as described in claim 1, characterized in that the peptide that can be decomposed by the hydrolase consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 5 to 8.

5. 2. The composition for purifying a target protein according to claim 1, wherein the target protein is selected from the group consisting of glycoproteins, cytokines, growth factors, blood factors, vaccines, hormones, enzymes, and antibodies.

6. The target proteins include interleukin-2, blood factor VII, blood factor VIII, blood factor IX, immunoglobulin, horseradish peroxidase (HRP), cytokines, α-interferon, β-interferon, γ-interferon, colony-stimulating factor (GM-CSF), human fibronectin extra domain B (EBD), bone morphogenetic protein (BMP), fibroblast growth factor (FGF), vascular endothelial growth factor (VGF), and vascular endothelial growth factor (VGF). Growth Factor (VEGF), Nerve Growth Factor (NGF), Epidermal Growth Factor (EGF), Insulin-like Growth Factor (IGF), Transforming Growth Factors (TGF-α and -β, TGF-α, -β), Brain-Derived Neurotrophic Factor (BDNF), Platelet-Derived Growth Factor (PDGF), Placental Growth Factor (Placental Growth Factor Factor (PIGF), Hepatocyte Growth Factor (HGF), Keratinocyte Growth Factor (KGF), Glucagon-Like Peptide-1 (GLP-1), Exendin, Somatostatin, LHRH (Luteinizing Hormone-Releasing Hormone), Adrenocorticotropic Hormone, Growth Hormone-Releasing Hormone (GHRH), Hormones, Oxytocin, Thymosin α-1alpha-1), corticotropin-releasing factor, calcitonin, bivalirudin, vasopressin, phospholipase-activating protein (PLAP), insulin, tumor necrosis factor (TNF), follicle-stimulating hormone, thyroid-stimulating hormone, antidiuretic hormone, pigmented hormone, parathyroid hormone, luteinizing hormone, calcitonin gene-related peptide (CGRP), enkephalin, somatomedin, erythropoietin, hypothalamic secretory factor, prolactin, chronic gonadotropin, growth hormone-releasing peptide (GHRP) 6. The composition for purifying a target protein according to claim 5, wherein the enzyme is selected from the group consisting of arginase, arginase deaminase, arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucidase, galactosidase, glucocerebrosidase, and glucoronidase.

7. The composition for purifying a target protein according to claim 1, wherein the target protein consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 19.

8. A cell transformed with the composition for purifying a target protein according to any one of claims 1 to 7.

9. 9. The transformed cell according to claim 8, wherein the cell is selected from the group consisting of Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, Salmonella typhimurium, Serratia marcescens, Pseudomonas, yeast, insect cells, CHO cell lines (Chinese Hamster Ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN, MDCK cell lines, and plant cells.

10. A) preparing an expression vector containing a nucleic acid encoding a fusion protein in which a CSQ tag and a target protein are fused via a peptide that can be degraded by a hydrolase; B) transfecting a host cell with the expression vector to obtain a transformant; C) expressing a fusion protein containing a CSQ tag and a target protein from the transformant; D) precipitating the fusion protein containing the CSQ tag and the target protein from the expressed transformant using calcium; and E) Separating the target protein from the fusion protein using a hydrolase; A method for expressing and purifying a target protein using a CSQ tag, comprising:

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