LTVLL LLLGVSGPWG QGQEPEGPSE VLPEESSGEE VPKEDGILVL SHHTLSLALQ
61 EHPALMVEFY APWCGHCKAL APEYSKAAAL LAAESASVTL AKVDGPAEPE LTKEFGVVGY
121 PTLKFFQNGN RTNPEEYTGP QKAEGIAEWL RRRVGPSAKR LEDEEDVQAL TDKWEVVVIG
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
[137]
2-2. temporary apparition
[138]
[139]
Transfection (1 mL) of each of the mCherry expression vectors expressed from 10 types of signal peptides was performed according to the CHO-S cell line Amaxa 4D-Nucleofector Protocol. Thereafter, intracellular fluorescence and fluorescence values ​​of fluorescent proteins secreted from the culture medium were measured on days 2 and 6 ( FIG. 3 ).
[140]
In the case of intracellular fluorescence, FACS (Accuri) was used to measure the average value in the histogram of the higher portion than the negative control (negative control; empty vector, pMaxGFP).
[141]
In the case of the fluorescence value of the fluorescent protein secreted into the culture medium, 100ul was sampled on the 2nd and 6th days, and then centrifuged to obtain only the supernatant. Fluorescence 587/610nm Multiple Reader ) was used for measurement.
[142]
It was confirmed that the signal peptides with high fluorescence values ​​measured in the culture medium were Cat, CC, Nuc, Clus, and Pig, and four secretory factors (Clus, Pig, Nuc, CC), SP7.2 to be used as a positive control, and a signal peptide with a high fluorescence value in cells (Proco) were selected as negative controls.
[143]
[144]
Example 3. Comparison of expression through Site-Specific Integration
[145]
[146]
3-1. Expression vector production for site-specific integration
[147]
[148]
The expression levels were quantitatively compared by inserting the mCherry sequences including the five signal peptides (Clus, Pig, Nuc, CC, Proco) selected in Example 2 and the control SP7.2 at specific positions in the CHO Genome. (FIGS. 4 and 5).
[149]
The insertion site was determined by Hprt Site, J.S Lee et al., 2015 , "Site-Specific integration in CHO cells mediated by CRISPR/Cas9 and homology-directed DNA repair pathway", Sci. Referring to Rep., 5, the Homology Arm sequence and sgRNA sequence were designed.
[150]
In the case of 5' Homology Arm, PCR was performed using CHO-S Genome as a template and primers containing Bg1II and NruI Enzyme Site, followed by digestion with Bg1II/NruI pcDNA3.1(+) vector ( Vector) was cloned.
[151]
In the case of the 3' Homology Arm, PCR is performed using the CHO-S Genome as a template and primers each containing SalI sites, and then SalI Single together with the vector into which the 5' Homology Arm is inserted. It was cut and cloned into the NeoR Gene Downstream (pcDNA3.1_hprt).
[152]
In order to confirm only the homology recombination and insertion into the genome, a Cmy-GFP-BHG pA fragment was prepared in the upstream part of the 5' Homology Arm for double selection and inserted with SpeI and Bg1II (pcDNA3.1_G_hprt).
[153]
In the case of GFP fragment, first, NcoI/XbaI in pcDNA3.1(+) Vector is inserted into MCS, and then PCR is performed using a primer containing SpeI and Bg1II restriction site. was inserted using the Bg1II site.
[154]
[155]
Using the completed pcDNA3.1_G_hprt Vector, the mCherry gene sequence containing the signal peptide sequence was cut with KpnI and XhoI and inserted into the MCS part.
[156]
[157]
As a result, CMV-EGFP-pA-5' Hprt Homology Arm - CMV - Signal Peptide Candidate - mCherry - BGH pA - NeoR Selection Marker Cassette - pcDNA3.1-based expression vector containing an expression cassette in the form of 3'Hprt Homology Arm was produced.
[158]
[159]
3-2. Site-Specific Integration
[160]
[161]
In order to insert the six types of site-specific integration vectors prepared above into Hprt sites in the CHO-S genome, they were knocked-in using CRISPR-Cas9. sgRNA 240 ng, cas9 Protein 1250 ng, and Donor Vector 1 μg were mixed with Nucleofector Solution to prepare 50 μl Mixture, respectively.
[162]
After dissolving CHO-S 1x10 6 cells in 50 μl of Nucleofector and mixing with the previously prepared mixture, the final 100 μl was subjected to electroporation.
[163]
After electroporation (Electrophoration), the mixture was mixed with 0.5㎖ medium, added to 2.5㎖ medium, and incubated in a 6-well plate at 36.5℃, CO 2 5% conditions.
[164]
[165]
After 2 days, selection was performed in CD CHO Media containing Zeneticin (0.5 mg/L), and subculture was performed until the viability was recovered to 90%.
[166]
After recovering 90% of viability, adjust the cell concentration to 3x10 5 cells/mL and incubate 4 mL each in duplicate in 6 wells, and measure Vi-Cell and measure the medium fluorescence value (587 nm/610 nm) every 2-3 days. was measured (FIG. 5).
[167]
As a result, it was confirmed that the CC, Clus, and Pig secretory sequences were expressed higher than the control SP7.2.
[168]
[169]
Example 4. Anti-PD-1 Antibody Expression and Mass Analysis
[170]
[171]
4-1. Construction of expression vector for anti-PD1 antibody production
[172]
[173]
After comparing the expression ability of signal peptides through Site Specific Integration, 4 types of signal peptides (CC, Pig, Clus, SP7.2) including high expression CC, Clus, and Pig ) to express the fused Anti-PD-1 antibody. As the target protein, the Pembrolizumab (Keytruda®) antibody sequence was used.
[174]
[175]
After synthesizing a DNA sequence that matches the amino acid sequence of the light chain and the heavy chain, a sequence fused with each signal peptide sequence was prepared through overlap PCR.
[176]
After restriction with BamHI and XhoI for the light chain and AscI and NotI for the heavy chain, pTz-D1G1 Vector (KR 10-1038126B1 promoter of pcDNA3.1(+)) included).
[177]
[178]
pcb_SP7.2_Pem
[179]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO:33)-Pem Light Chain (SEQ ID NO:58)-XhoI restriction site]-(C-terminal)'/'(N-terminal)-[AscI Restriction site - signal peptide (SEQ ID NO: 33) - Pem Heavy Chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'
[180]
[181]
pcb_Clus_Pem
[182]
'(N-terminal)-[BamHI restriction site-signal peptide (SEQ ID NO:4)-Pem Light Chain (SEQ ID NO:58)-XhoI restriction site]-(C-terminal)'/'(N-terminal)-[AscI Restriction site - signal peptide (SEQ ID NO: 4) - Pem Heavy Chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'
[183]
[184]
pCB_CC_Pem
[185]
'(N-terminal) - [BamHI restriction site - signal peptide (SEQ ID NO: 2) - Pem Light Chain (SEQ ID NO: 58) - XhoI restriction site] - (C-terminal)' / '(N-terminal) - [AscI Restriction site - signal peptide (SEQ ID NO: 2) - Pem Heavy Chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'
[186]
[187]
pCB_Pig_Pem
[188]
'(N-terminal) - [BamHI restriction site - signal peptide (SEQ ID NO: 5) - Pem Light Chain (SEQ ID NO: 58) - XhoI restriction site] - (C-terminal)' / '(N-terminal) - [AscI Restriction site - signal peptide (SEQ ID NO: 5) - Pem Heavy Chain (SEQ ID NO: 59) - NotI restriction site] - (C-terminus)'
[189]
[190]
4-2. Expression of Anti-PD1 Antibody
[191]
[192]
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 ExpiCHO Expression Medium (Thermo Fisher Scientific; 30 mL) was cultured for 12 days (Fed-Batch Culture; Day 1 & Day 5 Feeding) to express the fusion polypeptide Pembrolizumab.
[193]
[194]
4-3. Anti-PD1 antibody purification and mass analysis
[195]
[196]
The fusion polypeptide produced through the recombinant vector expression was purified by ProteinA. Specifically, the recovered culture solution was filtered with a 0.22 μm filter, and a column packed with ProteinA resin (Hitrap MSS, GE Healthcare, 11-0034-93) was mounted on AKTA TM Avant25 (GE Healthcare Life Sciences), and PBS buffer was added. The column was equilibrated by flow.
[197]
After injecting the filtered culture solution into the column, PBS buffer was again flowed to wash the column. After washing the column, an elution buffer (Citrate Buffer, pH 3.5) was flowed into the column to elute the target protein. The eluate was prepared using an Amicon Ultra Filter Device (MWCO 30K, Merck) and a centrifuge. After concentration was carried out using a buffer exchange with PBS.
[198]
[199]
Quantitative analysis of the fusion polypeptide was performed by measuring the absorbance at 280 nm and 340 nm in a UV Spectrophotometer (G113A, Agilent Technologies), and using the following formula. For the extinction coefficient of each material, a theoretically calculated value (1.404) using the amino acid sequence was used.
[200]
[201]

[202]
[203]
(*Extinction Coefficient (0.1%): Assuming that the protein concentration is 0.1% (1 g/L) and all cysteines in the primary sequence are oxidized to form disulfide bonds, Theoretical absorbance, calculated using the ProtParam tool (https://web.expasy.org/protparam/).
[204]
[205]
The purified target proteins were checked for mis-cleavage of the signal peptide in the N-Term portion of the protein using Q-TOF MS (FIG. 6). After dilution to a concentration of 1 mg/mL, PNGaseF was treated, and after treatment with 6M guanidine and DTT, Q-TOF MS (RMM-MT-001: ACQUITY UPLC+Q-TOF SYNAPT G2 (Waters)) was loaded.
[206]
As a result, it was confirmed that 100% cleavage was performed at the predicted cleavage site.
[207]
[208]
Through this, the signal peptide, which is a CHO cell-derived protein secretion factor of the present invention, improves productivity by increasing the expression level of the recombinant protein, and 100% cleavage at the cleavage site predicted through mass analysis. ), suggesting that it can be a powerful genetic tool to solve the problem of mis-cleavage of existing protein secretion factors.
[209]
[210]
[211]
From the above description, those skilled in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.
Claims
[Claim 1]
A protein secretion factor consisting of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.
[Claim 2]
The protein secretion factor according to claim 1, wherein the protein is an endogenous protein or a foreign protein.
[Claim 3]
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; an expression cassette to which it is operably linked.
[Claim 4]
The method of claim 3, wherein the target protein is an antibody, antibody fragment (Fab or ScFv), fusion protein, scaffold protein (Protein Scaffold), human growth hormone, serum protein, immunoglobulin, cytokine, α- , β- or γ-interferon, 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 secretion factor, growth differentiation factor, cell adhesion protein ), prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, Arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucodase, galactosidase , glucocerebrosidase and glucuronidase.
[Claim 5]
According to claim 3, wherein the nucleic acid sequence encoding 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 is SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15, the expression cassette.
[Claim 6]
According to claim 3, wherein the expression cassette further comprises a nucleic acid sequence encoding any one of the 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, and SEQ ID NO: 10 which is an expression cassette.
[Claim 7]
The expression cassette according to claim 3, wherein the expression cassette expresses the target protein without additional amino acids from which the nucleic acid sequence encoding the secretory factor has been removed when expressed in cells.
[Claim 8]
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; an expression vector for secretion of the target protein comprising an expression cassette to which the target protein is operably linked.
[Claim 9]
The method of claim 8, wherein the target protein is an antibody, antibody fragment (Fab or ScFv), fusion protein, scaffold protein (Protein Scaffold), human growth hormone, serum protein, immunoglobulin, cytokine, α- , β- or γ-interferon, 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 secretion factor, growth differentiation factor, cell adhesion protein ), prolactin, chronic gonadotropin, tissue plasminogen activator, growth hormone releasing peptide (GHPR), thymic humoral factor (THF), asparaginase, arginase, Arginine deaminase, adenosine deaminase, peroxide dismutase, endotoxinase, catalase, chymotrypsin, lipase, uricase, adenosine diphosphatase, tyrosinase, bilirubin oxidase, glucose oxidase, glucodase, galactosidase , which is selected from the group consisting of glucocerebrosidase and glucuronidase, an expression vector.
[Claim 10]
According to claim 8, wherein the 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 is SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 is SEQ ID NO: 15, the expression vector.
[Claim 11]
The method of claim 8, wherein the expression vector further comprises a nucleic acid sequence encoding any one of the 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, and SEQ ID NO: 10 that, an expression vector.
[Claim 12]
The expression vector according to claim 8, wherein the expression vector expresses the target protein without additional amino acids from which the nucleic acid sequence encoding the secretory factor has been removed when expressed in cells.
[Claim 13]
A transformant cell into which the expression vector of any one of claims 8 to 12 is introduced into a host cell.
[Claim 14]
The transformant cell according to claim 13, wherein the host cell is a Chinese hamster ovary cell (CHO cell).
[Claim 15]
i) 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; culturing a transformant cell comprising an expression vector for secretion of the target protein, comprising an expression cassette to which the target protein is operably linked; and ii) recovering the target protein from the culture or culture supernatant of the cultured cells.
[Claim 16]
The method of claim 15, further comprising the step of purifying the recovered target protein.
[Claim 17]
The method according to claim 15, wherein the host cell is a Chinese hamster ovary cell (CHO cell).
[Claim 18]
The method according to claim 15, wherein 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 is cleaved at the N-terminal cleavage site of the target protein, A method for producing a target protein.
[Claim 19]
The method according to claim 15, wherein the target protein is the target protein itself without additional amino acids from which the nucleic acid sequence encoding the protein secretion factor has been removed.