CHO cell-derived protein secretory factor and expression vector containing the same

Abstract

The present invention relates to: a protein secretion factor derived from a CHO cell, 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, a transformed cell into which the expression vector is introduced, and a method for producing a target protein using the transformed cell.

Description

Technical Field

[0001] 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, a transformed cell into which the expression vector is introduced, and a method for producing a target protein using the transformed cell. Background Art

[0002] For the useful protein components that are difficult to obtain in vivo, recombinant proteins can be produced on a large scale by gene recombination technology using microorganisms or animal cell systems. 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 masses, and productivity is reduced due to separation and purification difficulties. On the other hand, soluble proteins with correct protein folding can be easily obtained by extracellular secretion. Therefore, in order to obtain more suitable extracellular secretion in terms of protein yield and quality control, optimized recombinant protein expression systems are important.

[0003] In order 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.

[0004] As for the signal peptide, it is located at the N-terminal region of the recombinant protein to be produced, and is therefore 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.

[0005] Therefore, it is important to select a signal peptide that does not cause mis-cleavage and can induce high expression.

[0006] Meanwhile, conventional signal peptides mostly use human-derived signal peptides, and CHO cells are mainly used as expression host cells. Signal peptides can be used in combination between host cells, but this may cause quality problems.

[0007] Under these circumstances, the present inventors have made many efforts to improve the expression level in CHO cells and solve the problem of mis-cleavage. As a result, they developed a new signal peptide, which is a polypeptide consisting of the 17th to 31st amino acid sequence derived from CHO cells, and completed the present invention by confirming that the signal peptide can significantly improve expression and 100% cleave at the cleavage site to prevent mis-cleavage. Summary of the invention

[0008] [Technical issues]

[0009] One object of the present invention is to provide a protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0010] Another object of the present invention is to provide 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.

[0011] Another object of the present invention is to provide 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 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 the target protein are operably linked.

[0012] Another object of the present invention is to provide a transformed cell, wherein the expression vector is introduced into the host cell.

[0013] Another object of the present invention is to provide a method for producing a target protein, the method comprising:

[0014] 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 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 the target protein are operably linked; and

[0015] ii) recovering the target protein from the culture medium or culture supernatant of the cultured cells.

[0016] [Technical solution]

[0017] The present invention will be described in more detail below. At the same time, each explanation and exemplary embodiment disclosed herein can be applied to other explanations and exemplary embodiments. That is to say, all combinations of various different factors disclosed herein belong to the scope of the present invention. In addition, the scope of the present invention should not be limited by the specific disclosures provided below.

[0018] Furthermore, those skilled in the art may be able to identify or ascertain, using no more than routine experimentation, numerous equivalents to the specific aspects of the invention described herein. Additionally, such equivalents are intended to be encompassed by the present invention.

[0019] In order to achieve the above object, one aspect of the present invention provides a novel 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.

[0020] The "protein secretion factor" of the present invention refers to a factor that is linked to a target protein to induce the extracellular secretion of the target protein, and may be composed of a polypeptide. The protein secretion factor can promote the secretion of a target protein that is an endogenous protein and / or an exogenous protein, and specifically can promote the extracellular secretion of a light chain and / or a heavy chain of an antibody, but is not limited thereto.

[0021] The protein secretion factor in the present invention can be used interchangeably with "signal sequence" or "signal peptide (SP)".

[0022] The protein secretion factor of the present invention may have an 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 an 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.

[0023] In a specific embodiment of the present invention, the protein secretion factor may be derived from CHO cells, but is not limited thereto. When used herein, the term "CHO ​​cell" is a Chinese hamster ovary cell, and may be a host cell commonly used for transformation in the art. In addition, the CHO cell-derived protein secretion factor may be selected to increase the expression level in CHO cells as host cells.

[0024] In the present invention, the protein secretion factor composed of the amino acid sequence of SEQ ID NO: 1 may be cathepsin B (Cat), and may be used interchangeably with the Cat secretion sequence in the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 2 may be a CC motif chemokine (CC), and may be used interchangeably with the CC secretion sequence in the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 3 may be nucleosidexin-2 (NUC), and may be used interchangeably with the NUC secretion sequence in the present invention.

[0025] In addition, the protein secretion factor composed of the amino acid sequence of SEQ ID NO: 4 of the present invention may be clusterin (Clus), and may be used interchangeably with the Clus secretion sequence of the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 5 may be pigment epithelium-derived factor (Pig), and may be used interchangeably with the Pig secretion sequence of the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 6 may be procollagen C-endopeptidase enhancer 1 (Proco), and may be used interchangeably with the Proco secretion sequence of the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 7 may be sulfhydryl oxidase (Sulf), and may be used interchangeably with the Sulf secretion sequence of the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 8 may be lipoprotein lipase (Lip), and may be used interchangeably with the Lip secretion sequence of the present invention. The protein secretion factor composed of the amino acid sequence of SEQ ID NO: 9 may be nestin-1 (Nid), and may be used interchangeably with the Nid secretion sequence of 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.

[0026] The nucleic acid sequence encoding the cathepsin B signal peptide composed of the amino acid sequence of SEQ ID NO: 1 can be the polynucleotide sequence of SEQ ID NO: 11, the nucleic acid sequence encoding the CC motif chemokine signal peptide composed of the amino acid sequence of SEQ ID NO: 2 can be the polynucleotide sequence of SEQ ID NO: 12, and the nucleic acid sequence encoding the nucleoside-2 signal peptide composed of the amino acid sequence of SEQ ID NO: 3 can be the polynucleotide sequence of SEQ ID NO: 13.

[0027] 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 sulfhydryl oxidase (Sulf) signal peptide consisting of the amino acid sequence of SEQ ID NO: 9 may be the polynucleotide sequence of SEQ ID NO: 11. The nucleic acid sequence of the protein disulfide isomerase (Pro) signal peptide composed of the amino acid sequence of NO:10 may be the polynucleotide sequence of SEQ ID NO:20.

[0028] Although the protein secretory factor of the present invention is described as a "secretory factor composed of a specific amino acid sequence", it is clear that as long as the secretory factor has the same or corresponding activity as the secretory factor composed of the amino acid sequence of the corresponding sequence number, it does not exclude mutations that may occur through the addition of meaningless sequences upstream or downstream of the amino acid sequence, mutations that may occur naturally, or silent mutations thereof. Even if the sequence addition or mutation exists, it also falls within the scope of the present invention.

[0029] For example, as long as the secretory factor identical to or corresponding to the nucleic acid molecule consisting of the polynucleotide can function as a signal peptide, a nucleic acid sequence showing 85% or more, particularly 90% or more, more particularly 95% or more, even more particularly 98% or more or even more particularly 99% or more homology and / or identity with the above sequence can also be included in the present invention without limitation. In addition, it is obvious that a nucleic acid sequence having a deletion, modification, substitution or addition in a part of the sequence can also be included in the scope of the present invention, as long as the nucleic acid sequence has such homology.

[0030] When used herein, the term "homology" or "identity" refers 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 often used interchangeably.

[0031] The sequence homology or identity of conservative polynucleotide or polypeptide sequence can be determined by standard comparison algorithms and can be used together with the default gap penalty established by the program used. Basically, it is usually expected that homologous or identical sequences will hybridize with all or at least about 50%, 60%, 70%, 80% or 90% or more of the total length of the sequence under medium or high stringency conditions. Polynucleotides containing degenerate codons rather than codons in the hybrid polypeptide are also considered.

[0032] Whether any two polynucleotide or polypeptide sequences have homology, similarity or identity can be determined by a known computer algorithm, 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 performed 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 the GCG program package (Devereux, J. et al., Nucleic Acids Research 12: 387 (1984)), BLASTP, BLASTN, FASTA (Atschul, SF 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).

[0033] Homology, similarity or identity of polynucleotides or polypeptides can be determined by comparing sequence information using, for example, a 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. In summary, the GAP program defines homology, similarity or identity as the value obtained by dividing the number of similarly aligned symbols (i.e., nucleotides or or amino acids) by the total number of symbols in the shorter of the two sequences. Default parameters for the GAP program may include (1) a unary comparison matrix (containing 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 0.10 penalty for each symbol in each gap (or a gap open penalty of 10 and a gap extension penalty of 0.5); and (3) no penalty for end gaps. Thus, when used herein, the terms "homology" or "identity" refer to the relatedness between sequences.

[0034] 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.

[0035] The "protein secretion factor" of the present invention is as described above.

[0036] When used herein, the term "target protein" may refer to a protein endogenously expressed in a host cell or a protein expressed by an exogenous gene introduced therein. 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.

[0037] The target protein can be an antibody, an antibody fragment (Fab or ScFv), a fusion protein, a protein scaffold, human growth hormone, a serum protein, an immunoglobulin, a 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), a growth factor, a hormone, calcitonin, calcitonin gene-related peptide (CGRP), enkephalin, somatomedin, erythropoietin, hypothalamic releasing factor, growth differentiation factor, or the like. 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, endotoxin enzyme, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucosidase, galactosidase, glucocerebrosidase or glucuronidase, and specifically, it can be a heavy chain protein or a light chain protein of an antibody, but is not limited thereto.

[0038] When used herein, the term "operably linked" refers to the functional connection 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 connection can be prepared using gene recombination techniques known in the art, and the site-specific DNA connection can be prepared using a ligase known in the art, but is not limited thereto.

[0039] When used in this article, the term "expression cassette" refers to a sequence that regulates one or more genes and their expression, such as a nucleic acid sequence comprising any combination of various cis-acting transcriptional regulatory elements. The expression cassette of the present invention may also include various elements, such as nucleic acid sequences such as promoters and enhancers that are recognized in the art as necessary for expression regulation, 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 a gene and the expression of its transcription product, is generally 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 connected to the coding sequence. In addition, the expression cassette may include a 3' untranslated region, which includes a polyadenylation site at the 3' end.

[0040] The expression cassette of the present invention can be a combination of polynucleotides that allows extracellular secretion and expression of the target protein in a host cell 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, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 and a gene encoding a target protein.

[0041] 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 consisting of an amino acid sequence of SEQ ID NO: 11 to SEQ ID NO: 20.

[0042] The "protein secretion factor", "target protein", "operable link" and "expression cassette" of the present invention are as described above.

[0043] In the present invention, the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 11 may be cathepsin B (Cat), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 12 may be a CC motif chemokine (CC), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 13 may be nucleoside-2 (Nuc), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 14 may be clusterin (Clus), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 15 may be pigment epithelium-derived factor (Pig), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 16 may be procollagen C-endopeptidase enhancer 1 (Proco), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 17 may be sulfhydryl oxidase (Sulf), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 18 may be lipoprotein lipase (Lip), the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 19 may be lipoprotein lipase (Lip), and the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 20 may be lipoprotein lipase (Lip). The protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 19 may be nestin-1 (Nid), and the protein secretion factor encoded by the polynucleotide sequence of SEQ ID NO: 20 may be protein disulfide isomerase (Pro).

[0044] Another aspect of the present invention provides 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 consisting of an 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 the target protein are operably linked.

[0045] The "protein secretion factor", "target protein", "operable link" and "expression cassette" of the present invention are as described above.

[0046] 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, the extracellular secretion of the target protein is induced.

[0047] When used in this article, the term "expression vector" generally refers to a double-stranded DNA fragment as a vector, into which a target DNA fragment encoding a target protein is inserted. Expression vectors used to express proteins in the art can be used without restriction. Once the expression vector enters the host cell, the expression vector can be replicated independently of the host chromosomal DNA, and the inserted target DNA can be expressed. In order to increase the expression level of the transfected gene in the host cell, the transfected gene must be operably connected to the transcription and translation control sequences that operate in the selected expression host cell.

[0048] There is no particular limitation on the expression vector used in the present invention, as long as it can be replicated in a host cell, and any vector known in the art can be used. Examples of commonly used vectors may include natural or recombinant plasmids, cosmids, viruses, and bacteriophages. For example, as phage vectors or cosmid vectors, pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A, etc., can be used, and as plasmid vectors, vectors based on pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, pET, etc. can be used. Specifically, the vector may be a pTZ-based vector, but is not limited thereto.

[0049] In a specific embodiment of the present invention, an expression vector for secreting a target protein is prepared by operably linking a nucleic acid sequence encoding a protein secretion factor consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 to a gene encoding the target protein on the basis of a pTz-D1G1 vector (including a variant of the promoter of Korean Patent No. 10-1038126) (Example 4).

[0050] 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.

[0051] Another aspect of the present invention provides a transformed cell, wherein the expression vector is introduced into a host cell.

[0052] The "expression vector" of the present invention is as described above.

[0053] As used herein, the term "transformation" refers to the process of introducing a vector including 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.

[0054] As long as the polynucleotide of the conversion can be expressed in the host cell, it is not important whether it is inserted into the chromosome of the host cell and located therein or located outside the chromosome, and 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 including all elements necessary for self-expression. The expression cassette can routinely include a promoter, a transcription termination signal, a ribosome binding domain and a translation termination signal that are operably connected to the polynucleotide.

[0055] The method for transforming the vector of the present invention includes any method for introducing nucleic acid into cells, 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.

[0056] When used herein, the term "host cell" refers to a eukaryotic cell into which a nucleic acid molecule having the activity of a protein secretion factor of the present invention is introduced and in which a signal peptide can be used. The host cell may 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.

[0057] In the present invention, an example of the host cell may be an animal host cell, and specifically, it may be a Chinese hamster ovary cell (CHO cell), but is not limited thereto.

[0058] In a specific embodiment of the present invention, Chinese Hamster Ovary (CHO) cells, which are widely used in the production of recombinant proteins, are used as host cells (Example 4).

[0059] When used herein, the term "transformant" refers to a transformed animal cell, in which an expression vector comprising a signal peptide 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 target protein is introduced into a CHO cell as a host cell.

[0060] 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 (ie, antibody) as a target protein (Example 4).

[0061] Another aspect of the present invention provides a method for producing a target protein, the method comprising:

[0062] 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 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 the target protein are operably linked; and

[0063] ii) recovering the target protein from the culture medium or culture supernatant of the cultured cells.

[0064] The "protein secretion factor", "target protein", "operable connection", "expression cassette", "expression vector for secreting target protein", "host cell" and "transformant" of the present invention are as described above.

[0065] As used herein, the term "culture" refers to the process of growing transformed cells under suitable artificially controlled environmental conditions. In the present invention, the method of producing a target protein using CHO cells as host cells can be carried out using methods widely known in the art. Specifically, the culture can be carried out in a continuous manner by a batch process, a fed-batch process or a repeated fed-batch process, but is not limited thereto.

[0066] The culture medium used for cultivation should meet the needs of specific cell strains in a suitable manner. The carbon source that can be used in the present invention can include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, oils and fats such as soybean oil, sunflower seed 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 to this. These substances can be used alone or in combination.

[0067] The nitrogen source that can be used in the present invention can include peptone, yeast extract, meat extract, malt extract, corn steep liquor, defatted soybean cake and urea or inorganic compound, for example ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, but is not limited thereto. These nitrogen sources can also be used alone or in combination.

[0068] The phosphorus source that can be used in the present invention can include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salt, but is not limited thereto. In addition, the culture medium can 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 batches or continuously during the culture period.

[0069] Alkaline compounds such as sodium hydroxide, potassium hydroxide or ammonia or acidic compounds 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, defoamers such as fatty acid polyethylene glycol esters can be used to suppress the formation of foam. In order to maintain the culture medium under an aerobic state, oxygen or 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.

[0070] In a specific embodiment of the present invention, the recombinant expression vector (i.e., pCB-SP7.2-Pem, pCB-Clus-Pem, pCB-Pig-Pem, and pCB-CC-Pem) is introduced into CHO host cells (ExpiCHO-S TM cells) and cultured in 30 mL of ExpiCHO expression medium (CHO expression medium) by a fed-batch culture method for 12 days (Example 4).

[0071] The method for producing a target protein of the present invention may include a step of recovering the target protein from the culture medium. When used herein, the term "recovery" is a process of obtaining a target protein from a culture medium, and can be performed using methods known in the art such as centrifugation, filtration, anion exchange chromatography, crystallization, HPLC, etc., but the method is not limited thereto.

[0072] The recovery step may include a purification process, and those skilled in the art may select and use, as desired, in various known purification processes. For example, host cells may be separated from culture medium or the culture supernatant of host cells by conventional chromatographic 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 reversed phase HPLC. In addition, when the desired protein is a fusion protein with a specific tag, marker or chelating component, it may be purified by a specific binding partner or drug. The purified protein may be cut into desired protein regions, such as removing the secretory factor, or it may remain intact. Proteins in the desired form including additional amino acids may be produced by cutting the fusion protein during the cutting process.

[0073] The 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 of the present invention can be a secretion factor that is accurately cleaved at the N-terminal cleavage site of the target protein.

[0074] 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 a signal peptidase. However, due to the problem of mis-cleavage, existing protein secretion factors may often reduce the quality of the target protein. The "mis-cleavage" refers to the phenomenon that the signal peptide is not completely degraded at the correct position, and the signal peptide sequence is partially retained at the N-terminus of the target protein.

[0075] In a specific embodiment of the present invention, the cleavage of the protein secretion factor (signal peptide) was confirmed by Q-TOF MS mass spectrometry using the purified target protein. As a result, it was confirmed that 100% cleavage was observed at the predicted cleavage site.

[0076] Therefore, the expression vector comprising the protein secretion factor (signal peptide) of the present invention improves the productivity of the target protein through the efficient expression and secretion of the recombinant protein, and can be a powerful genetic tool to solve the problem of mis-cleavage.

[0077] [Beneficial Effects]

[0078] The protein secretion factor, i.e., signal peptide, of the present invention can significantly improve the productivity of recombinant protein by high-level expression, and is expected to be used as a powerful genetic tool that can solve the problem of mis-cleavage of conventional signal peptides by 100% cleavage at the cleavage site. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] FIG. 1 is a diagram showing signal peptide prediction using SignalP4.1.

[0080] Figure 2 This figure shows the expression level of signal peptide-mCherry confirmed by transient expression.

[0081] Figure 3 is a diagram showing a map of vectors used for site-specific integration.

[0082] Figure 4 is a graph comparing the expression levels of mCherry in site-specifically integrated cells.

[0083] FIG5 is a graph showing mass data demonstrating cleavage of anti-PD-1 antibodies fused to SP7.2 and Clus. DETAILED DESCRIPTION

[0084] 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.

[0085] Example 1. Preparation of a new signal peptide sequence derived from CHO cells

[0086] 1-1. CHO HCP quality analysis

[0087] Four types of MassPREP™ protein digest standards (ADH, BSA, PHO and ENL) were added to CHO cell culture medium (DXB11) treated with trypsin as described below. In the four types of MassPREP™ 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 a 2D LC (high pH RP / low pH RP)-Q-TOF (UDMSe) method.

[0088] For each fraction, MS data (UDMSe) were obtained by direct injection into a 2D column in a Q-TOF MS. The MS data (UDMSe) of the 10 fractions obtained in the above manner were merged into one data using ProteinLynx Global Server (PLGS, Ver. 3.0.2) software. Then, HCPs were identified using the merged data of each sample and the Chinese hamster protein database, and the concentration of each HCP was calculated using PHO as an internal standard.

[0089] 1-2. Selection of signal peptides from CHO HCP data

[0090] The proteins were arranged in order of high abundance and extracted from the CHO genome database ( http: / / chogenome.org) The amino acid sequence of each protein was confirmed. The amino acid sequence thus obtained was input into the SignalP4.1 server (http: / / www.cbs.dtu.dk / services / SignalP / ) to predict the presence of secretory proteins and signal peptide sequences (Table 1, Figure 1 and Table 4).

[0091] [Table 1]

[0092] HCP secretory proteins predicted by SignalP4.1

[0093]

[0094]

[0095] Example 2. Selection of CHO-derived efficient signal peptides by transient expression

[0096] 2-1. Preparation of recombinant protein expression vector for transient expression

[0097] In order to confirm whether the 10 signal peptides selected in Example 1 can be used as universal secretion factors, mCherry (pmCherry vector, Clontech, 632522) protein was selected as the target protein.

[0098] The polynucleotide sequence of the gene encoding the mCherry protein is shown in Table 2.

[0099] [Table 2]

[0100]

[0101]

[0102] Based on the gene encoding the mCherry protein, PCR was performed using the signal peptide sequence identified from the CHO HCP quality data and primers containing KpnI / XhoI, and mCherry expressed by 10 signal peptide sequences was constructed.

[0103] When the length of the signal peptide was long, the primer was divided into two and PCR was performed twice. The mCherry PCR products containing 10 signal peptide sequences were cut with KpnI and XhoI and then cloned into pcDNA3.1(+) (Invitrogen, catalog number V790-20) to construct an expression vector.

[0104] In addition, in order to use as a positive control, an mCherry protein expression vector fused with a SP7.2 signal peptide (Korean Patent Laid-Open Publication No. 10-2015-0125402A), 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.

[0105] [Table 3]

[0106]

[0107]

[0108] [Table 4]

[0109]

[0110]

[0111]

[0112]

[0113]

[0114]

[0115]

[0116]

[0117]

[0118] 2-2. Transient expression

[0119] According to the Amaxa 4D-nucleofector protocol for CHO-S cell lines, each mCherry expression vector expressed by 10 signal peptides was transfected (1 mL). Then, on days 2 and 6, the fluorescence values ​​of intracellular fluorescence and fluorescent protein secreted from the culture medium were measured ( Figure 2 ).

[0120] In the case of intracellular fluorescence, FACS (Accuri) was used to measure the mean of the fractions higher than the negative control (empty vector, pMaxGFP) represented as a histogram.

[0121] In the case of the fluorescence value of the fluorescent protein secreted into the culture 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 plate reader.

[0122] It was confirmed that the signal peptides with high fluorescence values ​​measured in the culture medium were Cat, CC, Nuc, Clus and Pig. In addition, four secreted factors (Clus, Pig, Nuc and CC) showing higher expression than the positive control SP7.2 (SP7.2 was used as a positive control) and a signal peptide (Proco) with high fluorescence values ​​in cells were selected as negative controls.

[0123] Example 3. Comparison of expression by site-specific integration

[0124] 3-1. Construction of expression vector for site-specific integration

[0125] The mCherry sequences including the five 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 ).

[0126] The insertion site was set at the Hprt site, and the homology arm sequence and sgRNA sequence were designed with reference to JS Lee et al., 2015, "Site-Specific integration in CHO Cells mediated by CRISPR / Cas9 and homology-directed DNA repair pathway", Sci.Rep., 5.

[0127] In the case of the 5' homology arm, PCR was performed using primers containing Bg1II and NruI enzyme sites and CHO-S genome as a template. Then, it was cloned into pcDNA3.1(+) vector digested with Bg1II / NruI.

[0128] In the case of the 3' homology arm, PCR was performed using each primer containing a SalI site and CHO-S genome as a template, followed by SalI single cutting together with the vector inserted with the 5' homology arm and cloned into the downstream of the NeoR gene (pcDNA3.1_hprt).

[0129] To confirm that only those fragments that had undergone homologous recombination were inserted into the genome, a Cmy-GFP-BHG pA fragment was constructed in the upstream region of the 5' homology arm and inserted into SpeI and Bg1II (pcDNA3.1_G_hprt) for double selection.

[0130] 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. SpeI was then inserted into the vector containing the homology region (pcDNA_hprt) using the Bg1II site.

[0131] Using the completed pcDNA3.1_G_hprt vector, the mCherry gene sequence containing the signal peptide sequence was cut out with KpnI and XhoI and inserted into the MCS region.

[0132] As a result, a pcDNA3.1-based expression vector containing an expression cassette in the form of CMV-EGFP-pA-5'Hprt homology arm-CMV-signal peptide candidate-mCherry-BGH pA-NeoR selection marker cassette-3'Hprt homology arm was constructed.

[0133] 3-2. Site-specific integration

[0134] Knock-in using CRISPR-Cas9 to insert 6 types of vectors for site-specific integration in 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 mixed separately with the nucleofection solution to prepare a 50 μL mixture.

[0135] First, 1x10 6 CHO-S cells were lysed in 50 μL of nucleofection reagent and then mixed with the previously prepared mixture, and the final 100 μL mixture was electroporated.

[0136] After electroporation, the mixture was mixed with 0.5 mL of culture medium, added to 2.5 mL of culture medium, and cultured in a 6-well plate at 36.5° C. and 5% CO 2 .

[0137] After 2 days, selection was performed in CD CHO medium containing Zeneticin (0.5 mg / L), and the cells were subcultured until 90% viability was restored.

[0138] After 90% viability was restored, 4 mL of cells were plated in duplicate in 6-well plates at 3x10 5 The cells were cultured at a concentration of 10 cells / mL, and the fluorescence values ​​(587 nm / 610 nm) of Vi-Cell and culture medium were measured every 2 to 3 days ( Figure 4 ).

[0139] As a result, it was confirmed that CC, Clus, and Pig secretory peptides showed higher expression compared to the control SP7.2.

[0140] Example 4. Expression and quality analysis of anti-PD-1 antibodies

[0141] 4-1. Preparation of expression vector for producing anti-PD-1 antibody

[0142] After comparing the expression ability of signal peptides by site-specific integration, anti-PD-1 antibodies fused with 4 types of signal peptides (CC, Pig, Clus, and SP7.2), including CC, Clus, and Pig, which showed high expression, were expressed. Antibody sequences as target proteins.

[0143] DNA sequences corresponding to the amino acid sequences of the light and heavy chains were synthesized and subsequently generated by overlapping PCR fusions to each signal peptide sequence.

[0144] 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).

[0145] pCB_SP7.2_Pem

[0146] '(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)'

[0147] pCB_Clus_Pem

[0148] '(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)'

[0149] pCB_CC_Pem

[0150] '(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)'

[0151] pCB_Pig_Pem

[0152] '(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)'

[0153] 4-2. Expression of anti-PD1 antibodies

[0154] 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; fed on days 1 and 5) to express the fusion polypeptide (i.e., pembrolizumab).

[0155] 4-3. Purification and quality analysis of anti-PD1 antibodies

[0156] The fusion polypeptide produced by expression of the recombinant vector was purified by protein A. Specifically, the recovered culture solution was filtered with a 0.22 μm filter, and then a column filled with protein A resin (Hitrap MSS, GE Healthcare, 11-0034-93) was installed in AKTA TM Avant25 (GE Healthcare Life Sciences) was placed on the column, and PBS buffer was passed through to equilibrate the column.

[0157] After the filtered culture fluid was injected into the column, PBS buffer was passed through again to clean the column. After the cleaning of the column was completed, 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 filter device (MWCO 30K, Merck) and a centrifuge. After concentration, PBS was used for buffer exchange.

[0158] The quantitative analysis of the fusion polypeptide was performed by measuring the absorbance 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 was a theoretical value (1.404) calculated using the amino acid sequence.

[0159]

[0160] (*Extinction coefficient (0.1%): It is the theoretical absorbance at 280nm assuming that the protein concentration is 0.1% (1g / L) and all cysteines in the primary sequence are oxidized to form disulfide bonds. Calculated by ProtParam tool (https: / / web.expasy.org / protparam / ))

[0161] Using Q-TOF MS, the purified target protein was used to confirm the presence of miscleavage of the signal peptide at the N-terminus of the protein ( FIG. 5 ). After dilution to a concentration of 1 mg / mL, the protein was treated with PNGaseF, then with 6 M guanidine and DTT, and then loaded onto a Q-TOF MS (RMM-MT-001: ACQUITY UPLC+Q-TOF SYNAPT G2 (Waters)).

[0162] As a result, it was confirmed that 100% cleavage was observed at the predicted cleavage site.

[0163] Based on the results, the signal peptide as the CHO cell-derived protein secretion factor of the present invention improved productivity by increasing the expression level of the recombinant protein, and 100% cleavage was observed at the predicted cleavage site as confirmed by mass analysis, suggesting that the signal peptide of the present invention may be a powerful genetic tool that can solve the erroneous cleavage that is a problem of existing protein secretion factors.

[0164] Although the present invention has been described with reference to specific illustrative embodiments, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the technical spirit or essential features of the present invention. Therefore, the above-mentioned embodiments are considered to be illustrative rather than restrictive in all aspects. 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 changes and their equivalents derived from the meaning and scope of the present invention are included within the scope of the claims. <110> LG CHEM, LTD. <120> CHO cell-derived protein secretory factor and expression vector containing the same <130> OPA20173 <150> KR 10-2019-0158447 <151> 2019-12-02 <160> 59 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> PRT <213> unknown <220> <223> Signal peptide of cathepsin B (Cat) <400> 1 Met Trp Trp Ser Leu Ile Pro Leu Ser Cys Leu Leu Ala Leu Ala Ser 1 5 10 15 Ala <210> 2 <211> twenty four <212> PRT <213> unknown <220> <223> CC motif chemokine (CC) signal peptide <400> 2 Met Gln Phe Ser Ala Arg Thr Leu Leu Cys Leu Leu Leu Thr Val Ala 1 5 10 15 Ala Cys Ser Ile Tyr Val Leu Ala 20 <210> 3 <211> twenty four <212> PRT <213> unknown <220> <223> Signal peptide of nucleocin-2 (Nuc) <400> 3 Met Arg Trp Lys Ile Ile Gln Leu Gln Tyr Cys Phe Leu Leu Val Pro 1 5 10 15 Cys Met Leu Thr Ala Leu Glu Ala 20 <210> 4 <211> twenty one <212> PRT <213> unknown <220> <223> Clus signal peptide <400> 4 Met Lys Ile Leu Leu Leu Cys Val Gly Leu Leu Leu Thr Trp Asp Asn 1 5 10 15 Gly Met Val Leu Gly 20 <210> 5 <211> 19 <212> PRT <213> unknown <220> <223> Pigment epithelium-derived factor (Pig) signal peptide <400> 5 Met Gln Ala Leu Val Leu Leu Leu Trp Thr Gly Ala Leu Leu Gly His 1 5 10 15 Gly Ser Ser <210> 6 <211> twenty four <212> PRT <213> unknown <220> <223> Signal peptide of procollagen C-endopeptidase enhancer 1 (Proco) <400> 6 Met Leu Pro Ala Val Leu Thr Ser Leu Leu Gly Pro Phe Leu Val Ala 1 5 10 15 Trp Val Leu Pro Leu Ala Arg Gly 20 <210> 7 <211> 31 <212> PRT <213> unknown <220> <223> Signal peptide of sulfhydryl oxidase (Sulf) <400> 7 Met Arg Arg Cys Gly Arg His Ser Gly Ser Pro Ser Gln Met Leu Leu 1 5 10 15 Leu Leu Leu Pro Pro Leu Leu Leu Ala Val Pro Gly Ala Gly Ala 20 25 30 <210> 8 <211> 20 <212> PRT <213> unknown <220> <223> Signal peptide of lipoprotein lipase (Lip) <400> 8 Met Glu Ser Lys Ala Leu Leu Leu Val Ala Leu Gly Val Trp Leu Gln 1 5 10 15 Ser Leu Thr Ala 20 <210> 9 <211> 30 <212> PRT <213> unknown <220> <223> Signal peptide of nestin-1 (Nid) <400> 9 Met Leu Asp Ala Ser Gly Trp Lys Pro Ala Ala Trp Thr Trp Val Leu 1 5 10 15 Leu Leu Gln Leu Leu Leu Ala Gly Pro Gly Asp Cys Leu Ser 20 25 30 <210> 10 <211> twenty two <212> PRT <213> unknown <220> <223> Signal peptide of protein disulfide isomerase (Pro) <400> 10 Met Asp Asp Arg Leu Leu Thr Val Leu Leu Leu Leu Leu Gly Val Ser 1 5 10 15 Gly Pro Trp Gly Gln Gly 20 <210> 11 <211> 51 <212> DNA <213> unknown <220> <223> Gene encoding the signal peptide of cathepsin B (Cat) <400> 11 atgtggtggt ccttgattcc gctctcttgc ctgctggcac tggcaagtgc c 51 <210> 12 <211> 72 <212> DNA <213> unknown <220> <223> CC motif chemokine (CC) signal peptide gene <400> 12 atgcagttct ccgcaagaac gcttctgtgc ctgctactca cagttgctgc ctgtagcatc 60 tatgtgctgg cc 72 <210> 13 <211> 72 <212> DNA <213> unknown <220> <223> Nucleocin-2 (Nuc) signal peptide gene <400> 13 atgaggtgga agatcatcca gctacagtac tgttttcttt tggtcccgtg catgcttact 60 gctctggaag ct 72 <210> 14 <211> 63 <212> DNA <213> unknown <220> <223> Clus signal peptide gene <400> 14 atgaagattc tcctgttgtg cgtggggctg ctgctgacct gggacaatgg catggtcctg 60 gga 63 <210> 15 <211> 57 <212> DNA <213> unknown <220> <223> Pigment epithelium-derived factor (Pig) signal peptide gene <400> 15 atgcaggccc tggtgctact cctctggaca ggagccctgc ttgggcatgg cagcagc 57 <210> 16 <211> 72 <212> DNA <213> unknown <220> <223> Procollagen C-endopeptidase enhancer 1 (Proco) signal peptide gene <400> 16 atgctgcctg ctgtcctaac ctccctcctg gggccattcc ttgtggcctg ggtactgcct 60 cttgcccgag gc 72 <210> 17 <211> 93 <212> DNA <213> unknown <220> <223> The gene for the signal peptide of sulfhydryl oxidase (Sulf) <400> 17 atgaggaggt gcggccgcca ctcggggtcg ccgtcgcaga tgctactgct gctgctgccg 60 ccgctgctgc tcgcggtgcc cggcgctggc gcg 93 <210> 18 <211> 60 <212> DNA <213> unknown <220> <223> Lipoprotein lipase (Lip) signal peptide gene <400> 18 atggagagca aagccctgct cctggtggct ctggggagtgt ggctccagag tttgaccgcc 60 60 <210> 19 <211> 90 <212> DNA <213> unknown <220> <223> The gene for the signal peptide of nestin-1 (Nid) <400> 19 atgctggacg cgagcggctg gaagcccgcg gcgtggacat gggtgctgct gctgcagcta 60 ttgctggcgg ggcccggaga ctgcctgagc 90 <210> 20 <211> 66 <212> DNA <213> unknown <220> <223> Protein disulfide isomerase (Pro) signal peptide gene <400> 20 atggatgatc ggctcctgac agtgttgctg ctcctgctgg gtgtctcagg cccatgggga 60 caggga 66 <210> twenty one <211> 339 <212> PRT <213> unknown <220> <223> Cathepsin B (Cat) <400> twenty one Met Trp Trp Ser Leu Ile Pro Leu Ser Cys Leu Leu Ala Leu Ala Ser 1 5 10 15 Ala His Asn Lys Pro Ser Phe His Pro Leu Ser Asp Asp Leu Ile Asn 20 25 30 Tyr Ile Asn Lys Arg Asn Thr Thr Trp Gln Ala Gly Arg Asn Phe His 35 40 45 Asn Val Asp Ile Ser Tyr Leu Lys Arg Leu Cys Gly Thr Ile Met Gly 50 55 60 Gly Pro Lys Leu Pro Glu Arg Val Ala Phe Ala Glu Asp Met Glu Leu 65 70 75 80 Pro Glu Asn Phe Asp Ala Arg Glu Gln Trp Ser Asn Cys Pro Thr Ile 85 90 95 Lys Gln Ile Arg Asp Gln Gly Ser Cys Gly Ser Cys Trp Ala Phe Gly 100 105 110 Ala Val Gly Ala Met Ser Asp Arg Leu Cys Ile His Thr Asn Gly His 115 120 125 Val Asn Val Glu Val Ser Ala Glu Asp Leu Leu Thr Cys Cys Gly Ser 130 135 140 Gln Cys Gly Asp Gly Cys Asn Gly Gly Tyr Pro Ser Gly Ala Trp Asn 145 150 155 160 Phe Trp Ile Lys Lys Gly Leu Val Ser Gly Gly Leu Tyr Asn Ser His 165 170 175 Val Gly Cys Leu Pro Tyr Thr Ile Pro Pro Cys Glu His His Val Asn 180 185 190 Gly Ser Arg Pro Gln Cys Thr Gly Glu Gly Asp Thr Pro Lys Cys Thr 195 200 205 Lys Ser Cys Glu Ala Gly Tyr Ser Pro Ser Tyr Lys Glu Asp Lys His 210 215 220 Tyr Gly Tyr Thr Ser Tyr Ser Val Ser Asn Asn Glu Lys Glu Ile Met 225 230 235 240 Ala Glu Ile Tyr Lys Asn Gly Pro Val Glu Gly Ala Phe Thr Val Phe 245 250 255 Ser Asp Phe Leu Thr Tyr Lys Ser Gly Val Tyr Lys His Glu Ala Gly 260 265 270 Asp Ile Met Gly Gly His Ala Ile Arg Ile Leu Gly Trp Gly Val Glu 275 280 285 Asn Ser Val Pro Tyr Trp Leu Val Ala Asn Ser Trp Asn Val Asp Trp 290 295 300 Gly Asp Asn Gly Leu Phe Lys Ile Leu Arg Gly Glu Asp His Cys Gly 305 310 315 320 Ile Glu Ser Glu Ile Val Ala Gly Ile Pro Arg Thr Asp Leu Tyr Trp 325 330 335 Gly Arg Phe <210> 22 <211> 143 <212> PRT <213> Unknown <220> <223> C-C Motif Chemokine (CC) <400> 22 Met Gln Phe Ser Ala Arg Thr Leu Leu Cys Leu Leu Leu Thr Val Ala 1 5 10 15 Ala Cys Ser Ile Tyr Val Leu Ala Gln Pro Asp Ala Val Asn Ser Pro 20 25 30 Leu Thr Cys Cys Tyr Ser Phe Thr Ala Lys Arg Ile Pro Glu Lys Arg 35 40 45 Leu Glu Ser Tyr Lys Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala 50 55 60 Val Ile Phe Ile Thr Lys Leu Lys Arg Glu Ile Cys Ala Asp Pro Lys 65 70 75 80 Gln Asp Trp Val Gln Thr Tyr Thr Lys Lys Leu Asp Gln Ser Gln Ala 85 90 95 Lys Ser Glu Ala Ala Thr Val Tyr Lys Thr Ala Pro Leu Asn Ala Asn 100 105 110 Leu Thr His Glu Ser Ala Val Asn Ala Ser Thr Thr Ala Phe Pro Thr 115 120 125 Thr Asp Leu Arg Thr Ser Val Arg Val Thr Ser Met Thr Val Asn 130 135 140 <210> 23 <211> 420 <212> PRT <213> Unknown <220> <223> Nucleolin - 2 (Nuc) <400> 23 Met Arg Trp Lys Ile Ile Gln Leu Gln Tyr Cys Phe Leu Leu Val Pro 1 5 10 15 Cys Met Leu Thr Ala Leu Glu Ala Val Pro Ile Asp Val Asp Lys Thr 20 25 30 Lys Val His Asn Thr Glu Pro Val Glu Ser Ala Arg Ile Asp Pro Pro 35 40 45 Asp Thr Gly Leu Tyr Tyr Asp Glu Tyr Leu Lys Gln Val Ile Asp Val 50 55 60 Leu Glu Thr Asp Gln His Phe Arg Glu Lys Leu Gln Lys Ala Asp Ile 65 70 75 80 Glu Glu Ile Arg Ser Gly Arg Leu Ser Lys Glu Leu Asp Leu Val Ser 85 90 95 His His Val Arg Thr Lys Leu Asp Glu Leu Lys Arg Gln Glu Val Ala 100 105 110 Arg Leu Arg Met Leu Ile Lys Ala Lys Leu Asp Ser Leu Gln Asp Thr 115 120 125 Gly Met Asn His His Leu Leu Leu Lys Gln Phe Glu His Leu Asn His 130 135 140 Gln Asn Pro Asp Thr Phe Glu Ser Ser Asp Leu Asp Met Leu Ile Lys 145 150 155 160 Ala Ala Thr Ala Asp Leu Glu Gln Tyr Asp Arg Thr Arg His Glu Glu 165 170 175 Phe Lys Lys Tyr Glu Met Met Lys Glu His Glu Arg Arg Glu Tyr Leu 180 185 190 Lys Thr Leu Asn Glu Glu Lys Arg Lys Glu Glu Glu Ser Lys Phe Glu 195 200 205 Glu Met Lys Arg Lys His Glu Asn His Pro Lys Val Asn His Pro Gly 210 215 220 Ser Lys Asp Gln Leu Lys Glu Val Trp Glu Glu Ala Asp Gly Leu Asp 225 230 235 240 Pro Asn Asp Phe Asp Pro Lys Thr Phe Phe Lys Leu His Asp Val Asn 245 250 255 Asn Asp Gly Phe Leu Asp Glu Gln Glu Leu Glu Ala Leu Phe Thr Arg 260 265 270 Glu Leu Glu Lys Val Tyr Asp Pro Arg Asn Ala Glu Asp Asp Met Ile 275 280 285 Glu Met Glu Glu Glu Arg Leu Arg Met Arg Glu His Val Met Asn Glu 290 295 300 Ile Asp Asn Asn Lys Asp Arg Leu Val Thr Leu Glu Glu Phe Leu Arg 305 310 315 320 Ala Thr Glu Lys Lys Glu Phe Leu Glu Pro Asp Ser Trp Glu Thr Leu 325 330 335 Gly Gln Gln Gln Leu Phe Thr Glu Glu Glu Leu Lys Glu Tyr Glu Ser 340 345 350 Ile Ile Ala Met Gln Glu Asn Glu Leu Lys Lys Arg Ala Asp Glu Leu 355 360 365 Gln Lys Gln Lys Glu Glu Leu Gln Arg Gln His Asp His Leu Glu Ala 370 375 380 Gln Lys Gln Glu Tyr Gln Gln Ala Val Gln Gln Leu Glu His Lys Lys 385 390 395 400 Phe Gln Gln Gly Ile Ala Pro Ser Gly Pro Ala Gly Glu Leu Glu Phe 405 410 415 Lys Pro Arg Met 420 <210> 24 <211> 447 <212> PRT <213> Unknown <220> <223> Clusterin (Clus) <400> 24 Met Lys Ile Leu Leu Leu Cys Val Gly Leu Leu Leu Thr Trp Asp Asn 1 5 10 15 Gly Met Val Leu Gly Glu Gln Glu Val Ser Asp Asn Glu Leu Lys Glu 20 25 30 Met Ser Thr Gln Gly Ser Arg Tyr Ile Asn Lys Glu Ile Gln Asn Ala 35 40 45 Val Gln Gly Val Lys Gln Ile Lys Thr Leu Ile Glu Lys Thr Asn Glu 50 55 60 Glu Arg Lys Ser Leu Leu Asn Ser Leu Glu Glu Ala Lys Lys Lys Lys 65 70 75 80 Glu Asp Ala Leu Asp Asp Thr Arg Asp Thr Glu Met Lys Leu Lys Ala 85 90 95 Phe Pro Glu Val Cys Asn Glu Thr Met Met Ala Leu Trp Glu Glu Cys 100 105 110 Lys Pro Cys Leu Lys Gln Thr Cys Met Lys Phe Tyr Ala Arg Val Cys 115 120 125 Arg Ser Gly Ser Gly Leu Val Gly Arg Gln Leu Glu Glu Phe Leu Asn 130 135 140 Gln Ser Ser Pro Phe Tyr Phe Trp Met Asn Gly Asp Arg Ile Asp Ser 145 150 155 160 Leu Met Glu Ser Asp Arg Gln Gln Ser Gln Val Leu Asp Ala Met Gln 165 170 175 Asp Ser Phe Thr Arg Ala Ser Gly Ile Met Asp Met Leu Phe Gln Asp 180 185 190 Arg Phe Phe Thr His Glu Pro Gln Asp Thr His Tyr Phe Ser Pro Phe 195 200 205 Gly Phe Pro His Arg Arg Pro His Phe Leu Tyr Pro Lys Ser Arg Leu 210 215 220 Val Arg Ser Leu Ile Pro Leu Ser His Tyr Gly Pro Pro Ser Phe His 225 230 235 240 Asp Met Phe Gln Pro Phe Leu Glu Met Ile His Gln Ala Gln Gln Ala 245 250 255 Met Asp Val Gln Phe His Arg Pro Ala Phe Gln Phe Pro Asp Lys Gly 260 265 270 Leu Arg Glu Gly Glu Asp Asp Arg Ala Val Cys Lys Glu Ile Arg His 275 280 285 Asn Ser Thr Gly Cys Leu Lys Met Lys Gly Gln Cys Glu Lys Cys Gln 290 295 300 Glu Ile Leu Ser Val Asp Cys Ser Ala Asn Asn Pro Ala Gln Ala His 305 310 315 320 Leu Arg Gln Glu Leu Asn Asp Ser Leu Gln Met Ala Glu Arg Leu Thr 325 330 335 Gln Gln Tyr Asn Glu Leu Leu His Ser Leu Gln Thr Lys Met Leu Asn 340 345 350 Thr Ser Ser Leu Leu Glu Gln Leu Asn Glu Gln Phe Asn Trp Val Ser 355 360 365 Gln Leu Ala Asn Leu Thr Gln Gly Glu Asp Gln Tyr Tyr Leu Arg Val 370 375 380 Ser Thr Val Thr Thr His Ser Asn Asn Ser Glu Glu Pro Ser Arg Val 385 390 395 400 Thr Glu Val Val Val Lys Leu Phe Asp Ser Asp Pro Ile Thr Val Val 405 410 415 Leu Pro Glu Glu Val Ser Lys Asp Asn Pro Lys Phe Met Asp Thr Val 420 425 430 Ala Glu Lys Ala Leu Gln Glu Tyr Arg Lys Lys Ser Arg Ala Glu 435 440 445 <210> 25 <211> 378 <212> PRT <213> Unknown <220> <223> Pigment epithelium-derived factor (Pig) <400> 25 Met Gln Ala Leu Val Leu Leu Leu Trp Thr Gly Ala Leu Leu Gly His 1 5 10 15 Gly Ser Ser Gln Asn Val Ala Ser Ser Ser Glu Glu Gly Ser Pro Ala 20 25 30 Pro Asp Ser Thr Gly Glu Pro Val Glu Glu Glu Glu Asp Pro Phe Phe 35 40 45 Lys Val Pro Val Asn Lys Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr 50 55 60 Asp Leu Tyr Arg Leu Arg Ser Ser Ala Ser Pro Thr Ala Asn Val Leu 65 70 75 80 Leu Ser Pro Leu Ser Val Ala Thr Ala Leu Ser Ala Leu Ser Leu Gly 85 90 95 Ala Glu Gln Arg Thr Glu Ser Ile Ile His Arg Ala Leu Tyr Tyr Asp 100 105 110 Leu Ile Ser Asn Ser Asp Ile His Ser Thr Tyr Lys Glu Leu Leu Ala 115 120 125 Ser Val Thr Ala Pro Glu Lys Ser Leu Lys Ser Ala Ser Arg Ile Val 130 135 140 Phe Glu Arg Lys Leu Arg Val Arg Ser Ser Phe Val Ala Pro Leu Glu 145 150 155 160 Lys Ser Tyr Gly Thr Arg Pro Arg Ile Leu Thr Gly Asn Pro Arg Ile 165 170 175 Asp Leu Gln Glu Ile Asn Asn Trp Ile Gln Ala Gln Met Lys Gly Lys 180 185 190 Leu Ala Arg Ser Thr Arg Glu Met Pro Ser Ala Ile Ser Ile Leu Leu 195 200 205 Leu Gly Val Ala Tyr Phe Lys Gly Gln Trp Val Thr Lys Phe Asp Ser 210 215 220 Arg Lys Thr Ser Leu Gln Asp Phe His Leu Asp Glu Asp Arg Thr Val 225 230 235 240 Lys Val Pro Met Met Ser Glu Pro Lys Ala Ile Leu Arg Tyr Gly Leu 245 250 255 Asp Ser Asp Leu Asn Cys Lys Val Trp Glu His Gly Gly Trp Glu Gly 260 265 270 Ser Glu Arg Gly Arg Val Ser Ser Ile Arg Lys Ser Ile Trp Gly Tyr 275 280 285 Ser Lys Ile His Glu Leu Gln Ser Leu Phe Glu Ser Pro Asp Phe Ser 290 295 300 Lys Ile Thr Gly Lys Pro Val Lys Leu Thr Gln Val Glu His Arg Ala 305 310 315 320 Ala Phe Glu Trp Asn Glu Glu Gly Val Glu Thr Ser Pro Asn Pro Gly 325 330 335 Leu Gln Pro Val Arg Leu Thr Phe Pro Leu Asp Tyr His Leu Asn Gln 340 345 350 Pro Phe Ile Phe Val Leu Arg Asp Thr Asp Thr Gly Ala Leu Leu Phe 355 360 365 Ile Gly Lys Ile Leu Asp Pro Arg Gly Thr 370 375 <210> 26 <211> 468 <212> PRT <213> Unknown <220> <223> Procollagen C - endopeptidase enhancer 1 (Proco) <400> 26 Met Leu Pro Ala Val Leu Thr Ser Leu Leu Gly Pro Phe Leu Val Ala 1 5 10 15 Trp Val Leu Pro Leu Ala Arg Gly Gln Thr Pro Asn Tyr Thr Arg Pro 20 25 30 Val Phe Leu Cys Gly Gly Asp Val Thr Gly Glu Ser Gly Tyr Val Ala 35 40 45 Ser Glu Gly Phe Pro Asn Leu Tyr Pro Pro Asn Lys Lys Cys Ile Trp 50 55 60 Thr Ile Thr Val Pro Glu Gly Gln Thr Val Ser Leu Ser Phe Arg Val 65 70 75 80 Phe Asp Met Glu Leu His Pro Ser Cys Arg Tyr Asp Ala Leu Glu Val 85 90 95 Phe Ala Gly Ser Gly Thr Ser Gly Gln Arg Leu Gly Arg Phe Cys Gly 100 105 110 Thr Phe Arg Pro Ala Pro Val Val Ala Pro Gly Asn Gln Val Thr Leu 115 120 125 Arg Met Thr Thr Asp Glu Gly Thr Gly Gly Arg Gly Phe Leu Leu Trp 130 135 140 Tyr Ser Gly Arg Ala Thr Ser Gly Thr Glu His Gln Phe Cys Gly Gly 145 150 155 160 Arg Met Glu Lys Ala Gln Gly Thr Leu Thr Thr Pro Asn Trp Pro Glu 165 170 175 Ser Asp Tyr Pro Pro Gly Ile Ser Cys Ser Trp His Ile Ile Ala Pro 180 185 190 Ser Asp Gln Val Ile Met Leu Thr Phe Gly Lys Phe Asp Val Glu Pro 195 200 205 Asp Thr Tyr Cys Arg Tyr Asp Ser Val Ser Val Phe Asn Gly Ala Val 210 215 220 Ser Asp Asp Ser Lys Arg Leu Gly Lys Phe Cys Gly Asp Lys Ala Pro 225 230 235 240 Ser Pro Ile Ser Ser Glu Gly Asn Glu Leu Leu Val Gln Phe Val Ser 245 250 255 Asp Leu Ser Val Thr Ala Asp Gly Phe Ser Ala Ser Tyr Lys Thr Leu 260 265 270 Pro Arg Asp Ala Val Glu Lys Glu Leu Ala Pro Ser Pro Gly Glu Asp 275 280 285 Val Gln Leu Gly Pro Gln Ser Arg Ser Asp Pro Lys Thr Gly Thr Gly 290 295 300 Pro Lys Val Lys Pro Pro Ser Lys Pro Lys Phe Gln Pro Ala Glu Lys 305 310 315 320 Pro Glu Val Ser Pro Asp Thr Gln Glu Thr Pro Val Ala Pro Asp Pro 325 330 335 Pro Ser Ala Thr Cys Pro Lys Gln Tyr Lys Arg Leu Gly Thr Leu Gln 340 345 350 Ser Asn Phe Cys Ala Ser Ser Leu Val Val Thr Gly Thr Val Lys Thr 355 360 365 Met Val Arg Gly Pro Gly Glu Gly Leu Thr Val Thr Ile Ser Leu Leu 370 375 380 Gly Val Tyr Lys Ser Gly Gly Leu Asp Leu Pro Ser Pro Pro Thr Asp 385 390 395 400 Thr Ser Leu Lys Leu Tyr Val Pro Cys Arg Gln Met Pro Pro Met Lys 405 410 415 Lys Gly Ala Ser Tyr Leu Leu Met Gly Gln Val Glu Glu Asn Arg Gly 420 425 430 Pro Ile Leu Pro Pro Glu Ser Phe Leu Val Pro Tyr Lys Pro Asn Gln 435 440 445 Asp Gln Ile Leu Asn Asn Leu Arg Lys Arg Lys Cys Pro Ser Gln Pro 450 455 460 Arg Pro Ala Ala 465 <210> 27 <211> 571 <212> PRT <213> Unknown <220> <223> Sulfhydryl oxidase (Sulf) <400> 27 Met Arg Arg Cys Gly Arg His Ser Gly Ser Pro Ser Gln Met Leu Leu 1 5 10 15 Leu Leu Leu Pro Pro Leu Leu Leu Ala Val Pro Gly Ala Gly Ala Val 20 25 30 Gln Val Ser Val Leu Tyr Ser Ser Ser Asp Pro Val Thr Val Leu Asn 35 40 45 Ala Asn Thr Val Arg Ser Thr Val Leu Arg Ser Asn Gly Ala Trp Ala 50 55 60 Val Glu Phe Phe Ala Ser Trp Cys Gly His Cys Ile Ala Phe Ala Pro 65 70 75 80 Thr Trp Lys Glu Leu Ala Tyr Asp Val Arg Glu Trp Arg Pro Val Leu 85 90 95 Asn Leu Ala Val Leu Asp Cys Ala Glu Glu Thr Asn Thr Ala Val Cys 100 105 110 Arg Asp Phe Asn Ile Ser Gly Phe Pro Thr Val Arg Phe Phe Lys Ala 115 120 125 Phe Ser Lys Asn Gly Ser Gly Ile Thr Leu Pro Val Ala Asp Ala Ser 130 135 140 Val Glu Thr Leu Arg Arg Lys Leu Ile Asp Ala Leu Glu Ser His Ser 145 150 155 160 Asp Met Trp Ser Ser Ser Arg Pro Lys Leu Lys Pro Ala Lys Leu Val 165 170 175 Glu Ile Asn Glu Phe Phe Ala Glu Thr Asn Glu Asp Tyr Leu Val Leu 180 185 190 Ile Phe Glu Asp Lys Asp Ser Tyr Val Gly Arg Glu Val Thr Leu Asp 195 200 205 Leu Phe Gln His His Ile Pro Val His Arg Val Leu Asn Thr Glu Arg 210 215 220 Asn Ala Val Ser Lys Phe Gly Val Val Glu Phe Pro Ser Cys Tyr Leu 225 230 235 240 Leu Phe Arg Asn Gly Ser Phe Ser Arg Val Pro Val Val Met Glu Ser 245 250 255 Arg Leu Phe Tyr Thr Ser Tyr Leu Lys Gly Met Ser Gly Pro Ile Leu 260 265 270 Val Asp Pro Pro Thr Thr Thr Ile Ser Thr Asp Ala Pro Val Thr Thr 275 280 285 Asp Val Val Pro Thr Val Trp Lys Val Ala Asn His Ala Arg Ile Tyr 290 295 300 Met Ala Asp Leu Glu Ser Ser Leu His Tyr Ile Phe Leu Val Glu Val 305 310 315 320 Gly Lys Phe Ser Val Leu Glu Gly Gln Arg Leu Leu Ala Leu Lys Lys 325 330 335 Leu Val Ala Val Leu Ala Lys Tyr Phe Pro Gly Arg Pro Leu Ala Gln 340 345 350 Asn Phe Leu His Ser Ile His Asp Trp Leu Gln Arg Gln Gln Arg Lys 355 360 365 Lys Ile Pro Tyr Lys Phe Phe Arg Ala Ala Leu Asp Asn Arg Lys Glu 370 375 380 Gly Ile Val Leu Thr Glu Lys Val Asn Trp Val Gly Cys Gln Gly Ser 385 390 395 400 Lys Pro His Phe Arg Gly Phe Pro Cys Ser Leu Trp Ile Leu Phe His 405 410 415 Phe Leu Thr Val Gln Ala Ser Arg Tyr Ser Glu Asn His Pro Gln Glu 420 425 430 Pro Ala Asp Gly Gln Glu Val Leu Gln Ala Met Arg Ser Tyr Val Gln 435 440 445 Trp Phe Phe Gly Cys Arg Asp Cys Ala Glu His Phe Glu Asn Met Ala 450 455 460 Ala Ser Thr Met His Arg Val Arg Ser Pro Thr Ser Ala Val Leu Trp 465 470 475 480 Leu Trp Thr Ser His Asn Lys Val Asn Ala Arg Leu Ser Gly Ala Pro 485 490 495 Ser Glu Asp Pro Tyr Phe Pro Lys Val Gln Trp Pro Leu Arg Glu Leu 500 505 510 Cys Phe Asp Cys His Asn Glu Ile Asn Gly Arg Glu Pro Val Trp Asp 515 520 525 Leu Glu Ala Thr Tyr Arg Phe Leu Lys Ala His Phe Ser Ser Glu Asn 530 535 540 Ile Ile Leu Asp Thr Pro Val Ala Gly Leu Ala Thr Gln Arg Asn Pro 545 550 555 560 Gln Ile Leu Gly Ala Thr Pro Glu Pro His Met 565 570 <210> 28 <211> 473 <212> PRT <213> Unknown <220> <223> Lipoprotein lipase (Lip) <400> 28 Met Glu Ser Lys Ala Leu Leu Leu Val Ala Leu Gly Val Trp Leu Gln 1 5 10 15 Ser Leu Thr Ala Ser Gln Gly Xaa Ala Ala Ala Asp Gly Gly Arg Asp 20 25 30 Phe Thr Asp Ile Glu Ser Lys Phe Ala Leu Arg Thr Pro Asp Asp Thr 35 40 45 Ala Glu Asp Asn Cys His Leu Ile Pro Gly Ile Ala Glu Ser Val Ser 50 55 60 Asn Cys His Phe Asn His Ser Ser Lys Thr Phe Val Val Ile His Gly 65 70 75 80 Trp Thr Val Thr Gly Met Tyr Glu Ser Trp Val Pro Lys Leu Val Ala 85 90 95 Ala Leu Tyr Lys Arg Glu Pro Asp Ser Asn Val Ile Val Val Asp Trp 100 105 110 Leu Tyr Arg Ala Gln Gln His Tyr Pro Val Ser Ala Gly Tyr Thr Lys 115 120 125 Leu Val Gly Asn Asp Val Ala Arg Phe Ile Asn Trp Met Glu Glu Glu 130 135 140 Phe Asn Tyr Pro Leu Asp Asn Val His Leu Leu Gly Tyr Ser Leu Gly 145 150 155 160 Ala His Ala Ala Gly Val Ala Gly Ser Leu Thr Asn Lys Lys Val Asn 165 170 175 Arg Ile Thr Gly Leu Asp Pro Ala Gly Pro Asn Phe Glu Tyr Ala Glu 180 185 190 Ala Pro Ser Arg Leu Ser Pro Asp Asp Ala Asp Phe Val Asp Val Leu 195 200 205 His Thr Phe Thr Arg Gly Ser Pro Gly Arg Ser Ile Gly Ile Gln Lys 210 215 220 Pro Val Gly His Val Asp Ile Tyr Pro Asn Gly Gly Thr Phe Gln Pro 225 230 235 240 Gly Cys Asn Ile Gly Glu Ala Ile Arg Val Ile Ala Glu Arg Gly Leu 245 250 255 Gly Asp Val Asp Gln Leu Val Lys Cys Ser His Glu Arg Ser Ile His 260 265 270 Leu Phe Ile Asp Ser Leu Leu Asn Glu Glu Asn Pro Ser Lys Ala Tyr 275 280 285 Arg Cys Asn Ser Lys Glu Ala Phe Glu Lys Gly Leu Cys Leu Ser Cys 290 295 300 Arg Lys Asn Arg Cys Asn Asn Val Gly Tyr Glu Ile Asn Lys Val Arg 305 310 315 320 Ala Lys Arg Ser Ser Lys Met Tyr Leu Lys Thr Arg Ser Gln Met Pro 325 330 335 Tyr Lys Val Phe His Tyr Gln Val Lys Ile His Phe Ser Gly Thr Glu 340 345 350 Ser Asp Lys Gln Leu Asn Gln Ala Phe Glu Ile Ser Leu Tyr Gly Thr 355 360 365 Val Ala Glu Ser Glu Asn Ile Pro Phe Thr Leu Pro Glu Val Ser Thr 370 375 380 Asn Lys Thr Tyr Ser Phe Leu Ile Tyr Thr Glu Val Asp Ile Gly Glu 385 390 395 400 Leu Leu Met Met Lys Leu Lys Trp Lys Ser Asp Ser Tyr Phe Ser Trp 405 410 415 Ser Asp Trp Trp Ser Ser Pro Gly Phe Val Ile Glu Lys Ile Arg Val 420 425 430 Lys Ala Gly Glu Thr Gln Lys Lys Val Ile Phe Cys Ala Arg Glu Lys 435 440 445 Val Ser His Leu Gln Lys Gly Lys Asp Ser Ala Val Phe Val Lys Cys 450 455 460 His Asp Lys Ser Leu Lys Lys Ser Gly 465 470 <210> 29 <211> 1212 <212> PRT <213> unknown <220> <223> Nest-1 <400> 29 Met Leu Asp Ala Ser Gly Trp Lys Pro Ala Ala Trp Thr Trp Val Leu 1 5 10 15 Leu Leu Gln Leu Leu Leu Ala Gly Pro Gly Asp Cys Leu Ser Arg Gln 20 25 30 Glu Leu Phe Pro Phe Gly Pro Gly Gln Gly Asp Leu Glu Leu Glu Ala 35 40 45 Gly Asp Asp Val Val Ser Pro Ala Leu Glu Leu Ile Gly Glu Leu Ser 50 55 60 Phe Tyr Asp Arg Ser Asp Ile Thr Ser Val Tyr Val Thr Thr Asn Gly 65 70 75 80 Ile Ile Ala Met Ser Glu Pro Pro Ala Arg Glu Ser His Pro Gly Thr 85 90 95 Phe Pro Pro Ser Phe Gly Ser Val Ala Pro Phe Leu Ala Asp Leu Asp 100 105 110 Thr Thr Asp Gly Leu Gly Asn Val Tyr Tyr Arg Glu Asp Leu Ser Pro 115 120 125 Ser Ile Met Gln Met Ala Ala Glu Tyr Val Gln Arg Gly Phe Pro Glu 130 135 140 Val Pro Phe Gln Pro Thr Ser Val Val Val Val Thr Trp Glu Ser Val 145 150 155 160 Ala Pro Tyr Glu Gly Pro Ser Gly Ser Ser Ala Gln Glu Gly Lys Arg 165 170 175 Asn Thr Phe Gln Ala Val Leu Ala Ser Ser Asn Ser Ser Ser Tyr Ala 180 185 190 Ile Phe Leu Tyr Pro Glu Asp Gly Leu Gln Phe Phe Thr Thr Phe Ser 195 200 205 Lys Lys Asp Glu Asn Gln Val Pro Ala Met Val Gly Phe Ser Gln Gly 210 215 220 Leu Val Gly Phe Leu Trp Arg Ser Asp Gly Ala Tyr Asn Ile Phe Ala 225 230 235 240 Asn Asp Arg Glu Ser Ile Glu Asn Leu Ala Lys Ser Ser Asn Ala Gly 245 250 255 His Gln Gly Val Trp Val Phe Glu Ile Gly Ser Pro Ala Thr Ala Lys 260 265 270 Gly Val Val Ser Ala Asp Val Asn Leu Gly Leu Asp Asp Asp Gly Ser 275 280 285 Asp Tyr Glu Asp Glu Glu Tyr Asp Leu Ala Thr Ser His Leu Gly Leu 290 295 300 Glu Asp Met Ala Thr Gln Pro Phe Pro Ser Pro Ser Pro Arg Arg Gly 305 310 315 320 Asn Thr His Pro His Asp Val Pro Arg Val Leu Ser Pro Ser Tyr Glu 325 330 335 Ala Thr Glu Arg Pro His Gly Ile Pro Thr Glu Arg Thr Arg Thr Phe 340 345 350 Gln Leu Pro Ala Glu Arg Phe His Gln Gln His Pro Gln Val Ile Asp 355 360 365 Val Asp Glu Val Glu Glu Thr Gly Ile Val Phe Ser Tyr Asn Ile Gly 370 375 380 Ser Gln Gln Thr Cys Ala Asn Asn Arg His Gln Cys Ser Val His Ala 385 390 395 400 Glu Cys Arg Asp Tyr Ala Thr Gly Phe Cys Cys Arg Cys Val Ala Asn 405 410 415 Tyr Thr Gly Asn Gly Arg Gln Cys Val Ala Glu Gly Ser Pro Gln Arg 420 425 430 Val Asn Gly Lys Val Lys Gly Arg Ile Phe Val Gly Asn Ser Gln Val 435 440 445 Pro Val Val Phe Glu Asn Thr Asp Leu His Ser Tyr Val Val Met Asn 450 455 460 His Gly Arg Ser Tyr Thr Ala Ile Ser Thr Ile Pro Glu Thr Val Gly 465 470 475 480 Tyr Ser Leu Leu Pro Leu Ala Pro Ile Gly Gly Ile Ile Gly Trp Met 485 490 495 Phe Ala Val Glu Gln Asn Gly Phe Lys Asn Gly Phe Ser Ile Thr Gly 500 505 510 Gly Glu Phe Thr Arg Gln Ala Glu Val Thr Phe Leu Gly His Pro Gly 515 520 525 Lys Leu Val Leu Lys Gln His Phe Ser Gly Ile Asp Glu His Gly His 530 535 540 Leu Thr Ile Asn Thr Glu Leu Asp Gly Arg Val Pro Gln Ile Pro Tyr 545 550 555 560 Gly Ser Ser Val His Ile Glu Pro Tyr Thr Glu Leu Tyr His Tyr Ser 565 570 575 Ser Ser Val Ile Thr Ser Ser Ser Thr Arg Glu Tyr Thr Val Thr Glu 580 585 590 Pro Asp Pro Asp Gly Thr Ala Pro Ser His Thr His Val Tyr Gln Trp 595 600 605 Arg Gln Thr Ile Thr Phe Gln Glu Cys Val His Asp Asp Ser Arg Pro 610 615 620 Ala Leu Pro Ser Thr Gln Gln Leu Ser Val Asp Ser Val Phe Val Leu 625 630 635 640 Tyr Asn Gln Glu Glu Arg Ile Leu Arg Tyr Ala Leu Ser Asn Ser Ile 645 650 655 Gly Pro Val Arg Glu Gly Ser Pro Asp Ala Leu Gln Asn Pro Cys Tyr 660 665 670 Ile Gly Thr His Gly Cys Asp Ser Asn Ala Ala Cys Arg Pro Gly Pro 675 680 685 Gly Thr Gln Phe Thr Cys Glu Cys Ser Ile Gly Phe Arg Gly Asp Gly 690 695 700 Gln Thr Cys Tyr Asp Ile Asp Glu Cys Ser Glu Gln Pro Ser Arg Cys 705 710 715 720 Gly Asn His Ala Ala Cys Asn Asn Ser Pro Gly Ala Tyr Leu Cys Glu 725 730 735 Cys Val Glu Gly Tyr His Phe Ser Asp Gly Gly Ile Cys Val Ala Asp 740 745 750 Val Asp Gln Arg Pro Ile Asn Tyr Cys Glu Thr Gly Leu His Asn Cys 755 760 765 Asp Ile Pro Gln Arg Ala Gln Cys Ile Tyr Met Gly Gly Ser Ser Tyr 770 775 780 Thr Cys Ser Cys Leu Pro Gly Phe Ser Gly Asp Gly Arg Ala Cys Gln 785 790 795 800 Asp Val Asp Glu Cys Gln Leu Ser Arg Cys His Pro Asp Ala Phe Cys 805 810 815 Tyr Asn Thr Pro Gly Ser Phe Thr Cys Gln Cys Lys Pro Gly Tyr Gln 820 825 830 Gly Asp Gly Phe Gln Cys Val Pro Gly Glu Val Gly Lys Thr Arg Cys 835 840 845 Gln Leu Glu Arg Glu His Ile Leu Gly Ala Ser Gly Val Ala Asp Ala 850 855 860 Gln Gln Pro Arg Leu Leu Gly Met Tyr Val Pro Gln Cys Asp Glu Tyr 865 870 875 880 Gly His Tyr Glu Pro Thr Gln Cys His His Gly Thr Gly Tyr Cys Trp 885 890 895 Cys Val Asp Arg Asp Gly Arg Glu Leu Glu Gly Thr Arg Thr Gln Pro 900 905 910 Gly Met Arg Pro Pro Cys Leu Ser Thr Val Ala Pro Pro Ile His Gln 915 920 925 Arg Pro Val Val Pro Thr Ala Val Ile Pro Leu Pro Pro Gly Thr His 930 935 940 Leu Leu Phe Ala Gln Thr Gly Lys Ile Glu Arg Leu Pro Leu Glu Gly 945 950 955 960 Asn Thr Met Lys Lys Thr Glu Ala Lys Ala Phe Phe His Ile Pro Ala 965 970 975 Lys Val Ile Ile Gly Leu Ala Phe Asp Cys Val Asp Lys Val Val Tyr 980 985 990 Trp Thr Asp Ile Ser Glu Pro Ser Ile Gly Arg Ala Ser Leu His Gly 995 1000 1005 Gly Glu Pro Thr Thr Ile Ile Arg Gln Asp Leu Gly Ser Pro Glu Gly 1010 1015 1020 Ile Ala Leu Asp His Leu Gly Arg Asn Ile Phe Trp Thr Asp Ser Gln 1025 1030 1035 1040 Leu Asp Arg Ile Glu Val Ala Arg Met Asp Gly Thr Gln Arg Arg Val 1045 1050 1055 Leu Phe Asp Thr Gly Leu Val Asn Pro Arg Gly Ile Val Thr Asp Ser 1060 1065 1070 Val Gly Gly Asn Leu Tyr Trp Thr Asp Trp Asn Arg Glu Asn Pro Lys 1075 1080 1085 Ile Glu Thr Ser Tyr Met Asp Gly Thr Asn Arg Arg Ile Leu Ala Gln 1090 1095 1100 Asp Asn Leu Gly Leu Pro Asn Gly Leu Thr Phe Asp Ala Phe Ser Ser 1105 1110 1115 1120 Gln Leu Cys Trp Val Asp Ala Gly Thr His Arg Ala Glu Cys Leu Asn 1125 1130 1135 Pro Ala Gln Pro Ser Ser Arg Lys Val Leu Glu Gly Leu Gln Tyr Pro 1140 1145 1150 Phe Ala Val Thr Ser Tyr Gly Lys Asn Leu Tyr Tyr Thr Asp Trp Lys 1155 1160 1165 Thr Asn Ser Val Ile Ala Met Asp Leu Ala Ile Ser Lys Glu Met Asp 1170 1175 1180 Ala Phe Thr Pro Thr Ser Arg Pro Gly Tyr Met Ala Ser Pro Leu Pro 1185 1190 1195 1200 Cys Pro Asn Ala Leu Lys Ala Thr Thr Thr Ala Gln 1205 1210 <210> 30 <211> 527 <212> PRT <213> unknown <220> <223> Proteomere <400> 30 Met Asp Asp Arg Leu Leu Thr Val Leu Leu Leu Leu Leu Gly Val Ser 1 5 10 15 Gly Pro Trp Gly Gln Gly Gln Glu Pro Glu Gly Pro Ser Glu Val Leu 20 25 30 Pro Glu Glu Ser Ser Gly Glu Glu Val Pro Lys Glu Asp Gly Ile Leu 35 40 45 Val Leu Ser His His Thr Leu Ser Leu Ala Leu Gln Glu His Pro Ala 50 55 60 Leu Met Val Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu 65 70 75 80 Ala Pro Glu Tyr Ser Lys Ala Ala Ala Leu Leu Ala Ala Glu Ser Ala 85 90 95 Ser Val Thr Leu Ala Lys Val Asp Gly Pro Ala Glu Pro Glu Leu Thr 100 105 110 Lys Glu Phe Gly Val Val Gly Tyr Pro Thr Leu Lys Phe Phe Gln Asn 115 120 125 Gly Asn Arg Thr Asn Pro Glu Glu Tyr Thr Gly Pro Gln Lys Ala Glu 130 135 140 Gly Ile Ala Glu Trp Leu Arg Arg Arg Val Gly Pro Ser Ala Lys Arg 145 150 155 160 Leu Glu Asp Glu Glu Asp Val Gln Ala Leu Thr Asp Lys Trp Glu Val 165 170 175 Val Val Ile Gly Phe Phe Gln Asp Leu Gln Gly Glu Asp Val Ala Thr 180 185 190 Phe Leu Ala Leu Ala Arg Asp Ala Leu Asp Ile Thr Phe Gly Phe Thr 195 200 205 Asp Gln Pro Gln Leu Phe Gln Lys Phe Gly Leu Thr Lys Asp Thr Val 210 215 220 Ile Leu Phe Lys Lys Phe Asp Glu Gly Arg Ala Asp Phe Pro Val Asp 225 230 235 240 Lys Asp Thr Gly Leu Asp Leu Gly Asp Leu Ser Arg Phe Leu Val Thr 245 250 255 His Ser Met His Leu Val Thr Glu Phe Asn Ser Gln Thr Ser Pro Lys 260 265 270 Ile Phe Ala Ala Lys Ile Leu Asn His Leu Leu Leu Phe Val Asn Lys 275 280 285 Thr Leu Ala Gln His Arg Glu Leu Leu Thr Asp Phe Arg Glu Ala Ala 290 295 300 Pro Pro Phe Arg Gly Gln Val Leu Phe Val Met Val Asp Val Ala Ala 305 310 315 320 Asp Asn Asp His Val Leu Asn Tyr Phe Gly Leu Lys Ala Glu Glu Ala 325 330 335 Pro Thr Leu Arg Leu Ile Asn Val Glu Thr Thr Lys Lys Tyr Ala Pro 340 345 350 Thr Gly Leu Val Pro Ile Thr Ala Ala Ser Val Ala Ala Phe Cys Gln 355 360 365 Ala Val Leu His Gly Gln Val Lys Pro Tyr Leu Leu Ser Gln Glu Ile 370 375 380 Pro Pro Asp Trp Asp Glu Arg Pro Val Lys Thr Leu Val Gly Lys Asn 385 390 395 400 Phe Glu Gln Val Ala Phe Asp Glu Thr Lys Asn Val Phe Val Lys Phe 405 410 415 Tyr Ala Pro Trp Cys Ser His Cys Lys Glu Met Ala Pro Ala Trp Glu 420 425 430 Ala Leu Ala Glu Lys Tyr Arg Asp Arg Glu Asp Ile Val Ile Ala Glu 435 440 445 Leu Asp Ala Thr Ala Asn Glu Leu Glu Ala Phe Ser Val His Gly Tyr 450 455 460 Pro Thr Leu Lys Phe Phe Pro Ala Gly Pro Asp Arg Lys Val Ile Glu 465 470 475 480 Tyr Lys Ser Thr Arg Asp Leu Glu Thr Phe Ser Lys Phe Leu Asp Ser 485 490 495 Gly Gly Asn Leu Pro Glu Glu Glu Pro Lys Glu Pro Ala Ile Ser Thr 500 505 510 Pro Glu Ile Gln Asp Asn Ser Thr Val Gly Pro Lys Glu Glu Leu 515 520 525 <210> 31 <211> 236 <212> PRT <213> Unknown <220> <223> mCherry <400> 31 Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe 1 5 10 15 Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe 20 25 30 Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr 35 40 45 Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp 50 55 60 Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His 65 70 75 80 Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe 85 90 95 Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val 100 105 110 Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys 115 120 125 Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys 130 135 140 Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly 145 150 155 160 Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly 165 170 175 His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val 180 185 190 Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser 195 200 205 His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly 210 215 220 Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys 225 230 235 <210> 32 <211> 711 <212> DNA <213> Unknown <220> <223> The gene of mCherry <400> 32 atggtgagca agggcgagga ggataacatg gccatcatca aggagttcat gcgcttcaag 60 gtgcacatgg agggctccgt gaacggccac gagttcgaga tcgagggcga gggcgagggc 120 cgcccctacg agggcaccca gaccgccaag ctgaaggtga ccaagggtgg ccccctgccc 180 ttcgcctggg acatcctgtc ccctcagttc atgtacggct ccaaggccta cgtgaagcac 240 cccgccgaca tccccgacta cttgaagctg tccttccccg agggcttcaa gtgggagcgc 300 gtgatgaact tcgaggacgg cggcgtggtg accgtgaccc aggactcctc cctgcaggac 360 ggcgagttca tctacaaggt gaagctgcgc ggcaccaact tcccctccga cggccccgta 420 atgcagaaga agaccatggg ctgggaggcc tcctccgagc ggatgtaccc cgaggacggc 480 gccctgaagg gcgagatcaa gcagaggctg aagctgaagg acggcggcca ctacgacgct 540 gaggtcaaga ccacctacaa ggccaagaag cccgtgcagc tgcccggcgc ctacaacgtc 600 aacatcaagt tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgaa 660 cgcgccgagg gccgccactc caccggcggc atggacgagc tgtacaagta a 711 <210> 33 <211> 22 <212> PRT <213> Unknown <220> <223> SP7.2 <400> 33 Met His Arg Pro Glu Ala Met Leu Leu Leu Leu Thr Leu Ala Leu Leu 1 5 10 15 Gly Gly Pro Thr Trp Ala 20 <210> 34 <211> 66 <212> DNA <213> Unknown <220> <223> Gene of SP7.2 <400> 34 atgcaccggc cagaggccat gctgctgctg ctcacgcttg ccctcctggg gggccccacc 60 tgggca 66 <210> 35 <211> 88 <212> DNA <213> The snowstorm <220> <223> Cat_SP_F <400> 35 agggggtacc gccaccatgt ggtggtcctt gattccgctc tcttgcctgc tggcactggc aagtgccgtg agcaagggcg 88 <210> 36 <211> 54 <212> DNA <213> The snowstorm <220> <223> CC_SP_F1 <400> 36 acagttgctg cctgtagcat ctatgtgctg gccgtgagca agggcgagga ggat <210> 37 <211> 78 <212> DNA <213> The snowstorm <220> <223> CC_SP_F2 <400> 37 agggggtacc gccaccatgc agttctccgc aagaacgctt ctgtgcctgc tactcacagt tgctgcctgt 78 <210> 38 <211> 52 <212> DNA <213> The snowstorm <220> <223> Nuc_SP_F1 <400> 38 ggtcccgtgc atgcttactg ctctggaagc tgtgagcaag ggcgaggagg at <210> 39 <211> 81 <212> DNA <213> Artificial sequence <220> <223> Nuc_SP_F2 <400> 39 agggggtacc gccaccatga ggtggaagat catccagcta cagtactgtt ttcttttggt 60 cccgtgcatg cttactgctc t 81 <210> 40 <211> 60 <212> DNA <213> Artificial sequence <220> <223> Clus_SP_F1 <400> 40 gggctgctgc tgacctggga caatggcatg gtcctgggag tgagcaaggg cgaggaggat 60 60 <210> 41 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Clus_SP_F2 <400> 41 agggggtacc gccaccatga agattctcct gttgtgcgtg gggctgctgc tgacctggga 60 caatg 65 <210> 42 <211> 90 <212> DNA <213> Artificial sequence <220> <223> Pig_SP_F <400> 42 ggtaccgcca ccatgcaggc cctggtgcta ctcctctgga caggagccct gcttgggcat 60 ggcagcagcg tgagcaaggg cgaggaggat 90 <210> 43 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Proco_SP_F1 <400> 43 ccttgtggcc tgggtactgc ctcttgcccg aggcgtgagc aagggcgagg aggat 55 <210> 44 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> Proco_SP_F2 <400> 44 agggggtacc gccaccatgc tgcctgctgt cctaacctcc ctcctggggc cattccttgt 60 ggcctgggta ctgcct 76 <210> 45 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> Sulf_SP_F1 <400> 45 atgctactgc tgctgctgcc gccgctgctg ctcgcggtgc ccggcgctgg cgcggtgagc 60 aagggcgagg aggat 75 <210> 46 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Sulf_SP_F2 <400> 46 agggggtacc gccaccatga ggaggtgcgg ccgccactcg gggtcgccgt cgcagatgct 60 actgctgctg ctgccgccgc t 81 <210> 47 <211> 58 <212> DNA <213> Artificial sequence <220> <223> Lip_SP_F1 <400> 47 ggtggctctg ggagtgtggc tccagagttt gaccgccgtg agcaagggcg aggaggat 58 <210> 48 <211> 62 <212> DNA <213> Artificial sequence <220> <223> Lip_SP_F2 <400> 48 agggggtacc gccaccatgg agagcaaagc cctgctcctg gtggctctgg gagtgtggct 60 cc 62 <210> 49 <211> 72 <212> DNA <213> Artificial sequence <220> <223> Nid_SP_F1 <400> 49 gggtgctgct gctgcagcta ttgctggcgg ggcccggaga ctgcctgagc cgtgagcaag 60 ggcgaggagg at 72 <210> 50 <211> 83 <212> DNA <213> Artificial sequence <220> <223> Nid_SP_F2 <400> 50 agggggtacc gccaccatgc tggacgcgag cggctggaag cccgcggcgt ggacatgggt 60 gctgctgctg cagctattgc tgg 83 <210> 51 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Pro_SP_F1 <400> 51 tgttgctgct cctgctgggt gtctcaggcc catggggaca gggagtgagc aagggcgagg 60 aggat 65 <210> 52 <211> 65 <212> DNA <213> Artificial sequence <220> <223> Pro_SP_F2 <400> 52 agggggtacc gccaccatgg atgatcggct cctgacagtg ttgctgctcc tgctgggtgt 60 ctcag 65 <210> 53 <211> 36 <212> DNA <213> Artificial sequence <220> <223> mCherry XhoR <400> 53 agggctcgag tttacttgta cagctcgtcc atgccg 36 <210> 54 <211> 36 <212> DNA <213> Artificial sequence <220> <223> Bgl_Hprt 5'F <400> 54 agggagatct caccagtttt cattttctgt gtgaga 36 <210> 55 <211> 42 <212> DNA <213> Artificial sequence <220> <223> Hprt 5'_NruR <400> 55 agggtcgcga attaggaata caaaataaat ctaggtcata gc 42 <210> 56 <211> 35 <212> DNA <213> Artificial sequence <220> <223> Sal_Hprt 3'F <400> 56 aggggtcgac cactatgtcg aggatttgga aaagg 35 <210> 57 <211> 34 <212> DNA <213> Artificial sequence <220> <223> Hprt 3'_SalR <400> 57 aggggtcgac gaggatggga gtgagagtga actg 34 <210> 58 <211> 218 <212> PRT <213> unknown <220> <223> Light chain of pembrolizumab <400> 58 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser 20 25 30 Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35 40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg 85 90 95 Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 59 <211> 447 <212> PRT <213> Unknown <220> <223> Heavy chain of pembrolizumab <400> 59 Gln Val Gln Leu Val Gln Ser Gly Val Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe 50 55 60 Lys Asn Arg Val Thr Leu Thr Thr Asp Ser Ser Thr Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445

Claims

A signal peptide consisting of the amino acid sequence of SEQ ID NO:

2.

2. An expression cassette, wherein a nucleic acid sequence encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 2 and a gene encoding a target protein are operably linked, wherein the target protein is pembrolizumab.

3. 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 IDNO: 2 and a gene encoding the target protein are operably linked, wherein the target protein is pembrolizumab.

4. A transformed cell, wherein the expression vector according to claim 3 is introduced into a host cell. The transformed cell according to claim 4 , wherein the host cell is a Chinese hamster ovary cell.

6. A method for producing a target protein, the method comprising: 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 signal peptide consisting of the amino acid sequence of SEQ ID NO: 2 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. The method according to claim 6 , further comprising purifying the recovered target protein.

8. The method of claim 6, wherein the cells are Chinese hamster ovary cells.

9. The method according to claim 6, wherein the signal peptide consisting of the amino acid sequence of SEQ ID NO: 2 is cleaved at the N-terminal cleavage site of the target protein.

10. The method according to claim 6, wherein the target protein is the target protein itself to which no additional amino acids are added, and the nucleic acid sequence encoding the signal peptide has been removed.

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