JP6131074B2 - Secretory signal sequence - Google Patents

Description

  The present invention relates to a signal sequence for efficiently secreting and producing alkaline phosphatase using mammalian cells into which an alkaline phosphatase gene has been introduced.

  Alkaline phosphatase (EC 3.1.3.1) is a phosphomonoesterase with low substrate selectivity that has an optimal pH for alkalinity. Many alkaline phosphatases have zinc at the active center and are known to exist widely in nature from prokaryotes such as E. coli to eukaryotic higher organisms such as mammals (Non-patent Document 1). .

  Alkaline phosphatase is a highly useful enzyme widely used in the fields of molecular biology and immunodiagnosis. For example, in the field of molecular biology, alkaline phosphatase derived from calf small intestine (Cal Intestinal phosphatase; hereinafter referred to as CIP), alkaline phosphatase derived from Arctic shrimp, alkaline phosphatase derived from Escherichia coli, etc. are dephosphorylated at the 5 ′ end of DNA or RNA. Used for oxidation. Alkaline phosphatase derived from human placenta (particularly modified to secretory type) is used as a reporter for determining the efficiency of introduction of a foreign gene into a host cell. On the other hand, in the immunodiagnosis field, CIP having a high specific activity is used as an antibody labeling enzyme.

  Alkaline phosphatase is involved in the absorption of nutrients in the intestinal tract of higher animals, and also has a function of dephosphorylating and detoxifying toxins derived from Gram-negative bacteria (for example, Non-Patent Document 2 and Non-Patent Document 3) Application development to treatment and diagnosis of bacterial infection or sepsis is also expected (for example, Patent Document 1 and Patent Document 2).

  Among alkaline phosphatases, CIP has high specific activity, so it is highly useful in the field of immunodiagnostics. Chinese hamster ovary cells (hereinafter referred to as CHO cells) (non-patent document 4) and methanol-assimilating yeast (patent document 3) Production of recombinant CIP is disclosed using as a host cell.

  CHO cells are the most widely used host cells for the purpose of producing heterologous proteins, and technological development for improving productivity has been actively performed. When a CHO cell is used to produce a secreted protein, it is considered that one of the bottlenecks related to productivity is a process in which a nascent polypeptide just translated from messenger RNA is secreted into the endoplasmic reticulum. In order to solve this problem, various signal sequences have been reported so far (for example, Patent Document 4 and Patent Document 5). However, a signal sequence suitable for the efficient secretory production of alkaline phosphatase using mammalian cells such as CHO cells has not been known so far.

Japanese National Patent Publication No. 9-502342 Japanese translation of PCT publication No. 2002-533679 Japanese Patent No. 3657895 Special table 2010-528634 International Publication 2008/000445 JP-A-6-284885 JP 2004-187852 A

McComb et al., Alkaline Phosphataes, Plenum Press, New York (1979). Poelstra et al., American J. MoI. Pathology, 151 (4), 1163-1169 (1997) Beumer et al. Pharmacology and Experimental Therapeutics, 307 (2), 737-744 (2003). Manes et al. Biol. Chem. 273 (36), 23353-23360 (1998) Tanaka et al., Biochem. Biophys. Res. Commun. 190 (3), 732-740 (1993) Masure et al., Eur. J. et al. Biochem. 218 (1), 129-141 (1993) Yasukawa et al., J. MoI. Biochem. 108, 673-676 (1990)

  An object of the present invention is to provide a signal sequence (signal peptide) for efficiently producing and secreting alkaline phosphatase using mammalian cells into which an alkaline phosphatase gene has been introduced.

  As a result of intensive studies to solve the above problems, the present inventors have efficiently used alkaline phosphatase in mammalian cells into which an alkaline phosphatase gene has been introduced by using a signal peptide of MMP-9 derived from mouse or Chinese hamster. The inventors have found that secretion production is possible, and have completed the present invention.

  That is, this invention includes the following aspects.

  (1) A signal peptide for secreting and producing alkaline phosphatase in a mammalian cell into which an alkaline phosphatase gene has been introduced, comprising the amino acid sequence set forth in SEQ ID NO: 1 or 2.

  (2) a polynucleotide encoding alkaline phosphatase, and an oligonucleotide encoding a signal peptide consisting of the amino acid sequence of SEQ ID NO: 1 or 2 linked in frame to the 5 ′ end of the polynucleotide, An expression vector for secretory production of alkaline phosphatase in mammalian cells.

  (3) The expression vector according to (2), wherein the alkaline phosphatase is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3.

  (4) The expression vector according to (3), comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 4 to 7.

  (5) The expression vector according to (4), comprising a polynucleotide comprising the base sequence described in any one of SEQ ID NOs: 8 to 13.

  (6) A cell capable of secretory production of alkaline phosphatase obtained by transfecting a mammalian cell with the expression vector according to any one of (2) to (5).

  (7) The cell according to (6), wherein the mammalian cell is a Chinese hamster ovary cell.

  (8) A method for producing alkaline phosphatase, wherein the cells according to (6) or (7) are cultured, and alkaline phosphatase is recovered from the obtained culture solution.

  Hereinafter, the present invention will be described in detail.

  The present invention relates to a mouse-derived MMP-9 signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or a Chinese hamster-derived MMP-9 signal peptide consisting of the amino acid sequence shown in SEQ ID NO: 2 as an alkaline phosphatase gene. It is characterized in that it is used as a signal peptide for secretory production of alkaline phosphatase in mammalian cells into which is introduced.

  MMP-9 is one of the proteolytic enzymes belonging to the matrix metalloprotease family. MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-11, MMP-12 , MMP-13, MMP-20, MMP-21, and MMP-28 are classified into secretory matrix metalloproteases. MMP-9 derived from mouse is the same as 105 kDa gelatinase obtained from the cell line of metastatic tumor (Non-patent document 5) and gelatinase B obtained from the cell line of macrophage (Non-patent document 6). The amino acid sequence is published (GenBank No. AAH46991). MMP-9 is also present in mammals other than mice, such as humans, monkeys, rats, Chinese hamsters, rabbits, dogs, sheep, pigs, horses, cows, and those derived from birds, amphibians, and fish Are known. Of these, the amino acid sequence of Chinese hamster MMP-9 has been revealed by genomic analysis of CHO cells (GenBank No. XM_003500575).

  In the present invention, the signal peptide refers to an oligopeptide consisting of about 20 amino acid residues having a function of targeting the nascent polypeptide just translated from messenger RNA to the endoplasmic reticulum, and in many cases, N Present on the terminal side. It is often absent from mature polypeptides because it undergoes cleavage by signal peptidases in the process of being secreted into the endoplasmic reticulum.

  In the present invention, the oligopeptide consisting of the amino acid sequence of SEQ ID NO: 1 used as a signal peptide for secreting and producing alkaline phosphatase in mammalian cells into which the alkaline phosphatase gene has been introduced is GenBank No. An oligopeptide corresponding to amino acid residues 1 to 19 in AAH46991. The oligopeptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 is GenBank No. XM_003500575 is an oligopeptide corresponding to the first to 19th amino acid residues.

  In the present invention, the expression vector refers to a circular or linear polynucleotide for functionally expressing a designed gene. In addition to a polynucleotide encoding a functionally expressed polypeptide, and a polynucleotide encoding a signal peptide linked in-frame to the 5 ′ end of the polynucleotide, a transcription activation sequence ( Promoter / enhancer), transcription termination sequence, marker genes such as drug resistance and auxotrophy, replication origin necessary for propagation as a plasmid in host cells, and the like. Here, linking in frame means arranging the polynucleotides encoding (poly) peptides so that they are translated in the same reading frame. Marker genes include drug resistance genes such as neomycin phosphotransferase gene (neomycin resistance gene), hygromycin B phosphotransferase gene (hygromycin B resistance gene), and blasticidin S deaminase gene (blasticidin resistance gene), glutamine Encodes selectable markers that can be expressed in mammalian cells used for transfection, such as genes related to auxotrophy such as synthetase gene, dihydrofolate reductase (dhfr) gene, genes that enable optical discrimination such as green fluorescent protein gene, etc. What is necessary is just to select suitably from the genes to be used.

  The expression vector of the present invention may contain a replication origin that works outside of animal cells, such as a colE1 replication origin that works in E. coli, and a selectable marker that can be expressed in E. coli (for example, β-lactamase gene (ampicillin / carbenicillin). A resistance gene)). By transforming Escherichia coli with the expression vector of the present invention containing the β-lactamase gene and the colE1 origin of replication described above, the recombinant E. coli is cultured in a medium containing carbenicillin. Can be prepared. For example, plasmid pOtiVEC-TOPO (manufactured by Life Technologies) or plasmid pECEdhfr (Non-patent Document 7) can be suitably used as a material for preparing the expression vector of the present invention.

  Although there is no limitation in particular in the origin of the alkaline phosphatase in this invention, the alkaline phosphatase (CIP) derived from the calf small intestine with a high specific activity is preferable. As an example of CIP, a polypeptide (GenBank No. AAN19391) comprising the amino acid sequence shown in SEQ ID NO: 3 or a derivative in which addition or deletion of an oligopeptide has occurred on the N-terminal side and / or C-terminal side of the polypeptide Alternatively, a derivative in which one to several amino acid residues in the polypeptide are substituted with other amino acid residues can be mentioned. In addition, since many mammalian-derived alkaline phosphatases including CIP are membrane-bound, in order to secrete them extracellularly, it is preferable to remove the GPI anchor region at 20 amino acid residues from the C-terminal side 20 of alkaline phosphatase (for example, In addition, the alkaline phosphatase which consists of an amino acid sequence of sequence number 3 is an alkaline phosphatase from which the GPI anchor was removed. Further, a tag sequence may be added to the C-terminal side in order to facilitate purification and detection of alkaline phosphatase that has been secreted to the outside of the cell by removal of the GPI anchor. Examples of tag sequences include His tags, Myc tags, HA tags, and FLAG tags.

  Polynucleotide encoding alkaline phosphatase, and polynucleotide encoding a signal peptide linked in-frame to the 5 'end of the polynucleotide are synthesized by enzymatic synthesis by polymerase chain reaction (PCR) using chemical synthesis or cDNA as a template. It can be synthesized by combining the above. When an expression vector is prepared using the polynucleotide, it is preferable to arrange a transcription activation sequence (promoter / enhancer) upstream thereof. The transcription activation sequence is not particularly limited as long as it functions in mammalian cells used for transfection, and even if it is constitutively activated, it can be regulated by external conditions. May be. For example, those that are constantly activated include human cytomegalovirus promoter (CMV promoter), SV40 early promoter, RSV promoter, SRα promoter, human elongation factor 1-α promoter, bovine growth hormone promoter, β-actin Examples of promoters include hamster ubiquitin / S27a promoter, and examples of those that can be regulated include TRE promoter (Clontech) and T-REX promoter (Invitrogen).

A polynucleotide encoding alkaline phosphatase and an oligonucleotide encoding a signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 linked in frame to the 5 ′ end of the polynucleotide, contained in the vector of the present invention As an example,
(A) a polynucleotide (eg, sequence) encoding a polypeptide (SEQ ID NO: 4) obtained by adding a mouse MMP-9 signal peptide (SEQ ID NO: 1) to the N-terminal side of CIP comprising the amino acid sequence set forth in SEQ ID NO: 3 A polynucleotide comprising the nucleotide sequence of No. 9 and SEQ ID NO: 11),
(B) A signal peptide (SEQ ID NO: 1) derived from mouse MMP-9 was added to the N-terminal side, and a His tag consisting of 6 histidines was added to the C-terminal side to CIP consisting of the amino acid sequence described in SEQ ID NO: 3. A polynucleotide encoding a polypeptide (SEQ ID NO: 5) (for example, a polynucleotide comprising the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 10),
(C) a polynucleotide encoding a polypeptide (SEQ ID NO: 6) obtained by adding a Chinese hamster MMP-9 signal peptide (SEQ ID NO: 2) to the N-terminal side of CIP comprising the amino acid sequence of SEQ ID NO: 3 (for example, A polynucleotide comprising the base sequence described in SEQ ID NO: 12), and (d) a Chinese hamster MMP-9 signal peptide (SEQ ID NO: 2) on the N-terminal side, and a His tag consisting of 6 histidines on the C-terminal side, A polynucleotide encoding a polypeptide (SEQ ID NO: 7) added to CIP consisting of the amino acid sequence shown in SEQ ID NO: 3 (for example, a polynucleotide consisting of the base sequence shown in SEQ ID NO: 13),
Can be given.

  Host cells to be transfected with the expression vector of the present invention are mammalian cells, human fetal kidney cells (for example, HEK293 cells), canine kidney cells (for example, MDCK cells), African green monkey kidney cells (for example, COS-7 cells) ), Mouse myeloma cells (for example, SP2 / 0 cells, P3U1 cells), and Chinese hamster ovary cells (CHO cells). Of these, CHO cells are preferred as host cells to be transfected with the expression vector of the present invention. There are no particular limitations on the CHO cell line, and examples include CHO-K1 cells (ATCC No. CCL-61), CHO-S cells, dh44 gene-deficient DG44 cells and DXB11 cells.

  In order to transfect mammalian cells with the expression vector of the present invention, a method commonly used by those skilled in the art, such as calcium phosphate coprecipitation method, DEAE dextran method, electroporation method, microinjection method and lipofection method, is selected as appropriate. Just do it. The expression vector of the present invention used for transfection may be a circular polynucleotide or a linear polynucleotide obtained by digestion with an appropriate restriction enzyme. For example, a recognition site for the restriction enzyme PvuI present in one place in the plasmid pOtiVEC-TOPO can be used for the purpose of linearizing the vector. In the above case, the polynucleotide inserted into pOtiVEC-TOPO may be designed so as not to include a restriction enzyme PvuI recognition site.

  Recombinant mammalian cells obtained by transfection with the expression vector of the present invention are monocloned by methods commonly used by those skilled in the art, and alkaline phosphatase activity in the culture medium obtained by culturing the monocloned cells. By selecting using as an index, recombinant cells having the desired alkaline phosphatase productivity can be selected. In addition, by introducing the expression vector of the present invention into the selected recombinant cells and culturing the recombinant cells that have been cloned from them, the recombinant cells with further increased alkaline phosphatase activity can be selected. Also good.

  When the expression vector of the present invention contains a selectable marker that can be co-expressed, the recombinant cell group expressing the selectable marker is selected by culturing in a selection medium corresponding to the selectable marker, After the selected cell group is cloned, it may be selected using the alkaline phosphatase activity in the culture medium as an index. As an example, the expression vector of the present invention containing a coexpressible dhfr gene is introduced into a dhfr gene-deficient CHO cell, and a group of cells expressing the dhfr gene is grown in a selective medium not containing hypoxanthine and thymidine. There is a method of culturing cells cloned from a cell group and selecting them using the alkaline phosphatase activity in the culture medium as an index. In addition, when the dhfr gene is used as a selection marker, the recombinant cells are cultured in a medium containing methotrexate (MTX), and the recombinant cells with further increased alkaline phosphatase activity are selected by inducing amplification of the introduced gene. May be.

  The alkaline phosphatase activity can be measured by, for example, a hydrolysis reaction using p-nitrophenyl phosphate as a substrate. As an example, p-nitrophenol produced by hydrolysis by alkaline phosphatase activity in a buffer solution of pH 9 to pH 10 (for example, 0.1 M glycine-sodium hydroxide buffer (pH 9.6)) at a reaction temperature of 25 ° C. Enzyme activity can be calculated by quantifying the absorbance at a wavelength of 405 nm. An appropriate amount of protein such as bovine serum albumin that has little influence on enzyme activity, a magnesium salt such as magnesium chloride, or a zinc salt such as zinc chloride may be added to the buffer solution. As a specific embodiment of the alkaline phosphatase activity measurement, 10 mM p-nitrophenyl phosphate disodium salt (hereinafter abbreviated as pNPP) is added to a microwell plate (manufactured by NUNC, catalog number 269620) to which 5 μL of the sample solution is added. 0.1 M glycine-sodium hydroxide buffer solution (pH 9.6) containing 0.1% bovine serum albumin (manufactured by SIGMA, catalog number A7030), 0.1 mM magnesium chloride, and 0.01 mM zinc chloride (PNPP substrate solution) was added at 100 μL, and the reaction was stopped by adding 100 μL of 0.5 M sodium hydroxide after incubation at 25 ° C. for a certain time, and the absorbance at 405 nm was measured using Infinite M200 (manufactured by Tecan Systems). Contains alkaline phosphatase after measurement There is a method of measuring by subtracting the absorbance of the measured value obtained in the same manner using a non-sample solution. The concentration of the sample solution and the reaction time may be determined experimentally, and the conditions may be determined so that the absorbance value is 2 or less, preferably 1.5 or less, more preferably 1 or less.

  Alkaline phosphatase can be quantified using an antibody. For example, when alkaline phosphatase is CIP, an anti-CIP antibody prepared using natural (extracted purified product from calf small intestine) CIP as an antigen is immobilized on a carrier (for example, a microwell plate), blocked using bovine serum albumin, Sample solution containing moderately diluted alkaline phosphatase is added and reacted for a certain period of time, followed by B / F (Bound / Free) separation, reaction with pNPP substrate solution, and known alkaline phosphatase bound to immobilized anti-CIP antibody The natural CIP at a concentration of 1 is measured as a standard sample and colorimetrically determined. After the B / F separation, after reacting for a certain time with an anti-CIP antibody labeled with an enzyme other than alkaline phosphatase other than the immobilized anti-CIP antibody, B / F separation is performed, It is also possible to measure the activity of the labeled enzyme, and perform colorimetric determination (so-called enzyme immunoassay) using natural CIP at a known concentration as a standard sample.

  In addition, when quantifying alkaline phosphatase to which an appropriate tag is added, measurement using an anti-tag antibody is possible. For example, in the case of alkaline phosphatase with a His tag added to the C-terminal side, a commercially available anti-His tag antibody that specifically recognizes the C-terminal His tag may be used, and an enzyme labeled with an enzyme other than the alkaline phosphatase. Instead of the labeled antibody, for example, by reacting with an anti-His tag antibody labeled with horseradish peroxidase for a certain period of time, B / F separation is performed, and then the peroxidase activity is measured by allowing the peroxidase substrate to act. It can be quantified.

  A medium for culturing a recombinant mammalian cell capable of secreting and producing alkaline phosphatase obtained by transfection with the expression vector of the present invention may be appropriately selected from those normally used by those skilled in the art. Roswell Park Memorial Institute (RPMI) 1640 medium, L-15 medium, Dulbecco's modified Eagle medium (DMEM; Dulbecco's modified Eagle's medium), Eagle's minimum essential medium (MEM; Eagle's minimum) Examples include a medium obtained by adding 5 to 10% volume of fetal bovine serum to Ham's F12 medium. In order to efficiently purify secreted alkaline phosphatase, a so-called serum-free medium without adding fetal calf serum may be used. When a serum-free medium is used, it is preferable to add insulin or transferrin that is necessary for the growth of many cells. Furthermore, growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), insulin-like growth factor I and II (IGFI and IGFII), prostaglandins, ceruloplasmin, high density lipoprotein, Low-density lipoprotein, bovine serum albumin, fatty acid, and the like may be added to the medium within a preferable range for growth of recombinant mammalian cells, secretion production of alkaline phosphatase, and purification of alkaline phosphatase. As a specific example, when the recombinant mammalian cell is a DG44 cell which is a dhfr gene-deficient CHO cell, glutamine is added to a medium containing hypoxanthine and thymidine, for example, CD CHO medium (trade name, manufactured by Life Technologies). In the case of DG44 cells obtained by transfection with the vector of the present invention containing the dhfr gene, a medium not containing hypoxanthine and thymidine, for example, CD OptiCHO medium (trade name, Life It is preferable to culture using a medium in which glutamine is added to Technologies). A nonionic surfactant such as Pluronic F68 (trade name, manufactured by Life Technologies) or a commercially available penicillin-streptomycin mixed solution may be appropriately added to the medium.

Externals such as container, temperature, CO ₂ concentration, shaking condition (aeration) when culturing recombinant mammalian cells capable of secreting and producing alkaline phosphatase obtained by transfection with the expression vector of the present invention There are no particular restrictions on the culture conditions. For example, when the recombinant mammalian cells are DG44 cells, they are cultured in a 200 mL flask containing 30 mL of culture solution at a temperature of 37 ° C., a CO ₂ concentration of 8%, and a shaking speed of 135 rpm. By doing so, recombinant mammalian cells can be cultured efficiently. Measurement of the cell density in the culture solution may be performed by appropriately selecting from methods usually used by those skilled in the art. For example, a culture solution in which cells are suspended is collected, and a commercially available trypan blue staining solution having the same volume as the collected culture solution (for example, trade name 0.4 w / v% Trypan Blue Solution, manufactured by Wako Pure Chemical Industries, Ltd.) The number of cells that are not mixed and stained (viable cells) may be measured and calculated using a solution hemocytometer under a microscope.

  In order to recover alkaline phosphatase from a culture solution of recombinant mammalian cells capable of secreting and producing alkaline phosphatase obtained by transfection with the expression vector of the present invention, an appropriate method selected by those skilled in the art can be selected. / Just do it in combination. Examples of the recovery method include concentration using an ultrafiltration membrane, ammonium sulfate salting out, hydrophobic chromatography, ion exchange chromatography, size exclusion chromatography, or a combination of the methods described above. In addition, when a tag is added to the C-terminal side of alkaline phosphatase, in addition to the method described above, purification may be performed by combining affinity chromatography using affinity for the tag.

  The present invention is characterized in that a signal peptide of MMP-9 derived from mouse or Chinese hamster is used as a signal peptide for secreting and producing alkaline phosphatase in mammalian cells into which alkaline phosphatase has been introduced. Alkaline phosphatase can be efficiently produced in the mammalian cells. The effect was that the amount of MMP-9 secreted by CHO cells was very small, and the amount of mouse MMP-9 secreted by CHO cells introduced with the mouse MMP-9 gene was also small. Even if it is compared with the prior art (Non-Patent Document 6), this effect is not expected at all.

Schematic diagram of plasmid pCIP1009H. Schematic diagram of plasmid pCIP6000H. Schematic diagram of plasmid pCIP6000. Schematic diagram of plasmid pCIP6200H2. Schematic diagram of plasmid pCIP6200-2. Schematic diagram of plasmid pCIP9200H. Schematic diagram of plasmid pCIP9200. Results of SDS-PAGE analysis of each fraction having alkaline phosphatase activity (Example 5). Results of SDS-PAGE analysis of each fraction having alkaline phosphatase activity (Example 7).

  In order to describe the present invention in more detail below, examples will be shown, but these examples show examples of the present invention, and the present invention is not limited to these examples.

Example 1 Preparation of Plasmid (Expression Vector) Plasmids (expression vectors) used in this study were prepared by the methods shown below.

(A) Plasmid pCIP1000
(A-1) An alkaline phosphatase (CIP) signal peptide derived from calf small intestine on the N-terminal side and a His tag consisting of 6 histidines on the C-terminal side are added to the CIP consisting of the amino acid sequence shown in SEQ ID NO: 3, respectively. An oligonucleotide comprising the sequence of SEQ ID NO: 18 using pCIP-1 (synthesized by OPERON Biotechnology), which is a plasmid containing the polynucleotide (SEQ ID NO: 17) encoding the polypeptide (SEQ ID NO: 16) as a template PCR was performed using Taq DNA polymerase (manufactured by Life Technologies) using Primer-1) and an oligonucleotide (Primer-2) having the sequence described in SEQ ID NO: 19 as a primer set.
(A-2) The obtained 1.5 kbp polynucleotide (DNA-1) was inserted into the cloning site of pOptiVEC-TOPO (Life Technologies), which is an expression vector using a CMV promoter.
(A-3) The base sequence of the plasmid into which the polynucleotide was inserted in (A-2) was examined. As a result, it was confirmed that this was a plasmid (5.924 kbp) into which a polynucleotide (SEQ ID NO: 21) encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 20 was inserted, and the plasmid was named pCIP1000.

(B) Plasmid pCIP1000H
Double digestion of 1.5 kbp polynucleotide (DNA-2) obtained by double digestion of pCIP-1 with restriction enzymes EcoRI and MluI and plasmid pCIP1000 prepared in (A) with restriction enzymes EcoRI and MluI The obtained 4.4 kbp polynucleotide (DNA-3) was ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.) to prepare plasmid pCIP1000H.

(C) Plasmid pCIP1200
(C-1) A plasmid comprising a polynucleotide (SEQ ID NO: 24) encoding a polypeptide (SEQ ID NO: 20) having a CIP signal peptide added to the N-terminal side of CIP consisting of the amino acid sequence set forth in SEQ ID NO: 3. PCIP-2 (synthesized by OPERON Biotechnologies) as a template, oligonucleotide (Primer-3) consisting of the sequence shown in SEQ ID NO: 22 and oligonucleotide (Primer-4) consisting of the sequence shown in SEQ ID NO: 23 as primers PCR was performed using Taq DNA polymerase (manufactured by Life Technologies) as a set. The amino acid sequence of the polypeptide translated from the polynucleotide consisting of the base sequence described in SEQ ID NO: 24 inserted in the plasmid pCIP-2 is the amino acid sequence described in SEQ ID NO: 21 inserted in the plasmid pCIP-1. Although it is the same as the amino acid sequence of the polypeptide translated from the polynucleotide comprising the base sequence (SEQ ID NO: 20), the codons are different and the base sequence homology between the polynucleotides is about 80%.
(C-2) The obtained 1.5 kbp polynucleotide (DNA-4) was double digested with restriction enzymes EcoRI and MluI, and the plasmid pCIP1000H prepared in (B) was doubled with restriction enzymes EcoRI and MluI. The 4.4 pbp polynucleotide (DNA-5) obtained by digestion was ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.) to prepare plasmid pCIP1200.

(D) Plasmid pCIP1009H
(D-1) Using the plasmid pCIP1200 prepared in (C) as a template, the oligonucleotide (Primer-5) consisting of the sequence shown in SEQ ID NO: 27 and the sequence shown in SEQ ID NO: 28 phosphorylated on the 5 ′ end side PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using the oligonucleotide (Primer-6) as a primer set to obtain a 0.75 kbp polynucleotide (DNA-6).
(D-2) Using the plasmid pCIP1000H prepared in (B) as a template, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 29 (Primer-7) and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 30 (Primer-8) PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.) As a primer set to obtain a 0.49 kbp polynucleotide (DNA-7).
(D-3) DNA-6 obtained in (D-1) and DNA-7 obtained in (D-2) are digested with restriction enzyme EcoRV, and DNA Ligation Kit Light Mix (manufactured by Takara Bio Inc.) After ligation using PCR, PCR was performed using Primer-5 (SEQ ID NO: 27) and Primer-8 (SEQ ID NO: 30) as a primer set to obtain a 1.2 kbp polynucleotide (DNA-8).
(D-4) A 0.55 kbp polynucleotide (DNA-9) obtained by double digesting DNA-8 obtained in (D-3) with restriction enzymes XbaI and SphI, and plasmid pCIP1000H as a restriction enzyme A 5.4 kbp polynucleotide (DNA-10) obtained by double digestion with XbaI and SphI is ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), and described in SEQ ID NO: 3. A polypeptide in which the first leucine of CIP consisting of the amino acid sequence is substituted with aspartic acid, a CIP signal peptide is added to the N-terminal side of the CIP, and a His tag consisting of 6 histidines is added to the C-terminal Plasmid p comprising a polynucleotide (SEQ ID NO: 26) encoding (SEQ ID NO: 25) IP1000HssD2 was prepared.
(D-5) Using the plasmid pCIP1000H prepared in (B) as a template, an oligonucleotide (Primer-9) consisting of the sequence shown in SEQ ID NO: 31 and an oligonucleotide (Primer-10) consisting of the sequence shown in SEQ ID NO: 32 PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.).
(D-6) Oligonucleotide (Primer-11) comprising the sequences of Primer-9 (SEQ ID NO: 31) and SEQ ID NO: 33 using the polynucleotide (DNA-11) obtained in (D-5) as a template PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.).
(D-7) Using the polynucleotide (DNA-12) obtained in (D-6) as a template, Primer-9 (SEQ ID NO: 31) and the sequence of SEQ ID NO: 34 phosphorylated on the 5 ′ end side PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.) Using the oligonucleotide (Primer-12) as a primer set, and finally a 0.55 kbp polynucleotide (DNA-13) was obtained.
(D-8) The polynucleotide obtained by digesting DNA-13 obtained in (D-7) with the restriction enzyme NdeI and the plasmid pCIP1000HsD2 prepared in (D-4) with the restriction enzymes NdeI and EcoRV. A 5.4 kbp polynucleotide (DNA-14) obtained by heavy digestion was ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), so that it was derived from mouse MMP-9 on the N-terminal side. A polynucleotide (SEQ ID NO: 5) encoding a polypeptide (SEQ ID NO: 5) obtained by adding a signal peptide (SEQ ID NO: 1), a His tag comprising 6 histidines to the C-terminal side, and a CIP comprising the amino acid sequence described in SEQ ID NO: 3 An expression vector pCIP1009H of the present invention containing SEQ ID NO: 8) was prepared. A schematic diagram of pCIP1009H is shown in FIG.

(E) Plasmid pECEdhfr2
(E-1) An oligonucleotide consisting of the sequence described in SEQ ID NO: 35 (Primer-13) and an oligonucleotide consisting of the sequence described in SEQ ID NO: 36 (Primer-14) using the plasmid pECEdhfr (Non-patent Document 7) as a template PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using as a primer set to obtain an amplification product (polynucleotide) (DNA-15) of 0.37 kbp.
(E-2) Using DNA-15 obtained in (E-1) as a template, Primer-13 (SEQ ID NO: 35) and oligonucleotide (Primer-15) comprising the sequences described in SEQ ID NO: 37 as a primer set PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) to obtain a 0.39 kbp amplification product (polynucleotide) (DNA-16).
(E-3) Using the plasmid pECEdhfr (Non-patent Document 7) as a template, an oligonucleotide (Primer-16) consisting of the sequence shown in SEQ ID NO: 38 and an oligonucleotide (Primer-17) consisting of the sequence shown in SEQ ID NO: 39 PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by TAKARA BIO INC.) As a primer set to obtain a 0.69 kbp amplification product (polynucleotide) (DNA-17).
(E-4) Using DNA-17 obtained in (E-3) as a template, oligonucleotide (Primer-18) and Primer-17 (SEQ ID NO: 39) comprising the sequence of SEQ ID NO: 40 as a primer set PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) to obtain a 0.71 kbp amplification product (polynucleotide) (DNA-18).
(E-5) a polynucleotide obtained by double digesting DNA-16 obtained in (E-2) with restriction enzymes MfeI and XbaI, and DNA-18 obtained in (E-4) A polynucleotide obtained by double digestion with XbaI and PciI, and a 2.8 kbp polynucleotide (DNA-19) obtained by double digestion of plasmid pECEdhfr (Non-patent Document 7) with restriction enzymes EcoRI and PciI; Was ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.) to produce plasmid pECEdhfr2 containing the base sequence described in SEQ ID NO: 41. The plasmid pECEdhfr2 eliminates the recognition site for the restriction enzyme EcoRI between the SV40 late promoter and the dhfr gene of the plasmid pECEdhfr (Non-patent Document 7), and the SV40 early to the SV40 polyA. This is a plasmid with the region replaced.

(F) Plasmid pCIP4000H
The 1.5 kbp polynucleotide (same as DNA-2) obtained by double digesting the plasmid pCIP1000H prepared in (B) with the restriction enzymes EcoRI and MluI, and the plasmid pECEDdhfr2 prepared in (E) are converted into the restriction enzyme EcoRI. And 3.9 kbp polynucleotide (DNA-20) obtained by double digestion with MluI using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), the CIP of the N-terminal side A polynucleotide (SEQ ID NO: 17) encoding a polypeptide (SEQ ID NO: 16) in which a signal peptide is added to a CIP consisting of the amino acid sequence shown in SEQ ID NO: 3 with a His tag consisting of 6 histidines on the C-terminal side. The contained plasmid pCIP4000H (5.415) kbp) was obtained.

(G) Plasmid pCIP5000H
(G-1) Using the plasmid pCIP1000H prepared in (B) as a template, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 44 (Primer-19) and an oligonucleotide consisting of the sequence shown in SEQ ID NO: 45 (Primer-20) PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using as a primer set to obtain an amplification product (polynucleotide) (DNA-21) of 0.8 kbp.
(G-2) Using DNA-21 obtained in (G-1) as a template, Primer-19 (SEQ ID NO: 44) and oligonucleotide (Primer-21) comprising the sequences described in SEQ ID NO: 46 as a primer set PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) to obtain an amplification product (polynucleotide) (DNA-22) of 0.8 kbp.
(G-3) An oligonucleotide consisting of DNA-19 obtained in (G-2) as a template, Primer-19 (SEQ ID NO: 44) and the sequence of SEQ ID NO: 47 phosphorylated on the 5 ′ end side ( PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using Primer-22) as a primer set to obtain a 0.8 kbp amplification product (polynucleotide) (DNA-23).
(G-4) obtained by digesting DNA-23 obtained in (G-3) with restriction enzyme SpeI and then double digesting plasmid pCIP1000HssD2 prepared in (D-4) with restriction enzymes SpeI and EcoRV Plasmid pCIP1014H was produced by ligation using a polynucleotide and DNA Ligation Kit Mighty Mix (Takara Bio Inc.).
(G-5) A 1.5 kbp polynucleotide obtained by double digesting pCIP1014H prepared in (G-4) with restriction enzymes EcoRI and MluI, and plasmid pECEDdhfr2 prepared in (E) with restriction enzyme EcoRI. The polynucleotide obtained by double digestion with MluI is ligated using DNA Ligation Kit Mighty Mix (manufactured by TAKARA BIO INC.) To convert the signal peptide of Cypridina-derived luciferase (CYPLF) to the N-terminal side, Plasmid pCIP5000H containing a polynucleotide (SEQ ID NO: 43) encoding a polypeptide (SEQ ID NO: 42) obtained by adding a His tag consisting of 6 histidines to the CIP consisting of the amino acid sequence shown in SEQ ID NO: 3 on the terminal side. Produced.

(H) Plasmid pCIP6000H
A 1.5 kbp polynucleotide (DNA-24) obtained by double digesting the plasmid pCIP1009H prepared in (D) with restriction enzymes EcoRI and MluI, and DNA-20 prepared in (F) are combined with DNA Ligation. By ligating using Kit Mighty Mix (manufactured by Takara Bio Inc.), a mouse MMP-9 signal peptide (SEQ ID NO: 1) is arranged on the N-terminal side, and a His tag consisting of 6 histidines on the C-terminal side. An expression vector pCIP6000H of the present invention containing a polynucleotide (SEQ ID NO: 8) encoding a polypeptide (SEQ ID NO: 5) added to CIP consisting of the amino acid sequence of No. 3 was prepared. A schematic diagram of pCIP6000H is shown in FIG.

(I) Plasmid pCIP6000
The 0.5 kbp polynucleotide (DNA-25) obtained by double digesting the plasmid pCIP1000 prepared in (A) with restriction enzymes HindIII and MluI, and the plasmid pCIP6000H prepared in (H) were converted into the restriction enzymes HindIII and MluI. The 4.9 kp polynucleotide (DNA-26) obtained by double digestion with ligation using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), whereby the amino acid sequence described in SEQ ID NO: 3 An expression vector pCIP6000 of the present invention comprising a polynucleotide (SEQ ID NO: 9) encoding a polypeptide (SEQ ID NO: 4) obtained by adding a mouse MMP-9 signal peptide (SEQ ID NO: 1) to the N-terminal side of CIP consisting of 5402 bp) was produced. A schematic diagram of pCIP6000 is shown in FIG.

(J) Plasmid pCIP6200H2
(J-1) Using an oligonucleotide (Primer-23) consisting of the sequence shown in SEQ ID NO: 48 as a template, an oligonucleotide consisting of the sequence shown in SEQ ID NO: 49 (Primer-24) and the sequence shown in SEQ ID NO: 50 PCR using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using the oligonucleotide (Primer-25) as a primer set and purifying the amplified product using agarose gel electrophoresis to produce a 0.07 kbp oligonucleotide (DNA-27) was obtained.
(J-2) Using a plasmid pCIP-2 (synthesized by OPERON Biotechnologies) as a template, an oligonucleotide (Primer-26) consisting of the sequence shown in SEQ ID NO: 51 and an oligonucleotide (Primer) consisting of the sequence shown in SEQ ID NO: 52 PCR is performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using -27) as a primer set, and the amplified product is purified using agarose gel electrophoresis to obtain a 0.58 kbp polynucleotide (DNA-28) Got.
(J-3) Using DNA-27 obtained in (J-1) and DNA-28 obtained in (J-2) as templates, Primer-24 (SEQ ID NO: 49) and Primer-27 (SEQ ID NO: 52) ) Was used as a primer set, and PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) to obtain an amplification product (polynucleotide) (DNA-29) of 0.6 kbp.
(J-4) Polynucleotide obtained by double digesting DNA-29 obtained in (J-3) with restriction enzymes EcoRI and EcoRV, and pCIP-2 double digested with restriction enzymes EcoRV and MluI The 0.9 kp polynucleotide (DNA-30) obtained in this way and the DNA-20 prepared in (F) were ligated using DNA Ligation Kit Mighty Mix (manufactured by TAKARA BIO INC.) To obtain an N-terminus. A polypeptide (SEQ ID NO: 5) having a mouse MMP-9 signal peptide (SEQ ID NO: 1) on the side and a His tag consisting of 6 histidines on the C-terminal side added to the CIP consisting of the amino acid sequence shown in SEQ ID NO: 3, respectively. The expression vector pCIP6200H2 of the present invention containing the polynucleotide (SEQ ID NO: 10) encoding) was prepared. A schematic diagram of pCIP6200H2 is shown in FIG.

(K) Plasmid pCIP6200-2
The plasmid pCIP1200 prepared in (C) was double-digested with restriction enzymes HindIII and MluI, and the 0.5 kp polynucleotide (DNA-31) obtained by double digestion with the plasmids pCIP6200H2 prepared in (J) was converted into the restriction enzymes HindIII and MluI. The 4.9 kp polynucleotide (DNA-32) obtained by double digestion with ligation using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), whereby the amino acid sequence described in SEQ ID NO: 3 The expression vector pCIP6200- of the present invention comprising a polynucleotide (SEQ ID NO: 11) encoding a polypeptide (SEQ ID NO: 4) obtained by adding a mouse MMP-9 signal peptide (SEQ ID NO: 1) to the N-terminal side of CIP consisting of 2 was produced. A schematic diagram of pCIP6200-2 is shown in FIG.

(L) Plasmid pCIP9200H
(L-1) Using an oligonucleotide (Primer-28) consisting of the sequence described in SEQ ID NO: 53 as a template, an oligonucleotide (Primer-29) consisting of the sequence described in SEQ ID NO: 54 and the sequence described in SEQ ID NO: 55 PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using the oligonucleotide (Primer-30) as a primer set to obtain a 0.07 kbp oligonucleotide (DNA-33).
(L-2) PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) using the plasmid pCIP1200 prepared in (C) as a template, Primer-26 (SEQ ID NO: 51) and Primer-27 (SEQ ID NO: 52) as a primer set. PCR was performed to obtain a 0.58 kbp oligonucleotide (DNA-34).
(L-3) DNA-33 obtained in (L-1) and DNA-34 obtained in (L-2) were used as templates, and Primer-29 (SEQ ID NO: 54) and Primer-27 (SEQ ID NO: 52) ) Was used as a primer set and PCR was performed using PrimeSTAR HS DNA polymerase (manufactured by Takara Bio Inc.) to obtain a 0.6 kbp polynucleotide (DNA-35).
(L-4) Polynucleotide (DNA-36) obtained by double digesting DNA-35 obtained in (L-3) with restriction enzymes EcoRI and EcoRV, and DNA- prepared in (J-4) 30 and DNA-20 prepared in (F) were ligated using DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.), so that the signal peptide of Chinese hamster MMP-9 (SEQ ID NO: 2) was placed on the N-terminal side. And a polynucleotide (SEQ ID NO: 13) encoding a polypeptide (SEQ ID NO: 7) in which a His tag consisting of 6 histidines is added to the CIP consisting of the amino acid sequence shown in SEQ ID NO: 3 on the C-terminal side. Thus, the expression vector pCIP9200H of the present invention was prepared. A schematic diagram of pCIP9200H is shown in FIG.

(M) Plasmid pCIP9200
A 0.9 kbp polynucleotide (DNA-37) obtained by double digesting the plasmid pCIP1200 prepared in (C) with restriction enzymes EcoRV and MluI, DNA-36 prepared in (L-4), ( The signal peptide of Chinese hamster MMP-9 to CIP consisting of the amino acid sequence of SEQ ID NO: 3 by ligating DNA-20 prepared in F) with DNA Ligation Kit Mighty Mix (manufactured by Takara Bio Inc.) An expression vector pCIP9200 of the present invention containing a polynucleotide (SEQ ID NO: 12) encoding a polypeptide (SEQ ID NO: 6) added with (SEQ ID NO: 2) on the N-terminal side was prepared. A schematic diagram of pCIP9200 is shown in FIG.

Example 2 Introduction into CHO cells and secretion of CIP (Part 1)
(1) Among the plasmids prepared in Example 1, (F) pCIP4000H, (G) pCIP5000H, (H) pCIP6000H was linearized by digestion with the restriction enzyme PvuI, and then FreeStyle MAX reagent (manufactured by Life Technologies) Was introduced into DG44 cells, which are Chinese hamster ovary cells (CHO cells).
(2) The DG44 cells into which the plasmid was introduced were treated with CD CHO medium (medium 1) containing 8 mM glutamine and 0.18% Pluronic F-68 (manufactured by Life Technologies) at a temperature of 37 ° C. and a CO ₂ concentration of 8 %, And cultured at a rotational shaking speed of 135 rpm for 2 days.
(3) The cultured cells are collected by centrifuging the culture solution at a low speed (900 rpm) and then suspended in a CD OptiCHO medium (medium 2) containing 8 mM glutamine and 0.18% Pluronic F-68 (medium exchange). (First time) The culture was continued for 3 days.
(4) In the same manner as in (3), the cultured cells were collected, suspended in the medium 2 (second medium exchange), and the culture was continued for 3 days.
(5) In the same manner as (3), the cultured cells were collected, suspended in the medium 2 (the third medium exchange), and the culture was continued for 4 days.
(6) The cell density and alkaline phosphatase activity of the cell group in the culture solution on the fourth day of (5) were measured. The alkaline phosphatase activity was quantified using a highly active alkaline phosphatase 13G (manufactured by Biozyme) derived from calf small intestine as a standard. The results are shown in Table 1. Compared with the signal peptide (SEQ ID NO: 14) of CIP itself and the signal peptide (SEQ ID NO: 15) of Cypridina-derived luciferase (hereinafter abbreviated as CYPLF) (Patent Document 7), the signal peptide (sequence of MMP-9 derived from mouse) It can be seen that the secretory production amount of CIP is improved when No. 1) is used.

実施例３ ＣＨＯ細胞への導入およびＣＩＰの分泌（その２）
（１）実施例１で作製したプラスミドのうち、（Ｊ）ｐＣＩＰ６２００Ｈ２、（Ｌ）ｐＣＩＰ９２００Ｈ、（Ｋ）ｐＣＩＰ６２００−２、（Ｍ）ｐＣＩＰ９２００を制限酵素ＰｖｕＩで消化することで直鎖化後、ＦｒｅｅＳｔｙｌｅ ＭＡＸ試薬（ライフテクノロジーズ社製）を使用したリポフェクション法によりチャイニーズハムスター卵巣細胞（ＣＨＯ細胞）であるＤＧ４４細胞に導入した。
（２）プラスミドを導入したＤＧ４４細胞を、８ｍＭのグルタミンおよび０．１８％のＰｌｕｒｏｎｉｃ Ｆ−６８（ライフテクノロジーズ社製）を含んだＣＤ ＣＨＯ培地（培地１）で、温度３７℃、ＣＯ_２濃度８％、回転振とう速度１３５ｒｐｍで２日間培養した。
（３）培養液を低速（９００ｒｐｍ）で遠心分離することで培養細胞を回収後、８ｍＭ グルタミンおよび０．１８％ Ｐｌｕｒｏｎｉｃ Ｆ−６８を含んだＣＤ ＯｐｔｉＣＨＯ培地（培地２）に懸濁して（培地交換１回目）、３日間培養を継続した。
（４）（３）と同様にして、培養細胞を回収後、培地２に懸濁して（培地交換２回目）、３日間培養を継続した。
（５）（３）と同様にして、培養細胞を回収後、培地２に懸濁して（培地交換３回目）、４日間培養を継続した。
（６）（３）と同様にして、培養細胞を回収後、培地２に懸濁して（培地交換４回目）、２日間培養を継続した。
（７）（６）の２日目の培養液中の細胞群の細胞密度とアルカリホスファターゼ活性を測定した。なおアルカリホスファターゼ活性の測定は、仔牛小腸由来の高活性アルカリホスファターゼ１３Ｇ（バイオザイム社製）を標準にして定量した。結果を表２に示す。マウス由来のＭＭＰ−９のシグナルペプチド（配列番号１）の代わりにチャイニーズハムスター由来のＭＭＰ−９のシグナルペプチド（配列番号２）を使用しても、ＣＩＰを効率的に分泌生産できることがわかる。

Example 3 Introduction into CHO cells and secretion of CIP (part 2)
(1) Among the plasmids prepared in Example 1, (J) pCIP6200H2, (L) pCIP9200H, (K) pCIP6200-2, (M) pCIP9200 was linearized by digestion with the restriction enzyme PvuI, and then FreeStyle MAX It was introduced into DG44 cells, which are Chinese hamster ovary cells (CHO cells), by a lipofection method using a reagent (Life Technologies).
(2) The DG44 cells into which the plasmid was introduced were treated with CD CHO medium (medium 1) containing 8 mM glutamine and 0.18% Pluronic F-68 (manufactured by Life Technologies) at a temperature of 37 ° C. and a CO ₂ concentration of 8 %, And cultured at a rotational shaking speed of 135 rpm for 2 days.
(3) The cultured cells are collected by centrifuging the culture solution at a low speed (900 rpm) and then suspended in a CD OptiCHO medium (medium 2) containing 8 mM glutamine and 0.18% Pluronic F-68 (medium exchange). (First time) The culture was continued for 3 days.
(4) In the same manner as in (3), the cultured cells were collected, suspended in the medium 2 (second medium exchange), and the culture was continued for 3 days.
(5) In the same manner as (3), the cultured cells were collected, suspended in the medium 2 (the third medium exchange), and the culture was continued for 4 days.
(6) In the same manner as (3), the cultured cells were collected and suspended in the medium 2 (the fourth medium exchange), and the culture was continued for 2 days.
(7) The cell density and alkaline phosphatase activity of the cell group in the culture medium on the second day of (6) were measured. The alkaline phosphatase activity was quantified using a highly active alkaline phosphatase 13G (manufactured by Biozyme) derived from calf small intestine as a standard. The results are shown in Table 2. It can be seen that CIP can be efficiently secreted and produced by using a Chinese hamster-derived MMP-9 signal peptide (SEQ ID NO: 2) instead of the mouse-derived MMP-9 signal peptide (SEQ ID NO: 1).

実施例４ ＣＩＰの生産（その１）
実施例２で検討した細胞のうち、（Ｈ）ｐＣＩＰ６０００Ｈを導入したＣＨＯ細胞を用いてアルカリホスファターゼの生産を試みた。
（１）実施例２に記載の方法に基づき（Ｈ）ｐＣＩＰ６０００ＨをＤＧ４４細胞に導入し、培地２を用いて増殖させた細胞群を限界希釈することで単クローン化し、細胞株＃４を得た。
（２）（１）で得られた細胞株＃４を濃度２５ｎＭから５００ｎＭのメトトレキサート（ＭＴＸ）を含んだ培地２で４日間培養後、培養液を低速（９００ｒｐｍ）で遠心分離することで細胞を回収し、各濃度のＭＴＸを含んだ培地２に懸濁して（培地交換１回目）、４日間培養を継続した。
（３）再度、各濃度のＭＴＸを含んだ培地２に交換（培地交換２回目）を行ない、さらに５日間培養した。
（４）ＭＴＸ濃度２５ｎＭで培養された細胞群から限界希釈による単クローン化を行ない、細胞株＃２５−２２を得た。
（５）（４）で得られた細胞株＃２５−２２を濃度７５ｎＭから５００ｎＭのＭＴＸを含んだ培地２で４日間培養後、培養液を低速（９００ｒｐｍ）で遠心分離して細胞を回収し、各濃度のＭＴＸを含んだ培地２に懸濁して（培地交換１回目）、６日間培養を継続した。
（６）再度、各濃度のＭＴＸを含んだ培地２に交換（培地交換２回目）を行ない、さらに６日間培養した。
（７）ＭＴＸ濃度７５ｎＭで培養された細胞群から限界希釈による単クローン化を行ない、細胞株＃７５−１３を得た。
（８）（７）で得られた細胞株＃７５−１３を培地２で培養し、３日から５日毎に培養液を採取し、採取した容量と同容量の培地２を加えて、培養を継続した。
（９）採取した培養液を１００００×ｇで２０分間遠心分離することで上清を回収した。
（１０）培養上清１．７Ｌに含まれるアルカリホスファターゼ活性を測定した。

Example 4 Production of CIP (Part 1)
Among the cells examined in Example 2, production of alkaline phosphatase was attempted using CHO cells into which (H) pCIP6000H was introduced.
(1) Based on the method described in Example 2, (H) pCIP6000H was introduced into DG44 cells, and a cell group grown using medium 2 was subjected to single dilution to obtain a cell line # 4. .
(2) After culturing the cell line # 4 obtained in (1) for 4 days in medium 2 containing methotrexate (MTX) at a concentration of 25 nM to 500 nM, the cells are obtained by centrifuging the culture solution at low speed (900 rpm). The cells were collected and suspended in the medium 2 containing MTX at each concentration (the first medium exchange), and the culture was continued for 4 days.
(3) The medium 2 containing each concentration of MTX was replaced again (second medium replacement), and further cultured for 5 days.
(4) Monocloning was performed by limiting dilution from a cell group cultured at an MTX concentration of 25 nM to obtain a cell line # 25-22.
(5) After culturing the cell line # 25-22 obtained in (4) for 4 days in medium 2 containing MTX at a concentration of 75 nM to 500 nM, the culture is centrifuged at a low speed (900 rpm) to recover the cells. The suspension was suspended in the medium 2 containing MTX at each concentration (the first medium exchange), and the culture was continued for 6 days.
(6) The medium 2 containing each concentration of MTX was replaced again (second medium replacement), and further cultured for 6 days.
(7) Monocloning was performed by limiting dilution from a cell group cultured at an MTX concentration of 75 nM to obtain a cell line # 75-13.
(8) The cell line # 75-13 obtained in (7) is cultured in the medium 2, the culture solution is collected every 3 to 5 days, the medium 2 having the same volume as the collected volume is added, and the culture is performed. Continued.
(9) The supernatant was recovered by centrifuging the collected culture at 10000 × g for 20 minutes.
(10) The alkaline phosphatase activity contained in 1.7 L of the culture supernatant was measured.

  As a result of quantitative determination using calf small intestine-derived highly active alkaline phosphatase 13G (manufactured by Biozyme) as a standard, the production amount of alkaline phosphatase was calculated to be 20 mg.

Example 5 Purification of CIP (Part 1)
Attempts were made to purify alkaline phosphatase produced in Example 4.
(1) To 1.3 L of the culture supernatant obtained in Example 4 (9), 5.2 g of activated carbon was added, stirred and mixed overnight at 4 ° C., and then centrifuged at 10000 × g for 10 minutes to obtain the supernatant. It was collected.
(2) The supernatant was filtered through a 0.22 μm nitrocellulose filter (culture supernatant fraction) and dialyzed against Tris buffer (20 mM Tris-HCl (pH 7.5)).
(3) The dialysate was applied to a HiPrep DEAE FF column (manufactured by GE Healthcare), and then alkaline phosphatase was eluted with a salt concentration gradient from 0 to 1 M NaCl.
(4) After adding ammonium sulfate to a fraction having alkaline phosphatase activity to a concentration of 1.2M, it was applied to HiTrap Phenyl HP (manufactured by GE Healthcare) and a salt with an ammonium sulfate concentration of 1.2M to 0M. Alkaline phosphatase was eluted with a concentration gradient.
(5) After dialyzing the fraction having alkaline phosphatase activity (Phenyl recovered fraction-1) with Tris buffer, the dialysate was applied to HiTrap DEAE FF (manufactured by GE Healthcare), and the NaCl concentration was adjusted from 0M to 1M. Alkaline phosphatase was eluted with a salt gradient.
(6) A fraction having alkaline phosphatase activity (DEAE recovered fraction-1) was applied to TSKgel G3000SWXL (manufactured by Tosoh Corporation) to elute alkaline phosphatase.
(7) A fraction having alkaline phosphatase activity (G3000 recovered fraction-1) was applied to HiTrap DEAE FF, and eluted with a salt concentration gradient from NaCl concentration 0 M to 1 M, and a fraction having alkaline phosphatase activity was recovered. (DEAE collection fraction-2).

  The result of SDS-PAGE analysis of each fraction having alkaline phosphatase activity is shown in FIG. In FIG. 8, lane M is a protein marker (manufactured by New England Biolabs, catalog number P7708S), lane number 1 is a culture supernatant fraction, lane number 2 is a phenyl recovery fraction-1, Number 3 is DEAE recovered fraction-1 and lane number 4 is G3000 recovered fraction-1.

  In addition, the alkaline phosphatase contained in DEAE recovery fraction-2 was 2.9 mg as a result of quantification using calf small intestine-derived highly active alkaline phosphatase 13G (manufactured by Biozyme) as a standard.

Example 6 Production of CIP (Part 2)
Among the cells examined in Example 3, production of alkaline phosphatase was attempted using (K) CHO cells into which pCIP6200-2 was introduced.
(1) Based on the method described in Example 3, (K) pCIP6200-2 was introduced into DG44 cells, a cell group grown using medium 2 was subjected to limiting dilution, and cell line # 11 was isolated. Obtained.
(2) The cell line # 11 obtained in (1) was cultured in the medium 2, the culture solution was collected every 3 to 5 days, the medium 2 having the same volume as the collected volume was added, and the culture was continued. .
(3) The supernatant was collected by centrifuging the collected culture at 10000 × g for 20 minutes.
(4) The alkaline phosphatase activity contained in 8 L of the culture supernatant was measured.

  As a result of quantitative determination using calf small intestine-derived highly active alkaline phosphatase 13G (manufactured by Biozyme) as a standard, the production amount of alkaline phosphatase was calculated to be 8 mg.

Example 7 Purification of CIP (Part 2)
Attempts were made to purify the alkaline phosphatase produced in Example 6.
(1) 32 g of activated carbon was added to 8 L of the culture supernatant obtained in Example 6 (3), stirred and mixed overnight at 4 ° C., and then centrifuged at 10,000 × g for 10 minutes to collect the supernatant.
(2) The supernatant was filtered through a 0.22 μm nitrocellulose filter and then concentrated with an ultrafiltration membrane pericon XL Biomax-50 (manufactured by Millipore) (culture supernatant concentrated fraction).
(3) The concentrated solution was applied to HiPrep DEAE FF (manufactured by GE Healthcare), and alkaline phosphatase was eluted with a salt concentration gradient from NaCl concentration 0M to 1M.
(4) After adding ammonium sulfate to a fraction having alkaline phosphatase activity (DEAE recovered fraction-3) to a concentration of 1.2 M, it was applied to HiTrap Butyl HP (manufactured by GE Healthcare) and an ammonium sulfate concentration of 1.2 M Alkaline phosphatase was eluted with a salt concentration gradient from 1 to 0M.
(5) The fraction having alkaline phosphatase activity (Butyl recovery fraction-1) was dialyzed with Tris buffer, dialyzed solution was applied to HiTrap DEAE FF (manufactured by GE Healthcare), and NaCl concentration from 0M to 1M Alkaline phosphatase was eluted with a salt gradient.
(6) After adding ammonium sulfate to a fraction having alkaline phosphatase activity to a concentration of 1.2 M, it was applied to HiTrap Phenyl HP (manufactured by GE Healthcare), and with a salt concentration gradient from 1.2 M to 0 M ammonium sulfate concentration. Alkaline phosphatase was eluted.
(7) The fraction having alkaline phosphatase activity (Phenyl recovered fraction-2) is dialyzed with Tris buffer, and the dialysate is applied to HiTrap DEAE FF (manufactured by GE Healthcare), with a NaCl concentration of 0M to 1M. Alkaline phosphatase was eluted with a salt gradient.
(8) A fraction having alkaline phosphatase activity (DEAE recovered fraction-4) was applied to TSKgel G3000SWXL (manufactured by Tosoh Corporation) to elute alkaline phosphatase.
(9) A fraction having alkaline phosphatase activity (G3000 recovered fraction-2) was applied to HiTrap DEAE FF, and eluted with a salt concentration gradient from NaCl concentration 0 M to 1 M, and a fraction having alkaline phosphatase activity was recovered. (DEAE recovered fraction-5).

  The results of SDS-PAGE analysis of each fraction having alkaline phosphatase activity are shown in FIG. In FIG. 9, lane M is a protein marker (manufactured by New England Biolabs, catalog number P7708S), lane number 1 is a culture supernatant concentrated fraction, lane number 2 is a phenyl recovery fraction-2. Lane number 3 is DEAE collection fraction-3 and lane number 4 is G3000 collection fraction-2.

  In addition, the alkaline phosphatase contained in DEAE recovery fraction-5 was quantified with a highly active alkaline phosphatase 13G (manufactured by Biozyme) derived from calf small intestine as a standard.

Example 8
10 μg each of alkaline phosphatase obtained in Examples 5 and 7 was separated by SDS-PAGE, a band corresponding to alkaline phosphatase was cut out according to a conventional method, transferred to a PVDF (polyvinylidene fluoride) membrane, and then from the N-terminal side. The amino acid sequence of 5 residues was analyzed.

  As a result, both were Leu-Ile-Pro-Ala-Glu (SEQ ID NO: 56), which was consistent with the N-terminal sequence described in SEQ ID NO: 3.

Claims (7)

A polynucleotide encoding alkaline phosphatase;
An oligonucleotide encoding a signal peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 2 linked in-frame to the 5 ′ end of the polynucleotide;
An expression vector for secretory production of alkaline phosphatase in mammalian cells. The expression vector according to claim 1 , wherein the alkaline phosphatase is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 3. The expression vector according to claim 2 , comprising a polynucleotide encoding a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs: 4 to 7. The expression vector according to claim 3 , comprising a polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 8 to 13. A cell capable of secretory production of alkaline phosphatase obtained by transfecting a mammalian cell with the expression vector according to any one of claims 1 to 4 . The cell according to claim 5 , wherein the mammalian cell is a Chinese hamster ovary cell. A method for producing alkaline phosphatase, wherein the cells according to claim 5 or 6 are cultured, and alkaline phosphatase is recovered from the obtained culture broth.

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