# Patent

## Title
Expression vector for animal cells including CSP-B 5′-SAR factor and method for producing recombinant proteins using same

## Abstract

The present invention relates to an expression vector for animal cells, comprising: (a) CSP-B (Cytotoxic Serine Protease-B) 5′-SAR (Scaffold or Matrix Attachment Region); (b) a promoter operable in animal cells; and (c) a polyadenylation sequence, and to a method for producing recombinant proteins using same. The vector of the present invention includes CSP-B 5′-SAR, and thus has the effect of overcoming the inhibition of gene expression according to the position of a foreign gene introduced into an animal cell, and significantly improving the expression rate of a target protein. The vector of the present invention effectively expresses recombinant proteins for drugs or antibodies in animal cells. The vector of the present invention and the method for producing recombinant proteins using same may be very usefully applied to the industrial mass production of drugs.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the U.S. national stage filing under 35 U.S.C. §371 of International Application PCT/KR2012/003995, filed May 21, 2012, which claims priority from Korean Patent Application 10-2011-0056685, filed Jun. 13, 2011.
TECHNICAL FIELD
The present invention relates to an expression vector for animal cells, including a 5′-scaffold or matrix attachment region (5′-SAR) of cytotoxic serine protease-B (CSP-B), and methods for producing recombinant proteins using the expression vectors.
BACKGROUND ART
The introduction of foreign genes coding recombinant proteins into animal cells such as Chinese hamster ovary (CHO) cells can produce protein drugs for clinical treatment.
A novel erythropoiesis stimulating protein (NESP), which is an erythropoiesis-stimulating factor, is also called Darbepoetin alfa, and is a protein drug obtained by adding two N-linked sugar chains to naturally occurring erythropoietin through genetic modification (Egrie and Browne, Br. J. Cancer, 84 Suppl. 1:3-10 (2001)). NESP promotes red blood cell production by stimulating hematopoietic stem cells and facilitating their differentiation into erythrocytes. Since the serum half-life of NESP is three times longer than that of the existing recombinant erythropoietin, equivalent therapeutic effect can be expected with fewer administrations when treating anemia of patients with chronic kidney disease.
Trastuzumab as an anti-malignant tumor agent is a humanized monoclonal antibody prepared using recombinant DNA technology, and selectively acts on human epidermal growth factor receptor 2 (HER2) on cell surfaces. HER2 overexpression is confirmed in 25 ̃30% of primary breast cancers and trastuzumab suppresses the proliferation of HER2-overexpressed human tumor cells.
Expression levels of foreign genes may be varied depending on their insertion sites in the animal cell chromosomes since foreign gene expression is influenced by surrounding regulator elements or the chromatin structure (Zahn-Zabal et al., J. Biotechnol, 87, 29-42 (2001)).
When chromatin factors that prevent surrounding chromatins from influencing foreign gene expression are used, the suppression of gene expression depending on the position (position effect) can be overcome and the possibility of isolation of animal cell clones that highly express recombinant proteins can be increased, thereby decreasing the preparation time of cell lines for producing drugs. Attempts to produce stable cell lines by using chromatin factors that can overcome the suppression of gene expression depending on the position have been made. The factors therefor are boundary element (BE), scaffold or matrix attachment region (SAR/MAR), locus control region (LCR), and the like.
SAR also called MAR is 300 ̃3000-bp DNA element. It has been known that SAR allows the chromatin to attach to proteins of the nuclear matrix and controls gene expression (Makrides (Ed.), Gene Transfer and Expression in Mammalian Cells. Elsevier. Chapter 10 (2003)). In addition, SAR can improve expression of foreign genes in transfected cell lines (Poljak et al., Nucleic Acids Res, 22, 4386-4394 (1994); Kalos and Fournier, Mol. Cell. Biol, 15, 198-207 (2005)).
Zahn-Zabal et al. had produced stable cells by introducing a chicken lysozyme 5′-MAR into a luciferase expression vector and transfecting CHO cells with the vector (Zahn-Zabal et al., J. Biotechnol, 87, 29-42 (2001)). The stable cells transfected with a vector containing lysozyme 5′-MAR exhibited a higher luciferase expression level as compared with stable cells transfected with a vector not containing MAR. In addition, the vector containing two copies of MAR exhibited a higher luciferase expression level than the vector containing one copy of lysozyme 5′-MAR. This result shows that the introduction of MAR into the animal cell expression vector can increase the target protein expression level.
Korean Patent Application No. 2000-0043996 discloses that the animal cell expression vector includes human β-globin MAR so that gene expression suppression occurring when foreign genes are introduced into animal cells can be overcome. This patent describes that, besides the human β-globin MAR, human interferon-β MAR (GenBank Accession #M83137) and CSP-B 3′-SAR (GenBank Accession #M62716) are used, and these three SARs/MARs can improve the expression level of β-galactosidase, which is a target protein. Further, this patent discloses that the β-globin MAR had excellent effects as compared with the interferon-β MAR or the CSP-B 3′-SAR.
Korean Patent Application No. 2001-0079227 discloses that the introduction of human interferon-β MAR into the animal cell expression vector can improve foreign gene expression and lead to efficient expression of recombinant proteins. In addition, Korean Patent Application No. 2007-0108451 discloses that the animal cell expression vector including two copies of human β-globin MAR can further improve the target protein expression level as compared with the vector including one copy of human β-globin MAR. This patent describes comparison results between one copy and two copies of MAR factors, but not comparison results between two copies and three copies of MAR factors.
Hanson and Ley found CSP-B 5′-SAR and CSP-B 3′-SAR from approximately 70 kb of the human chromosome 14q11.2 hematopoietic serine protease gene cluster and obtained their nucleotide sequences, and verified that these SARs bind to scaffolds derived from the cellular nucleus through in vitro experiment (Hanson and Ley, Blood, 79, 610-618 (1992)).
The CSP-B 5′-SAR and CSP-B 3′-SAR are registered as GenBank Accession #M62717 and GenBank Accession #M62716, respectively. They are 2429-bp long and 1233-bp long, respectively, and thus the CSP-B 5′-SAR is two folds longer than the 3′-SAR. Human chromosome 14 DNA sequence is registered as GenBank Accession #AL136018. In this sequence, the length between a transcription initiation codon for a gene coding the CSP-B protein and the 5′-SAR located upstream thereof is 1195 bp, and the length between a transcription termination codon of the gene for coding the CSP-B protein and the 3′-SAR located downstream thereof is 4543 bp.
Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosures of cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls and details of the present invention are explained more clearly.
DETAILED DESCRIPTION OF THE INVENTION
Technical Problem
The present inventors have made efforts to develop vectors capable of efficiently producing recombinant proteins in animal cells. As a result, the present inventors have established that recombinant proteins can be produced at excellent production efficiency when cytotoxic serine protease-B (CSP-B) 5′-scaffold or matrix attachment region (SAR) is incorporated in the vector, and then completed the present invention.
Therefore, an aspect of the present invention is to provide expression vectors for animal cells.
Another aspect of the present invention is to provide animal cells transfected with the expression vectors.
Still another aspect of the present invention is to provide a method for producing recombinant proteins.
Other purposes and advantages of the present invention will become clarified by the following detailed description of invention, claims, and drawings.
Technical Solution
In accordance with an aspect of the present invention, there is provided an expression vector for animal cells, the expression vector including: (a) a 5′-scaffold or matrix attachment region (5′-SAR) of cytotoxic serine protease-B (CSP-B); (b) a promoter operable in animal cells; and (c) a polyadenylation sequence.
The present inventors have made efforts to develop vectors capable of efficiently producing recombinant proteins in animal cells. As a result, the present inventors have established that recombinant proteins can be produced at excellent production efficiency when the 5′-scaffold or matrix attachment region (5′-SAR) of cytotoxic serine protease-B (CSP-B) is incorporated in the vector.
The expression vector for animal cells of the present invention includes CSP-B 5′-SAR; a promoter operable in animal cells; and a polyadenylation sequence.
The present invention is mainly characterized by using a vector including CSP-B 5′-SAR. CSP-B 5′-SAR used herein preferably includes a nucleotide sequence registered as GenBank accession #M62717, and its exemplary nucleotide sequence is described in SEQ ID NO: 1.
CSP-B 5′-SAR significantly increases the efficiency in production of recombinant proteins in the expression vector.
CSP-B 5′-SAR may be located upstream or downstream of the nucleotide sequence coding a protein to be expressed, and is preferably located downstream of the nucleotide sequence.
According to a preferable embodiment of the present invention, two copies of the 5′-SAR are continuously present on the vector.
According to a preferable embodiment of the present invention, the two copies of the 5′-SAR are continuously present downstream of a protein-coding nucleotide sequence to be expressed.
As proved in the following examples, CSP-B 5′-SAR further improves the target protein expression level in animal cells as compared with CSP-B 3′-SAR and β-globin MAR of the related art. For example, when one copy of CSP-B 5′-SAR, CSP-B 3′-SAR, and β-globin MAR were respectively introduced into vectors expressing β galactosidase as a target protein in CHO animal cells, the β galactosidase activities for the respective cases were measured to be 10.0-fold, 2.2-fold, and 8.7-fold higher as compared with when the SAR factor was not introduced (FIG. 7). That is, CSP-B 5′-SAR exhibits a relatively higher target protein expression level than other factors.
Further, similar results were obtained when effects of the SAR factor were verified by using NESP as a target protein. When CSP-B 5′-SAR and CSP-B 3′-SAR were introduced into vectors and then CHO cells were transfected with the vectors, the NESP expression levels for the respective cases were measured to be 11.2-fold and 3.2-fold higher as compared with when the SAR factor was not introduced (Table 3 and FIG. 9). The introduction of CSP-B 5′-SAR into vectors increased the frequency of formation of positive clones expressing NESP in the transfected cells, and also increased the NESP expression levels of randomly selected clones (Tables 4 and 5 and FIGS. 10 and 11).
Meanwhile, the effects of CSP-B 5′-SAR were verified by using an anti-HER2 antibody as a target protein. When two copies of CSP-B 5′-SAR were introduced into the vector and then CHO cells were transfected with the vector, the anti-HER2 antibody expression level was measured to be relatively higher than as compared with when the SAR factor was not introduced (Table 6 and FIG. 13). The introduction of SAR into vectors increased the anti-HER2 antibody expression level of randomly selected clones (FIGS. 14 and 15).
According to a preferable embodiment of the present invention, multiple copies of CSP-B 5′-SAR may be present in the expression vector. The production efficiency of recombinant proteins are further increased when multiple copies of CSP-B 5′-SAR are present in the expression vector than when one copy of CSP-B 5′-SAR is present in the expression vector. More preferably, two copies of CSP-B 5′-SAR may be present in the expression vector. As proved in the following examples, the production efficiency of recombinant proteins was more excellent when two copies of CSP-B 5′-SAR are present in the expression vector than when three copies of CSP-B 5′-SAR are present in the expression vector.
As proved in the following examples, as a result of verifying effects of CSP-B 5′-SAR according to the copy number of CSP-B 5′-SAR by introducing one, two, and three copies of CSP-B 5′-SAR into vectors, the continuous location of two copies of the SAR factor was measured to exhibit the highest β-galactosidase activity (FIG. 8). That is, the copy number of SAR factor in the vector influences the target protein expression level, and two copies of the SAR factor may be the most desirable.
In the case where multiple copies of CSP-B 5′-SAR are present in the expression vector, the multiple copies of CSP-B 5′-SAR may be located in a continuous or non-continuous manner. Preferably, the multiple copies of SAR factors may be continuously present, and more preferably, may be continuously present downstream of a nucleotide sequence coding a protein to be expressed.
According to a preferable embodiment of the present invention, the promoter operable in the animal cells may be a cytomegalovirus (CMV) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a Simian virus 40 (SV40) promoter, an SV40E1 promoter, a herpes simplex virus (HSV) tk promoter, a respiratory syncytial virus (RSV) promoter, an elongation factor 1-α (EF1-α) promoter, a metallothionein promoter, a β-actin promoter, a human interleukin-2 (IL-2) gene promoter, a human interferon (IFN) gene promoter, a human IL-4 gene promoter, a human lymphotoxin gene promoter, or human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter. The CMV promoter, SV40 promoter, SV40E1 promoter, EF1-α promoter, metallothionein promoter, or β-actin promoter is more preferable, and the CMV promoter is most preferable.
According to a preferable embodiment of the present invention, the vector of the present invention may be a nucleotide sequence coding a protein to be expressed. Preferable examples of the protein may include hormone, cytokine, antibody, peptide aptamer, Adnectin, affibody (U.S. Pat. No. 5,831,012), avimer ('Silverman, J. et al, Nature Biotechnology 23(12):1556 (2005)), or a Kunitz domain (Kunitz domain, Arnoux B et al., Acta Crystallogr. D Biol. Crystallogr. 58 (Pt 7):12524 (2002)), and (Nixon, A E, Current opinion in drug discovery & development 9(2):2618 (2006). More preferably, the protein to be expressed by using the vector of the present invention may be erythropoietin (EPO), EPO analogs, or anti-human epidermal growth factor receptor 2 (anti-HER2) antibodies. EPO, novel erythropoiesis stimulating proteins (NESPs; A30N, H32T, P87V, W88N, and P90T) as EPO analogs, or anti-HER2 antibodies may be most preferable.
The EPO analogs include mutants having modification of one or more amino acids from the human EPO amino acid sequence. The EPO analogs may be prepared by the mutagenesis through addition, deletion, or substitution of amino acid residues, whereby the glycosylation sites may be increased or may be changed. The EPO analogs have more carbohydrate chains than the human EPO, and include at least one additional glycosylation site. In addition, the EPO analogs exhibit a higher sialic acid level than the naturally occurring human EPO due to the additional glycosylation site. However, the protein secondary or tertiary structure, which is important in biological activity, is not changed by the additional glycosylation site. The EPO analogs may have one, two, or three additional sites for N-cosylation or O-glycosylation. For example, the leucine residue at position 69 is substituted with asparagine, which serves as a fourth site for N-glycosylation.
Examples of the EPO analogs to be expressed in the vectors of the present invention may be ones in which an additional site for glycosylation is introduced to at least one of positions 30, 51, 57, 69, 88, 89, 136, and 138 in the EPO sequence (e.g., sequence of GenBank Accession #M11319). More preferably, the EPO analogs may be NESPs including A30N, H32T, P87V, W88N, and P90T mutations.
According to a preferable embodiment of the present invention, the polyadenylation sequence includes a bovine growth hormone terminator, herpes simplex virus (HSV)-derived thymidine kinase (TK), or SV40-derived polyadenylation sequence.
A preferable construct of the vector of the present invention may be, in a 5′ to 3′ direction, a promoter operable in animal cells—an expression protein gene—a polyadenylation sequence—CSP-B 5′-SAR, and a more preferable construct is a promoter operable in animal cells—an expression protein gene—a polyadenylation sequence—CSP-B 5′-SAR—CSP-B 5′-SAR.
The vector of the present invention may further include a selection marker. Examples of the selection marker include resistance genes against antibiotics acting on eukaryotic cells, and preferably include resistance genes against neomycin, geneticin (G418), and kanamycin.
According to a preferable embodiment of the present invention, the vector of the present invention is used in expressing a target protein in animal cells. The animal cells include mammalian cells, rodent cells, avian cells, and insect cells. The animal cells more preferably include Chinese hamster ovary (CHO) cells, VERO cells, HeLa cells, W138 cells, baby hamster kidney (BHK) cells, COS cells, and Madin-Darby Canine Kidney (MDCK) cells. The CHO cells, VERO cells, HeLa cells, or MDCK cells are still more preferable, and the CHO cells are most preferable.
According to a preferable embodiment of the present invention, the vector of the present invention includes a dihydrofolate reductase (DHFR) coding sequence. The sequence is useful in vector amplification.
According to another aspect of the present invention, the present invention provides animal cells transfected with the foregoing expression vectors of the present invention.
According to still another aspect of the present invention, the present invention provides a method for producing recombinant proteins, the method including incubating the foregoing transfected animal cells of the present invention.
According to a preferable embodiment of the present invention, the method for producing recombinant proteins includes: (i) incubating the transfected animal cells of the present invention; and (ii) collecting recombinant proteins produced in the incubating.
In the related art, CSP-B 5′-SAR used herein was not used in an attempt to be introduced into expression vectors for animal cells to improve productivity in recombinant proteins for medical substances. As can be seen from the following examples, the introduction of CSP-B 5′-SAR into vectors can improve the target protein expression level in animal cells. By developing the method in which CSP-B 5′-SAR, a new factor that was not previously used, is introduced into vectors to produce recombinant proteins, the target protein expression efficiency can be further improved and the industrial productivity of protein medical substances can be increased.
According to reports in the related art, there were no attempts to introduce CSP-B 5′-SAR into expression vectors for animal cells to improve productivity of recombinant proteins for medical substances. Moreover, studies were not conducted that, in the case where one, two, and three copies of SAR factors are introduced in the vectors and then animal cells are transfected with the vectors, how many copies of SAR factors are included in the vector in order to achieve the highest productivity of recombinant proteins. Moreover, detailed preceding study results about the increase effect in the target gene expression level depending on the position of the SAR factor have not been disclosed.
When the expression vectors of the present invention are introduced into cells to produce transfected animal cells, the vectors may be introduced into cells by various methods. The vectors may be injected into animal cells by, for example, micro-injection (Capecchi, M. R., Cell, 22:479 (1980)), calcium phosphate precipitation (Graham, F. L. et al., Virology, 52:456 (1973)), electroporation (Neumann, E. et al., EMBO J., 1:841 (1982)), liposome-mediated transfection (Wong, T. K. et al., Gene, 10:87 (1980)), DEAE-dextran treatment (Gopal, Mol. Cell. Biol. 5:1188-1190 (1985)), gene bombardment (Yang et al., Proc. Natl. Acad. Sci., 87:9568-9572 (1990)), or the like.
The transfected animal cells may be incubated by various methods known in the art using protocols. As a medium usable herein, any medium that can be generally used to incubate animal cells may be employed. For example, Eagle's minimum essential medium (Eagles's MEM, Eagle, H. Science 130:432 (1959)), α-MEM (Stanner, C. P. et al., Nat. New Biol. 230:52 (1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519 (1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53:288 (1965)), F10 (Ham, R. G. Exp. Cell Res. 29:515 (1963)), Dulbecco's modification of Eagle's medium (DMEM, Dulbecco, R. et al., Virology 8:396 (1959)), a mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102:255 (1980)), Way-mouth's MB752/1 (Waymouth, C. J. Natl. Cancer Inst. 22:1003 (1959)), McCoy's 5A (McCoy, T. A., et al., Proc. Soc. Exp. Biol. Med. 100:115 (1959)), MCDB series (Ham, R. G. et al., In Vitro 14:11 (1978)), and the like may be used. Detailed descriptions of incubations and media of animal cells are disclosed in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R Liss, Inc., New York, which is incorporated by reference into the present specification.
Advantageous Effects
Features and advantages of the present invention are summarized as follows:
(a) The vectors of the present invention include CSP-B 5′-SAR, and can have effects of overcoming gene expression suppression depending on positions of foreign genes introduced into animal cells and significantly improve the target protein expression level.
(b) The vectors of the present invention can effectively express recombinant proteins for medical substances (e.g., EPO) or antibodies (e.g., anti-HER2 antibody) in animal cells.
(c) The vectors of the present invention and the method for producing recombinant proteins using the vectors can be very usefully employed in industrial mass production of medical substances.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 schematically shows a structure of a β-galactosidase expression vector. Here, lac Z represents a β-galactosidase gene; BGH pA represents a polyadenylation sequence of a bovine growth hormone gene; and TK pA represents a polyadenylation sequence of a thymidine kinase gene.
FIGS. 2a-2c schematically show three structures (a, b, and c) of β-galactosidase expression vectors including one copy of SAR/MAR factor.
FIGS. 3a-3d schematically show four structures (a, b, c, and d) of β-galactosidase expression vectors including two copies of SAR factors.
FIG. 4 schematically shows a structure of a β-galactosidase expression vector including three copies of CSP-B 5′-SAR.
FIGS. 5a-5b schematically show two (a and b) structures of NESP expression vectors.
FIGS. 6a-6b schematically show two (a and b) structures of NESP expression vectors including SAR/MAR factors.
FIG. 7 is a graph showing the β-galactosidase activity (relative) of cells transfected with each of vectors including one copy of SAR/MAR factor introduced thereinto.
FIG. 8 is a graph showing the β-galactosidase activity (relative) of cells transfected with each of vectors including one copy, two copies, and three copies of SAR factors introduced thereinto.
FIG. 9 is a graph showing the NESP (erythropoiesis-stimulating factor) expression level (relative) of cells transfected with each of vectors including SAR factors introduced thereinto.
FIG. 10 is a graph showing NESP expression levels of 24 clones randomly selected from cells transfected with the SAR factor-not-introduced pCLS07A1 vector.
FIG. 11 is a graph showing NESP expression levels of 24 clones randomly selected from cells transfected with the pCLS10A1f1 vector including CSP-B 5′-SAR introduced thereinto.
FIGS. 12a-12b schematically show two (a and b) structures of anti-HER2 antibody expression vectors.
FIG. 13 is a graph showing the anti-HER2 antibody expression level (relative) of cells transfected with the vector including two copies of SAR factors introduced thereinto.
FIG. 14 is a graph showing anti-HER2 antibody expression levels of 19 clones randomly selected from cells transfected with the SAR factor-not-introduced pCLS05H2 vector.
FIG. 15 is a graph showing anti-HER2 antibody expression levels of 19 clones randomly selected from cells transfected with the pCLS05H2f2 vector including two copies of CSP-B 5′-SAR introduced thereinto.



MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.
EXAMPLES 
Example 1
Preparation of SAR/MAR Factor
Preparation of Human CSP-B 5′-SAR and CSP-B 3′-SAR
For polymerase chain reaction (PCR) amplification of human CSP-B 5′-SAR DNA registered as GenBank Accession #M62717 and #AL136018, genomic DNA of human natural killer cells (NK cells, ATCC CRL-2407) was prepared. The genomic DNA was prepared from the NK cells using a DNA isolation kit (DNEASY® Blood & Tissue Kit, Qiagen, Cat. No. 69504), and was used as a template for 5′-SAR DNA PCR.
PCR was performed using the Cs5S300F primer (attct tcagc acctc cttaa ttttt ctccc; SEQ ID NO: 5) and the Cs5S300R primer (ccagg cagcc aaaga tcagt agttg tgttg; SEQ ID NO: 6) while using the genomic DNA of the NK cells as a template. The PCR was performed under the conditions of 35 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C.
Then, the PCR product was cloned into the PGEM-T® vector (Promega, Cat. No. A3600) to prepare the PGEMT®-CS5S.3.0K vector.
For preparation of human CSP-B 3′-SAR DNA registered as Gen Bank Accession #M62716 and #AL136018, PCR was performed using the 2kCspSF primer (tggtt ccttc attgg aaaag gaaaa cac; SEQ ID NO: 7) and the 2kCspSR primer (tccgc tgagg ctgtg cccac agcca cc; SEQ ID NO: 8) while using the genomic DNA of the NK cells as a template. Then, PCR was performed using the CspSF primer (ggatc ccatt ctcct tgatg tacta at; SEQ ID NO: 9) and the CspSR primer (gaatt caaac aactc aatag caaga aac; SEQ ID NO: 10) while using the primary PCR product as a template. The PCRs were performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 5 minutes at 72° C. The secondary PCR product was cloned into the PGEM-T® vector to prepare the PGEMT®-CS3S.1.2K vector.
Preparation of Human β-Globin MAR
For PCR amplification of human β-globin MAR DNA registered as GenBank Accession #L22754 and #NW—001838021, genomic DNA of human G-2 cells was prepared. The genomic DNA was prepared from the G-2 cells using a DNA isolation kit, and was used as a template for 5′ MAR DNA PCR. PCR was performed using the Bg5MF-100E-Nhel primer (aattg ctagc ttgta ttctg tttcg tgagg caagg ttt; SEQ ID NO: 11) and the Bg5MR-100R-Xhol primer (aattc tcgag ttcct ctcta tgttg gctca aatgt cct; SEQ ID NO: 12) while using the genomic DNA of the G-2 cells as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 3 minutes and 30 seconds at 72° C. Then, the PCR product was cloned into the PCDNATM3.3TOPO® vector (Invitrogen, Cat. No. K8300-01) to prepare the PCDNATM3.3-BG5M vector.
Example 2
Preparation of β-Galactosidase Expression Vector
Preparation of pC06
For preparation of the pC06 vector sequentially including the cytomegalovirus (CMV) promoter, multiple cloning sites (MCS), and the bovine growth hormone (BGH) polyadenylation sequence (pA), the following experiment was conducted. First, PCR was performed using the V6_F primer (aagct tggat ccgaa ttcat cgatg gccgg ccggt accct cgagc tgtgc cttct agttg ccagc; SEQ ID NO: 13) and the V6_R primer (gctag ctaga gcccc agctg gttct ttccg; SEQ ID NO: 14) while using the pcDNA3.1(−) vector (Invitrogen, Cat. No. V795-20) as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Then, the PCR product was cloned into the PCDNATM3.3TOPO® vector (Invitrogen) to prepare the pC06 vector.
Preparation of pC04′
For preparation of the pC04′ sequentially including the Simian virus 40 (SV40) promoter, the Kozak sequence (gccatc), the dihydrofolate reductase (dhfr) gene, the herpes simplex virus thymidine kinase (HSV-TK) pA, the following experiment was conducted. First, PCR was performed using the PsvApaDraF primer (aaaat tgggc cccac tacgt gctgt ggaat gtgtg tcagt taggg t; SEQ ID NO: 15) and the PsvKzDHR primer (tggtc gaacc atgat ggcgc gaaac gatcc tcatc ctgtc tct; SEQ ID NO: 16) while using the PCDNATM3.3 vector as a template. Then, PCR was performed using the DHKzPsvF primer (ggatc gtttc gcgcc atcat ggttc gacca ttgaa ctgca tcg; SEQ ID NO: 17) and the TKNheNdeR primer (tgtgt gcata tggct agcga taaca atttc acaca ggaaa cag; SEQ ID NO: 18) while using the POPTIVECTM-TOPO® vector as a template. The PCRs were performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Then, these two PCR products were ligated through overlapping PCR, and cloned between the Apal and Ndel restriction sites of the PGEM-T® vector, to prepare the pC04′ vector.
Preparation of pC04
For preparation of the pC04 vector in which one 72-bp enhancer (ggtgt ggaaa gtccc caggc tcccc agcag gcaga agtat gcaaa gcatg catct caatt agtca gcaac ca; SEQ ID NO: 19) was removed from the SV40 promoter included in the pC04′ vector (SV40dEI promoter), the following experiment was conducted. First, the pC04′ vector was digested with the restriction enzyme Sphl, and then the thus obtained large DNA fragment was self-ligated, to prepare the pC04 vector.
Preparation of pCLS07 Vector
For preparation of the pCLS07 vector including the SV40dE1 promoter, the Kozak sequence, the dhfr gene, and TK pA, which were inserted between the BGH pA and the SV40 promoter of the pC06 vector, the following experiment was conducted. First, PCR was performed using the PsvApaDraF primer and the TKNheNdeR primer while using the pC04 vector as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute and 20 seconds at 72° C. Then, this PCR product and the pC06 vector were digested with the restriction enzymes Dralll and Nhel, respectively, and the thus obtained two large DNA fragments are ligated to prepare the pCLS07 vector.
Preparation of pCLS08 Vector
For preparation of the pCLS08 vector by inserting MCS into the Nhel restriction site of the pCLS07 vector, the following experiment was conducted. First, PCR was conducted using the NhAsE1F primer (aattg ctagc atata gcgat cgcgc aggac agctt ccgac agcag ggcca gg; SEQ ID NO: 20) and the BbPsSbNhE1R primer (aattg ctagc atata cctgc aggta tatgg gccca tatag ctgag gttga atgag aatat cactg tccca gacac; SEQ ID NO: 21) while using the erythropoietin gene cloned into the PGEM-T® vector as a template, to prepare MCS. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 15 seconds at 72° C. Then, this PCR product was cloned into the Nhel restriction site of the pCLS07 vector to prepare the pCLS08 vector.
Preparation of pCLS08G1 Vector
For preparation of the pCLS08G1 vector by inserting the lac Z and lac Y genes between the Hindlll and Xhol restriction sites of the pCLS08 vector, the following experiment was conducted. First, PCR was performed using the galHinF primer (aatta agctt ctcgc gcaac ctatt ttccc ctcga ac; SEQ ID NO: 22) and the galXhoR primer (aattc tcgag ccgag tttgt cagaa agcag accaa ac; SEQ ID NO: 23) while using the pSV-β-galactosidase vector (Promega, Cat. No. TB094) as a template, to amplify the lac Z and lac Y genes. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 3 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between Hindlll and Xhol restriction sites of the pCLS08 vector to prepare the pCLS08G1 vector.
Preparation of pCLS09G1 Vector
For preparation of the pCLS09G1 vector by exchanging the MCS between two Nhel restriction sites of the pCLS08G1 vector with another MCS, the following experiment was conducted. First, PCR was performed using the NhSbE1F primer (aattg ctagc atata cctgc aggtt gaatg agaat atcac tgtcc cagac a; SEQ ID NO: 24) and the NhAsSfPsE1R primer (aattg ctagc atata gcgat cgcta tatgg ccatg atggc catat agggc ccagg acagc ttccg acagc agggc caggc; SEQ ID NO: 25) while using the erythropoietin gene cloned into the PGEM-T® vector as a template, to prepare new MCS. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 15 seconds at 72° C. Then, the existing MCS between the two Nhel restriction sites of the pCLS80G1 vector was removed, and the new MCS generated through the PCR was cloned thereinto, to prepare pCLS09GI vector. The map of the pCLS09G1 vector was shown in FIG. 1.
Example 3
Preparation of β-Galactosidase Expression  Vector Including SAR/MAR Factor
Preparation of pCLS09G1t1 Vector
For preparation of the pCLS09G1t1 vector by inserting the CSP-B 3′-SAR between the Sbfl and PspOMI restriction sites of the pCLS09G1 vector, the following experiment was conducted. First, PCR was conducted using the cs3sSbf1F primer (aattc ctgca gggga tccca ttctc cttga tgtac taat; SEQ ID NO: 26) and the cs3sPsp1R primer (aattg ggccc gaatt caaac aactc aatag caaga aac; SEQ ID NO: 27) while using the pGEMT-CS3S.1.2k vector as a template, to amplify CSP-B 3′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the SbfI and PspOMI restriction sites of the pCLS09G1 vector to prepare the pCLS09G1t1 vector.
Preparation of pCLS09G1f1 Vector
For preparation of the pCLS09G1f1 vector by inserting the CSP-B 5′-SAR between the Sbfl and PspOMI restriction sites of the pCLS09G1 vector, the following experiment was conducted. First, PCR was conducted using the cs5sSbf1F primer (aattc ctgca gggaa ttcct aaaca gagca attag gtaag; SEQ ID NO: 28) and the cs5sPsp1R primer (aattg ggccc gaatt ccagt gtaaa cgtct tcctt gt; SEQ ID NO: 29) while using the pGEMT-CS5S.3.0k vector as a template, to amplify CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the SbfI and PspOMI restriction sites of the pCLS09G1 vector to prepare the pCLS09G1f1 vector.
Preparation of pCLS09G1g1 Vector
For preparation of the pCLS09G1g1 vector by inserting the β-globin MAR between the Sbfl and PspOMI restriction sites of the pCLS09G1 vector, the following experiment was conducted. First, PCR was performed using the glmSbf1F primer (aattc ctgca ggttg tattc tgttt cgtga ggcaa ggttt; SEQ ID NO: 30) and the glmPsp1R primer (aattg ggccc ttcct ctcta tgttg gctca aatgt cct; SEQ ID NO: 31) while using the pcDNA3.3-Bg5M vector as a template, to amplify the β-globin MAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 3 minutes and 20 seconds at 72° C. Then, this PCR product was cloned between the SbfI and PspOMI restriction sites of the pCLS09G1 vector to prepare the pCLS09G1g1 vector.
Preparation of pCLS09G1t2 Vector
For preparation of the pCLS09G1t2 vector by inserting CSP-B 3′-SAR between the PspOMI and SfiI restriction sites of the pCLS09G1t1 vector, the following experiment was conducted. First, PCR was performed using the cs3sPsp2F primer (aattg ggccc ggatc ccatt ctcct tgatg tacta at; SEQ ID NO: 32) and the cs3sSfi2R primer (aattg gccat gatgg ccgaa ttcaa acaac tcaat agcaa gaaac; SEQ ID NO: 33) while using the pGEMT-CS3S.1.2k vector as a template, to amplify CSP-B 3′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the PspOMI and SfiI restriction sites of the pCLS09G1t1 vector to prepare the pCLS09G1t2 vector.
Preparation of pCLS09G1f2 Vector
For preparation of the pCLS09G1f2 vector by inserting CSP-B 5′-SAR between the PspOMI and SfiI restriction sites of the pCLS09G1f1 vector, the following experiment was conducted. First, PCR was performed using the cs5sPsp2F primer (aattg ggccc gaatt cctaa acaga gcaat taggt aag; SEQ ID NO: 34) and the cs5sSfi2R primer (aattg gccat gatgg ccgaa ttcca gtgta aacgt cttcc ttgt; SEQ ID NO: 35) while using the pGEMT-CS5S.3.0k vector as a template, to amplify CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the PspOMI and SfiI restriction sites of the pCLS09G1f1 vector to prepare the pCLS09G1f2 vector.
Preparation of pCLS09G1tf Vector
For preparation of the pCLS09G1tf vector by inserting CSP-B 5′-SAR between the PspOMI and SfiI restriction sites of the pCLS09G1t1 vector, the following experiment was conducted. First, PCR was performed using the cs5sPsp2F primer (aattg ggccc gaatt cctaa acaga gcaat taggt aag; SEQ ID NO: 34) and the cs5sSfi2R primer (aattg gccat gatgg ccgaa ttcca gtgta aacgt cttcc ttgt; SEQ ID NO: 35) while using the pGEMT-CS5S.3.0k vector as a template, to amplify CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the PspOMI and SfiI restriction sites of the pCLS09G1t1 vector to prepare the pCLS09G1tf vector.
Preparation of pCLS09G1f3 Vector
For preparation of the pCLS09G1f3 vector by inserting the CSP-B 5′-SAR into the BglII restriction site between the CMV promoter and the bla promoter of the pCLS09G1f2 vector, the following experiment was conducted. First, PCR was performed using the cs5sBgl3F primer (aatta gatct gaatt cctaa acaga gcaat taggt aag; SEQ ID NO: 36) and the cs5sBgl3R primer (aatta gatct gaatt ccagt gtaaa cgtct tcctt gt; SEQ ID NO: 37) while using the pGEMT-CS5S.3.0k vector as a template, to amplify CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned into the BglII restriction site of the pCLS09G1f2 vector to prepare the pCLS09G1f3 vector.
Preparation of pCLS09G1f4 Vector
For preparation of the pCLS09G1f4 vector, in which the SAR factors are located at both sides of the lac Z and lac Y genes, by inserting CSP-B 5′-SAR into the BglII restriction site between the CMV promoter and the bla promoter of the pCLS09G1f1 vector, the following experiment was conducted. First, PCR was performed using the cs5sBgl3F primer (aatta gatct gaatt cctaa acaga gcaat taggt aag; SEQ ID NO: 36) and the cs5sBgl3R primer (aatta gatct gaatt ccagt gtaaa cgtct tcctt gt; SEQ ID NO: 37) while using the pGEMT-CS5S.3.0k vector as a template, to amplify CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned into the BglII restriction site of the pCLS09G1f1 vector to prepare the pCLS09G1f4 vector.
Example 4
β-Galactosidase Expression Using SAR/MAR Factor-Introduced Vector
Transfection Using pCLS09G1, pCLS09G1f1, pCLS09G1t1, or pCLS09G1g1 Vector
In order to verify the effect due to the respective introduction of one copy of CSP-B 5′-SAR, CSP-B 3′-SAR, and β-globin MAR into the vectors, CHO DG44 cells (Invitrogen, Cat. No. 12609) were transfected as follows.
Adhesion culture type DG44 cells were dispensed in a 6-well plate at 4×105 cells per well. After 24 hours, it was confirmed that cells completely adhered to the plate bottom. 500 μl of the OPTI-MEM® medium (Gibco, Cat. No. 31985-070) was dispensed into each of three sterilized tubes, and then 2 μg of the prepared pCLS09G1, pCLS09G1f1, pCLS09G1t1, or pCLS09G1g1 vectors were mixed therewith, followed by mild pipetting. 500 μl of the OPTI-MEM® medium was dispensed into each of three sterilized tubes. 10 μl of LIPOFECTAMINE® 2000 (Invitrogen, Cat. No. 11668-027) was mixed therewith, followed by mild pipetting, and then left for 5 minutes at room temperature. DNA and a reagent solution were mixed, and then left at room temperature for 20 minutes. This mixture was put into wells of a 6-well plate prepared for each DNA, followed by well mixing.
Verification on β-Galactosidase Activity of Cell Pool Transfected with pCLS09G1, pCLS09G1f1, pCLS09G1t1, or pCLS09G1g1
DG44 cells were transfected with the pCLS09G1, pCLS09G1f1, pCLS09G1t1, or pCLS09G1g1 vector. After 24 hours, the medium was exchanged with a medium containing 10% FBS and 500 μg/ml of GENETICIN®. Medium exchange was performed every three or four days, and the cells were cultured under the conditions of 37° C. and 5% CO2 for three weeks, thereby preparing a stable cell pool.
A β-galactosidase analysis kit (Stratagen, Cat. No. 200383) was used to verify β-galactosidase activity on the stable cells transfected, and then obtain β-galactosidase activities according to vectors and relative β-galactosidase activities based on the pCLS09G1 vector. As shown in Table 1 and FIG. 7, the β-galactosidase activity was increased for the SAR/MAR factor-introduced vectors as compared with the SAR/MAR factor-not-introduced vector. In addition, the pCLS09G1f1 vector including CSP-B 5′-SAR exhibited higher β-galactosidase activity than pCLS09G1g1 including β-globin MAR. Therefore, it is preferable to introduce the CSP-B 5′-SAR into vectors to increase the target protein expression level.










TABLE 1







β-galactosidase
Relative β-


Vector used for
Introduced
activity
galactosidase


transfection
SAR/MAR
(units/mg)
activity


















pCLS09G1
None
3.4
1.0


pCLS09G1f1
CSP-B 5′-SAR
34.3
10.0


pCLS09G1t1
CSP-B 3′-SAR
7.4
2.2


pCLS09G1g1
P-globin MAR
29.8
8.7









Verification on β-Galactosidase Activity of Cell Pool Transfected with pCLS09G1f1, pCLS09G1f2, pCLS09G1f3, or pCLS09G1f4

The following experiment was conducted to verify that the CSP-B 5′-SAR increased the target protein expression levels and investigate the change in the target protein expression level depending on the copy number of SAR factors introduced into the vector. DG44 cells were transfected with the pCLS09G1f1, pCLS09G1f2, pCLS09G1f3, or pCLS09G1f4 vector and then selected with the Geneticin-containing medium, thereby obtaining cell pools. The β-galactosidase activity on the stable cells transfected was verified. The β-galactosidase activities depending on the vectors were obtained. As shown in Table 2 and FIG. 8, the vector including two copies of SAR factors introduced thereinto exhibited higher β-galactosidase activity than the vectors including one copy or three copies of SAR factors introduced thereinto. That is, the increase in the target protein expression levels due to the SAR factor is varied depends on the copy number of SAR factor, and it was verified that the introduction of two copies of SAR factor into vectors can lead to the maximum increase in the target protein expression level.









TABLE 2








β-galactosidase
Relative β-


Vector used
activity
galactosidase


for
(units/mg)
activity
















transfection
Introduced SAR
Set 1
Set 2
Set 3
Mean
Set 1
Set 2
Set 3
Mean



















pCLS09G1f1
One copy of
116
30
50
65
1.0
1.0
1.0
1.0



CSP-B 5′-SAR


pCLS09G1f2
Continuous two
147
103
107
119
1.3
3.4
2.1
2.3



copies of CSP-B



5′-SAR


pCLS09G1f3
Three copies of
12
36
19
22
0.1
1.2
0.4
0.6



CSP-B 5′-SAR


pCLS09G1f4
Non-continuous
112
112
66
97
1.0
3.7
1.3
2.0



two copies of



CSP-B 5′-SAR









Example 5
Preparation of NESP Expression Vector
Preparation of Erythropoietin Gene
For the cloning of the 582-bp erythropoietin gene registered as GenBank Accession #M11319 (human fetal liver genomic erythropoietin gene) and GenBank Accession #NM000799 (human erythropoietin mRNA), the following experiment was conducted. First, PCR was performed using the IE1 ATGF primer (atggg ggtgc acgaa tgtcc tgcct; SEQ ID NO: 38) and the 1E1TGAR primer (tcatc tgtcc cctgt cctgc aggcc t; SEQ ID NO: 39) while using the Human liver MARATHON®-Ready cDNA library (BD, Cat. No. 7407-1) as a template. The PCR was performed under the conditions of 40 cycles of 10 seconds at 98° C., 30 seconds at 58° C., and 1 minute at 72° C. Then, this PCR product was cloned into the PGEM-T® vector.
Preparation of NESP Gene
For preparation of genes of erythropoietin analogs, NESPs (A30N, H32T, P87V, W88N, and P90T), as described in claim 3 of Korean Patent Application No. 1995-0701453, the following experiment was conducted. First, for preparation of genes of erythropoietin analogs A30N and H32T, PCR was performed using the M13R primer (gaaac agcta tgacc atg; SEQ ID NO: 40) and the IA1g1R primer (ctcat tcaag ctgca tgttt catta cagcc cgtcg tgat; SEQ ID NO: 41) and PCR was performed using the M13F primer (caggg ttttc ccagt cacga; SEQ ID NO: 42) and the IA1g1F primer (atcac gacgg gctgt aatga aacat gcagc ttgaa tgag; SEQ ID NO: 43), while using the erythropoietin gene cloned into the pGEM-T vector as a template. The PCRs were performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 40 seconds at 72° C. Then, these two PCR products were ligated through overlapping PCR, and then cloned between the Ndel and Sphl restriction sites of the pGEM-T vector.
In order to mutate the genes of the analogs A30N and H32T into genes of the analogs A30N, H32T, P87V, W88N, and P90T and attach the Kozak sequence (gccacc) before the transcription initiation codon, PCR was performed using the M13R primer and the IA1G2R primer (cacat gcagc tgcag tgtct cattc acctg ggaag agttg ac; SEQ ID NO: 44) and PCR was performed using the M13F primer and the IA1G2F primer (gtcaa ctctt cccag gtgaa tgaga cactg cagct gcatg tg; SEQ ID NO: 45), while using the genes of the analogs, A30N and H32T, cloned into the pGEM-T vector as a template. The PCRs were performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 40 seconds at 72° C. Then, overlapping PCR was performed using the IE1KzATGHinF primer (aatta agctt gccac catgg gggtg cacga atgtc ctgcc t; SEQ ID NO: 46) and the IE1TGAXhoR primer (aattc tcgag tcatc tgtcc cctgt cctgc aggcc t; SEQ ID NO: 47) while using these two PCR products as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 50 seconds at 72° C.
Preparation of pCLS07A1 Vector
The PCR product of the NESP genes were cloned between the HindIII and XhoI restriction sites of the MSC of the pCLS07 vector, to prepare the pCLS07A1 vector.
Example 6
Preparation of NESP Expression Vector Including SAR Factor
Preparation of pCLS10A1 Vector
For preparation of the pCLS10A1 vector by inserting MCS for the SAR or MAR factor introduction into the Nhel restriction site of the pCLS07A1 vector capable of exhibiting NESP in animal cells, the following experiment was conducted. First, PCR was performed using the NhAsf1F primer (aattg ctagc atata ggcgc gccaa cttga ttagg gtgat ggttc acgta g; SEQ ID NO: 48) and the NhClPaPsf1R primer (aattg ctagc atata atcga ttata tttaa ttaaa tatag ggccc ttgag tgttg ttcca gtttg gaaca aga; SEQ ID NO: 49) while the pcDNA3.1(−) vector as a template, to obtain MCS. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 15 seconds at 72° C. This PCR product was cloned into the NheI restriction site of the pCLS07A1 vector to prepare the pCLS10A1.
Preparation of pCLS10A1t1 Vector
For preparation of the pCLS10A1t1 vector by inserting the CSP-B 3′-SAR between the Ascl and PspOMI restriction sites of the pCLS10A1 vector, the following experiment was conducted. First, PCR was performed using the cs3sAsc1F primer (aattg gcgcg ccgga tccca ttctc cttga tgtac taat; SEQ ID NO: 50) and the cs3sPsp1R primer (aattg ggccc gaatt caaac aactc aatag caaga aac; SEQ ID NO: 51) while using the PGEMT®-CS3S.1.2K vector as a template, to amplify CSP-B 3′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the Ascl and PspOMI restriction sites of the pCLS10A1 vector to prepare the pCLS10A1 t1 vector.
Preparation of pCLS10A1f1 Vector
For preparation of the pCLS10A1f1 vector by inserting the CSP-B 5′-SAR between the Ascl and PspOMI restriction sites of the pCLS10A1 vector, the following experiment was conducted. First, PCR was performed using the cs5sAsc1F primer (aattg gcgcg ccgaa ttcct aaaca gagca attag gtaag; SEQ ID NO: 52) and the cs5sPsp1R primer (aattg ggccc gaatt ccagt gtaaa cgtct tcctt gt; SEQ ID NO: 53) while using the PGEM-T®CS5S.3.0.K vector as a template, to amplify the CSP-B 5′-SAR. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 2 minutes and 30 seconds at 72° C. Then, this PCR product was cloned between the Ascl and PspOMI restriction sites of the pCLS10A1 vector to prepare the pCLS10A1f1 vector.
Example 7
NESP Expression Using SAR Factor-Introduced Vector
Verification on NESP Expression Level of DG44 Cell Pool Transfected with pCLS07A1, pCLS10A1f1, or pCLS10A1t1
In order to verify whether the effect of the SAR factor, which was confirmed through the β-galactosidase activity experiment, is also applied to expressions of other recombinant proteins, the following experiment was conducted. DG44 cells were transfected with the pCLS07A1, pCLS10A1f1, or pCLS10A1t1 vector including the target protein NESP and the SAR factor, and selected with the GENETICIN®-containing medium, thereby obtaining a cell pool. The NESP expression level was verified by enzyme-linked immunosorbent assay (ELISA). As shown in Table 3 and FIG. 9, it was verified that the introduction of the SAR factor increased the expression level. In particular, the NESP expression level was increased by 11.2 fold when CSP-B 5′-SAR was introduced downstream of the NESP gene than when CSP-B 5′-SAR was not introduced.










TABLE 3







NESP
Relative NESP


Vector used for
Introduced
expression level
expression


transfection
SAR/MAR
(μg/Ml)
level


















pCLS07A1
None
0.7
1.0


pCLS10A1f1
CSP-B 5′-SAR
7.9
11.2


pCLS10A1t1
CSP-B 3′-SAR
2.3
3.2









Verification on Frequency of Formation of Positive Clones Expressing NESP

In order to verify effects of the SAR factor of overcoming gene expression suppression depending on positions of foreign genes, DG44 cells were transfected with the pCLS07A1, pCLS10A1t1, or pCLS10A1f1 vector, and single colonies were created by using a 96-well plate. The colony formation frequency for each vector was confirmed through microscopic observation. The culture liquid was taken up and the expression level of NESP was measured, thereby obtaining the frequency of formation of positive clones expressing NESP for each vector. As shown in Tables 4 and 5, 82% of colony formation frequency was shown when the SAR factor was not introduced, and 90% and 50% of wells showed colony formation for the introductions of CSP-B 5′-SAR and CSP-B 3′-SAR, respectively. Further, the frequency of positive clones was approximately 40% for non-introduction of SAR factors and the introduction of CSP-B 3′-SAR, and increased to 59% for the introduction of CSP-B 5′-SAR. That is, it can be verified that the introduction of CSP-B 5′-SAR overcome gene expression suppression depending on positions of foreign genes and thus more clones expressing the target protein NESP were formed.











TABLE 4








Number of





Total
colony-
Colony


Vector used for
Introduced
number of
forming
formation


transfection
SAR/MAR
wells
wells
frequency (%)







pCLS07A1
None
240
197
82


pCLS10A1f1
CSP-B 5′-
240
217
90



SAR


pCLS10A1t1
CSP-B 3′-
240
119
50



SAR




















TABLE 5







Total
Number of
Frequency of


Vector used for
Introduced
number of
Positive
positive


transfection
SAR/MAR
colonies
clones
clones (%)



















pCLS07A1
None
197
89
45


pCLS10A1f1
CSP-B 5′-
217
127
59



SAR


pCLS10A1t1
CSP-B 3′-
119
51
43



SAR









Verification on NESP Expression Level of Single Clone

Among positive clones on the 96-well plate, of which NESP expression was confirmed, 24 positive clones were arbitrarily selected for each vector and subcultured in a 24-well plate. The culture liquid was taken up and the expression level was measured. As shown in FIGS. 10 and 11, the number of high-expression clones that exhibited an expression level of 10 μg/Ml or higher among 24 arbitrarily selected positive clones was 13 for the CSP-B 5′-SAR-introduced vector and 5 for the SAR factor-not introduced vector. That is, it was verified that the frequency of high-expression clones increased when CSP-B 5′-SAR was introduced into the vector.
Example 8
Preparation of Anti-HER2 Expression Vector
Preparation of pC01 Vector
For preparation of the pC01 vector sequentially including a neomycin-resistant gene, an ampicillin-resistant gene, a CMV promoter, MCS, and BGH pA, the following experiment was conducted. First, PCR was performed using the V1_F primer (aagct tcctc agcat cgatg gccgg ccgga tccct gtgcc ttcta gttgc cagcc atctg t; SEQ ID NO: 54) and the V1_R primer (tagag cccca gctgg ttctt tccgc ctcag; SEQ ID NO: 55) while using the pcDNA3.1(−) vector as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Then, this PCR product was cloned into the pcDNA3.3-TOPO vector to prepare the pC01 vector.
Preparation of pC02 Vector
For preparation of the pC02 vector sequentially including a CMV promoter, the MCS of the pC01 vector and another MCS, and BGH pA, the following experiment was conducted. First, PCR was performed using the V2_F primer (gaatt ctgta caggt acccc tgcag gctcg agctg tgcct tctag ttgcc agcca tctgt; SEQ ID NO: 56) and the V2_R primer (tagag cccca gctgg ttctt tccgc ctcag; SEQ ID NO: 57) while using the pcDNA3.1(−) vector as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Then, this PCR product was cloned into the pcDNA3.3-TOPO vector to prepare the pC02 vector.
Preparation of pC03 Vector
For preparation of the pC03 vector sequentially including the neomycin-resistant gene, the ampicillin-resistant gene, the CMV promoter, the first MCS, and the BGH pA of the pC01 vector and the CMV promoter, the second MCS, and the BGH pA of the pC02 vector, the following experiment was conducted. First, the pC01 vector was digested with restriction enzymes DraIII and AgeI, thereby obtaining a large DNA fragment. Then, PCR was performed using the PcmvAgeF primer (aatct gaccg gtgtt aggcg ttttg cgctg cttcg cg; SEQ ID NO: 58) and the BGHNheDraR primer (ttact acact acgtg gatcg agcta gctag agccc cagct ggttc tttcc g; SEQ ID NO: 59) while using the pC02 vector as a template. This PCR product was digested with restriction enzymes DraIII and AgeI. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Last, this PCR product was ligated to the DNA fragment obtained by digesting the pC01 vector with restriction enzymes DraIII and AgeI, thereby preparing the pC03 vector.
Preparation of pCLS05 Vector
For preparation of the pCLS05 vector including the SV40dE1 promoter, the Kozak sequence, the DHFR sequence, and TK pA inserted between the BGH pA and the SV40 promoter of the 140pC03 vector, the following experiment was conducted. First, PCR was performed using the PsvApaDraF primer and the TKNheNdeR primer while using the pC04 vector as a template. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute and 30 seconds at 72° C. Then, this PCR product and the pC03 vector were digested with restriction enzymes Dralll and Nhel, and the thus obtained two large DNA fragments are ligated to prepare the pCLS05 vector.
Preparation of pCLS05H1 Vector
For preparation of the pCLS05H1 vector by inserting the anti-HER2 antibody heavy chain gene between EcoRI and Xhol restriction sites of the pCLS05 vector, the following experiment was conducted. First, PCR was performed using the H1ssF primer (ctctt cttgg tagca acagc tacag gtgtc cactc cgagg tccaa ctggt cgaaa gcggt gga; SEQ ID NO: 60) and the H1TGAXhoR primer (aattc tcgag tcatt taccc ggaga caggg agagg ctctt; SEQ ID NO: 61) while using the synthesized anti-HER2 antibody gene as a template, to amplify a heavy chain gene having a part of the signal sequence. PCR was performed using the H1ssEcoF primer (aattg aattc gccac catgg gatgg agctg tatca tcctc ttctt ggtag caaca gctac agg; SEQ ID NO: 62) and the H1TGAXhoR primer (SEQ ID NO: 61) while using this PCR product as a template, to amplify a heavy chain gene having the Kozak sequence and the signal sequence. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute and 30 seconds at 72° C. Then, this PCR product was cloned into the PGEM-T® vector and then digested with restriction enzymes EcoRI and Xhol, to obtain the heavy chain gene having the Kozak sequence and the signal sequence, which was then cloned between the EcoRI and Xhol restriction sites of the pCLS05 vector, thereby preparing the pCLS05H1 vector.
Preparation of pCLS05H2 Vector
For preparation of the pCLS05H2 vector by inserting the anti-HER2 antibody light chain gene between HindIII and BamHI restriction sites of the pCLS05H1 vector, the following experiment was conducted. First, PCR was performed using H2ssF primer (ctctt cttgg tagca acagc tacag gtgtc cactc cgata tccag atgac ccaga gtccc tct; SEQ ID NO: 63) and the H2TGABamR primer (aattg gatcc tcaac actct cccct gttga agctc tttgt; SEQ ID NO: 64) while using the synthesized anti-HER2 gene as a template, to amplify a light chain gene having a part of the signal sequence. PCR was performed using the H2ssHinF primer (aatta agctt gccac catgg gatgg agctg tatca tcctc ttctt ggtag caaca gctac agg; SEQ ID NO: 65) and the H2TGABamR primer (SEQ ID NO: 64) while using this PCR product as a template, to amplify a light chain gene having the Kozak sequence and the signal sequence. The PCR was performed under the conditions of 30 cycles of 10 seconds at 98° C., 30 seconds at 60° C., and 1 minute at 72° C. Then, this PCR product was cloned into the pGEM-T vector and then digested with restriction enzymes HindIII and BamHI, to obtain a light chain gene having the Kozak sequence and the signal sequence, which was then cloned between the HindIII and BamHI restriction sites of the pCLS05H1 vector, thereby preparing the pCLS05H2 vector. The map of the pCLS05H2 vector is shown in FIG. 12a. 
Example 9
Preparation of Anti-HER2 Expression Vector Including SAR Factor
Preparation of pCLS05H2f2 Vector
For preparation of the pCLS05H2f2 vector by introducing two copies of CSP-B 5′-SAR between the heavy chain gene and the DHFR gene of the pCLS05H2 vector that can express the anti-HER2 antibody in animal cells, the following experiment was conducted. The pCLS05H2 vector and the pCLS09G1f2 vector were digested with restriction enzymes XhoI and BssHII, and the thus obtained two large DNA fragments were ligated to prepare the pCLS05H2f2 vector. The map of the pCLS05H2f2 vector is shown in FIG. 12b. 
Example 10
Anti-HER2 Antibody Expression Using SAR Factor-Introduced Vector
Verification on Anti-HER2 Antibody Expression Level of DG44 Cell Pool Transfected with pCLS05H2 or pCLS05H2f2 Vector
DG44 cells were transfected with the pCLS05H2 vector including a gene of the target protein anti-HER2 antibody and the pCLS05H2f2 vector including the antibody gene and the SAR factor, respectively, and then selected with GENETICIN®-containing medium, to obtain cell pools. The anti-HER2 antibody expression levels for the respective vectors were verified by enzyme-linked immunosorbent assay (ELISA). As shown in Table 6 and FIG. 13, it was confirmed that the anti-HER2 antibody expression level was 30-fold higher when two copies of SAR factors were introduced than when the SAR factor was not introduced. The GENETICIN® resistance was exhibited and the surviving cells were relatively more numerous when the pCLS05H2f2 vector including the SAR factor was used.










TABLE 6









Anti-HER2 antibody
Relative anti-HER2


Vector used

expression level
antibody expression


for
Introduced
(μg/Ml)
level
















transfection
SAR
Set 1
Set 2
Set 3
Mean
Set 1
Set 2
Set 3
Mean



















pCLS05H2
None
0.29
0.50
0.26
0.35
1.0
1.0
1.0
1.0


pCLS05H2f2
CSP-B
6.74
8.22
13.6
9.54
22.7
16.2
52.6
30.5



Continuous



two copies



of 5′-SAR









Verification on Anti-HER2 Antibody Expression Level of Single Clone

Positive clones on the 96-well plate, of which anti-HER2 antibody expression was confirmed, were arbitrarily selected, and subcultured in a 24-well plate. The culture liquid was taken up and the expression level was measured. As shown in FIGS. 14 and 15, the number of high-expression clones that exhibited an expression level of 5 μg/Ml or higher among 19 arbitrarily selected positive clones was 10 for the vector to which two copies of CSP-B 5′-SAR were introduced and 6 for the vector to which the SAR factor was not introduced. That is, it was verified that the frequency of high-expression clones relatively increased when CSP-B 5′-SAR factor was introduced into the vector.
Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

## Claims

1. An expression vector for animal cells, comprising:
a protein-coding nucleotide sequence to be expressed, wherein the protein is a Novel Erythropoiesis Stimulating Protein (NESP) or an anti-human epidermal growth factor receptor 2 (anti-HER2) antibody;
a 5′-scaffold attachment region (5′-SAR) of cytotoxic serine protease-B (CSP-B), wherein the 5′-SAR is present in two copies downstream of the protein-coding nucleotide sequence to be expressed, and the two copies of the 5′-SAR are consecutively present in the vector;
a promoter operable in animal cells; and
a polyadenylation sequence.

2. The expression vector according to claim 1, wherein the promoter operable in the animal cells is a cytomegalovirus (CMV) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, a Simian virus 40 (SV40) promoter, an SV40E1 promoter, a herpes simplex virus (HSV) tk promoter, a respiratory syncytial virus (RSV) promoter, an elongation factor 1-α (EF1-α) promoter, a metallothionein promoter, a β-actin promoter, a human interleukin-2 (IL-2) gene promoter, a human interferon (IFN) gene promoter, a human IL-4 gene promoter, a human lymphotoxin gene promoter, or a human granulocyte-macrophage colony-stimulating factor (GM-CSF) gene promoter.

3. The expression vector according to claim 1, wherein the polyadenylation sequence is a bovine growth hormone polyadenylation sequence, an HSV thymidine kinase (TK) polyadenylation sequence, or an SV40 polyadenylation sequence.

4. The expression vector according to claim 1, wherein the animal cells are Chinese hamster ovary (CHO) cells, VERO cells, HeLa cells, WI38 cells, baby hamster kidney (BHK) cells, COS cells, or Madin-Darby canine kidney (MDCK) cells.

5. An isolated animal cell transfected with the expression vector according to claim 1.

6. A method for preparing a recombinant protein, comprising incubating the transfected animal cell of claim 5 and expressing the NESP or anti-HER2 antibody.