WO2014017851A2 - Nouvelles régions de fixation à la matrice (mar) et procédé de production d'une protéine cible à l'aide de celles-ci - Google Patents

Nouvelles régions de fixation à la matrice (mar) et procédé de production d'une protéine cible à l'aide de celles-ci Download PDF

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WO2014017851A2
WO2014017851A2 PCT/KR2013/006679 KR2013006679W WO2014017851A2 WO 2014017851 A2 WO2014017851 A2 WO 2014017851A2 KR 2013006679 W KR2013006679 W KR 2013006679W WO 2014017851 A2 WO2014017851 A2 WO 2014017851A2
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mar
recombinant vector
pcahlhip
promoter
target protein
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WO2014017851A3 (fr
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Sun Kyu Kim
Min Ji Ko
Yu Bin Choi
Eun A Kim
Dong In Kim
Kyung Duk Moon
Sang Kyung Park
Dong Heon Lee
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Hanwha Chemical Corporation
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

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  • the present invention relates to novel matrix attachment regions (MARs), a recombinant vector including the same, a transformant transformed with the recombinant vector, and a method for producing a target protein by culturing the transformant.
  • MARs novel matrix attachment regions
  • Chromatin constituting the mammalian chromosome is divided into extended euchromatin and condensed heterochromatin depending on the degree of condensation.
  • Most actively transcribed genes are located within euchromatin, because the condensed structure of DNA becomes loose to allow access of proteins such as RNA polymerase to genes for expression.
  • heterochromatin is a chromatin that is tightly packed by deacetylation, and genes of heterochromatin are usually not expressed, or expressed in a small amount under tight control (Razin et al., 2007, J. Mol. Biol. 369: 597-607). Therefore, when a foreign gene is introduced and expressed in cells, gene expression levels and persistence depend on whether the foreign gene is placed in euchromatin or heterochromatin.
  • the protein expression level and stability depend on the location of the gene insert on the chromosome of animal cell. Thus, for overexpression of a therapeutic protein, numerous animal cell clones having the therapeutic protein-encoding gene incorporated at different sites are acquired, and then a clone showing high-level constitutive expression should be selected therefrom.
  • MARs matrix attachment regions
  • MARs matrix attachment regions
  • CHO Chinese hamster ovary
  • the present inventors have made many efforts to identify a novel MAR element capable of improving production of foreign proteins. As a result, they identified MAR elements having the base sequences represented by SEQ ID NOs: 1 to 13, and found that the MAR elements are able to improve expression levels of foreign proteins, thereby completing the present invention.
  • An object of the present invention is to provide a novel MAR (matrix attachment region) element for improving protein expression.
  • Another object of the present invention is to provide a recombinant vector including the MAR element.
  • Still another object of the present invention is to provide a transformant that is transformed with the recombinant vector.
  • Still another object of the present invention is to provide a method for producing a target protein using the MAR element.
  • the novel MAR element of the present invention is able to remarkably increase protein production of host cells, and thus can be used for stably producing a foreign protein in animal cells in a high yield.
  • FIG. 1 shows a preparation process of the pLNCG vector, which is disclosed in Example 1, and a constitution thereof;
  • FIG. 2 shows a preparation process of the light chain expression vector, pCALSN which is disclosed in Example 4.
  • FIG. 3 shows a preparation process of the pCAhLSN vector containing the adalimumab light chain gene, which is disclosed in Example 4, and a constitution thereof;
  • FIG. 4 shows a preparation process of the heavy chain expression vector, pCAHIG which is disclosed in Example 4.
  • FIG. 5 shows a preparation process of the pCAhHIG vector containing the adalimumab heavy chain gene, which is disclosed in Example 4, and a constitution thereof;
  • FIG. 6 shows a preparation process of the pCAhLHIG vector containing the adalimumab light chain and heavy chain genes, which is disclosed in Example 4, and a constitution thereof;
  • FIG. 7 shows a preparation process of the pCAhLHIP vector which is disclosed in Example 4, and a constitution thereof;
  • FIG. 8 shows a preparation process of the pCAhLHIP-Kx/Ky antibody expression vector which is disclosed in Example 5, and a constitution thereof;
  • FIG. 9 is a graph showing the protein expression-increasing effect of the novel MAR element, in which pCAhLHIP of the FIG. 9 represents antibody production of CHO-K1 cell line pool transformed with empty vector, and pCAhLHIP-Kx/Ky represents antibody production of CHO-K1 cell line pool transformed with the vector having the novel MAR element of the present invention.
  • the present invention provides a MAR (matrix attachment region) element for improving protein expression, which has a base sequence represented by one selected from the group consisting of SEQ ID NOs: 1 to 13.
  • MAR matrix attachment region
  • the phrase "improving protein expression” means that protein expression of a target gene affected by the MAR element of the present invention is facilitated to improve production of the protein.
  • target gene refers to a nucleic acid molecule encoding a protein, the expression of which is desired to be increased, and any target gene can be used without limitation, as long as it is typically used in the art.
  • target genes useful in the medical and industrial fields may include hormones, hormone analogues, enzymes, enzyme inhibitors, receptors and receptor fragments, antibodies and antibody fragments, monoclonal antibodies, structural proteins, toxin proteins or the like, but the target genes usable in the present invention are not limited to these examples.
  • the MAR element of the present invention may include all the sequences having a homology, preferably 70% homology, more preferably 80% homology, much more preferably 90% homology, and most preferably 95% homology to the base sequence represented by one selected from the group consisting of SEQ ID NOs: 1 to 13, as long as it is able to improve expression levels of the target gene and a protein encoded thereby.
  • the term "homology” refers to the identity between two base sequences, and can be determined by a method widely known in the art, such as BLAST 2.0, which calculates the parameters, score, identity, and similarity.
  • the MAR element of the present invention may be a nucleotide sequence that is isolated and purified from the mouse genome.
  • a GFP-expressing retrovirus was used in order to identify the novel MAR element for improving the expression levels of foreign proteins in animal cells.
  • NIH3T3 cells were infected with the GFP-expressing retrovirus, and then high GFP-expressing cells were obtained from the cells containing a single copy of GFP.
  • the sequences around the retroviral gene insert were cloned, followed by sequencing analysis. Based on the determined sequences, the accurate insert site and the sequences of 100 kb surrounding the insert site on the mouse genome were obtained by BLAST, and then novel MAR elements represented by SEQ ID NOs: 1 to 13 were newly identified from the sequences using a MAR finder (see Examples 1 to 5).
  • the present invention provides a recombinant vector including the MAR element of the present invention.
  • the term "recombinant vector” is an expression vector capable of expressing a target protein in a suitable host cell, and refers to a gene construct containing essential regulatory elements operably linked to express a gene insert.
  • the recombinant vector may be an animal cell expression vector, and it may be any animal cell expression vector known in the art without limitation.
  • the recombinant vector of the present invention may include the MAR element of the present invention as an essential element, and may further include a promoter sequence typically used in the art.
  • promoter refers to an untranslated DNA sequence that contains the binding site for polymerase and has an ability to initiate transcription of a gene located downstream into mRNA.
  • the promoter usable in the present invention may be any promoter used in the art, and for example, selected from the group consisting of a CMV promoter, an LTR promoter, an EF ⁇ promoter, a SV40 promoter and a TK promoter, but is not limited thereto.
  • the CMV (Cytomegalovirus) promoter was used.
  • the recombinant vector of the present invention may include the MAR element of the present invention as an essential element, and further include a polynucleotide encoding a protein and a promoter.
  • the polynucleotide and the promoter may be operably linked to each other.
  • a recombinant vector including the MAR element of the present invention, the CMV promoter, and the genes of the light and heavy chains of an antibody protein, adalimumab (Humira) is provided, and has a cleavage map of FIG. 8.
  • operably linked means that a nucleotide sequence of the promoter and a nucleotide sequence encoding the target protein are functionally connected to perform general functions.
  • the operable linkage to the recombinant vector may be prepared using a genetic recombinant technique well known in the art, and site-specific DNA cleavage and ligation may be carried out using enzymes generally known in the art.
  • the MAR element of the present invention may be also operably linked to the nucleotide sequence encoding the target protein and the promoter sequence.
  • the recombinant vector of the present invention may include two MAR elements, and the MAR elements may be located at 5' upstream and 3' downstream of the polynucleotide encoding the protein, respectively.
  • the two MAR elements may be selected from the group consisting of two MAR elements represented by SEQ ID NO: 2, two MAR elements represented by SEQ ID NOs: 4 and 5, two MAR elements represented by SEQ ID NOs: 6 and 7, and two MAR elements represented by SEQ ID NO: 13.
  • pCAhLHIP-K2/K2 pCAhLHIP-K5/K4, pCAhLHIP-K7/K6, and pCAhLHIP-K13/K13 were prepared as recombinant vectors including two MAR elements in the above combinations.
  • the recombinant vector of the present invention may include a selection marker.
  • the selection marker is to select the cells transformed with the vector.
  • a selectable marker which confers a selectable phenotype such as drug resistance, nutritional auxotrophy, resistance to a cytotoxic agent or expression of a surface protein, can be used. Because cells expressing the selection marker can only survive under the environment treated with a selective agent, it is possible to select the transformed cells.
  • the present invention provides a transformant that is transformed with the recombinant vector of the present invention.
  • transformation means a method that the MAR element and a gene are introduced into a host cell to be expressed in the host cell.
  • the transformation method may be any transformation method, and easily performed according to the typical method in the art.
  • the transformation method typically used in the art such as such as a CaCl 2 precipitation, a Hanahan method that is an improved CaCl 2 method by using DMSO (dimethyl sulfoxide) as a reducing material, electroporation, calcium phosphate precipitation, protoplast fusion, agitation using silicone carbide fiber, Agrobacterium-mediated transformation, PEG- and lipofectamine-mediated transformation, may be used without limitation.
  • the host cell may be a eukaryotic cell, preferably an animal cell, and more preferably a mammalian cell line.
  • the mammalian cell line may include human cells such as a human embryonic kidney cell line (293 cell or subcloned 293 cell by suspension culture, Graham et al., J. Gen Virol 36, 59 (1977)), a human cervical carcinoma cell line (HELA), a human lung cell line (W138), a human hepatoma cell line (Hep G2, HB 8065); rodent cells such as a baby hamster kidney cell (BHK), a Chinese hamster ovary cell line (CHO), a mouse Sertoli cell line (TM4, Mather, Biol.
  • human embryonic kidney cell line (293 cell or subcloned 293 cell by suspension culture, Graham et al., J. Gen Virol 36, 59 (1977)
  • HELA human cervical carcinoma cell line
  • W138 human lung cell line
  • Hep G2, HB 8065 human
  • a mouse mammary tumor cell line MMT 060562, ATCC CCL51
  • other mammalian cells such as a monkey kidney CV1 cell line transformed with SV40 (COS-7), a monkey kidney cell line (CV1), an Africa green monkey kidney cell line (VERO-76), a dog kidney cell line (MDCK), a buffalo rat liver cells (BRL 3A); and myeloma-derived hybridomas (e.g., NS0).
  • the host cell may be CHO cell.
  • CHO cell line was transformed with each of the recombinant vectors of the present invention, pCAhLHIP-K2/K2, pCAhLHIP-K5/K4, pCAhLHIP-K7/K6, and pCAhLHIP-K13/K13 so as to prepare the transformants, pCAhLHIP-K2, pCAhLHIP-K5/k4, pCAhLHIP-K7/K6, and pCAhLHIP/K13, which were deposited at the Korean Collection for Type Cultures (KCTC) in the Korea Research Institute of Bioscience and Biotechnology on Mar. 19, 2012 under the Accession Nos. KCTC 12165BP, KCTC 12166BP, KCTC 12167BP and KCTC.
  • KCTC 12165BP Korean Collection for Type Cultures
  • the present invention provides a method for producing the target protein using the MAR element of the present invention.
  • the method according to the present invention includes the steps of:
  • target protein means a polypeptide having several amino acids that are desired to be produced in the transformed cells.
  • the target protein may be selected from the group consisting of hormones, hormone analogues, enzymes, enzyme inhibitors, receptors and receptor fragments, antibodies and antibody fragments, monoclonal antibodies, structural proteins, and toxin proteins, but the target protein of the present invention is not limited to these examples.
  • the antibody may be an antibody having an activity of binding to TNF (tumour necrosis factor alpha), and examples of the antibody include Adalimumab (Humira), Infliximab (Remocade), Golimumab (CNTO 148) or the like.
  • TNF tumor necrosis factor alpha
  • examples of the antibody include Adalimumab (Humira), Infliximab (Remocade), Golimumab (CNTO 148) or the like.
  • production of the target protein using the animal cells may be performed using a proper medium and culture conditions known in the art.
  • the culturing procedures can be readily adjusted by those skilled in the art according to the selected strain.
  • the recombinant vector may further include a tag sequence for protein purification.
  • the tag sequence for protein purification may include glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA) and 6x His (hexahistidine; Qiagen, USA), but the type of the sequence needed for purification of the target protein is not limited to these examples.
  • the target protein may be recovered from the culture broth or the animal cells obtained from the culture process by using the typical separation technique, for example, fractionation on an immunoaffinity column or an ion-exchange column, precipitation, reverse phase HPLC, chromatography, SDS-PAGE, and gel filtration.
  • typical separation technique for example, fractionation on an immunoaffinity column or an ion-exchange column, precipitation, reverse phase HPLC, chromatography, SDS-PAGE, and gel filtration.
  • antibody-production ability of the CHO-K1 cell transformed with the recombinant vector that includes the MAR element of the present invention, the CMV promoter, and the genes of the light and heavy chains of adalimumab (Humira) and has the cleavage map of FIG. 8 was examined.
  • the CHO-K1 cell having the MAR element of the present invention showed approximately 2.4-fold to 6.3-fold higher antibody-production ability than the CHO-K1 cell having no MAR element (see FIG. 9).
  • the pAcGFP1-N2 vector (Clonetech) was digested with restriction enzymes, BamHI and NotI to obtain a GFP gene fragment.
  • the pLNCX2 vector (Clonetech) was digested with restriction enzymes, BglII and NotI, and then the previously obtained GFP gene fragment was inserted thereto and ligated with each other, thereby preparing a retrovirus vector harboring the GFP gene, pLNCG (FIG. 1).
  • NIH3T3 cells were prepared in a 100 mm-culture plate at a density of 2 ⁇ 10 6 one day before retroviral transfection. After removing the medium of NIH3T3 cells, 3 ml of a fresh medium and 2 ml of the retrovirus-containing medium were mixed with each other, and 8 ⁇ g/ml polybrene was added thereto, and the mixture was applied to NIH3T3 cells, followed by incubation for 24 hours. The medium of the retroviral-transfected NIH3T3 cells was replaced with the new medium, and the cells were treated with 10 ⁇ g/ml of puromycin for 3 weeks to remove non-infected cells. From the retroviral-transfected NIH3T3 cells where the non-infected cells were removed, the top 5% GFP-expressing cells were isolated by FACSAria three times.
  • the isolated high GFP-expressing cells were used to generate the clones by limiting dilution.
  • the cells were proliferated, and then high GFP-expressing clones were selected by FACS.
  • genomic DNAs were isolated from the high GFP-expressing clones using a QIAamp kit (QIAGEN).
  • Real-time PCR was performed to analyze the copy number of GFP on genomic DNAs.
  • Real-time PCR was performed using a SYBR green DNA kit (Roche) according to the instructions, and each 10 ng/ml of the genomic DNAs were prepared and used as samples.
  • PCR was performed under the conditions of 95°C for 10 minutes and 45 cycles of 95°C for 10 seconds, 59°C for 10 seconds, and 72°C for 15 seconds with a single fluorescence detection in each cycle. After completion of PCR, the temperature was reduced to 65 C, and then slowly increased by 0.1°C per second to perform continuous acquisition of fluorescence data during a melting curve analysis for detection of non-specific amplification. At this time, the pLNCG vector was used as a standard.
  • the primer sequences used are given in the following Table 1.
  • a single NIH3T3 cell is known to have approximately 6.2 pg of genomic DNA, and thus the copy number of GFP gene per cell was calculated by dividing the total copy number of GFP obtained from real-time PCR by 8045 which is the number of cells corresponding to 50 ng of DNA, thereby selecting clones having a single copy of GFP gene per cell.
  • SP specific primers
  • PCR was performed using a GeneAmp® PCR system 9700 (Applied Biosystems).
  • 0.3 ⁇ l of 10 ⁇ M SP3 primer, 0.8 ⁇ l of 50 ⁇ M AD primer, 2 ⁇ l of 25 ng/ml genomic DNA, 6.9 ⁇ l of ultra pure water, and 10 ⁇ l of Extaq premix (Takara) were added, and the reaction was performed under the following conditions: the first round of 1 cycle of 92°C for 3 minutes and 95°C for 1 minute; the second round of 5 cycles of 94°C for 30 seconds, 65°C for 1 minute, and 72°C for 2 minutes; the third round of 1 cycle of 94°C for 30 seconds, 25°C for 2 minutes, ramping (10%) until 72°C, 72°C for 2 minutes; the fourth round of 15 cycles of 94°C for 30 seconds, 65°C for 1 minute, 72°C for 2 minutes, 94°C for 30 seconds, 65°C for 1 minute, 72°C for 2 minutes, 94°C for 30 seconds
  • a 1/40 dilution of the primary PCR product was used as a template (1 ⁇ l), and 0.4 ⁇ l of 10 ⁇ M SP2 primer, 0.8 ⁇ l of 50 ⁇ M AD primer, 7.8 ⁇ l of ultra pure water, and 10 ⁇ l of Extaq premix (Takara) were added, and the reaction was performed under the following conditions: 12 cycles of 94°C for 30 seconds, 65°C for 1 minute, 72°C for 2 minutes, 94°C for 30 seconds, 65°C for 1 minute, 72°C for 2 minutes, 94°C for 30 seconds, 45°C for 1 minute, and 72°C for 2 minutes, and further reaction at 72°C for 5 minutes and then the temperature was reduced to 4°C.
  • tertiary PCR reaction 0.5 ⁇ l of the secondary PCR product was used as a template, and 1 ⁇ l of 10 ⁇ M SP1 primer, 2 ⁇ l of 50 ⁇ M AD primer, 21.5 ⁇ l of ultra pure water and 25 ⁇ l of Extaq premix (Takara) were mixed to prepare a reaction mixture, and the reaction was performed under the following conditions: 20 cycles of 94°C 30 seconds, 45°C 1 minute, and 72°C for 2 minutes, and further reaction at 72°C for 5 minutes and then the temperature was reduced to 4°C.
  • the retroviral insertion site on the mouse (Mus musculus) chromosome was examined by BLASTn, and the surrounding 100 kb of genomic DNA sequences were obtained. Thereafter, 13 candidate MAR elements were identified from the genomic DNA sequences using a MAR finder (http://genomecluster.secs.oakland.edu/MarWiz/) program.
  • the MAR elements have the base sequences represented by SEQ ID NOs: 1 to 13, respectively, and designated as K1 to K13, respectively.
  • BAC clones including the novel MAR sequences was purchased from Invitrogen, and used as a template to perform PCR for DNA cloning of MAR.
  • the BAC clones and primers used are given in the following Table 3.
  • the pCALSN vector (Korean Patent Application No. 10-2012-0057027) was digested with NheI and NotI to remove the light chain gene of trastuzumab, and the light chain gene of adalimumab digested with NheI and NotI was inserted thereto to prepare a pChLSN vector.
  • the light chain expression vector pCALSN is a vector including CMV promoter-AI intron-trastuzumab light chain gene-poly A fragment, which was prepared as in FIG. 2.
  • the pChLSN vector was digested with NdeI (located within CMV promoter) and NheI (located between CMV promoter and adalimumab light chain gene), and the AI fragment containing a part of the CMV promoter obtained by PCR using pCALSN as a template was digested with NdeI and XbaI, and inserted thereto to prepare pCAhLSN (FIG. 3).
  • the primer sequences used for obtaining the AI fragment containing a part of the CMV promoter are given in the following Table 4.
  • the pCAHIG vector (Korean Patent Application No. 10-2012-0057027) was digested with NheI and NotI to remove the heavy chain gene of trastuzumab, and the heavy chain gene of adalimumab digested with NheI and NotI was inserted thereto to prepare pChHIG.
  • the heavy chain expression vector pCAHIG is a vector including CMV promoter-AI intron-trastuzumab heavy chain gene-IRES-GS-poly A fragment, which was prepared as in FIG. 4.
  • the pChHIG vector was digested with NdeI (located within CMV promoter) and NheI (located between CMV promoter and adalimumab heavy chain gene), and the AI fragment containing a part of the CMV promoter obtained by PCR using pCALSN as a template was digested with NdeI and XbaI, and inserted thereto to prepare pCAhHIG (FIG. 5).
  • a CMV-AI-L-polyA fragment was obtained from pCAhLSN by PCR, and then digested with SacII and MluI, and inserted into the pCA201HIG digested with SacII and MluI to prepare pCAhLHIG (FIG. 6).
  • PCR was performed using CMV_Ac and AflII_Ac primers under the conditions of 94°C for 5 minutes, and 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute and 30 seconds, and further reaction at 72°C for 7 minutes.
  • the primer sequences used are given in the following Table 5.
  • the pCAhLHIG vector was digested with MluI, and then ligated by klenow fill-in to remove the MluI restriction enzymes site from the vector and thus prepare a pCAhLHIH-MK vector (FIG. 7).
  • PCR was performed using pCAhLHIH-MK as a template and the primer of Table 6 to prepare a fragment EcoRV-205MK-SfiI containing a plasmid backbone from BGH poly, which was inserted into the PCR cloning vector, TOPcloner Blunt V2 (Enzynomics).
  • the pCAhLHIG(-) vector was digested with the restriction enzymes, EcoRV and SalI.
  • One end of the PCR product SalI-puro-AflII was digested with SalI, and inserted thereto to prepare a pCAhLHIP vector.
  • the pCA205LHIP vector has the AscI restriction site located before the first CMV promoter inducing the transcription of the light chain gene so that the novel MAR element is cloned and inserted thereto, and has the MluI restriction site located behind PolyA which is connected to puromycin gene-IRES-heavy chain gene (FIG. 7).
  • vectors were prepared.
  • the novel MAR elements were inserted in combinations of K2/K2, K5/K4, K7/K6 and K13/K13 before and after the gene to be expressed, and the resultants were inserted into antibody expression vectors, respectively.
  • the MAR element inserted before the gene was inserted backwards.
  • the pCAhLHIP vector was digested with MluI, and the novel MAR elements, K2, K4, K6 and K13 digested with MluI were inserted thereto, respectively to give pCAhLHIP-/Ky (Ky represents the MAR element inserted after the gene, and y represents the number of the MAR element, 2, 4, 6 or 13).
  • pCAhLHIP-/Ky was digested with AscI, and the novel MAR K2, K5, K7, and K13 digested with MluI were inserted thereto, so as to prepare antibody expression vectors pCAhLHIP-Kx/Ky (Kx represents the MAR element inserted before the gene, and x represents the number of the MAR element) containing the novel MAR before and after the expression gene (FIG. 8).
  • the antibody expression vectors thus prepared were pCAhLHIP-K2/K2 having two K2 MAR elements, pCAhLHIP-K5/K4 having K5 and K4 MAR elements, pCAhLHIP-K7/K6 having K7 and K6 MAR element, and pCAhLHIP-K13/K13 having two K13 MAR elements.
  • CHO-K1 cells were plated in a 100 mm-culture dish containing 10 ml of medium, followed by transformation using 24 ⁇ g of DNA and 60 ⁇ l of lipofectamine (Invitrogen).
  • CHO-K1 cells were transduced with the empty vector pCAhLHIP and the novel MAR-containing vector pCAhLHIP-Kx/Ky, namely, pCAhLHIP-K2, pCAhLHIP-K5/K4, pCAhLHIP-K7/K6, and pCAhLHIP-K13/K13, respectively.
  • the cells were treated with 10 ⁇ g/ml puromycin, and cell growth was observed until the cells showed normal growth pattern. After cell growth for the selection period of approximately 2 weeks, expression levels of the cell pools were determined, and compared between the vectors. The expression levels were determined by ELISA using Anti-Fc, and PCD (Picograms/cell/day) was calculated to determine the expression level per cell. Then, the relative expression levels thereof were compared, and the results are shown in FIG. 9.
  • the cell pools transformed with the vectors having the novel MAR elements of the present invention showed approximately 2.4-fold to 6.3-fold higher antibody production than the cell pool transformed with the vector having no MAR element (see FIG. 9), indicating that the novel MAR elements of the present invention can be effectively used for producing foreign proteins in animal cells in a high yield.
  • CHO cell lines transformed with the antibody expression vectors of the present invention pCAhLHIP-K2/K2, pCAhLHIP-K5/K4, pCAhLHIP-K7/K6, and pCAhLHIP-K13/K13 were designated as pCAhLHIP-K2, pCAhLHIP-K5/k4, pCAhLHIP-K7/K6, and pCAhLHIP/K13, respectively and they were deposited at the Korean Collection for Type Cultures (KCTC) in the Korea Research Institute of Bioscience and Biotechnology on Mar. 19, 2012 under the Accession Nos. KCTC 12165BP, KCTC 12166BP, KCTC 12167BP and KCTC 12168BP.
  • the novel MAR element of the present invention is able to remarkably increase protein production of host cells, and thus can be used for stably producing a foreign protein in animal cells in a high yield.

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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Plant Pathology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne de nouvelles régions de fixation à la matrice (MAR), un vecteur recombinant les comprenant, un transformant transformé par le vecteur recombinant et un procédé de production d'une protéine cible par la mise en culture du transformant.
PCT/KR2013/006679 2012-07-27 2013-07-25 Nouvelles régions de fixation à la matrice (mar) et procédé de production d'une protéine cible à l'aide de celles-ci WO2014017851A2 (fr)

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KR1020120082822A KR20140015999A (ko) 2012-07-27 2012-07-27 신규 ＭＡＲｓ 및 이를 이용하여 목적 단백질을 생산하는 방법
KR10-2012-0082822 2012-07-27

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WO2014017851A2 true WO2014017851A2 (fr) 2014-01-30
WO2014017851A3 WO2014017851A3 (fr) 2014-05-08

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EP3159411A4 (fr) * 2014-06-17 2017-06-07 Korea Research Institute Of Bioscience And Biotechnology Vecteur comprenant un fragment de gène pour l'amélioration de l'expression de protéines recombinantes et utilisation associée
CN107058387A (zh) * 2017-04-13 2017-08-18 新乡医学院 一种适合hek293细胞的双顺反子表达载体及其制备方法、表达系统、应用
CN110343718A (zh) * 2018-04-03 2019-10-18 新乡医学院 一种高效稳定的细胞表达载体、表达系统及其制备方法、应用

Families Citing this family (1)

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KR101629564B1 (ko) * 2014-09-30 2016-06-13 한국생명공학연구원 목적 유전자 발현 증가용 재조합 벡터 및 이의 이용

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KR20070118563A (ko) * 2005-03-04 2007-12-17 주식회사 셀트리온 １ 카피 이상의 ｍａｒ ｄｎａ 서열이 유전자의전사종결영역의 3′말단에 삽입된 동물세포 발현 벡터 및그를 이용한 외래 유전자의 발현 방법
KR20090053893A (ko) * 2006-08-23 2009-05-28 셀렉시스 에스. 에이. 전사를 증가시키기 위한 기질부착부위(ｍａｒｓ) 및 그의 용도
US20100226912A1 (en) * 2007-05-21 2010-09-09 Vivalis RECOMBINANT PROTEIN PRODUCTION IN AVIAN EBx® CELLS
WO2012046255A2 (fr) * 2010-10-08 2012-04-12 Cadila Healthcare Limited Vecteur d'expression pour l'expression à haut niveau de protéines recombinantes

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KR20070118563A (ko) * 2005-03-04 2007-12-17 주식회사 셀트리온 １ 카피 이상의 ｍａｒ ｄｎａ 서열이 유전자의전사종결영역의 3′말단에 삽입된 동물세포 발현 벡터 및그를 이용한 외래 유전자의 발현 방법
KR20090053893A (ko) * 2006-08-23 2009-05-28 셀렉시스 에스. 에이. 전사를 증가시키기 위한 기질부착부위(ｍａｒｓ) 및 그의 용도
US20100226912A1 (en) * 2007-05-21 2010-09-09 Vivalis RECOMBINANT PROTEIN PRODUCTION IN AVIAN EBx® CELLS
WO2012046255A2 (fr) * 2010-10-08 2012-04-12 Cadila Healthcare Limited Vecteur d'expression pour l'expression à haut niveau de protéines recombinantes

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3159411A4 (fr) * 2014-06-17 2017-06-07 Korea Research Institute Of Bioscience And Biotechnology Vecteur comprenant un fragment de gène pour l'amélioration de l'expression de protéines recombinantes et utilisation associée
US9902969B2 (en) 2014-06-17 2018-02-27 Korea Research Institute Of Bioscience And Biotechnology Vector comprising gene fragment for enhancement of recombinant protein expression and use thereof
CN107058387A (zh) * 2017-04-13 2017-08-18 新乡医学院 一种适合hek293细胞的双顺反子表达载体及其制备方法、表达系统、应用
CN107058387B (zh) * 2017-04-13 2019-09-06 新乡医学院 一种适合hek293细胞的双顺反子表达载体及其制备方法、表达系统、应用
CN110343718A (zh) * 2018-04-03 2019-10-18 新乡医学院 一种高效稳定的细胞表达载体、表达系统及其制备方法、应用

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WO2014017851A3 (fr) 2014-05-08

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