CHO cell-derived protein secretion factor and expression vector comprising same

Abstract

The present invention relates to a CHO cell-derived protein secreting factor and an expression vector comprising the same. Specifically, the present invention relates to a CHO cell-derived protein secretion factor, an expression cassette in which a nucleic acid sequence encoding the protein secretion factor and a gene encoding a target protein are operably linked, an expression vector comprising the expression cassette, and a transformed cell into which the expression vector is introduced, and a method for producing a target protein using the transformed cell.

Description

[0001] This application is a divisional application of the application with an international filing date of December 1, 2020, international application number PCT / KR2020 / 017413, which entered the Chinese national phase on May 31, 2022, application number 202080083199.2, and invention title "CHO cell-derived protein secretion factor and expression vector containing the same". Technical Field

[0002] The present invention relates to a CHO cell-derived protein secretion factor, an expression cassette in which a nucleic acid sequence encoding the protein secretion factor and a gene encoding a target protein are operably linked, an expression vector containing the expression cassette, a transformed cell into which the expression vector is introduced, and a method for producing a target protein using the transformed cell. Background Art

[0003] For useful protein components that are difficult to obtain in vivo, recombinant proteins can be produced on a large scale using microbial or animal cell systems through genetic recombination technology. Recombinant proteins can be regulated so that they can be expressed intracellularly or secreted extracellularly. However, the disadvantage of intracellular expression is that proteins often accumulate as insoluble aggregates, and productivity is reduced due to difficulties in separation and purification. On the other hand, soluble proteins with correct protein folding can be easily obtained through extracellular secretion. Therefore, an optimized recombinant protein expression system is important for obtaining more suitable extracellular secretion in terms of protein yield and quality control.

[0004] To produce recombinant proteins, components such as host cells, genes of interest, expression vectors, selection markers, promoters, and signal peptide sequences are essential. The quality and productivity of recombinant proteins vary with the selection of these components.

[0005] Regarding the signal peptide, it is located at the N-terminal region of the recombinant protein to be produced, so it is related to the expression level of the recombinant protein and is a component that allows extracellular secretion. Depending on which signal peptide is used, differences in expression levels can be observed, and due to incorrect cleavage of the signal peptide, the signal peptide sequence may remain at the N-terminal of the protein, affecting the quality of the recombinant protein.

[0006] Therefore, it is important to select a signal peptide that does not cause incorrect cleavage and can induce high expression.

[0007] Meanwhile, most conventional signal peptides use signal peptides of human origin, and CHO cells are mainly used as expression host cells. Signal peptides can be used in combination between host cells, but quality problems may occur.

[0008] In these cases, the inventors of the present invention have made many efforts to improve the expression level in CHO cells and solve the problem of incorrect cleavage. As a result, they have developed a new signal peptide, which is a polypeptide composed of the amino acid sequences at positions 17 to 31 derived from CHO cells, and have completed the present invention by confirming that the signal peptide can significantly improve expression and is cleaved 100% at the cleavage site to prevent incorrect cleavage. Summary of the Invention

[0009] [Technical Problem]

[0010] An object of the present invention is to provide a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0011] Another object of the present invention is to provide an expression cassette in which a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked.

[0012] Another object of the present invention is to provide an expression vector for secreting a target protein, the expression vector comprising an expression cassette in which a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked.

[0013] Another object of the present invention is to provide a transformed cell in which the expression vector is introduced into a host cell.

[0014] Another object of the present invention is to provide a method for producing a target protein, the method comprising:

[0015] i) culturing a transformed cell comprising an expression vector for secreting a target protein, the expression vector comprising an expression cassette in which a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked; and

[0016] ii) recovering the target protein from the culture medium or culture supernatant of the cultured cells.

[0017] [Technical Solution]

[0018] The present invention will be described in more detail hereinafter. Meanwhile, each of the explanations and exemplary embodiments disclosed herein can be applied to other explanations and exemplary embodiments. That is, all combinations of the various different factors disclosed herein fall within the scope of the present invention. In addition, the scope of the present invention should not be limited by the specific disclosures provided hereinafter.

[0019] In addition, those of ordinary skill in the art may recognize or confirm many equivalents of specific aspects of the present invention described herein using only routine experimentation. In addition, these equivalents are also intended to be included in the present invention.

[0020] To achieve the above object, one aspect of the present invention provides a new protein secretion factor derived from CHO cells. Specifically, the present invention provides a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. More specifically, the protein secretion factor may consist of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto.

[0021] The "protein secretion factor" of the present invention refers to a factor that is linked to a target protein to induce extracellular secretion of the target protein and may consist of a polypeptide. The protein secretion factor can promote the secretion of the target protein as an endogenous protein and / or an exogenous protein, and specifically can promote the extracellular secretion of the light chain and / or heavy chain of an antibody, but is not limited thereto.

[0022] The protein secretion factor in the present invention can be used interchangeably with a "signal sequence" or "signal peptide (SP)".

[0023] The protein secretion factor of the present invention may have the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, but is not limited thereto. In addition, the protein secretion factor of the present invention may also include a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10, but is not limited thereto.

[0024] In a specific embodiment of the present invention, the protein secretion factor may be derived from CHO cells, but is not limited thereto. As used herein, the term "CHO cell" is a Chinese hamster ovary cell and can be a host cell commonly used for transformation in the art. In addition, the protein secretion factor derived from CHO cells can be selected to improve the expression level in CHO cells as host cells.

[0025] In the present invention, the protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1 may be cathepsin B (Cat) and can be used interchangeably with the Cat secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 2 may be C-C motif chemokine (CC) and can be used interchangeably with the CC secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 3 may be nucleolin-2 (NUC) and can be used interchangeably with the NUC secretion sequence in the present invention.

[0026] In addition, the protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 4 in the present invention may be clusterin (Clus) and can be used interchangeably with the Clus secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 5 may be pigment epithelium-derived factor (Pig) and can be used interchangeably with the Pig secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 6 may be procollagen C-endopeptidase enhancer 1 (Proco) and can be used interchangeably with the Proco secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 7 may be thiol oxidase (Sulf) and can be used interchangeably with the Sulf secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 8 may be lipoprotein lipase (Lip) and can be used interchangeably with the Lip secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 9 may be nestin-1 (Nid) and can be used interchangeably with the Nid secretion sequence in the present invention. The protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 10 may be protein disulfide isomerase (Pro) and can be used interchangeably with the Pro secretion sequence in the present invention.

[0027] The nucleic acid sequence encoding the cathepsin B signal peptide consisting of the amino acid sequence of SEQ ID NO: 1 may be the polynucleotide sequence of SEQ ID NO: 11, the nucleic acid sequence encoding the C-C motif chemokine signal peptide consisting of the amino acid sequence of SEQ ID NO: 2 may be the polynucleotide sequence of SEQ ID NO: 12, and the nucleic acid sequence encoding the nucleolin-2 signal peptide consisting of the amino acid sequence of SEQ ID NO: 3 may be the polynucleotide sequence of SEQ ID NO: 13.

[0028] In addition, the nucleic acid sequence encoding the clusterin signal peptide consisting of the amino acid sequence of SEQ ID NO: 4 may be the polynucleotide sequence of SEQ ID NO: 14, the nucleic acid sequence encoding the pigment epithelium-derived factor (Pig) signal peptide consisting of the amino acid sequence of SEQ ID NO: 5 may be the polynucleotide sequence of SEQ ID NO: 15, the nucleic acid sequence encoding the procollagen C-endopeptidase enhancer 1 (Proco) signal peptide consisting of the amino acid sequence of SEQ ID NO: 6 may be the polynucleotide sequence of SEQ ID NO: 16, the nucleic acid sequence encoding the sulfhydryl oxidase (Sulf) signal peptide consisting of the amino acid sequence of SEQ ID NO: 7 may be the polynucleotide sequence of SEQ ID NO: 17, the nucleic acid sequence encoding the lipoprotein lipase (Lip) signal peptide consisting of the amino acid sequence of SEQ ID NO: 8 may be the polynucleotide sequence of SEQ ID NO: 18, the nucleic acid sequence encoding the nestin-1 (Nid) signal peptide consisting of the amino acid sequence of SEQ ID NO: 9 may be the polynucleotide sequence of SEQ ID NO: 19, and the nucleic acid sequence encoding the protein disulfide isomerase (Pro) signal peptide consisting of the amino acid sequence of SEQ ID NO: 10 may be the polynucleotide sequence of SEQ ID NO: 20.

[0029] Although the protein secretion factor of the present invention is described as a "secretion factor consisting of a specific amino acid sequence", it is obvious that as long as the secretion factor has the same or corresponding activity as the secretion factor consisting of the amino acid sequence of the corresponding serial number, it does not exclude mutations that may occur through the addition of nonsense sequences upstream or downstream of the amino acid sequence, mutations that may occur naturally, or silent mutations thereof. Even if such sequence addition or mutation exists, it falls within the scope of the present invention.

[0030] For example, as long as a secreted factor that is identical or corresponding to a nucleic acid molecule composed of the polynucleotide can function as a signal peptide, nucleic acid sequences having a homology and / or identity of 85% or higher, particularly 90% or higher, more particularly 95% or higher, even more particularly 98% or higher or even more particularly 99% or higher with the above sequences can also be included in the present invention without limitation. In addition, it is obvious that nucleic acid sequences having deletions, modifications, substitutions or additions in a part of the sequences can also be included within the scope of the present invention as long as the nucleic acid sequences have such homology.

[0031] As used herein, the terms "homology" or "identity" refer to the degree of relatedness between two given amino acid sequences or nucleic acid sequences and can be expressed as a percentage. The terms "homology" and "identity" are generally used interchangeably with each other.

[0032] The sequence homology or identity of a conserved polynucleotide or polypeptide sequence can be determined by standard alignment algorithms and can be used with the default gap penalties established by the program used. Basically, it is generally expected that homologous or identical sequences hybridize with all or at least about 50%, 60%, 70%, 80% or 90% or more of the full length of the sequence under medium or high stringency conditions. Polynucleotides containing degenerate codons instead of the codons in the hybridizing polypeptide are also contemplated.

[0033] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by known computer algorithms such as the "FASTA" program (Pearson et al., (1988) [Proc. Natl. Acad. Sci. USA 85]: 2444), using default parameters. Alternatively, it can be determined by the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453), which is carried out using the Needleman program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.0.0 or subsequent versions), or using the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S.F. et al., J MOLEC BIOL 215]: 403 (1990); Guide to Huge Computers, edited by Martin J. Bishop, Academic Press, San Diego, 1994 and [CARILLO et al., (1988) SIAM J Applied Math 48: 1073). For example, homology, similarity or identity can be determined using BLAST or ClustalW of the National Center for Biotechnology Information (NCBI).

[0034] The homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, the GAP computer program, such as Needleman et al., (1970), J Mol Biol. 48: 443, as disclosed in Smith and Waterman, Adv. Appl. Math (1981) 2:482. Generally speaking, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of symbols that are similar and aligned (i.e., nucleotides or amino acids) by the total number of symbols in the shorter of the two sequences. The default parameters for the GAP program can include (1) a unitary comparison matrix (with values of 1 for identities and 0 for non-identities) and the weighted comparison matrix of Gribskov et al., (1986), Nucl. Acids Res. 14:6745, as disclosed in Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358 (1979) (or the EDNAFULL substitution matrix (EMBOSS version of NCBI NUC4.4)); (2) a penalty of 3.0 for each gap and an additional penalty of 0.10 for each symbol in the gap (or a gap opening penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for terminal gaps. Thus, as used herein, the term "homology" or "identity" refers to the relatedness between sequences.

[0035] Another aspect of the present invention provides an expression cassette, wherein a nucleic acid sequence encoding a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked.

[0036] The "protein secretion factor" of the present invention is as described above.

[0037] As used herein, the term "target protein" can refer to a protein that is endogenously expressed in a host cell or a protein expressed by an exogenous gene introduced into it. There is no particular limitation on the type of target protein, as long as the extracellular secretion efficiency is improved by the signal peptide sequence of the present invention.

[0038] The target protein can be an antibody, antibody fragment (Fab or ScFv), fusion protein, protein scaffold, human growth hormone, serum protein, immunoglobulin, cytokine, α-, β- or γ-interferon, granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), phospholipase-activating protein (PLAP), insulin, tumor necrosis factor (TNF), growth factor, hormone, calcitonin, calcitonin gene-related peptide (CGRP), enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, growth differentiation factor, cell adhesion protein, prolactin, chorionic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase or glucuronidase, and specifically, it can be the heavy chain protein or light chain protein of an antibody, but is not limited thereto.

[0039] As used herein, the term "operably linked" refers to the functional linkage between the above gene sequence, promoter sequence and signal peptide sequence to initiate and mediate the transcription of the nucleic acid sequence encoding the protein secretion factor of the present application and the gene encoding the target protein. The operable linkage can be prepared using gene recombination techniques known in the art, and site-specific DNA ligation can be prepared using ligases known in the art, but is not limited thereto.

[0040] As used herein, the term "expression cassette" refers to a sequence that regulates the expression of one or more genes, such as a nucleic acid sequence comprising any combination of various different cis-acting transcriptional regulatory elements. The expression cassette of the present invention can also include various different elements, such as nucleic acid sequences necessary for expression regulation recognized in the art, such as promoters and enhancers, as well as nucleic acid sequences encoding protein secretion factors and target proteins. The sequence that regulates the expression of a gene, that is, the sequence that regulates the transcription of the gene and the expression of its transcription product, is usually referred to as a "regulatory unit". Most regulatory units are located upstream of the coding sequence of the target gene so that it is operably linked to the coding sequence. In addition, the expression cassette can include a 3' untranslated region, which includes a polyadenylation site at the 3' end.

[0041] The expression cassette of the present invention can be a combination of polynucleotides in which a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked, allowing extracellular secretion and expression of the target protein in a host cell.

[0042] Another aspect of the present invention provides an expression cassette in which a gene encoding a target protein is operably linked to a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 11 to SEQ ID NO: 20.

[0043] The "protein secretion factor", "target protein", "operably linked" and "expression cassette" of the present invention are as described above.

[0044] In the present invention, the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 11 can be cathepsin B (Cat), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 12 can be C-C motif chemokine (CC), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 13 can be nucleolin-2 (Nuc), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 14 can be clusterin (Clus), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 15 can be pigment epithelium-derived factor (Pig), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 16 can be procollagen C-endopeptidase enhancer 1 (Proco), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 17 can be thiol oxidase (Sulf), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 18 can be lipoprotein lipase (Lip), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 19 can be nestin-1 (Nid), and the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 20 can be protein disulfide isomerase (Pro).

[0045] Another aspect of the present invention provides an expression vector for secreting a target protein, the expression vector comprising an expression cassette in which a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein are operably linked.

[0046] The "protein secretion factor", "target protein", "operably linked", and "expression cassette" of the present invention are as described above.

[0047] As used herein, the term "expression vector for secreting a target protein" refers to an expression vector in which the protein secretion factor and the gene encoding the target protein are operably linked so that when the vector is introduced into a host cell and expressed therein, extracellular secretion of the target protein is induced.

[0048] As used herein, the term "expression vector" generally refers to a double-stranded DNA fragment that serves as a vector into which a target DNA fragment encoding a target protein is inserted. Expression vectors used in the art for expressing proteins can be used without limitation. Once the expression vector enters a host cell, the expression vector can replicate independently of the host chromosomal DNA and can express the inserted target DNA. In order to increase the expression level of the transfected gene in the host cell, the transfected gene must be operably linked to transcriptional and translational control sequences that function in the selected expression host cell.

[0049] There is no particular limitation on the expression vector used in the present invention as long as it can replicate in a host cell, and any vector known in the art can be used. Examples of commonly used vectors can include natural or recombinant plasmids, cosmids, viruses, and phages. For example, as phage vectors or cosmid vectors, pWE15, M13, λMBL3, λMBL4, λⅨII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A, etc. can be used, while as plasmid vectors, vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used. Specifically, the vector can be a pTZ-based vector, but is not limited thereto.

[0050] In a specific embodiment of the present invention, the expression vector for secreting a target protein is prepared by operably linking a nucleic acid sequence encoding a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 to a gene encoding a target protein on the basis of the pTz-D1G1 vector (a variant of the promoter including Korean Patent No. 10-1038126) (Example 4).

[0051] The expression vector may further include a nucleic acid sequence encoding a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0052] Another aspect of the present invention provides a transformed cell, wherein the expression vector is introduced into a host cell.

[0053] The "expression vector" of the present invention is as described above.

[0054] As used herein, the term "transformation" refers to the process of introducing a vector comprising a polynucleotide encoding a target polypeptide into a host cell, thereby allowing the protein encoded by the polynucleotide to be expressed in the host cell.

[0055] As long as the transformed polynucleotide can be expressed in the host cell, it does not matter whether it is inserted into and located within the chromosome of the host cell or is extrachromosomal; both situations can be included. In addition, the polynucleotide includes DNA and RNA encoding the target polypeptide. The polynucleotide can be introduced in any form as long as it can be introduced into the host cell and expressed therein. For example, the polynucleotide can be introduced into the host cell in the form of an expression cassette, which is a gene construct comprising all the elements necessary for its own expression. The expression cassette can conventionally include a promoter operably linked to the polynucleotide, a transcription termination signal, a ribosome binding domain, and a translation termination signal.

[0056] The method of transforming the vector of the present invention includes any method of introducing nucleic acid into a cell and can be carried out by selecting suitable standard techniques known in the art according to the host cell. For example, transformation can be carried out by particle bombardment, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) technology, DEAE-dextran technology, cationic liposome technology, lithium acetate-DMSO technology, but the method is not limited thereto.

[0057] As used herein, the term "host cell" refers to a eukaryotic cell into which a nucleic acid molecule having the activity of the protein secretion factor of the present invention is introduced and which can act as a signal peptide therein. The host cell can include, for example, well-known eukaryotic hosts such as yeast, insect cells such as Spodoptera frupperda, and animal cells such as CHO, COS1, COS7, BSC1, BSC40, and BMT10, but is not limited thereto.

[0058] In the present invention, an example of the host cell can be an animal host cell, and specifically, it can be a Chinese hamster ovary cell (CHO cell), but is not limited thereto.

[0059] In a specific embodiment of the present invention, Chinese hamster ovary (CHO) cells widely used in the production of recombinant proteins are used as host cells (Example 4).

[0060] As used herein, the term "transformant" refers to a transformed animal cell, which includes an expression vector consisting of a signal peptide and a target protein composed of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5, and is introduced into CHO cells as host cells.

[0061] In a specific embodiment of the present invention, it was confirmed that the transformant increased the expression levels of the light chain and heavy chain of pembrolizumab (i.e., an antibody) as the target protein (Example 4).

[0062] Another aspect of the present invention provides a method for producing a target protein, the method comprising:

[0063] i) culturing a transformed cell comprising an expression vector for secreting a target protein, the expression vector comprising an expression cassette, wherein a nucleic acid sequence encoding a protein secretion factor composed of the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 and a gene encoding the target protein are operably linked; and

[0064] ii) recovering the target protein from the culture medium or culture supernatant of the cultured cells.

[0065] The "protein secretion factor", "target protein", "operably linked", "expression cassette", "expression vector for secreting a target protein", "host cell", and "transformant" of the present invention are as described above.

[0066] As used herein, the term "culturing" refers to the process of growing transformed cells under suitable artificially controlled environmental conditions. In the present invention, the method for producing a target protein using CHO cells as host cells can be carried out using methods widely known in the art. Specifically, the culturing can be carried out continuously by a batch process, a fed-batch process, or a repeated fed-batch process, but is not limited thereto.

[0067] The culture medium for culturing should meet the requirements of a specific cell line in a suitable manner. The carbon sources that can be used in the present invention may include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil, fatty acids such as palmitic acid, stearic acid, and linoleic acid, alcohols such as ethanol, and organic acids such as gluconic acid, acetic acid, and pyruvic acid, but are not limited thereto. These substances can be used alone or in combination.

[0068] The nitrogen sources that can be used in the present invention may include peptone, yeast extract, meat extract, malt extract, corn steep liquor, defatted soybean cake, and urea or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate, but are not limited thereto. These nitrogen sources can also be used alone or in combination.

[0069] The phosphorus sources that can be used in the present invention may include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts, but are not limited thereto. In addition, the culture medium may contain metal salts required for growth, such as magnesium sulfate or iron sulfate. Finally, in addition to the above substances, growth essential substances such as amino acids and vitamins can also be used. In addition, suitable precursors can be used in the culture medium. These substances can be appropriately added to the culture medium in a batch or continuous manner during the culture.

[0070] An alkaline compound such as sodium hydroxide, potassium hydroxide, or ammonia water or an acidic compound such as phosphoric acid or sulfuric acid can be added to the culture medium in a suitable manner to adjust the pH of the culture medium. In addition, an antifoaming agent such as polyethylene glycol fatty acid ester can be used to inhibit the formation of foam. In order to maintain the culture medium in an aerobic state, oxygen or an oxygen-containing gas can be injected into the culture medium. The temperature of the culture medium can generally be 20 °C to 45 °C, preferably 25 °C to 40 °C, but can be changed according to conditions and is not limited thereto.

[0071] In a specific embodiment of the present invention, the recombinant expression vectors (i.e., pCB-SP7.2-Pem, pCB-Clus-Pem, pCB-Pig-Pem, and pCB-CC-Pem) are introduced into CHO host cells (ExpiCHO-S TM cells) and cultured for 12 days by a fed-batch culture method in 30 mL of ExpiCHO expression medium (CHO expression medium) (Example 4).

[0072] The method for producing a target protein of the present invention may include the step of recovering the target protein from the culture medium. As used herein, the term "recovery" is the process of obtaining the target protein from the culture medium, and can be carried out using methods known in the art such as centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc., but the method is not limited thereto.

[0073] The recovery step may include a purification process, and those skilled in the art can select and use among various different known purification processes as needed. For example, the host cells can be separated from the culture medium or the culture supernatant of the host cells by conventional chromatography methods such as immunoaffinity chromatography, receptor affinity chromatography, hydrophobic interaction chromatography, lectin affinity chromatography, size exclusion chromatography, cation or anion exchange chromatography, high performance liquid chromatography (HPLC), and reverse phase HPLC. In addition, when the desired protein is a fusion protein with a specific tag, marker, or chelating component, it can be purified by a specific binding ligand or drug. The purified protein can be cleaved into the desired protein region, for example, removing the secretion factor, or it can remain as it is. The desired form of the protein including additional amino acids can be produced by cleaving the fusion protein during the cleavage process.

[0074] The protein secretion factor of the present invention consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5 can be a secretion factor that is accurately cleaved at the N-terminal cleavage site of the target protein.

[0075] The signal peptide is located at the N-terminal region of the recombinant protein to be produced, and when the target protein is transported, the signal peptide is degraded by signal peptidase. However, due to the problem of incorrect cleavage, existing protein secretion factors often may reduce the quality of the target protein. The "incorrect cleavage" refers to the phenomenon that the signal peptide is not completely degraded at the correct position, and a part of the signal peptide sequence remains at the N-terminal of the target protein.

[0076] In a specific embodiment of the present invention, using the purified target protein, the cleavage of the protein secretion factor (signal peptide) was confirmed by a Q-TOF MS mass spectrometer. As a result, it was confirmed that 100% cleavage was observed at the predicted cleavage site.

[0077] Therefore, the expression vector containing the protein secretion factor (signal peptide) of the present invention improves the productivity of the target protein through the high-efficiency expression and secretion of the recombinant protein, and can be a powerful genetic tool for solving the problem of incorrect cleavage.

[0078] [Advantageous Effects]

[0079] The protein secretion factor, i.e., the signal peptide, of the present invention can significantly improve the productivity of the recombinant protein through high-level expression, and is expected to be used as a powerful genetic tool, which can solve the problem of incorrect cleavage of conventional signal peptides by 100% cleavage at the cleavage site. Brief Description of the Drawings

[0080] Figure 1 is a diagram for predicting the signal peptide using SignalP4.1.

[0081] Figure 2 This is a figure for confirming the expression level of signal peptide-mCherry through transient expression.

[0082] Figure 3 This is a figure showing the vector map for site-specific integration.

[0083] Figure 4 This is a figure comparing the expression levels of mCherry in site-specific integration cells.

[0084] Figure 5 is a figure showing mass data, confirming the cleavage of the anti-PD-1 antibody fused with SP7.2 and Clus. Detailed Description of the Invention

[0085] Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are provided for illustrative purposes only, and the scope of the present invention is not intended to be limited to or by these examples.

[0086] Example 1. Preparation of a New Signal Peptide Sequence Derived from CHO Cells

[0087] 1-1. CHO HCP Quality Analysis

[0088] Four types (ADH, BSA, PHO, and ENL) of MassPREPTM protein digest standards were added to the CHO cell culture medium (DXB11) treated with trypsin as described below. Among the four types of MassPREPTM protein digest standards, PHO was used as an internal standard for calculating the concentration of each host cell protein (HCP). The host cell protein (HCP) of each sample was analyzed using the 2D LC (high pH RP / low pH RP)-Q-TOF (UDMSe) method.

[0089] For each fraction, MS data (UDMSe) was obtained by directly injecting it into the 2D column in the Q-TOF MS. The MS data (UDMSe) of 10 fractions obtained in the above manner were combined into one data using ProteinLynx Global Server (PLGS, Ver. 3.0.2) software. Then, the HCP was identified using the combined data of each sample and the Chinese hamster protein database, and the concentration of each HCP was calculated using PHO as an internal standard.

[0090] 1-2. Selection of Signal Peptide from CHO HCP Data

[0091] The proteins were arranged in descending order of concentration, and from the CHO genomic database ( http: / / chogenome.org)Confirm the amino acid sequence of each protein. Input the obtained amino acid sequences into the SignalP 4.1 server (http: / / www.cbs.dtu.dk / services / SignalP / ) to predict the presence of secreted proteins and signal peptide sequences (Table 1, Figure 1, and Table 4).

[0092]

[0093] Example 2. Selection of highly efficient CHO-derived signal peptides by transient expression

[0094] 2-1. Preparation of recombinant protein expression vectors for transient expression

[0095] To confirm whether the 10 signal peptides selected in Example 1 can be used as general secretion factors, mCherry (pmCherry vector, Clontech, 632522) protein was selected as the target protein.

[0096] The polynucleotide sequence of the gene encoding the mCherry protein is shown in Table 2.

[0097]

[0098] Based on the gene encoding the mCherry protein, PCR was performed using the signal peptide sequences identified from the CHO HCP quality data and primers containing KpnI / XhoI, and mCherry expressed by 10 signal peptide sequences was constructed.

[0099] When the length of the signal peptide is long, the primers are divided into two, and PCR is performed twice. The mCherry PCR products containing 10 signal peptide sequences are digested with KpnI and XhoI, and then cloned into pcDNA3.1(+) (Invitrogen, catalog number V790-20) to construct an expression vector.

[0100] In addition, to be used as a positive control, an mCherry protein expression vector fused with the SP7.2 signal peptide (Korean Patent Publication No. 10-2015-0125402 A), which is a known secretion factor, was prepared in the same manner. The sequence of the SP7.2 signal peptide is shown in Table 3.

[0101]

[0102]

[0103] 2-2. Transient expression

[0104] Each mCherry expression vector expressed by 10 signal peptides was transfected (1 mL) according to the CHO-S cell line Amaxa 4D-nucleofector protocol. Then, on days 2 and 6, the intracellular fluorescence and the fluorescence value of the fluorescent protein secreted into the medium were measured ( Figure 2 ).

[0105] In the case of intracellular fluorescence, FACS (Accuri) was used to measure the average value of the part represented by a histogram that was higher than the negative control (empty vector, pMaxGFP).

[0106] In the case of the fluorescence value of the fluorescent protein secreted into the medium, 100 μL was sampled on days 2 and 6, then centrifuged to obtain only the supernatant, and the fluorescence was measured at 587 / 610 nm using a multi-wavelength microplate reader.

[0107] It was confirmed that the signal peptides with high fluorescence values measured in the medium were Cat, CC, Nuc, Clus, and Pig. In addition, four secretory factors (Clus, Pig, Nuc, and CC) showing higher expression than the positive control SP7.2 (SP7.2 was used as the positive control) and one signal peptide (Proco) with a high fluorescence value in the cells were selected as negative controls.

[0108] Example 3. Comparison of expression by site-specific integration

[0109] 3-1. Construction of expression vectors for site-specific integration

[0110] The mCherry sequences including the 5 signal peptides (Clus, Pig, Nuc, CC, and Proco) selected in Example 2 and the control SP7.2 were consistently inserted into specific sites of the CHO genome to quantitatively compare the expression levels ( Figure 3 and 4 ).

[0111] The insertion site is set at the Hprt locus, and the homologous arm sequences and sgRNA sequences are designed with reference to J.S Lee et al., 2015, "Site-Specific integration in CHO Cells mediated by CRISPR / Cas9 and homology-directed DNA repair pathway", Sci. Rep., 5.

[0112] In the case of the 5' homologous arm, PCR was performed using primers containing Bg1Ⅱ and NruI enzyme sites and the CHO-S genome as a template. Then, it was cloned into the pcDNA3.1(+) vector digested with Bg1II / NruI.

[0113] In the case of the 3' homologous arm, PCR was performed using each primer containing a SalI site and the CHO-S genome as a template, and then it was singly digested with SalI together with the vector inserted with the 5' homologous arm, and it was cloned downstream of the NeoR gene (pcDNA3.1_hprt).

[0114] To confirm that only those fragments that have undergone homologous recombination are inserted into the genome, a Cmy-GFP-BHG pA fragment was constructed in the upstream region of the 5' homologous arm and inserted into SpeI and Bg1Ⅱ (pcDNA3.1_G_hprt) for dual selection.

[0115] In the case of the GFP fragment, it was first inserted into the MCS of the pcDNA3.1(+) vector using NcoI / XbaI, and then PCR was performed using primers containing SpeI and Bg1II restriction sites. Then, SpeI was inserted into the vector containing the homologous region (pcDNA_hprt) using the Bg1II site.

[0116] Using the completed pcDNA3.1_G_hprt vector, the mCherry gene sequence containing the signal peptide sequence was cut off with KpnI and XhoI and inserted into the MCS region.

[0117] As a result, a pcDNA3.1-based expression vector containing an expression cassette was constructed, and the expression cassette takes the form of CMV-EGFP-pA-5' Hprt homologous arm-CMV-signal peptide candidate-mCherry-BGH pA-NeoR selection marker cassette-3'Hprt homologous arm.

[0118] 3-2. Site-Specific Integration

[0119] Knock-in was performed using CRISPR-Cas9 to insert six types of vectors for site-specific integration at the Hprt locus into the CHO-S genome. 240 ng of sgRNA, 1,250 ng of cas9 protein, and 1 µg of donor vector were independently mixed with the nucleofection solution to prepare a 50 µL mixture.

[0120] First, 1x10 6 CHO-S cells were lysed in 50 µL of nucleofection reagent, then mixed with the previously prepared mixture, and then the final 100 µL mixture was electroporated.

[0121] After electroporation, the mixture was mixed with 0.5 mL of medium, added to 2.5 mL of medium, and cultured in a 6-well plate at 36.5 °C and 5% CO2.

[0122] After 2 days, selection was carried out in CD CHO medium containing Zeneticin (0.5 mg / L), and then the cells were passaged until 90% viability was restored.

[0123] After 90% viability was restored, 4 mL of cells were cultured in duplicate in a 6-well plate at a concentration of 3x10 5 cells / mL, and the fluorescence values (587 nm / 610 nm) of Vi-Cell and the medium were measured every 2 to 3 days ( Figure 4 ).

[0124] As a result, it was confirmed that the CC, Clus, and Pig secretion peptides showed higher expression compared to the control SP7.2.

[0125] Example 4. Expression and Quality Analysis of Anti-PD-1 Antibody

[0126] 4-1. Preparation of Expression Vector for Producing Anti-PD-1 Antibody

[0127] After comparing the expression ability of signal peptides by site-specific integration, anti-PD-1 antibodies fused with four types of signal peptides (CC, Pig, Clus, and SP7.2) including those showing high expression were expressed. The pembrolizumab (Keytruda®) antibody sequence was used as the target protein.

[0128] DNA sequences corresponding to the amino acid sequences of the light and heavy chains were synthesized, and then sequences fused with each signal peptide sequence were generated by overlap PCR.

[0129] In the case of the light chain, the amino acid sequence was restricted with BamHI and XhoI, and in the case of the heavy chain, the amino acid sequence was restricted with AscI and NotI, and then the antibody was inserted into the pTz-D1G1 vector, which is a variant of pcDNA3.1(+) (including the promoter of Korean Patent No. 10-1038126B1).

[0130] pCB_SP7.2_Pem

[0131] '(N-terminus) - [BamHI restriction site - signal peptide (SEQ ID NO: 33) - Pem light chain (SEQ ID NO: 58) - XhoI restriction site] - (C-terminus)' / '(N-terminus) - [AscI restriction site - signal peptide (SEQ ID NO: 33) - Pem heavy chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'

[0132] pCB_Clus_Pem

[0133] '(N-terminus) - [BamHI restriction site - signal peptide (SEQ ID NO: 4) - Pem light chain (SEQ ID NO: 58) - XhoI restriction site] - (C-terminus)' / '(N-terminus) - [AscI restriction site - signal peptide (SEQ ID NO: 4) - Pem heavy chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'

[0134] pCB_CC_Pem

[0135] '(N-terminus) - [BamHI restriction site - signal peptide (SEQ ID NO: 2) - Pem light chain (SEQ ID NO: 58) - XhoI restriction site] - (C-terminus)' / '(N-terminus) - [AscI restriction site - signal peptide (SEQ ID NO: 2) - Pem heavy chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'

[0136] pCB_Pig_Pem

[0137] '(N - terminus) - [BamHI restriction site - signal peptide (SEQ ID NO: 5) - Pem light chain (SEQ ID NO: 58) - XhoI restriction site] - (C - terminus)' / '(N - terminus) - [AscI restriction site - signal peptide (SEQ ID NO: 5) - Pem heavy chain (SEQ ID NO: 59) - NotI restriction site] - (C - terminus)'

[0138] 4 - 2. Expression of anti - PD1 antibody

[0139] The prepared recombinant expression vectors pCB - SP7.2 - Pem, pCB - Clus - Pem, pCB - Pig - Pem, and pCB - CC - Pem were introduced into ExpiCHO - S TM cells (Thermo Fisher Scientific), and cultured in ExpiCHO expression medium (Thermo Fisher Scientific; 30 mL) for 12 days (fed - batch culture; feeding on day 1 and day 5) to express the fusion polypeptide (i.e., pembrolizumab).

[0140] 4 - 3. Purification and quality analysis of anti - PD1 antibody

[0141] The fusion polypeptide produced by the expression of the recombinant vector was purified by Protein A. Specifically, the recovered culture broth was filtered through a 0.22 μm filter, and then a column packed with Protein A resin (Hitrap MSS, GE Healthcare, 11 - 0034 - 93) was installed on AKTA TM Avant25 (GE Healthcare Life Sciences), and PBS buffer was passed through to equilibrate the column.

[0142] After injecting the filtered culture broth into the column, PBS buffer was passed through again to wash the column. After the column washing was completed, the elution buffer (citrate buffer, pH 3.5) was passed through the column to elute the target protein. The eluate was concentrated using an Amicon Ultra filtration device (MWCO 30K, Merck) and a centrifuge. After concentration, buffer exchange was performed using PBS.

[0143] The quantitative analysis of the fusion polypeptide was performed by measuring the absorbance values at 280 nm and 340 nm using a UV spectrophotometer (G113A, Agilent Technologies), and using the following calculation formula. The extinction coefficient of each material is the theoretical value (1.404) calculated using the amino acid sequence.

[0144]

[0145] (*Extinction coefficient (0.1%): It is the theoretical absorbance value at 280 nm assuming a protein concentration of 0.1% (1 g / L) and all cysteines in the primary sequence are oxidized to form disulfide bonds. Calculated using the ProtParam tool (https: / / web.expasy.org / protparam / )

[0146] The purified target protein was used to confirm the presence of incorrect cleavage of the signal peptide at the N-terminus of the protein using Q-TOF MS (Figure 5). After dilution to a concentration of 1 mg / mL, the protein was treated with PNGaseF, then with 6M guanidine and DTT, and then loaded onto Q-TOF MS (RMM-MT-001: ACQUITY UPLC + Q-TOF SYNAPT G2 (Waters)).

[0147] As a result, 100% cleavage was confirmed at the predicted cleavage site.

[0148] Based on the results, the signal peptide of the CHO cell-derived protein secretion factor of the present invention improves productivity by increasing the expression level of the recombinant protein, and 100% cleavage was confirmed at the predicted cleavage site by mass analysis, suggesting that the signal peptide of the present invention may be a powerful genetic tool that can solve the problem of incorrect cleavage of existing protein secretion factors.

[0149] Although the present invention has been described with reference to specific illustrative embodiments, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without departing from the technical spirit or essential characteristics of the present invention. Therefore, the above embodiments are considered illustrative in all respects and not restrictive. In addition, the scope of the present invention is defined by the claims rather than the specific embodiments, and it should be understood that all modifications or variations derived from the meaning and scope of the present invention and their equivalents are included within the scope of the claims.

Claims

1. A signal peptide, which consists of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

3.

2. The signal peptide according to claim 1, wherein the target protein of the signal peptide is an endogenous protein.

3. The signal peptide according to claim 1, wherein the target protein of the signal peptide is an exogenous protein.

4. An expression cassette, wherein the nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and the gene encoding the target protein are operably linked.

5. The expression cassette according to claim 4, wherein the target protein is selected from the group consisting of antibodies, antibody fragments, fusion proteins, protein scaffolds, human growth hormone, serum proteins, immunoglobulins, cytokines, α-, β- or γ-interferon, granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor, phospholipase activating protein, insulin, tumor necrosis factor, growth factors, hormones, calcitonin, calcitonin gene-related peptide, enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, growth differentiation factor, cell adhesion proteins, prolactin, chorionic gonadotropin, tissue plasminogen activator, ghrelin, thymic humoral factor, asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase and glucuronidase.

6. The expression cassette according to claim 5, wherein the antibody fragment is selected from Fab or ScFv.

7. The expression cassette according to claim 4, wherein the nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 consists of the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:

15.

8. The expression cassette according to claim 4, wherein the expression cassette further comprises a nucleic acid sequence encoding any one of the signal peptides consisting of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:

10.

9. The expression cassette according to claim 4, wherein when expressed in a cell, the expression cassette expresses the target protein as it is, wherein no additional amino acids are added to the target protein and the nucleic acid sequence encoding the signal peptide has been removed.

10. An expression vector for secreting a target protein, the expression vector comprising an expression cassette, wherein a nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding the target protein are operably linked.

11. The expression vector according to claim 10, wherein the target protein is selected from the group consisting of antibodies, antibody fragments, fusion proteins, protein scaffolds, human growth hormone, serum proteins, immunoglobulins, cytokines, alpha-, beta- or gamma-interferon, granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor, phospholipase-activating protein, insulin, tumor necrosis factor, growth factors, hormones, calcitonin, calcitonin gene-related peptide, enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, growth differentiation factor, cell adhesion proteins, prolactin, chorionic gonadotropin, tissue plasminogen activator, ghrelin, thymic humoral factor, asparaginase, arginase, arginine deaminase, adenosine deaminase, superoxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase and glucuronidase.

12. The expression vector according to claim 11, wherein the antibody fragment is selected from Fab or ScFv.

13. The expression vector according to claim 10, wherein the nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 consists of the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:

15.

14. The expression vector according to claim 10, wherein the expression vector further comprises a nucleic acid sequence encoding any one of the signal peptides consisting of the amino acid sequences of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO:

10.

15. The expression vector according to claim 10, wherein the expression vector expresses the target protein as it is when expressed in a cell, wherein no additional amino acids are added to the target protein and the nucleic acid sequence encoding the signal peptide has been removed.

16. A transformed cell, wherein the expression vector according to any one of claims 10 to 15 is introduced into a host cell.

17. The transformed cell according to claim 16, wherein the host cell is a Chinese hamster ovary cell.

18. A method for producing a target protein, the method comprising: i) Culturing a transformed cell comprising an expression vector for secreting a target protein, said expression vector comprising an expression cassette in which a nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding the target protein are operably linked; and ii) Recovering the target protein from the culture medium or culture supernatant of the cultured cells.

19. The method according to claim 18, further comprising purifying the recovered target protein.

20. The method according to claim 18, wherein the host cell is a Chinese hamster ovary cell.

21. The method according to claim 18, wherein the signal peptide consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 is cleaved at the N-terminal cleavage site of the target protein.

22. The method according to claim 18, wherein the target protein is the target protein itself without additional amino acids added, and the nucleic acid sequence encoding the signal peptide has been removed.

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