CHO CELL-DERIVED PROTEIN SECRETORY FACTORS AND EXPRESSION VECTORS COMPRISING THE SAME

Abstract

The present invention relates to a CHO cell-derived protein secretory factor, an expression cassette in which a nucleic acid sequence encoding the protein secretory 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.

Claims

1. A protein secretory factor consisting of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

2. The protein secretory factor of claim 1, wherein the protein is an endogenous protein or a foreign protein.

3. An expression cassette in which a nucleic acid sequence encoding a protein secretory 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 a target protein are operably linked.

4. The expression cassette of claim 3, wherein the target protein is selected from the group consisting of 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, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucodase, galactosidase, glucocerebrosidase, and glucuronidase.

5. The expression cassette of claim 3, wherein the nucleic acid sequence encoding the protein secretory 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 consists of the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

6. The expression cassette of claim 3, wherein the expression cassette further comprises a nucleic acid sequence encoding any one of protein secretory factors consisting of an 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.

7. The expression cassette of claim 3, wherein the expression cassette expresses a target protein in which no additional amino acids have been added, from which the nucleic acid sequence encoding the secretory factor has been removed, when expressed in a cell, as it is.

8. An expression vector for secreting a target protein, comprising the expression cassette of claim 3.

9. The expression vector of claim 8, wherein the target protein is selected from the group consisting of 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, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucodase, galactosidase, glucocerebrosidase, and glucuronidase.

10. The expression vector of claim 8, wherein the nucleic acid sequence encoding the protein secretory 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 consists of the nucleic acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.

11. The expression vector of claim 8, wherein the expression vector further comprises a nucleic acid sequence encoding any one of protein secretory factors consisting of an 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.

12. The expression vector of claim 8, wherein the expression vector expresses a target protein in which no additional amino acids have been added, from which the nucleic acid sequence encoding the secretory factor has been removed, when expressed in a cell, as it is.

13. A transformed cell in which the expression vector of claim 8 is introduced into a host cell.

14. The transformed cell of claim 13, wherein the host cell is a Chinese hamster ovary cell (CHO cell).

15. A method for producing a target protein, comprising: i) culturing a transformed cell comprising an expression vector for secreting a target protein, which includes an expression cassette in which a nucleic acid sequence encoding a protein secretory 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 a target protein are operably linked; and ii) recovering the target protein from the culture medium or culture supernatant of the cultured cells.

16. The method of claim 15, further comprising purifying the recovered target protein.

17. The method of claim 15, wherein the host cell is a Chinese hamster ovary cell (CHO cell).

18. The method of claim 15, wherein the protein secretory 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 is cleaved at the N-terminal cleavage site of the target protein.

19. The method of claim 15, wherein the target protein is a target protein itself in which no additional amino acids have been added, from which the nucleic acid sequence encoding the secretory factor has been removed.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0078] FIGS. 1(a) to 1(j) are graphs is a graph predicting the signal peptides using SignalP4.1.

[0079] FIG. 2 is a diagram confirming the expression levels of signal peptide-mCherry through temporary expression.

[0080] FIG. 3 is a diagram showing a vector map for site specific integration.

[0081] FIG. 4 is a diagram comparing the expression levels of mCherry in the site-specific integrated cells.

[0082] FIGS. 5(a) to 5(c) are graphs is a graph showing mass data that confirms the cleavage of an anti-PD-1 antibody fused with SP7.2 and Clus.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0083] Hereinafter, the present invention will be described in more detail by way of Examples. However, these Examples are given for illustrative purposes only, and the scope of the invention is not intended to be limited to or by these Examples

Example 1. Preparation of Novel Signal Peptide Sequences Derived from CHO Cells

1-1. CHO HCP Mass Analysis

[0084] Four types (ADH, BSA, PHO, and ENL) of MassPREPTM Protein Digest Standard were added to the culture medium of CHO cells (DXB11) treated with trypsin as follows. Among the four types of MassPREPTM Protein Digest Standard, PHO was used as an internal standard for calculating the concentration of each host cell protein (HCP). Each sample was analyzed for host cell protein (HCP) using a 2D LC (high pH RP/Low pH RP)-Q-TOF (UDMSe) method.

[0085] MS data (UDMSe) was obtained for each fraction by injecting directly into Q-TOF MS in the 2D column. 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. Thereafter, the HCP was 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.

1-2. Selection of Signal Peptides From CHO HCP Data

[0086] The proteins were arranged in the order of high concentrations, and the amino acid sequence of each protein was confirmed from the CHO Genome Database (http://chogenome.org). The thus-obtained amino acid sequences were entered into SignalP4.1 Server (http://www.cbs.dtu.dk/services/SignalP/) to predict the presence of secretory proteins and signal peptide sequences (Table 1, FIGS. 1 and Table 4).

TABLE-US-00001 HCP Secretion Protein Predicted via SignalP4.1 Protein SEQ ID NO: SP (Signal Peptide) Sequence SEQ ID NO: DNA Sequence Cathepsin B (Cat) 1 MWWSLIPLSC LLALASA 11 ATG TGG TGG TCC TTG ATT CCG CTC TCT TGC CTG CTG GCA CTG GCA AGT GCC C—C motif chemokine 2 MQFSARTLLC LLLTVAACSI YVLA 12 ATG CAG TTC TCC GCA AGA ACG CTT CTG TGC CTG CTA CTC ACA GTT GCT GCC TGT AGC ATC TAT GTG CTG GCC Nucleobindin-2 (Nuc) 3 MRWKIIQLQY CFLLVPCMLT ALEA 13 ATG AGG TGG AAG ATC ATC CAG CTA CAG TAC TGT TTT CTT TTG GTC CCG TGC ATG CTT ACT GCT CTG GAA GCT Clusterin (Clus) 4 MKILLLCVGL LLTWDNGMVL G 14 ATG AAG ATT CTC CTG TTG TGC GTG GGG CTG CTG CTG ACC TGG GAC AAT GGC ATG GTC CTG GGA Pigment epithelium-derived factor (Pig) 5 MQALVLLLWT GALLGHGSS 15 ATG CAG GCC CTG GTG CTA CTC CTC TGG ACA GGA GCC CTG CTT GGG CAT GGC AGC AGC Procollagen C-endopeptida se enhancer 1 (Proco) 6 MLPAVLTSLL GPFLVAWVLP LARG 16 ATG CTG CCT GCT GTC CTA ACC TCC CTC CTG GGG CCA TTC CTT GTG GCC TGG GTA CTG CCT CTT GCC CGA GGC Sulfhydryl oxidase (Sulf) 7 MRRCGRHSGS PSQMLLLLLP PLLLAVPGAG A 17 ATG AGG AGG TGC GGC CGC CAC TCG GGG TCG CCG TCG CAG ATG CTA CTG CTG CTG CTG CCG CCG CTG CTG CTC GCG GTG CCC GGC GCT GGC GCG Lipoprotein lipase (Lip) 8 MESKALLLVA LGVWLQSLTA 18 ATG GAG AGC AAA GCC CTG CTC CTG GTG GCT CTG GGA GTG TGG CTC CAG AGT TTG ACC GCC Nidogen-1 (Nid) 9 MLDASGWKPA AWTWVLLLQL LLAGPGDCLS 19 ATG CTG GAC GCG AGC GGC TGG AAG CCC GCG GCG TGG ACA TGG GTG CTG CTG CTG CAG CTA TTG CTG GCG GGG CCC GGA GAC TGC CTG AGC Protein disulfide-isom erase (Pro) 10 MDDRLLTVLL LLLGVSGPWG QG 20 ATG GAT GAT CGG CTC CTG ACA GTG TTG CTG CTC CTG CTG GGT GTC TCA GGC CCA TGG GGA CAG GGA

Example 2. Selection of CHO-Derived High Efficiency Signal Peptides via Temporary Expression

2-1. Preparation of Recombinant Protein Expression Vectors for Temporary Expression

[0087] In order to confirm whether the 10 types of signal peptides selected in Example 1 can be used as general secretory factors, mCherry (pmCherry Vector, Clontech, 632522) protein was selected as the target protein.

[0088] The sequence of the polynucleotide of the gene encoding the mCherry protein is shown in Table 2.

TABLE-US-00002 Protein SEQ ID NO: Amino Acid Sequence SEQ ID NO: DNA Sequence mCherry 31 MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK 32 ATGGTGAGCAAGGGCGAGGAGGATAACATGGCCATCATCAAGGAGTTCATGCGCTTCAAGGTGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCTCAGTTCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATCCCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGTGAAGCTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATCAAGCAGAGGCTGAAGCTGAAGGACGGCGGCCACTACGACGCTGAGGTCAAGACCACCTACAAGGCCAAGAAGCCCGTGCAGCTGCCCGGCGCCTACAACGTCAACATCAAGTTGGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAACGCGCCGAGGGCCGCCACTCCACCGGCGGCATGGACGAGCTGTACAAGTAA

[0089] Based on the gene encoding the mCherry protein, PCR was performed using the signal peptide sequences identified from the CHO HCP Mass Data and primers containing Kpnl/Xhol, and the mCherry expressed by the 10 types of signal peptide sequences was constructed.

[0090] 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 the 10 types of signal peptide sequences were cleaved with Kpnl and Xhol and then cloned into pcDNA3.1 (+) (Invitrogen, Cat. No. V790-20) to construct expression vectors.

[0091] Additionally, for use as a positive control, a mCherry protein expression vector fused with the SP7.2 signal peptide (Korean Patent Laid-Open Publication 10-2015-0125402 A), which is a known secretory factor, was prepared in the same manner. The sequence of the SP7.2 signal peptide is shown in Table 3.

TABLE-US-00003 Protein SEQ ID NO: SP Sequence SEQ ID NO: DNA Sequence SP7.2 33 MHRPEAMLLLLTLALLGGPTWA 34 ATGCACCGGCCAGAGGCCATGCTGCTGCTGCTCACGCTTGCCCTCCTGGGGGGCCCCACCTGGGCA

TABLE-US-00004 Protein GeneBank No. Amino Acid Sequence Expected SP Sequence from SignalP4.1 DNA Sequence Clusterin XP_007643887.1 1 MKILLLCVGL LLTWDNGMVL GEQEVSDNEL KEMSTQGSRY INKEIQNAVQ GVKQIKTLIE 61 KTNEERKSLL NSLEEAKKKK EDALDDTRDT EMKLKAFPEV CNETMMALWE ECKPCLKQTC 121 MKFYARVCRS GSGLVGRQLE EFLNQSSPFY FWMNGDRIDS LMESDRQQSQ VLDAMQDSFT 181 RASGIMDMLF QDRFFTHEPQ DTHYFSPFGF PHRRPHFLYP KSRLVRSLIP LSHYGPPSFH 241 DMFQPFLEMI HQAQQAMDVQ FHRPAFQFPD KGLREGEDDR AVCKEIRHNS TGCLKMKGQC 301 EKCQEILSVD CSANNPAQAH LRQELNDSLQ MAERLTQQYN ELLHSLQTKM LNTSSLLEQL 361 NEQFNWVSQL ANLTQGEDQY YLRVSTVTTH SNNSEEPSRV TEVWKLFDS DPITWLPEE 421 VSKDNPKFMD TVAEKALQEY RKKSRAE MKILLLCVGL LLTWDNGMVL G ATG AAG ATT CTC CTG TTG TGC GTG GGG CTG CTG CTG ACC TGG GAC AAT GGC ATG GTC CTG GGA Sulfhydryl oxidase XP_007639037.1 1 MRRCGRHSGS PSQMLLLLLP PLLLAVPGAG AVQVSVLYSS SDPVTVLNAN TVRSTVLRSN 61 GAWAVEFFAS WCGHCIAFAP TWKELAYDVR EWRPVLNLAV LDCAEETNTA VCRDFNISGF 121 PTVRFFKAFS KNGSGITLPV ADASVETLRR KLIDALESHS DMWSSSRPKL KPAKLVEINE 181 FFAETNEDYL VLIFEDKDSY VGREVTLDLF QHHIPVHRVL NTERNAVSKF GWEFPSCYL 241 LFRNGSFSRV PWMESRLFY TSYLKGMSGP ILVDPPTTTI STDAPVTTDV VPTVWKVANH 301 ARIYMADLES SLHYIFLVEV GKFSVLEGQR LLALKKLVAV LAKYFPGRPL AQNFLHSIHD 361 WLQRQQRKKI PYKFFRAALD NRKEGIVLTE KVNWVGCQGS KPHFRGFPCS LWILFHFLTV 421 QASRYSENHP QEPADGQEVL QAMRSYVQWF FGCRDCAEHF ENMAASTMHR VRSPTSAVLW 481 LWTSHNKVNA RLSGAPSEDP YFPKVQWPLR ELCFDCHNEI NGREPVWDLE ATYRFLKAHF 541 SSENIILDTP VAGLATQRNP QILGATPEPH M MRRCGRHSGS PSQMLLLLLP PLLLAVPGAG A ATG AGG AGG TGC GGC CGC CAC TCG GGG TCG CCG TCG CAG ATG CTA CTG CTG CTG CTG CCG CCG CTG CTG CTC GCG GTG CCC GGC GCT GGC GCG Cathepsin B XP_003498026.1 1 MWWSLIPLSC LLALASAHNK PSFHPLSDDL INYINKRNTT WQAGRNFHNV DISYLKRLCG 61 TIMGGPKLPE RVAFAEDMEL PENFDAREQW SNCPTIKQIR DQGSCGSCWA FGAVGAMSDR 121 LCIHTNGHVN VEVSAEDLLT CCGSQCGDGC NGGYPSGAWN FWIKKGLVSG GLYNSHVGCL 181 PYTIPPCEHH VNGSRPQCTG EGDTPKCTKS CEAGYSPSYK EDKHYGYTSY SVSNNEKEIM 241 AEIYKNGPVE GAFTVFSDFL TYKSGVYKHE AGDIMGGHAI RILGWGVENS VPYWLVANSW 301 NVDWGDNGLF KILRGEDHCG IESEIVAGIP RTDLYWGRF MWWSLIPLSC LLALASA ATG TGG TGG TCC TTG ATT CCG CTC TCT TGC CTG CTG GCA CTG GCA AGT GCC Nucleobindin-2 XP_003513452.1 1 MRWKIIQLQY CFLLVPCMLT ALEAVPIDVD KTKVHNTEPV ESARIDPPDT GLYYDEYLKQ 61 VIDVLETDQH FREKLQKADI EEIRSGRLSK ELDLVSHHVR TKLDELKRQE VARLRMLIKA 121 KLDSLQDTGM NHHLLLKQFE HLNHQNPDTF ESSDLDMLIK AATADLEQYD RTRHEEFKKY 181 EMMKEHERRE YLKTLNEEKR KEEESKFEEM KRKHENHPKV NHPGSKDQLK EVWEEADGLD 241 PNDFDPKTFF KLHDVNNDGF LDEQELEALF TRELEKVYDP RNAEDDMIEM EEERLRMREH 301 VMNEIDNNKD RLVTLEEFLR ATEKKEFLEP DSWETLGQQQ LFTEEELKEY ESIIAMQENE 361 LKKRADELQK QKEELQRQHD HLEAQKQEYQ QAVQQLEHKK FQQGIAPSGP AGELEFKPRM MRWKIIQLQY CFLLVPCMLT ALEA ATG AGG TGG AAG ATC ATC CAG CTA CAG TAC TGT TTT CTT TTG GTC CCG TGC ATG CTT ACT GCT CTG GAA GCT Procollagen C-endopeptidase enhancer 1 XP_003510679.1 1 MLPAVLTSLL GPFLVAWVLP LARGQTPNYT RPVFLCGGDV TGESGYVASE GFPNLYPPNK 61 KCIWTITVPE GQTVSLSFRV FDMELHPSCR YDALEVFAGS GTSGQRLGRF CGTFRPAPVV 121 APGNQVTLRM TTDEGTGGRG FLLWYSGRAT SGTEHQFCGG RMEKAQGTLT TPNWPESDYP 181 PGISCSWHII APSDQVIMLT FGKFDVEPDT YCRYDSVSVF NGAVSDDSKR LGKFCGDKAP 241 SPISSEGNEL LVQFVSDLSV TADGFSASYK TLPRDAVEKE LAPSPGEDVQ LGPQSRSDPK 301 TGTGPKVKPP SKPKFQPAEK PEVSPDTQET PVAPDPPSAT CPKQYKRLGT LQSNFCASSL 361 VVTGTVKTMV RGPGEGLTVT ISLLGVYKSG GLDLPSPPTD TSLKLYVPCR QMPPMKKGAS 421 YLLMGQVEEN RGPILPPESF LVPYKPNQDQ ILNNLRKRKC PSQPRPAA MLPAVLTSLL GPFLVAWVLP LARG ATG CTG CCT GCT GTC CTA ACC TCC CTC CTG GGG CCA TTC CTT GTG GCC TGG GTA CTG CCT CTT GCC CGA GGC C—C motif chemokine XP_003495840.1 1 MQFSARTLLC LLLTVAACSI YVLAQPDAVN SPLTCCYSFT AKRIPEKRLE SYKRITSSKC 61 PKEAVIFITK LKREICADPK QDWVQTYTKK LDQSQAKSEA ATVYKTAPLN ANLTHESAVN 121 ASTTAFPTTD LRTSVRVTSM TVN MQFSARTLLC LLLTVAACSI YVLA ATG CAG TTC TCC GCA AGA ACG CTT CTG TGC CTG CTA CTC ACA GTT GCT GCC TGT AGC ATC TAT GTG CTG GCC Lipoprotein lipase XP_003499976.1 1 MESKALLLVA LGVWLQSLTA SQGXAAADGG RDFTDIESKF ALRTPDDTAE DNCHLIPGIA 61 ESVSNCHFNH SSKTFVVIHG WTVTGMYESW VPKLVAALYK REPDSNVIW DWLYRAQQHY 121 PVSAGYTKLV GNDVARFINW MEEEFNYPLD NVHLLGYSLG AHAAGVAGSL TNKKVNRITG 181 LDPAGPNFEY AEAPSRLSPD DADFVDVLHT FTRGSPGRSI GIQKPVGHVD IYPNGGTFQP 241 GCNIGEAIRV IAERGLGDVD QLVKCSHERS IHLFIDSLLN EENPSKAYRC NSKEAFEKGL 301 CLSCRKNRCN NVGYEINKVR AKRSSKMYLK TRSQMPYKVF HYQVKIHFSG TESDKQLNQA 361 FEISLYGTVA ESENIPFTLP EVSTNKTYSF LIYTEVDIGE LLMMKLKWKS DSYFSWSDWW 421 SSPGFVIEKI RVKAGETQKK VIFCAREKVS HLQKGKDSAV FVKCHDKSLK KSG MESKALLLVA LGVWLQSLTA ATG GAG AGC AAA GCC CTG CTC CTG GTG GCT CTG GGA GTG TGG CTC CAG AGT TTG ACC GCC Nidogen-1 XP_003507635.2 1 MLDASGWKPA AWTWVLLLQL LLAGPGDCLS RQELFPFGPG QGDLELEAGD DWSPALELI 61 GELSFYDRSD ITSVYVTTNG IIAMSEPPAR ESHPGTFPPS FGSVAPFLAD LDTTDGLGNV 121 YYREDLSPSI MQMAAEYVQR GFPEVPFQPT SWWTWESV APYEGPSGSS AQEGKRNTFQ 181 AVLASSNSSS YAIFLYPEDG LQFFTTFSKK DENQVPAMVG FSQGLVGFLW RSDGAYNIFA 241 NDRESIENLA KSSNAGHQGV WVFEIGSPAT AKGVVSADVN LGLDDDGSDY EDEEYDLATS 301 HLGLEDMATQ PFPSPSPRRG NTHPHDVPRV LSPSYEATER PHGIPTERTR TFQLPAERFH 361 QQHPQVIDVD EVEETGIVFS YNIGSQQTCA NNRHQCSVHA ECRDYATGFC CRCVANYTGN 421 GRQCVAEGSP QRVNGKVKGR IFVGNSQVPV VFENTDLHSY VVMNHGRSYT AISTIPETVG 481 YSLLPLAPIG GIIGWMFAVE QNGFKNGFSI TGGEFTRQAE VTFLGHPGKL VLKQHFSGID 541 EHGHLTINTE LDGRVPQIPY GSSVHIEPYT ELYHYSSSVI TSSSTREYTV TEPDPDGTAP 601 SHTHVYQWRQ TITFQECVHD DSRPALPSTQ QLSVDSVFVL YNQEERILRY ALSNSIGPVR 661 EGSPDALQNP CYIGTHGCDS NAACRPGPGT QFTCECSIGF RGDGQTCYDI DECSEQPSRC 721 GNHAACNNSP GAYLCECVEG YHFSDGGICV ADVDQRPINY CETGLHNCDI PQRAQCIYMG 781 GSSYTCSCLP GFSGDGRACQ DVDECQLSRC HPDAFCYNTP GSFTCQCKPG YQGDGFQCVP 841 GEVGKTRCQL EREHILGASG VADAQQPRLL GMYVPQCDEY GHYEPTQCHH GTGYCWCVDR 901 DGRELEGTRT QPGMRPPCLS TVAPPIHQRP VVPTAVIPLP PGTHLLFAQT GKIERLPLEG 961 NTMKKTEAKA FFHIPAKVII GLAFDCVDKV VYWTDISEPS IGRASLHGGE PTTIIRQDLG 1021 SPEGIALDHLGRNIFWTDSQ LDRIEVARMD GTQRRVLFDT GLVNPRGIVT DSVGGNLYWT 1081 DWNRENPKIE TSYMDGTNRR ILAQDNLGLP NGLTFDAFSS QLCWVDAGTH RAECLNPAQP 1141 SSRKVLEGLQ YPFAVTSYGK NLYYTDWKTN SVIAMDLAIS KEMDAFTPTS RPGYMASPLP 1201 CPNALKATTT AQ MLDASGWKPA AWTWVLLLQL LLAGPGDCLS ATG CTG GAC GCG AGC GGC TGG AAG GCG GCG GCG TGG ACA TGG GTG CTG CTG CTG CAG CTA TTG CTG GCG GGG CCC GGA GAC TGC CTG AGC Pigment epithelium-derived factor XP_003515170.1 1 MQALVLLLWT GALLGHGSSQ NVASSSEEGS PAPDSTGEPV EEEEDPFFKV PVNKLAAAVS 61 NFGYDLYRLR SSASPTANVL LSPLSVATAL SALSLGAEQR TESIIHRALY YDLISNSDIH 121 STYKELLASV TAPEKSLKSA SRIVFERKLR VRSSFVAPLE KSYGTRPRIL TGNPRIDLQE 181 INNWIQAQMK GKLARSTREM PSAISILLLG VAYFKGQWVT KFDSRKTSLQ DFHLDEDRTV 241 KVPMMSEPKA ILRYGLDSDL NCKVWEHGGW EGSERGRVSS IRKSIWGYSK IHELQSLFES 301 PDFSKITGKP VKLTQVEHRA AFEWNEEGVE TSPNPGLQPV RLTFPLDYHL NQPFIFVLRD 361 TDTGALLFIG KILDPRGT MQALVLLLWT GALLGHGSS ATG CAG GCC CTG GTG CTA CTG CTG TGG ACA GGA GCC CTG CTT GGG CAT GGC AGC AGC Protein disulfide-isomerase XP_003501525.1 1 MDDRLLTBVLL LLLGVSGPWG QGQEPEGPSE VLPEESSGEE VPKEDGILVL SHHTLSLALQ 61 EHPALMVEFY APWCGHCKAL APEYSKAAAL LAAESASVTL AKVDGPAEPE LTKEFGWGY 121 PTLKFFQNGN RTNPEEYTGP QKAEGIAEWL RRRVGPSAKR LEDEEDVQAL TDKWEWVIG 181 FFQDLQGEDV ATFLALARDA LDITFGFTDQ PQLFQKFGLT KDTVILFKKF DEGRADFPVD 241 KDTGLDLGDL SRFLVTHSMH LVTEFNSQTS PKIFAAKILN HLLLFVNKTL AQHRELLTDF 301 REAAPPFRGQ VLFVMVDVAA DNDHVLNYFG LKAEEAPTLR LINVETTKKY APTGLVPITA 361 ASVAAFCQAV LHGQVKPYLL SQEIPPDWDE RPVKTLVGKN FEQVAFDETK NVFVKFYAPW 421 CSHCKEMAPA WEALAEKYRD REDIVIAELD ATANELEAFS VHGYPTLKFF PAGPDRKVIE 481 YKSTRDLETF SKFLDSGGNL PEEEPKEPAI STPEIQDNST VGPKEEL MDDRLLTVLL LLLGVSGPWG QG ATG GAT GAT CGG CTC CTG ACA GTG TTG CTG CTC CTG CTG GGT GTC TCA GGC CCA TGG GGA CAG GGA

2-2. Temporary Expression

[0092] Each of the mCherry expression vectors expressed by the 10 types of signal peptides were transfected (1 mL) according to the CHO—S cell line Amaxa 4D-Nucleofector protocol. Thereafter, on the 2.sup.nd and 6.sup.th days, the fluorescence values of the intracellular fluorescence and the fluorescent proteins secreted from the culture medium were measured (FIG. 3).

[0093] In the case of the intracellular fluorescence, FACS (Accuri) was used to measure the average value in the histogram of the portion higher than that of the negative control (empty vector, pMaxGFP).

[0094] In the case of the fluorescence values of the fluorescent proteins secreted into the culture medium, 100 .Math.0 was sampled on the 2.sup.nd and 6.sup.th days, and then centrifuged to obtain the supernatant only, and fluorescence was measured at 587/610 nm using a multiple reader.

[0095] It was confirmed that the signal peptides with a high fluorescence value measured in the culture medium were Cat, CC, Nuc, Clus, and Pig. Additionally, the four types of secretory factors (Clus, Pig, Nuc, and CC) which showed the expression higher than the positive control SP7.2, SP7.2 to be used as the positive control, and one type of signal peptide with a high fluorescence value in cells (Proco) were selected as a negative control.

Example 3. Comparison of Expression Through Site-Specific Integration

3-1. Construction of Expression Vectors for Site-Specific Integration

[0096] The mCherry sequences including the 5 types of signal peptides (Clus, Pig, Nuc, CC, and Proco) selected in Example 2 and the control SP7.2 were identically inserted into a specific site of the CHO genome to quantitatively compare the expression levels (FIGS. 4 and 5).

[0097] The insertion site was set at the Hprt Site, and a homology arm sequence and sgRNA sequence were 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.

[0098] In the case of the 5′ homology arm, PCR was performed using primers containing Bg1 II and Nrul enzyme sites along with the CHO—S genome as a template. Thereafter, it was cloned into a pcDNA3.1(+) vector digested with Bg1II/Nrul.

[0099] In the case of 3′ homology arm, PCR was conducted using each primer containing Sall site along with the CHO—S genome as a template, and then Sall single cut was made along with the vector inserted with 5′ homology arm, and it was cloned into the downstream of the NeoR gene (pcDNA3.1_hprt).

[0100] In order to confirm only those that have been undergone homology recombination and inserted into the genome, Cmy-GFP-BHG pA fragments were constructed in the upstream region of the 5′ homology arm and inserted into Spel and Bg1II (pcDNA3.1_G_hprt) for double selection.

[0101] In the case of the GFP fragment, it was first inserted into the MCS of the pcDNA3.1(+) Vector with Ncol/Xbal, and then PCR was performed using primers containing Spel and Bg1II restriction sites. Thereafter, Spel was inserted using the Bg1II site into the vector (pcDNA_hprt) containing the homology.

[0102] Using the completed pcDNA3.1_G_hprt Vector, the mCherry gene sequences containing the signal peptide sequence was cut with Kpnl and Xhol and inserted into the MCS region.

[0103] As a result, the pcDNA3.1-based expression vector containing the 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.

3-2. Site-Specific Integration

[0104] Knock-in was performed using CRISPR-Cas9 in order to insert the 6 types of vectors for site specific integration into the Hprt Site in the CHO-S genome. 240 ng of sgRNA, 1,250 ng of cas9 protein, and 1 .Math.g of donor vector were independently mixed with Nucleofector solution to prepare 50 .Math.L mixture.

[0105] CHO-S 1×10.sup.6 cells were first dissolved in 50 .Math.L of Nucleofector and then mixed with the previously prepared mixture, and subsequently, the final 100 .Math.L of the mixture was subjected to electroporation.

[0106] After performing the electroporation, the mixture was mixed with 0.5 mL of the medium, added to 2.5 mL of the medium, and cultured in a 6 well-plate at 36.5° C. and 5% CO.sub.2.

[0107] After 2 days, selection was performed in the CD CHO media containing Zeneticin (0.5 mg/L), and then the cells were sub-cultured until 90% of viability was recovered.

[0108] After 90% of viability was recovered, 4 mL of the cells were cultured in duplicate in a 6 well-plate at a concentration of 3×10.sup.5 cells/mL, and the Vi-Cell and the medium fluorescence values (587 nm/610 nm) were measured every 2 to 3 days (FIGS. 5).

[0109] As a result, it was confirmed that the CC, Clus, and Pig secretory peptides showed higher expression than that of the control SP7.2.

Example 4. Expression of Anti PD-1 Antibody and Mass Analysis

4-1. Preparation of Expression Vectors for Production of Anti PD-1 Antibody

[0110] After comparing the expression ability of signal peptides through site specific integration, anti-PD-1 antibodies fused with the 4 types of signal peptides (CC, Pig, Clus, and SP7.2) including CC, Clus, and Pig showing high expression were expressed. The Pembrolizumab (Keytruda®) antibody sequence was used as the target protein.

[0111] DNA sequences corresponding to the amino acid sequences of the light chain and the heavy chain were synthesized, and subsequently, sequences fused with each of the signal peptide sequences were produced through overlap PCR.

[0112] In the case of the light chain, the amino acid sequences were restricted with BamHI and Xhol, and in the case of the heavy chain, the amino acid sequences were restricted with AscI and Notl, and then the antibodies were inserted into the pTz-D1G1 vector, a variant of pcDNA3.1 (+) (including the promoter of KR Patent No. 10-1038126B1).

pCB SP7.2 Pem

[0113] ‘(N-terminal) - [BamHI Restriction Site - Signal Peptide (SEQ ID NO: 33) -Pem Light Chain (SEQ ID NO: 58) - Xhol Restriction Site] - (C-terminal)’ / ‘(N-terminal) - [AscI Restriction Site - Signal Peptide (SEQ ID NO: 33) - Pem Heavy Chain (SEQ ID NO: 59) - Notl Restriction Site] - (C-terminal)’

pCB Clus Pem

[0114] ‘(N-terminal) - [BamHI Restriction Site - Signal Peptide (SEQ ID NO: 4) - Pem Light Chain (SEQ ID NO: 58) - Xhol Restriction Site] - (C-terminal)’ / ‘(N-terminal) -[AscI Restriction Site - Signal Peptide (SEQ ID NO: 4) - Pem Heavy Chain (SEQ ID NO: 59) - Notl Restriction Site] - (C-terminal)’

pCB CC Pem

[0115] ‘(N-terminal) - [BamHI Restriction Site - Signal Peptide (SEQ ID NO: 2) - Pem Light Chain (SEQ ID NO: 58) - Xhol Restriction Site] - (C-terminal)’ / ‘(N-terminal) -[AscI Restriction Site - Signal Peptide (SEQ ID NO: 2) - Pem Heavy Chain (SEQ ID NO: 59) - Notl Restriction Site] - (C-terminal)’

pCB Pig Pem

[0116] ‘(N-terminal) - [BamHI Restriction Site - Signal Peptide (SEQ ID NO: 5) - Pem Light Chain (SEQ ID NO: 58) - Xhol Restriction Site] - (C-terminal)’ / ‘(N-terminal) -[AscI Restriction Site - Signal Peptide (SEQ ID NO: 5) - Pem Heavy Chain (SEQ ID NO: 59) - Notl Restriction Site] - (C-terminal)’

4-2. Expression of Anti-PD1 Antibody

[0117] The prepared recombinant expression vectors pCB-SP7.2-Pem, pCB-Clus-Pem, pCB-Pig-Pem and pCB-CC-Pem were introduced into ExpiCHO-S™ cells (Thermo Fisher Scientific), and cultured in the ExpiCHO expression medium (Thermo Fisher Scientific; 30 mL) for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding) to express the fusion polypeptide (i.e., Pembrolizumab).

4-3. Purification of Anti-PD1 Antibody and Mass Analysis

[0118] The fusion polypeptide produced through the expression of the recombinant vectors was purified by ProteinA. Specifically, the recovered culture solution was filtered with a 0.22 .Math.m filter, and then a column packed with ProteinA resin (Hitrap MSS, GE Healthcare, 11-0034-93) was mounted on AKTA™ Avant25 (GE Healthcare Life Sciences) and a PBS buffer was flowed through to equilibrate the column.

[0119] After the filtered culture solution was injected into a column, a PBS buffer was flowed through again to wash the column. After washing of the column was completed, an elution buffer (citrate buffer, pH 3.5) was flowed through the column to elute the target protein. The eluate was concentrated using the Amicon Ultra filter device (MWCO 30K, Merck) and a centrifuge. After the concentration was performed, buffer exchange was performed with PBS.

[0120] 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 employing the following calculation equation. The extinction coefficient of each material was a value theoretically calculated using the amino acid sequence (1.404).

[00001]$\begin{array}{l}\text{Protein Concentration}\left(\text{mg}/\text{mL}\right)=\\ \frac{\text{Absorbance}\left({\text{A}}_{280\text{nm}}-{\text{A}}_{340\text{nm}}\right)}{*\text{Extinction Coefficient}}\times \text{Dilution Factor}\end{array}$

(*Extinction Coefficient (0.1 %): It is a theoretical absorbance at 280 nm under assumption that the protein concentration is 0.1% (1 g/L), and all cysteines on the primary sequence are oxidized to form a disulfide bond. Calculated via ProtParam tool (https://web.expasy.org/protparam/)

[0121] The purified target proteins were used to confirm the presence of mis-cleavage of signal peptides at the N-terminus of the proteins using Q-TOF MS (FIG. 6). After dilution to a concentration of 1 mg/mL, the proteins were treated with PNGaseF, followed by 6 M Guanidine and DTT, and then loaded onto Q-TOF MS (RMM-MT-001: ACQUITY UPLC+Q-TOF SYNAPT G2 (Waters)).

[0122] As a result, it was confirmed that 100% cleavage was observed at the predicted cleavage sites.

[0123] Based on the results, the signal peptide, which is the CHO cell-derived protein secretory factor of the present invention, improves productivity by increasing the expression level of the recombinant proteins, and by confirming through mass analysis that 100% cleavage was observed at the predicted cleavage sites, it implies that the signal peptide of the present invention can be a powerful genetic tool which can solve the mis-cleavage, which is the problem of the existing protein secretory factors.

[0124] While the present invention has been described with reference to the particular illustrative embodiments, it will be understood by those skilled in the art to which the present invention pertains 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 embodiments described above are considered to be illustrative in all respects and not restrictive. Furthermore, the scope of the present invention is defined by the appended claims rather than the detailed description, and it should be understood that all modifications or variations derived from the meanings and scope of the present invention and equivalents thereof are included in the scope of the appended claims.