Procedure for the production of protein

专利摘要:

公开号:ES2697535T9
申请号:ES10786229T
申请日:2010-06-10
公开日:2019-03-29
发明作者:Koichi Kawakami；Keina Yamaguchi；Risa Ogawa；Masayoshi Tsukahara
申请人:Kyowa Hakko Kirin Co Ltd；Inter University Research Institute Corp Research Organization of Information and Systems；
IPC主号:

专利说明:

[0001] Procedure for the production of protein.
[0002] Technical field
[0003] The present invention relates to a method for producing a protein of interest, comprising introducing a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and transposon sequences in both ends of the gene fragment, in a mammalian cell in suspension, integrating the gene fragment inserted between a pair of transposon sequences in a chromosome of the mammalian cell in order to obtain a mammalian cell capable of expressing the protein of interest, and culturing the mammalian cell in suspension, and a mammalian cell capable of expressing the protein of interest.
[0004] Background of the technique
[0005] The production of exogenous proteins by recombinant DNA techniques is used in various industries, such as the pharmaceutical industry and the food industry. In most cases, the production of recombinant proteins is carried out by introducing an expression vector comprising a nucleotide sequence encoding a protein of interest in a host, such as Escherichia coli, a yeast, a cell of insect, a plant cell and an animal cell, selecting a transformant in which the expression vector is integrated into the chromosome, and further culturing the cell line under appropriate culture conditions.
[0006] However, in order to develop a host that can efficiently produce an exogenous protein, it is necessary to select a host cell with good productivity for each protein of interest, for which a further technical innovation of the exogenous protein production techniques is desired. for the individual host.
[0007] In bacterial systems, such as Escherichia coli and yeast systems, different from animal cells, post-translational modifications, such as saccharide chain modifications, are difficult to achieve in many cases and, thus, cause a problem during the production of a protein that presents its activity.
[0008] Because the produced protein is subjected to a post-translational modification, such as phosphorylation and the addition of saccharide chains in the insect system, this system has the merit that the protein can be expressed with its original physiological activity. However, because the structure of the saccharide chain of the secreted protein is different from that of the mammalian derived cells, antigenicity and the like become a problem when applying the protein to pharmaceutical use.
[0009] In addition, because a recombinant virus is used in the insect cell system by introducing an exogenous gene, there is a problem that the inactivation and containment of the virus is required from a security point of view.
[0010] In the animal cell system, post-translational modifications, such as phosphorylation, saccharide chain addition and folding, can be carried out on proteins derived from higher animals, including humans, in a manner more similar to those produced in the living body. Such precise post-translational modifications are necessary to recreate the physiological activity originally possessed by a protein in its recombinant protein, and a protein production system is usually applied in which a mammalian cell is used as a host to pharmaceutical products and the like which require said protein. physiological activity.
[0011] However, a protein expression system in which a mammalian cell is used as a host generally exhibits low productivity and also in many cases causes a stability problem of the introduced genes. Improving the productivity of a protein through the use of a mammalian cell in culture as a host is not only very important to produce drugs for treatment, diagnostic agents and the like, but it also makes a great contribution to the research and development of the same. In this way, it is urgent to develop a gene expression system that enables the easy obtaining of a high productivity cell line using a mammalian cell in culture, in particular a Chinese hamster ovary cell (CHO cell) as a host.
[0012] A transposon is a transposable genetic element that can be transferred from one locus to another locus on the chromosome. A transposon is a powerful tool for the study of molecular biology and genetics and is used for a purpose, such as mutagenesis, gene capture and the preparation of transgenic individuals, in insects or nematodes (eg, Drosophila melanogaster or Caenorhabditis elegans ) and plants. However, the development of such a technique has been delayed for vertebrate animals, including mammalian cells.
[0013] However, in recent years transposons have been reported to exhibit activities also in vertebrate animals, and some of them have been shown to exhibit activity in mammalian cells, such as cells derived from mouse and human. Typical examples include transposons Tol1 (patent reference No. 1) and Tol2 (non-patent reference No. 1 and non-patent reference No. 13) cloned from medaka fish (ciprinodontid), 'Sleeping Beauty' reconstructed from a non-autonomous transposon existing in the genome of an Onchorhynchus fish (non-patent reference n ° 2), the artificial transposon 'Frog prince' (non-patent reference n ° 3), which is derived from the frog and a piggyBac transposon (non-patent reference no. 4), which is derived from an insect.
[0014] These DNA transposons have been used for mutagenesis, gene capture, preparation of transgenic individuals, expression of drug-resistant proteins and the like, as a gene transfer tool to produce a novel phenotype in a mammalian cell genome (references no. Patent No. 5 to 12).
[0015] In the case of insects, a procedure has been studied in which an exogenous gene is introduced into the silkworm chromosome using the PiggyBac transposon derived from a lepidopteran insect to express the protein encoded by said exogenous gene and has been given to know a protein production process using the above-mentioned techniques (patent reference n ° 2).
[0016] However, because the protein of interest expressed does not have a sufficient level of expression and is produced throughout the body of the silkworm, it causes an economic problem due to the need for an advanced purification technique to recover the expressed exogenous protein. in a highly purified form from body fluid that includes a large amount of contaminated proteins.
[0017] In addition, an example is known in which a protein related to resistance to G418 is expressed in a mammalian cell by using the medapo-derived transposon Tol2 (non-patent reference no. 2)
[0018] A minimal cis sequence and a highly repetitive sequence have been identified in the subterminal region of the Tol2 transposon that are essential for transposition (non-patent reference no. 14).
[0019] A technique for selecting cells in which a gene has been transferred in a stable state by the use of a new drug resistance gene as a stable marker and a technique for obtaining cells in which a gene is expressed at a high level are given to know in the patent reference n ° 3.
[0020] List of references
[0021] Patent Literature
[0022] Patent Reference No. 1 WO2008 / 072540
[0023] Patent Reference No. 2 Published Unexamined Japanese Patent Application No. 2001-532188 Patent Reference No. 3 Published Japanese Patent Application No. 2002-262879
[0024] Non-patent literature
[0025] Reference No. Patent No. 1 Nature 383, 30 (1996)
[0026] Non-Patent Reference No. 2 Cell 91, 501-510 (1997)
[0027] Non-Patent Reference No. 3 Nucleic Acids Res, 31, 6873-6881 (2003)
[0028] Reference no of patent n ° 4 Insect Mol.Biol.5, 141-151 (1996)
[0029] Non-Patent Reference No. 5 Genetics.166, 895-899 (2004)
[0030] Non-Patent Reference No. 6 PLoS Genet, 2, e169 (2006)
[0031] Non-Patent Reference No. 7 Proc. Natl. Acad. Sci. USA 95, 10769-10773 (1998)
[0032] Non-Patent Reference No. 8 Proc. Natl. Acad. Sci. USA 98: 6759-6764 (2001)
[0033] Non-Patent Reference No. 9 Nature 436, 221-6 (2005)
[0034] Non-Patent Reference No. 10 Nucleic Acids Res., 31.6873-6881 (2003)
[0035] Non-Patent Reference No. 11 Nucleic Acids Res., 35, e87 (2007)
[0036] Reference no of patent n ° 12 Proc Natl. Acad. Sci. USA, 103, 15008-15013 (2006)
[0037] Non-Patent Reference No. 13 Genome Biology, 8 suppl I, S7.1-S7.10 (2007)
[0038] Non-Patent Reference No. 14 Genetics 174, 639-649 (2006)
[0039] Summary of the invention
[0040] Technical problem
[0041] In order to produce and analyze a protein of interest, it is necessary to select a cell line that expressing a protein of interest stably and at elevated level using a cell in mammalian derived culture, although the preparation and culture of the cell that produces the protein of interest requires considerable effort and time.
[0042] Furthermore, although the expression of a protein of interest in a mammalian cell is known through the use of a transposon sequence, the preparation of a cell that can express a protein of interest at high level is not known and thus can be used as a system for producing the protein by using a transposon sequence, a method of preparing a mammalian cell that can produce a protein of interest at high level by using a transposon sequence and a method of producing a protein. protein by using the cell.
[0043] As mentioned above, it has been necessary to express a protein of interest in a high amount by establishing a protein production system that can produce at a high level a protein of interest by utilizing a mammalian cell in culture of efficiently and in a short time. In this manner, the objects of the invention are to provide a cell capable of expressing at high level a protein of interest that can be established efficiently and a method for producing the protein of interest through the use of the cell.
[0044] Solution to problems
[0045] In order to solve the aforementioned problems, in the context of the present invention, exhaustive studies have been carried out and it has been discovered as a result that a mammalian cell capable of expressing a protein of high level can be efficiently prepared. interest by introducing a protein expression vector comprising a gene fragment comprising a DNA encoding the protein of interest and a selectable marker gene and transposon sequences at both ends of the gene fragment, in a mammalian cell in suspension, and integration of the gene fragment inserted between a pair (two) of the transposon sequences into a chromosome of the mammalian cell. In addition, it has been discovered that the protein of interest can be produced efficiently by using the cell and thus the invention has been carried out.
[0046] Detailed description of the invention
[0047] Specifically, the invention is as set forth below:
[0048] A method for producing a protein of interest, comprising introducing a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, in a cell of CHO in suspension able to survive and proliferate in a medium without serum; introducing an expression vector (b) comprising a DNA encoding a transposase that recognizes the transposon sequences and possesses transfer activity of the gene fragment inserted between the transposon sequences on a chromosome into the CHO cell; integrating the inserted gene fragment between the transposon sequences in a chromosome of the CHO cell to obtain one of said CHO cells capable of expressing the protein of interest, and culturing the CHO cell in suspension.
[0049] 2. A method described in item 1 above, to produce a protein of interest, comprising:
[0050] (A) simultaneously introducing the expression vectors (a) and (b) into the CHO cell,
[0051] (B) transiently expressing the transposase from the expression vector introduced in step (A) to integrate the gene fragment inserted between the transposon sequences in a chromosome of the CHO cell in order to obtain a CHO cell in suspension able to express the protein of interest, and
[0052] (C) culturing in suspension the CHO cell in suspension capable of expressing the protein of interest obtained in step (B) to produce the protein of interest.
[0053] 3. A method for obtaining a suspension CHO cell capable of expressing a protein of interest, comprising introducing a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and , at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3, in a suspension CHO cell capable of surviving and proliferating in a serum-free medium; introducing an expression vector (b) comprising a coding DNA of a transposase that recognizes the transposon sequences and possesses transfer activity of the gene fragment inserted between the transposon sequences on a chromosome within the CHO cell; and integrating the inserted gene fragment between a pair of transposon sequences, into a chromosome of the CHO cell.
[0054] 4. The method described in any of the aforementioned items 1 to 3, wherein the CHO cell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO / DG44, Pro-3 and CHO- two.
[0055] 5. The method described in any of the aforementioned items 1 to 4, wherein the selectable marker gene is a cycloheximide resistance gene.
[0056] 6. The method described in item 5 above, wherein the cycloheximide resistance gene is a gene encoding a mutant of the human ribosomal protein L36a.
[0057] 7. The method described in item 6 above, wherein the mutant is a mutant in which the proline at position 54 of the human ribosomal protein L36a is replaced by another amino acid.
[0058] 8. The procedure described in item 7 above, in which the other amino acid is glutamine.
[0059] 9. A suspension CHO cell capable of surviving and proliferating in a serum-free medium and producing a protein of interest, wherein the cell comprises an expression vector (a) comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No. 15 or the nucleotide sequences of Tol2 shown in SEQ ID No. 2 and SEQ ID No. 3 and an expression vector (b) comprising a DNA encoding a transposase (a transferase) that recognizes the transposon sequences and possesses transfer activity of the inserted gene fragment between the transposon sequences in a chromosome to integrate the gene fragment inserted between the transposon sequences in the chromosome of the CHO cell.
[0060] 10. The cell described in item 9 above, wherein the CHO cell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO / DG44, Pro-3 and CHO-S;
[0061] 11. The cell described in item 9 or 10 above, wherein the selectable marker gene is a resistance gene to cycloheximide.
[0062] 12. The cell described in item 11 above, wherein the cycloheximide resistance gene is a gene encoding a mutant of the human ribosomal protein L36a.
[0063] The method described in item 12 above, wherein the mutant is a mutant in which the proline at position 54 of the human ribosomal protein L36a is replaced by another amino acid.
[0064] 14. The cell described in item 13 above, wherein the other amino acid is glutamine, and 15. Use of a protein expression vector (a) comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences shown in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences shown in SEQ ID No. 2 and SEQ ID No. 3 and an expression vector (b) comprising a DNA encoding a transposase that recognizes the transposon sequences and possesses transfer activity of the gene fragment inserted between the transposon sequences in a chromosome to integrate the gene fragment inserted between the transposon sequences in the chromosome of a suspended CHO cell capable of surviving and proliferating in a serum-free medium.
[0065] Advantageous effects of the invention
[0066] According to the protein production process of the invention, a protein of interest can be efficiently produced by the use of a mammalian cell. In addition, the cell of the invention can be used as a protein production cell to produce a recombinant protein with high efficiency.
[0067] BRIEF DESCRIPTION OF THE DRAWINGS
[0068] [Figure 1] Figure 1 depicts a schematic illustration of a transposon vector for expressing an anti-human influenza M2 antibody. Tol2-L represents a left-side transposon of Tol2 (SEQ ID No. 2), Tol2-R represents a right-side transposon of Tol2 (SEQ ID No. 3); CMV represents a CMV promoter; poly A represents a polyadenylation site; Hc represents an H chain cDNA of human antibody; Lc represents a human antibody L chain cDNA, and CHX-r represents a cycloheximide resistance gene.
[0069] [Figure 2] Figure 2 depicts a schematic illustration of an expression vector of human influenza anti-M2 antibody. CMV represents a CMV promoter; poly A represents a polyadenylation site; Hc represents an H chain cDNA of human antibody; Lc represents an L chain cDNA of human antibody and CHX-r represents a cycloheximide resistance gene.
[0070] [Figure 3] Figure 3 represents a schematic illustration of a Tol2 transposase expression vector. CAGGS represents a CAGGS promoter; Poly A represents a polyadenylation site and TPase cDNA represents the cDNA of a Tol2 transposase.
[0071] [Figure 4A] Figure 4A represents the result of examining the level of expression of a human influenza anti-M2 antibody in a CHO-K1 cell in suspension by using a Tol2 transposon vector to express an anti-human influenza M2 antibody . The ordinate shows the produced level of antibody (| jg / ml) and the abscissa shows the number of transgenic clones of the CHO-K1 cell in suspension.
[0072] [Figure 4B] Figure 4B represents the result of examining the level of expression of a human influenza anti-M2 antibody in an adhesive CHO-K1 cell by using a Tol2 transposon vector to express an anti-human influenza M2 antibody. The ordinate shows the produced level of antibody (jg / ml) and the abscissa shows the number of transgenic clones of the adhesive CHO-K1 cell.
[0073] [Figure 5] Figure 5 depicts a schematic illustration of a Tol1 transposon vector for expressing an anti-human influenza M2 antibody. Tol1-L represents a left-side transposon of Tol1 (SEQ ID No. 14), Tol1-R represents a right-side transposon of Tol2 (SEQ ID No. l5); CMV represents a CMV promoter; poly A represents a polyadenylation site; Hc represents an H chain cDNA of human antibody; Lc represents a human antibody L chain cDNA, and CHX-r represents a cycloheximide resistance gene.
[0074] [Figure 6] Figure 6 represents a schematic illustration of a Tol1 transposase expression vector. CAGGS represents a CAGGS promoter; Poly A represents a polyadenylation site and TPase cDNA represents the cDNA of a Tol1 transposase.
[0075] [Figure 7] Figure 7 depicts the result of examining the level of expression of a human influenza anti-M2 antibody in a suspended CHO-K1 cell by using a Tol1 transposon vector to express an anti-M2 human influenza antibody . The ordinate shows the produced antibody level (jg / ml) and the abscissa shows the number of transgenic clones of the CHO-K1 cell in suspension.
[0076] The invention relates to a method for producing a protein of interest, comprising introducing a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and transposon sequences at both ends of the gene fragment, in a mammalian cell in suspension, integrating the gene fragment inserted between a pair (two) of transposon sequences in a chromosome of the mammalian cell in order to obtain a mammalian cell capable of expressing said protein. interest, and to cultivate in suspension the mammalian cell. Examples of the method for producing a protein of interest of the present invention include a process, comprising steps (A) to (C) below:
[0077] (A) a step of simultaneous introduction of the following expression vectors, (a) and (b), into a mammalian cell in suspension:
[0078] (a) an expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and transposon sequences at both ends of the gene fragment, (b) an expression vector comprising a DNA encoding a transposase that recognizes the transposon sequences and possesses transfer activity of a gene fragment inserted between a pair of the transposon sequences into a chromosome,
[0079] (B) a step of transient expression of the transposase from the expression vector introduced in step (A) to integrate the gene fragment inserted between a pair of the transposon sequences in a chromosome of the mammalian cell in order to obtain a mammalian cell in suspension capable of expressing the protein of interest, and
[0080] (C) a suspension culture step of the mammalian cell in suspension capable of expressing the protein of interest obtained in step (B) to produce the protein of interest.
[0081] In addition, the present invention relates to a mammalian cell in suspension capable of producing a protein of interest, wherein a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a protein of interest is introduced. selectable marker gene and transposon sequences at both ends of the gene fragment, to integrate the gene fragment inserted between a pair of the transposon sequences on a chromosome.
[0082] In addition, the present invention relates to a mammalian cell in suspension capable of producing a protein of interest, wherein an expression vector (a) comprises a gene fragment comprising a DNA encoding a protein of interest and a gene selectable marker and transposon sequences at both ends of the gene fragment, and an expression vector (b) comprising a DNA encoding a transposase (a transferase) that recognizes the transposon sequences and possesses transfer activity of the gene fragment inserted between a pair of transposon sequences to a chromosome to integrate the gene fragment inserted between a pair of transposon sequences within the chromosome.
[0083] The term "transposon" in the present specification is a transposable genetic element and refers to a gene unit that moves in a chromosome or from a chromosome to another chromosome (transposition) maintaining a certain structure.
[0084] The transposon comprises a gene unit of repeated transposon sequences (also referred to as inverted repeat sequences (IR sequences) or terminal inverted repeat sequences (IRT sequences)) that are located in the same direction or in the reverse direction at both ends of the gene unit , and a nucleotide sequence encoding a transposase that recognizes the transposon sequence to transfer a gene present between the transposon sequences.
[0085] The transposase translated from the transposon can transfer a DNA by recognizing transposon sequences from both ends of the transposon, cutting out the inserted DNA fragment between a pair of the transposon sequences and inserting the fragment into the site to be transferred .
[0086] The term "transposon sequence" as used herein refers to the nucleotide sequence of a transposon recognized by a transposase and has the same meaning as the IR sequence or the IRT sequence. A DNA comprising the nucleotide sequence can comprise an imperfect repeat fraction with the proviso that it can be transferred (inserted into another position in the genome) by the activity of a transposase, and comprises a transposase-specific transposase sequence.
[0087] As the transposon sequence to be used in the invention, a nucleotide sequence derived from a pair of natural or artificial DNA transposons that can be recognized by a transposase and transposed to mammalian cells is used.
[0088] The nucleotide sequence derived from a DNA-type transposon is a pair of nucleotide sequences derived from the transposon1 or transposon2 derived from the medaka fish.
[0089] The nucleotide sequences of the Tol2 and Tol1 transposons derived from the medaka fish are shown in SEQ ID No. 6 and SEQ ID No. 13, respectively.
[0090] Examples of a nucleotide sequence derived from a pair of Tol2 transposons include the nucleotide sequence at positions 1 to 2.229 and the nucleotide sequence between positions 4,148 and 4,682 in the nucleotide sequence of the Tol2 transposon shown in SEC ID n ° 6 of the sequence listing. As the nucleotide sequence derived from a pair of Tol2 transposons, the nucleotide sequence between positions 1 and 200 (SEQ ID No. 2) (hereinafter referred to as the "Tol2-L sequence") and the nucleotide sequence between the positions are used. 2,285 and 2,788 (SEQ ID No. 3) (hereinafter referred to as "Tol2-R sequence") in the nucleotide sequence of the Tol2 transposon shown in SEQ ID No. 1 of the Sequence Listing.
[0091] Examples of a nucleotide sequence derived from a pair of Tol1 transposons include the nucleotide sequence comprising a nucleotide sequence between positions 1 and 157 and the nucleotide sequence between positions 1,748 and 1,855 in the nucleotide sequence of the nucleotide. Tol1 transposon shown in SEQ ID No. 13 of the Sequence Listing.
[0092] As the nucleotide sequence derived from a pair of Tol1 transposons, the nucleotide sequence is used between positions 1 and 200 (SEQ ID No. 14) (hereinafter referred to as "Tol1-L sequence") and the nucleotide sequence between positions 1,351 and 1,855 (SEQ ID No. 15) (hereinafter referred to as "Tol1- sequence") R ") in the nucleotide sequence of the Tol2 transposon shown in SEQ ID No. 1 of the Sequence Listing.
[0093] Examples of the transposon sequence include the transposon sequences, the transfer reactions of which are controlled by using a partial sequence of a transposon sequence derived from the aforementioned transposon, by adjusting the length of the sequence of nucleotides and by modifying the nucleotide sequence due to addition, deletion or substitution.
[0094] With respect to the control of the transfer reaction of a transposon, the transfer reaction can be accelerated or suppressed by acceleration or deletion, respectively, of the recognition of the transposon sequence by a transposase.
[0095] The term "transposase" as used herein refers to an enzyme that recognizes nucleotide sequences that present transposon sequences and transfer a DNA present between the nucleotide sequences within one chromosome or from the chromosome to another chromosome.
[0096] Examples of the transposase include Tol1 and Tol2, which is derived from the medaka fish, 'Sleeping Beauty' reconstructed from a non-autonomous transposon present in the genome of an Onchorhynchus fish , the artificial transposon 'Frog prince', which is derives from the frog, and the PiggyBac transposon, which is derived from an insect.
[0097] As transposase, a native enzyme can be used, and any transposase in which a portion of its amino acids have been substituted, removed, inserted and / or added can be used, provided that the same transposase transfer activity is maintained. By controlling the enzymatic activity of the transposase, the transfer reaction of the DNA between the transposon sequences can be controlled.
[0098] In order to analyze whether or not it possesses a transfer activity similar to that of the transposase, it can be measured by the 2-component analysis system disclosed in Japanese Unexamined Patent Application Publication No. 235575/2003.
[0099] By way of illustration, it can be analyzed whether a non-autonomous Tol2 element can be transferred and inserted or not inserted into a mammalian cell chromosome by the activity of a transposase, by separate use of a plasmid comprising a Tol2 transposon with Tols transposase deletion ( non-autonomous transposon derived from Tol2) and a plasmid comprising the Tol2 transposase.
[0100] The term "non-autonomous transposon" as used herein refers to a transposon that has lost a transposase present within the transposon and that, therefore, its autonomous transference can not be carried out. The non-autonomous transposon can transfer the inserted DNA between the transposon sequences of the non-autonomous transposon to the chromosome of the host cell in the case that a transposase protein, an mRNA encoding the transposase protein or a DNA encoding the protein is allowed transposase are present simultaneously in the cell.
[0101] The transposase gene refers to a gene encoding a transposase. In order to improve its expression efficiency in a mammalian cell, a sequence that fits a space between the Kozak consensus sequence (Kozak M., Nucleic Acids Res. 12: 857-872, 1984) or a binding sequence ribosomal, the Shine-Dalgarno sequence and the start codon, of appropriate distance (eg, from 6 to 18 bases), can be connected to a site located upstream of the ATG translation start codon of the gene.
[0102] According to the method of the invention, in order to integrate a gene fragment comprising a DNA coding for the protein of nterés and a selectable marker gene in an expression vector on the chromosome of a host cell, a host is introduced into the host cell. expression vector comprising the gene fragment comprising a DNA encoding the protein of interest and a selectable marker gene and transposon sequences at both ends of the gene fragment, and allowing a transposase to act on the transposon sequences comprised in the vector of expression that has been introduced into the cell. In order to allow a transposase to act on the transposon sequences comprised in the expression vector that has been introduced into the cell, the transposase can be injected into the cell, or an expression vector can be introduced which comprises a DNA encoding the transposase in the host cell together with an expression vector comprising a DNA encoding the protein of interest and a selectable marker gene. In addition, by introducing an RNA encoding a transposase gene into the host cell, the transposase can be expressed in the cell.
[0103] The expression vector is not particularly limited. Any expression vector can be used by optionally selecting among the expression vectors known to the person skilled in the art, according to the host cell into which an expression vector comprising a transposase gene, and the like is introduced.
[0104] In order to produce a protein consisting of two or more polypeptides by the method of the invention, the DNA can be integrated into the chromosome of the cell by integrating a DNA encoding two or more polypeptides into the same or different expression vectors , followed by the introduction of the expression vectors in a host cell.
[0105] The transposase can be inserted into an expression vector to express it together with the protein of interest, or it can be inserted into a different vector of the expression vector. The transposase can be allowed to act transiently or can be allowed to act continuously, although it is preferred to allow the transposase to act transiently in order to prepare a cell for stable production.
[0106] As a method to allow the transposase acts transiently examples is included a method comprising preparing an expression vector comprising a DNA encoding the transposase and an expression vector comprising a D ⁿ encoding a protein of interest, followed by the introduction of both expression plasmids simultaneously into a host cell.
[0107] The term "expression vector" as used herein refers to an expression vector that will be used for introduction into a mammalian cell in order to express a protein of interest. The expression vector used in the invention has a structure in which at least one pair of transposon sequences are present on both sides of an expression cassette.
[0108] The term "expression cassette" as used herein refers to a nucleotide sequence that presents a region of control of gene expression necessary to express a protein of interest and a sequence encoding the protein of interest. Examples of the gene expression control region include an enhancer, a promoter and a terminator. The expression cassette may contain a selectable marker gene.
[0109] Any promoter can be used, with the proviso that it can function in an animal cell. Examples include an IE (early immediate) gene promoter from cytomegalovirus (CMV), the SV40 early promoter, a retrovirus promoter, a metallothionein promoter, a heat shock promoter, the SRa promoter, the virus from the Moloney murine leukemia, an intensifier and the like. In addition, an enhancer of the human CMV IE gene can be used in conjunction with the promoter.
[0110] The term "selectable marker gene" refers to another marker gene that can be used to distinguish a cell into which a plasmid vector has been introduced relative to a cell that does not have the vector. Examples of the selectable marker gene include a drug resistance gene (a neomycin resistance gene, a DHFR gene, a puromycin resistance gene, a blasticidin resistance gene, a hygromycin resistance gene and a gene of resistance to cycloheximide (published Japanese Unexamined Patent Application No. 262879/2002), fluorescence and bioluminescence marker genes (such as green fluorescent protein, GFP) and the like.
[0111] In the invention, preferred selectable marker is a drug resistance gene and a particularly preferred selectable marker is a cycloheximide resistance gene. In addition, by performing a gene modification of the selectable marker gene, the drug resistance performance and the luminescence yield of the selectable marker protein can also be modified. Cycloheximide (hereinafter sometimes referred to as CHX) is an inhibitor of protein synthesis and as examples of the use of the CHX resistance gene as a selectable marker gene, cases of yeast cells are known (Kondo K., J. Bacteriol 177 (24): 7171-7177, 1995) and animal cells (published Japanese Unexamined Patent Application No. 262879/2002).
[0112] In the case of animal cells, it has been found that resistance to cycloheximide is provided by a transformant that expresses a protein encoded by the nucleotide sequence shown in SEQ ID No. 7 of the Sequence Listing, in which the proline in the position 54 of the L36a subunit of the human ribosomal protein encoded by the nucleotide sequence shown in SEQ ID No. 5 of the Sequence Listing has been replaced by glutamine.
[0113] The method for introducing the aforementioned protein expression vector comprising a transposon sequence, a plasmid vector expressing a transposase and RNA is not particularly limited. Examples include calcium phosphate transfection, electroporation, a liposome method, a gene gun method, and the like.
[0114] Examples of the method for directly introducing a transposase in the form of a protein include microinjection or endocytosis for introduction into a cell. Gene transfer can be carried out by the procedure described in the Shin Idenshi Kogaku Handbook (New Genetic Engineering Handbook), edited by Masami Muramatsu and Tadashi Yamamoto, published by Yodo-sha, ISBN 9784897063737.
[0115] The host cell is a mammalian cell in suspension. The mammalian cell is a Chinese hamster ovary cell, CHO cell (Journal of Experimental Medicine 108: 945, 1958; Proc. Natl. Acad. Sci. USA 601275, 1968); Genetics 55: 513, 1968); Chromosoma 41: 129, 1973); Methods in Cell Science 18: 115, 1996); Radiation Research 148: 260, 1997); Proc. Natl. Acad. Sci. USA 77: 4216, 1980; Proc. Natl. Acad. Sci. 60: 1275, 1968); Cell 6: 121, 1975); Molecular Cell Genetics, appendix I and II (pages 883 to 900). Examples of the CHO cell include CHO / DG44, CHO-K1 (ATCC No. CCL-61), DUKXB11 (ATCC No. CCL-9096), Pro-5 (ATCC No. CCL-1781), c Ho -S (Life Technologies, cat # 11619), Pro-3 and CHO cell sub-strains.
[0116] In addition, the aforementioned host cell can also be used in the protein production process of the invention by its modification so that it is suitable for the production of proteins, by modification of the chromosomal DNA, the introduction of an exogenous gene, and the like. .
[0117] In addition, in order to control the structure of the saccharide chain bound to a protein of interest that must be produced, Lec13, which acquires resistance to lectins [Somatic Cell and Molecular Genetics 12:55, 1986] and a CHO cell from which The α1,6-fucosyltransferase gene (documents No. WO2005 / 35586 and No. WO2002 / 31140) can also be used as the host cell.
[0118] The protein of interest can be any protein with the proviso that it can be expressed by the method of the invention. Specifically, examples include a human serum protein, a peptide hormone, a growth factor, a cytokine, a blood coagulation factor, a protein from the fibrinolysis system, an antibody and partial fragments of various proteins, and the like.
[0119] Preferred examples of the protein of interest include a monoclonal antibody, such as a chimeric antibody, a humanized antibody and a human antibody; Fc fusion protein and protein bound to albumin, and a fragment thereof.
[0120] An effector activity of a monoclonal antibody obtained by the method of the present invention can be controlled by various methods. For example, known methods are a method for controlling an amount of fucose (hereinafter referred to as "nuclear fucose") that is linked to N-acetylglucosamine (GlcNAc) by a-1,6 linkage at a reducing end of a linked saccharide chain by N of complex type that is linked to asparagine (Asn) at position 297 of an Fc region of an antibody (documents No. WO2005 / 035586, No. WO2002 / 31140 and No. WO00 / 61739), a method for controlling an effector activity of a monoclonal antibody by modifying one or more amino acid groups of an Fc region of the antibody, and the like. The effector activity of the monoclonal antibody produced by the method of the present invention can be controlled by the use of any of the methods.
[0121] "Effector activity" refers to an antibody-dependent activity that is induced by an Fc region of an antibody. As an effector activity, antibody dependent cellular cytotoxicity (ADCc activity), complement dependent cytotoxicity (CDC activity), antibody dependent phagocytosis (ADP activity) by phagocytic cells, such as macrophages or dendritic cells, and the like are known.
[0122] In addition, by controlling the nuclear fucose content of a complex-type N-linked saccharide chain of the Fc region of a monoclonal antibody, an effector activity of the antibody can be increased or decreased.
[0123] As a method of reducing the fucose content that is attached to a complex-bound N-linked saccharide chain to Fc of the antibody, an antibody to which fucose is not bound can be obtained by expression of an antibody using a CHO cell which is deficient in a gene encoding α1,6-fucosyltransferase. The antibody to which fucose is not bound has a high ADCC activity.
[0124] On the other hand, as a method to increase the content of fucose that is bound to a saccharide chain linked by N-type complex to Fc of an antibody, an antibody to which fucose is bound can be obtained, by the expression of an antibody using a cell host in which a gene encoding α1,6-fucosyltransferase has been introduced. The antibody to which fucose is attached has a lower ADCC activity than the antibody to which fucose is not bound.
[0125] In addition, by modifying one or more amino acid residues in an Fc region of an antibody, it can increase or decrease ADCC activity or CDC activity. For example, the CDC activity of an antibody can be increased by using the amino acid sequence of the Fc region described in document No.2007 / 0148165.
[0126] In addition, the ADCC activity or CDC activity of an antibody can be increased or reduced by modification of the amino acid as indicated in US Patent Nos. 6,737,056, No. 7,297,775 or No. 7,317,091. The term "mammalian cell in suspension" in the present invention refers to a cell that does not adhere to a cell culture anchor coating that facilitates the adhesion of cells in the culture, such as microbeads, a culture vessel to the cell. tissue culture (also referred to as tissue culture or adhesion culture container, and the like) and the like, and can survive and grow by suspension in the culture liquid.
[0127] In the case that the cell does not adhere to the cell culture anchor, it can survive and grow in individual cell state in the culture liquid or survive and grow in a cellular mass state formed by agglutination of two or more cells.
[0128] In addition, as a mammalian cell in suspension for use in the present invention, a cell that can survive and grow in a serum-free medium that does not contain bovine fetal serum (hereinafter referred to as FCS) and the like, while in suspension in the culture liquid without adhering to the cell culture anchor is preferable, and a mammalian cell that can survive and grow in suspension in a protein-free medium containing no protein is more preferable.
[0129] As a culture vessel for tissue culture, it can be any culture vessel, such as a flask, a Petri dish and the like, with the proviso that a coating for the adhesion culture is applied therein. Specifically, for example, whether or not it is a mammalian cell in suspension can be confirmed by use of a commercially available tissue culture flask (manufactured by Greiner), an adhesion culture flask (manufactured by Sumitomo Bakelite) and the like.
[0130] As a suspension mammalian cell for use in the present invention, it can be a CHO cell prepared by further adapting a CHO cell that originally had the growth property in suspension, to suspension culture, or a CHO cell in suspension prepared by adapting an adhesive CHO cell to the suspension culture conditions. Examples of cells that originally had the growth property in suspension include the HO-S cell (manufactured by Invitrogen) and the like.
[0131] The "suspension mammalian cell prepared by adaptation of a mammalian adhesive cell to the above-mentioned suspension culture conditions" can be prepared by the procedure described in Mol. Biotechnol. 15 (3): 249-57, 2000, or by the procedure shown below, and can be prepared by establishing a cell that exhibits a suspension growth property and a survival property similar to those present prior to adaptation to suspension culture or higher than those present before adaptation to suspension culture (J. Biotechnol. 130 (3): 289-90, 2007).
[0132] The expression "similar to those present before adaptation to suspension culture" refers to the survival ratio, the proliferation rate (doubling time) and the like, of the cell adapted to the suspension culture are substantially similar to those of the cell before adaptation to suspension culture.
[0133] Examples of the method for adapting a mammalian cell in suspension to the suspension culture conditions according to the present invention include the following procedure. The serum content of a medium containing serum is reduced to 1/10 and the subculture is repeated at a relatively high cell concentration. Upon reaching the level of survival and proliferation of the mammalian cell, the serum content is further reduced and the subculture is repeated. By this method, a mammalian cell in suspension can be prepared which can survive and proliferate under serum-free conditions.
[0134] In addition, a mammalian cell in suspension can also be prepared by a method comprising culturing with the addition of a suitable nonionic surfactant, such as Pluronic-F68 or the like, to the culture liquid.
[0135] In the present invention, as a property of the mammalian cell in suspension, when growing in suspension 2 × 10 5 cells / ml, the cell concentration after cultivation for 3 or 4 days preferably of 5 × 10 5 cells / ml or more, more preferably of 8 × 10 5 cells / ml or higher, particularly preferably 1 × 10 6 cells / ml or more, most preferably 1.5 × 10 6 cells / ml or more.
[0136] In addition, the doubling time of the mammalian cell in suspension of the present invention preferably it is 48 hours or less, more preferably 24 hours or less, particularly preferably 18 hours or less, most preferably 11 hours or less.
[0137] Examples of the medium for suspension culture include commercially available medium, such as CD-CHO medium (manufactured by Invitrogen), EX-CELL ^{3 2 5-P f} medium (manufactured by SAFC Biosciences), SFM4CHO medium (manufactured by HyClone) and similar. In addition, it can also be obtained by mixing saccharides, amino acids and similar acids which are necessary for the culture of mammalian cells.
[0138] The mammalian cell in suspension can be cultured using a culture vessel that can be used for suspension culture under culture conditions capable of suspension culture. Examples of the culture vessel include a 96 well plate for cell culture (manufactured by Corning), a T flask (manufactured by Becton Dickinson), an Erlenmeyer flask (manufactured by Corning) and the like.
[0139] With respect to culture conditions, for example, it can be grown statically in a 5% CO ² atmosphere at a culture temperature of 37 ° C. Agitation culture equipment, such as a culture equipment for exclusive use in suspension culture, Wave Bioreactor (manufactured by GE Healthcare Bioscience) can also be used.
[0140] With respect to the suspension culture conditions of a mammalian cell in suspension using the Wave Bioreactor equipment, the cell can be cultured by the procedure described on the GE Healthcare Bioscience website http://www.gelifesciences.co.jp /tech-support/manual/pdf/cellcult/wave-03-16.pdf. In addition to the agitation culture, the culture can also be used by rotating agitation equipment, such as a bioreactor. Culturing with a bioreactor can be carried out by the method described in Cytotechnology 52: 199-207, 2006, and the like.
[0141] In the present invention, by using a cell line different from the mammalian cells in suspension, any cell line can be used provided that it is a mammalian cell line adapted to the suspension culture by the aforementioned method and is a lineage. cell that can be used in the protein production process of the present invention.
[0142] The purification of the protein of interest produced by the mammalian cell in suspension is carried out by separating the protein of interest for impurities other than the protein of interest in a culture liquid or cell homogenate containing the protein of interest. . Examples of the separation process include centrifugation, dialysis, precipitation with ammonium sulfate, column chromatography, a filter and the like. The separation can be carried out based on the difference of physical-chemical properties of the protein of interest and impurities and based on the difference in its affinity for the column carrier.
[0143] The procedure for purifying the protein of interest can be carried out, for example, by the procedure described in Protein Experimentation Note (the first volume) - Extraction, Separation and Expression of Recombinant Protein (translation of a textbook written in Japanese) ( edited by Masato Okada and Kaori Miyazaki, published by Yodo-sha, ISBN 9784897069180).
[0144] The present invention has been described above showing preferred embodiments thereof for the sake of easy understanding. Hereinafter, the present invention is further described specifically on the basis of examples, although the above-mentioned explanations and the examples below are provided solely by way of non-limiting example. According to the foregoing, the scope of the invention is not limited to the embodiments and examples that are specifically described herein, but is limited exclusively by the claims. Various experimental techniques related to genetic recombination described hereinafter, such as cloning and the like, were carried out according to the genetic engineering techniques described in Molecular Cloning, 2nd edition, edited by J. Sambrook, E.F. Frisch and T. Maniatis, and Current Protocols in Molecular Biology, edited by Frederick M. Ausubel et al., Published by Current Protocols, and the like.
[0145] Examples
[0146] [Example 1]
[0147] Preparation of transposon vector for the expression of human influenza anti-M2 antibody
[0148] A plasmid containing a gene expression cassette for mammalian cells comprising an arbitrary human antibody gene and a drug resistance marker gene inserted between a pair of Tol2 transposon sequences as a plasmid vector for protein expression was used. .
[0149] Each DNA of the genes used was chemically and artificially synthesized based on a known nucleotide sequence or obtained by preparing the primers for the two terminal sequences and carrying out a PCR using an appropriate DNA source as a template. In order to carry out the gene manipulation subsequently, a restriction site for a restriction enzyme was added to the end of the primer.
[0150] Among the nucleotide sequences of the non-autonomous Tol2 transposon disclosed in Japanese Unexamined Patent Application Publication No. 235575/2003 (SEQ ID No. 1), the nucleotide sequence between positions 1 and 200 (Tol2 sequence) was used. -L) (SEQ ID No. 2) and the nucleotide sequence between positions 2,285 and 2,788 (Tol2-R sequence) (SEQ ID No. 3).
[0151] Each synthetic DNA fragment comprising a pair of transposon sequences (manufactured by Takara Bio Inc.) was prepared by the following procedure. A DNA fragment comprising a nucleotide sequence in which a recognition sequence of the restriction enzyme NruI was bound to both the 5'-terminal and the 3'-terminal end of the Tol2-R sequence was prepared. Next, a DNA fragment comprising a nucleotide sequence was prepared in which a restriction enzyme recognition sequence Fsel was attached to the 5'-terminal end of the Tol2-L sequence and an AscI restriction enzyme was attached to the 3'-terminal end thereof.
[0152] Next, DNA fragments prepared in this way comprising the sequence Tol2-R and the sequence Tol2-L were inserted into the expression vector N5LG1-M2-Z3 (document No. WO2006 / 061723) comprising a coding nucleotide sequence. of an amino acid sequence of the human influenza anti-M2 antibody Z3G1.
[0153] The vector N5LG1-M2-Z3 (document No. WO2006 / 061723) into which a nucleotide sequence (SEQ ID No. 8) encoding the H chain of the human influenza anti-M2 antibody Z3G1 (ATCC no. of deposit PTA-5968, deposited on March 13, 2004, American Type Culture Collection, Manassas, VA, USA) and a nucleotide sequence (SEQ ID No. 10 and SEQ ID No. 11) coding for the L chain (SEC ID No. 9) thereof were inserted under the control of the CMV enhancer / promoter as an antibody gene expression cassette.
[0154] The DNA fragment comprising the Tol2-R sequence was inserted into the restriction enzyme NruI site of the N5LG1-M2-Z3 vector, on the 5'-terminal side of a gene fragment comprising the gene expression cassette of the antibody and a resistance marker gene. Next, the fragment of a Dn comprising the sequence Tol2-L was inserted into the restriction enzyme sites Fsel and AscI on the 3'-terminal side.
[0155] In addition, a transposon vector was constructed to express a human influenza anti-M2 antibody (Figure 1) by inserting a cycloheximide resistance gene cassette connected to a nucleotide sequence (SEQ ID NO: 5) encoding a cycloheximide resistance gene (a gene in which the proline at position 54 of the human ribosomal protein L36a has been replaced by glutamine) at the Fsel recognition site of vector N5LG1-M2-Z3 connected to the Tol2 transposon sequence, under the control of the CMV enhancer / promoter.
[0156] On the other hand, a vector that did not contain transposon sequences was called an expression vector of human influenza anti-M2 antibody and was used as the control vector (Figure 2).
[0157] [Example 2]
[0158] Preparation of transposase expression vector
[0159] The transposase was expressed using an expression vector independent of the expression vector of the antibody of interest. That is, a gene that was coding for a Tol2 transposase derived from medaka fish (SEQ ID No. 4) downstream of the CAGGS promoter from a pCAGGS vector (Gene 108: 193-200, 1991) was inserted and used as the vector of expression (figure 3).
[0160] [Example 3]
[0161] (1) Preparation of CHO cell in suspension
[0162] An adhesive CHO cell that had been cultured using a-MEM medium (manufactured by Invitrogen) containing 10% serum (FCS) was detached and recovered by treatment with trypsin and cultured under agitation at 37 ° C in an incubator with 5% CO ² using a fresh a-MEM medium containing 10% FCS. Several days later, the growth of these cells was confirmed and the culture was then carried out with shaking by seeding them in a-MEM medium containing 5% FCS at a concentration of 2 × 10 5 cells / ml.
[0163] Several days later, inoculation was carried out in a similar manner using the α-MEM medium containing 5% FCS. Finally, a cell adapted to the suspension culture was prepared by repetition of the subculture and culture under agitation using a-MEM medium without serum and it was confirmed that the cells had the same growth capacity as in the case of the culture in the presence of serum.
[0164] (2) Preparation of antibody producing CHO cell
[0165] The transposon vector for expressing the human influenza anti-M2 antibody prepared in Examples 1 and 2 (hereinafter referred to as the transposon vector) and the Tol2 transposase expression vector pCAGGS-T2TP (Figure 3; Kawakami K. and Noda T., Genetics 166: 895-899, 2004) were used as expression vectors. In addition, as the control, the expression vector of human influenza anti-M2 antibody that did not present transposon sequences was used.
[0166] By introducing the aforementioned expression vectors into the CHO-K1 cell adapted to the suspension culture (American Type Culture Collection cat No. CCL-61) or the HEK293 cell (FreeStyle 293F cell, manufactured by Invitrogen), the frequencies of obtaining clones resistant to cycloheximide were compared.
[0167] Each cell (4x106 cells) in 400 μl of PBS and the transposon vector were suspended to express the human influenza anti-M2 antibody (10 pg) and the transposase expression vector Tol2 (25 pg) directly in the form of circular DNA by electroporation In this regard, in order to express the transposase Tol2 transiently, the expression vector of the Tol2 transposase was introduced directly in the form of circular DNA in order to avoid its integration in the chromosome of the host.
[0168] In addition, as a control, the expression vector of human influenza anti-M2 antibody (10 pg) was linearized with a restriction enzyme and then introduced into each cell, according to the standard method of gene transfer by electroporation.
[0169] Electroporation was carried out using a 4 mm hollow width cuvette (manufactured by Bio-Rad), using an electroporator (Gene Pulser Xcell System (manufactured by Bio-Rad)) under the conditions of 300 V voltage, 500 pF electrostatic capacity and at room temperature.
[0170] After transfection by electroporation, each cell was seeded in three 96-well plates and cultured in a CO ² incubator for 3 days using the EX-CELL 325-PF medium manufactured by sAfC Biosciences for the CHO cell and the FreeStyle medium. -293 (manufactured by Invitrogen) for the HEK293 cell.
[0171] Then, from the day of media exchange on the 4th day of transfection, 3 pg / ml of cycloheximide was added to the medium so that the cells were cultured in the presence of cycloheximide, followed by cultivation for 3 weeks, carrying out simultaneously the media exchange every week.
[0172] After culturing for 3 weeks, the number of wells in which cycloheximide-resistant colonies were found was counted. The results are shown in Tables 1 and 2.
[0173] Table 11
[0174] Table 1. Comparison of numbers of cells resistant to cycloheximide (CHO cells)
[0175] Vector Vector conventional transposon
[0176] Test 1 155/288 0/288
[0177] Test 2 100/288 0/288
[0178] Test 3 94/288 0/288
[0179] ÍTabla 21
[0180] Table 2. Comparison of numbers of cells resistant to cycloheximide (HEK293 cells)
[0181] Vector Vector conventional transposon
[0182] Test 1 0/288 0/288
[0183] Test 2 0/288 0/288
[0184] Test 3 0/288 0/288
[0185] As shown in Table 1, each transposon vector of human influenza anti-M2 antibody expression or human influenza anti-M2 antibody expression vector was introduced into the suspended CHO-K1 cell. As a result, no transformants resistant to cycloheximide were obtained from the cell introduced with the human influenza anti-M2 antibody expression vector as in the case of the other cell lines, but cycloheximide-resistant transformants were obtained from the cell into which transposon vector had been introduced to express influenza anti-M2 antibody human with a high frequency.
[0186] On the other hand, as shown in Table 2, no cycloheximide-resistant transformants were obtained by introducing the transposon vector to express human influenza anti-M2 antibody and human influenza anti-M2 antibody expression vector in the HEK293 cell .
[0187] Based on these results, it was found that the protein coding gene and the cycloheximide resistance gene that had been inserted between a pair of transposon sequences were efficiently introduced into the chromosome of the host cell, i.e., a mammalian cell in the host cell. suspension.
[0188] (3) Examination of antibody production by CHO cell in suspension and adhesive CHO cell In order to examine the efficiency of antibody production by a suspension CHO cell or an adhesive CHO cell, they were examined the amounts of the antibodies produced by the respective cell lines. As the CHO cell in suspension, the CHO-K1 cell in suspension adapted to the suspension culture was used. In addition, as an adhesive CHO cell, the adhesive CHO-K1 cell was used before adaptation to the suspension culture.
[0189] The transposon vector of expression of the human influenza anti-M2 antibody (10 | jg) and the transposase expression vector Tol2 (25 jg) was introduced into the suspended CHO-K1 cell and into the adhesive CHO-K1 cell , respectively, by electroporation. Then, the CHO-K1 cell in suspension was seeded and the CHO-K1 cell adhesive in three 96-well plates for each cell.
[0190] A medium for the suspension cells (EX-CELL 325-PF, manufactured by SAFC Biosciences) was used for the suspended CHO-K1 cell, and the a-MEM medium containing 10% serum was used for the cell of CHO-K1 adhesive. Each cell was cultured in a CO ² incubator for 3 days. From the day of media exchange on the 4th day of transfection, 3 μg / ml of cycloheximide was added to the medium so that the cells were cultured in the presence of cycloheximide and the cells were further cultured for 3 weeks. In this case, the media exchange was carried out every week.
[0191] For the CHO-K1 cell in suspension, 1x106 cells were seeded in a 6-well plate and cultured under stirring in a CO ² incubator for 3 days and the amount of human influenza anti-M2 antibody protein was measured by HPLC. using the culture supernatant.
[0192] For the adhesive CHO-K1 cell, the media exchange was carried out after the cells reached the confluence in a 6-well plate (2x106 cells) and 3 days after the static culture, the amount of antibody protein was measured by HPLC. using the culture supernatant.
[0193] The concentration of the antibody in the culture supernatant was measured following the procedure described in Yeast Res. 7: 1307-1316, 2007. The results are shown in Figures 4A and 4B.
[0194] As shown in Figure 4A, a large number of cells was obtained which showed a markedly elevated antibody expression level when using the CHO-K1 cell adapted to the suspension culture. On the other hand, as shown in Figure 4B, only cells showing an expression level at the HPLC detection limit (5 jg / ml) or lower were obtained when using the adhesive CHO-K1 cell.
[0195] Based on these results, it was found that, for the expression of a protein of interest using a transposon vector, the protein of interest can be expressed at a high level by using a mammalian cell in suspension.
[0196] Furthermore, it is discovered from the results of Examples 1 to 3 that the method of the invention can be used as a novel method to produce a protein of interest by efficiently preparing a producer cell that can express an exogenous gene at high level using a mammalian cell in suspension adapted to the suspension culture.
[0197] [Example 4]
[0198] Preparation of Tol1 transposon vector for the expression of human influenza anti-M2 antibody
[0199] In the same manner as in Example 1, a plasmid containing a gene expression cassette for mammalian cells, comprising an arbitrary human antibody gene and a drug resistance marker gene, was used as the protein expression plasmid vector. inserted between a pair of Tol1 transposon sequences.
[0200] Each DNA of the genes used was chemically and artificially synthesized based on a known sequence information or obtained by preparing the primers for the two terminal sequences and carrying out a PCR using an appropriate DNA source as a template. For gene manipulation to be carried out subsequently, a site cut by a restriction enzyme was added to the end of the primer.
[0201] Among the non-autonomous Tol1 transposon nucleotide sequences shown in SEQ ID No. 13 of the Sequence Listing (document No. WO2008 / 072540), the nucleotide sequence between positions 1 and 200 (Tol1-L sequences) was used ( SEQ ID No. 14) and the nucleotide sequence between positions 1,351 and 1,855 (Tol1-R sequence) (SEQ ID No. 15) as the transposon sequences.
[0202] Each of the synthetic DNA fragments comprising each pair of transposon sequences was prepared by the following procedure. A DNA fragment comprising a nucleotide sequence in which an NruI restriction enzyme recognition sequence was connected to both the 5'-terminal end and the 3'-terminal end of the Tol1-R sequence was prepared. Next, a DNA fragment comprising a nucleotide sequence in which a recognition sequence of a restriction enzyme Fsel was connected to the 5'-terminal end of the Tol1-L sequence and an AscI restriction enzyme site that was connected to the 3'-terminal end of it.
[0203] Next, the DNA fragments prepared in this manner comprising the sequence of Tol1-R and the sequence of Tol1-L were inserted into the expression vector N5LG1-M2-Z3. The DNA fragment comprising the Tol1-R sequence was inserted into the NruI restriction enzyme site of vector N5LG1-M2-Z3, present on the 5'-terminal side of a gene fragment comprising the gene expression cassette of the vector. antibody and a resistance marker gene, and the DNA fragment comprising the sequence of Tol1-L was inserted into the sites of the restriction enzymes Fsel and AscI present on the 3'-terminal side.
[0204] In addition, a Tol1 transposon vector was constructed to express an anti-human influenza M2 antibody (FIG. 5) by inserting a cycloheximide resistance gene cassette connected to a cycloheximide resistance gene (a gene in which the proline at position 54 of the human ribosomal protein L36a has been mutated to glutamine) at the Fsel recognition site of the N5LG1-M2-Z3 vector connected to the Tol1 transposon sequence, under the control of the CMV enhancer / promoter.
[0205] [Example 5]
[0206] Preparation of Tol1 transposase expression vector
[0207] The transposase was expressed using an expression vector independent of the expression vector of the antibody of interest. That is, a gene expression cassette of the Tol1 transposase connected to a DNA fragment encoding a Tol1 transposase derived from medaka fish containing the nucleotide sequence shown in SEQ ID No. 16 of the Sequence Listing was inserted into pBluescriptlI SK (+) (manufactured by Stratagene) under the control of the CMV enhancer / promoter and used as expression vector pTol1-ase (Figure 6).
[0208] [Example 6]
[0209] (1) Preparation of antibody producing CHO cell
[0210] The transposon vector Tol1 to express the human influenza anti-M2 antibody (hereinafter referred to as the Tol1 transposon vector) and the Tol1 pTol1-asa transposase expression vector of Examples 4 and 5 were used as the expression vectors. In addition, the CHO-K1 cell prepared by the suspension culture adaptation in the same manner as in Example 3 (1) was used as the cell.
[0211] The aforementioned expression vectors were introduced into the CHO-K1 cell adapted to the suspension culture and the frequency of obtaining clones resistant to cycloheximide was measured. The CHO-K1 cell adapted to the suspension culture (4x106 cells) was suspended in 400 μl PBS and the Tol1 transposon vector to express the human influenza anti-M2 antibody (10 pg) and the Tol1 transposase expression vector (50 pg) were co-transfected directly in the form of circular DNA by electroporation. In order to carry out the transient expression of the Tol1 transposase, the expression vector of the Tol1 transposase was introduced directly in the form of circular DNA in order to avoid integration into the chromosome of the host.
[0212] Electroporation was carried out using a 4 mm hollow width cuvette (manufactured by Bio-Rad), using an electroporator (Gene Pulser Xcell System (manufactured by Bio-Rad)) under the conditions of 300 V voltage, 500 pF electrostatic capacity and at room temperature.
[0213] After transfection by electroporation, each cell was seeded in two 96-well plates and cultured in a CO ² incubator for 3 days using the EX-CELl 325-PF medium (manufactured by ^s A ^f C Biosciences) for the cell of CHO. Then, from the day of media exchange on the 4th day of transfection, 3 pg / ml of cycloheximide was added to the medium so that the cells were cultured in the presence of cycloheximide, followed by cultivation for 3 weeks, carrying out simultaneously the media exchange every week.
[0214] After culturing for 3 weeks, the number of wells in which cycloheximide-resistant colonies were found was counted. The results are shown in Table 3. Each of the tests 1 to 3 in Table 3 shows the result of carrying out the gene transfer three times.
[0215] ÍTabla 31
[0216] Vector Tol1 transposon
[0217] Test 1 133/192
[0218] Test 2 67/192
[0219] Test 3 122/1992
[0220] As shown in Table 3, by introducing the transposon vector Tol1 to express the human influenza anti-M2 antibody in the suspended CHO-K1 cell, cycloheximide-resistant transformants were obtained at a high frequency in a similar manner to Example 3, in which the transposon vector Tol2 had been introduced to express the human influenza anti-M2 antibody.
[0221] It was found, based on these results, that the antibody gene and the cycloheximide resistance gene inserted between a pair of transposon sequences were efficiently transduced into the interior of the chromosome of the host cell, i.e., the mammalian cell in suspension as well. in the case of the use of the Tol1 transposon
[0222] (2) Examination of antibody production by CHO-K1 cell in suspension
[0223] The efficiency of antibody production of the CHO-K1 cell in suspension was examined using the CHO-K1 cell in suspension. The transposon vector for expressing the human influenza anti-M2 antibody (10 pg) and the transposase Tol1 expression vector (50 pg) were introduced by electroporation into the CHO-K1 cell in suspension adapted to the suspension culture.
[0224] The cells were then seeded in two respective 96-well plates and cultured for 3 days in a CO ² incubator using EX-CELl 325-PF suspension culture medium. After the exchange of medium on the 4th day after electroporation, the cells were cultured for 3 weeks in the presence of 3 pg / ml of cycloheximide. In this case, the media exchange was carried out every week.
[0225] For the CHO-K1 cell in suspension, 1x106 cells were seeded in a 6-well plate and cultured under stirring in a CO ² incubator for 3 days and the amount of human influenza anti-M2 antibody protein was measured by HPLC. using the culture supernatant.
[0226] The concentration of the antibody in the culture supernatant was measured following the procedure described in Yeast Res. 7: 1307-1316, 2007. The results are shown in Figure 7.
[0227] As shown in Figure 7, a large number of cells showing a markedly elevated antibody expression level were obtained in case the Tol1 transposon was also used. From this result it was found that, similarly to the case of the use of the nucleotide sequence derived from the Tol2 transposon, a mammalian cell in suspension capable of expressing at high level the protein of interest when using a sequence could also be obtained. of nucleotides derived from the Tol1 transposon as the transposon sequence.
[0228] The present application is based on the Japanese application n ° 2009-140626, filed on June 11, 2009 and in the provisional application of US patent n ° 61 / 186,138, filed on June 11, 2009.
[0229] Industrial applicability
[0230] By the process for producing the protein of the present invention, a protein of interest can be efficiently produced using a mammalian cell in suspension. The cell of the present invention can be used as a protein producing cell for the production of a recombinant protein.
[0231] Free text of the sequence listing
[0232] SEQ ID No. 1. Description of artificial sequence: nucleotide sequence of non-autonomous Tol2 transposon

权利要求:
Claims (15)
[1]
A method for producing a protein of interest, comprising introducing a protein expression vector (a) comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment , a pair of transposon sequences which are the nucleotide sequences of Tol1 represented in SEQ ID No. 14 and SEQ ID No. 15 or the nucleotide sequences of Tol 2 shown in SEQ ID No. 2 and SEQ ID No. 3, in a CHO cell in suspension capable of surviving and proliferating in a serum-free medium; introducing an expression vector (b) comprising a DNA encoding a transposase that recognizes the transposon sequences and exhibits an activity of transferring a gene fragment inserted between the transposon sequences in a chromosome in the CHO cell; integrating the inserted gene fragment between the transposon sequences in a chromosome of the CHO cell to obtain said CHO cell capable of expressing the protein of interest; and culturing the CHO cell in suspension.
[2]
The method according to claim 1, comprising:
(A) simultaneously introducing the expression vectors (a) and (b) into the CHO cell,
(B) transiently expressing the transposase from the expression vector introduced in step (A) to integrate the gene fragment inserted between the transposon sequences in a chromosome of the CHO cell to obtain said CHO cell in suitable suspension to express the protein of interest, and
(C) culturing in suspension the CHO cell in suspension capable of expressing the protein of interest obtained in step (B) to produce the protein of interest.
[3]
3. Method for obtaining a suspended CHO cell capable of expressing a protein of interest, comprising introducing a protein expression vector comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences depicted in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences depicted in SEQ ID No. 2 and SEQ. ID No. 3, in a suspension CHO cell able to survive and proliferate in a serum-free medium; introducing an expression vector (b) comprising a DNA encoding a transposase that recognizes the transposon sequences and exhibits an activity of transferring a gene fragment inserted between the transposon sequences in a chromosome in the CHO cell; and integrating the inserted gene fragment between the transposon sequences, into a chromosome of the CHO cell.
[4]
4. Process according to any of claims 1 to 3, wherein the CHO cell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO / DG44, Pro-3 and CHO-S.
[5]
The method according to any of the preceding claims, wherein the selectable marker gene is a cycloheximide resistance gene.
[6]
The method according to claim 5, wherein the cycloheximide resistance gene is a gene encoding a mutant of the human ribosomal protein L36a.
[7]
The method according to claim 6, wherein the mutant is a mutant in which the proline at position 54 of the human ribosomal protein L36a is replaced with another amino acid.
[8]
8. The method according to claim 7, wherein the other amino acid is glutamine.
[9]
9. CHO cell in suspension able to survive and proliferate in a serum-free medium and to produce a protein of interest, said cell comprising an expression vector (a) comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences which are the Tol1 nucleotide sequences depicted in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences depicted in FIG. SEQ ID No. 2 and SEQ ID No. 3 and an expression vector (b) comprising a DNA encoding a transposase (a transferase) that recognizes the transposon sequences and exhibits an activity of transferring the inserted gene fragment between the sequences of transposon in a chromosome to integrate the gene fragment inserted between the transposon sequences in a chromosome of the CHO cell.
[10]
The cell according to claim 9, wherein the CHO cell is at least one selected from CHO-K1, CHO-K1SV, DUKXB11, CHO / DG44, Pro-3 and CHO-S.
[11]
11. A cell according to claim 9 or 10, wherein the selectable marker gene is a cycloheximide resistance gene.
[12]
The cell according to claim 11, wherein the cycloheximide resistance gene is a gene encoding a mutant of the human ribosomal protein L36a.
[13]
The cell according to claim 12, wherein the mutant is a mutant in which the proline at position 54 of the human ribosomal protein L36a is replaced with another amino acid.
[14]
The cell according to claim 13, wherein the other amino acid is glutamine.
[15]
15. Use of a protein expression vector (a) comprising a gene fragment comprising a DNA encoding a protein of interest and a selectable marker gene and, at both ends of the gene fragment, a pair of transposon sequences that are the Tol1 nucleotide sequences represented in SEQ ID No. 14 and SEQ ID No. 15 or the Tol2 nucleotide sequences depicted in SEQ ID No. 2 and SEQ ID No. 3 and an expression vector (b) comprising a DNA encoding a transposase that recognizes the transposon sequences and exhibits an activity of transferring a gene fragment inserted between the transposon sequences in a chromosome, to integrate the gene fragment inserted between the transposon sequences in a chromosome of a CHO cell in suspension able to survive and proliferate in a medium without serum.

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法律状态:

优先权:

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申请号 | 申请日 | 专利标题

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US18613809P| true| 2009-06-11|2009-06-11|

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JP2009140626|2009-06-11|

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PCT/JP2010/059881|WO2010143698A1|2009-06-11|2010-06-10|Process for production of protein|

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