WO2025141778A1 - Novel promoter - Google Patents

Abstract

Provided is a novel promoter for expressing a recombinant protein with a mammalian cell as a host.　Exemplified is a promoter constituted by a polynucleotide in which a partial region of a CAG promoter sequence is deleted, wherein, in a CAG promoter of SEQ ID NO: 1, nucleic acids are deleted in one or both of the region (a) of positions 469-571 and the region (b) of positions 799-1563.

Description

New Promoter

The present invention relates to a promoter for expressing a recombinant protein in a mammalian cell as a host. The present invention also relates to a method for producing a recombinant protein using the promoter.

　In order to express recombinant proteins using mammalian cells as hosts, it is common to introduce an expression vector incorporating the gene of the target protein into the host cells and culture them. Protein expression vectors contain sequences necessary for gene transcription and translation, such as promoters, terminators, and translation initiation signals. Various factors are involved in the production of recombinant proteins, such as the sequences of these regions in the expression vector and the culture conditions of the recombinant cells. The selection of the promoter is particularly important, as it affects the expression level of the recombinant protein. Several plasmid vectors and viral vectors that use the powerful CAG promoter are commercially available.

The CAG promoter is constructed as a hybrid promoter by combining a CMV enhancer with a modified chicken-derived β-actin promoter. The wild-type CAG promoter is described in Gene 108:193-199 "Efficient Selection for High-Expression Transfectants with a Novel Eukaryotic Vector" (Non-Patent Document 1). The information contained in this document is incorporated herein by reference. The CAG promoter is also described in JP 3-168087 A.

The DNA sequence of the wild-type CAG promoter is shown in SEQ ID NO:1.
Japanese Patent No. 5670330 (WO2010/015079) discloses an improved CAG promoter.

Patent No. 5670330 (WO2010/015079) Patent Publication No. 3-168087

Niwa et al., 1991, Gene 108:193-199

The objective of the present invention is to provide a highly versatile promoter that is superior in many ways to the existing high expression promoter, the CAG promoter, for recombinant protein production using mammalian cells.

The present inventors conducted extensive research to develop a promoter that is superior to conventional CAG promoters. The present inventors then identified a site in the CAG promoter that can be deleted, and by deleting said site, discovered a new modified CAG promoter that is easy to use and has high expression levels. This promoter is referred to as the "promoter of the present invention."

The promoter of the present invention includes the CAG promoter shown in SEQ ID NO:1, which has the following:
(a) 469-571;
(b) 799～1563
The present invention also includes a promoter in which the nucleic acid of either or both of the above regions has been deleted.

The invention disclosed in this application includes the following aspects.
(1) An isolated polynucleotide consisting of a sequence in which at least the region from base 469 to base 571 of the nucleotide sequence set forth in SEQ ID NO:1 is deleted, or consisting of a nucleotide sequence having 90% or more identity thereto.
(2) The polynucleotide according to (1) above, which consists of the nucleotide sequence set forth in SEQ ID NO:2 or a nucleotide sequence having 90% or more identity thereto.
(3) An isolated polynucleotide consisting of a sequence in which at least the region from base 799 to base 1563 of the sequence set forth in SEQ ID NO:1 is deleted, or consisting of a nucleotide sequence having 90% or more identity thereto.
(4) The polynucleotide according to (3) above, which consists of the nucleotide sequence set forth in SEQ ID NO: 3 or a nucleotide sequence having 90% or more identity thereto.
(5) An isolated polynucleotide consisting of a sequence in which at least the following regions (a) and (b) are deleted from the nucleotide sequence set forth in SEQ ID NO: 1, or consisting of a nucleotide sequence having 90% or more identity thereto:
(a) the region from base 469 to base 571; and (b) the region from base 799 to base 1563.
(6) The polynucleotide according to (5) above, which consists of the sequence set forth in SEQ ID NO: 4 or a nucleotide sequence having 90% or more identity thereto.
(7) A promoter consisting of the polynucleotide according to any one of (1) to (6) above.
(8) A vector comprising the promoter described in (7) above.
(9) A vector comprising the promoter and a gene encoding the protein described in (7) above.
(10) A transformed cell obtained by transforming a mammalian cell with the vector according to (9) above.
(11) The transformed cell according to (10), wherein the mammalian cell is a CHO cell.
(12) A method for producing a recombinant protein, comprising the steps of producing a recombinant protein by culturing the transformed cell described in (11) above, and recovering the recombinant protein produced from the resulting culture.
(13) A method according to (12) above, which is a method for producing an antibody, wherein the vector comprises a promoter consisting of a sequence set forth in any one of SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and a gene encoding an antibody, and the gene encodes an amino acid sequence including the VL or an amino acid sequence including the VH of the antibody, or the gene encodes an amino acid sequence including the VL and an amino acid sequence including the VH of the antibody.
(14) An isolated polynucleotide fragment having promoter function, the polynucleotide fragment having an array having at least 70%, at least 80%, at least 90% or 100% identity to any of the arrays set forth in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.
(15) The vector according to (9) above, wherein the protein is an antibody heavy chain (H chain) or light chain (L chain).
(16) A cell comprising a gene incorporating a promoter consisting of a nucleotide sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.
(17) The cell according to (16) above, wherein the gene encodes an antibody polypeptide.
(18) A method for producing an antibody using a cell containing a gene incorporating a promoter consisting of a nucleotide sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

The present inventors have identified the minimum required sequence of the CAG promoter and developed a promoter that is excellent in the expression level of foreign genes and is easy to handle. The promoter of the present invention has a wide host range, similar to the wild-type CAG promoter, and cell lines that stably express foreign genes can be established using various types of cells derived from various animals. Furthermore, the promoter of the present invention is easier to grow and maintain than the wild-type CAG promoter. For example, the promoter of the present invention can be easily amplified by PCR because the GC-rich region 469 to 571 is missing from the DNA sequence of the wild-type CAG promoter (SEQ ID NO: 1). Since the DNA fragment containing the promoter of the present invention can be amplified by PCR, the need for subcloning using an intermediate plasmid can be avoided. Therefore, by using the promoter of the present invention, an expression vector into which a foreign gene is introduced can be easily constructed.

The promoter of the present invention can be used in the production of antibody pharmaceuticals produced using animal cells constructed using recombinant gene technology as a cell substrate for production. In addition, the present invention is highly versatile and can become a platform technology for the production of not only antibodies but also various other protein pharmaceuticals.

1 shows a circular DNA containing the sequence of the promoter of the present invention shown in SEQ ID NO:2. 1 shows a circular DNA containing the sequence of the promoter of the present invention shown in SEQ ID NO:3. 1 shows the expression of luciferase by the promoter of the present invention and the wild-type CAG promoter. PCR amplification of the promoter fragment is shown. 1 shows expression of monoclonal antibodies using the promoter of the present invention and the wild-type CAG promoter.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in practicing or testing the present invention, the preferred methods and materials are described herein. All publications mentioned below are incorporated herein by reference.

The promoter of the present invention is a promoter that can be easily used and improves the expression level of a foreign gene in a mammalian cell by deleting a part of an existing CAG promoter. Specifically, the promoter is a promoter in which the nucleic acid of one or more of the following regions (a) or (b) is deleted from the CAG promoter sequence shown in SEQ ID NO: 1:
(a) 469th T to 571st C
(b) 799th G to 1563th C

A specific example of the promoter of the present invention is a promoter consisting of the sequence set forth in SEQ ID NO:2, in which the nucleotides at positions 469 to 571 of the nucleotide sequence set forth in SEQ ID NO:1 have been deleted.

A specific example of the promoter of the present invention is a promoter consisting of the sequence set forth in SEQ ID NO:3, in which the nucleotides at positions 799 to 1563 of the nucleotide sequence set forth in SEQ ID NO:1 have been deleted.

A specific example of the promoter of the present invention is a promoter consisting of the sequence set forth in SEQ ID NO:4, in which the nucleotides at positions 469 to 571 and 799 to 1563 of the nucleotide sequence set forth in SEQ ID NO:1 have been deleted.

SEQ ID NO:2 and SEQ ID NO:3 were prepared by removing a portion of the CAG promoter sequence from a circular DNA (plasmid) in which the CAG promoter of SEQ ID NO:1 was cloned. SEQ ID NO:4 of the present invention was prepared by artificial synthesis. Details of the preparation of the promoter of the present invention will be described later.

The promoter of the present invention can be easily amplified by PCR because the DNA polynucleotide fragment lacks the GC-rich region 469-571. In contrast, the wild-type CAG promoter contains a GC-rich region and cannot be amplified by PCR as is.

The promoter of the present invention can be cloned as a polynucleotide fragment or inserted into a circular or linear plasmid and maintained and stored as is. Alternatively, the promoter of the present invention can be inserted into an appropriate vector for cloning, and the vector can be maintained and stored in a state where it is incorporated into a microorganism such as E. coli.

To produce a recombinant protein using the promoter of the present invention, it is preferable to use an expression vector containing the promoter of the present invention and a polynucleotide encoding the recombinant protein. When producing a recombinant antibody protein using a mammalian cell as a host, a plasmid vector is generally used as the expression vector.

However, the vector to which the promoter of the present invention can be applied may be any vector that can express a target protein in animal cells, cell-free protein translation systems, etc., and there are no particular limitations on the type.

The expression vector may further include components that allow stable protein expression when incorporated into mammalian cells. The expression vector preferably has an appropriate restriction enzyme site that allows incorporation of a gene encoding a target protein downstream of the promoter of the present invention. The expression vector preferably also includes a marker gene for selecting or visualizing cells that have been transformed by incorporating the protein expression vector, and another promoter that expresses the marker gene is located upstream of the marker gene. The other promoter is generally a promoter other than the promoter of the present invention. The expression vector may further include components that allow the vector to be maintained in E. coli in order to facilitate cloning in E. coli.

There are no particular limitations on the recombinant proteins that can be expressed using the promoter of the present invention. Examples of pharmaceutical proteins include antibody proteins (immunoglobulins), various enzymes, and human-derived receptor proteins.

In one embodiment, the promoter of the present invention is used in the production of antibody pharmaceuticals. In the process of producing antibody pharmaceuticals using gene recombination technology, a gene expression construct (expression vector) is constructed by inserting the gene of the target antibody into a vector suitable for recombinant protein expression. The expression vector is then introduced (transfected) into a host cell to obtain an antibody-producing cell.

An antibody gene may contain a nucleic acid encoding an amino acid sequence including VL (e.g., a light chain amino acid sequence) and an amino acid sequence including VH (e.g., a heavy chain amino acid sequence). When expressing a recombinant antibody protein, the nucleic acid encoding the amino acid sequence including VL and the nucleic acid encoding the amino acid sequence including VH are each placed downstream of the promoter of the present invention. The nucleic acid encoding the amino acid sequence including VL and the nucleic acid encoding the amino acid sequence including VH may be included in the same expression vector. The nucleic acid encoding the amino acid sequence including VL and the nucleic acid encoding the amino acid sequence including VH may be included in separate expression vectors.

There are no particular limitations on the mammalian cells used to express recombinant proteins using the promoter of the present invention. Examples include Chinese hamster ovary (CHO) cells (K1 strain, DG44 strain, DXB11 strain), human embryonic kidney-derived cells (HEK cells), human leukemia cell-derived cells (HL-60 cells), human cervical cancer-derived cells (HeLa cells), human leukemia T cell-derived cells (Jurkat), African green monkey kidney cell-derived cells (COS cells), mouse myeloma cells such as Sp2/0 cells and NS0 cells, and induced pluripotent stem cells (iPSCs).

Recombinant proteins produced using CHO cells have been confirmed to be safe for use as pharmaceuticals, and are currently a common technique. Therefore, one embodiment of using the promoter of the present invention to produce a protein as an active ingredient of a pharmaceutical is a recombinant protein expression system using CHO cells. The promoter of the present invention can be suitably used to construct a CHO cell line that highly expresses an antibody.

Means of delivering genes into cells are well known in the art and may be appropriately selected according to the cells used as hosts. A commercially available gene transfer system may be used to transform cells with an expression vector containing the promoter of the present invention. Methods for introducing an expression vector into mammalian cells include methods using transfection reagents such as electroporation and lipofection, and methods using viral vectors. Methods for inserting a foreign gene into the host genome of a mammalian cell include random integration, targeted integration (site-specific gene insertion using recombinase, a sequence-specific recombination enzyme), transposon vectors, and methods using site-specific nucleases. Site-specific gene transfer methods into specific positions in the host genome are expected to be a method for efficiently obtaining cells with excellent production of the target protein and excellent passage stability.

When developing a recombinant protein into a pharmaceutical product, the following steps are carried out: microorganisms or cells into which the gene for the desired protein has been introduced are cultured, and the desired protein is then extracted, purified, concentrated, and formulated.

　The recombinant protein can be produced with high efficiency by transforming mammalian cells with an expression vector containing the promoter of the present invention and the gene for the target protein to obtain a transformant (transformed cell) capable of producing the recombinant protein, culturing the transformant, and recovering the recombinant protein produced from the culture.

When recovering a recombinant protein from a culture of a transformant, a purification procedure using chromatography such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, etc. may be combined, and the recombinant protein can be recovered with high efficiency and high purity by such procedures.

In an additional aspect of the present invention, a purified or isolated polynucleotide fragment (DNA) having promoter function is proposed, the polynucleotide fragment (DNA) having a sequence having at least 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%, 97%, 98%, 99% or more identity to the nucleotide sequence set forth in SEQ ID NO:4. Here, when the expression induced by a promoter consisting of these DNAs (i.e., having 70% or more identity to the nucleotide sequence described in SEQ ID NO: 4) shows an expression level equal to or higher than that induced by a wild-type CAG promoter consisting of the DNA described in SEQ ID NO: 1, specifically, when the expression shows 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more, the promoter is said to have a "promoter function". The expression efficiency referred to here refers to the expression efficiency when the promoter and the promoter described in SEQ ID NO: 1 are each bound to substantially the same reporter gene, transfected into substantially the same cell line, and cultured under the same conditions. Typically, it refers to the expression efficiency when cultured under optimal conditions for expressing the reporter gene by the promoter.

The present invention will be specifically explained below using examples. Note that these examples are for the purpose of explaining the present invention and do not limit the scope of the present invention.

[Construction of the promoter of the present invention]
A circular DNA containing the sequence of the promoter of the present invention shown in SEQ ID NO: 2 is shown in Figure 1. A circular DNA containing the promoter of the present invention was prepared by inverse PCR using primer CAGDel3F (AGCCAATCAGAGCGGCGC: SEQ ID NO: 5) and primer CAGDel3R2 (ATTAAAAAATAAATAAATACAAAATTGGGGGTGG: SEQ ID NO: 6) to delete 103 base pairs from positions 469 to 571, and ligating the resulting fragment using T4 ligase.

　A circular DNA containing the promoter sequence of the present invention shown in SEQ ID NO:3 is shown in Figure 2. The 765 base pairs from 799 to 1563 were removed using the restriction enzymes AfeI and SacII, and ligated using T4 ligase to produce a circular DNA containing the promoter of the present invention.

The promoter of the present invention shown in SEQ ID NO:4 was produced by artificially synthesizing a DNA fragment consisting of a sequence in which base pairs 469 to 571 and 799 to 1563 were deleted using the method described above.

Example 1: Luciferase Expression
The luciferase reporter gene was introduced downstream of a promoter lacking nucleotides 469-571 of SEQ ID NO:1 (i.e., SEQ ID NO:2), a promoter lacking nucleotides 799-1563 of SEQ ID NO:3, or a wild-type CAG promoter. The plasmid was transfected into CHO cells using a site-specific gene insertion method (Targeted integration system) using recombinase, and stable expressing cell pools were selected, and luciferase expression was evaluated in fed-batch culture for 14 days (n=3). Luciferase expression was measured on days 3, 5, 7, 10, 12, and 14 and normalized to luciferase expressed under the control of wild-type CAG.

The results are shown in Figure 3. The promoter of sequence number 2 did not affect reporter gene expression compared to wild-type CAG. On the other hand, the promoter of sequence number 3 induced reporter gene expression that was approximately 40% higher from 7 to 14 days compared to wild-type CAG.

Example 2: PCR amplification of deleted promoter fragments
The promoter of SEQ ID NO:4 was amplified by PCR using two different polymerases and primer sets.

The results are shown in Figure 4. When the promoter of sequence number 4 was used as a template, optimization of the PCR reaction was not required, and the desired PCR product (approximately 860 bp) was obtained under all conditions. On the other hand, wild-type CAG did not amplify the desired product (approximately 1730 bp) under any conditions. These results confirmed that the promoter of sequence number 4 can be amplified by PCR.

Example 3: Expression of STA551
STA551 is a switch antibody that uses Switch-Ig (registered trademark) developed by Chugai Pharmaceutical Co., Ltd. It is activated by recognizing adenosine triphosphate (ATP), which is believed to be present in high concentrations in tumor tissues, as a switch molecule, and binds to the target antigen, CD137.

We created a plasmid containing four copies of the L chain and two copies of the H chain of STA551 using sequence number 4 or wild-type CAG as the promoter.

The wild-type CAG plasmid was prepared in two steps. First, to introduce ligation/homology sites, each of the L/H genes was subcloned into an intermediate plasmid containing wild-type CAG and the appropriate homology sites for ligation using a standard cloning method. Then, after amplifying and purifying each intermediate plasmid, all fragments were combined to construct a single expression plasmid using the homology/ligation method. This process requires two E. coli transformation steps and takes at least 5 days.

On the other hand, the plasmid containing the promoter of sequence number 4 was prepared in one step without the need for an intermediate plasmid, because the fragment containing sequence number 4 can be amplified by PCR. A DNA fragment containing the promoter of sequence number 4, the L/H gene, and a homology site for ligation was prepared by PCR using appropriate primers. The construction of the plasmid containing the promoter of sequence number 4 requires only one E. coli transformation step and can be constructed in just three days.

The plasmid was transfected into CHO cells using a site-specific gene insertion method (targeted integration system) using recombinase, and a stable expression cell pool was selected. STA551 expression was evaluated on days 10, 12, and 14 of a 14-day fed-batch culture (n=3). STA551 expression was comparable for the two promoters.

These results confirm that the use of the promoter of sequence number 4 facilitates the construction of complex plasmids and that the promoter of sequence number 4 can be used to express monoclonal antibodies at a level at least equivalent to that of wild-type CAG.

While the above describes a preferred embodiment of the present invention, it is recognized and understood that various modifications may be made thereto, and the appended claims are intended to cover all such modifications without departing from the spirit and scope of the present invention.

The promoter sequences described in this invention are shown.
Sequence number 1_CAG promoter
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTTAATTATTTTGTGCAG
CGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGC
GGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT
TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGG
CGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCC
CGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTC
CTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTG
AAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGT
GCGTGTGTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGC
GCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCG
GGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTG
TGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGC
ACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGC
GTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG
CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCG
GCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGG
CGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCA
CCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGG
AGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC
CGCGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTG
TGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGC
TCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAccggt

SEQ ID NO:2_469-571 (Del3)
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTATAATAGCCAATCAGAG
CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA
GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGC
CGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG
GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTC
TTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAG
CGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAGCGCCGTGTGCGGCTCCGCGCT
GCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCG
CGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGGGGAACAA
AGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGG
GCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGC
GGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGT
GGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGG
CGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATG
GTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATC
TGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAG
GAAGGAAATGGGCGGGGAGGGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCT
CCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGG
GTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTT
CTTCTTTTTCCTACAGCTCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGG
CAAAccggt

SEQ ID NO:3_799-1563 (Del12)
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTTAATTATTTTGTGCAG
CGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGC
GGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGT
TTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGG
CGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCC
CGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTC
CTCCGGGCTGTAATTAGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGT
TCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCT
TCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCA
AAccggt

Sequence numbers 4_469-571,799-1563 (eCAPE)
GTCGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATA
GCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC
CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG
GGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTAC
ATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG
CCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACG
TATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCA
TCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTATAATAGCCAATCAGAG
CGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAA
GCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGC
CGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGG
GCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGGGGGGACGGCTGCCTTCGGGGGGG
ACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAA
CCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT
GTCTCATCATTTTGGCAAAccggt

Claims (18)

An isolated polynucleotide consisting of a sequence in which at least the region from base 469 to base 571 of the nucleotide sequence set forth in SEQ ID NO:1 is deleted, or consisting of a nucleotide sequence having 90% or more identity thereto. The polynucleotide according to claim 1, which consists of the nucleotide sequence set forth in SEQ ID NO:2 or a nucleotide sequence having 90% or more identity thereto. An isolated polynucleotide consisting of a sequence in which at least the region from base 799 to base 1563 of the nucleotide sequence set forth in SEQ ID NO:1 is deleted, or consisting of a nucleotide sequence having 90% or more identity thereto. The polynucleotide according to claim 3, which consists of the nucleotide sequence set forth in SEQ ID NO:3 or a nucleotide sequence having 90% or more identity thereto. An isolated polynucleotide consisting of a sequence in which at least the following regions (a) and (b) are deleted from the nucleotide sequence set forth in SEQ ID NO: 1, or consisting of a nucleotide sequence having 90% or more identity thereto:
(a) the region from base 469 to base 571; and (b) the region from base 799 to base 1563. The polynucleotide according to claim 5, which consists of the nucleotide sequence set forth in SEQ ID NO:4 or a nucleotide sequence having 90% or more identity thereto. A promoter comprising a polynucleotide according to any one of claims 1 to 6. A vector comprising the promoter according to claim 7. A vector comprising the promoter and a gene encoding the protein according to claim 7. A transformed cell obtained by transforming a mammalian cell with the vector according to claim 9. The transformed cell according to claim 10, wherein the mammalian cell is a CHO cell. A method for producing a recombinant protein, comprising the steps of producing a recombinant protein by culturing the transformed cell according to claim 11, and recovering the recombinant protein produced from the resulting culture. The method according to claim 12, which is a method for producing an antibody, wherein the vector comprises a promoter consisting of a nucleotide sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a gene encoding the antibody, and the gene encodes an amino acid sequence including the VL or an amino acid sequence including the VH of the antibody, or the gene encodes an amino acid sequence including the VL and an amino acid sequence including the VH of the antibody. An isolated polynucleotide fragment having promoter function, the polynucleotide fragment having a sequence having at least 70%, at least 80%, at least 90% or 100% identity to any of the sequences set forth in SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The vector according to claim 9, wherein the protein is an antibody heavy chain (H chain) or light chain (L chain). A cell containing a gene incorporating a promoter consisting of a nucleotide sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. The cell of claim 16, wherein the gene encodes an antibody polypeptide. A method for producing an antibody using cells containing a gene incorporating a promoter consisting of a nucleotide sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.