Method for producing protein

Abstract

Provided is a method for producing a useful protein, the method comprising a step for cultivating, in a dark place, transformed rice into which a polynucleotide encoding a useful protein has been introduced.

Description

Protein Production Methods

[0001] The present invention relates to a method for producing a protein.

[0002] Traditionally, microorganisms or animals have been used to produce proteins, but this has many issues, such as being costly and having the risk of contamination by pathogens, toxins, etc.

[0003] On the other hand, when producing proteins using plants, costs can be kept low because only inorganic salts, water, light, and moderate temperature are required for plant growth, and there is no risk of contamination with pathogens or toxins, and safety is often guaranteed based on past experience as a food source. Also, when proteins are produced from the edible parts of plants, it is possible to directly consume the plants themselves without separating and purifying the produced protein. Furthermore, when proteins are produced from plant seeds, etc., they can be stored for long periods at room temperature.

[0004] While protein production in plants has these advantages, it also has the disadvantage of a lower protein production capacity compared to animals or microorganisms. To overcome this disadvantage, research has been conducted to increase the amount of foreign proteins produced in plants (Non-Patent Documents 1 to 4). Non-Patent Document 1 discloses a method for accumulating foreign proteins in chloroplasts, which have lower protease activity than the cytoplasm. Non-Patent Document 2 describes the relationship between sequences around the translation initiation codon and improved productivity of foreign proteins, and Non-Patent Documents 3 and 4 describe the relationship between terminators and improved productivity of foreign proteins.

[0005] Trends Biotechnol 2006, 24, 426-432J Biosci Bioeng 2010, 109, 170-173Plant Biotechnol 2014, 31, 191- 194Plant Cell Physiol 2010, 51, 328-332

[0006] However, when considering applying the method described in the above document on a commercial scale, a large amount of equipment such as lighting would be required, leaving room for improvement in terms of cost.

[0007] If proteins can be produced in the dark using plants, it will be possible to reduce electricity costs for lighting, etc. An objective of the present invention is to provide a method for producing useful proteins in the dark using rice.

[0008] To solve the above problems, the inventors conducted extensive research and found that a useful protein can be produced by cultivating transformed rice in the dark. The present invention was completed based on these findings and includes the following broad aspects. [Item 1] A method for producing a useful protein, comprising cultivating transformed rice into which a polynucleotide encoding a useful protein has been introduced in the dark. [Item 2] The production method according to Item 1, wherein the cultivation period in the dark is 4 days or more. [Item 3] The production method according to Item 1 or 2, wherein the cultivation period in the light is 15% or less of the total cultivation period. [Item 4] The production method according to any one of Items 1 to 3, wherein the transformed rice is cultivated in a medium containing at least one compound selected from the group consisting of a nitrogen-containing compound, a potassium-containing compound, a sulfur-containing compound, and a sugar. [Item 5] The production method according to any one of Items 1 to 4, wherein the useful protein is at least one compound selected from the group consisting of GFP, lysostaphin, and cholera toxin B subunit. [Item 6] A therapeutic drug for bovine mastitis, comprising lysostaphin produced by the method of any one of Items 1 to 4. [Item 7] A method for producing a therapeutic drug for bovine mastitis, comprising the step of producing lysostaphin by the method of any one of Items 1 to 4. [Item 8] A vaccine, comprising the cholera toxin B subunit produced by the method of any one of Items 1 to 4. [Item 9] A method for producing a vaccine, comprising the step of producing cholera toxin B subunit by the method of any one of Items 1 to 4.

[0009] According to the present invention, useful proteins can be produced in the dark using rice, thereby enabling the production of useful proteins at low cost.

[0010] A schematic diagram of the structure of the pBsGFP vector is shown. RB: right border, NPTII: kanamycin resistance gene, UBI pro: ubiquitin promoter, GFP: green fluorescent protein gene, NOS ter: nopaline synthase gene terminator, HPT: hygromycin resistance gene, LB: left border. A schematic diagram of the structure of the pBP3DLYS vector is shown. LB: left border, HPT: hygromycin resistance gene, 3D pro: RAmy3D promoter, 3D: RAmy3D exocrine signal, Lys: lysostaphin gene, 3D ter: RAmy3D terminator, RB: right border. A schematic diagram of the structure of the pB35SspLYS vector is shown. RB: right border, NPTII: kanamycin resistance gene, 35S pro: cauliflower mosaic virus 35S promoter, 3D: RAmy3D exocytic signal, Lys: lysostaphin gene, NOS ter: nopaline synthase gene terminator, HPT: hygromycin resistance gene, LB: left border. A schematic diagram of the structure of the pBspCTB vector is shown. RB: right border, NPTII: kanamycin resistance gene, Ubi pro: ubiquitin promoter, 3A: RAmy3A exocytic signal, H: 6xHis tag, CTB: cholera toxin B subunit gene, HSP ter: heat shock protein terminator, HPT: hygromycin resistance gene, LB: left border. A schematic diagram of the structure of the pBcpCTB vector is shown. RB: right border, NPTII: kanamycin resistance gene, Ubi pro: ubiquitin promoter, cp: chloroplast targeting signal, H: 6x His tag, CTB: cholera toxin B subunit gene, HSP ter: heat shock protein terminator, HPT: hygromycin resistance gene, LB: left border. The figure shows the amount of GFP protein per total soluble protein of the pBsGFP line. For rice plants 12 days after germination, the figures show bands observed in Western blotting using 10 μg of total soluble protein, and the relative intensity of each band in Western blotting. For both water and sucrose + MS medium, the band intensity is shown as a relative value, with the intensity in bright light set at 1. The figure shows the amount of GFP protein per individual of the pBsGFP line.The figures show the relative intensities of bands observed in Western blotting using 15 μl of protein extract, CBB staining, and Western blotting for rice plants on the 12th day after germination. The band intensities under each cultivation condition are shown as relative values, with water in bright light set to 1. The figures show the results of Western blotting and CBB staining for the pBP3DLYS1 line. PC: positive control. The figures show the amount of LYS (lysostaphin) produced by the pBP3DLYS1 line. The amount of LYS produced is calculated by relative values ​​of the intensity of each Western blotting band to the signal of the positive control (PC), which is 40 ng of LYS in the lane on the far right. The figures show the percentage of LYS in the total soluble protein amount of the pBP3DLYS1 line. The figures show the results of Western blotting and CBB staining for the pB35SspLYS line. PC: positive control. The figures show the amount of LYS produced by the pB35SspLYS line. The amount of LYS produced was calculated by relative measurement of the density of each Western blotting band to the signal of the positive control (PC), 10 ng of LYS, in the lane at the far right. The percentage of LYS in the total soluble protein amount for the pB35SspLYS strain is shown. The results of Western blotting and CBB staining for the pBspCTB strain are shown. PC: positive control. The amount of CTB (cholera toxin B subunit) produced by the pBspCTB strain is shown. The amount of CTB produced was calculated by relative measurement of the density of each Western blotting band to the signal of the positive control (PC), 10 ng of CTB, in the lane at the far right. The percentage of CTB in the total soluble protein amount for the pBspCTB strain is shown. The results of Western blotting and CBB staining for the pBcpCTB strain are shown. PC: positive control. The amount of CTB (cholera toxin B subunit) produced by the pBcpCTB strain is shown. The CTB production amount was calculated by calculating the density of each band in Western blotting relative to the signal of the positive control (PC), which is 10 ng of CTB in the rightmost lane. The percentage of CTB in the total soluble protein amount of the pBcpCTB strain is shown. A schematic diagram of the construction of the pR3D-LYS vector is shown.RB: right border, UBI pro: ubiquitin promoter, 3D: RAmy3D exocytosis signal, Lys: lysostaphin gene, HSP ter: heat shock protein gene terminator, HPT: hygromycin resistance gene, LB: left border. A schematic diagram of the structure of the pR3D-LYS vector is shown. RB: right border, UBI pro: ubiquitin promoter, 3D: RAmy3D exocytosis signal, Lys: lysostaphin gene, KDEL: endoplasmic reticulum retention sequence, HSP ter: heat shock protein gene terminator, HPT: hygromycin resistance gene, LB: left border. A schematic diagram of the structure of the pR3D-LYS vector is shown. RB: right border, UBI pro: ubiquitin promoter, cp: Rubisco small subunit chloroplast targeting sequence, Lys: lysostaphin gene, HSP ter: heat shock protein gene terminator, HPT: hygromycin resistance gene, LB: left border. This shows the amount of lysostaphin and the results of Western blotting and CBB staining in Example 4. WT: wild-type rice. The graph shows the amount of lysostaphin produced per fresh weight, calculated from the relative value of the density of each band in Western blotting to the signal of the positive control (LYS 50 ng, 30 ng, and 10 ng) in the rightmost lane. The average amount of lysostaphin in Example 4 is shown. "**" indicates a significant difference at the 1% level.

[0011] As used herein, the singular forms (a, an, the, etc.) include both the singular and the plural unless otherwise specified herein or clearly contradictory in context. As used herein, "comprise" is a concept that also encompasses "consist essentially of" and "consist of."

[0012] The present invention provides a method for producing a useful protein. The method of the present invention comprises the step of cultivating in the dark transformed rice plants into which a polynucleotide encoding a useful protein has been introduced.

[0013] The rice variety used in the present invention is not particularly limited, and examples thereof include Kasalath, Nipponbare, Yamadanishiki, and Kusayutaka. The rice transformed for the present invention may be a wild species or a cultivated species. It may also be a genetically modified or genome-edited version of these.

[0014] The transformed rice of the present invention is a rice plant transformed with a polynucleotide encoding a useful protein, enabling the plant to produce the useful protein. Examples include rice plants genetically modified by recombinant DNA technology to be able to produce the useful protein, and rice plants modified to produce the useful protein by introducing a viral vector. The polynucleotide encoding the useful protein is preferably introduced into rice as a nucleic acid molecule (e.g., T-DNA, plasmid, etc.) containing the polynucleotide encoding the useful protein. The polynucleotide encoding the useful protein is not particularly limited and may be derived from an organism other than rice or may be artificially produced. The artificially produced polynucleotide may be, for example, a polynucleotide of a chimeric gene in which two or more genes are joined together, or a polynucleotide of a mutant gene in which a gene possessed by an organism is mutated. The mutant gene may be, for example, one in which some bases in the base sequence have been deleted or substituted. Furthermore, the base sequence may also be one in which a partial base sequence has been inserted.

[0015] To overexpress a useful protein in rice, it is preferable to functionally link a polynucleotide encoding the useful protein downstream of a promoter in a nucleic acid molecule containing the polynucleotide encoding the useful protein. Examples of such promoters include, but are not limited to, the 35S promoter of cauliflower mosaic virus (CaMV), the rice amylase gene RAmy3D promoter, the maize ubiquitin promoter, and the rice actin promoter.

[0016] Alternatively, a useful protein can be overexpressed to accumulate in a specific organelle such as the chloroplast or endoplasmic reticulum, or to be secreted extracellularly. To this end, a suitable signal peptide can be added to a nucleic acid molecule containing a polynucleotide encoding the useful protein. Examples of signal peptides include the RAmy3D exocrine signal peptide, the RAmy3A exocrine signal peptide, the chloroplast targeting signal peptide, the KDEL endoplasmic reticulum localization sequence, the CIN1 invertase, and the signal peptide of the DUF26-like 33KD secretory protein.

[0017] Examples of terminators to be linked downstream of a polynucleotide encoding a useful protein include, but are not limited to, the Agrobacterium-derived nopaline synthase gene (NOS) terminator, the rice amylase gene RAmy3D terminator, and the rice heat shock protein (HSP) terminator.

[0018] The polynucleotide encoding a useful protein is preferably incorporated into the nucleic acid molecule together with a marker gene. Examples of marker genes include drug resistance genes such as hygromycin resistance genes, bialaphos resistance genes, chlorsulfuron resistance genes, and herbicide resistance genes such as ALS(G95A). A reporter gene may also be incorporated into the nucleic acid molecule. Examples of reporter genes used to confirm the expression location in a plant include the luciferase gene, GFP (green fluorescent protein) gene, and RFP (red fluorescent protein) gene.

[0019] In one embodiment, when GFP is produced using the method of the present invention, a nucleic acid molecule comprising a polynucleotide encoding GFP preferably contains a promoter such as a ubiquitin promoter, and a terminator such as a nopaline synthase gene terminator or a heat shock protein gene terminator.

[0020] In one embodiment, when lysostaphin is produced using the method of the present invention, a nucleic acid molecule comprising a polynucleotide encoding lysostaphin preferably comprises a promoter such as a ubiquitin promoter, a RAmy3D promoter, or a cauliflower mosaic virus 35S promoter. Furthermore, it preferably comprises a signal peptide such as a RAmy3D exocrine signal peptide or a RAmy3A exocrine signal peptide, and an endoplasmic reticulum retention sequence such as a KDEL sequence. Furthermore, it preferably comprises a terminator such as a RAmy3D terminator, a nopaline synthase gene terminator, or a heat shock protein gene terminator.

[0021] In one embodiment, when producing cholera toxin B subunit using the method of the present invention, a nucleic acid molecule comprising a polynucleotide encoding the cholera toxin B subunit preferably contains a promoter such as a ubiquitin promoter, a signal peptide such as an RAmy3A exocrine signal, an RAmy3D exocrine signal, or a chloroplast targeting signal, and a terminator such as a heat shock protein terminator.

[0022] A known method can be used to introduce an expressible nucleic acid molecule containing a polynucleotide encoding a useful protein into rice cells, such as the Agrobacterium method, particle gun method, polyethylene glycol method, and electroporation.

[0023] Once a transformed rice plant is obtained by the above-described nucleic acid molecule introduction technique, it is possible to obtain offspring (progeny individuals) through sexual or asexual reproduction. Furthermore, mass production of plants is also possible from propagation materials such as seeds, cuttings, stems, calli, and protoplasts from the plant, its offspring, or clones. The transformed rice of the present invention includes progeny plants such as the "T0 generation," which is the regenerated generation that has been transformed, and the "T1 generation," which is the self-pollinated seeds of the T0 generation plants, as well as hybrid plants and their progeny plants obtained by crossbreeding these plants as a single parent.

[0024] In one embodiment, the transformed rice of the present invention is preferably a plant obtained by the above-mentioned nucleic acid molecule introduction technique, which has been subcultured for a certain number of generations and repeatedly selected using a predetermined marker or the like as an indicator, so that the introduced gene is stably maintained throughout the plant.

[0025] In the present invention, a useful protein is a protein used as a useful substance, and is preferably a soluble protein. Examples of such proteins include peptides, vaccines, antibodies, enzymes, hormones, and functional proteins. More specifically, useful proteins include antibacterial proteins (e.g., lysostaphin, persulcatusin, CCL28 (chemokine)), viral proteins used as vaccines (e.g., influenza hemagglutinin (HA) protein), adjuvants (e.g., cholera toxin B subunit), hematopoietic factors (e.g., granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, thrombopoietin), and growth factors (e.g., epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), and nerve growth factor (NGF)). , basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), etc.), cytokines (e.g., interferon, interleukin (IL)-1, IL-6, etc.), monoclonal antibodies or fragments thereof, tissue plasminogen activator, urokinase, serum albumin, blood coagulation factor VIII, leptin, insulin, stem cell growth factor (SCF), protease, lipase, cellulase, amylase, albumin, peptidase, luciferase, lactamase, collagen, gelatin, lactoferrin, transferrin, green fluorescent protein (GFP), β-glucuronidase (GUS), etc.

[0026] The production method of the present invention includes a step of cultivating the above-mentioned transformed rice in a dark place. In a preferred embodiment, the transformed rice is cultivated from seeds. Cultivation in a dark place allows the protein to be produced from the starch in the endosperm.

[0027] The cultivation period in a dark place is preferably 4 days or more, more preferably 7 days or more, and particularly preferably 10 days or more. The upper limit of the cultivation period in a dark place is not particularly limited, but is, for example, 20 days or less, preferably 15 days or less. The cultivation period in a dark place is preferably 85% or more of the entire cultivation period (the period from sowing to the end of cultivation), more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more. In a preferred embodiment, the cultivation period in a dark place is 100% of the entire cultivation period.

[0028] The cultivation period under light conditions is preferably 15% or less of the total cultivation period, more preferably 10% or less, even more preferably 5% or less, and particularly preferably 1% or less. In a preferred embodiment, the cultivation period under light conditions is 0% of the total cultivation period. The cultivation period under light conditions is preferably 2 days or less, more preferably 1 day or less. The light source used under light conditions is not particularly limited, but examples thereof include sunlight, fluorescent lamps, LEDs, inorganic and organic EL, etc.

[0029] The cultivation temperature is preferably 10° C. or higher, more preferably 20° C. or higher, and is preferably 40° C. or lower, more preferably 35° C. or lower.

[0030] The medium preferably contains at least one selected from the group consisting of a nitrogen-containing compound, a potassium-containing compound, a sulfur-containing compound, and a sugar, more preferably two or more, even more preferably three or more, and particularly preferably four or more. In a preferred embodiment, the medium contains a nitrogen-containing compound, a potassium-containing compound, and a sulfur-containing compound. In a preferred embodiment, the medium contains a nitrogen-containing compound, a potassium-containing compound, a sulfur-containing compound, and a sugar.

[0031] Nitrogen-containing compounds include compounds containing nitrogen atoms, such as nitrates (e.g., potassium nitrate, ammonium nitrate, etc.), ammonium salts (e.g., ammonium nitrate, ammonium chloride, etc.), and amino acids (e.g., glycine, aspartic acid, arginine, etc.). Among these, nitrates or ammonium salts are preferred, and nitrates are more preferred. The content of nitrogen-containing compounds in the medium is preferably 300 to 18,000 mg / L, and more preferably 1,000 to 10,000 mg / L.

[0032] Examples of potassium-containing compounds include compounds containing potassium atoms, such as potassium salts (e.g., potassium nitrate, potassium iodide, potassium chloride, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, etc.). The content of the potassium-containing compound in the medium is preferably 150 to 12,000 mg / L, more preferably 500 to 5,000 mg / L.

[0033] Examples of sulfur-containing compounds include compounds containing sulfur atoms, such as sulfates (e.g., magnesium sulfate, manganese sulfate, zinc sulfate, copper sulfate, etc.). The content of sulfur-containing compounds in the medium is preferably 10 to 1500 mg / L, more preferably 100 to 700 mg / L.

[0034] Examples of sugars include sucrose and glycol, with sucrose being preferred. The sugar content in the medium is preferably 1 to 100 g / L, more preferably 10 to 50 g / L.

[0035] The pH of the medium is preferably 5.0 to 6.5.

[0036] Preferred media include MS medium (Murashige and Skoog (1962) Physiol. Plant. 15, 473-497), N6 medium (Chu et al. (1975) Sci. Sin. (Peking) 18, 659-668), AA medium (Toriyama and Hinata (1985) Plant Sci. 41, 179-183), MS medium supplemented with sugar, N6 medium supplemented with sugar, and AA medium supplemented with sugar, with MS medium supplemented with sucrose and N6 medium supplemented with sucrose being more preferred.

[0037] A preferred method for extracting the protein produced by the method of the present invention is to obtain a fraction containing the useful protein from the grown plant, and then separate and purify the useful protein by a suitable method.

[0038] A wide variety of known buffer solutions can be used as the extraction solution for protein extraction. Examples include TBS buffer, glycine buffer, citrate buffer, acetate buffer, phosphate buffer, MES buffer, TES buffer, and borate buffer. Known protease inhibitors, such as benzamide, PMSF, AEBSF, antipain, chymostatin, leupeptin, pepstatin A, and EDTA, can be used during protein extraction. Known antioxidants, such as DTT, GSH, β-mercaptoethanol, TCEP, cysteine, mercaptoethylamine, and mercaptopropionic acid, can also be used during protein extraction. Known surfactants, such as Tween 20, Triton X-100, NP-40, CHAPS, CTAB, sodium cholate, sodium deoxycholate, and SDS, can also be used.

[0039] The extracted protein can be purified by known techniques, such as affinity chromatography, ion exchange chromatography, gel filtration chromatography, hydrophobic interaction chromatography, immobilized metal affinity chromatography, reverse phase chromatography, and hydroxyapatite chromatography, which may be used in appropriate combination.

[0040] In one embodiment, the useful protein produced by the present invention can be used as a raw material for pharmaceuticals, industrial products, etc. In the production method of the present invention, useful proteins are produced in the dark using rice, which reduces utility costs and enables useful proteins to be produced at lower cost than conventional methods.

[0041] In one embodiment, there is provided a method for producing a therapeutic drug for bovine mastitis, comprising a step of producing lysostaphin by the production method of the present invention. The therapeutic drug for bovine mastitis contains lysostaphin obtained by the method of the present invention. Lysostaphin is a protein that has antibacterial activity against Staphylococcus aureus, a major causative bacterium of bovine mastitis. The method of the present invention enables efficient production of lysostaphin, and is also useful in producing a therapeutic drug for bovine mastitis.

[0042] In one embodiment, there is provided a method for producing a vaccine, comprising the step of producing a cholera toxin B subunit by the production method of the present invention. The vaccine comprises the cholera toxin B subunit obtained by the method of the present invention. The vaccine is preferably a vaccine for livestock. The cholera toxin B subunit is an adjuvant used in vaccines. The method of the present invention enables efficient production of the cholera toxin B subunit, which is useful in producing a vaccine. The method can also be used as an orally ingested vaccine.

[0043] The present invention will be specifically described below with reference to examples, but these examples are not intended to limit the scope of the present invention.

[0044] [Example 1] Green Fluorescent Protein (GFP) [Materials and Methods] (Green Fluorescent Protein (GFP)-Transduced Rice) The next generation of selfed rice (Nipponbare) was produced by introducing the pBsGFP vector (Figure 1), which expresses GFP under the control of the cauliflower mosaic virus 35S promoter. The specific production method is described below.

[0045] The sequence encoding GFP was inserted in the sense orientation between the 35S promoter and the NOS terminator of the binary vector.

[0046] This binary vector was introduced into rice callus using the Agrobacterium method, and regenerated plants were obtained from the transformed callus using standard methods. Seeds obtained by self-pollination from the regenerated plants were sown, and seeds were further obtained by self-pollination. The regenerated plants and their self-pollinated offspring were cultivated in a transgenic greenhouse.

[0047] (Sowing) The seeds of the above transgenic rice were removed from the husks and placed in a 50 ml plastic tube. The surface was sterilized by shaking for 20 minutes in a solution of sodium hypochlorite (2.5% available chlorine concentration) containing 2-3 drops of 20% (v / v) Triton X-100. This procedure was repeated once or twice, and the seeds were washed several times with DDW in a clean bench until no more bubbles were produced. Ten seeds were then sowed in a 9 cm diameter, 2 cm high Petri dish. The Petri dish was lined with a sheet of filter paper and wrapped in parafilm. Four conditions were investigated: light and dark conditions with water, and light and dark conditions with 3% sucrose and MS medium (Murashige and Skoog Mixed Salts for Medium, Nippon Pharmaceutical Co., Ltd.). All liquid volumes were adjusted to 5 ml. The plants were cultivated at 28°C for 12 days under full light conditions using fluorescent lamps, and under total darkness conditions where the petri dishes were covered with aluminum foil to block out light.

[0048] (Total soluble protein extraction) Total soluble protein was extracted from rice seedlings that were ready for extraction 4 to 14 days after germination. Five of the ten rice seedlings with the best growth were selected, and the seeds were removed. The entire plant was wiped with Kimwipes to remove excess moisture, and its fresh weight was measured. After measuring the fresh weight, each rice seedling was frozen in liquid nitrogen and crushed using a mortar and pestle. TBS was added and the seedlings were thoroughly crushed. The amount of TBS added was 0.5 ml or 1.0 ml, depending on the size of the seedling. The solution and sediment in the mortar were transferred to a 1.5 ml tube. This was used as the extract and stored frozen at -20°C.

[0049] (Protein Measurement) Total soluble protein concentration was calculated using the Bradford method (Bradford, 1976). Bovine serum albumin (BSA) was used to create a calibration curve. The extract was thawed on ice, mixed thoroughly, and centrifuged at 15,000 rpm for 5 minutes at 4°C. 10 μl of the supernatant, 790 μl of DDW, and 200 μl of Protein Assay (Bio-Rad) were mixed thoroughly to make a 1 ml solution. After allowing to stand at room temperature for 15 minutes, the entire volume was transferred to a cuvette and the absorbance (595 nm) was measured. A BSA solution for the calibration curve was prepared (Table 1), and a calibration curve was created from the absorbance. The total soluble protein concentration of the sample was calculated using this calibration curve, and the protein amount was calculated by multiplying this by the volume of the protein solution.

[0050]

[0051] (SDS-PAGE) The supernatant of the protein extract was mixed with loading buffer and placed in boiling water for 5 minutes to heat-denaturate the protein. This was applied to a 12% polyacrylamide gel and electrophoresed at 20 mA until the dye in the loading buffer migrated to the end of the gel. The running buffer was SDS-PAGE buffer (0.025 M Tris, 0.192 M glycine, 0.1% (w / v) SDS).

[0052] (CBB staining) After electrophoresis, the polyacrylamide gel was immersed in Quick CBB Plus (Wako Pure Chemical Industries, Ltd.) for 30 minutes or more and stained by horizontal shaking. After staining, the gel was destained by repeated horizontal shaking using IEW several times and then photographed.

[0053] (Western Blotting (WB)) 8 x 10 cm for one gel after electrophoresis 2 Proteins were transferred from the gel to the membrane using one Immun-Blot PVDF membrane (Bio-Rad) and four pieces of filter paper slightly larger than the membrane. The four filter papers were soaked in transfer buffer (0.047 M Tris, 0.039 M glycine, 0.0375% (w / v) SDS, 20% (v / v) methanol), and the PVDF membrane was soaked in methanol. The membrane was placed in the tray of a Trans-Blot Turbo Transfer System (Bio-Rad) in the following order, ensuring no air was trapped between the two filter papers, the membrane, the electrophoresed gel, and the two filter papers. The tray was then covered with a lid and transferred at 25 V and 0.1 mA for 1 hour. After transfer, the membrane was washed with TBS-T buffer (50 mM Tris-HCl (pH 8.0), 0.9% (w / v) NaCl, 0.5% Tween-20) by shaking for 10 minutes, then sealed in a hybribag together with 20 ml of blocking buffer (TBS-T buffer containing 1% BSA) and shaken at room temperature for 1 hour for blocking.

[0054] After blocking, the membrane was sealed in a hybribag with 15 ml of primary antibody solution diluted as follows, and shaken at room temperature for 2 hours or left to stand overnight at 4°C. LYS: Anti-LYS diluted 1 / 3000 with TBS-T buffer. CTB: Anti-cholera toxin antibody rabbit serum (Bio Academia) diluted 1 / 2000 with TBS-T buffer.

[0055] After primary antibody incubation, the membrane was immersed in TBS-T buffer and washed three times by shaking for 5 minutes. For LYS and CTB, the membrane was then sealed in a hybribag with 15 ml of secondary antibody solution (Anti-Rabbit IgG (H+L) HRP Conjugate (Bio-Rad) diluted 1:2500 in TBS-T buffer) and shaken at room temperature for 1 hour. After secondary antibody incubation, the membrane was similarly immersed in TBS-T buffer and washed three times by shaking for 5 minutes. For LYS and CTB, the membrane was placed in a hybribag and mixed with equal parts luminol and hydrogen peroxide (5 ml each) from Immobilon Western Chemiluminescent HRP Substrate (Merck). After permeation for 5 minutes at room temperature, the membrane was placed on a tray for WB imaging, covered with clear film to prevent air intrusion, and the signal was detected using a ChemiDocTouchMP (Bio-Rad). From the results obtained by WB, the production amount was calculated from the PC concentration using the density of each band using ImageJ.

[0056] Western blotting was performed on the 12th day of growth, when total protein content is thought to be highest, using protein extracts from rice seedlings grown in water and Suc+MS medium under light and darkness. The results are shown in Figures 6 and 7.

[0057] In the dark, the amount of GFP protein per 10 μg of total soluble protein was 1.3 times higher in water alone and 1.2 times higher in sucrose + MS medium, compared to the amount in the light (Figure 6).

[0058] In a comparison at the individual level using 15 μl of protein extract, the amount of GFP protein in water in the dark was 1.6 times higher, in sucrose + MS medium in the dark was 14.7 times higher, and in sucrose + MS medium in the light was 5.6 times higher, compared to water in the light (Figure 7).

[0059] [Example 2] Lysostaphin (LYS) [Materials and Methods] (Lysostaphin (LYS)-introduced Rice) The selfed progeny of rice (Nipponbare) introduced with the vector pBP3DLYS (Figure 2) that expresses LYS under the control of the RAmy3D promoter, and the selfed progeny of rice (Nipponbare) introduced with the vector pB35SspLYS (Figure 3) that expresses LYS under the control of the 35S promoter were produced. The specific production method is described below.

[0060] Single-stranded DNA encoding the RAmy3D signal peptide (sense and antisense strands) was synthesized and annealed to form double-stranded DNA. This DNA was cloned into a plasmid containing a chemically synthesized lysostaphin-encoding sequence to generate a lysostaphin-encoding sequence with the RAmy3D signal sequence at its N-terminus. This sequence and the RAmy3D promoter were amplified by PCR, and the two PCR products were ligated in a second PCR and cloned into a binary vector (Figure 2).

[0061] The sequence encoding lysostaphin with the RAmy3D signal sequence added was amplified by PCR and cloned between the 35S promoter and NOS terminator of a binary vector (Fig. 3).

[0062] These binary vectors were introduced into rice calli using the Agrobacterium method, and regenerated plants were obtained from the transformed calli according to standard methods. The regenerated plants were cultivated in a recombinant temperature room and seeds were obtained by self-pollination. The sowing of the obtained seeds and subsequent procedures were carried out in the same manner as in Example 1.

[0063] Western blotting was performed on rice seedlings grown under four different conditions (water and Suc+MS medium) in the light and dark conditions on day 12, when total protein content is thought to be highest, using protein extracts. Lysostaphin production at the individual level and the proportion of lysostaphin in total soluble protein were compared. Western blotting was performed on two different promoter-driven strains: the pBP3DLYS1-1a strain (hereafter referred to as the pBP3D strain), which expresses LYS under the RAmy3D promoter, and the pB35SspLYS#6 strain (hereafter referred to as the pB35S strain), which expresses LYS under the 35S promoter. The results are shown in Figures 8 to 13.

[0064] In the case of the pBP3D strain, LYS production was highest under the dark / Suc + MS condition at 1129 ng / ml, followed by the light / Suc + MS condition at 911 ng / ml, with the dark / water condition being the lowest. On the other hand, the proportion of LYS in the total soluble protein was equally high under the light / water and dark / Suc + MS conditions at 0.29%, with the light / Suc + MS condition being the lowest (Figures 8-10).

[0065] The pB35Ssp strain, like the pBP3D strain, produced the highest amount of LYS under the dark / Suc + MS condition (512 ng / ml), followed by the light / Suc + MS condition (415 ng / ml). The percentage of LYS relative to the total soluble protein was highest under the dark / water condition and lowest under the light / Suc + MS condition. The results showed that dark was higher than light under both the water and Suc + MS conditions (Figures 11-13).

[0066] Both the pBP3D and pB35Ssp lines showed the highest LYS production under dark and Suc + MS conditions, indicating that dark production is higher than light under Suc + MS conditions. Comparing overall production between lines, the pBP3D line produced approximately twice as much LYS as the pB35Ssp line under dark and Suc + MS conditions. Although both lines accumulate LYS through exocrine secretion via the RAmy3D signal, the RAmy3D promoter, whose expression is stimulated by sugar starvation rather than systemic expression via the 35S promoter, is more compatible with gradual growth conditions, where the plant absorbs sugars and nutrients from the medium, possibly resulting in differences in production between lines using different promoters. Regarding the percentage of LYS in total soluble protein, the pBP3D line showed the highest percentage under light and water conditions, while the pB35Ssp line showed the highest percentage under dark and water conditions. This result reflects the fact that total soluble protein is lower under water conditions than under Suc + MS conditions. For the pBP3D strain, there was almost no difference in the LYS ratio of total soluble protein between light / water and dark / Suc + MS conditions, suggesting that dark / Suc + MS conditions, which produce the highest amount of LYS, are the most suitable conditions. On the other hand, for the pB35Ssp strain, while dark / Suc + MS conditions were the highest in terms of production amount alone, dark / water was the highest in terms of the ratio of LYS to total soluble protein. This suggests that the results can be optimized by changing the conditions depending on factors such as nutrient costs and planting density.

[0067] [Example 3] Cholera toxin B subunit (CTB) [Materials and Methods] (Rice introduced with cholera toxin B subunit (CTB)) The selfed progeny of rice (Nipponbare) introduced with vector pBspCTB (Figure 4), which expresses CTB under the ubiquitin promoter and has an exocrine signal, and the selfed progeny of rice (Nipponbare) introduced with vector pBcpCTB (Figure 5), which expresses CTB under the ubiquitin promoter and has a chloroplast targeting signal, were produced. The specific production method is described below.

[0068] Single-stranded DNA encoding the RAmy3A signal peptide, sense and antisense, was synthesized and annealed to form double-stranded DNA. This DNA was cloned into a plasmid containing a sequence encoding a chemically synthesized CTB with a 6xHis tag attached to its N-terminus, producing a sequence encoding a 6xHis-tagged CTB with the RAmy3A signal sequence attached to its N-terminus. This sequence was cleaved from the plasmid with restriction enzymes and cloned into a binary vector (Figure 4).

[0069] The sequence encoding the RubisCO chloroplast targeting sequence was amplified by PCR and cloned into a plasmid containing a sequence encoding CTB with a chemically synthesized 6xHis tag attached to its N-terminus, generating a sequence encoding 6xHis-tagged CTB with the RubisCO chloroplast targeting sequence attached to its N-terminus (Fig. 5).

[0070] These binary vectors were introduced into rice calli using the Agrobacterium method, and regenerated plants were obtained from the transformed calli according to standard methods. The regenerated plants were cultivated in a recombinant temperature room and seeds were obtained by self-pollination. The sowing of the obtained seeds and subsequent procedures were carried out in the same manner as in Example 1.

[0071] Western blotting was performed on the 12th day of growth, when total protein content is thought to be highest, using protein extracts from rice seedlings grown under four different media conditions (water and Suc+MS medium) in the light and dark conditions. CTB production at the individual plant level and the proportion of CTB in total soluble protein were compared. Western blotting was performed on two lines with different transport signals: pBspCTB#15 (hereafter referred to as the pBsp line), which expresses CTB under the ubiquitin promoter and has the exocrine signal of RAmy3D, and pBcpCTB#4 (hereafter referred to as the pBcp line), which expresses CTB under the ubiquitin promoter and has the chloroplast transport signal. The results are shown in Figures 14 to 19.

[0072] For the pBsp strain, CTB production was highest in the dark with Suc+MS, and under water conditions, production was higher in the dark than in the light, resulting in a dark > light result. The proportion of CTB in the total soluble protein was highest in the dark with water at 0.15%, and lowest at about 1 / 10 of that with Suc+MS in the light, with all other conditions at 0.05% (Figures 14-18).

[0073] For the pBcp strain, the dark concentration was nearly twice as high as that for the light concentration under both water and Suc + MS conditions. As with the pBsp strain, the dark and water concentration was the highest at 0.10%, and although the Suc + MS concentration was less than one-fifth of that, the dark concentration was higher than the light concentration under both water and Suc + MS conditions (Figures 17-19).

[0074] For both the pBsp and pBcp strains, CTB production was higher in the dark under both water and Suc + MS conditions, indicating increased production under Suc + MS conditions, similar to LYS. However, the proportion of CTB in the total soluble protein was overwhelmingly higher under water than under Suc + MS (due to the lower total soluble protein content under water). This suggests that, similar to the pB35Ssp strain of LYS, CTB production can be achieved by increasing the number of plants grown per unit area and cultivating them in water alone, thereby reducing nutrient costs and cleaning costs due to algae growth.

[0075] Example 4 Lysostaphin (LYS) Materials and Methods (Lysostaphin (LYS)-Introduced Rice) The following plants were produced: a self-pollinated offspring of rice (Nipponbare) introduced with the vector pR3D-LYS (Figure 20) that expresses LYS under the control of a ubiquitin promoter; a self-pollinated offspring of rice (Nipponbare) introduced with the vector pR3D-LYS-ER (Figure 21) that expresses LYS under the control of a ubiquitin promoter; and a self-pollinated offspring of rice (Nipponbare) introduced with the vector pBcp-LYS201 (Figure 22) that expresses LYS under the control of a ubiquitin promoter. Specific production methods are described below.

[0076] The sequence encoding lysostaphin with the RAmy3D signal peptide added was amplified by PCR, and this sequence was cloned into a binary vector (FIG. 20).

[0077] A sequence encoding lysostaphin to which the RAmy3D signal peptide was added was amplified by PCR using primers containing a sequence encoding the KDEL sequence at the C-terminus, and this sequence was cloned into a binary vector (FIG. 21).

[0078] The sequence encoding the chloroplast targeting sequence and the sequence encoding lysostaphin were each amplified by PCR, and these two PCR products were ligated in a second PCR and then cloned into a binary vector (Figure 22).

[0079] These binary vectors were then introduced into rice calli using the Agrobacterium method, and regenerated plants were obtained from the transformed calli by standard methods. The regenerated plants were cultivated in a transgenic temperature chamber and self-pollinated to obtain seeds.

[0080] (Seeding) The seeds of the transgenic rice described above were dehusked and placed in a 50 ml plastic tube. The surface was sterilized by shaking for 20 minutes in a solution of sodium hypochlorite (2.5% available chlorine concentration) containing 2-3 drops of 20% (v / v) Triton X-100. This procedure was repeated once or twice, and the tubes were washed several times with DDW in a clean bench until no more bubbles were formed. Ten seeds were then sown in a 9 cm diameter, 2 cm high Petri dish. Each dish was lined with a single sheet of filter paper and wrapped in parafilm. Sucrose + MS medium was used for cultivation under both light and dark conditions. All volumes were adjusted to 5 ml. The seeds were grown at 28°C for 12 days under full light conditions (fluorescent light) or total darkness (dark conditions) (covered with aluminum foil).

[0081] Total soluble protein was extracted from four to seven individuals under each condition, and lysostaphin production was measured by Western blotting using an anti-lysostaphin antibody. Analytical procedures were performed as described in Example 1. The results are shown in Figures 23 and 24. In Figure 23, no bands were observed in dark / endoplasmic reticulum (e), light / apoplast (g), and light / endoplasmic reticulum (c and e). These are individuals in which the transgene was not present due to segregation. CBB staining indicates that equal amounts of protein were applied. Figure 24 shows the average lysostaphin production for apoplast-, endoplasmic reticulum-, and plastid-localized individuals, excluding individuals without the transgene. For apoplast- and endoplasmic reticulum-localized individuals, lysostaphin production was higher in the dark than in the light. For plastid-localized individuals (differentiated into etioplasts in the dark and chloroplasts in the light), lysostaphin production was higher in the light than in the dark. These results revealed that the amount of lysostaphin produced was greater in the dark when the cells were localized in the endoplasmic reticulum as well as the apoplast, and that the amount of lysostaphin produced was greater when the cells were localized in the endoplasmic reticulum than when the cells were localized in the apoplast.

Claims

1. A method for producing a useful protein, which comprises the step of cultivating in the dark transformed rice plants into which a polynucleotide encoding a useful protein has been introduced.

2. The method of claim 1, wherein the cultivation period in a dark place is 4 days or more.

3. The method of claim 1, wherein the cultivation period under light is 15% or less of the total cultivation period.

4. The production method according to claim 1, wherein the transformed rice is cultivated in a medium containing at least one compound selected from the group consisting of nitrogen-containing compounds, potassium-containing compounds, sulfur-containing compounds, and sugars.

5. The production method according to claim 1, wherein the useful protein is at least one selected from the group consisting of GFP, lysostaphin, and cholera toxin B subunit.

6. A therapeutic agent for bovine mastitis, comprising lysostaphin produced by the method according to any one of claims 1 to 4.

7. A method for producing a therapeutic agent for bovine mastitis, comprising the step of producing lysostaphin by the method according to any one of claims 1 to 4.

8. A vaccine comprising the cholera toxin B subunit produced by the method according to any one of claims 1 to 4.

9. A method for producing a vaccine, comprising the step of producing cholera toxin B subunit by the method according to any one of claims 1 to 4.

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