Method for synthesizing secretory protein

Abstract

To provide a novel technique capable of stably and efficiently producing a secretory protein that exhibits activity in vivo.SOLUTION: The present invention for solving the above problem is a method for producing a secretory protein, the method comprising introducing an adenoviral vector encoding the secretory protein into a malignant tumor-derived cultured cell line.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] The present invention relates to a method for biosynthesis of a secretory protein. [Background technology]

[0002] In vivo nerve growth factors and angiogenic factors are functionally active molecules whose expression in the body is strictly controlled in time and space. When this control starts to break down, it causes various diseases, but it is predicted that supplementing these active molecules will alleviate the diseases to some extent. However, since the protein structure of the active molecules, including their post-translational modifications, is species-specific, there is little evidence that extracting them from other species and administering them to the human body would be beneficial. Human recombinant proteins can be expressed in mammalian cells to obtain molecules equivalent to those in the human body, but it is often difficult to obtain even microgram amounts.

[0003] To overcome the shortage of biomolecules in the human body, it is difficult to synthesize equivalent molecules in large quantities outside the body. This problem affects basic research, the creation of research antibodies, and clinical research. The main factors are the three-dimensional structure and folding of proteins (Non-Patent Document 1), the lack of intracellular auxiliary proteins and folding factors (Non-Patent Document 2), the difficulty of post-translational modification (Non-Patent Document 3), and the complexity and diversity of proteins (Non-Patent Document 4).

[0004] Successful examples include recombinant protein production (Non-Patent Document 5), cell-free translation systems (Non-Patent Document 6), and protein production by synthetic biology (Non-Patent Document 7). However, these technologies have quality and quantity issues. For example, in recombinant protein production, post-translational modification and folding may not be performed properly (Non-Patent Document 8). In cell-free translation systems, post-translational modification and proper folding may not be guaranteed, making mass production difficult (Non-Patent Document 9). In synthetic biology, optimization of gene circuits and metabolic pathways affects the amount of protein produced, and if the gene circuits and metabolic pathways are not efficient, mass production of proteins may be difficult (Non-Patent Document 10). These are synthesis methods that consider the balance between quality and quantity, but it is still considered most effective to use mammalian cells to synthesize protein molecules that are more equivalent to human biomolecules. However, there are also several issues with the synthesis of human physiologically active proteins using mammalian cells. First, production efficiency tends to be low and costs are high, which is due to the slow growth rate of mammalian cells and the difficulty of maintenance and management (Non-Patent Document 11). Next, the selection and stabilization of an appropriate cell line is important, but can be difficult (Non-Patent Document 12). In addition, control of post-translational modifications can be difficult, as different cell lines and culture conditions can result in different glycosylation patterns (Non-Patent Document 13). Protein secretion and recovery also pose challenges, as protein recovery and purification inside and outside the cell can be difficult (Non-Patent Document 14). Furthermore, there is a risk of viral contamination, and strict virus testing and removal processes are required to ensure the safety of the production process (Non-Patent Document 15). As described above, the technology for synthesizing biologically active proteins in vitro has many problems, even for a single expression system. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Anfinsen, CB (1973). Principles that govern the folding of protein chains. Science, 181(4096), 223-230. [Non-Patent Document 2] Hartl, FU, Bracher, A., & Hayer-Hartl, M. (2011). Molecular chaperones in protein folding and proteostasis. Nature, 475(7356), 324-332. [Non-Patent Document 3] Walsh, CT, Garneau-Tsodikova, S., & Gatto, GJ Jr. (2005). Protein posttranslational modifications: the chemistry of proteome diversifications. Angewandte Chemie International Edition, 44(45), 7342-7372. [Non-Patent Document 4] Dill, KA, & MacCallum, JL (2012). The protein-folding problem, 50 years on. Science, 338(6110), 1042-1046. [Non-Patent Document 5] Goeddel, DV, Kleid, DG, Bolivar, F., Heyneker, HL, Yansura, DG, Crea, R., ... & Riggs, AD (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proceedings of the National Academy of Sciences, 76(1), 106-110. [Non-Patent Document 6] Shimizu, Y., Inoue, A., Tomari, Y., Suzuki, T., Yokogawa, T., Nishikawa, K., & Ueda, T. (2001). Cell-free translation reconstituted with purified components. Nature Biotechnology, 19(8), 751-755. [Non-Patent Document 7] Cameron, DE, Bashor, CJ, & Collins, JJ (2014). A brief history of synthetic biology. Nature Reviews Microbiology, 12(5), 381-390. [Non-Patent Document 8] Gupta, SK, & Shukla, P. (2018). Microbial platform technology for recombinant antibody fragment production: A review. Critical Reviews in Microbiology, 44(1), 1-13. [Non-Patent Document 9] Zemella, A., Thoring, L., Hoffmeister, C., & Kubick, S. (2015). Cell-free protein synthesis: pros and cons of prokaryotic and eukaryotic systems. Chembiochem, 16(17), 2420-2431. [Non-Patent Document 10] Nielsen, J., & Keasling, JD (2016). Engineering cellular metabolism. Cell, 164(6), 1185-1197. [Non-Patent Document 11] Wurm, FM (2004). Production of recombinant protein therapeutics in cultivated mammalian cells. Nature biotechnology, 22(11), 1393-1398. [Non-Patent Document 12] Birch, JR, & Racher, AJ (2006). Antibody production. Advanced drug delivery reviews, 58(5-6), 671-685. [Non-Patent Document 13] Stanley, P., & Schachter, H. (2010). Mammalian N-glycan branching and disease. In Essentials of Glycobiology (pp. 215-224). Cold Spring Harbor Laboratory Press. [Non-Patent Document 14] Shukla, AA, & Thommes, J. (2010). Recent advances in large-scale production of monoclonal antibodies and related proteins. Trends in biotechnology, 28(5), 253-261. [Non-Patent Document 15] Merten, OW (2002). Virus contaminations of cell cultures-A biotechnological view. Cytotechnology, 39(2), 91-116. Summary of the Invention [Problem to be solved by the invention]

[0006] When normal cells are used to produce active secretory proteins in vivo, they must be maintained for long periods of time by continuously feeding them expensive culture nutrients due to their low proliferation ability, resulting in high costs and very low production efficiency. Furthermore, when a plasmid encoding a biomolecule is introduced into cultured cells, it is difficult to stably and efficiently produce the biomolecule. In view of the above, an objective of the present invention is to provide a novel technique capable of stably and efficiently producing a secretory protein that exhibits activity in vivo. [Means for solving the problem]

[0007] The present invention that solves the above problems is as follows. [1] A method for producing a secreted protein, comprising introducing an adenovirus vector encoding the secreted protein into a cultured cell line derived from a malignant tumor. [2] The method according to [1], wherein the animal species from which the secretory protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species. [3] The method according to [1] or [2], wherein the secretory protein is derived from a human and the malignant tumor-derived cultured cell line is derived from a human. [4] The method according to any one of [1] to [3], comprising culturing the malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced in a serum-free medium. [5] The secretory protein is secreted from a specific tissue, The malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue. The method according to any one of [1] to [4]. [6] The secretory protein is a growth factor having heparin-binding activity and / or lectin-binding activity, The method according to any one of [1] to [5], comprising purifying the secretory protein from a culture supernatant of a malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced, by affinity chromatography using a carrier carrying heparin and / or lectin. [7] The method according to any one of [1] to [6], wherein the secretory protein is netrin and the malignant tumor-derived cultured cell line is a cultured glioma cell line. [8] A composition comprising the secretory protein produced by the method according to any one of [1] to [7]. [9] The composition described in [8], which is any one of a pharmaceutical composition, a food composition, a cosmetic composition, and an aquaculture feed composition.

[10] The composition described in [8], which is a pharmaceutical composition for treating a disease in which a decreased amount of the secretory protein in the body is observed, comprising the secretory protein as an active ingredient.

[11] The composition described in claim 10, wherein the secretory protein is netrin and the disease is ischemic cerebral infarction, Alzheimer's disease, or Parkinson's disease. Effect of the Invention

[0008] According to the present invention, a secretory protein can be stably produced in large quantities. [Brief description of the drawings]

[0009] [Figure 1] Microscopic photograph (x100) showing the confluent state of human glioma (astrocytoma) U-373MG. [Diagram 2] (Left) The elution fraction of human netrin-1 produced by heparin column chromatography was divided into six parts, and a part of the fraction was subjected to SDS-PAGE (CBB staining) and Western blotting (anti-human netrin-1 antibody) to confirm the purification of the target glycoprotein, human netrin-1. (Right) 1 microgram of netrin-1 purified according to the protocol of the invention after scaling up was confirmed by SDS-PAGE (CBB staining). It was confirmed that secretion and expression into the culture supernatant and purification were possible with good reproducibility. [Diagram 3] FIG. 1 shows the angiogenic activity of human netrin-1 on human umbilical vein endothelial cells (HUVEC) confirmed by migration assay. [Figure 4] The culture supernatant was purified from serum-free culture of a stable expression cell line produced by introducing pCDNA-3.1(+)-human netrin-1 plasmid expression vector into the human non-small cell lung cancer cell line H1299, and confirmed by SDS-PAGE (CBB staining) and Western blotting (anti-human netrin-1 antibody) as in Figure 2. Impurities can be confirmed in the purified protein (CBB staining) compared to the adenovirus expression system using U373-MG cells. [Diagram 5] This figure shows that human netrin-1 expression vectors using pcDNA3.1(+) plasmid were transfected into various malignant tumor cells, and expression was confirmed in the same manner as in the test in Figure 4. In actual human tissues, netrin-1 is often expressed in the large intestine in addition to the brain, but is hardly expressed in colon cancer cell lines HCT116 and LS174T, etc. Depending on the cell line, the gene introduction rate by transfection is overwhelmingly lower than that of adenovirus. [Figure 6] FIG. 1 shows the results of confirmation tests of the kinase activities of Src, Akt and MAPK, which demonstrate that the produced human netrin-1 has survival signaling activity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention relates to a method for producing a secretory protein. Specifically, an adenovirus vector encoding a secretory protein is introduced into a cultured cell line derived from a malignant tumor, and the secretory protein is produced in the cell line. Since the secretory protein is secreted into the culture supernatant of the cell line, the secretory protein can be obtained by purifying the culture supernatant.

[0011] When normal cells are used to synthesize secretory proteins in vivo, they have low proliferation ability and must be maintained for long periods of time by continuously feeding them expensive culture nutrients, resulting in high costs and very low production efficiency. In order to solve such problems, the present invention uses a cultured cell line derived from a malignant tumor. Unlike normal cells, the established cultured cell line derived from a malignant tumor has a significantly faster proliferation rate and proliferates three-dimensionally without the occurrence of contact inhibition, which is the proliferation control caused by cell contact. Therefore, according to the present invention, it is possible to realize the mass synthesis of proteins stably and at low cost.

[0012] Furthermore, in order to synthesize active proteins in vivo, it is necessary to reproduce the post-translational modifications that occur in vivo, such as glycosylation and disulfide bonds, but this cannot be reproduced in a protein synthesis system using E. coli. The present invention uses cell lines, rather than E. coli, for the synthesis of secreted proteins, and is capable of producing proteins that are active in vivo.

[0013] In addition, when culturing normal human cells for a long period of time, the risk of viral infection increases, as normal cells are highly susceptible to viral infection, and once infected, their proliferation ability is significantly affected. To overcome this problem, the present invention uses an adenovirus vector having a strong expression promoter in a malignant tumor cell line with high proliferation ability for biomolecule synthesis, thereby reducing the effects of viral infection.

[0014] As described above, by using an adenovirus vector and a malignant tumor cell line for the synthesis of secreted proteins, the present invention makes it possible to improve production efficiency, reduce costs, stabilize culture, control specific post-translational modifications, and reduce the risk of infection.

[0015] It is known that post-translational modifications such as glycosylation have species specificity. Therefore, it is preferable that the animal species from which the secretory protein to be introduced by the adenovirus vector is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species. This allows the production of a secretory protein derived from the same animal species that has activity in the living body of the same animal species.

[0016] The type of the malignant tumor cell line in the present invention is not particularly limited. For example, the following cell lines can be mentioned as human malignant tumor cell lines: Brain and nervous system tumor cell lines, such as glioma cell lines, such as U-373MG, U-343MG, NCH421K, and NCH644, astrocytoma cell lines, such as CCF-STTG1, and neuroblastoma cell lines, such as SK-N-LO; breast cancer cell lines such as BT-20, COLO-824, and ZR-75-1; Intestinal and colon cancer cell lines, such as colon adenocarcinoma cell lines, such as Caco-2, COLO-205, and duodenal adenocarcinoma cell lines, such as HuTu-80; bladder cancer cell lines such as 5637, ECV-304, and HB-CLS-1; Skin cancer cell lines such as cutaneous melanoma cell lines such as A-375, A-431, cutaneous sarcoma cell lines such as HS1-CLS, dermal fibroblast-like cell lines such as WS1; Hepatoma cell lines, such as epithelial hepatoma cell lines, such as Hep-G2, PLC-PRF-5, and endothelial hepatoma cell lines, such as SK-HEP-1; leukemia cell lines such as Burkitt's lymphoma cell lines such as EB1, acute lymphocytic leukemia (ALL) cell lines such as CCRF-CEM, erythroblastic leukemia cell lines such as HEL-92.1.7, acute myeloid leukemia (AML) cell lines such as HL-60, T-cell lymphoma cell lines such as HSB, B-lymphocyte cell lines such as RPMI-1788, monocytic leukemia cell lines such as THP-1, lymphoblastoid cell lines such as TK6; submandibular gland adenoma cell lines such as A64-CLS, thyroid cancer cell lines such as C-643, Struma sarcoma cell lines such as CLS-117, oral cancer cell lines such as CLS-354, esophageal cancer cell lines such as COLO-680N, pharyngeal cancer cell lines such as Detroit-562, HNSCC (oropharynx, tonsil) cell lines such as HNO41, HNSCC (oral cavity; tongue) cell lines such as HNO97, HNSCC (larynx) cell lines such as HNO210, HNSCC (tongue) cell lines such as HNO223, nasal septum carcinoma cell lines such as RPMI-2650, head and neck cancer cell lines (larynx, pharynx, nasal cavity, thyroid) such as retinoblastoma cell lines such as Y-79; Lung cancer cell lines such as A-427, A-549, Cal-1, CLS-54, small cell lung cancer cell lines such as DMS-79, lung histiocytoma cell lines such as GCT, lung fibroblast cell lines such as HEL-299, lung mesothelioma cell lines such as H-Put-1, large cell lung carcinoma cell lines such as LCLC-97TM1; Osteosarcoma cell lines such as Ewing sarcoma cell lines such as CADO-ES1, MHH-ES1, osteosarcoma cell lines such as HOS (TE-85), KHOS-240S, osteochondrosarcoma cell lines such as SW-1353; Gastric cancer cell lines, such as gastric adenocarcinoma cell lines, such as AGS, CLS-145, and gastric cancer cell lines, such as HGC-27, KATO-III; renal cancer cell lines such as renal embryonic cell lines such as 293 (HEK-293), renal cancer cell lines such as 769-P, 786-0, A-498, renal clear cell carcinoma cell lines such as RCC (KTCTL-13), renal (Wilms tumor) cell lines such as SK-NEP-1; adrenal cancer cell lines such as NCI-H295R, Huamun adrenal adenocarcinoma cell lines such as SW-13; pancreatic cancer cell lines such as pancreatic adenocarcinoma cell lines such as AsPC-1, Capan-1, DAN-G, and pancreatic cancer cell lines such as Panc-1; Rhabdomyosarcoma cell lines such as A-204, A-673, and HS-729; soft tissue tumor cell lines such as fibrosarcoma cell lines such as HT-1080, fibrosarcoma cell lines such as SW-684, male liposarcoma cell lines such as SW-872, synovial sarcoma cell lines such as SW-982; Reproductive tract-related tumor cell lines (uterus, placenta, ovary) such as endometrial adenocarcinoma cell lines, e.g. AN3 CA; placental choriocarcinoma cell lines, e.g. BEWO; cervical carcinoma cell lines, e.g. CaSki, DEER-186, CERV-215; ovarian adenocarcinoma cell lines, e.g. NIH:OVCAR-3, OAW-42; ovarian teratocarcinoma cell lines, e.g. PA-1; vulvar leiomyosarcoma cell lines, e.g. SK-LMS-1; uterine leiomyosarcoma cell lines, e.g. SK-UT-1, SK-UT-1B; Prostate cancer cell lines such as DU-145, LNCaP, and PC-3.

[0017] Although cell lines derived from human malignant tumors have been exemplified above, many types of cell lines derived from malignant tumors of other animal species are also commercially available, and these can be used for the present invention without limitation.

[0018] Adenovirus introduction into host cells is mediated by the Coxsackie-Adenovirus Receptor (CAR), and therefore the malignant tumor-derived cell line used is preferably one that expresses the CAR protein.

[0019] Furthermore, even in cell lines that do not express the CAR protein, transduction with an adenoviral vector can be performed by introducing a vector encoding the CAR protein and expressing the CAR protein. A kit for introducing a CAR protein and improving the transduction efficiency of an adenovirus vector is commercially available, and may be used. Examples of such commercially available products include CAR Receptor Booster (Clontech) and ViraDuctin adenovirus gene transfer kit (Cell biolabs).

[0020] The type of secretory protein produced in the present invention is not particularly limited, and may be any protein that is released extracellularly, such as an extracellular matrix protein, a blood protein, a digestive enzyme, or a hormone. The present invention is preferably applied for the production of growth factors or cytokines.

[0021] Examples of secretory proteins include insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), angiopoietin, angiostatin, granulocyte colony stimulating factor (G-CSF), erythropoietin (EPO), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (AFGF), and fibroblast growth factor (GFGF). aFGF, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), insulin growth factor I and II (IGF-I and TGF-II), any one of the transforming growth factor a superfamily including TGFa, activin, inhibin or bone morphogenetic protein (BMP) BMP1-15, any one of the neuregulin family including neuregulin 1-4, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin These include hormones and growth and differentiation factors including NT-3 and NT-4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), Neurturin, Agrin, any one of the Semaphorin / Collapsin family, Netrins including Netrin-1 and Netrin-2, Hepatocyte Growth Factor (HGF), Ephrin, Noggin, Sonic hedgehog (SHH), and the like.

[0022] In the present invention, a preferred embodiment is one in which an adenovirus vector encoding a secretory protein secreted from a specific tissue is introduced into a malignant tumor-derived cultured cell line derived from a malignant tumor in the specific tissue. This makes it possible to mimic the cell type-specific post-translational modifications that occur in vivo, and to produce active secreted proteins.

[0023] In a specific embodiment of the present invention, an adenoviral vector encoding netrin, more specifically netrin-1, is introduced into a glioma-derived cultured cell line. When producing netrin as an active ingredient of a pharmaceutical to be administered to humans, an adenovirus vector encoding human netrin, more specifically human netrin-1, is introduced into a cultured cell line derived from a human glioma.

[0024] Furthermore, it has been confirmed in the Examples that netrin produced and purified by the method of the present invention can be stably stored at 4°C for more than one year while retaining its activity.

[0025] The adenovirus vector encoding the secretory protein used in the present invention can be prepared by a conventional method. For example, the adenovirus vector can be prepared by the COS-TPC method or the full-length DNA transfer method. The COS-TPC method is a method in which a recombinant cosmid incorporating a target cDNA or expression cassette and a parental virus DNA-terminal protein complex (DNA-TPC) are co-transfected into 293 cells, and a recombinant adenovirus is prepared by utilizing homologous recombination that occurs in the 293 cells (Miyake, S., Makimura, M., Kanegae, Y., Harada, S., Takamori, K., Tokuda, C., and Saito, I. (1996) Proc. Natl. Acad. Sci. USA, 93, 1320.). On the other hand, the full-length DNA transfer method is a method in which recombinant adenovirus is produced by restriction digestion of recombinant cosmids into which a gene of interest has been inserted, followed by transfection into 293 cells (Terashima Miho, Kondo Koki, Kanegae Hiromi, Saito Izumi (2003) Experimental Medicine 21(7)931.). The COS-TPC method can be performed using the Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.) and Adenovirus genome DNA-TPC (Takara Bio Inc.). The full-length DNA transfer method can also be performed using the Adenovirus Expression Vector Kit (Dual Version) (Takara Bio Inc.).

[0026] In addition, a FLAG tag or 3xFLAG may be added to the secretory protein to be incorporated into the adenovirus vector, which can facilitate purification using an affinity column or the like. A fluorescent protein such as GFP may also be added. This makes it easy to confirm by a fluorescence microscope that the secretory protein is expressed in cells infected with the adenovirus vector.

[0027] In the present invention, the above-mentioned adenovirus vector is introduced into a cultured cell line derived from a malignant tumor. There is no particular limitation on the method of introduction, and it can be carried out by a conventional method.

[0028] The cultured cells transfected with the adenovirus vector can be cultured by a known method, i.e., in a CO2 incubator using a medium suitable for the malignant tumor-derived cultured cell line used, with the medium being replaced as needed.

[0029] The medium for seeding the cultured cells can be any known medium appropriate for the type of cultured cells, such as D-MEM / F12 medium or glutamine-supplemented RPMI 1640 medium. The medium may be supplemented with reagents commonly used in the art, such as HEPES, EDTA, antibiotics (eg, penicillin, gentamicin, streptomycin / penicillin, etc.), and bovine serum.

[0030] In a preferred embodiment of the present invention, a serum-free medium is used as the medium. By culturing in a serum-free medium, the synthesized protein can be easily collected. Furthermore, when the secretory protein produced by the present invention is provided as a pharmaceutical product, by culturing in a serum-free medium, contamination with biomolecules such as heterologous proteins derived from serum can be prevented, and a protein preparation with guaranteed pharmaceutical quality can be produced.

[0031] The proliferation rate of the malignant tumor-derived cultured cell line used in the present invention is not affected even when cultured in a serum-free medium.

[0032] When a growth factor is used as a secretory protein to be introduced into a cultured cell line derived from a malignant tumor by an adenovirus vector, the growth factor secreted into the culture supernatant exerts autocrine activity, and therefore advantageous effects can be obtained when the cell line is cultured in a serum-free medium. In other words, by using a serum-free medium, it is possible to synthesize a secretory protein without containing exogenous serum components, and in particular, growth factor proteins can continue to provide nutrients to the cells that are synthesizing the protein by autocrine. Therefore, cells infected with an adenovirus vector expressing a target growth factor can continue to synthesize the target protein and survive in a serum-free medium without cell death due to nutrient deficiency in the absence of serum. Regarding the activity of the biosynthesized growth factor, it has been confirmed that a growth signal is transmitted in an experiment in which the generated growth factor was added to cultured cells in a serum-free environment (see Figure 6).

[0033] Since the target protein in the present invention is a secretory protein, the protein synthesized in the above-mentioned malignant tumor-derived cultured cell line is secreted into the culture supernatant. The secretory protein present in the culture supernatant obtained after the culture can be purified by known methods, such as affinity chromatography using a column carrying a substance (specific ligand) having affinity for the target protein, ion exchange chromatography or chromatofocusing for isolating the protein by utilizing surface charging, hydrophobic interaction chromatography for isolating the protein by utilizing its hydrophobicity, or gel filtration chromatography for isolating the protein by its size. The purification may be carried out once or multiple times.

[0034] In a preferred embodiment of the present invention, the secreted protein of interest is purified by affinity chromatography.

[0035] The column to be used can be appropriately selected depending on the secretory protein to be synthesized. For example, when the secretory protein has heparin binding activity, a heparin column can be used as the column.

[0036] There are very few growth factors that do not have heparin-binding activity, except for erythropoietin (EPO) and granulocyte-colony stimulating factor (G-CSF). Therefore, the present invention, which includes a purification step by affinity chromatography using a carrier carrying heparin, can be applied to the production of almost all growth factors including polymer dimers (insulin, VEGF group, HGF group, FGF group, NGF, etc.).

[0037] Furthermore, a lectin column carrying a lectin, which is a sugar-recognizing protein, may be used as the column. Since most proteins secreted outside cells are glycoproteins, a lectin column can be suitably used for purifying the secretory protein produced by the present invention.

[0038] The lectin column may be used in combination with a heparin column, which allows for higher affinity chromatography to be performed, thereby obtaining a highly purified protein.

[0039] The synthesis method according to the present invention may include other known steps in addition to the steps described above.

[0040] The secretory protein produced by the method of the present invention has post-translational modifications such as glycosylation equivalent to those of the same protein active in vivo, and therefore the secretory protein produced by the method of the present invention is preferably incorporated into various compositions to be administered or ingested by a living body. That is, the present invention also relates to a composition containing the secreted protein produced by the above-mentioned method.

[0041] Specific examples of the composition of the present invention include a pharmaceutical composition, a food composition, a cosmetic composition, and a feed composition for aquaculture.

[0042] The pharmaceutical composition of the present invention contains the secretory protein produced by the above-mentioned method as an active ingredient. The pharmaceutical composition of the present invention is preferably used for treating a disease in which a decrease in the amount of the secretory protein in the body is observed.

[0043] For example, when netrin is produced by the method of the present invention, it can be used as an active ingredient in a pharmaceutical composition for treating ischemic cerebral infarction, Alzheimer's dementia, or Parkinson's disease, which are diseases in which a decrease in the amount of netrin secreted is observed.

[0044] The pharmaceutical composition of the present invention is preferably administered via a parenteral route, and may be in the form of, for example, an injection, eye drop, nasal, pulmonary, or transdermal dosage form. The specific formulation for each dosage form can be appropriately designed depending on the applicable disease and the disease site.

[0045] A specific example of the food composition of the present invention is cultured meat, which can be provided in the form of a cultured meat food product containing the protein produced by the above-mentioned method.

[0046] When the present invention is provided in the form of a cosmetic composition, it is preferable to select a growth factor having the effect of regenerating skin tissue and preventing aging as the secretory protein to be blended as an active ingredient. The cosmetic composition is also preferably in the form of an external preparation for skin.

[0047] The animals to which the feed composition for aquaculture of the present invention is administered are not particularly limited, but examples include fish and mammals, and the feed composition for aquaculture of the present invention can be applied to aquaculture and livestock farming. In this case, it is preferable that the animal species from which the secretory protein produced by the method of the present invention is derived, the animal species from which the malignant tumor-derived cultured cell line is derived, and the animal species to which the feed composition for aquaculture of the present invention is administered are the same or closely related species. In addition, it is preferable that the secretory protein to be incorporated in the feed composition for aquaculture of the present invention is a growth factor.

[0048] Furthermore, when the present invention is applied as a composition for experimental reagents, examples of the composition include a form in which the composition is added to a medium when culturing cultured cells or tissues, such as various stem cells such as iPS cells, primary cells, passaged cells, and established cell lines.

[0049] Furthermore, since the secretory protein produced by the method of the present invention has a structure that is active in vivo, it is possible to prepare high-titer antibodies by using this as an antigen. EXAMPLES

[0050] Hereinafter, an example will be given in which netrin-1, a human axon guidance factor and angiogenesis factor, was produced as a secretory protein.

[0051] (1) Cultivation of human malignant tumor-derived cell lines U-373MG cells (ATCC), a human malignant tumor cell line derived from glial cells that express netrin-1 in vivo, were cultured at 3.0 × 10 6 The cells were seeded on eight 15 cm diameter plastic culture dishes. The culture medium used was DMEM containing antibiotics for culture and 10% FBS (20 mL / dish). The seeded cells were cultured at 37°C under 5% CO2 for 24 hours, and once it was confirmed that the cells had settled, they were then infected with the adenovirus vector. (The number of cells at this time was 1.0 × 10 7 It was confirmed in advance that the number of cells was approximately

[0052] (2) Adenovirus vector infection The cells were infected with an adenovirus vector (Adenovirus Dual Expression Kit, Takara Bio) encoding human netrin-1 cDNA (NCBI Gene ID: 9423) at an MOI of 5, and further cultured for 24 hours.

[0053] (3) Serum-free culture and collection of culture supernatant 24 hours after infection, the cells were confirmed to be confluent under a microscope (Figure 1), and the culture medium was aspirated and discarded. The cultured cells were then washed three times with 10 mL PBS(-), and the culture medium was replaced with 30 mL serum-free DMEM (containing antibiotics), and culture was continued at 37°C under 5% CO2. After 72 hours of serum-free culture, the entire serum-free culture medium was collected and stored at 4°C. An additional 30 mL of serum-free culture medium was added and culture was continued. When the cells began to float, the culture medium was similarly collected and stored at 4°C.

[0054] (4) Preparation of culture supernatant containing the target secreted protein The culture supernatant stored at 4°C was subsequently subjected to all operations at 4°C. The culture solution after recovery was centrifuged at 1000×g for 15 minutes, and the supernatant was centrifuged at 10,000×g for 20 minutes. The supernatant after centrifugation was passed through a 0.22 μm filter using a peristaltic pump. The supernatant after passing through the filter was subjected to affinity column chromatography.

[0055] (5) Affinity column purification All of the culture supernatant was pumped onto heparin column #1 using a peristaltic pump and adsorbed at a flow rate of 500 μL / min. After all of the culture supernatant was adsorbed, 30 mL of washing buffer #2 was poured in and washed. After washing, 10 mL of elution buffer #3 was poured in and the eluted fraction was obtained, centrifuged and concentrated using an ultrafiltration membrane (50 kDa or more), and then stored at 4°C. #1 Heparin column: HiTrapTM Heparin HP (Amersham Biosciences 17-0406-01) #2 washing buffer: 10mM phosphate buffer (pH 7.0) #3 elution buffer: 1M NaCl in 10mM phosphate buffer (pH 7.0)

[0056] (6) Quantification, storage and confirmation of purified protein Because the eluted fraction contained a high concentration of salt, it was diluted with DDW and the protein was quantified by the Bradford method. After quantification, the eluted fraction was adjusted to 1 mg / mL with elution buffer and stored at 4°C (it was possible to store it at 4°C for one year while maintaining its activity).

[0057] 20 μg (5 μg for silver staining) was dissolved in 5×SDS-PAGE sample buffer, boiled for 2 minutes, and then subjected to SDS-PAGE on an 8% acrylamide gel, followed by CBB staining (or silver staining). A single band of human netrin-1 protein was confirmed with a molecular weight of approximately 80 kDa (Figure 2). This method produces a 1.0×10 7 Approximately 100-120 μg of human netrin-1 was produced from U373-MG cells (one 15 cm diameter culture dish).

[0058] (7) Confirmation of biomolecule activity Because netrin-1 not only acts as an axon guidance factor but also has angiogenic activity, we performed a migration assay (Park, KW, 2004) using human umbilical vein endothelial cells (HUVECs). As a positive control, human VEGF was used to confirm the activity of netrin-1 for angiogenesis (Figure 3).

[0059] (8) Comparison with expression systems using plasmid-transfected stable expression strains In order to prove that the expression system of the present invention is superior to other currently existing biosynthetic methods using plasmid expression in mammalian cells, a pCDNA-3.1(+) plasmid expression vector encoding human netrin-1 was introduced into the human non-small cell lung cancer cell line H1299, which is easy to create a stable expression line, and a cell line that stably and highly expresses human netrin-1 was established by antibiotic selection.

[0060] As with the above-mentioned U373-MG, the established human netrin-1 stably expressing H1299 cell clone was cultured under serum-free conditions, and human netrin-1 secreted into the culture supernatant was purified (FIG. 4).

[0061] It was found that about 1 / 4 of the amount of U-373MG could be recovered from a similar culture volume, but since cell selection resulted in a decrease in proliferation ability, the plasmid-based expression system cannot be said to be a stable biosynthetic system.

[0062] In addition, we created stable expression lines using plasmids from cell lines derived from malignant tumors of multiple tissues, and confirmed the expression levels. However, we were unable to confirm stable expression in most cells, which suggests the efficiency of forced expression using adenovirus vectors (Figure 5).

[0063] (9) Confirmation of growth factor activity We confirmed whether the netrin-1 produced by the above method has a signal transduction function as a growth factor by examining the kinase activity of Src, Akt, and MAPK. After culturing the human non-small cell lung cancer cell line H1299 in a culture medium containing serum, the culture medium was replaced with a serum-free medium, and after 24 hours of starvation, the produced netrin-1 was added to the culture medium at 500 ng / mL. The cells were frozen and collected over time, and the cell lysates were subjected to SDS-PAGE and Western blotting. When the kinase activity of each was confirmed with a phosphorylation antibody, it was shown that Src and Akt were activated over time after the addition of netrin-1. This demonstrated that the netrin-1 synthesized and purified by adenovirus has survival signaling activity (Figure 6). [Industrial Applicability]

[0064] The present invention can be applied to techniques for producing proteins as active ingredients in medicines, foods, cosmetics, aquaculture feed, and experimental reagents.

Claims

1. A method for producing a secreted protein, comprising introducing an adenovirus vector encoding the secreted protein into a cultured cell line derived from a malignant tumor.

2. The method according to claim 1 , wherein the animal species from which the secretory protein is derived and the animal species from which the malignant tumor-derived cultured cell line is derived are the same animal species.

3. The method according to claim 1 , wherein the secreted protein is derived from a human and the malignant tumor-derived cultured cell line is derived from a human.

4. The method according to claim 1 , comprising culturing the malignant tumor-derived cultured cell line into which the adenoviral vector has been introduced in a serum-free medium.

5. The secretory protein is one that is secreted from a specific tissue, The malignant tumor-derived cultured cell line is derived from a malignant tumor of the specific tissue. The method of claim 1.

6. the secretory protein is a growth factor having heparin-binding activity and / or lectin-binding activity, The method according to claim 1, comprising purifying the secreted protein from the culture supernatant of a malignant tumor-derived cultured cell line into which the adenovirus vector has been introduced by affinity chromatography using a carrier carrying heparin and / or lectin.

7. The method according to claim 1 , wherein the secretory protein is netrin, and the malignant tumor-derived cultured cell line is a glioma cultured cell line.

8. A composition comprising the secreted protein produced by the method according to any one of claims 1 to 7.

9. The composition according to claim 8, which is any one of a pharmaceutical composition, a food composition, a cosmetic composition, an aquaculture feed composition, and a composition for use as a laboratory reagent.

10. The composition according to claim 8, which is a pharmaceutical composition for treating a disease in which a decrease in the amount of the secretory protein in the body is observed, comprising the secretory protein as an active ingredient.

11. The composition according to claim 10, wherein the secretory protein is netrin, and the disease is ischemic cerebral infarction, Alzheimer's dementia, or Parkinson's disease.

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