Method for recovering target protein and method for producing target protein

Abstract

To provide a method for recovering a target protein from a plant body in high efficiency, and to provide a method for producing the target protein using the method.SOLUTION: A method for collecting target proteins from a plant body expressing the target proteins, the method comprising immersing the plant body in a buffer solution containing a chelating agent at a concentration equal to or higher than that of 15mM, and disrupting the plant body.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for recovering and producing a target protein. [Background technology]

[0002] Methods have been developed for heterologously expressing a target protein in a plant using recombinant DNA technology or the like, and recovering the expressed target protein from the plant. To recover the target protein from the plant, it is necessary to disrupt the cell wall. Methods for disrupting the cell wall include physically disrupting the cell wall and decomposing the cell wall using digestive enzymes. Patent Document 1, for example, discloses a method for physically disrupting the cell wall, in which fluidized biomass is exposed to high-frequency pulses and shear force, and then a liquid fraction is obtained. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Special Publication No. 2016-535996 Summary of the Invention [Problem to be solved by the invention]

[0004] However, according to the studies of the present inventors, conventional methods for physically disrupting cell walls have not been able to sufficiently disrupt the cell walls, making it difficult to efficiently recover the target protein. Furthermore, methods for decomposing cell walls using digestive enzymes require a long time for the enzymatic reaction and are costly, making them unsuitable for industrial production of the target protein.

[0005] In view of the above circumstances, an object of the present invention is to provide a method for recovering a target protein from a plant with high efficiency, and a method for producing a target protein using said method. [Means for solving the problem]

[0006] The present inventors have discovered that a target protein can be efficiently recovered from a plant expressing the target protein by combining immersing the plant in a buffer containing 15 mM or more of a chelating agent and crushing the plant.

[0007] The present disclosure provides, for example, the inventions described in the following [1] to [6]. [1] A method for recovering a target protein from a plant expressing the target protein, comprising: A method comprising immersing a plant body in a buffer containing 15 mM or more of a chelating agent, and crushing the plant body. [2] The method according to [1], which comprises immersing the plant body in a buffer solution containing 15 mM or more of a chelating agent, and then crushing the plant body. [3] The method according to [1], which comprises, after crushing the plant body, immersing the plant body in a buffer solution containing 15 mM or more of a chelating agent. [4] The method according to any one of [1] to [3], wherein the chelating agent is an aminocarboxylic acid chelating agent or a hydroxycarboxylic acid chelating agent. [5] The aminocarboxylic acid chelating agent is ethylenediaminetetraacetic acid, The method according to [4], wherein the hydroxycarboxylic acid chelating agent is citric acid. [6] Preparing a plant that expresses the target protein; and A method for producing a target protein, comprising recovering the target protein from the plant by the method according to any one of [1] to [5]. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide a method for recovering a target protein from a plant with high efficiency, and a method for producing a target protein using said method. [Brief explanation of the drawings]

[0009] [Figure 1]FIG. 1 shows the results of agarose gel electrophoresis of samples collected from plants expressing C-reactive protein by immersing the plants in a buffer containing EDTA and then crushing the plants. [Figure 2] (A) A graph showing the results of agarose gel electrophoresis of a sample collected from a plant expressing a nucleocapsid protein by immersing the plant in a buffer containing EDTA and then crushing the plant. (B) A graph showing the results of Western blotting assay of a sample collected from a plant expressing a nucleocapsid protein by immersing the plant in a buffer containing EDTA and then crushing the plant. [Figure 3] (A) A graph showing the results of agarose gel electrophoresis of a sample recovered from a plant expressing a nucleocapsid protein by disrupting the plant and then immersing the plant in a buffer containing EDTA. (B) A graph showing the results of Western blotting assay of a sample recovered from a plant expressing a nucleocapsid protein by disrupting the plant and then immersing the plant in a buffer containing EDTA. [Figure 4] (A) A graph showing the results of agarose gel electrophoresis of a sample collected from a plant expressing a nucleocapsid protein by immersing the plant in a buffer containing CyDTA and then crushing the plant. (B) A graph showing the results of Western blotting assay of a sample collected from a plant expressing a nucleocapsid protein by immersing the plant in a buffer containing CyDTA and then crushing the plant. [Figure 5] FIG. 10 shows the results of Western blotting assay of samples collected from plants expressing nucleocapsid protein by immersing the plants in a buffer containing citric acid and then disrupting the plants. [Figure 6] FIG. 1 shows the results of a Western blotting assay of samples collected from plants expressing human serum proteins by crushing the plants and then immersing them in a buffer containing EDTA. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, embodiments of the present disclosure will be described in detail.

[0011] [Method for recovering target protein] The method for recovering a target protein from a plant expressing the target protein according to this embodiment includes immersing the plant in a buffer containing 15 mM or more of a chelating agent and crushing the plant.

[0012] The target protein is not limited as long as it can be expressed in the plant body, and may be expressed in the cytoplasm, apoplast, or organelle in the plant body. As used herein, "apoplast" refers to the portion of plant tissue outside the cell membrane, i.e., the entire cell wall and intercellular space. The target protein may be, for example, a naturally occurring protein, a modified naturally occurring protein, or an artificially designed protein. The origin of the target protein is also not limited, and may be, for example, derived from animals (including mammals such as humans), plants, filamentous fungi, bacteria, or yeast. The use of the target protein is also not limited, and may be, for example, a protein used in medical applications, the food industry, or the chemical industry.

[0013] Examples of target proteins include acute phase proteins, enzymes, antibodies, antigens, epitopes, growth factors, hormones, cytokines, transcription factors, receptors, or partial peptides thereof. For example, the target protein may be selected from the group consisting of acute phase proteins and antigens.

[0014] Examples of acute phase proteins include C-reactive protein, serum amyloid A, fibrinogen, haptoglobin, α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, etc., and may be C-reactive protein. C-reactive protein can be expressed in the apoplast in plants.

[0015] Examples of enzymes include oxidases, reductases, lipases (eg, phospholipases), proteases, kinases, phosphatases, cellulases, steroid synthesis enzymes, methylases, demethylases, collagenases, transglutaminases, glycosidases, and chitinases.

[0016] Examples of antibodies include complete antibodies, Fab, F(ab'), F(ab')2, Fc, Fc fusion proteins, heavy chains (H chains), light chains (L chains), single-chain Fvs (scFvs), sc(Fv)2, disulfide-linked Fvs (sdFvs), and diabodies.

[0017] Examples of antigens and epitopes include proteins derived from filamentous fungi, proteins derived from bacteria, proteins derived from viruses, and proteins derived from human serum (human serum proteins). When the antigens and epitopes are antigenic proteins or epitopes used as vaccines, they are not particularly limited as long as they are immunogenic, and examples include proteins derived from pathogenic filamentous fungi, proteins derived from pathogenic bacteria, and proteins derived from pathogenic viruses. Examples of viral proteins include nucleocapsid proteins. Nucleocapsid proteins can be expressed in the cytoplasm of plants.

[0018] Examples of growth factors include epidermal growth factor (EGF), insulin-like growth factor (IGF), transforming growth factor (TGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), erythropoietin (EPO), thrombopoietin (TPO), and hepatocyte growth factor (HGF).

[0019] Examples of hormones include peptide and protein hormones.

[0020] Examples of cytokines include interleukins (IL), hematopoietic factors (CSF, EPO, TPO), interferons (IFNα, IFNβ, IFNγ), tumor necrosis factors (TNF), growth factors (EGF, FGF, PDGF), and chemokines (IL-8).

[0021] Examples of transcription factors include general transcription factors, upstream transcription factors, and inducible transcription factors.

[0022] Examples of receptors include G protein-coupled receptors, ion channel receptors, and cytokine receptor superfamily receptors.

[0023] The target protein may also be a fusion protein in which two or more proteins are bound together, which may be a fusion protein of two or more heterologous proteins or a fusion protein of two or more homologous proteins.

[0024] The molecular weight of the target protein is not particularly limited, and may be, for example, 1 kDa to 1000 kDa, 1 kDa to 500 kDa, 1 kDa to 250 kDa, 1 kDa to 100 kDa, or 1 kDa to 75 kDa.

[0025] The protein of interest may be a monomer, dimer, trimer or multimer.

[0026] The target protein may be tagged to facilitate detection and / or purification of the target protein. The tag may be a known tag, such as a histidine tag.

[0027] Plants expressing a target protein can be produced by known methods, such as agroinfiltration, plant virus vectors, the magnICON® system, and particle gun technology, and may also be the magnICON® system.

[0028] When using the magnICON® system, a plant expressing a target protein can be produced by introducing a cDNA of the genome of tobacco mosaic virus (TMV) or potato virus X (PVX) into which a gene encoding the target protein has been inserted into a T-DNA vector, and then infecting the plant with Agrobacterium transformed with the resulting T-DNA vector. Infection of the plant with the Agrobacterium may be carried out by contacting the plant with an infiltration buffer. Examples of infiltration buffers include those containing the Agrobacterium, 5 to 20 mM 2-morpholinoethanesulfonic acid, and 5 to 20 mM MgSO4, with a pH of 4.5 to 6.5, and an OD of 100. 660 It may be a buffer solution in which the pH is 0.0005 to 0.6.

[0029] When using the agroinfiltration method, Agrobacterium is transformed with T-DNA into which a gene encoding a target protein has been inserted, and the Agrobacterium is then infected into a plant, thereby producing a plant that expresses the target protein.

[0030] When using the plant virus vector method, a plant that expresses a target protein can be produced by inoculating the plant with RNA obtained from cDNA of the plant virus genome into which a gene encoding the target protein has been inserted, thereby infecting the plant. Examples of such virus vectors include TMV vectors, PVX vectors, plum pox virus (PPV) vectors, alfalfa mosaic virus (AIMV) vectors, cucumber mosaic virus (CMV) vectors, cowpea mosaic virus (CPMV) vectors, and zucchini yellow mosaic virus (ZYMV) vectors.

[0031] When using the particle gun method, metal microparticles coated with nucleic acid encoding the target protein are fired at high speed, and the gene encoding the target protein is introduced into the cells, thereby producing a plant that expresses the target protein.

[0032] Plants used for transformation are not particularly limited, and examples include plants belonging to the Solanaceae family (e.g., tobacco, eggplant, tomato, bell pepper, chili pepper), Rosaceae family (e.g., rose, strawberry), Brassicaceae family (e.g., Arabidopsis thaliana, rapeseed, Chinese cabbage, cabbage, radish, rapeseed), Asteraceae family (e.g., chrysanthemum, garland chrysanthemum, lettuce), Chenopodiaceae family (e.g., spinach, sugar beet), Poaceae family (e.g., wheat, rice, barley, corn), and Fabaceae family (e.g., soybean, adzuki bean, kidney bean, broad bean). The plant may be, for example, a plant of the Solanaceae or Brassicaceae family, a plant of the genus Nicotiana, or Nicotiana benthamiana, which may be 3 to 10 weeks old or 4 to 7 weeks old.

[0033] The target protein can be expressed in the cytoplasm, apoplast, or organelle by known methods. For example, a target protein that naturally expresses in the cytoplasm, apoplast, or organelle when expressed in a plant body may be used, or a target protein targeted to be expressed in the cytoplasm, apoplast, or organelle may be used. The targeting may be achieved, for example, by linking a DNA sequence encoding a signal peptide that directs localization to the cytoplasm, apoplast, or organelle to the DNA sequence encoding the target protein.

[0034] The method for recovering a target protein from a plant expressing the target protein according to this embodiment includes soaking the plant in a buffer containing 15 mM or more of a chelating agent and crushing the plant, but may also include crushing the plant after soaking the plant in a buffer containing 15 mM or more of a chelating agent, or may include soaking the plant in a buffer containing 15 mM or more of a chelating agent after crushing the plant.

[0035] Examples of plant bodies that can be used include leaves, roots, shoots, stems, flowers, fruits, embryos, seedlings, and parts thereof, as well as combinations thereof. However, leaves or leaf parts are preferred. Leaf parts are preferably leaf fragments. Furthermore, leaf fragments are preferred when the plant body is immersed in a buffer solution containing 15 mM or more of a chelating agent and then crushed. Leaf fragments are more preferred when the plant body is crushed and then immersed in a buffer solution containing 15 mM or more of a chelating agent. Leaf fragments may be, for example, fragments obtained by hollowing out a leaf with a cork poler, or fragments obtained by cutting a leaf with a cutter or razor. Leaf fragments having a diameter of 1.5 to 3 cm obtained by hollowing out a leaf with a cork poler are preferred.

[0036] The plant body may be a fresh plant body (e.g., fresh leaves), or may be a plant body that has been treated by freezing, freeze-thawing, drying, etc., as long as the present invention can be carried out. The plant body may be frozen by cooling it to -200 to 0°C or -100 to -50°C.

[0037] The chelating agent is not particularly limited, but examples thereof include aminocarboxylic acid chelating agents, hydroxycarboxylic acid chelating agents, hydroxamic acid chelating agents, phosphonic acid chelating agents, etc. From the viewpoint of further improving the recovery efficiency of the target protein, aminocarboxylic acid chelating agents or hydroxycarboxylic acid chelating agents are preferred. One type of chelating agent may be used alone, or two or more types may be used in combination.

[0038] The aminocarboxylic acid chelating agent is a chelating agent having an amino group and a carboxyl group. Examples of the aminocarboxylic acid chelating agent include ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), trans-1,2-diaminocyclohexanetetraacetic acid, diethylenetriaminepentaacetic acid (DTPA), N-(2-hydroxyethyl)ethylenediaminetriacetic acid (HEDTA), triethylenetetraminehexaacetic acid (TTHA), and salts thereof, among which EDTA or its salts are preferred, and EDTA is more preferred.

[0039] The hydroxycarboxylic acid chelating agent is a chelating agent having a hydroxy group and a carboxyl group, and examples of the hydroxycarboxylic acid chelating agent include citric acid, malic acid, lactic acid, tartaric acid, gluconic acid, and salts thereof, with citric acid or a salt thereof being preferred, and citric acid being more preferred.

[0040] The hydroxamic acid chelating agent is a chelating agent having a hydroxamic acid group (—CO—NHOH). Examples of the hydroxamic acid chelating agent include acetohydroxamic acid, octanohydroxamic acid, benzenesulfohydroxamic acid, and salts thereof.

[0041] A phosphonic acid chelating agent is a chelating agent having a phosphono group (—PO(OH) 2 ). Examples of the phosphonic acid chelating agent include etidronic acid, nitrilotris(methylenephosphonic acid), and salts thereof.

[0042] The buffer solution is not particularly limited and known buffer solutions can be used, including, for example, Tris buffer, Tris-HCl buffer, phosphate buffer, acetate buffer, and histidine buffer, with Tris buffer being preferred. The Tris (trishydroxymethylaminomethane) concentration in the Tris buffer may be 30 to 300 mM or 60 to 150 mM. The pH of the buffer may be, for example, 3 to 11, 3 to 10, 5 to 9.5, or 7 to 9.5. For example, when citric acid, gluconic acid, or a salt thereof is used as the chelating agent, the pH may be 3 to 10, 5 to 9.5, or 7 to 9.5.

[0043] The concentration of the chelating agent in the buffer solution containing the chelating agent is 15 mM or more, but from the viewpoint of further improving the recovery efficiency of the target protein, it is preferably 18 mM or more, 20 mM or more, 23 mM or more, 25 mM or more, 28 mM or more, 30 mM or more, 35 mM or more, 40 mM or more, 45 mM or more, 50 mM or more, 75 mM or more, 100 mM or more, 150 mM or more, or 200 mM or more, and the upper limit of these concentrations may be 2000 mM, 1500 mM, 1000 mM, 750 mM, 500 mM, 300 mM, or 200 mM.

[0044] The amount of the buffer solution containing the chelating agent used for the above-mentioned immersion may be 0.5 to 10 parts by mass, or may be 1.5 to 6 parts by mass, per part by mass of the plant body.

[0045] The above-mentioned immersion may be carried out under conditions where the buffer solution containing the chelating agent and the plant body are at 0 to 25°C, or under conditions where the buffer solution containing the chelating agent and the plant body are at 0 to 5°C.

[0046] When the plant bodies are immersed in a buffer containing 15 mM or more of a chelating agent and then disrupted, the plant bodies may be disrupted 3 to 2 hours or 5 to 30 minutes after the immersion. When the plant bodies are immersed in a buffer containing 15 mM or more of a chelating agent after being disrupted, the immersion may be maintained for 3 to 2 hours or 5 to 30 minutes.

[0047] The plant body can be crushed by a conventional method, for example, using a mortar and pestle, a bead mill, ultrasonic waves, a homogenizer, a blender, etc., and preferably using a mortar and pestle or a bead mill.

[0048] The plant body may be crushed with a mortar and pestle after cooling the plant body and the mortar and pestle to, for example, −196° C. to 0° C. or −196° C. to −30° C. The plant body may be crushed with a mortar and pestle until the plant body is in a powder form.

[0049] When crushing a plant body using a bead mill, for example, the diameter of the beads may be 2 mm to 5 mm or 2 mm to 3 mm, the crushing time per cycle may be 1 to 60 seconds or 1.5 to 20 seconds, the number of cycles may be 1 to 10 or 2 to 5, and the peripheral speed of the agitator may be 2 to 20 m / s or 3 to 12 m / s.

[0050] When the plant bodies are crushed after being immersed in a buffer solution containing 15 mM or more of a chelating agent, the crushing is preferably carried out using a bead mill, and when the plant bodies are crushed and then immersed in a buffer solution containing 15 mM or more of a chelating agent, the crushing is preferably carried out using a mortar and pestle.

[0051] When the plant body is immersed in a buffer containing 15 mM or more of a chelating agent and then disrupted, the disrupted product may be allowed to stand at 0 to 25° C. or 0 to 5° C. for 1 to 120 minutes or 5 to 60 minutes. During the standing time, the disrupted product may be in contact with the buffer containing 15 mM or more of a chelating agent.

[0052] The method for recovering a target protein from a plant expressing the target protein according to this embodiment may include immersing the plant in a buffer solution containing 15 mM or more of a chelating agent, crushing the plant, and then isolating and purifying the target protein.

[0053] Separation and purification of the target protein can be performed by conventional methods, but for example, separation of the target protein is preferably performed by centrifugation, which may be performed at a centrifugal force of 2000 to 32000 × g or 4000 to 16000 × g at a rotation radius of 1 to 60 minutes or 1.5 to 20 minutes, at a temperature of 0.5 to 10°C or 0.5 to 5°C.

[0054] The method for recovering a target protein from a plant expressing the target protein according to this embodiment allows for highly efficient recovery of the target protein from the plant. For example, if the amount of target protein recovered by the method is greater than the amount of target protein recovered by a similar method except that a buffer containing less than 15 mM of chelating agent (e.g., 10 mM, 5 mM, or 1 mM) or no chelating agent is used instead of a buffer containing 15 mM or more of chelating agent, the method can be considered to be capable of highly efficient recovery of the target protein from the plant. For example, if the amount of target protein recovered by the former method is, for example, 1.1-fold or more, 2-fold or more, 3-fold or more, 4-fold or more, 5-fold or more, 6-fold or more, 7-fold or more, 8-fold or more, 9-fold or more, 10-fold or more, 15-fold or more, or 20-fold or more, compared to the amount of target protein recovered by the latter method, the former method can be considered to be capable of highly efficient recovery of the target protein from the plant. The amounts of target protein recovered by both methods can be compared using standard methods, such as polyacrylamide gel electrophoresis or Western blotting assay.

[0055] [Method for producing a target protein] The method for producing a target protein according to this embodiment includes preparing a plant that expresses the target protein, and recovering the target protein from the plant that expresses the target protein according to this embodiment using a method for recovering the target protein.

[0056] The target protein may be the same as the target protein in the above-mentioned "Method for recovering a target protein." Preparation of a plant expressing a target protein may be performed by producing a plant expressing a target protein by the method for producing a plant expressing a target protein in the above-mentioned "Method for recovering a target protein." [Example]

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0058] In the following examples, target proteins were recovered from N. benthamiana plants in which the target proteins were expressed, and the recovery efficiency was evaluated. The production of N. benthamiana plants, infiltration of Agrobacterium transformants into N. benthamiana, recovery of leaves from N. benthamiana, and recovery of target proteins from the leaves were carried out by the methods described below.

[0059] 1. Preparation of Nicotiana benthamiana (1) Seeding Seedling pots (TS-10.5, Tokai Kasei Co., Ltd.) were filled to the brim with soil, and 2–3 Nicotiana benthamiana seeds were sown per pot. Several of these seedling pots were then arranged in a medium-sized seedling box (AZ-032, Anzen Kogyo Co., Ltd.), bottom-watered for 30 minutes, and covered with agricultural soil cover (Paopao 90, Mitsubishi Chemical Agri Dream Co., Ltd.).

[0060] (2) Growth The N. benthamiana plants were then grown for 14 days under the following environmental conditions. During this period, the agricultural bed covering material was removed 5 to 7 days after sowing, and after the N. benthamiana plants germinated, they were thinned to one plant per seedling pot. After thinning, when the plants were two weeks old, the medium-sized seedling raising boxes were placed on cultivation shelves and grown under the following environmental and nutrient solution conditions until they were five weeks old, and then subjected to the following [2. Infiltration of Agrobacterium-transformed plants into N. benthamiana using the magnICON® system] or [3. Leaf collection]. At this time, 32 seedling raising pots were placed per cultivation shelf. <Environmental conditions> -Temperature: 22℃ - Light cycle: 16 hours day / 8 hours night -Relative humidity: 30~80% -CO2 concentration: 400~700ppm -Light source: High-output 4950lm HF fluorescent lamp, FHF32EX-NH (Toshiba Lighting & Technology Corporation) <Nutrient solution conditions> The nutrient solution supplied to the cultivation shelves was a 1:1 volumetric mixture of fertilizer A solution (OAT House No. 1 (OAT Agrio Co., Ltd.) 150 g / L) and fertilizer B solution (OAT House No. 2 (OAT Agrio Co., Ltd.) 100 g / L), with an electrical conductivity (EC) of 1.8 mS / cm and a pH of 6.0.

[0061] 2. Infiltration of Agrobacterium transformants into Nicotiana benthamiana using the magnICON® system (1) Preparation of Agrobacterium transformants cDNA of the genome of a plant virus (Tobacco Mosaic Virus (TMV) or Potato Virus X (PVX)) described below, into which a gene encoding a target protein described below had been inserted, was introduced into a T-DNA vector, and Agrobacterium transformed with the resulting T-DNA vector was prepared.

[0062] (2) Cultivation of Agrobacterium The day before infiltration, 5 mL of LB liquid medium was placed in each test tube, and a 20 mg / mL rifampicin solution and a 50 mg / mL kanamycin solution were added to a final concentration of 50 μg / mL and 25 μg / mL, respectively. 100 μL of a glycerol stock of Agrobacterium transformants was then added, and the mixture was cultured at 28°C for approximately 24 hours with shaking (230 rpm) to obtain a culture solution.

[0063] (3) Infiltration buffer adjustment The culture medium from (2) above was collected and diluted 10-fold with phosphate-buffered saline (PBS; 10x PBS Liquide Concentre (OmniPur®) diluted 10-fold with purified water and stored refrigerated) to obtain a diluted solution. The OD value of the diluted solution, which is the optical density measured at an absorbance of 660 mm, was measured using a simple optical density (OD) monitor (mini photo 518R, Taitec Co., Ltd.), and confirmed to be 2.0 or higher. 5.3 L of purified water, 130 mL of 0.5 M 2-(N-morpholino)ethanesulfonic acid (MES, pH 5.4), and 65 mL of 1 M MgSO4 were added to a wide-mouth bucket, and the bucket was placed in a vacuum desiccator (300G, AS ONE Corporation) and stirred. Then, the final concentrations of MES and MgSO4 were adjusted to 10 mM, pH 5.4, and OD 660 The infiltration buffer was adjusted to 0.002.

[0064] (4) Viral infiltration Infiltration was performed on 5-week-old N. benthamiana plants as described above in [1. Preparation of plant biomass] using the following procedure. The roots of the N. benthamiana plants were placed facing upward, and the above-ground parts of the plants were immersed in the infiltration buffer described in (3) above. A stick was placed over the opening of the bucket as a stopper to prevent the soil from being submerged in the infiltration buffer. A vacuum pump and a vacuum desiccator were connected with a hose, and the vacuum pump was operated with the exhaust cock fully open to suction pressure of -0.092 MPaG, infecting the N. benthamiana plants by infiltration with the virus. The exhaust cock was then closed, the vacuum pump was removed from the vacuum desiccator, and the exhaust cock was suddenly opened. After the pressure was restored, the N. benthamiana plants were removed from the infiltration buffer, and the surface of the leaves was gently wiped dry.

[0065] [3. Leaf Collection] N. benthamiana plants that had or had not been subjected to the above-mentioned [2. Infiltration of Agrobacterium transformants into N. benthamiana using the magnICON® system] were cultured for 7 days under the same environmental conditions as those described in [1. Preparation of plant biomass], including temperature, light cycle, relative humidity, CO2 concentration, and light source. Leaves were then harvested from the former and latter N. benthamiana plants and stored at -80°C to identify infected and wild-type leaves, respectively. For harvesting, leaves or fragments cut into 1.6 cm diameter pieces using a cork polarizer were collected in bead mill tubes (Thermo Fisher Scientific, 2 mL Tough Microtubes 2.8 mm Ceramic). The leaves were subjected to the following procedure: [4-1. Recovery of target proteins by crushing leaves and immersion in buffer solution], and the cork polarizer-cut fragments were subjected to the following procedure: [4-2. Recovery of target proteins by crushing leaves and immersion in buffer solution].

[0066] [4-1. Recovery of target protein by crushing leaves and immersing in buffer solution] The mortar, pestle, and spoon were pre-cooled with liquid nitrogen. The infected leaves stored at -80°C were placed in the mortar and ground with the pestle and mortar until powdered. The weight of the 1.5 mL tube (Weight 1) was measured using a scale. An appropriate amount of the ground material was placed in the 1.5 mL tube using the spoon, and the weight of the tube containing the ground material (Weight 2) was measured. A buffer solution (100 mM Tris, chelating agent described below, pH 9.0) was added to the 1.5 mL tube in an amount three times the weight of the ground material (Weight 2 - Weight 1). The tube was then refrigerated (approximately 4°C) for 15 minutes and then centrifuged at 8,000 × g for 5 minutes at 4°C in a centrifuge (Tomy Seiko Co., Ltd., Multi Spin). 100 μL of the supernatant was collected in a 1.5 mL tube as the extract. The extract was stored at -80°C. 75 μL of the above extract was placed in a 1.5 mL tube, and 25 μL of sample buffer (20 μL of 1 M DTT mixed with 180 μL of 4× Laemmli sample buffer) was added. The mixture was heated in a heat block set to 95°C for 5 minutes to prepare the sample for analysis.

[0067] 4-2. Recovery of target protein by crushing leaves after immersion in buffer solution Approximately 0.1 g of leaf fragments, excised with a cork pole, were placed in a bead mill tube and allowed to stand on ice for 10 minutes. 300 μL of ice-cold buffer solution (100 mM Tris, chelating agent, pH 9.0) was then added to the tube. After allowing the tube to stand on ice for 30 minutes, the tube was placed in a bead mill (Thermo Fisher Scientific, Bead Mill 24) and homogenized using a program consisting of 2.8 mm beads, a 6 m / s agitator peripheral speed, 5 seconds of grinding, three cycles, and a 1-second cycle interval. The tube was then allowed to stand on ice for 30 minutes. After allowing the tube to stand, the mixture was centrifuged at 8000 × g for 10 minutes at 4°C in a centrifuge (Tomy Seiko Co., Ltd., Multi Spin), and the supernatant was collected as the extract in a 1.5 mL tube. The extract was then stored at -80°C. 75 μL of the above extract was placed in a 1.5 mL tube, and 25 μL of sample buffer (20 μL of 1 M DTT mixed with 180 μL of 4× Laemmli sample buffer) was added. The mixture was heated in a heat block set to 95°C for 5 minutes to prepare the sample for analysis.

[0068] [Recovery of AC-reactive proteins by disruption after immersion in EDTA-containing buffer] Using C-reactive protein as the target protein, TMV as the plant virus, and a buffer containing 100 mM EDTA as the buffer, analytical samples were obtained by the procedures described above in [1. Preparation of N. benthamiana plants], [2. Infiltration of N. benthamiana with Agrobacterium transformants using the magnICON® system], [3. Leaf collection], and [4-2. Recovery of target protein by immersion in buffer and subsequent crushing of leaves] (Example 1). Furthermore, analytical samples were obtained by the same procedures as in Example 1, except that [2. Infiltration of N. benthamiana with Agrobacterium transformants using the magnICON® system] was omitted and a buffer containing no chelating agent was used as the buffer (Comparative Example 1). Furthermore, analytical samples were obtained by the same procedures as in Example 1, except that a buffer containing no chelating agent was used as the buffer (Comparative Example 2). The target proteins and chelating agents used in Example 1 and Comparative Examples 1 and 2 are summarized in Table 1. The analytical samples of Example 1, Comparative Example 1, and Comparative Example 2 were subjected to polyacrylamide gel electrophoresis according to a standard method, and the results are shown in Figure 1. In Table 1, "Lane No." refers to the lane number in Figure 1. Note that C-reactive protein is a protein expressed in the apoplast of Nicotiana benthamiana.

[0069] [Table 1]

[0070] As shown in Figure 1, in Comparative Example 1, where proteins were recovered from wild-type leaves using a buffer solution containing no chelating agent, no C-reactive protein was detected by polyacrylamide gel electrophoresis. Furthermore, in Example 1, where proteins were recovered from infected leaves using a buffer solution containing 100 mM EDTA, the approximately 24 kDa C-reactive protein band was stronger than in Comparative Example 2, where proteins were recovered from infected leaves using a buffer solution containing no chelating agent. These results demonstrate that immersing plant bodies in a buffer solution containing a certain concentration of EDTA and then disrupting the plants improves the extraction efficiency of the target protein. Furthermore, C-reactive protein is a pentameric protein, with a molecular weight of approximately 120 kDa. The approximately 24 kDa band observed above is likely due to dissociation of C-reactive protein into monomers upon addition of sample buffer to the extract.

[0071] B. Recovery of nucleocapsid protein by disruption after immersion in EDTA-containing buffer. The target protein was a histidine-tagged nucleocapsid protein, the plant virus was TMV, and the buffer solution contained 5, 20, 25, 30, 40, 50, or 100 mM EDTA. Analytical samples were obtained according to the procedures described above in [1. Preparation of N. benthamiana plants], [2. Infiltration of Agrobacterium transformants into N. benthamiana plants using the magnICON® system], [3. Leaf collection], and [4-2. Recovery of target protein by crushing leaves after immersion in buffer solution]. The cases where the EDTA concentrations in the buffer solution were 5, 20, 25, 30, 40, 50, and 100 mM were designated Comparative Example 5 and Examples 2 to 7, respectively. In addition, an analytical sample (Comparative Example 3) was obtained using the same procedures as in Comparative Example 5 and Examples 2 to 7, except that the above-mentioned step (2. Infiltration of Agrobacterium transformants into Nicotiana benthamiana using the magnICON® system) was not performed and a buffer containing no chelating agent was used as the buffer. In addition, an analytical sample (Comparative Example 4) was obtained using the same procedures as in Comparative Example 5 and Examples 2 to 7, except that a buffer containing no chelating agent was used as the buffer. Table 2 summarizes the target proteins and chelating agents used in Examples 2 to 7 and Comparative Examples 3 to 5. The analytical samples of Examples 2 to 7 and Comparative Examples 3 to 5 were subjected to polyacrylamide gel electrophoresis and Western blotting assays using anti-histidine tag antibodies according to standard methods. The results are shown in Figures 2(A) and 2(B), respectively. In Table 2, "lane number" refers to the lane number in Figures 2(A) and 2(B). The histidine-tagged nucleocapsid protein is a protein expressed in the cytoplasm of Nicotiana benthamiana.

[0072] [Table 2]

[0073] As shown in Figures 2(A) and (B), in Comparative Example 3, in which proteins were recovered from wild-type leaves using a chelating agent-free buffer, polyacrylamide gel electrophoresis and Western blotting assays failed to detect nucleocapsid protein. Furthermore, in Examples 2 to 7, in which proteins were recovered from infected leaves using buffers containing 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 100 mM EDTA, respectively, the approximately 46 kDa nucleocapsid protein band in polyacrylamide gel electrophoresis and Western blotting assays was stronger than in Comparative Example 4, in which proteins were recovered from infected leaves using a chelating agent-free buffer, and Comparative Example 5, in which proteins were recovered from infected leaves using a buffer containing 5 mM EDTA. These results demonstrate that immersing plant bodies in a buffer containing 15 mM or more EDTA and then disrupting the plants improves the efficiency of target protein extraction.

[0074] [C. Recovery of nucleocapsid protein by disruption and immersion in EDTA-containing buffer] The target protein was a histidine-tagged nucleocapsid protein, the plant virus was TMV, and the buffer solution contained 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, or 100 mM EDTA. Analytical samples were obtained according to the procedures described above in [1. Preparation of N. benthamiana plants], [2. Infiltration of Agrobacterium transformants into N. benthamiana plants using the magnICON® system], [3. Leaf collection], and [4-1. Collection of target protein by immersion in buffer after crushing leaves]. The cases in which the EDTA concentrations in the buffer solutions were 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 100 mM were designated Examples 8 to 13, respectively. In addition, analytical samples were obtained using the same procedures as in Examples 8 to 13, except that the above-mentioned step [2. Infiltration of Agrobacterium transformants into Nicotiana benthamiana using the magnICON® system] was omitted and a buffer containing no chelating agent was used as the buffer (Comparative Example 6). In addition, analytical samples were obtained using the same procedures as in Examples 8 to 13, except that a buffer containing no chelating agent was used as the buffer (Comparative Example 7). Table 3 summarizes the target proteins and chelating agents used in Examples 8 to 13 and Comparative Examples 6 and 7. The analytical samples used in Examples 8 to 13 and Comparative Examples 6 and 7 were subjected to polyacrylamide gel electrophoresis and Western blotting assays using anti-histidine tag antibodies according to standard methods. The results are shown in Figures 3(A) and 3(B). In Table 3, "lane number" refers to the lane number in Figures 3(A) and 3(B). The histidine-tagged nucleocapsid protein is a protein expressed in the cytoplasm of Nicotiana benthamiana.

[0075] [Table 3]

[0076] As shown in Figures 3(A) and (B), in Comparative Example 6, in which proteins were recovered from wild-type leaves using a buffer solution containing no chelating agent, no nucleocapsid protein was detected by polyacrylamide gel electrophoresis and Western blotting assay. Furthermore, in Examples 8 to 13, in which proteins were recovered from infected leaves using buffer solutions containing 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 100 mM EDTA, respectively, the band corresponding to the approximately 46 kDa nucleocapsid protein in polyacrylamide gel electrophoresis and Western blotting assay was stronger than that in Comparative Example 7, in which proteins were recovered from infected leaves using a buffer solution containing no chelating agent. These results demonstrate that the efficiency of target protein extraction is improved by disrupting the plant bodies and then immersing them in a buffer solution containing EDTA at a concentration of 15 mM or higher.

[0077] [D. Recovery of nucleocapsid protein by disruption after immersion in a buffer containing CyDTA] The target protein was a histidine-tagged nucleocapsid protein, the plant virus was TMV, and the buffer solution contained 5, 20, 25, 30, 40, 50, or 100 mM trans-1,2-cyclohexanediaminetetraacetic acid monohydrate (CyDTA). Analytical samples were obtained according to the procedures described above: (1) Preparation of N. benthamiana plants, (2) Infiltration of Agrobacterium transformants into N. benthamiana plants using the magnICON® system, (3) Leaf collection, and (4-2) Recovery of target protein by crushing leaves after immersion in buffer. The cases where the CyDTA concentrations in the buffer solution were 5, 20, 25, 30, 40, 50, or 100 mM were designated Comparative Example 10 and Examples 14-19, respectively. An analytical sample (Comparative Example 8) was obtained using the same procedures as in Comparative Example 10 and Examples 14 to 19, except that the above-mentioned step (2. Infiltration of Agrobacterium transformants into Nicotiana benthamiana using the magnICON® system) was omitted and a buffer containing no chelating agent was used as the buffer. An analytical sample (Comparative Example 9) was obtained using the same procedures as in Comparative Example 10 and Examples 14 to 19, except that a buffer containing no chelating agent was used as the buffer. Table 4 summarizes the target proteins and chelating agents used in Examples 14 to 19 and Comparative Examples 8 to 10. The analytical samples from Examples 14 to 19 and Comparative Examples 8 to 10 were subjected to polyacrylamide gel electrophoresis and Western blotting assays using anti-histidine tag antibodies according to standard methods. The results are shown in Figures 4(A) and (B). In Table 4, "lane number" refers to the lane number in Figures 4(A) and (B). The histidine-tagged nucleocapsid protein is a protein that is expressed in the cytoplasm of Nicotiana benthamiana.

[0078] [Table 4]

[0079] As shown in Figures 4(A) and (B), in Comparative Example 8, in which proteins were recovered from wild-type leaves using a chelating agent-free buffer, polyacrylamide gel electrophoresis and Western blotting assays failed to detect nucleocapsid protein. Furthermore, in Examples 14 to 19, in which proteins were recovered from infected leaves using buffers containing 20 mM, 25 mM, 30 mM, 40 mM, 50 mM, and 100 mM CyDTA, respectively, the approximately 46 kDa nucleocapsid protein band in polyacrylamide gel electrophoresis and Western blotting assays was stronger than in Comparative Example 9, in which proteins were recovered from infected leaves using a chelating agent-free buffer, and Comparative Example 10, in which proteins were recovered from infected leaves using a buffer containing 5 mM CyDTA. These results demonstrate that the efficiency of target protein extraction can be improved by immersing plant bodies in a buffer containing 15 mM or more of CyDTA, an aminocarboxylic acid chelating agent similar to EDTA, followed by disruption of the plant bodies.

[0080] [E. Recovery of nucleocapsid protein by disruption after immersion in a citrate-containing buffer] The target protein was a histidine-tagged nucleocapsid protein, the plant virus was TMV, and the buffer solution contained 20 mM, 50 mM, 100 mM, or 200 mM citric acid. Analytical samples were obtained according to the procedures described above in [1. Preparation of N. benthamiana plants], [2. Infiltration of Agrobacterium transformants into N. benthamiana plants using the magnICON® system], [3. Leaf collection], and [4-2. Recovery of target protein by crushing leaves after immersion in buffer solution]. Examples 20 to 23 were used, respectively, with citric acid concentrations of 20 mM, 50 mM, 100 mM, and 200 mM. Analytical samples were also obtained according to the same procedures as in Examples 20 to 23, except that a buffer containing no chelating agent was used (Comparative Example 11). The target proteins and chelating agents used in Examples 20 to 23 and Comparative Example 11 are summarized in Table 5. The analytical samples of Examples 20 to 23 and Comparative Example 11 were subjected to Western blotting assay using an anti-histidine tag antibody according to a standard method, and the results are shown in Figure 5. In Table 5, "Lane No." refers to the lane number in Figure 5. The histidine-tagged nucleocapsid protein is a protein expressed in the cytoplasm of Nicotiana benthamiana.

[0081] [Table 5]

[0082] As shown in Figure 5, the nucleocapsid protein bands in the Western blotting assays of Examples 20 to 23, in which proteins were recovered from infected leaves using buffers containing 20 mM, 50 mM, 100 mM, and 200 mM citric acid, were stronger than those in Comparative Example 11, in which proteins were recovered from infected leaves using a buffer containing no chelating agent. These results demonstrate that the efficiency of target protein extraction can be improved by disrupting the plant bodies, then immersing the plants in a buffer containing 15 mM or more of citric acid, a hydroxycarboxylic acid chelating agent different from EDTA, an aminocarboxylic acid chelating agent.

[0083] [F. Recovery of human serum proteins by immersion in EDTA-containing buffer after disruption] The target protein was a histidine-tagged human serum protein, the plant virus was TMV, and the buffer solution contained 20 mM, 50 mM, 100 mM, or 200 mM EDTA. Analytical samples were obtained according to the procedures described above in [1. Preparation of N. benthamiana plants], [2. Infiltration of Agrobacterium transformants into N. benthamiana plants using the magnICON® system], [3. Leaf collection], and [4-1. Recovery of target protein by immersion in buffer after crushing leaves]. Examples 24 to 27 were used, respectively, with EDTA concentrations of 20 mM, 50 mM, 100 mM, and 200 mM. Analytical samples were also obtained according to the same procedures as in Examples 24 to 27, except that a buffer containing no chelating agent was used (Comparative Example 12). The target proteins and chelating agents used in Examples 24 to 27 and Comparative Example 12 are summarized in Table 6. The analytical samples from Examples 24 to 27 and Comparative Example 12 were subjected to Western blotting assay using an anti-histidine tag antibody according to a standard method, and the results are shown in Figure 6. In Table 6, "Lane No." refers to the lane number in Figure 6. The histidine-tagged human serum proteins are proteins expressed in the apoplast of Nicotiana benthamiana.

[0084] [Table 6]

[0085] As shown in Figure 6, the human serum protein bands in the Western blotting assays of Examples 24 to 27, in which proteins were recovered from infected leaves using buffers containing 20 mM, 50 mM, 100 mM, and 200 mM EDTA, were stronger than those in Comparative Example 12, in which proteins were recovered from infected leaves using a buffer containing no chelating agent. These results demonstrate that disrupting the plant bodies and then immersing them in a buffer containing EDTA at a concentration of 15 mM or more improves the extraction efficiency of proteins other than C-reactive protein and nucleocapsid protein.

Claims

1. A method for recovering a target protein from a plant expressing the target protein, comprising: A method comprising immersing a plant body in a buffer containing 15 mM or more of a chelating agent, and crushing the plant body.

2. The method according to claim 1, comprising crushing the plant body after immersing the plant body in a buffer containing 15 mM or more of a chelating agent.

3. 2. The method of claim 1, further comprising, after crushing the plant body, immersing the plant body in a buffer containing 15 mM or more of a chelating agent.

4. The method according to any one of claims 1 to 3, wherein the chelating agent is an aminocarboxylic acid-based chelating agent or a hydroxycarboxylic acid-based chelating agent.

5. the aminocarboxylic acid chelating agent is ethylenediaminetetraacetic acid; 5. The method of claim 4, wherein the hydroxycarboxylic acid chelating agent is citric acid.

6. providing a plant that expresses the target protein; and A method for producing a target protein, comprising recovering the target protein from the plant by the method according to any one of claims 1 to 3.

7. The method according to claim 6 , wherein the chelating agent is an aminocarboxylic acid chelating agent or a hydroxycarboxylic acid chelating agent.

8. the aminocarboxylic acid chelating agent is ethylenediaminetetraacetic acid; 8. The method of claim 7, wherein the hydroxycarboxylic acid chelating agent is citric acid.

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