Method for enhancing folding of disulfide-bonded protein in escherichia coli

Abstract

The present invention relates to a method for enhancing the folding of disulfide-bonded proteins in E. coli. The method for activating a disulfide-bonded protein in E coli according to the present invention enables a disulfide bond-requiring protein to be stably folded and activated in E coli without a separate refolding process by utilizing an artificial protein endowed with redox capability. Accordingly, the method allows for the efficient production of disulfide-bonded proteins, which have been difficult to express in E coli in the past, thereby providing advantages for large-scale industrial application and pharmaceutical development involving disulfide-bonded proteins.

Description

A method for enhancing the folding of disulfide proteins in Escherichia coli

[0001] The present invention relates to a method for enhancing the folding of disulfide proteins in E. coli.

[0002] Escherichia coli (E. coli) has long been one of the most widely used host systems for the production of various recombinant proteins. E. coli boasts a well-established technological infrastructure and extensive experience in genetic engineering, molecular biology, and protein engineering. Furthermore, the following advantages make E. coli an attractive expression host.

[0003] E. coli has a short generation time (typically 20-30 minutes), resulting in rapid growth and rapid mass cultivation. Optimizing expression vectors, in particular, allows for the mass production of recombinant proteins in a relatively short period of time. Its nutritional requirements are relatively simple, and growth and expression can be achieved using inexpensive media, making it economical. Furthermore, its expression and purification processes are simpler than those of other cell lines (e.g., yeast and animal cells), and there are numerous established protocols.

[0004] Furthermore, a variety of plasmid vectors, promoters, and induction systems have been developed, facilitating expression optimization. Simple transformation and recombination techniques facilitate the creation of desired variants or mutations, facilitating genetic manipulation. These features have enabled E. coli to play a crucial role in the production of a wide range of proteins, from pharmaceuticals (e.g., insulin, interferon, etc.) to industrial enzymes and research reagents (e.g., antibody fragments).

[0005] However, E. coli has several limitations, primarily due to the environmental and biological characteristics of protein expression. In particular, the following problems are frequently observed when expressing complex proteins containing disulfide bonds. Specifically, the E. coli cytoplasm is regulated to maintain a reduced state, making disulfide bond formation within proteins difficult or unstable. Incomplete disulfide bond formation can lead to protein misfolding or denaturation, making it difficult for the protein to function properly. Furthermore, highly expressed proteins that fail to fold properly often form insoluble aggregates. Aggregated proteins typically lose biological activity and require an additional denaturation-refolding process. This process is time-consuming and expensive, and can lead to reduced yields or loss of protein function under denaturing conditions.

[0006] Proteins require various auxiliary proteins (chaperones, disulfide bond isomerases, etc.) to form complex structures. However, E. coli lacks the necessary cofactors for protein folding. Therefore, its native chaperones or isomerases may not be able to properly fold certain proteins, potentially reducing the quality of the produced proteins.

[0007] That is, proteins with multiple disulfide bonds generally do not fold well within E. coli cells, and due to the reducing environment within the cell, disulfide bond proteins cannot form proper folding within E. coli cells. Therefore, in order to produce these proteins, proteins existing in insoluble inclusion bodies of E. coli are separated and subjected to a complex recombination and refolding process, or they are produced using expensive animal cells.

[0008] While E. coli is widely utilized in industry and research as a low-cost, high-efficiency protein expression system, numerous challenges remain to be overcome for the efficient production of proteins requiring disulfide bonds. Existing approaches to address these limitations (e.g., targeting the periplasm, using mutant strains with oxidative environments, and co-expression of chaperones) have shown some success, but their universal application to all types of proteins is limited.

[0009] Accordingly, the inventors of the present invention, after much effort, have developed a technology for producing a protein having multiple disulfide bonds that does not fold well in existing E. coli in a water-soluble state without a refolding process in E. coli using an artificial protein having two heme molecules, thereby completing the present invention.

[0010] One object of the present invention is to provide a signal peptide for expression of a target protein comprising any one sequence selected from the group consisting of sequence numbers 1 to 3.

[0011] Another object of the present invention is to provide a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3.

[0012] Another object of the present invention is to provide a nucleic acid construct comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide being composed of any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and a gene for the target protein.

[0013] Another object of the present invention is to provide an expression vector comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide being composed of any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and a gene for the target protein.

[0014] Another object of the present invention is to provide a host cell comprising an expression vector comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and a gene for the target protein.

[0015] Another object of the present invention is to provide a method for expressing a target protein, comprising the steps of (a) culturing a host cell according to the present invention to produce a target protein; and (b) isolating the produced target protein.

[0016] To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0017] In addition, the terminology used in the present invention is for the purpose of description only and should not be construed as limiting. The singular expression includes the plural expression unless the context clearly indicates otherwise. In the present invention, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0018] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0019] Proteins containing multiple disulfide bonds generally do not fold properly within E. coli cells. Due to the reductive environment within the cell, disulfide-bonded proteins cannot fold properly. Therefore, to produce these proteins, proteins are either isolated from insoluble inclusion bodies of E. coli and subjected to a complex reassembly and refolding process, or they are produced using expensive animal cells.

[0020] Accordingly, the inventors of the present invention developed a technology for producing a protein having multiple disulfide bonds that does not fold well in existing E. coli in a soluble state without a refolding process in E. coli by using an artificial protein having two heme molecules.

[0021] This is a method that utilizes the principle that heme molecules promote oxidation and reduction reactions in protein folding. Using this technology, various proteins that were previously expressed as insoluble inclusion bodies in E. coli, such as vascular endothelial growth factor (VEGF), stem cell growth factor protein R-spondin1 furin domain, Epidermal Growth Factor (EGF), Thymic stromal lymphopoietin (TSLP), Thymic stromal lymphopoietin Receptor ectodomain (TSLPR ectodomain), Human Serum Albumin (HSA), antibody ScFv-Fc (Single chain fragment variable-fragment crystallizable) fusion protein, and Bone morphogenetic protein 2 (BMP2), were purified into a water-soluble state, and the folding and activity of the proteins were confirmed.

[0022] Accordingly, the present invention provides a signal peptide for expression of a target protein comprising any one sequence selected from the group consisting of sequence numbers 1 to 3.

[0023] In addition, the present invention provides a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3. The sequence encoding the signal peptide for expression of the target protein may be comprised of any one nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4 to 6, but any sequence capable of expressing the signal peptide of SEQ ID NOs: 1 to 3 may be applied, and is not limited thereto.

[0024] The amino acid sequence of the signal peptide according to the above sequence numbers 1 to 3 and an exemplary nucleic acid sequence encoding the same can be referred to Table 1 below.

[0025]

[0026] In addition, as a preferred embodiment for carrying out the present invention, the present invention provides a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and a nucleic acid construct comprising a gene for the target protein.

[0027] In a more preferred embodiment, the target protein may be a protein having disulfide bonds, and specifically, a protein having 2, 3, 4, 5, 6, 7, 8, 9, 10 or more disulfide bonds.

[0028] These target proteins include any protein that can be expressed by an E. coli expression system.

[0029] As a preferred example, the target protein may be any one selected from the group consisting of VEGF (Vascular Endothelial Growth Factor), stem cell growth factor protein R-spondin1 furin domain, EGF (Epidermal Growth Factor), TSLP (Thymic stromal lymphopoietin), TSLPR ectodomain (Thymic stromal lymphopoietin Receptor ectodomain), HSA (Human Serum Albumin), antibody ScFv-Fc (Single chain Fragment variable-Fragment crystallizable) fusion protein, and BMP2 (Bone morphogenetic protein 2).

[0030] In the present invention, the 'nucleic acid structure' includes all of them, regardless of whether they are inserted into the chromosome of the host cell or located outside the chromosome, as long as they can be expressed within the host cell.

[0031] In addition, in the present invention, the 'nucleic acid (=polynucleotide)' includes DNA and RNA encoding a target protein. The nucleic acid may be introduced into a host cell in any form, as long as it can be expressed. For example, the nucleic acid may be introduced into a host cell in the form of an expression cassette, which is a genetic construct containing all elements necessary for autonomous expression. The expression cassette typically includes a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the nucleic acid. The expression cassette may be in the form of an expression vector capable of self-replication. In addition, the nucleic acid may be introduced into a host cell in its own form and operably linked to a sequence necessary for expression in the host cell.

[0032] Accordingly, as another preferred embodiment for carrying out the present invention, the present invention provides an expression vector comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of SEQ ID NOs: 1 to 3; and a gene for the target protein.

[0033] As used herein, the term "vector" refers to a DNA construct containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, may integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein.

[0034] For the purposes of the present invention, it is preferred to use a plasmid vector. A typical plasmid vector that can be used for this purpose may have a structure that includes (a) an origin of replication that allows efficient replication to include tens to hundreds of plasmid vectors per host cell, (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site that allows insertion of a foreign DNA fragment.

[0035] Even if an appropriate restriction enzyme cleavage site does not exist, the vector and foreign DNA can be easily ligated using a synthetic oligonucleotide adaptor or linker according to conventional methods.

[0036] Additionally, a gene is operably linked when it is placed in a functional relationship with another nucleic acid sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that gene expression is enabled when an appropriate molecule (e.g., a transcriptional activating protein) binds to the regulatory sequence(s). For example, DNA for a pre-sequence or a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.

[0037] These sequences are joined by ligation at convenient restriction enzyme sites. If such sites do not exist, conventional synthetic oligonucleotide adaptors or linkers can be used.

[0038] In the present invention, the term "transformation" refers to the introduction of a specific foreign DNA strand from outside the cell into the cell. A host microorganism or recombinant microorganism containing the introduced DNA strand is called a "transformed microorganism." "Transformation," which means introducing DNA into a host cell so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration, refers to introducing a vector containing a polynucleotide encoding a target protein into a host cell or integrating a polynucleotide encoding a target protein into the chromosome of the host cell so that the protein encoded by the polynucleotide can be expressed in the host cell.

[0039] The transformed polynucleotide may include any of these, whether integrated into the chromosome of the host cell or located extrachromosomally, as long as it can be expressed within the host cell.

[0040] The expression vector of the present invention may be operably linked to a promoter. As a preferred example, the expression vector may include a T7 promoter, but is not limited thereto. The promoter used according to the present invention may be a constitutive promoter or a regulatory promoter, but is not limited thereto. In addition to the promoters described above, any promoter commonly available in the art may be used without limitation. That is, the expression vector according to the present invention includes a nucleic acid construct as described above, and the nucleic acid construct is positioned after the promoter for expression, but is not designed to affect the promoter itself.

[0041] In a more preferred embodiment, the expression vector may further comprise a protease cleavage site sequence, an affinity tag sequence, or both.

[0042] For example, the protease cleavage site sequence may be any one selected from the group consisting of a TEV cleavage site sequence, a thrombin cleavage site sequence, a factor Xa cleavage site sequence, an enteropeptidase cleavage site sequence, and a rhinovirus 3C protease cleavage site sequence, but any protease cleavage site sequence commonly used in the art may be applied without limitation thereto.

[0043] The affinity tag sequence may be any one selected from the group consisting of AviTag, streptadiene-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag and Myc-tag, but any tag sequence known in the art that can easily purify an expressed protein may be applied without limitation thereto.

[0044] In addition, as another preferred embodiment for carrying out the present invention, the present invention provides a host cell comprising an expression vector according to the present invention.

[0045] Preferably, the host cell may be any one selected from the group consisting of E. coli, Corynebacterium glutamicum and Bacillus subtillis, and as an example, the E. The E. coli may be any one selected from the group consisting of BL21(DE3), BL21(DE3) RIL, BL21(DE3) RIPL, BL21 Al, HMS174(DE3), SHuffle® T7(E. coliK12, C3026J), SHuffle® T7 Express (E. coliB, C3029J), SHuffle® T7 ExpresslysY(E. coliB, C3030J), DH5ct, W31 10, B834, Origami, Rosetta, NovaBlue(DE3), Lemo21(DE3), T7, ER2566, and C43(DE3).

[0046] In the embodiment of the present invention, E. coli was used, but the present invention is not limited thereto, and any type of microorganism may be used as long as the target protein can be sufficiently expressed.

[0047] Of course, not all vectors are equally effective in expressing the DNA sequences of the present invention, and similarly, not all hosts are equally effective in the same expression system. However, those skilled in the art can appropriately select and apply various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, when selecting a vector, consideration should be given to the host, as the vector must replicate within it. The vector's copy number, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0048] The transformed host cell can be transformed according to any conventionally known method. In addition, in the present invention, a conventionally known genetic manipulation method can be used as a method for inserting the gene into the chromosome of the host cell, and in one embodiment, a method using a retroviral vector, an adenovirus vector, an adeno-associated virus vector, a herpes simplex virus vector, a poxvirus vector, a lentivirus vector, or a non-viral vector is also possible.

[0049] In addition to the method using an expression vector, the transformation method of the present invention may also use a method of directly inserting the nucleic acid construct into the chromosome of a host cell. Generally, electroporation, lipofection, ballistics, virosomes, liposomes, immunoliposomes, polycations or lipid:nucleic acid conjugates, naked DNA, artificial virons, chemically facilitated DNA introduction, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, lithium acetate-DMSO method, etc. may be used. Sonoporation, for example, a method using the Sonitron 2000 system (Rich-Mar), can also be used for nucleic acid delivery, and other representative nucleic acid delivery systems include methods from Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc. (Rockville, Maryland), and BTX Molesular Syetem (Holliston, MA). Lipofection methods are described in U.S. Patent No. 5,049,386, U.S. Patent No. 4,946,787 and U.S. Patent No. 4,897,355, and lipofection reagents are commercially available, for example, TRANSFECTAM. TM and LIPOFECTIN TMCationic or neutral lipids suitable for effective receptor-recognition lipofection of polynucleotides include Felgner's lipids (WO91 / 17424 and WO91 / 16024), which can be delivered into cells via ex vivo introduction and into target tissues via in vivo introduction.

[0050] In addition, as another preferred embodiment for carrying out the present invention, the present invention provides a method for expressing a target protein, comprising the steps of (a) culturing a host cell according to the present invention to produce a target protein; and (b) isolating the produced target protein.

[0051] In addition, the step (b) may be to separate the target protein secreted into the surrounding cytoplasm, but is not limited thereto.

[0052] If this technology is applied in the future, it will be possible to easily produce high-value-added protein drugs with numerous disulfide bonds from E. coli at relatively low cost, and thus it is expected to be used as a biopharmaceutical production technology with wide application.

[0053] The method for activating disulfide bond proteins in E. coli according to the present invention utilizes an artificial protein with redox capabilities to enable proteins requiring disulfide bonds to be stably folded and activated in E. coli without a separate refolding process, thereby efficiently producing disulfide bond proteins that were previously difficult to express in E. coli, thereby enabling large-scale industrial use of disulfide bond proteins and development of pharmaceuticals.

[0054] Figure 1 shows the results of producing VEGF-A (vascular endothelial growth factor A) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, and E: Elution after Ni-NTA).

[0055] Figure 2 shows the results of producing R-spondin1 WT furin domain protein in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, and E: Elution after Ni-NTA).

[0056] Figure 3 shows the results of analyzing the folding of VEGF-A and R-spondin 1 purine domain proteins purified after folding in E. coli.

[0057] Figure 4 shows the results of producing epidermal growth factor (EGF) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, and Sup: Supernatant after Sonication & Centrifugation).

[0058] Figure 5 shows the results of producing TSLP (thymic stromal lymphopoietin) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, and E: Elution after Ni-NTA).

[0059] Figure 6 shows the results of producing the TSLPR ectodomain (thymic stromal lymphopoietin receptor ecto-domain) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, E1: First eluate after Ni-NTA (1 st Elution after Ni-NTA) and E2: first elution after Ni-NTA (2 ndElution after Ni-NTA)).

[0060] Figure 7 shows the results of producing HSA (human serum albumin) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, E1: First eluate after Ni-NTA (1 st Elution after Ni-NTA) and E2: first elution after Ni-NTA (2 nd Elution after Ni-NTA)).

[0061] Figure 8 shows the results of producing BMP2 (bone morphogenetic protein 2) in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, and E: Elution after Ni-NTA).

[0062] Figure 9 shows the results of producing antibody ScFv-Fc fusion proteins in E. coli (BI: Before Induction, AI: After Induction, P: Pellet after Sonication & Centrifugation, Sup: Supernatant after Sonication & Centrifugation, and E: Elution after Ni-NTA).

[0063] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0064] Example 1. Production of VEGF-A (Vascular Endothelial Growth Factor A) in E. coli

[0065] 1. Preparation of protein

[0066] To linearize the pET-21a(+) vector, it was digested with NdeI / EcoRI restriction enzymes, electrophoresed on a 1.0% agarose gel, and purified using a gel extraction kit (Macherey-Nagel). Next, the 'insertion DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized for the codon of E. coliB.

[0067] Specifically, in order to express the active form of VEGF-A (Accession: P15692), only the DNA sequence for 95 amino acids from amino acid 219 (Glu) to amino acid 313 (Lys) among the sequences registered in uniprot was inserted into the target protein part of the inserted DNA and DNA synthesis was ordered.

[0068] In addition, the 'inserted DNA' was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the NdeI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0069] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0070] The sequences used for subcloning are summarized in Table 2 below.

[0071] 명칭Sequence(N term → C term or 5' → 3')서열번호4D2GSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKNGSVSPSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKN1GGATCGCCAGAACTGCGCGAGAAACACCGTGCGTTAGCCGAACAAGTGTACGCCACAGGCCAAGAAATGCTGAAGAACACGAGCAATTCGCCGGAACTTCGCGAGAAACATCGTGCTCTGGCAGAACAGGTGTATGCGACTGGCCAGGAAATGCTGAAAAACGGGTCTGTAAGTCCGTCACCTGAACTGCGGGAGAAACACCGCGCTTTGGCCGAACAGGTTTACGCAACCGGTCAGGAGATGCTCAAGAACACCTCCAATAGCCCGGAACTGCGTGAGAAACATCGCGCATTAGCGGAACAAGTCTATGCGACCGGTCAGGAAATGTTGAAAAAT4Long LinkerLEDYDIPTT7TEV cleavage siteENLYFQGS8additional sequence(Front of NdeI restriction enzyme)GAAGGAGATATA9additional sequence(Back of EcoRI restrictionenzyme)GAGCTCCGTCGA10VEGF-AMEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKH11ATGGAGGTCGTCAAGTTCATGGACGTTTATCAGCGCTCTTACTGTCATCCAATTGAGACGCTGGTCGACATCTTCCAGGAATA TCCGGACGAGATCGAATATATTTTTTAAACCATCATGCGTGCCTCTGATGCGTTGTGGGGGTTGTTGCAATGACGAGGGATTGGAGTGTGTACCGACCGAAGAATCTAATATCACAATGCAGATTATGCGTATCAAACCACATCAAGGACAACACATCGGTGAAATGTCTTTTCTTCAACACAATAAAATGCGAATGTCGCCCCAAGCAC12

[0072] 2. Cultivation of E. coli

[0073] The manufactured protein expression vector was transformed into E. coli DE3 RIL and cultured in 4 L LB medium (containing 100 ㎍ / ㎖ of antibiotic Ampicillin) at 37°C.

[0074] Cells were grown until OD600 reached 0.8, at which point protein expression was induced by adding 1 mM IPTG.

[0075] Subsequent expression was carried out by shaking culture at 18°C ​​for 17 hours, thereby ensuring stable protein production.

[0076] 3. Sonication

[0077] The expressed cells were first suspended in Lysis Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 5 mM β-Mercaptoethanol, and 25 mM Imidazole, and then disrupted by sonication.

[0078] Ultrasonic treatment was applied in a cycle of operating (ON) for 1 second and stopping (OFF) for 2 seconds under conditions of 65% amplitude within a total volume of 100 ml, and the treatment was repeated 6 times for 6 minutes.

[0079] Next, the crushed solution was centrifuged at 4°C and 13,000 g of centrifugal force for 40 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0080] 4. Protein purification

[0081] In the purification step, IMAC (Immobilized metal affinity chromatography) was performed using a Histrap HP Ni-NTA column (5 ml).

[0082] Specifically, the solution loaded onto the column was washed with 50 ml of Wash Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 5 mM β-Mercaptoethanol, and 25 mM Imidazole to remove impurities. Thereafter, the purification process was completed by eluting the protein using 150 ml of Elution Buffer in which the Imidazole concentration was increased to 275 mM in the same basic buffer.

[0083] The eluted VEGF was adjusted to a concentration of 1 mg / ml and subjected to SDS-PAGE. The protein bands on the acrylamide gel were compared to determine the final purity of the purified VEGF after staining and destaining with Coomassie blue.

[0084] As shown in Fig. 1, when experiments were performed on a vascular endothelial growth factor (VEGF) target protein with three disulfide bonds known to be expressed insoluble in E. coli, high water solubility was confirmed in E. coli, and it was easily purified.

[0085] In addition, since vascular endothelial growth factor exists as a dimer to be active, multimer formation was analyzed using the SEC-MALS technique, and it was analyzed that it had the exact molecular weight of a dimer.

[0086] Example 2. Production of R-spondin 1 purine domain in E. coli

[0087] 1. Preparation of protein

[0088] To linearize the pET-21a(+) vector, it was digested with NdeI / EcoRI restriction enzymes, electrophoresed on a 1.0% agarose gel, and purified using a gel extraction kit (Macherey-Nagel). Next, the 'insertion DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized for the codon of E. coliB.

[0089] Specifically, in order to express the active form of disulfide-bonded R-spondin 1 (Accession: Q2MKA7), only the DNA sequence for 104 amino acids from amino acid 40 (Cys) to amino acid 143 (Ser) of the R-spondin 1 purine domain among the sequences registered in uniprot was inserted into the target protein part of the inserted DNA and DNA synthesis was ordered.

[0090] In addition, the 'inserted DNA' was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the NdeI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0091] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0092] The sequences used for subcloning are summarized in Table 3 below.

[0093] 명칭Sequence(N term → C term or 5' → 3')서열번호4D2GSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKNGSVSPSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKN1GGATCGCCAGAACTGCGCGAGAAACACCGTGCGTTAGCCGAACAAGTGTACGCCACAGGCCAAGAAATGCTGAAGAACACGAGCAATTCGCCGGAACTTCGCGAGAAACATCGTGCTCTGGCAGAACAGGTGTATGCGACTGGCCAGGAAATGCTGAAAAACGGGTCTGTAAGTCCGTCACCTGAACTGCGGGAGAAACACCGCGCTTTGGCCGAACAGGTTTACGCAACCGGTCAGGAGATGCTCAAGAACACCTCCAATAGCCCGGAACTGCGTGAGAAACATCGCGCATTAGCGGAACAAGTCTATGCGACCGGTCAGGAAATGTTGAAAAAT4Long LinkerLEDYDIPTT7TEV cleavage siteENLYFQGS8additional sequence(Front of NdeI restriction enzyme)GAAGGAGATATA9additional sequence(Back of EcoRI restriction enzyme)GAGCTCCGTCGA10R-spondin 1 furindomainMCAKGCELCSEVNGCLKCSPKLFILLERNDIRQVGVCLPPSCPPGYFDARNPDMNKCIKCKIEHCEACFSHNFCTKCKEGLYLHKGRCYPACPEGSSAANGTMECS13ATGTGCGCCAAAGGCTGCGAATTGTGTTCGGAGGTAAACGGCTGCCTGAAATGCTCCCCGAAACTTTTTATCCTTTTAGAACGTAATGATATTCGTCAAGTT GGCGTCTGTTTGCCCTCTTGCCCACCTGGATACTTTGATGCGCGTAATCCCGATATGAACAAATGCATTAAGTGTAAGATTGAGCACTGTGAGGCTTGCTTCAGCCACAACTTCTGCACCAAGTGTAAGGAGGGCTTGTACCTTCACAAAGGCCGTTGTTATCCTGCATGTCCTGAAGGCTCTAGTGCAGCAAATGGGACAATGGAATGCTCG14

[0094] 2. Cultivation of E. coli

[0095] The manufactured protein expression vector was transformed into E. coli DE3 Clearcoli and cultured in 4.5 L LB medium (containing 100 ㎍ / ㎖ of antibiotic Ampicillin) at 37°C.

[0096] Cells were grown until OD600 reached 0.7, at which point protein expression was induced by adding 1 mM IPTG.

[0097] Subsequent expression was carried out by shaking culture at 20°C for 16 hours, thereby ensuring stable protein production.

[0098] 3. Sonication

[0099] The expressed cells were first suspended in Lysis Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 2 mM β-Mercaptoethanol, 25 mM Imidazole, and 1 mM PMSF, and then disrupted by sonication.

[0100] Ultrasonic treatment was performed in a cycle of operating (ON) for 1 second and stopping (OFF) for 2 seconds under conditions of 70% amplitude within a total volume of 200 ml, and the treatment was repeated 6 times for 15 minutes.

[0101] Next, the crushed solution was centrifuged at 4°C with a centrifugal force of 12,000 g for 50 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0102] 4. Protein purification

[0103] In the purification step, IMAC (Immobilized metal affinity chromatography) was performed using a Histrap HP Ni-NTA column (5 ml).

[0104] First, the solution loaded onto the column was washed with 160 mL of Wash Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 2 mM β-Mercaptoethanol, and 25 mM Imidazole to remove impurities.

[0105] Afterwards, the purification process was completed by eluting the protein using 135 ml of Elution Buffer in which the Imidazole concentration was increased to 275 mM in the same basic buffer.

[0106] The eluted protein was subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the final purity of the purified R-spondin1 furin domain.

[0107] As shown in Fig. 2, when experiments were performed on the R-spondin1 furin domain, which has eight disulfide bonds and is known to be expressed insoluble in E. coli, high water solubility was also confirmed in E. coli.

[0108] Experimental Example 1. Folding Analysis of VEGF-A Protein, R-Spondin 1 Purine Domain Protein, and HSA Protein Purified after Folding in E. coli

[0109] Thermal denaturation of proteins was measured by circular dichroism (CD) experiments using a J1500 spectropolarimeter (Jasco, Tokyo, Japan).

[0110] The protein used for structural analysis was prepared at a concentration of 0.5 mg / mL, and the sample was dissolved in a buffer solution consisting of 2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄, and 137.9 mM NaCl.

[0111] CD spectra were measured at 0.5 nm intervals in the range of 200 nm to 250 nm. The average value calculated from the measured data was expressed in the form of θ(mdeg), and the melting temperature (Tm) of the protein was determined as the midpoint between the lowest and highest θ values ​​obtained when the temperature varied from 20°C to 95°C under conditions of a wavelength of 215 nm.

[0112] As shown in Fig. 3, when CD analysis was performed to confirm folding after purification, all of the VEGF-A protein, HSA protein, and R-spondin1 protein purified after folding in E. coli showed CD results with an activated form, and subsequent thermal stability Tm analysis confirmed that folding was smoothly performed into a stabilized form of protein at 70.34°C, 75.13°C, and 70.6°C, respectively.

[0113] Example 3. Production of EGF (Epidermal Growth Factor) in E. coli

[0114] 1. Protein purification

[0115] Specifically, the pET-21a(+) vector was linearized by digestion with NdeI / EcoRI restriction enzymes, followed by electrophoresis on a 1.0% agarose gel and purification using a gel extraction kit (Macherey-Nagel). Next, the 'insertion DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized for the codon of E. coliB.

[0116] To express the active form of EGF (Accession: Q6QBS2), the DNA sequence was inserted into the target protein portion of the insert DNA and DNA synthesis was ordered.

[0117] In addition, the 'inserted DNA' was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the NdeI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0118] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0119] The sequences used for subcloning are summarized in Table 4 below.

[0120] 명칭Sequence(N term → C term or 5' → 3')서열번호4D2GSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKNGSVSPSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKN1GGATCGCCAGAACTGCGCGAGAAACACCGTGCGTTAGCCGAACAAGTGTACGCCACAGGCCAAGAAATGCTGAAGAACACGAGCAATTCGCCGGAACTTCGCGAGAAACATCGTGCTCTGGCAGAACAGGTGTATGCGACTGGCCAGGAAATGCTGAAAAACGGGTCTGTAAGTCCGTCACCTGAACTGCGGGAGAAACACCGCGCTTTGGCCGAACAGGTTTACGCAACCGGTCAGGAGATGCTCAAGAACACCTCCAATAGCCCGGAACTGCGTGAGAAACATCGCGCATTAGCGGAACAAGTCTATGCGACCGGTCAGGAAATGTTGAAAAAT4Long LinkerLEDYDIPTT7TEV cleavage siteENLYFQGS8additional sequence(Front of NdeI restriction enzyme)GAAGGAGATATA9additional sequence(Back of EcoRI restriction enzyme)GAGCTCCGTCGA10EGFNSDSECPLSHDGYCLHDGVCMYIEALDKYACNCVVGYIGERCQYRDLKWWE15AATAGTGACTCCGAGTGCCCATTGAGCCATGACGGTTATTGTCTTCACGATGGTGTGTGCATGTATATCGAAGCGCTTGACAAATATGCGTGCAACTGCGTTGTCGGCTACATCGGGGAGCGTTGTCAATACCGTGATTTGAAGTGGTGGGAA16

[0121] 2. 대장균의 배양

[0122] The manufactured protein expression vector was transformed into SHuffle®T7 Express lysY Competent E. Coli and cultured in 100 ml LB medium (containing 100 μg / ml of antibiotic Ampicillin) at 37°C.

[0123] Cells were grown until OD600 reached 0.6, at which point protein expression was induced by adding 1 mM IPTG.

[0124] Subsequent expression was carried out by shaking culture at 15°C for 17 hours, which ensured stable protein production.

[0125] 3. Sonication

[0126] The expressed cells were first suspended in Lysis Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 25 mM Imidazole, and 1 mM PMSF, and then disrupted by sonication.

[0127] Ultrasonic treatment was applied in a cycle of operating (ON) for 1 second and stopping (OFF) for 2 seconds under conditions of 35% amplitude within a total volume of 10 ml, and the treatment for 3 minutes was repeated 6 times.

[0128] Next, the crushed solution was centrifuged at 4°C with a centrifugal force of 13,000 g for 20 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0129] The supernatant was subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the solubility of the purified EGF.

[0130] 4. Protein purification

[0131] In the purification step, IMAC (Immobilized metal affinity chromatography) was performed using a Histrap HP Ni-NTA column (5 ml).

[0132] First, the solution loaded onto the column was washed with 160 mL of Wash Buffer consisting of 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10% glycerol, 2 mM β-Mercaptoethanol, and 25 mM Imidazole to remove impurities.

[0133] Afterwards, the purification process was completed by eluting the protein using 135 ml of Elution Buffer in which the Imidazole concentration was increased to 275 mM in the same basic buffer.

[0134] The eluted protein was subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the final purity of the purified R-spondin1.

[0135] As shown in Fig. 4, when experiments were performed on EGF with three disulfide bonds, which is known to be expressed insoluble in E. coli, high water solubility was also confirmed in E. coli.

[0136] Example 4. Production of TSLP (Thymic stromal lymphopoietin) and TSLPR (Thymic stromal lymphopoietin Receptor) in E. coli

[0137] 1. Preparation of protein

[0138] To linearize the pET-21a(+) vector, it was digested with NdeI / EcoRI restriction enzymes, electrophoresed on a 1.0% agarose gel, and purified using a gel extraction kit (Macherey-Nagel). Next, the 'insertion DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized for the codon of E. coliB.

[0139] Specifically, in order to express the active form of TSLP (Acceesion: Q969D9), the DNA sequence for 115 amino acids from amino acid 29 (Thy) to amino acid 143 (Ser) among the sequences registered in uniprot and the DNA sequence for 199 amino acids from amino acid 23 (Gln) to amino acid 221 (Thr) of the extracellular domain of TSLPR (Acceesion: Q9HC73) were inserted into the target protein portion of the DNA and DNA synthesis was ordered.

[0140] In addition, the 'inserted DNA' was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the NdeI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0141] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0142] The sequences used for subcloning are summarized in Table 5 below.

[0143]

[0144] 2. E. coli culture

[0145] The prepared protein expression vector was transformed into SHuffle®T7 Express lysY Competent E. Coli and cultured in 200 ml LB medium (containing 100 μg / ml of antibiotic Ampicillin) at 30°C.

[0146] Cells were grown until OD600 reached 0.6, at which point protein expression was induced by adding 1 mM IPTG.

[0147] Subsequent expression was carried out by shaking culture at 16°C for 20 hours, which ensured stable protein production.

[0148] 3. Sonication

[0149] The expressed cells were first suspended in Lysis Buffer consisting of 30 mM Tris-HCl pH7.5, 150 mM NaCl, and 5% glycerol, and then disrupted by sonication.

[0150] Ultrasonic treatment was applied in a cycle of operating (ON) for 1 second and stopping (OFF) for 3 seconds under conditions of 60% amplitude within a total volume of 10 ml, and the treatment for 2 minutes was repeated 3 times.

[0151] Next, the crushed solution was centrifuged at 4°C with a centrifugal force of 15,000 g for 60 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0152] 4. Protein purification

[0153] In the purification step, nickel affinity chromatography (Ni-NTA) was used using an open column. 1 ml of nickel beads was added to 9 ml of the supernatant, and the protein was bound to the beads at 4°C for 30 minutes before being loaded onto an open column.

[0154] The solution loaded onto the column was washed with 4.5 ml of Wash Buffer consisting of 30 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, and 25 mM Imidazole to remove impurities.

[0155] Afterwards, the purification process was completed by eluting the protein using 500*㎕ of Elution Buffer in which the Imidazole concentration was increased to 250 mM in the same basic buffer.

[0156] The eluted proteins were subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the final purity of the purified TSLP and TSLPR.

[0157] As shown in Figures 5 and 6, when experiments were performed on TSLP and TSLPR, which have three disulfide bonds and are known to be expressed insoluble in E. coli, high solubility in E. coli was also confirmed.

[0158] Example 5. Production of HSA (Human Serum Albumin) and BMP2 (Bone Morphogenetic Protein 2) in E. coli

[0159] 1. Preparation of protein

[0160] To linearize the pET-21a(+) vector, it was digested with NdeI / EcoRI restriction enzymes, electrophoresed on a 1.0% agarose gel, and purified using a gel extraction kit (Macherey-Nagel). Next, the 'insertion DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized for the codon of E. coliB.

[0161] Specifically, in order to express the active form of HSA (Accession: P02768), the DNA sequence for 591 amino acids from amino acid 19 (Arg) to amino acid 609 (Lys) among the sequences registered in uniprot and the DNA sequence for 114 amino acids from amino acid 283 (Gln) to amino acid 396 (Arg) of BMP2 (Accession: P12643) were inserted into the target protein part of the insert DNA and DNA synthesis was ordered.

[0162] In addition, the 'inserted DNA' was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the NdeI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0163] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0164] The sequences used for subcloning are summarized in Table 6 below.

[0165]

[0166] 2. Cultivation of E. coli

[0167] The prepared protein expression vector was transformed into Shuffle®T7 Express Competent E. coli and cultured in 100 ml LB medium (containing 100 μg / ml of the antibiotic Ampicillin) at 30°C.

[0168] Cells were grown until OD600 reached 0.7, at which point protein expression was induced by adding 1 mM IPTG.

[0169] Subsequent expression was carried out by shaking culture at 15°C for 17 hours, which ensured stable protein production.

[0170] 3. Sonication

[0171] The expressed cells were first suspended in Lysis Buffer consisting of 1X Dulbecco's Phosphate Buffered Saline (2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄, 137.9 mM NaCl) and then disrupted by sonication.

[0172] Ultrasonic treatment was applied in a cycle of operating (ON) for 1 second and stopping (OFF) for 2 seconds under conditions of 30% amplitude within a total volume of 200 ml, and the 2-minute treatment was repeated 6 times.

[0173] Next, the crushed solution was centrifuged at 4°C and 13,000 g of centrifugal force for 40 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0174] 4. Protein purification

[0175] In the purification step, nickel affinity chromatography (Ni-NTA) was used using an open column. 1 ml of nickel beads was added to 9 ml of the supernatant, and the protein was bound to the beads at 4°C for 30 minutes before being loaded onto an open column.

[0176] The solution loaded onto the column was washed with 25 ml of Wash Buffer consisting of 1X Dulbecco's Phosphate Buffered Saline (2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄, 137.9 mM NaCl) to remove impurities.

[0177] Afterwards, the purification process was completed by eluting the protein using 2 ml of Elution Buffer in which the Imidazole concentration was increased to 250 mM in the same basic buffer.

[0178] The eluted protein was subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the final purity of the purified HSA.

[0179] As shown in Figures 7 and 8, when experiments were performed on HSA with seventeen disulfide bonds and BMP2 with seven disulfide bonds, which are known to be expressed insoluble in E. coli, high water solubility was also confirmed in E. coli.

[0180] Example 6. Production of ScFv-Fc fusion protein (single chain fragment variable - fragment crystallizable fusion protein) in E. coli

[0181] 1. Preparation of protein

[0182] The pET-21a(+) vector, which contains a 6x His tag, a redox artificial protein (4D2), a long linker, and a recognition and cleavage site for Tev protease, was linearized by digestion with BamHI / EcoRI restriction enzymes, followed by electrophoresis on a 1.0% agarose gel and purification using a gel extraction kit (Macherey-Nagel). Then, the 'insert DNA' to be inserted into the vector was constructed, and the DNA sequence was optimized to the codon of E. coliB.

[0183] Specifically, to express ScFv-Fc, the amino acid sequence of the heavy chain portion (Fab-heavy chain) of the camel Fab (Fragment antigen-binding region) and the amino acid sequence of Fc were connected with a Glycine and Serine linker to form a single amino acid chain, and then inserted into the target protein portion of the inserted DNA to order DNA synthesis.

[0184] In addition, the 'inserted DNA' was designed to consist only of the restriction enzyme site and the target protein from the N-terminus, and when inserted into the backbone vector cut with BamHI / EcoRI restriction enzymes, it was designed to consist of a 6x His tag, a redox artificial protein (4D2), a long linker, a recognition and cleavage site of Tev protease, and the target protein from the N-terminus, just like other proteins. After designing the 5' and 3' ends of the 'inserted DNA' to include 12 additional sequences before and after the BamHI / EcoRI restriction enzyme sequences present in the backbone vector, it was synthesized using a DNA oligomer synthesis service (Integrated Dna Technologies, USA).

[0185] Afterwards, the prepared linear vector and insert DNA were recombined using an Infusion-based subcloning kit (Takara, Korea) and subcloned by transforming into E. coli. The T7 primer was used for sequencing to confirm the inserted DNA.

[0186] The sequences used for subcloning are summarized in Table 7 below.

[0187] 명칭Sequence(N term → C term or 5' → 3')서열번호4D2GSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKNGSVSPSPELREKHRALAEQVYATGQEMLKNTSNSPELREKHRALAEQVYATGQEMLKN1GGATCGCCAGAACTGCGCGAGAAACACCGTGCGTTAGCCGAACAAGTGTACGCCACAGGCCAAGAAATGCTGAAGAACACGAGCAATTCGCCGGAACTTCGCGAGAAACATCGTGCTCTGGCAGAACAGGTGTATGCGACTGGCCAGGAAATGCTGAAAAACGGGTCTGTAAGTCCGTCACCTGAACTGCGGGAGAAACACCGCGCTTTGGCCGAACAGGTTTACGCAACCGGTCAGGAGATGCTCAAGAACACCTCCAATAGCCCGGAACTGCGTGAGAAACATCGCGCATTAGCGGAACAAGTCTATGCGACCGGTCAGGAAATGTTGAAAAAT4Long LinkerLEDYDIPTT7TEV cleavage siteENLYFQGS8additional sequence(Front of BamHI restriction enzyme)CTGTATTTTCAG25additional sequence(Back of EcoRI restriction enzyme)GAGCTCCGTCGA10ScFv-Fc fusionproteinAAQPAMAEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYRMYWVRQPPGKGLEWVSAISAGGGSTYYGDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCANRAGWGMGDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSQTVVTQEPSLSVSPGGTVTLTCGLSSGSVTASNYPGWFQQTPGQAPRALIYSTNDRHSGVPSRFSGSISGNKAALTITGAQPEDEADYYCALDIGDITEFGGGTHLTVLGQPGPGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK26ACAATTCAACCTACCGCGTGGTGAGTGTGTTGACGGTATTACATCAGGATTGGTTGAACGGCAAGGAGTATAAGTGTAAAGTTTCAAATAAGGCCTTGCCAGCCCCAATTGAGAAAACTATCTCGAAAGCCAAAGGGCAGCCTCGTGAACCCCAAGTCTATACCCTGCCGCCTAGTCGTGATGAGTTGACAAAAAACCAAGTGTCCTTGACCTGCTTAGTAAAGGGCTTTTATCCATCTGACATCGCAGTGGAATGGGAGAGCAACGGTCAACCCGAAAATAACTACAAAACTACACCGCCCGTGTTGGATAGCGATGGTTCTTTCTTTTTGTACAGTAAGTTAACTGTTGACAAGTCGCGTTGGCAACAAGGCAACGTGTTCTCATGTAGTGTAATGCACGAGGCCCTGCATAACCACTACACCCAGAAATCTCTTTCGTTATCGCCAGGTAAG27

[0188] 2. 대장균의 배양

[0189] The manufactured protein expression vector was transformed into E. coli BL21 (DE3) and cultured in 500 ml LB medium (containing 100 ㎍ / ml of antibiotic Ampicillin) at 37°C.

[0190] Cells were grown until OD600 reached 0.7, at which point protein expression was induced by adding 1 mM IPTG.

[0191] Subsequent expression was carried out by shaking culture at 18°C ​​for 17 hours, which ensured stable protein production.

[0192] 3. Sonication

[0193] The expressed cells were first suspended in Lysis Buffer consisting of 1X Dulbecco's Phosphate Buffered Saline (2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄, 137.9 mM NaCl) and then disrupted by sonication.

[0194] Ultrasonic treatment was applied in a cycle of operating (ON) for 1 second and stopping (OFF) for 2 seconds under conditions of 60% amplitude within a total volume of 20 ml, and the 2-minute treatment was repeated 3 times.

[0195] Next, the crushed solution was centrifuged at 4°C with a centrifugal force of 13,000 g for 20 minutes, and the supernatant containing the dissolved protein was secured during this process.

[0196] 4. Protein purification

[0197] In the purification step, nickel affinity chromatography (Ni-NTA) was used using an open column. 1 ml of nickel beads was added to 9 ml of the supernatant, and the protein was bound to the beads at 4°C for 30 minutes before being loaded onto an open column.

[0198] The solution loaded onto the column was washed with 25 ml of Wash Buffer consisting of 1X Dulbecco's Phosphate Buffered Saline (2.67 mM KCl, 1.47 mM KH₂PO₄, 8.06 mM Na₂HPO₄, 137.9 mM NaCl) to remove impurities.

[0199] Afterwards, the purification process was completed by eluting the protein using 2 ml of Elution Buffer in which the Imidazole concentration was increased to 250 mM in the same basic buffer.

[0200] The eluted protein was subjected to SDS-PAGE, Coomassie blue staining and destaining, and the protein bands on the acrylamide gel were compared to confirm the final purity of the purified ScFv-Fc fusion protein.

[0201] As shown in Fig. 9, when experiments were performed on an ScFv-Fc fusion protein with four disulfide bonds, which is known to be expressed insoluble in E. coli, high water solubility was also confirmed in E. coli.

Claims

1. A signal peptide for expression of a target protein comprising any one sequence selected from the group consisting of sequence numbers 1 to 3.

2. A nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of sequence numbers 1 to 3.

3. In the second paragraph, the nucleic acid is a nucleic acid consisting of any one sequence selected from the group consisting of sequence numbers 4 to 6.

4. A nucleic acid structure comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide being composed of any one sequence selected from the group consisting of sequence numbers 1 to 3; and a gene for the target protein.

5. A nucleic acid structure in claim 4, wherein the target protein is a protein having a disulfide bond.

6. An expression vector comprising a nucleic acid encoding a signal peptide for expression of a target protein, the signal peptide comprising any one sequence selected from the group consisting of sequence numbers 1 to 3; and a gene for the target protein.

7. An expression vector according to claim 6, wherein the expression vector further comprises a protease cleavage site sequence, an affinity tag sequence, or both.

8. An expression vector according to claim 7, wherein the protease cleavage site sequence is any one selected from the group consisting of a TEV cleavage site sequence, a thrombin cleavage site sequence, a factor Xa cleavage site sequence, an enteropeptidase cleavage site sequence, and a rhinovirus 3C protease cleavage site sequence.

9. An expression vector in claim 7, wherein the affinity tag sequence is any one selected from the group consisting of AviTag, streptadiene-tag, polyhistidine (His6)-tag, FLAG-tag, HA-tag, and Myc-tag.

10. An expression vector according to claim 6, wherein the expression vector is operably linked to a promoter.

11. A host cell comprising an expression vector according to any one of claims 6 to 10.

12. In claim 11, the host cell is any one selected from the group consisting of E. coli, Corynebacterium glutamicum, and Bacillus subtillis.

13. In the 12th paragraph, the E. coli is a host cell selected from the group consisting of BL21(DE3), BL21(DE3) RIL, BL21(DE3) RIPL, BL21 Al, HMS174(DE3), SHuffle® T7(E. coliK12, C3026J), SHuffle® T7 Express (E. coliB, C3029J), SHuffle® T7 ExpresslysY(E. coliB, C3030J), DH5ct, W31 10, B834, Origami, Rosetta, NovaBlue(DE3), Lemo21(DE3), T7, ER2566, and C43(DE3). 14.(a) A step of culturing a host cell of any one of claims 11 to 13 to produce a target protein; and (b) A method for expressing a target protein, comprising the step of isolating the generated target protein.

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