WO2014157429A1 - タンパク質の製造方法 - Google Patents
タンパク質の製造方法 Download PDFInfo
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- WO2014157429A1 WO2014157429A1 PCT/JP2014/058702 JP2014058702W WO2014157429A1 WO 2014157429 A1 WO2014157429 A1 WO 2014157429A1 JP 2014058702 W JP2014058702 W JP 2014058702W WO 2014157429 A1 WO2014157429 A1 WO 2014157429A1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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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
- C12N15/67—General methods for enhancing the expression
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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
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- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
Definitions
- the present application relates to a recombinant cell for enhancing the expression of a protein from a foreign gene in a recombinant cell, and an invention using such a cell. More specifically, the ability to synthesize or secrete the target protein using cells with enhanced expression of p180 protein and / or SF3b4 protein, or cells having such characteristics is enhanced. And as a result, to provide a method for producing a protein.
- a cis-element is used in a vector for expression of the target protein. The use of is also a feature.
- Biotechnology drugs applying genetic recombination technology in recent years, especially when the antibody drug market is expanding rapidly, the burden on medical costs is also a concern, and protein production is more efficient than before. There is a constant need for the development of manufacturing technology for biotechnology drugs that can be performed at lower cost.
- Escherichia coli and the like can produce the target protein at low cost, they cannot perform post-translational modifications such as sugar chain modification and are not suitable for the production of glycoproteins.
- Escherichia coli has a property that it is easy to make inclusion bodies containing the produced protein, and obtaining a target protein requires a further solubilization process after synthesis, which has a drawback of high work load.
- glycoproteins such as antibodies
- the added sugar chain affects the water solubility of the target protein, protease resistance, tissue targeting, and also its biological activity.
- the production technology using animal cells is required, and in recent years, the technology has greatly advanced. Under such circumstances, many of the current antibody drugs are produced in Chinese Hamster Ovary (CHO) cells, but optimization of the manufacturing process remains an important issue.
- Endoplasmic reticulum Proteins secreted extracellularly from eukaryotic cells, including mammalian cells, are synthesized in the endoplasmic reticulum, which is an intracellular organelle partitioned by the inner membrane.
- the endoplasmic reticulum is roughly divided into two types: a rough endoplasmic reticulum that has a ribosome on the surface, which is a protein synthesizer composed of a large RNA-protein complex, and a smooth endoplasmic reticulum that has no ribosome.
- the detailed formation mechanism is not clear.
- the gene of the target protein is in the control of a promoter that exhibits high transcriptional activity in the expression vector, and its mRNA is expressed at a high level. It is expected that However, even under such circumstances, there are often cases where the mRNA level does not correlate with the protein expression level itself, and one reason for this is low translation efficiency on the endoplasmic reticulum membrane.
- mRNA can be provided in a form that is easy to use for the translation device on the endoplasmic reticulum membrane, as in the case of fibroblasts, This means that there is still room for further enhancement of protein synthesis ability.
- Non-patent Document 1 In order to activate collagen synthesis, it is not sufficient for the mRNA to be present on the endoplasmic reticulum, and it is necessary to form polysomes with high translation efficiency.
- Non-patent document 2 A protein that promotes localization, a splicing factor 3B subunit 4 (SF3b4) protein, was newly found. Then, when a cell that highly expresses both SF3b4 protein and p180 protein was created, the localization of mRNA on the endoplasmic reticulum increased significantly in cells with such characteristics, and the secretory function of cultured cells was increased. Clarified that it is possible to As a result, the present invention has been completed.
- SF3b4 splicing factor 3B subunit 4
- the inventors of the present invention have an object in a cell in which the expression of the full-length p180 protein or a part thereof and the expression of a protein that promotes the localization of the endoplasmic reticulum (ER) of mRNA are enhanced in the cell. As a result, it was shown that a recombinant cell with enhanced ability to synthesize or secrete protein can be provided.
- ER endoplasmic reticulum
- the inventors of the present invention also enhance the expression of the protein that promotes localization of the endoplasmic reticulum (ER) of mRNA in the second embodiment of the present invention, by increasing the expression of the full-length p180 protein or a part thereof.
- the ability to synthesize or secrete the target protein by transforming a nucleic acid molecule encoding the target protein or increasing the amount of the target protein produced in the recombinant cell As a result, it has been shown that a method for producing a protein can be provided.
- such a characteristic recombinant cell is provided, or by using such a characteristic recombinant cell, the ability to synthesize or secrete the target protein is enhanced. It has been shown that the above-described problems can be solved.
- the p180 protein is (A) It consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of human-derived p180 protein (SEQ ID NO: 2), and has a function of promoting polysome formation on the endoplasmic reticulum membrane in cells. protein, (B) In the amino acid sequence of human-derived p180 protein (SEQ ID NO: 2), it consists of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added.
- a protein having a function of promoting polysome formation (C) consisting of an amino acid sequence defined by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of a gene encoding human p180 protein (SEQ ID NO: 1) on the endoplasmic reticulum membrane in the cell
- a protein having a function of promoting polysome formation on the intracellular endoplasmic reticulum membrane and (e) a nucleotide sequence complementary to the nucleotide sequence of a gene encoding human p180 protein (SEQ ID NO: 1),
- the full length or part of the mammalian p180 protein is human p180 protein (SEQ ID NO: 2), mouse p180 protein (GenBank Accession No. NP_077243), rat p180 protein (GenBank Accession No. XP_230637), Chinese hamster. p180 protein (GenBank Accession No. XM_003496471), canine p180 protein (GenBank Accession No. NP_001003179), equine p180 protein (GenBank Accession No. XP_001915027), monkey p180 protein (GenBank Accession No.
- XP_002798281 chimpanzee p180 protein (GenBank Accession No XP_514527), porcine p180 protein (GenBank Accession No. XP_001926148), or a part thereof, the recombinant cell according to [3].
- [5] A portion containing the amino acid sequence corresponding to the region consisting of amino acids 27 to 157 of the protein having the amino acid sequence shown in SEQ ID NO: 2 (human p180 protein), 623 to 737 amino acids Selected from a portion containing an amino acid sequence corresponding to the region consisting of amino acids, a portion containing an amino acid sequence corresponding to the region consisting of amino acids 738 to 944, and a portion containing an amino acid sequence corresponding to the region consisting of amino acids 945 to 1540
- the recombinant cell according to any one of [1] to [4].
- the protein that promotes the endoplasmic reticulum (ER) localization of mRNA is the full-length splicing factor 3B subunit 4 (SF3b4) protein or a portion thereof (full-length amino acid sequence 424 AA of SEQ ID NO: 4; RRM1) 13.
- the recombinant cell according to any one of [1] to [5], which is selected from the group consisting of SEQ ID NO: 4 of 13 to 91 AA; and SEQ ID NO: 4 of RRM2.
- SF3b4 protein (I) a protein comprising an amino acid sequence having at least 70% sequence identity with the human-derived SF3b4 protein amino acid sequence (SEQ ID NO: 4) and having a function of promoting the localization of mRNA to the endoplasmic reticulum, (Ii) In the amino acid sequence of human-derived SF3b4 protein (SEQ ID NO: 4), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and localization of mRNA to the endoplasmic reticulum A protein having a function of promoting (Iii) consisting of an amino acid sequence defined by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence (SEQ ID NO: 3) of the gene encoding the human-derived SF3b4 protein, and containing the mRNA into the endoplasmic reticulum A protein having a function of promoting localization, (Iv) consisting of an amino acid sequence defined by
- mammalian SF3b4 protein or a part thereof includes human SF3b4 protein (SEQ ID NO: 4), mouse SF3b4 protein (GenBank Accession No. NP_694693.1), rat SF3b4 protein (GenBank Accession No. NP_001011951.1). ), Chinese hamster SF3b4 protein (GenBank Accession No. XP_003498680.1), canine SF3b4 protein (GenBank Accession No. XP_540295.3), horse SF3b4 protein (GenBank Accession No. XP_001488649.2), monkey SF3b4 protein (GenBank Accession No.
- NP_001097793.1 chimpanzee SF3b4 protein (GenBank Accession No. XP_513768.2), porcine SF3b4 protein (GenBank Accession No. XP_001926524.1), or a part thereof, the recombinant cell according to [8].
- the ability to synthesize or secrete a protein as a target product is enhanced by transforming a nucleic acid molecule encoding the protein as a target product or increasing the amount of protein produced as a target product.
- the recombinant cell according to any one of [1] to [9].
- Recombinant cells that enhance the expression of the full-length p180 protein or a portion thereof, enhance the expression of proteins that promote the endoplasmic reticulum (ER) localization of mRNA, or enhance the expression of both of these proteins A method for producing a protein as a target product by transforming a nucleic acid molecule encoding the protein as the target product or increasing the production amount of the protein as the target product.
- the full length or part of the mammalian p180 protein is human p180 protein (SEQ ID NO: 2), mouse p180 protein (GenBank Accession No. NP_077243), rat p180 protein (GenBank Accession No. XP_230637), Chinese hamster. p180 protein (GenBank Accession No. XM_003496471), canine p180 protein (GenBank Accession No. NP_001003179), equine p180 protein (GenBank Accession No. XP_001915027), monkey p180 protein (GenBank Accession No.
- XP_002798281 chimpanzee p180 protein (GenBank Accession No XP_514527), porcine p180 protein (GenBank Accession No. XP_001926148), or a part thereof, the method according to [13].
- the portion of the mammalian p180 protein comprises a portion comprising the region consisting of amino acids 27 to 157 of the protein having the amino acid sequence shown in SEQ ID NO: 2 (human p180 protein), consisting of amino acids 623 to 737
- the protein that promotes endoplasmic reticulum (ER) localization of mRNA is the full-length splicing factor 3B subunit 4 (SF3b4) protein or a portion thereof (full-length amino acid sequence 424 AA of SEQ ID NO: 4; RRM1
- SF3b4 full-length splicing factor 3B subunit 4
- RRM1 full-length amino acid sequence 424 AA of SEQ ID NO: 4; RRM1
- [17] The method according to [16], wherein the SF3b4 protein is derived from a mammal.
- mammalian SF3b4 protein or a part thereof includes human SF3b4 protein (SEQ ID NO: 4), mouse SF3b4 protein (GenBank Accession No. NP_694693.1), rat SF3b4 protein (GenBank Accession No. NP_001011951.1) ), Chinese hamster SF3b4 protein (GenBank Accession No. XP_003498680.1), canine SF3b4 protein (GenBank Accession No. XP_540295.3), horse SF3b4 protein (GenBank Accession No. XP_001488649.2), monkey SF3b4 protein (GenBank Accession No.
- the recombinant cell is a cell line represented by accession number NITE BP-01753 (CHO 3D5), accession number NITE BP-1535 (CHO YA7), or accession number NITE ABP-01811 (CHO 1B2).
- accession number NITE BP-01753 (CHO 3D5)
- accession number NITE BP-1535 (CHO YA7)
- accession number NITE ABP-01811 (CHO 1B2).
- RNA-binding protein is recognized / bound (or alternatively) downstream of the promoter and upstream of the start codon of the nucleotide sequence of the DNA encoding the target protein.
- the cis-element is recognized / bound (or interacts) with an RNA recognition motif (RRM) type RNA binding protein.
- RRM RNA recognition motif
- the nucleotide sequence of the cis-element is derived from the nucleotide sequence of the 5 ′ untranslated region of the type I collagen gene, the sequence derived from the nucleotide sequence of the 5 ′ untranslated region of the fibronectin gene, matrix metalloprotease 14 ( MMP14) a sequence derived from the nucleotide sequence of the 5 ′ untranslated region of the gene, a sequence derived from the nucleotide sequence of the 5 ′ untranslated region of the prolyl 4-hydroxylase A2 (P4HA2) gene, a sequence of the prolyl 4-hydroxylase A1 (P4HA1) gene
- MMP14 matrix metalloprotease 14
- P4HA2 prolyl 4-hydroxylase A2
- P4HA1 prolyl 4-hydroxylase A1
- nucleotide sequence of the cis-element is 1 to 102 nucleotides, 1 to 78 nucleotides, 1 to 78 nucleotides of SEQ ID NO: 5, 60 nucleotides, 61-126 nucleotides, 16-57 nucleotides, 79-126 nucleotides, 103-126 nucleotides, 58-78 nucleotides, 51-78 nucleotides, 1-27 nucleotides, 70-78 nucleotides
- Expression cells may be intact host cells, cells with enhanced expression of p180 protein or its portion, cells with enhanced expression of SF3b4 protein or its portion, or both. The method according to any one of [22] to [28], wherein the cells are both enhanced.
- Recombinants of the invention with enhanced expression of the full-length p180 protein or part thereof and / or expression of a protein that promotes endoplasmic reticulum (ER) localization of mRNA (eg, full-length SF3b4 protein or part thereof)
- ER endoplasmic reticulum
- mRNA eg, full-length SF3b4 protein or part thereof
- Figure 1 shows p180 protein expression and mRNA endoplasmic reticulum (ER) localization in each cell (CHO cell, CHO (3D5 cell, CHO 5g cell, and CHO YA7 cell) prepared in Example 1. It is a figure which shows the result of having investigated the expression of SF3b4 protein which is a protein to promote by a Western blot.
- Figure 2 compares the results of SEAP protein expression when exogenously introduced into the cells prepared in Example 1 is an expression plasmid for human placenta-derived secretory alkaline phosphatase (SEAP), a secretion marker.
- SEAP human placenta-derived secretory alkaline phosphatase
- FIG. 3 shows the expression of SEAP ⁇ ⁇ ⁇ ⁇ mRNA in the membrane fraction of each cell prepared in Example 1 when a human placenta-derived secretory alkaline phosphatase (SEAP) expression plasmid, which is a secretion marker, is introduced exogenously. It is the figure which showed the degree of localization compared.
- FIG. 4 is a schematic diagram of an insert part of an expression vector containing a cis-element (A) and a diagram showing the results of evaluating the secretory activation ability of the cis-element (B and C).
- FIG. 5 is a diagram showing changes in protein secretory activity when collagen is expressed.
- FIG. 6 is a diagram showing that secretory activation of an antibody occurs by inserting a cis-element.
- FIG. 7 is a diagram comparing the effects of kozak sequence and cis-element # 1 on secretory activation using CHO cells and CHO YA7 cells.
- Figure 8 shows the relationship between cis-element structure and protein expression enhancement using cis-element # 1, cis-element # 2, cis-element # 3, and cis-element # 4
- FIG. 10 shows the effect of the substitution, deletion, and insertion on the activity of the motif in the nucleotide of the motif GAN 1- (X) n -ACN 2 .
- FIG. 11 is a diagram showing that the production of collagen is markedly suppressed with the suppression of SF3b4 expression.
- FIG. 12 is a diagram showing that the weight of COL1A1 cDNA in the polysome fraction shifts to the high density side when the cis-element is included compared to the case where the cis-element is not included.
- the inventors of the present invention in the first embodiment of the present invention, in the cell, the expression of the full-length p180 protein or a part thereof and / or the expression of the full-length splicing factor 3B subunit 4 (SF3b4) protein or a part thereof in the cell, It was shown that a recombinant cell with enhanced ability to synthesize or secrete a protein as a target substance in the cell can be provided.
- SF3b4 full-length splicing factor 3B subunit 4
- the full-length or part of the p180 protein and / or the full-length or part of the SF3b4 protein are both expressed in the cell, whereby the target protein on the endoplasmic reticulum membrane in the cell is expressed.
- Polysome formation involving mRNA which is an expression product of the encoding nucleic acid molecule, can be promoted.
- the polysome refers to one molecule of mRNA bound to a plurality of ribosomes present on the endoplasmic reticulum membrane in the cell.
- the first aspect of the above-described recombinant cell of the present invention is that expression of p180 protein, particularly mammalian p180 protein, or its portion is enhanced in the cell.
- the p180 protein is an essential endoplasmic reticulum membrane protein and is a protein that is highly expressed in secretory tissues and can promote polysome formation.
- mouse p180 protein has 87% similarity
- rat p180 protein has 87% similarity
- Chinese hamster P180 protein has 88% similarity
- dog p180 protein has 91% similarity
- equine p180 protein has 89% similarity
- monkey p180 protein has 91-92% similarity
- chimpanzee p180 protein has 98% similarity
- porcine p180 protein is known to have 86% similarity, respectively, and all mammalian p180 proteins have amino acid similarity within 84% or more, otherwise It has been reported that even if the range is extended to the p180 protein of an organism, it is contained within the range of 76% or more in terms of amino acid similarity.
- p180 protein when referring to “p180 protein”, (A) It consists of an amino acid sequence having at least 70% sequence identity with the amino acid sequence of human-derived p180 protein (SEQ ID NO: 2), and has a function of promoting polysome formation on the endoplasmic reticulum membrane in cells. protein, (B) In the amino acid sequence of human-derived p180 protein (SEQ ID NO: 2), it consists of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added.
- a protein having a function of promoting polysome formation consisting of an amino acid sequence defined by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence of a gene encoding human p180 protein (SEQ ID NO: 1) on the endoplasmic reticulum membrane in the cell
- a protein having a function of promoting polysome formation consisting of an amino acid sequence defined by a nucleotide sequence in which one or several nucleotides have been deleted, substituted, or added in the nucleotide sequence (SEQ ID NO: 1) of a gene encoding human-derived p180 protein
- a protein having a function of promoting polysome formation on the endoplasmic reticulum membrane in the cell consisting of an amino acid sequence defined by a nucleotide sequence in which one or several nucleotides have been deleted, substituted, or added in the nucleotide sequence (SEQ ID NO: 1) of a gene encoding human-derived p180 protein
- the% sequence identity value May select any value between 70% and 100%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Percent sequence identity values such as 96%, 97%, 98%, 99%, 100%, etc. can be selected.
- the% value of sequence identity is: You can choose any value between 70% and 100%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% % Values of sequence identity such as 97%, 98%, 99%, 100%, etc. can be selected.
- nucleotides when “one or several nucleotides are deleted, substituted or added”, it means that the number of nucleotides to be deleted, substituted or added is about 1 to 10. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid numbers can be selected.
- a protein having a target function can be defined without introducing a stop codon by such “deletion, substitution, or addition of one or several nucleotides”.
- the phrase “under stringent conditions” can be easily determined by a person skilled in the art having general techniques based on, for example, the length of the nucleotide sequence of a gene.
- Basic conditions are described in 5 ⁇ SSC as described in, for example, Current Protocols in Molecular Biology Vol.1 (John Wiley and Sons, Inc.) and Molecular Cloning 2nd edition (Sambrook et al. (1989)).
- Hybridization conditions such as 5 ⁇ Denhardt's solution, 1% SDS, 25 ° C. to 68 ° C., several hours to overnight can be mentioned.
- the hybridization temperature is more preferably 45 ° C. to 68 ° C. (without formamide) or 30 ° C.
- nucleic acid molecules containing nucleotide sequences having a certain identity or higher can be cloned by appropriately setting hybridization conditions such as formamide concentration, salt concentration and temperature. All cloned nucleic acid molecules are included within the scope of the present invention.
- the full length of the human p180 protein is a protein having the amino acid sequence shown in SEQ ID NO: 2 (GenBank Accession No No. AB287347), which is encoded by the nucleotide sequence shown in SEQ ID NO: 1 (GenBank Accession No.AB287347).
- the mouse p180 protein described above is encoded by the nucleotide sequence shown by GenBank Accession No. NP_077243
- the rat p180 protein is encoded by the nucleotide sequence shown by GenBank Accession No. XP_230637
- Chinese hamster p180 protein is a protein having the amino acid sequence shown in SEQ ID NO: 2 (GenBank Accession No. AB287347), which is encoded by the nucleotide sequence shown in SEQ ID NO: 1 (GenBank Accession No.AB287347).
- the mouse p180 protein described above is encoded by the nucleotide sequence shown by GenBank Accession No. NP_077243
- a region consisting of amino acids 27 to 157 a region consisting of amino acids 623 to 737 of a protein having the amino acid sequence shown in SEQ ID NO: 2 (human p180 protein)
- a part containing any of the region consisting of amino acids 738 to 944 and the region consisting of amino acids 945 to 1540 polysome formation on the endoplasmic reticulum membrane can be promoted (patent) Reference 1).
- part of p180 protein when referring to “part of p180 protein”, a part comprising an amino acid sequence corresponding to a region consisting of amino acids 27 to 157 of a protein having the amino acid sequence shown in SEQ ID NO: 2 (human p180 protein) Including a portion containing an amino acid sequence corresponding to the region consisting of amino acids 623 to 737, a portion including an amino acid sequence corresponding to the region consisting of amino acids 738 to 944, and an amino acid sequence corresponding to the region consisting of amino acids 945 to 1540 It means a part such as a part. Those containing such moieties can exhibit the ability to promote polysome formation.
- the portion of the p180 protein thus defined includes a region consisting of amino acids 27 to 157 of the above-mentioned protein having the amino acid sequence shown in SEQ ID NO: p2 (human p180 protein), 623 to 737 In addition to the region comprising amino acid No. 7, the region comprising amino acids 738 to 944, the portion comprising the region comprising amino acids 945 to 1540, etc., adjacent to the C-terminal side of the N-terminal transmembrane domain of human p180 protein MTB-2 domain, or highly basic N-terminal region containing a ribosome-binding repeat domain, highly basic tandem repeat domain, or microtubule binding and bundling domain (MTB-1 domain), etc. It is included as an example (Patent Document 1).
- the amino acid sequences of human p180 protein and other mammalian p180 proteins are generally highly conserved. Therefore, among the amino acid sequences of the proteins shown in the above (a) to (e), the amino acid sequence of the fragment containing the corresponding part or region can also be used as the “part of p180 protein”.
- the recombinant cell of the present invention described above is characterized in that the expression of a protein that promotes the localization of mRNA endoplasmic reticulum (ER) is enhanced in the cell.
- Proteins that promote such endoplasmic reticulum (ER) localization of mRNA include SF3b4 proteins, particularly the full-length mammalian SF3b4 protein or portions thereof (eg, full-length amino acid sequence 424 AA of SEQ ID NO: 4; RRM1 SEQ ID NO: 4 is 13-91 AA; RRM2 is SEQ ID NO: 4, 100-179 AA; C-terminal region 180-424 AA.), Etc. are included.
- the SF3b4 protein is normally detected only in the nucleus, but most fibroblasts that actively secrete collagen are detected in the nucleus, but are examined in more detail. As a result, it was revealed that a part of SF3b4 protein exists in the membrane fraction containing endoplasmic reticulum in the cytoplasm.
- the SF3b4 protein (also called SAP49 / SF3b49) is classified into the RNA recognition motif (RRM) type RNA binding protein (RBP) family by containing two RNA recognition motifs (RRM) on the amino terminal side, and the carboxy terminal side. Is a substance having a proline-rich domain whose function is unknown.
- RNA recognition motifs RRM1, RRM2
- RRM1 and RRM2 RNA recognition motifs
- yeast SF3b4 protein when compared with human SF3b4 protein, it shows 100% similarity in all mammalian species examined in mammals, and in other species also yeast SF3b4 protein Are known to have 40-54% similarity and the insect SF3b4 protein 63-81% similarity, respectively, and are reported to be highly conserved proteins in all organisms.
- SF3b4 protein when referred to as “SF3b4 protein”, (I) a protein comprising an amino acid sequence having at least 70% sequence identity with the human-derived SF3b4 protein amino acid sequence (SEQ ID NO: 4) and having a function of promoting the localization of mRNA to the endoplasmic reticulum, (Ii) In the amino acid sequence of human-derived SF3b4 protein (SEQ ID NO: 4), consisting of an amino acid sequence in which one or several amino acids are deleted, substituted, or added, and localization of mRNA to the endoplasmic reticulum A protein having a function of promoting (Iii) consisting of an amino acid sequence defined by a nucleotide sequence having at least 70% sequence identity with the nucleotide sequence (SEQ ID NO: 3) of the gene encoding the human-derived SF3b4 protein, and containing the mRNA into the endoplasmic reticulum A protein having a function of promoting localization, (Iv)
- the% sequence identity value when “having at least 70% sequence identity with the amino acid sequence of human-derived SF3b4 protein (SEQ ID NO: 4)”, the% sequence identity value May select any value between 70% and 100%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Percent sequence identity values such as 96%, 97%, 98%, 99%, 100%, etc. can be selected.
- the% value of sequence identity is: You can choose any value between 70% and 100%, for example, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% % Values of sequence identity such as 97%, 98%, 99%, 100%, etc. can be selected.
- nucleotides when “one or several nucleotides are deleted, substituted or added”, it means that the number of deleted, substituted or added nucleotides is about 1 to 10. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid numbers can be selected.
- a protein having a target function can be defined without introducing a stop codon by such “deletion, substitution, or addition of one or several nucleotides”.
- the full length of the human SF3b4 protein is a protein having the amino acid sequence shown in SEQ ID NO: 4 (GenBank Accession No No. NP_005841.1), and this protein is encoded by the nucleotide sequence shown in SEQ ID NO: 3 (GenBank Accession No. NP_005841.1).
- the mouse SF3b4 protein described above is encoded by the nucleotide sequence represented by GenBank Accession No. NP_694693.1
- the rat SF3b4 protein is encoded by the nucleotide sequence represented by GenBank Accession No. NP_001011951.1.
- the hamster SF3b4 protein is encoded by the nucleotide sequence shown by GenBank Accession No.
- the dog SF3b4 protein is encoded by the nucleotide sequence shown by GenBank Accession No. XP_540295.3, Encoded by the nucleotide sequence shown by GenBank Accession No. XP_001488649.2, monkey SF3b4 protein is encoded by the nucleotide sequence shown by GenBank Accession No. NP_001097793.1, and chimpanzee SF3b4 protein is GenBank Accession No.
- the pig SF3b4 protein is encoded by the nucleotide sequence shown by GenBank Accession No. XP_001926524.1, encoded by the nucleotide sequence shown by XP — 513768.2.
- mRNA precursor transcribed from the DNA is converted into an amino acid by splicing. Introns without genetic information are removed and converted to mature mRNA.
- the process is carried out by spliceosome, a large complex composed of small nuclear RNA (snRNA) -protein.
- snRNA small nuclear RNA
- splicing factors including SF3b4 protein have any function at the protein translation level, but our analysis has shown that SF3b4 protein is significantly present in the membrane fraction containing endoplasmic reticulum in the cytoplasm. Increased and associated mRNA-bound SF3b4 protein interacts with the coiled-coil domain of p180 protein to promote mRNA localization to the endoplasmic reticulum, resulting in increased secretion of cultured cells It was clarified that it can be made.
- Fibrosis a condition in which various tissues are injured and fibrotic for a long period of time, is a disease with a poor prognosis because the cause, detailed onset mechanism, and effective treatment are unknown.
- idiopathic pulmonary fibrosis is thought to increase the fibrosis due to an increase in collagen and other abnormalities for the repair of alveolar epithelial damage caused by various stimuli and an abnormal repair reaction. No effective treatment has been established. In such pathological conditions, no clue for preventing an abnormal increase in collagen was obtained, but SF3b4 protein, which was previously thought to function as a splicing factor in the present invention, was synthesized / secreted in collagen.
- collagen synthesis can be suppressed by inhibiting or suppressing the function of SF3b4. Since the suppression of SF3b4 expression is thought to be possible by administration of its specific shRNA, etc., and its functional inhibition is thought to be possible by various drugs that inhibit the splicing process, accumulation of abnormal collagen in fibrosis can be achieved by inhibiting the function of SF3b4 or suppressing the expression. It was shown that it could be suppressed to prevent fibrosis from becoming serious.
- the cells that can be used to produce the recombinant cells in the present invention may be any cells that are suitable for protein expression.
- the source cells include CHO cells, Mammal-derived cells such as HEK293 cells and HeLa cells can be used. These cells are transfected with the full-length p180 protein or part thereof and / or the full-length SF3b4 protein or part thereof as described above using methods commonly used in the art. The full length of p180 protein or part thereof and / or the full length of SF3b4 protein or part thereof can be expressed in the cells.
- a transformation method generally used in the art should be used. Can do.
- DNA encoding the full length of p180 protein or a portion thereof and / or DNA encoding the full length of SF3b4 protein or a portion thereof for example, in an expression vector such as pcDNA, pEGFP, pCAGGS, etc.
- the respective expression vectors can be used after being transformed into cells.
- a cell line derived from CHO cell, CHO 5g cell, a sequence derived from the nucleotide sequence of the 5 ′ untranslated region of a gene stably expressing SF3b4 protein As a recombinant cell, a CHOD3D5 cell line derived from a CHO cell, and a CHO YA7 cell derived from a CHO cell as a recombinant cell in which the expression of these two proteins was simultaneously enhanced (see below) (See Example 1), these cell lines were deposited with the Patent Microorganism Deposit Center of the National Institute of Product Evaluation Technology (Accession number NITE BP-01753 for CHO 3D5 cells), Accession number NITE BP-1535 for CHO YA7 cells , Or receipt number for CHOB1B2 cells NITE ABP-01811).
- the inventors of the present invention also provide a nucleic acid molecule encoding a protein of interest in a recombinant cell in which the expression of the full-length p180 protein or a portion thereof and / or the expression of the full-length SF3b4 protein or a portion thereof is enhanced. That the ability to synthesize or secrete the target protein can be enhanced by increasing the production amount of the target protein or the production amount of the target protein, thereby providing a method for producing the protein. Indicated.
- the protein as the target product for enhancing the synthetic ability or secretory ability and as a result producing the protein may be any protein intended to be produced by a biotechnological method.
- glycoproteins can be mentioned as the target protein, and examples thereof include, but are not limited to, antibodies, collagen, fibronectin, laminin and the like.
- the inventors of the present invention in an expression unit for expressing a protein of interest, starts the nucleotide sequence of DNA encoding the protein of interest as downstream of the promoter.
- a method for increasing the expression level of a target protein in an expression system cell by inserting a cis-element upstream of a codon.
- Patent Document 1 Non-Patent Document 2
- the amount of protein synthesized / secreted in cells transfected with the DNA encoding the protein of interest regardless of whether the gene transcript encoding the protein of interest is expressed at a high level.
- mRNA is not provided in a form that is easy to use for the translation device on the endoplasmic reticulum membrane in the cells used.
- the cis-element present in the 5 ′ untranslated region of the collagen gene has an action of increasing the expression level of the protein. More specifically, the RRM protein that recognizes the cis-element sequence in the 5 'untranslated region of the mature mRNA binds to it, thereby allowing the mRNA to be deposited on the membrane of the endoplasmic reticulum, where the secreted protein is synthesized. It was thought to have functions to enhance transport / localization and further increase translation efficiency.
- the cis-element confirmed in the present invention was clarified from the result of analysis of the type 1 collagen gene, and the nucleotide sequence of this cis-element is present in the 5 ′ untranslated region of the type 1 collagen gene. It became clear.
- the nucleotide sequence of such a cis-element can include a sequence derived from the nucleotide sequence of the 5 ′ untranslated region of the type I collagen gene, but as long as it has a desired effect, Sequence derived from the nucleotide sequence of the 5 'untranslated region of the fibronectin gene, Sequence derived from the nucleotide sequence of the 5' untranslated region of the matrix metalloproteinase 14 (MMP14) gene, 5 'of the prolyl 4-hydroxylase A2 (P4HA2) gene
- MMP14 matrix metalloproteinase 14
- P4HA2 prolyl 4-hydroxylase A2
- the structural features of the cis-element that can be used in the present invention include 9 to 12 nucleotides in the 5 ′ untranslated region of the gene contained in the expression plasmid for expressing the protein of interest.
- the motif “GAN 1- (X) n -ACN 2 ” (n 3 to 6) (N 1 and N 2 may independently be any nucleotide of A, T, C, G) )) Or one or more.
- Specific examples of motifs include those present as natural cis-elements, which are characterized by N 1 being G and N 2 being A or G or C .
- the nucleotide sequence of the cis-element is any one selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 8. Can be used.
- An expression plasmid containing such a cis-element was prepared not only in an intact host cell, but also in a cell prepared by the present invention in which expression of the full-length p180 protein or a portion thereof was enhanced, splicing factor 3B subunit 4 (SF3b4) It can be used in cells in which expression of the full-length protein or a part thereof is enhanced, or cells in which both of these expressions are enhanced.
- SF3b4 splicing factor 3B subunit 4
- Example 1 Preparation of plasmids for cell lines co-expressing SF3b4 protein, or p180 protein and SF3b4 protein
- Establishment of cell lines co-expressing SF3b4 protein, or p180 protein and SF3b4 protein requires the nucleic acid encoding p180 protein.
- the expression plasmid containing and the expression plasmid containing the nucleic acid encoding the SF3b4 protein were separately prepared and sequentially transfected into CHO cells.
- An expression plasmid pEF-SF3b4 encoding the full-length human SF3b4 protein was prepared according to the following method. That is, after a cDNA sequence encoding the full length of human SF3b4 protein (GenBank Accession No. NP_005841.1) is amplified by PCR, it is inserted and linked to the KpnI-EcoRV site of pEF1 / Myc-His vector (Life Technologies), Plasmid pEF-SF3b4 was obtained.
- the expression plasmid pEF-CHO-SF3b4 encoding the full length Chinese hamster SF3b4 protein was prepared according to the following method. That is, total RNA was extracted from CHO cells, and a cDNA sequence encoding the full length Chinese hamster SF3b4 protein (GenBank Accession No. XP_003498680.1) was amplified by PCR. Thereafter, the plasmid was inserted and ligated into the KpnI-EcoRV site of the pEF1 / Myc-His vector to obtain a plasmid pEF-CHO-SF3b4.
- An expression plasmid pEF-Cis encoding a human cis element sequence (for example, cis-elements # 1 to # 11) was prepared according to the following method. That is, a nucleic acid sequence encoding a human cis-element (for example, in the case of cis-element # 1, SEQ ID NO: 5) is amplified by PCR, and then is added to the bglII-HindIII site of a pEGFP vector (manufactured by clontech). The plasmid prCMV-cis # -SEAP was obtained by insertion ligation. Details of the expression vector containing each cis-element are described in Example 9.
- a plasmid expressing human p180 and Chinese hamster SF3b4 was transfected into CHO cells by the lipofection method. Thereafter, the drug was selected by culturing in the presence of 300 ⁇ g / ml of Hygromycin ⁇ ⁇ . After culturing for 14 days, a colony of a cell line resistant to Hygromycin was isolated, and a cell line CHO 1B2 cell that stably co-expressed human p180 protein and Chinese hamster SF3b4 protein was established.
- Patent Microorganism Depositary Center NPMD
- receipt number NITE ABP-01811, receipt date 4 March 2014 Patent Microorganism Depositary Center
- CHO 5g cells stably express p180 protein
- CHO 3D5 cells stably express SF3b4 protein
- CHO YA7 cells CHO 1B2 cells express p180 protein and SF3b4 protein.
- 5% CHO 5g cells, CHO 3D5 cells, CHO YA7 cells, and CHO 1B2 cells in Dulbecco's modified Eagle's medium (DMEM) containing 5% fetal calf serum The cells were cultured at 37 ° C. in the presence of CO 2 . As control cells, CHO cells were also cultured.
- DMEM Dulbecco's modified Eagle's medium
- CHO 3D5 cells expressed p180 protein at a level below the detection limit, as in CHO cells, but highly expressed SF3B4 protein (lane 2).
- CHO 5g cells expressed high levels of p180 protein and SF3b4 protein was found at a low level of endogenous expression (lane 3).
- CHO YA7 cells and CHO 1B2 cells both expressed p180 protein and SF3b4 protein at high levels (lane 4, lane 6).
- Example 2 Secretion activation by expression of p180 protein and / or expression of SF3b4 protein
- Cell line CHO 5g cell expressing p180 protein prepared in Example 1 cell line CHO 3D5 cell expressing SF3b4 protein
- the cell line CHO YA7 cells co-expressing p180 protein and SF3b4 protein were examined for expression of p180 protein and / or SF3b4 protein and activation of protein secretion.
- SEAP human placenta-derived secretory alkaline phosphatase
- a secretion marker was constructed according to the following method. That is, a cDNA fragment encoding the full length of SEAP protein (GenBank Accession No. NP_001623.3) is inserted and linked to the NheI-XhoI site of an expression vector for mammalian cells (pEGFP-C3, manufactured by Clontech), and the SEAP protein expression plasmid prCMV -Got SEAP.
- CHO 3D5, CHO 5g, CHO YA7, and CHO cells were each expressed as a plasmid pEF1-expressing SEAP expression plasmid and ⁇ -galactosidase for internal standardization.
- LacZ (Life Technologies) was co-transfected using Lipofectamine LTX reagent (Life Technologies). After culturing in DMEM containing 0.1% fetal bovine serum for 20 hours, a culture supernatant and a substrate solution containing p-Nitrophenyl phosphate (pNPP, manufactured by Sigma) were mixed.
- pNPP p-Nitrophenyl phosphate
- the absorbance at a wavelength of 405 nm was measured with an absorptiometer.
- the ⁇ -galactosidase activity of the cell fraction was measured according to a conventional method of ⁇ -Galactosidase Enzyme Assay System (manufactured by promega).
- Fig. 2 shows the SEAP activity normalized by ⁇ -galactosidase measurements.
- CHO 5g cells expressing p180 protein alone and CHO 3D5 cells expressing SF3b4 protein alone the SEAP secretion activity in the culture supernatant was significantly increased 1.7 to 2.0 times compared to CHO (Fig. 2).
- SEAP activity was further increased to 3.1 times compared to CHO cells.
- the SEAP activity ratio of CHO 1B2 cells when the SEAP activity of CHO cells was set to 1 was 3.1 times, and the SEAP activity was significantly increased. From this, it was shown that Chinese hamster SF3b4 having high similarity with human SF3b4 has a secretion enhancing activity equivalent to that of human SF3b4.
- Example 3 Promotion of localization of mRNA to membrane fraction by co-expression of p180 protein and SF3b4 protein
- Cell line CHO 5g cell expressing p180 protein prepared in Example 1 cell line CHO expressing SF3b4 protein Using 3D5 cells and the cell line CHO YA7 cells that co-express p180 protein and SF3b4 protein, promote expression of p180 protein and / or SF3b4 protein and localization of mRNA to membrane fraction I investigated the relationship.
- CHO YA7 cells and control cells were transfected with the above SEAP expression plasmid using lipofectamine LTX, and after 40 hours, they were fractionated into a cytoplasmic fraction and a membrane fraction.
- the fractionation method was performed according to the method described in Non-Patent Document 1 (Ueno, et al., (2010) J Biol Chem 285, 29941-29950). RNA was extracted from each fraction in accordance with the usual method of Trisol-LS reagent (Life Technologies). Thereafter, quantitative PCR was performed using SEAP-specific primers to quantify the amount of SEAP mRNA (FIG. 3).
- CHO-3D5 cells CHO-5g cells, CHO-YA7 cells, and CHO cells
- the total amount of mRNA which is the sum of the cytoplasm and membrane fraction
- about 30% of the total mRNA was present in the membrane fraction in CHO cells, CHO ⁇ 3D5 cells, and CHO 5g cells, whereas about 70% was localized in the membrane fraction in CHO ⁇ ⁇ YA7 cells.
- the localization of mRNA was significantly shifted from the cytoplasm to the membrane fraction.
- Example 4 Construction of an expression vector in which various cis-elements are inserted into a secretory alkaline phosphatase expression unit
- the cell line CHO 3D5 cell expressing SF3b4 protein prepared in Example 1 and p180 protein Using the cell line CHO YA7 cells co-expressing the SF3b4 protein, the structure of the expression plasmid was examined for the purpose of further increasing the protein expression efficiency.
- RNA derived from human fibroblasts was prepared according to the method described in Non-Patent Document 1 (Ueno, et al., (2010) J Biol Chem 285, 29941-29950), and RT-PCR was performed as a template.
- primers SEQ ID NO: 15, SEQ ID NO: 16
- BglII and HindIII recognition sequences were added on the 5 'and 3' sides during amplification
- cis-element # 1 derived from human type I collagen ⁇ 1 SEQ ID NO: 5 was amplified.
- the amplified fragment was treated with BglII-HindIII and inserted and ligated into the BglII-HindIII site between prCMV-SEAP CMV promoter and SEAPSORF.
- an expression plasmid prCMV-cis # 1-SEAP in which cis-element # 1 was inserted between the CMV promoter and the initiation methionine of SEAP was obtained (FIG. 4A).
- Example 5 Activation of protein secretion by cis-element
- the effect of the expressed protein on secretion was examined using the expression plasmid containing the cis-element prepared in Example 4.
- the two expression plasmids, prCMV-cis # 1-SEAP and prCMV-SEAP prepared in Example 4 were transfected using Lipofectamine LTX reagent (manufactured by Life technologies).
- the cells the four cell lines prepared in Example 1 were used. After culturing in DMEM containing 0.1% FBS for 20 hours, the culture supernatant and a substrate solution containing a fluorescent substrate 4-Methylumbelliferyl phosphate (4-MUP, Sigma) were mixed.
- the fluorescence intensity (excitation light 360 nm, fluorescence 440 nm) was measured with a fluorometer.
- total mRNA is extracted from the cell fraction using Trizol reagent (Lifetechnologies), quantitative PCR is performed using SEAP-specific primers, and the amount of SEAP cDNA is determined. Quantified.
- the SEAP activity ratio of prCMV-cis # 1-SEAP to prCMV-SEAP corrected with the amount of SEAP cDNA is shown in FIGS. 4B and 4C.
- the SEAP activity increased 2.7 times by the insertion of cis-element # 1 in CHO cells.
- the SEAP activity increased by 3.0 to 3.2 times by the insertion of cis-element # 1. From these results, it was shown that by inserting cis-element # 1 into the expression unit, protein synthesis and secretion amount per transcript can be increased in various CHO cells.
- Example 6 Activation of collagen secretion by cis-element # 1
- the effect on collagen expression was examined using an expression plasmid containing a cis-element.
- An expression plasmid for human type I collagen ⁇ 1 was constructed according to the following method. That is, a cDNA fragment encoding the entire length of COL1A1 (GenBank Accession No. NM_000088.3) was inserted and linked to the NheI-XhoI site of a mammalian cell expression vector (pEGFP-C3, manufactured by Clontech), and under the control of the CMV promoter. A plasmid prCMV-COL1A1 expressing COL1A1 (1-5297) containing cis-element # 1 was obtained.
- COL1A1 the gene fragments from 127 to 4251 and 127 to 5297 encoding only the full length of COL1A1 ORF were similarly amplified to construct prCMV-COL1A1-ORF and prCMV-COL1A1-ORF-UTR did.
- expression plasmids for human type II collagen ⁇ 1 (COL2A1) and human type III collagen ⁇ 1 (COL3A1) were constructed according to the following method. That is, a cDNA fragment encoding the full length of COL2A1 (GenBank Accession No. NP_001835.3) or COL3A1 (GenBank Accession No No.
- NP_000081.1 is cis-element # 1 in an expression vector for mammalian cells (pcDNA, manufactured by Invitrogen).
- the plasmid pcDNA-cis # 1-COL2A1 and COL3A1 (1 to 4401) that express COL2A1 (1 to 4464) under the control of the CMV promoter are expressed by ligation into the pcR-cis # 1 EcoRV-NotI site. Plasmid pcDNA-cis # 1-COL3A1 was obtained.
- prCMV-COL1A1 was transfected into the three cell lines prepared in Example 1 by the lipofection method. After culturing in DMEM containing 0.1% fetal bovine serum and 200 ⁇ M ascorbic acid for 40 hours, the amount of COL1A1 procollagen in the culture supernatant was analyzed by Western blotting (FIG. 5A).
- Example 1 Furthermore, in order to investigate the secreted amount of collagen that formed homotrimers, the three cell lines prepared in Example 1 were combined with pcDNA-cis # 1-COL2A1, pcDNA-cis # 1-COL3A1 or prCMV prepared in Example 6. -COL1A1 and prCMV-COL1A1-ORF were transfected by the lipofection method. After culturing in DMEM containing 2% fetal bovine serum and 200 ⁇ M ascorbic acid for 72 hours, the culture supernatant was collected and acidified by adding HCl to 0.1 ⁇ N, and pepsin (SIGMA to 0.5 mg / ml). And digestion reaction was performed at 4 ° C. for 16 hours.
- cis-element # 1 enhances the expression of collagen molecules in various CHO cells, and when cis-element # 1 is used in the presence of SF3b4 protein and / or p180 protein, the amount of collagen secretion that maintains a triple helical structure was shown to increase further.
- Example 7 Effect of enhancing expression of antibody molecule by cis-element The influence of cis-element on antibody expression was examined as follows.
- Antibody heavy chain and light chain full length expression plasmids were constructed according to the following method. That is, the antibody heavy chain (Heavy chain, HC) and light chain (Light chain, LC) encoded by anti-IL-8 antibody expression plasmid (p6G425V11N35A.choSD, ATCC 209552) are artificial gene synthesis service (MBL) Was synthesized. Thereafter, the full length of the heavy chain ORF was inserted and linked to the NheI-SpeI site of the pEF1 / Myc-His vector, and the full length of the light chain ORF was linked to the KpnI-EcoRV site of the pEF1 / Myc-His vector.
- MBL gene synthesis service
- the light chain expression cassette was excised with ClaI, inserted and ligated into the ClaI site of the heavy chain expression vector, and an anti-IL-8 antibody (heavy chain, light chain) co-expression plasmid pEF-HC-LC was constructed. Furthermore, an expression plasmid pEF-cis # 1-HC-LC in which cis # 1 was inserted upstream of the heavy chain and light chain ORF of this plasmid was also constructed.
- the three cell lines prepared in Example 1 were transfected with pEF-HC-LC and pEF-cis # 1-HC-LC by the lipofection method. After culturing in DMEM containing 0.1% fetal bovine serum for 96 hours, the amount of antibody production in the culture supernatant was quantified by ELISA using Human IgG ELISA Quantitation Set (Bethyl). As a result, the amount of antibody secretion increased 2.7 times by the insertion of cis-element # 1 in CHO cells. In each of CHOD3D5 cells and CHO YA7 cells, the amount of antibody secretion increased 2.5 and 1.8 times by the insertion of cis-element # 1 (FIG. 6A). From these results, it was shown that the amount of antibody secretion can be increased in various CHO cells by inserting cis-element # 1 into the expression unit.
- cis-element # 1 effectively acts on antibody production, and that its activity acts more remarkably in the presence of SF3b4 protein, p180 protein, or both.
- Example 8 Comparison of secretory activation effect of kozak sequence and cis-element # 1
- secretory activation exhibited by a kozak sequence known as a consensus sequence involved in translation initiation in eukaryotic mRNA. The effect was compared with the secretory activation effect exhibited by the cis-element.
- Expression plasmids prCMV-SEAP-kozak, prCMV-cis # 1-SEAP, in which the 6-bp sequence TCCTGC immediately before the start methionine codon ATG of prCMV-SEAP and prCMV-cis # 1-SEAP prepared in Example 4 was replaced with GCCACC -kozak was constructed by the following method. That is, PCR was performed using a SEAP-specific primer, and a SEAP fragment (1-132) with GCCACC added just before ATG was amplified.
- Example 9 Effect of enhancing protein expression by adding cis-elements Using the expression plasmids containing various cis-elements to the cells prepared in Example 1, details of cis-element sequences that have protein expression-enhancing effects investigated.
- the expression vector prCMV-cis # 2-SEAP in which the cis-element sequence of human fibronectin gene, cis-element # 2 (SEQ ID NO: -6), was inserted into prCMV-SEAP was constructed according to the following method. Specifically, RNA derived from human fibroblasts according to the method described in Non-Patent Document 1 (Ueno, et al., (2010) J Biol Chem 285, 29941-29950) was used as a template for RT-PCR. went.
- a fragment containing cis-element # 2 was amplified using primers (SEQ ID NO: 13 and SEQ ID NO: 14) to which BglII and HindIII recognition sequences were added on the 5 ′ and 3 ′ sides, respectively.
- the amplified fragment was treated with BglII-HindIII, and then inserted and ligated to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and SEAP ORF.
- an expression plasmid was obtained in which cis-element # 2 was inserted between the CMV promoter and the initiation methionine of SEAP.
- prCMV An expression vector prCMV in which cis-element sequence and cis-element # 3 of human type I collagen ⁇ 1 gene and cis-element sequence and cis-element # 4 of human-derived fibronectin gene are inserted into prCMV-SEAP -cis # 3-SEAP etc. were constructed according to the following method.
- a primer containing the cis-element of cis-element # 3 (SEQ ID NO: 9, SEQ ID NO: 10) or a primer containing the cis-element of cis-element # 4 (SEQ ID NO: 11, SEQ ID NO : 12) was heat-treated at 95 ° C for 10 minutes and then gradually lowered to 25 ° C to anneal the two primers to prepare various linkers.
- Various linkers were inserted and linked to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and the SEAP-ORF.
- Expression vectors prCMV-cis # 5-SEAP and prCMV-cis # 6-SEAP in which cis-elements # 5 and # 6 were inserted into prCMV-SEAP were constructed according to the following method. Using cis-element # 1 partial sequence with primers (SEQ ⁇ ⁇ ⁇ ⁇ ⁇ ID ⁇ NO: 28 and 29, SEQ ID NO: 30 and 31) to which BglII and HindIII recognition sequences were added, respectively, cis-element # 5 (1-60) A fragment containing cis-element # 6 (61-126) was amplified.
- the amplified fragment was treated with BglII-HindIII, and then inserted and ligated to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and SEAP ORF.
- expression plasmids in which cis-element # 5 and cis-element # 6 were inserted between the CMV promoter and the initiation methionine of SEAP were obtained.
- the expression vector prCMV-cis # 7-SEAP in which cis-element # 7 was inserted into prCMV-SEAP was constructed according to the following method. After synthesizing the COL2A1 gene-derived sequence, it was inserted and linked to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and the SEAPSORF using the BglII and HindIII recognition sequences added to the ends. As a result, an expression plasmid was obtained in which cis-element # 7 was inserted between the CMV promoter and the initiation methionine of SEAP.
- a primer containing the cis-element of cis-element # 8 (SEQ ID NO: 32, SEQ ID NO: 33), a primer containing the cis-element of cis-element # 9 (SEQ ID NO: 34, SEQ ID NO : 35), primer containing cis-element of cis-element # 10 (SEQ ID NO: 36, SEQ ID NO: 37) is heat-treated at 95 °C for 10 minutes and then gradually lowered to 25 °C to anneal the two primers
- Various linkers were prepared. Various linkers were inserted and linked to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and SEAP-ORF.
- the expression vector prCMV-cis # 11-SEAP in which cis-element # 11 was inserted into prCMV-SEAP was constructed according to the following method. Using a primer (SEQ ID NO: 38, SEQ ID NO: 39) to which a partial sequence of cis-element # 1 was added with BglII and HindIII recognition sequences, a fragment containing cis-element # 11 (1-113) Amplified. The amplified fragment was treated with BglII-HindIII, and then inserted and ligated to the BglII-HindIII site between the CMV promoter of prCMV-SEAP and SEAP ORF. As a result, expression plasmids in which cis-element # 11 (SEQ ID No: 27) was inserted between the CMV promoter and the initiation methionine of SEAP were obtained.
- prCMV-cis # 1-SEAP In CHO YA7 cells, prCMV-cis # 1-SEAP, prCMV-cis # 2-SEAP, prCMV-cis # 3-SEAP, prCMV-cis # 4-SEAP, prCMV-cis # 5-SEAP, prCMV-cis # 6 -SEAP, prCMV-cis # 7-SEAP, prCMV-cis # 8-SEAP, prCMV-cis # 9-SEAP, prCMV-cis # 10-SEAP, prCMV-cis # 11-SEAP, prCMV-SEAP .
- the SEAP activity in the culture supernatant after 20 hours was measured by the method described in Example 5.
- adding cis-elements # 1 to # 11 into the expression unit allows the SF3b4 protein, which has a protein expression enhancing effect, to be localized on the endoplasmic reticulum membrane, via which cell secretion occurs. It was shown that the ability can be enhanced.
- Example 11 Effect of base substitution and insertion of motif sequence The effects of base substitution and insertion within a motif on expression enhancing activity were examined.
- motif GAN 1- (X) n -ACN 2 in Cis-element # 3 mutants derived from cis-element # 3 with N 1 and N 2 as A, G, C, and T, respectively, are described in Example 8. It constructed by the same method (FIG. 10A).
- a mutant having a motif inserted into the polyA and polyC sequences and a control were also constructed (FIG. 10A). According to the method described in Example 5, the secretory activity of each element was evaluated.
- Example 12 Decrease in collagen secretion accompanying suppression of SF3b4 expression The effect on the secretion of collagen accompanying suppression of SF3b4 expression was examined.
- Human fetal lung-derived fibroblasts (HEL) were transfected with siRNA against SF3b4 (Life technologies, human SF3b4 siRNA HSS115684) according to the standard method of Oligofectamine (Life technologies). The cells were cultured for 4 days under the conditions of 0.1% FBS-DMEM and 200 ⁇ M ascorbic acid phosphate. Thereafter, the medium was collected, and the amount of COL1A1 procollagen in the culture supernatant and the amount of SF3b4 protein in the cell fraction were analyzed according to the method of Example 6.
- Example 13 Effect on collagen secretion in p180 / SF3b4 co-expressing floating cell line
- a CHO-S cell line stably coexpressing human p180 protein and human SF3b4 was established by the following method.
- CHO-S cells (Life technologies) were transfected with pCDNA-p180 / 54R by the lipofection method and cultured in the presence of Zeocin 300 ⁇ g / ml for 14 days for drug selection.
- a colony of Zeocin-resistant cell line was isolated, and pEF-SF3b4 was transfected by lipofection method, and drug selection was performed under the condition of Hygromycin 600 ⁇ g / ml.
- a cell line colony resistant to both Zeocin and Hygromycin was isolated, and a CHO-S-derived cell line 54 # 160 stably co-expressing human p180 protein and human SF3b4 was established.
- prCMV-COL1A1 and prCMV-COL1A1-ORF were transfected into CHO-S cells and the prepared 54 # 160 cells, respectively, by the lipofection method. After culturing in serum-free, CD FortiCHO medium (Life technologies) containing 8 mM L-glutamine for 96 hours, the amount of COL1A1 procollagen in the culture supernatant was analyzed by Western blotting. When prCMV-COL1A1 containing cis-element # 1 was transfected, the amount of procollagen in the control CHO-S cell was increased to about 3.5 times in 54 # 160 cells. When prCMV-COL1A1-ORF not containing cis-element # 1 was transfected, the amount of procollagen in the culture supernatant was below the detection limit by Western blotting.
- cis-element # 1 can enhance the synthesis / secretion of collagen, a macromolecule, under serum-free conditions in suspended CHO-S cells. Furthermore, cis-element # 1, SF3b4 protein and p180 It was shown that the secretory activity of suspended CHO cells can be remarkably enhanced by three protein factors.
- Example 14 Weight shift of mRNA in polysome by cis-element # 1 CHO YA7 cells were transfected with prCMV-COL1A1 and prCMV-COL1A1-ORF, respectively, by the lipofection method. After 40 hours, the membrane fraction was prepared according to the method described in Example 3. The obtained membrane fraction was subjected to 15-50% sucrose density gradient centrifugation to fractionate the polysome fraction. MRNA was extracted from each fraction according to the method described in Example 5, and the amount of COL1A1 cDNA was quantified by quantitative PCR. Further, the amount of secreted procollagen at this time was analyzed by the method described in Example 6.
- the COL1A1 cDNA in the polysome fraction has a distribution with a peak at fraction 24 in the case of prCMV-COL1A1-ORF that does not contain the cis element, whereas it has a peak at fraction 26 when it contains cis # 1.
- the distribution shifted to a higher density fraction (Fig. 12).
- procollagen secretion increased 4.9 times that of the control in the presence of cis-element # 1.
- cis-element # 1 has the ability to cause mRNA weight shift, and this weight shift was confirmed to correlate with increased expression.
- the recombinant cell of the present invention in which the expression of the full length of p180 protein or a part thereof and the expression of the full length of SF3b4 protein or a part thereof is enhanced is transformed with the DNA encoding the target protein, the recombinant cell of the present invention It has been revealed that the ability to synthesize or secrete proteins increases dramatically.
- SEQ ID NO: 1 Nucleotide sequence encoding human p180 SEQ ID NO: 2: Amino acid sequence of human p180 protein SEQ ID NO: 3: Nucleotide sequence encoding human SF3b4 SEQ ID NO: 4: Amino acid sequence of human SF3b4 protein SEQ ID NO: 5: cis-element # 1 SEQ ID NO: 6: cis-element # 2 SEQ ID NO: 7: cis-element # 3 SEQ ID NO: 8: cis-element # 4 SEQ ID NO: 9: Primer containing cis-element of cis-element # 3 SEQ ID NO: 10: Primer containing cis-element of cis-element # 3 SEQ ID NO: 11: cis- of cis-element # 4 Primer containing the element SEQ ID NO: 12: Primer containing the cis-element of cis-element # 4 SEQ ID NO: 13: Contains the
Abstract
Description
[2] p180タンパク質が、
(a)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(b)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(c)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(d)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、および
(e)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
からなる群から選択される、[1]に記載の組換え細胞。
[3] p180タンパク質が哺乳動物由来のものである、[1]または[2]に記載の組換え細胞。
[4] 哺乳動物のp180タンパク質の全長またはその部分が、ヒトp180タンパク質(SEQ ID NO: 2)、マウスp180タンパク質(GenBank Accession No. NP_077243)、ラットp180タンパク質(GenBank Accession No. XP_230637)、チャイニーズハムスターp180タンパク質(GenBank Accession No. XM_003496471)、イヌp180タンパク質(GenBank Accession No. NP_001003179)、ウマp180タンパク質(GenBank Accession No. XP_001915027)、サルp180タンパク質(GenBank Accession No. XP_002798281)、チンパンジーp180タンパク質(GenBank Accession No. XP_514527)、ブタp180タンパク質(GenBank Accession No. XP_001926148)、またはそれらの部分である、[3]に記載の組換え細胞。
[5] p180タンパク質の部分が、SEQ ID NO: 2に示されるアミノ酸配列を有するタンパク質(ヒトp180タンパク質)の27~157番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、623~737番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、738~944番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、945~1540番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、から選択される、[1]~[4]のいずれか1項に記載の組換え細胞。
[6] mRNAの小胞体(ER)局在化を促進するタンパク質が、スプライシング因子3Bサブユニット4(SF3b4)タンパク質の全長またはその部分(SEQ ID NO: 4の全長アミノ酸配列424 AA;RRM1であるSEQ ID NO: 4の13~91 AA;.RRM2であるSEQ ID NO: 4の100、からなる群から選択される、[1]~[5]のいずれか1項に記載の組換え細胞。
[7] SF3b4タンパク質が、
(i)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(ii)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iii)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iv)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(v)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
からなる群から選択される、[6]のいずれか1項に記載の組換え細胞。
[8] SF3b4タンパク質が哺乳動物由来のものである、[6]または[7]に記載の組換え細胞。
[9] 哺乳動物のSF3b4タンパク質の全長またはその部分が、ヒトSF3b4タンパク質(SEQ ID NO: 4)、マウスSF3b4タンパク質(GenBank Accession No. NP_694693.1)、ラットSF3b4タンパク質(GenBank Accession No. NP_001011951.1)、チャイニーズハムスターSF3b4タンパク質(GenBank Accession No. XP_003498680.1)、イヌSF3b4タンパク質(GenBank Accession No. XP_540295.3)、ウマSF3b4タンパク質(GenBank Accession No. XP_001488649.2)、サルSF3b4タンパク質(GenBank Accession No. NP_001097793.1)、チンパンジーSF3b4タンパク質(GenBank Accession No. XP_513768.2)、ブタSF3b4タンパク質(GenBank Accession No. XP_001926524.1)、またはそれらの部分である、[8]に記載の組換え細胞。
[10] 目的物としてのタンパク質の合成能または分泌能が、目的物としてのタンパク質をコードする核酸分子を形質転換するかまたは目的物としてのタンパク質の産生量を増加させることにより亢進された、[1]~[9]のいずれか1項に記載の組換え細胞。
[11] 受託番号NITE BP-01753(CHO 3D5)、受託番号NITE BP-1535(CHO YA7)、または受領番号NITE ABP-01811(CHO 1B2)により表される、細胞株。
[12] p180タンパク質の全長またはその部分の発現を亢進させ、mRNAの小胞体(ER)局在化を促進するタンパク質の発現を亢進させ、またはこれらのタンパク質両方の発現を亢進させた組換え細胞において、目的物としてのタンパク質をコードする核酸分子を形質転換するかまたは目的物としてのタンパク質の産生量を増加させることによる、目的物としてのタンパク質を製造する方法。
[13] p180タンパク質が哺乳動物由来のものである、[12]に記載の方法。
[14] 哺乳動物のp180タンパク質の全長またはその部分が、ヒトp180タンパク質(SEQ ID NO: 2)、マウスp180タンパク質(GenBank Accession No. NP_077243)、ラットp180タンパク質(GenBank Accession No. XP_230637)、チャイニーズハムスターp180タンパク質(GenBank Accession No. XM_003496471)、イヌp180タンパク質(GenBank Accession No. NP_001003179)、ウマp180タンパク質(GenBank Accession No. XP_001915027)、サルp180タンパク質(GenBank Accession No. XP_002798281)、チンパンジーp180タンパク質(GenBank Accession No. XP_514527)、ブタp180タンパク質(GenBank Accession No. XP_001926148)、またはそれらの部分である、[13]に記載の方法。
[15] 哺乳動物のp180タンパク質の部分が、SEQ ID NO: 2に示されるアミノ酸配列を有するタンパク質(ヒトp180タンパク質)の27~157番アミノ酸からなる領域を含む部分、623~737番アミノ酸からなる領域を含む部分、738~944番アミノ酸からなる領域を含む部分、945~1540番アミノ酸からなる領域を含む部分、から選択される、[13]または[14]に記載の方法。
[16] mRNAの小胞体(ER)局在化を促進するタンパク質が、スプライシング因子3Bサブユニット4(SF3b4)タンパク質の全長またはその部分(SEQ ID NO: 4の全長アミノ酸配列424 AA;RRM1であるSEQ ID NO: 4の13~91 AA;.RRM2であるSEQ ID NO: 4の100、からなる群から選択される、[12]~[15]のいずれか1項に記載の方法。
[17] SF3b4タンパク質が哺乳動物由来のものである、[16]に記載の方法。
[18] 哺乳動物のSF3b4タンパク質の全長またはその部分が、ヒトSF3b4タンパク質(SEQ ID NO: 4)、マウスSF3b4タンパク質(GenBank Accession No. NP_694693.1)、ラットSF3b4タンパク質(GenBank Accession No. NP_001011951.1)、チャイニーズハムスターSF3b4タンパク質(GenBank Accession No. XP_003498680.1)、イヌSF3b4タンパク質(GenBank Accession No. XP_540295.3)、ウマSF3b4タンパク質(GenBank Accession No. XP_001488649.2)、サルSF3b4タンパク質(GenBank Accession No. NP_001097793.1)、チンパンジーSF3b4タンパク質(GenBank Accession No. XP_513768.2)、ブタSF3b4タンパク質(GenBank Accession No. XP_001926524.1)、またはそれらの部分である、[17]に記載の方法。
[19] 組換え細胞が、受託番号NITE BP-01753(CHO 3D5)、受託番号NITE BP-1535(CHO YA7)、または受領番号NITE ABP-01811(CHO 1B2)により表される細胞株である、[12]~[18]のいずれか1項に記載の方法。
[20] 目的物としてのタンパク質が、糖タンパク質である、[12]~[19]のいずれか1項に記載の方法。
[21] 目的物としてのタンパク質が、コラーゲン、フィブロネクチンまたは抗体である、[20]に記載の方法。
[22] 目的物としてのタンパク質を発現するための発現ユニット中、プロモータの下流でかつ目的物としてのタンパク質をコードするDNAのヌクレオチド配列の開始コドンの上流に、RNA結合タンパク質が認識/結合(あるいは相互作用)するcis-エレメントを挿入することにより、発現系細胞内における目的物としてのタンパク質の発現量を増大させる方法。
[23] cis-エレメントが、RNA認識モチーフ(RRM)型RNA結合タンパク質が認識/結合(あるいは相互作用)するものである、[22]に記載の方法。
[24] cis-エレメントが、SF3b4タンパク質のRNA認識モチーフ(RRM)が認識/結合(あるいは相互作用)するものである、[23]に記載の方法。
[25] cis-エレメントのヌクレオチド配列が、1または数個の9mer~12merの配列モチーフGAN1-(X)n-ACN2(n=3~6)(N1およびN2は独立してA、T、C、Gのいずれのヌクレオチドであってもよい))を含むものである、[22]~[24]のいずれかに記載の方法。
[26] cis-エレメントのヌクレオチド配列が、1または数個の9mer~12merの配列モチーフ(GAG-(X) n-ACV(n=3~6)(VはAまたはGまたはCを示す)、SEQ ID NO: 17~20)を含むものである、[25]に記載の方法。
[27] cis-エレメントのヌクレオチド配列が、タイプIコラーゲン遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、フィブロネクチン遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、マトリックスメタロプロテアーゼ14(MMP14)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、プロリル4-ヒドラキシラーゼA2(P4HA2)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、プロリル4-ヒドラキシラーゼA1(P4HA1)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、からなる群から選択される配列である、[22]~[26]のいずれか1項に記載の方法。
[28] cis-エレメントのヌクレオチド配列が、SEQ ID NO: 5の全長またはSEQ ID NO: 7の全長の他、SEQ ID NO: 5のうち1~102番ヌクレオチド、1~78番ヌクレオチド、1~60番ヌクレオチド、61~126番ヌクレオチド、16~57番ヌクレオチド、79~126番ヌクレオチド、103~126番ヌクレオチド、58~78番ヌクレオチド、51~78番ヌクレオチド、1~27番ヌクレオチド、70~78番ヌクレオチドからなる群から選択されるいずれかの配列である[22]~[27]のいずれか1項に記載の方法。
[29] 発現系細胞が、無傷の宿主細胞、p180タンパク質の全長またはその部分の発現が亢進された細胞、SF3b4タンパク質の全長またはその部分の発現が亢進された細胞、またはこれらの両方の発現がともに亢進された細胞である、[22]~[28]のいずれか1項に記載の方法。
[30] SF3b4を機能阻害または発現抑制することにより、コラーゲン合成を抑制し、異常コラーゲンによる肺胞上皮、線維症の重症化を防止するための医薬組成物。
(a)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(b)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(c)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(d)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(e)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
のことをいう。
C-末端域である180~424 AA.)、等が含まれる。
(i)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(ii)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iii)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iv)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(v)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
のことをいう。
プラスミドの調製
SF3b4タンパク質、またはp180タンパク質とSF3b4タンパク質を共発現する細胞株の樹立は、p180タンパク質をコードする核酸を含む発現プラスミドとSF3b4タンパク質をコードする核酸を含む発現プラスミドとを別個に調製し、CHO細胞に対して順番にトランスフェクトすることにより、行った。
ヒトp180タンパク質を安定に発現するCHO細胞の樹立は、特許文献1に記載の方法に準じて行った。即ち、CHO細胞にヒトp180タンパク質発現プラスミドpcDNA-p180/54Rをリポフェクション法によりトランスフェクションした後、Zeocin 400μg/ml存在下で培養し、薬剤選択を行った。10日間培養後、Zeocinに耐性を持つ細胞株のコロニーを分離し、p180タンパク質を安定に発現する細胞株CHO 5g細胞を樹立した。
ヒトSF3b4タンパク質を安定に発現するCHO細胞を調製するため、CHO細胞にヒトSF3b4タンパク質発現プラスミドpEF-SF3b4をリポフェクション法によりトランスフェクションした後、G418 400μg/ml存在下で培養し、薬剤選択を行った。10日間培養後、G418に耐性を持つ細胞株のコロニーを分離し、SF3b4タンパク質を安定に発現する細胞株CHO 3D5細胞を樹立した(独立行政法人製品評価技術基盤機構特許微生物寄託センター(NPMD)、千葉県木更津市かずさ鎌足2-5-8(日本)、受託番号NITE BP-01753、受託日2013年11月21日)。
次に、ヒトp180タンパク質とヒトSF3b4タンパク質とを安定に共発現するCHO細胞を樹立するため、細胞株CHO 5g細胞に対して、SF3b4タンパク質発現プラスミドpEF-SF3b4をリポフェクション法によりトランスフェクションした。その後、G418 400μg/ml、Zeocin 100μg/ml存在下で培養し、薬剤選択を行った。14日間培養後、G418とZeocinに耐性を持つ細胞株のコロニーを分離し、ヒトp180タンパク質とヒトSF3b4タンパク質とを安定に共発現する細胞株CHO YA7細胞を樹立した(独立行政法人製品評価技術基盤機構特許微生物寄託センター(NPMD)、千葉県木更津市かずさ鎌足2-5-8(日本)、受託番号NITE BP-1535、受託日2013年2月13日)。
CHO 5g細胞がp180タンパク質を安定に発現していること、CHO 3D5細胞がSF3b4タンパク質を安定に発現していること、そしてCHO YA7細胞およびCHO 1B2細胞がp180タンパク質とSF3b4タンパク質とを共発現していることをそれぞれ確認するため、CHO 5g細胞、CHO 3D5細胞、CHO YA7細胞、CHO 1B2細胞のそれぞれを、5%ウシ胎児血清を含むダルベッコ改変イーグル培地(DMEM)中で、5%CO2存在下、37℃で培養した。コントロール細胞として、CHO細胞も併せて培養した。
実施例1において作製したp180タンパク質を発現する細胞株CHO 5g細胞、SF3b4タンパク質を発現する細胞株CHO 3D5細胞、およびp180タンパク質とSF3b4タンパク質とを共発現する細胞株CHO YA7細胞を使用して、p180タンパク質の発現および/またはSF3b4タンパク質の発現と、タンパク質分泌の活性化について調べた。
実施例1において作製したp180タンパク質を発現する細胞株CHO 5g細胞、SF3b4タンパク質を発現する細胞株CHO 3D5細胞、およびp180タンパク質とSF3b4タンパク質とを共発現する細胞株CHO YA7細胞を使用して、p180タンパク質の発現および/またはSF3b4タンパク質の発現と、mRNAの膜画分への局在化の促進との関係について調べた。
本実施例においては、実施例1において作製したSF3b4タンパク質を発現する細胞株CHO 3D5細胞、およびp180タンパク質とSF3b4タンパク質とを共発現する細胞株CHO YA7細胞を使用して、タンパク質の発現効率を更に上げることを目的として、発現プラスミドの構造を検討した。
実施例4において作製したcis-エレメントを含む発現プラスミドにより、発現タンパク質の分泌への影響を検討した。実施例4で作成した2種の発現プラスミド、prCMV-cis#1-SEAPおよびprCMV-SEAPを、LipofectamineLTX試薬(Life technologies社製)を用いてトランスフェクションした。細胞は実施例1で作製した4種の細胞株を用いた。0.1%FBSを含むDMEM中で20時間培養した後、培養上清と蛍光基質4-Methylumbelliferyl phosphate (4-MUP、Sigma社製)を含む基質溶液を混合した。室温で30分反応後、蛍光強度(励起光360 nm、蛍光440nm)を蛍光光度計にて測定した。またトランスフェクション効率をSEAP cDNA総量で補正するため、細胞画分からTrizol試薬(Lifetechnologies社製)を用いて全mRNAを抽出し、SEAP特異的なプライマーを用いて定量的PCRを行い、SEAP cDNA量を定量した。SEAP cDNA量で補正したprCMV-cis#1-SEAPのprCMV-SEAPに対するSEAP活性比を図4B、図4Cに示す。
本実施例においては、cis-エレメントを含む発現プラスミドにより、コラーゲン発現への影響を検討した。
cis-エレメントによる抗体発現への影響を以下のように検討した。
本実施例においては、真核細胞のmRNA中において翻訳の開始に関与する共通配列として知られるkozak配列が示す分泌活性化効果と、cis-エレメントが示す分泌活性化効果とを比較することを目的として行った。
実施例1において作製した細胞に対して、各種cis-エレメントを含む発現プラスミドを使用して、タンパク質発現増強作用のあるcis-エレメント配列の詳細を検討した。
cis-エレメント#1に同定されたモチーフ配列GAN1-(X)n-ACN2について、有効な鎖長数nの検討を以下のように行った。GAG-(X) n-ACV(VはAまたはGまたはCを示す)について、nを1-9merまで変化させたcis-エレメント#3由来の変異体を実施例8と同様の方法で構築した(図9A)。またモチーフを欠失させた変異体(motif delete)も併せて構築した。各エレメントのSEAP分泌活性を評価するため、実施例5に記載の方法に従い、それぞれの分泌活性を評価した。その結果、n=3~6の場合に、cis-エレメント#3と同等の活性が得られた(図9B)。以上の結果より、本系において分泌活性化に重要となるcis-エレメント内のモチーフはGAN1-(X)n-ACN2のX鎖長nが3~6残基であることが判明した。
モチーフ内の塩基置換および挿入による発現増強活性への影響を検討した。Cis-エレメント#3内のモチーフGAN1-(X)n-ACN2について、N1およびN2をそれぞれA、G、C、Tとしたcis-エレメント#3由来の変異体を実施例8と同様の方法で構築した(図10A)。またpolyA配列、polyC配列にモチーフを挿入した変異体とコントロールも併せて構築した(図10A)。実施例5に記載の方法に従い、各エレメントの分泌活性を評価した。その結果、cis-エレメント#3のN2をGからA、C、Tに置換したときのSEAP活性は、いずれもcis-エレメント#3と同等の活性が得られた(図10B)。cis-エレメント#3のN1をGからA、C、Tに置換したときのSEAP活性は、いずれもcis-エレメント#3と同等の活性を示した(図10B)。またモチーフを挿入したエレメントもcis-エレメント#3と同等の活性を有していた。以上の結果から、高い活性を有するモチーフとしてGAN1-(X)n-ACN2(N1およびN2はA、G、C、Tのいずれの塩基でもよく、n=3~6の整数)であると確認された。
SF3b4の発現抑制に伴ったコラーゲン分泌量への影響を検討した。ヒト胎児肺由来繊維芽細胞(HEL)に、Oligofectamine(Life technologies)の定法に従ってSF3b4に対するsiRNA(Life technologies, human SF3b4 siRNA HSS115684)をトランスフェクションした。0.1%FBS-DMEM、アスコルビン酸リン酸エステル200μMの条件下で4日間培養した。その後、培地を回収し、培養上清中のCOL1A1プロコラーゲン量ならびに細胞画分のSF3b4タンパク質量を実施例6の方法に準じて解析した。その結果、細胞内のSF3b4発現量がコントロールの20%に低下した時、分泌されたCOL1A1プロコラーゲン量は10%に低下した(図11)。以上の結果より、SF3b4の発現抑制に伴いコラーゲンの産生が顕著に抑制されることが示された。
ヒトp180タンパク質とヒトSF3b4を安定共発現するCHO-S細胞株を以下の方法により樹立した。CHO-S細胞(Life technologies 社)に、pCDNA-p180/54Rをリポフェクション法によりトランスフェクションし、Zeocin 300μg/ml存在下で14日間培養し、薬剤選択を行った。Zeocin 耐性細胞株のコロニーを分離しpEF-SF3b4をリポフェクション法により、トランスフェクションし、Hygromycin 600μg/mlの条件で薬剤選択を行った。14日間培養後、Zeocin およびHygromycinの両者に耐性を持つ細胞株のコロニーを分離し、ヒトp180タンパク質とヒトSF3b4を安定に共発現するCHO-S由来細胞株54#160を樹立した。
CHO YA7細胞にprCMV-COL1A1、prCMV-COL1A1-ORFをそれぞれリポフェクション法にてトランスフェクションした。40時間後、実施例3に記載の方法に従い、膜画分を調製した。得られた膜画分を15~50%のショ糖密度勾配遠心分離に供し、ポリソーム画分を分画した。各画分から実施例5に記載の方法に準じてmRNAを抽出し、定量的PCRによりCOL1A1 cDNA量を定量した。また、このときの分泌プロコラーゲン量を実施例6に記載の方法で解析した。その結果、ポリソーム画分中のCOL1A1 cDNAは、cisエレメントを含まないprCMV-COL1A1-ORFの場合、フラクション24をピークとする分布であるのに対し、cis#1を含む場合はフラクション26をピークとした分布となり、より高密度の画分へと分布がシフトしていた(図12)。さらにピークのシフトに伴って、プロコラーゲンの分泌量はcis-エレメント#1存在下ではコントロールの4.9倍に増加していた。p180およびSF3b4存在下においてcis-エレメント#1はmRNAの重量化シフトを起こす能力を有し、この重量化シフトは発現増加と相関する事が確認された。
SEQ ID NO: 2:ヒトp180タンパク質のアミノ酸配列
SEQ ID NO: 3:ヒトSF3b4をコードするヌクレオチド配列
SEQ ID NO: 4:ヒトSF3b4タンパク質のアミノ酸配列
SEQ ID NO: 5:cis-エレメント#1
SEQ ID NO: 6:cis-エレメント#2
SEQ ID NO: 7:cis-エレメント#3
SEQ ID NO: 8:cis-エレメント#4
SEQ ID NO: 9:cis-エレメント#3のcis-エレメントを含むプライマー
SEQ ID NO: 10:cis-エレメント#3のcis-エレメントを含むプライマー
SEQ ID NO: 11:cis-エレメント#4のcis-エレメントを含むプライマー
SEQ ID NO: 12:cis-エレメント#4のcis-エレメントを含むプライマー
SEQ ID NO: 13:5'側にBglII認識配列を付加した、cis-エレメント#2のcis-エレメントを含むプライマー
SEQ ID NO: 14:3'側にHindIII認識配列を付加した、cis-エレメント#2のcis-エレメントを含むプライマー
SEQ ID NO: 15:5'側にBglII認識配列を付加した、cis-エレメント#1を増幅するためのプライマー
SEQ ID NO: 16:3'側にHindIII認識配列を付加した、cis-エレメント#1を増幅するためのプライマー
SEQ ID NO: 17:cis-エレメントのモチーフ(9mer)
SEQ ID NO: 18:cis-エレメントのモチーフ(10mer)
SEQ ID NO: 19:cis-エレメントのモチーフ(11mer)
SEQ ID NO: 20:cis-エレメントのモチーフ(12mer)
SEQ ID NO: 21:cis-エレメント#5
SEQ ID NO: 22:cis-エレメント#6
SEQ ID NO: 23:cis-エレメント#7
SEQ ID NO: 24:cis-エレメント#8
SEQ ID NO: 25:cis-エレメント#9
SEQ ID NO: 26:cis-エレメント#10
SEQ ID NO: 27:cis-エレメント#11
SEQ ID NO: 28:5'側にBglII認識配列を付加した、cis-エレメント#5のcis-エレメントを含むプライマー
SEQ ID NO: 29: 3'側にHindIII認識配列を付加した、cis-エレメント#5のcis-エレメントを含むプライマー
SEQ ID NO: 30: 5'側にBglII認識配列を付加した、cis-エレメント#6のcis-エレメントを含むプライマー
SEQ ID NO: 31: 3'側にHindIII認識配列を付加した、cis-エレメント#6のcis-エレメントを含むプライマー
SEQ ID NO: 32: cis-エレメント#8のcis-エレメントを含むプライマー
SEQ ID NO: 33: cis-エレメント#8のcis-エレメントを含むプライマー
SEQ ID NO: 34: cis-エレメント#9のcis-エレメントを含むプライマー
SEQ ID NO: 35: cis-エレメント#9のcis-エレメントを含むプライマー
SEQ ID NO: 36: cis-エレメント#10のcis-エレメントを含むプライマー
SEQ ID NO: 37: cis-エレメント#10のcis-エレメントを含むプライマー
SEQ ID NO: 38: 5'側にBglII認識配列を付加した、cis-エレメント#11のcis-エレメントを含むプライマー
SEQ ID NO: 39: 3'側にHindIII認識配列を付加した、cis-エレメント#11のcis-エレメントを含むプライマー
Claims (30)
- 細胞において、p180タンパク質の全長またはその部分の発現、mRNAの小胞体(ER)局在化を促進するタンパク質の発現、またはこれらの両方の発現がともに亢進された、細胞内での目的物としてのタンパク質の合成能または分泌能が亢進された組換え細胞。
- p180タンパク質が、
(a)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(b)ヒト由来のp180タンパク質のアミノ酸配列(SEQ ID NO: 2)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(c)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
(d)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、および
(e)ヒト由来のp180タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 1)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、細胞内の小胞体膜上でのポリソーム形成を促進する機能を有するタンパク質、
からなる群から選択される、請求項1に記載の組換え細胞。 - p180タンパク質が哺乳動物由来のものである、請求項1または2に記載の組換え細胞。
- 哺乳動物のp180タンパク質の全長またはその部分が、ヒトp180タンパク質(SEQ ID NO: 2)、マウスp180タンパク質(GenBank Accession No. NP_077243)、ラットp180タンパク質(GenBank Accession No. XP_230637)、チャイニーズハムスターp180タンパク質(GenBank Accession No. XM_003496471)、イヌp180タンパク質(GenBank Accession No. NP_001003179)、ウマp180タンパク質(GenBank Accession No. XP_001915027)、サルp180タンパク質(GenBank Accession No. XP_002798281)、チンパンジーp180タンパク質(GenBank Accession No. XP_514527)、ブタp180タンパク質(GenBank Accession No. XP_001926148)、またはそれらの部分である、請求項3に記載の組換え細胞。
- p180タンパク質の部分が、SEQ ID NO: 2に示されるアミノ酸配列を有するタンパク質(ヒトp180タンパク質)の27~157番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、623~737番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、738~944番アミノ酸からなる領域に対応するアミノ酸配列、945~1540番アミノ酸からなる領域に対応するアミノ酸配列を含む部分、から選択される、請求項1~4のいずれか1項に記載の組換え細胞。
- mRNAの小胞体(ER)局在化を促進するタンパク質が、スプライシング因子3Bサブユニット4(SF3b4)タンパク質の全長またはその部分(SEQ ID NO: 4の全長アミノ酸配列424 AA;RRM1であるSEQ ID NO: 4の13~91 AA;.RRM2であるSEQ ID NO: 4の100、からなる群から選択される、請求項1~5のいずれか1項に記載の組換え細胞。
- SF3b4タンパク質が、
(i)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)と少なくとも70%の配列同一性を有するアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(ii)ヒト由来のSF3b4タンパク質のアミノ酸配列(SEQ ID NO: 4)において、1または数個のアミノ酸が欠失、置換、または付加されたアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iii)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と少なくとも70%の配列同一性を有するヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(iv)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)において、1または数個のヌクレオチドが欠失、置換、または付加されたヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
(v)ヒト由来のSF3b4タンパク質をコードする遺伝子のヌクレオチド配列(SEQ ID NO: 3)と相補的なヌクレオチド配列と、ストリンジェントな条件下でハイブリダイズ可能であるヌクレオチド配列により規定されるアミノ酸配列からなり、mRNAの小胞体への局在化を促進させる機能を有するタンパク質、
からなる群から選択される、請求項6に記載の組換え細胞。 - SF3b4タンパク質が哺乳動物由来のものである、請求項6または7に記載の組換え細胞。
- 哺乳動物のSF3b4タンパク質の全長またはその部分が、ヒトSF3b4タンパク質(SEQ ID NO: 4)、マウスSF3b4タンパク質(GenBank Accession No. NP_694693.1)、ラットSF3b4タンパク質(GenBank Accession No. NP_001011951.1)、チャイニーズハムスターSF3b4タンパク質(GenBank Accession No. XP_003498680.1)、イヌSF3b4タンパク質(GenBank Accession No. XP_540295.3)、ウマSF3b4タンパク質(GenBank Accession No. XP_001488649.2)、サルSF3b4タンパク質(GenBank Accession No. NP_001097793.1)、チンパンジーSF3b4タンパク質(GenBank Accession No. XP_513768.2)、ブタSF3b4タンパク質(GenBank Accession No. XP_001926524.1)、またはそれらの部分である、請求項8に記載の組換え細胞。
- 目的物としてのタンパク質の合成能または分泌能が、目的物としてのタンパク質をコードする核酸分子を形質転換するかまたは目的物としてのタンパク質の産生量を増加させることにより亢進された、請求項1~9のいずれか1項に記載の組換え細胞。
- 受託番号NITE BP-01753(CHO 3D5)、受託番号NITE BP-1535(CHO YA7)、または受領番号NITE ABP-01811(CHO 1B2)により表される、細胞株。
- p180タンパク質の全長またはその部分の発現を亢進させ、mRNAの小胞体(ER)局在化を促進するタンパク質の発現を亢進させ、またはこれらのタンパク質両方の発現を亢進させた組換え細胞において、目的物としてのタンパク質をコードする核酸分子を形質転換するかまたは目的物としてのタンパク質の産生量を増加させることによる、目的物としてのタンパク質を製造する方法。
- p180タンパク質が哺乳動物由来のものである、請求項12に記載の方法。
- 哺乳動物のp180タンパク質の全長またはその部分が、ヒトp180タンパク質(SEQ ID NO: 2)、マウスp180タンパク質(GenBank Accession No. NP_077243)、ラットp180タンパク質(GenBank Accession No. XP_230637)、チャイニーズハムスターp180タンパク質(GenBank Accession No. XM_003496471)、イヌp180タンパク質(GenBank Accession No. NP_001003179)、ウマp180タンパク質(GenBank Accession No. XP_001915027)、サルp180タンパク質(GenBank Accession No. XP_002798281)、チンパンジーp180タンパク質(GenBank Accession No. XP_514527)、ブタp180タンパク質(GenBank Accession No. XP_001926148)、またはそれらの部分である、請求項13に記載の方法。
- 哺乳動物のp180タンパク質の部分が、SEQ ID NO: 2に示されるアミノ酸配列を有するタンパク質(ヒトp180タンパク質)の27~157番アミノ酸からなる領域を含む部分、623~737番アミノ酸からなる領域を含む部分、738~944番アミノ酸からなる領域を含む部分、945~1540番アミノ酸からなる領域を含む部分、から選択される、請求項13または14に記載の方法。
- mRNAの小胞体(ER)局在化を促進するタンパク質が、スプライシング因子3Bサブユニット4(SF3b4)タンパク質の全長またはその部分(SEQ ID NO: 4の全長アミノ酸配列424 AA;RRM1であるSEQ ID NO: 4の13~91 AA;.RRM2であるSEQ ID NO: 4の100、からなる群から選択される、請求項12~15のいずれか1項に記載の方法。
- SF3b4タンパク質が哺乳動物由来のものである、請求項16に記載の方法。
- 哺乳動物のSF3b4タンパク質の全長またはその部分が、ヒトSF3b4タンパク質(SEQ ID NO: 4)、マウスSF3b4タンパク質(GenBank Accession No. NP_694693.1)、ラットSF3b4タンパク質(GenBank Accession No. NP_001011951.1)、チャイニーズハムスタートSF3b4タンパク質(GenBank Accession No. XP_003498680.1)、イヌSF3b4タンパク質(GenBank Accession No. XP_540295.3)、ウマSF3b4タンパク質(GenBank Accession No. XP_001488649.2)、サルSF3b4タンパク質(GenBank Accession No. NP_001097793.1)、チンパンジーSF3b4タンパク質(GenBank Accession No. XP_513768.2)、ブタSF3b4タンパク質(GenBank Accession No. XP_001926524.1)、またはそれらの部分である、請求項17に記載の方法。
- 組換え細胞が、受託番号NITE BP-01753(CHO 3D5)、受託番号NITE BP-1535(CHO YA7)、または受領番号NITE ABP-01811(CHO 1B2)により表される細胞株である、請求項12~18のいずれか1項にに記載の方法。
- 目的物としてのタンパク質が、糖タンパク質である、請求項12~19のいずれか1項に記載の方法。
- 目的物としてのタンパク質が、コラーゲン、フィブロネクチンまたは抗体である、請求項20に記載の方法。
- 目的物としてのタンパク質を発現するための発現ユニット中、プロモータの下流でかつ目的物としてのタンパク質をコードするDNAのヌクレオチド配列の開始コドンの上流に、RNA結合タンパク質が認識/結合(あるいは相互作用)するcis-エレメントを挿入することにより、発現系細胞内における目的物としてのタンパク質の発現量を増大させる方法。
- cis-エレメントが、RNA認識モチーフ(RRM)型RNA結合タンパク質が認識/結合(あるいは相互作用)するものである、請求項22に記載の方法。
- cis-エレメントが、SF3b4タンパク質のRNA認識モチーフ(RRM)が認識/結合(あるいは相互作用)するものである、請求項23に記載の方法。
- cis-エレメントのヌクレオチド配列が、1または数個の9mer~12merの配列モチーフGAN1-(X)n-ACN2(n=3~6)(N1およびN2は独立してA、T、C、Gのいずれのヌクレオチドであってもよい))を含むものである、請求項22~24のいずれか1項に記載の方法。
- cis-エレメントのヌクレオチド配列が、1または数個の9mer~12merの配列モチーフ(GAG-(X) n-ACV(n=3~6)(VはAまたはGまたはCを示す)、SEQ ID NO: 17~20)を含むものである、請求項25に記載の方法。
- cis-エレメントのヌクレオチド配列が、タイプIコラーゲン遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、フィブロネクチン遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、マトリックスメタロプロテアーゼ14(MMP14)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、プロリル4-ヒドラキシラーゼA2(P4HA2)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、プロリル4-ヒドラキシラーゼA1(P4HA1)遺伝子の5'非翻訳領域のヌクレオチド配列に由来する配列、からなる群から選択される配列である、請求項22~26のいずれか1項に記載の方法。
- cis-エレメントのヌクレオチド配列が、SEQ ID NO: 5の全長またはSEQ ID NO: 7の全長の他、SEQ ID NO: 5のうち1~102番ヌクレオチド、1~78番ヌクレオチド、1~60番ヌクレオチド、61~126番ヌクレオチド、16~57番ヌクレオチド、79~126番ヌクレオチド、103~126番ヌクレオチド、58~78番ヌクレオチド、51~78番ヌクレオチド、1~27番ヌクレオチド、70~78番ヌクレオチドからなる群から選択されるいずれかの配列である、請求項22~27のいずれか1項に記載の方法。
- 発現系細胞が、無傷の宿主細胞、p180タンパク質の全長またはその部分の発現が亢進された細胞、SF3b4タンパク質の全長またはその部分の発現が亢進された細胞、またはこれらの両方の発現がともに亢進された細胞である、請求項22~28のいずれか1項に記載の方法。
- SF3b4を機能阻害または発現抑制することにより、コラーゲン合成を抑制し、異常コラーゲンによる肺胞上皮、線維症の重症化を防止するための医薬組成物。
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