WO2009048197A1 - Système vecteur pour expression élevée de protéines dans des cellules mammaliennes - Google Patents

Système vecteur pour expression élevée de protéines dans des cellules mammaliennes Download PDF

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WO2009048197A1
WO2009048197A1 PCT/KR2007/006133 KR2007006133W WO2009048197A1 WO 2009048197 A1 WO2009048197 A1 WO 2009048197A1 KR 2007006133 W KR2007006133 W KR 2007006133W WO 2009048197 A1 WO2009048197 A1 WO 2009048197A1
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vector
expression
protein
gene
proteins
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Young Woo Park
Semi Kim
Kwang Pyo Lee
Hyo Jeong Hong
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Korea Research Institute Of Bioscience And Biotechnology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/63—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • 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
    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/60—Vectors comprising a special origin of replication system from viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/10—Vectors comprising a special translation-regulating system regulates levels of translation
    • C12N2840/105—Vectors comprising a special translation-regulating system regulates levels of translation enhancing translation

Definitions

  • the present invention relates to a vector system for high expression of proteins.
  • the most preferred method to obtain a protein having physiological activity is the expression of the target protein in animal cells and the extracellular secretion of the same.
  • the protein expressed in animal cells has advantages of protein folding and post-translational modification such as glycosylation and sulfation, suggesting that is most likely the native form.
  • the representative system for protein expression using animal cells is the stable expression system characterized by integration of a target gene into animal cell chromosome.
  • a dhfr (dihydrofolate reductase) knock-out CHO cell line, DG44 is preferably used.
  • a target gene is cloned into a vector having dhfr gene, which is introduced into DG44.
  • the culture medium is treated with G418 and G418 resistant colonies are selected to obtain stable transfectants .
  • the clone obtained above is cultured with gradual increase of MTX (methotrexate), so that copies of the intracellular recombinant gene are increased and thereby the expression of the target protein is increased.
  • the culture medium confirmed to have a protein of each clone secreted therein is taken and the protein expression therein is measured, leading to the selection of highly expressed clones.
  • EBV Epstein Barr virus
  • the plasmid having oriP the origin of replication of EBV, facilitates high-copy extrachromosomal replication in any EBNAl expressing environment. And as a result, high expression of proteins is facilitated even by transient transfection.
  • EBNAl nuclear antigen 1 The cell line expressing EBNAl (EBV nuclear antigen 1) is exemplified by 293E cell line.
  • Vectors having oriP are exemplified by pMEP4, pCEP4 (Invitrogen, USA), etc. Dr.
  • CMV immediate-early enhancer/promoter region has been known to induce protein expression highly and consistently in various cell lines.
  • Chimeric intron region is composed of 5' -donor site of the first intron of human beta-globin gene and 3' -acceptor site of the intron of immunoglobulin heavy chain variable region.
  • the intron adjacent to cDNA insert has an effect of increasing the expression by the cDNA insertion (Buchman and Berg, Molecular and Cellular Biology, Vol.8, No.10, p.4395-4405, 1988; Brondyk, pCI and pSI Mammalian Expression Vectors. Promega Notes 49, 7-11, 1994).
  • the vector having CMV immediate-early enhancer/promoter region and chimeric intron is exemplified by pCI-neo mammalian expression vector (Promega cat. E1841).
  • this vector system has a disadvantage of low protein expression rate, compared with the conventional stable cell lines.
  • the present inventors tried to solve the above problems of the conventional stable transfection vector and transient transfection vector.
  • the present inventors constructed a gene construct by combining oriP region, CMV immediate-early enhancer/promoter region, chimeric intron region, kozak sequence and 2 stop codons and further completed this invention by confirming that transfection with the above gene construct resulted in the high expression of recombinant proteins.
  • the expression vector of the present invention shortens the construction time of the conventional stable cell line and increases the expression of a protein, which has been a weakness of the conventional EBV based vector, so that it can be effectively used for the production of a target protein with high efficiency. [Disclosure] [Technical Problem]
  • the present invention provides a gene expression cassette prepared by serial fusion of oriP region, CMV immediate-early enhancer/promoter region, chimeric intron region, kozak sequence and 2 stop codons in that order.
  • the present invention also provides a vector for high expression of proteins containing the gene expression cassette .
  • the present invention further provides an animal cell line for high expression of proteins prepared by transfecting a host animal cell with the vector for high expression of proteins.
  • the present invention provides a production method of a protein using the vector for high expression of proteins.
  • the vector for high expression of proteins containing the gene expression cassette of the present invention is the expression system for proteins in animal cells with high speed and high efficiency, and has advantages of reducing costs, compared with the conventional stable cell line establishment, and increasing yields of proteins. Therefore, this system of the invention is very effective in further advancement of protein production and antibody production in animal cells along with the advancement of techniques thereof, in studies of protein structures and functions, and in reducing costs for protein production, suggesting that it facilitates protein production within a short period with high efficiency with less costs.
  • Fig.l is the map of vector #1.
  • Fig. 2 is the map of the expression vector for the protein production in animal cells: CMVp: CMV promoter; leader: leader sequence; OriP : EBV origin of replication (oriP); CMV I. E: CMV immediate-early enhancer/promoter ; and intron : chimeric intron.
  • Fig. 3 is a set of photographs illustrating the comparison of protein expressions/secretions by vector #1 - vector #4:
  • Vector #4-4d loading of 15 j ⁇ , of culture medium recovered on the 4 th day of culture with vector #4;
  • Fig. 4 illustrates the comparison between the conventional stable cell line method and the novel transient transfection method:
  • 293E cells (2X10 6 ) were transiently transfected with vector #4. The medium was replaced with a serum-free medium and the culture medium was recovered each day.
  • the present invention provides a gene expression cassette in which oriP region, CMV immediate-early enhancer/promoter region, chimeric intron region, kozak sequence and 2 stop codon sequences are fused stepwise.
  • the gene expression cassette can additionally include one or more polynucleotides selected from the group consisting of nucleotides encoding leader sequence, restriction enzyme sites, Fc gene and myc gene, but not always limited thereto.
  • the kozak sequence of the expression vector is preferably GCCGCC or GCCACC.
  • the leader sequence of the expression vector is preferably preprotrypsin leader sequence or mouse immunoglobulin heavy chain leader sequence (Berntzen et al, Journal of Immunological Methods 298:94-104, 2005), but not always limited thereto, and any leader sequence known to those in the art that is applicable in protein expression can be used in this invention.
  • the restriction enzyme sites of the expression vector can be one or more sites selected from the group consisting of Act II, Ace I, Acc65 I, AfI II, AfI III, Alw44 I, AlwN I, Ava I, Ava II, Avr II, BaI I, BamE I, Ban II, Bbe I, Bbs I, Bbu I, BgI II, Bsa I, BsaA I, BsaB I, BsaM I, Bsm I, Bsp ⁇ I, BspM I, BsrG I, BssE II, BssS I, Bst98 I, BstX I, BstZ I, CfrlO I, CIa I, Csp I, Csp45 I, Dra I, Dra II, Dra III, Dsa I, £ ⁇ agr I, Ear I, E 1 ClHK I, .Eco42 I, EcoCR I, E 1 CoR I, Ehe I, Fsp I,
  • the present invention provides a vector for high expression of proteins containing the gene expression cassette .
  • the present inventors used a vector containing DHFR gene as a form of the conventional CMV promoter-leader sequence-target gene cloning site-Fc (see Fig. 1). Then, oriP gene originated from pMEP4 vector (Invitrogen, USA) was inserted therein (see Fig. 2, vector #2). CMV promoter of vector #1 was substituted with CMV immediate-early enhancer/promoter and chimeric intron region of pCl-neo mammalian expression vector (see Fig. 2, vector #3).
  • CMV promoter region of vector #2 was substituted with CMV immediate-early enhancer/promoter region and chimeric intro region of #3 and kozak sequence and stop codon were added thereto, resulting in the construction of vector #4 (see Fig. 2, vector #4).
  • Protein expressions were compared by using the above 4 vectors. Transient transfection was performed in 293E cell line, followed by examination of protein expression. As a result, vector #4 was more effective in inducing protein expression/secretion than vectors #1 - #3 (see Fig. 3). With vector #4, the protein expression was gradually increased, while with vectors #1 - #3, protein expression was not detected or if detected, the protein expression was reduced from the 8 th day (see Fig. 3A). The protein expression level was divided by beta-actin level indicating GFP/cells, the index for transfection efficiency, leading to the quantification of the protein expression. As a result, vector #4 induced higher expression of the protein, compared with vectors #1 - #3 (see Fig. 3C).
  • the present inventors constructed stable cell line with DG44 cells by using vector #1, followed by investigation of the protein expression.
  • the inventors also treated 293E cell line with vector #4 and investigated the protein expression therein. And the results were compared by the same manner as described above. As a result, high expression of proteins was confirmed when vector #4 was used, compared with when the conventional method was used. And the high expression of proteins was more peculiar in a serum free medium. Therefore, it was confirmed that the vector for high expression of proteins of the present invention facilitates high expression of proteins and extends duration of the expression, compared with the conventional system.
  • the present invention also provides an animal cell line for high expression of proteins prepared by transfecting a host animal cell with the vector for high expression of proteins.
  • the host cell herein is preferably 293E cell line having EBNA-I but not always limited thereto, and any animal cells transformed with EBNA-I expression vector that can be used for protein production can be used in this invention .
  • the present invention provides a production method of a protein using the vector for high expression of proteins.
  • This method is composed of the following steps: 1) inserting the gene expression cassette into the expression vector: 2) inserting a target protein gene into the expression vector of step 1); 3) transiently transfecting a host cell with the expression vector containing the target gene prepared in step 2 ) ; 4 ) culturing the host cell transfected in step 3); 5) replacing the medium for the host cell culture of step 4) with a serum free medium; 6) obtaining the serum free medium of step 5); and 7) purifying the target protein from the medium obtained in step 6 ) .
  • the expression vector of step 1) is preferably one or more vectors selected from the group consisting of pcDNA3 (Invitrogen, USA), pcDNA 3.1 (Invitrogen, USA), pCI vector (Promega, USA), mammalian expression vector (Sigma, USA) and pCMV-Tag epitope tagging mammalian vector (Stratagene, USA), but not always limited thereto, and any expression vector useful for the expression in animal cells, known to those in the art, can be used.
  • the insertion of the target protein gene of step 2 ) is preferably performed by the conventional method well-known to those in the art.
  • the host cell of step 3) is preferably an animal cell line, and 293E cell line having EBNA-I is preferred, but not always limited thereto and any animal cells transformed with EBNA-I expression vector and applicable for the production of a protein can be used in this invention.
  • the medium for cell culture of step 4) is properly selected according to the host cell and a commercial medium is preferred.
  • the purification of the target protein in step 7) is performed by the conventional method well-known to those in the art.
  • Example 1 Construction of an expression vector
  • the present inventors constructed a vector for expression/secretion containing DHFR gene in the form of CMV promoter-leader sequence-target gene cloning site-Fc- myc (vector of Fig. 1 and vector #1 of Fig. 2).
  • a target gene is cloned in this vector and expressed in animal cells thereafter, the target protein is expressed as Fc fused protein and secreted in the culture solution in that form.
  • vector #1 of the present invention extracellular region of AxI (genebankBC032229 , 33 rd amino acid - 426 th amino acid) was cloned as a target protein.
  • the construction of the vector was as follows. At first, PCR was performed with a forward primer (5 '-gag etc gga tec atg gga tgg age tat ate ate etc ttt ttg gta gca aca get aca gat gtc cac teg cag ggg gcc gtg ggg gcc gaa gaa agt ccc ttc gtg gg-3 ' : SEQ. ID. NO: 1) composed of ' BamHI-leader sequence-Sfil-AXL N-terminal sequence' and a reverse primer (5 '-atg atg get age ggc cga
  • AXL was used as a template and the prepared reaction mixture was denatured at 94 ° C for 4 minutes , followed by 25 cycles of denaturation at 94 ° C for 30 seconds , annealing at 55 ° C for 30 seconds, polymerization at 72 ° C for 4 minutes, and final extension at 72 ° C for 10 minutes . ) .
  • Another PCR was performed with a forward primer (5 '-teg gcc get age cat cat cat cat cac cac cac age ggc ctg gtg ccg cgcggc age gac aaa act cac aca tgc c-3 ' : SEQ. ID.
  • the vector was introduced into bacteria for transformation, followed by clone selection.
  • the vector constructed above was digested with Xhol/Xbal and purified.
  • PCR was performed with a forward primer (5"-ctg tga etc gag att ccg ccc etc tec ctc-3 ' : SEQ. ID. NO: 5) composed of ⁇ XhoI-IRES initial sequence' and a reverse primer (5'-ggg ccc tct aga tta gtc ttt ctt etc gta gac ttc-3 ' : SEQ. ID.
  • Ligation was performed as follows. 100 ng of each vector and DNA fragment for insertion were mixed with 1 unit of ligase (Roche), followed by reaction at 16 ° C for 16 hours. After ligation, E. coli XLl-Blue (Stratagene, USA) was transformed with the same, followed by selection from ampicillin containing plate. The product was digested with proper restriction enzymes to obtain plasmids containing a target DNA fragment, followed by DNA sequencing for final confirmation. All the restriction enzymes were purchased from New England Biolabs (USA) and reacted according to the manufacturer's instruction. oriP gene of pMEP4 (Invitrogen, USA) was introduced into vector #1 (vector #2 of Fig. 2). The introduction was performed as follows.
  • PCR was performed with a forward primer (5 '-tat ata age tta tgg gat gga get ata tca-3' : SEQ. ID. NO: 7) in which HindIII site was attached to leader- target gene-Fc-myc region of vector #1 and a reverse primer (5 '-tat ata tac teg agt cac agg tct tec tc-3' : SEQ. ID.
  • vector #3 by substituting CMV promoter region of vector #1 with CMV immediate-early enhancer/promoter region and chimeric intro region (vector #3 of Fig. 2).
  • CMV immediate-early enhancer/promoter region and chimeric intron region referred to pCl-neo mammalian expression vector (Promega cat. No. E1841, USA).
  • the gene substitution was performed as follows. PCR was performed with a forward primer (5 1 - tat agt cga cat ggg atg gag gag eta tat cat c-3 ' : SEQ. ID.
  • the PCR product was digested with Sail and Xhol, which was ligated into pCI-neo vector (Promega, USA) digested with Xhol. Then, the vector was introduced into bacteria for transformation, followed by clone selection.
  • the present inventors constructed vector #4 by substituting CMV promoter region of vector #2 with CMV immediate-early enhancer/promoter region and chimeric intron region of vector #3 (vector #4 of Fig. 2).
  • the inventors added kozak sequence in front of initiation codon of leader sequence with expecting kozak sequence could help the protein expression and added one more stop codon for translation termination.
  • pCI-neo vector Promega, USA was digested with BgIII and Xhol and the resultant fragment containing CMV immediate-early enhancer/promoter region and chimeric intron region was inserted in vector #2 by ligation.
  • PCR was performed with a forward primer (5 r -ata tat gtc gac gcc ace atg gga tgg age tat ate-3' : SEQ. ID. NO: 11) in which Sail site and kozak sequence were attached to leader-target gene-Fc-myc region of vector #1 and a reverse primer (5 '-tat ata etc gag eta tea cag gtc ttc etc aga-3' : SEQ. ID. NO: 12) in which Xhol site and one additional stop codon were attached.
  • the PCR product was digested with Sail and Xhol, which was ligated in the above construct digested with Xhol. Then, the vector was introduced into bacteria for transformation, followed by clone selection.
  • the present inventors transformed animal cells with the 4 different vectors constructed in Example 1 by transient transfection. Then, the expressions of AxI gene cloned in each vector and extracellular secretion of proteins were compared.
  • Western blotting was performed as follows. 30 ⁇ i of the culture medium containing the protein secreted or 30 ⁇ g of cell lysate was mixed with SDS sample buffer, which was heated and proceeded to 10% SDS-PAGE gel. The separated protein proceeded to nitrocellulose membrane, followed by blocking with 5% skim milk. Then, the reactant was reacted with anti-Fc antibody (1:1000: Pierce), anti-actin antibody (1:1000: Santa Cruz) or anti-GFP antibody (1:1000: Santa Cruz), and horseradish peroxidase- conjugated secondary antibody. Analysis was performed with ECL kit (ECL Plus, Amersham, USA) according to the manufacturer's instruction.
  • vector #4 demonstrated to be more effective in inducing high expression of proteins and in extending the protein expression, compared with vectors #1, #2 and #3 (Fig. 3).
  • the protein expression was finally quantified by dividing the amount of secreted protein (Fig. 3A) by beta-actin level indicating GFP/cells, the index for transfection efficiency (Fig. 3C). Mock was not transfected and as expected, the expression of the target protein was not detected by Western blotting (Fig. 3A).
  • Vector #4 was more effective in inducing high expression of proteins than vectors #1, #2 and #3. When cells were transfected with vector #1, the target protein expression became decrease from the forth day of experiment.
  • vector #4 containing oriP region of vector #2 and CMV immediate-early enhancer/promoter and chimeric intron region of vector #3 was superior in inducing high expression of proteins, which was higher expression than the sum of the remaining three vector caused expressions. This seems to be because that vector #4 not only contains major regions of vector #2 and vector #3, in relation to the protein expression, but also contains kozak sequence and stop codons.
  • DG44 cell line was cultured in DMEM/F12 (Gibco, USA) supplemented with 10% FBS, and then transfected with vector
  • Stable clones were selected using 500 ⁇ g/V ⁇ i of G418. MTX concentration was gradually increased until it reached 700 nM to increase the amount or protein expressed and secreted.
  • the serum-free medium for the recovery of secreted proteins was purchased from Cambrex (USA) .
  • the protein level in the selected clone culture medium exhibiting the highest expression rate was compared with the protein level in 293E cell line transfected with vector #4 constructed in Example ⁇ 2-l>, which was performed on the 2 nd , the 4 th and the 6 th day. The comparison was performed by the same manner as described in Example ⁇ 2-l>.
  • the protein expression rate in 293E cell line transfected with vector #4 of the present invention was higher than that in the conventional cell line (Fig. 4).
  • the protein expression in the conventional cell line could not be continued long after replacing the culture medium with a serum-free medium, whereas the protein expression in 293E cell line transfected with vector #4 of the present invention was continued for a long while (Figs. 3 and 4).

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Abstract

Système vecteur pour expression élevée de protéines dans des cellules animales, ce système contenant la cassette d'expression génique dans laquelle fusionnent par étapes et dans l'ordre ci-après la région oriP, la région activateur/promoteur immédiat-précoce CMV, la région intron chimère, la séquence kozak et deux codons d'arrêt. Ledit système est très efficace pour réduire les coûts et le temps requis dans la production de protéines cibles, par rapport aux techniques classiques.
PCT/KR2007/006133 2007-10-10 2007-11-30 Système vecteur pour expression élevée de protéines dans des cellules mammaliennes WO2009048197A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015195049A1 (fr) * 2014-06-18 2015-12-23 Agency For Science, Technology And Research Nouveaux promoteurs pour une expression de haut niveau
CN106459999A (zh) * 2014-06-18 2017-02-22 新加坡科技研究局 用于高水平表达的新型启动子
US10563222B2 (en) 2014-06-18 2020-02-18 Agency For Science, Technology And Research Promoters for high level expression

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