Method for recovering and producing a target protein

Abstract

Disclosed is a method for recovering a target protein expressed in an apoplast that includes a step for injecting a buffer solution into a plant body from a stoma, water stoma, or cross section of a plant that expresses a target protein in the apoplast and a step for extracting the buffer solution inside the plant body from a stoma, water stoma, or cross section.

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 expressing a target protein in a plant body by recombinant gene technology, etc., and recovering the expressed target protein. In conventional recovery and production of a target protein using a plant body, for example, as described in Non-Patent Document 1, a method of crushing the leaves of the plant to extract most of the plant components has often been used.

[0003] However, in conventional methods involving the disruption of plant bodies, the extract obtained by disrupting the plant bodies contains insoluble foreign matter derived from plants such as plant residues and soluble impurities in addition to the target protein, and therefore a purification step is required to remove these. As described in Non-Patent Document 2, when attempting to increase the amount of target protein extracted, the amount of unnecessary plant components increases, which increases the burden on this purification step. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Marillonnet et al. “In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium.”, Proc Natl Acad Sci US A., 2004 Oct 26;101(43):15546 [Non-Patent Document 2] Dixon et al. “The Impact of Six Critical Impurities on Recombinant Protein Recovery and Purification from Plant Hosts”, Molecular Pharming: Applications, Challenges, and Emerging Areas, 2018, 137-180 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a method for recovering a target protein and a method for producing a target protein that do not require disruption of the plant body. [Means for solving the problem]

[0006] The present inventors have found that it is possible to recover a target protein expressed in the apoplast without disrupting the plant by injecting a buffer solution into the plant through the stomata, water pores or cross section of the plant body expressing the target protein in the apoplast and extracting the buffer solution from the inside of the plant body through the stomata, water pores or cross section. The present invention is based on this novel finding.

[0007] That is, the present invention relates to, for example, the following inventions. [1] Injecting a buffer solution into the plant body through the stomata, water pores or cross section of the plant body expressing the target protein in the apoplast; and A step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross section. A method for recovering a target protein expressed in the apoplast, comprising the steps of: [2] The method according to [1], wherein the step of injecting the buffer solution into the plant body through the stomata, water pores or cross-section includes injecting the buffer solution by applying pressure or vacuum. [3] The method according to [1] or [2], wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section comprises extracting the buffer solution by applying pressure or pressure reduction. [4] The step of injecting a buffer solution into the plant body through a stomata, a water hole or a cross section includes injecting the buffer solution by applying pressure or pressure; The step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section includes extracting the buffer solution by applying pressure or pressure; The method according to [1], which comprises repeating two or more sets of a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section and a step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section. [5] The method according to any of [1] to [4], wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the plant body through the stomata, water pores or cross-section. [6] The method according to any of [1] to [5], wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the inside of the plant body through the stomata, water pores or cross-section. [7] The method according to [2] or [4], wherein injecting the buffer solution into the plant body by pressurization or depressurization includes pressurizing or depressurizing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container. [8] The method according to [3] or [4], wherein extracting the buffer solution from within the plant body by pressurization or decompression includes pressurizing or decompressing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container. [9] Providing a plant that expresses a protein of interest in the apoplast; Injecting a buffer solution into the plant body through the stomata, water holes or cross-section of the plant body; and Extracting a buffer solution from inside the plant body through the stomata, water pores or cross section; A method for producing a target protein, comprising:

[10] The method according to [9], wherein the step of injecting the buffer solution into the plant body through the stomata, water pores or cross-section comprises injecting the buffer solution by applying pressure or vacuum.

[11] The method according to [9] or

[10] , wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section comprises extracting the buffer solution by applying pressure or vacuum.

[12] The step of injecting a buffer solution into the plant body through a stomata, a water hole or a cross section includes injecting the buffer solution by applying pressure or pressure; The step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section includes extracting the buffer solution by applying pressure or pressure; The method described in [9], which comprises repeating two or more sets of a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section and a step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section.

[13] The method according to any of [9] to

[12] , wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the plant body through the stomata, water pores or cross-section.

[14] The method according to any of [9] to

[13] , wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the inside of the plant body through the stomata, water pores or cross-section.

[15] The method according to

[10] or

[12] , wherein injecting the buffer solution into the plant body by pressurization or depressurization includes pressurizing or depressurizing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container.

[16] The method according to

[11] or

[12] , wherein extracting the buffer solution from within the plant body by pressurization or decompression includes pressurizing or decompressing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container.

[17] Injecting a buffer solution into the inside of a leaf through a stomata or water pores of a leaf of a plant body expressing a target protein in the apoplast; A step of extracting the buffer solution from inside the leaf through the stomata or water pores. A method for recovering a target protein expressed in the apoplast, comprising the steps of:

[18] The method according to

[17] , wherein the step of injecting the buffer solution into the inside of the leaf through the stomata or water pores includes injecting the buffer solution by applying pressure or vacuum.

[19] The method according to

[17] or

[18] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises extracting the buffer solution by applying pressure or vacuum.

[20] The step of injecting the buffer solution into the inside of the leaf through the stomata or water pores includes injecting the buffer solution by applying pressure or vacuum; The step of extracting the buffer solution from inside the leaf through the stomata or water pores includes extracting the buffer solution by applying pressure or vacuum; The method described in

[17] , comprising repeating two or more sets of steps each consisting of a step of injecting a buffer solution into the inside of the leaf through the stomata or water pores and a step of extracting the buffer solution from the inside of the leaf through the stomata or water pores. [twenty one] The method according to any of

[17] to

[20] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises immersing the leaves of the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the leaf through the stomata or water pores. [twenty two] The method according to any of

[17] to

[21] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises immersing the leaves of the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the leaf through the stomata or water pores. [twenty three] The method according to any one of [7] to

[22] , wherein the target protein is an enzyme. [twenty four] The method according to

[18] or

[20] , wherein injecting the buffer solution into the plant body by pressurization or depressurization comprises pressurizing or depressurizing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container. [twenty five] The method according to

[19] or

[20] , wherein extracting the buffer solution from within the plant body by pressurization or decompression comprises pressurizing or decompressing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container.

[26] Providing a plant that expresses a protein of interest in the apoplast; Injecting a buffer solution into the inside of the leaf through the stomata or water holes of the leaf of the plant body; Extracting the buffer solution from inside the leaf through the stomata or water pores; A method for producing a target protein, comprising:

[27] The method according to

[26] , wherein the step of injecting the buffer solution into the inside of the leaf through the stomata or water pores includes injecting the buffer solution by applying pressure or vacuum.

[28] The method according to

[26] or

[27] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises extracting the buffer solution by applying pressure or vacuum.

[29] The step of injecting the buffer solution into the inside of the leaf through the stomata or water pores includes injecting the buffer solution by applying pressure or vacuum; The step of extracting the buffer solution from inside the leaf through the stomata or water pores includes extracting the buffer solution by applying pressure or vacuum; The method described in

[26] , comprising repeating two or more sets of steps each consisting of a step of injecting a buffer solution into the inside of the leaf through the stomata or water pores and a step of extracting the buffer solution from the inside of the leaf through the stomata or water pores.

[30] The method according to

[26] or

[27] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises immersing the leaves of the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the leaf through the stomata or water pores.

[31] The method according to

[24] , wherein the step of extracting the buffer solution inside the leaf through the stomata or water pores comprises immersing the leaves of the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the leaf through the stomata or water pores.

[32] The method according to any one of

[26] to

[31] , wherein the target protein is an enzyme.

[33] The method according to

[27] or

[29] , wherein injecting the buffer solution into the plant body by pressurization or depressurization comprises pressurizing or depressurizing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container.

[34] The method according to

[28] or

[29] , wherein extracting the buffer solution from within the plant body by pressurization or decompression comprises pressurizing or decompressing using a gas, liquid, a press, or a combination thereof in a pressure-resistant container. Effect of the Invention

[0008] According to the present invention, it is possible to provide a method for recovering a target protein and a method for producing a target protein, which do not require disruption of a plant body.

[0009] If the amount of unnecessary plant-derived components extracted during extraction of a target protein is reduced, the burden on the purification step is reduced. According to the present invention, since the disruption of the plant body is not required, the purification step of removing unnecessary plant components contained in the extract obtained by disrupting the plant body can be omitted. According to the present invention, the amount of the target protein can be obtained equal to or greater than that of the conventional extraction method in which the plant body is disrupted while reducing unnecessary plant components, and therefore the target protein can be efficiently extracted and recovered. [Brief description of the drawings]

[0010] [Figure 1] The results of SDS-PAGE (a) and Western blot analysis (b) of the homogenate extract and the pressurized / vacuum extract are shown in Fig. 1. Lane 1 is the pressurized / vacuum extract, lane 2 is the homogenate extract, and the arrows indicate the position of the phosphodiesterase band. [Diagram 2] The results of SDS-PAGE (a) and Western blot analysis (b) of the disruption extract and osmotic extract are shown in Figure 2. Lane 1 is the disruption extract, lane 2 is the osmotic extract, and the arrows indicate the position of the phosphodiesterase band. [Diagram 3]Schematic diagrams illustrating pressurization or depressurization using gas, liquid, or a press in a pressure-resistant container. Pressure can be increased by adding gas in the direction of the downward arrow into a pressure-resistant container containing a plant immersed in a buffer solution, and depressurization can be achieved by removing gas in the direction of the upward arrow (a). Pressure can be increased by adding liquid in the direction of the downward arrow into a pressure-resistant container containing a plant immersed in a buffer solution, and depressurization can be achieved by removing liquid in the direction of the upward arrow (b). In a pressure-resistant container containing a plant immersed in a buffer solution, the gas and / or liquid can be compressed and pressurized by narrowing the sealed space containing the plant immersed in the buffer solution by pressing in the direction of the downward arrow, and depressurization can be achieved by expanding the sealed space in the direction of the upward arrow or using a pressure reducing valve (c). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] [Method for recovering target protein] A method for recovering a target protein according to one embodiment of the present invention includes the steps of injecting a buffer solution into a plant body through a stomata, water pore or cross section of the plant body expressing the target protein in the apoplast, and extracting the buffer solution from the inside of the plant body through the stomata, water pore or cross section, wherein the target protein to be recovered is the target protein expressed in the apoplast.

[0012] As used herein, the term "apoplast" refers to the part of plant tissue that is outside the cell membrane, that is, the total of the cell wall and intercellular spaces.

[0013] The target protein is not limited as long as it can be expressed in the apoplast of a plant body, and may be, for example, a naturally occurring protein, a protein derived from a naturally occurring protein, or an artificially designed protein. Examples of the target protein include 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 enzymes, antigens, and antibodies, or may be an enzyme. 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 yeasts.

[0014] Examples of the enzyme include oxidases, reductases, lipases (e.g., phospholipases), proteases, kinases, phosphatases, cellulases, steroid synthesis enzymes, methylases, demethylases, collagenases, transglutaminases, glycosidases, and chitinases. For example, the enzyme may be selected from the group consisting of phospholipases, esterases, and oxidases, and may be an esterase or a phosphodiesterase.

[0015] 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 (scFv), sc(Fv)2, disulfide-linked Fvs (sdFv), and diabodies.

[0016] Examples of antigens and epitopes include proteins derived from filamentous fungi, bacteria, and viruses. In the case of antigenic proteins or epitopes used as vaccines, there are no particular limitations as long as they have immunogenicity, and examples of such antigenic proteins include proteins derived from pathogenic filamentous fungi, proteins derived from pathogenic bacteria, and proteins derived from pathogenic viruses.

[0017] 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).

[0018] Hormones include, for example, peptide and protein hormones.

[0019] 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).

[0020] Examples of transcription factors include general transcription factors, upstream transcription factors, and inducible transcription factors.

[0021] Receptors include, for example, G protein-coupled receptors, ionotropic receptors, and the cytokine receptor superfamily.

[0022] The target protein may also be a fusion protein in which two or more proteins are bound together. The fusion protein of two or more proteins may be a fusion protein of two or more heterologous proteins, or a fusion protein of two or more homogeneous proteins.

[0023] The molecular weight of the target protein is not particularly limited, and may be, for example, 1 kDa to 100 kDa, 1 kDa to 75 kDa, 1 kDa to 50 kDa, or 1 kDa to 10 kDa. In addition, the molecular weight of the target protein may be, for example, 500 kDa or less, 300 kDa or less, 200 kDa or less, 100 kDa or less, 75 kDa or less, or 50 kDa or less.

[0024] The protein of interest may be a monomer, dimer, trimer or multimer.

[0025] The protein of interest may be tagged to facilitate detection and / or purification of the protein of interest.

[0026] A plant expressing a target protein can be produced by known methods, such as the agroinfiltration method, the plant virus vector method, the magnICON (registered trademark) system, and the particle gun method.

[0027] When using the agroinfiltration method, Agrobacterium is transformed with a T-DNA containing an inserted gene encoding a target protein, and the Agrobacterium is then infected into a plant to obtain a plant that expresses the target protein.

[0028] When using the plant virus vector method, a plant expressing a target protein can be obtained by inoculating a plant with RNA obtained from cDNA of a plant virus genome into which a gene encoding a target protein has been inserted, as a vector, to infect the plant. Examples of such virus vectors include tobacco mosaic virus (TMV) vectors, plum pox virus (PPV) vectors, potato virus X (PVX) vectors, alfalfa mosaic virus (AIMV) vectors, cucumber mosaic virus (CMV) vectors, cowpea mosaic virus (CPMV) vectors, and zucchini yellow mosaic virus (ZYMV) vectors.

[0029] When using the magnICON (registered trademark) system, a cDNA of the TMV or PVX genome into which a gene encoding a target protein has been inserted is introduced into a T-DNA vector, and a plant body that expresses the target protein can be obtained by infecting the plant body with Agrobacterium transformed with the resulting T-DNA vector.

[0030] When using the particle gun method, metal microparticles coated with a nucleic acid encoding a target protein are fired at high speed, and the gene encoding the target protein is introduced into the cells, thereby obtaining a plant body expressing the target protein.

[0031] 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 Legume family (e.g., soybean, adzuki bean, kidney bean, broad bean). For example, the plant body may be a plant of the Solanaceae or Brassicaceae family, a plant of the Nicotiana or Arabidopsis genus, such as Nicotiana benthamiana, Nicotiana tabacum, or Arabidopsis thaliana.

[0032] The target protein can be expressed in the apoplast by a known method. For example, a target protein that is naturally expressed in the apoplast when expressed in a plant body may be used, or a target protein targeted to be expressed in the apoplast (cell wall and / or intercellular space) may be used. Examples of proteins that are expressed in the apoplast include albumin, antibodies, and antigens. A method for expressing a target protein in the apoplast that is normally expressed outside the apoplast may include, for example, linking a DNA sequence encoding a signal peptide that directs extracellular localization to a DNA sequence encoding the target protein. Examples of signal peptides that direct extracellular localization include the rice amylase signal peptide (McCormick 1999, Proc Natl Acad Sci USA 96:703-708), tobacco plant pathogenesis-related protein (PRP), and secretion targeting signals derived from calreticulin protein.

[0033] (A step of injecting a buffer solution into the plant body through the stomata, water pores or cross section of the plant body expressing the target protein in the apoplast) The buffer solution is injected into the apoplast inside the plant body by injecting the buffer solution into the inside of the plant body through the stomata, water pores, or cross-sections present in the plant body. The stomata or water pores are usually present on the underside of the plant leaves. Alternatively, the buffer solution is injected into the apoplast inside the plant body by injecting the buffer solution into the inside of the plant body through a cut cross-section of the plant body. By injecting the buffer solution into the apoplast, the target protein expressed in the apoplast migrates into the injected buffer solution.

[0034] As the plant body, for example, leaves, roots, shoots, stems, flowers, fruits, embryos, seedlings, and combinations thereof can be used, but when the buffer solution is injected from the stomata or water pores, it is preferable that the plant body includes leaves, and when the buffer solution is injected from the cross section, it is preferable that the plant body has a cut surface. Here, since the invention according to this embodiment does not include crushing the plant body expressing the target protein, the plant body after cutting maintains a part of its morphology. Cutting the plant body may be, for example, cutting 1 to 3 places of the plant body, or cutting the plant body or leaves into 1 / 10 to 1 / 2 (volume), and the plant body may be a cut piece. The plant body can be cut, for example, using a cutter and a razor.

[0035] The plant body to be used may be a fresh plant body (for example, fresh leaves), or a plant body that has been subjected to treatments such as freezing, thawing, and drying, so long as the present invention can be carried out.

[0036] Examples of the method of injecting a buffer solution into the plant body from a stoma, a water hole or a cross section include a method of directly injecting a buffer solution into the plant body from a stoma, a water hole or a cross section, a method of injecting using pressurization or reduced pressure, and a method of injecting using osmotic pressure. From the viewpoint of improving the extraction efficiency of the target protein, the method of injecting a buffer solution into the plant body from a stoma, a water hole or a cross section is preferably a method of injecting using pressurization or reduced pressure. In addition, the method of injecting a buffer solution into the plant body from a stoma, a water hole or a cross section may be a method of injecting a buffer solution into the leaf from a stoma or water hole on the back of the leaf, and from the viewpoint of improving the extraction efficiency of the target protein, it is preferable to inject a buffer solution into the leaf from a stoma or a water hole directly, or a method of injecting using pressurization or reduced pressure.

[0037] Examples of the method for directly injecting the buffer solution into the plant body from the stomata, water hole, or cross section include a method in which the tip of a syringe filled with the buffer solution is placed against the stomata, water hole, or cross section, and the plunger is moved toward the tip of the syringe to inject the buffer solution into the plant body from the stomata, water hole, or cross section. In this case, the plunger may be moved manually or by applying other mechanical external force.

[0038] An example of a method of injection using pressure is a method of injecting the buffer solution into the plant body from the stomata, water holes, or cross section by pressurizing the inside of a container that contains a plant body immersed in the buffer solution. Pressurization inside a container (e.g., a pressure-resistant container) can be performed, for example, by injecting gas into the container, or by compressing the gas inside the container. When the container is a syringe, for example, the pressurization can be performed by sealing one side of the syringe and moving a plunger (with the inside of the plunger sealed) from the other side toward the tip of the syringe, or by injecting gas from the other side. The pressure inside the pressurized container is not particularly limited as long as it is higher than atmospheric pressure and can inject the buffer solution into the plant body from the stomata, water holes, or cross section, but may be, for example, 0.01 to 5 MPa, 0.01 to 1 MPa, 0.01 to 0.5 MPa, or 0.01 to 0.1 MPa. The pressurization time is not particularly limited, and may be, for example, 0 to 5 minutes, 0 to 1 minute, 0 to 0.5 minutes, 1 second to 5 minutes, 1 second to 1 minute, or 1 second to 0.5 minutes.

[0039] An example of the method of injection using reduced pressure is a method in which the pressure in a container (e.g., a pressure-resistant container, a desiccator) in which a plant body immersed in a buffer solution is contained is reduced, and then the pressure is restored to inject the buffer solution into the plant body through the stomata, water holes, or cross-sections. The pressure in the container can be reduced, for example, by removing the air in the container, and the pressure can be restored by stopping the reduction in pressure and returning the air to the container. When the container is a syringe, the pressure can be reduced, for example, by sealing one side of the syringe and moving the plunger from the other side in the direction opposite to the tip of the syringe, or by removing the air in the plunger from the other side. The pressure in the reduced container is not particularly limited as long as it is lower than atmospheric pressure and the buffer solution can be injected into the plant body through the stomata, water holes, or cross-sections by restoring the pressure, but may be, for example, -0.01 to -0.1 MPa or -0.01 to -0.05 MPa. The decompression time is not particularly limited and may be, for example, 0 to 5 minutes, 0 to 1 minute, 0 to 0.5 minutes, 1 second to 5 minutes, 1 second to 1 minute, or 1 second to 0.5 minutes. The pressure recovery time is also not particularly limited and may be, for example, 0 to 5 minutes, 0 to 1 minute, 0 to 0.5 minutes, 1 second to 5 minutes, 1 second to 1 minute, or 1 second to 0.5 minutes.

[0040] Injecting the buffer solution into the plant body by pressurization or depressurization may include pressurizing or depressurizing using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container (e.g., a pressure-resistant tank). When air pressure is used, for example, a pressure-resistant container containing a plant immersed in a buffer solution can be pressurized by adding a gas, and depressurized by removing the gas (FIG. 3(a)). When a liquid is used, for example, a pressure-resistant container containing a plant immersed in a buffer solution can be pressurized by adding a liquid, and depressurized by removing the liquid (FIG. 3(b)). When a press is used, for example, a sealed space containing the plant immersed in a buffer solution can be narrowed by a press in a pressure-resistant container containing a plant immersed in a buffer solution, thereby compressing the gas and / or liquid to pressurize the plant, and conversely, the sealed space can be widened or a pressure reducing valve can be used to depressurize the plant (FIG. 3(c)).

[0041] An example of a method of injection using osmotic pressure is immersing a plant body in a buffer solution having an osmotic pressure lower than the concentration of the liquid contained in the apoplast inside the plant body. For example, a method of immersing a plant body in a buffer solution having a lower concentration than the concentration of the liquid contained in the apoplast inside the plant body. The concentration of the buffer solution may be, for example, a salt concentration or a sugar concentration.

[0042] As the buffer, for example, a general buffer such as a Tris buffer, a phosphate buffer, or a citrate buffer can be used. In the present invention, the buffer is not particularly limited, but can be appropriately set by a person skilled in the art while considering the influence on each protein characteristic such as hydration, stability, activity, etc. that may be given to the target protein. The concentration of the Tris buffer may be, for example, 10 to 300 mM, or 10 to 100 mM. The concentration of the phosphate buffer may be, for example, 10 to 200 mM. The concentration of the citrate buffer may be, for example, 10 to 200 mM. The pH of the buffer may be, for example, pH 3 to 11, pH 5 to 9, or pH 7 to 9.

[0043] The buffer may contain a salt. Examples of salts contained in the buffer include common salts such as sodium chloride, magnesium chloride, and sodium phosphate. The type and concentration of inorganic salts can be appropriately determined by those skilled in the art while taking into consideration the effects on each protein characteristic, such as hydration, stability, and activity, that can be imparted to the target protein. The salt concentration of the buffer may be, for example, 1 to 3000 mM or 1 to 1000 mM. When the buffer is injected into the inside of the plant body through the stomata, water pores, or cross section using osmotic pressure, the salt concentration of the buffer in which the plant body is immersed is preferably lower than the salt concentration of the liquid contained in the apoplast inside the plant body.

[0044] The buffer may contain sugar. Examples of sugars that can be used in the buffer include common sugars such as sucrose, trehalose, and maltose. The type and concentration of sugar can be appropriately determined by those skilled in the art, taking into consideration the effects on the hydration, stability, activity, and other protein characteristics that can be imparted to the target protein. The sugar concentration of the buffer may be, for example, 0.1 to 50% or 0.1 to 10%. When the buffer is injected into the plant body through the stomata, water pores, or cross-section using osmotic pressure, the sugar concentration of the buffer in which the plant body is immersed is preferably lower than the sugar concentration of the liquid contained in the apoplast inside the plant body.

[0045] The buffer solution may contain additives, such as chelating agents such as EDTA, EGTA, NTA, and DTPA, antioxidants such as α-mercaptoethanol, ascorbate, sodium metabisulfate, and dithiothreitol, surfactants such as SDS, CTAB, CHAPS, and TritonX-100, denaturants such as urea and guanidine hydrochloride, amino acids such as aspartic acid and glutamic acid, alcohols such as glycerol and isopropanol, enzymatic proteins such as cellulase, protease, and pectinase, and nonenzymatic proteins such as expansin.

[0046] The buffer may be one that does not contain an additive. By adding an additive such as a chelating agent, a step of removing the additive using a column or the like may be required after the buffer is extracted from the pores, water pores, or cross section. The present invention has the advantage that a step of removing the additive is not required because the target protein can be extracted using a buffer that does not contain an additive.

[0047] The temperature at which this step is carried out is not particularly limited, and may be, for example, 1 to 50° C., 1 to 30° C., 1 to 15° C., or 4 to 50° C. To prevent denaturation of the target protein, a temperature of 30° C. or lower is preferable.

[0048] The buffer solution used in this step may be referred to as the first buffer solution, and the buffer solution used in the step of "extracting the buffer solution from inside the plant body from the stomata, water pores, or cross section" described below may be referred to as the second buffer solution. The first buffer solution and the second buffer solution may be the same or different. The second buffer solution may be prepared by further adding salts, etc. to the first buffer solution.

[0049] When the buffer solution is injected into the interior of the plant body through the stomata, water pores or cross section while the plant body is immersed in the buffer solution, the "step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section" described below may be carried out while the plant body is still immersed in the buffer solution, or the "step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section" described below may be carried out after separating the plant body from the buffer solution.

[0050] This step may further include, after injecting the buffer solution into the inside of the plant body, for example, removing the plant body from the container and removing the buffer solution adhering to the surface of the plant body.

[0051] (Step of extracting buffer solution from inside the plant body through stomata, water holes or cross sections) The target protein expressed in the apoplast is contained in the buffer solution injected into the plant body through the stomata, water pores, or cross section. Therefore, by extracting the buffer solution from inside the plant body through the stomata, water pores, or cross section, the target protein can be recovered in a form contained in the buffer solution.

[0052] Examples of the method for extracting a buffer solution from inside a plant body from a stomata, water pore or cross section include a method for extracting a buffer solution directly from a stomata, water pore or cross section, a method for extracting using pressurization or reduced pressure, a method for extracting using osmotic pressure, and a method for extracting using centrifugal force. From the viewpoint of improving the extraction efficiency of a target protein, the method for extracting a buffer solution from inside a plant body from a stomata, water pore or cross section is preferably a method for extracting using pressurization or reduced pressure, or a method for extracting using osmotic pressure. In addition, the method for extracting a buffer solution from inside a plant body from a stomata, water pore or cross section may be a method for extracting a buffer solution from inside a leaf from a stomata or water pore, and from the viewpoint of improving the extraction efficiency of a target protein, it is preferably a method for extracting using pressurization or reduced pressure, or a method for extracting using osmotic pressure. The step of extracting a buffer solution from inside a plant body from a stomata, water pore or cross section may be one other than extraction using centrifugal force.

[0053] An example of a method for directly extracting a buffer solution from a stomata, water pore, or cross section of a plant body is to place the tip of an empty syringe against the stomata, water pore, or cross section of the plant body and move the plunger in the opposite direction to the tip of the syringe to extract the buffer solution from the stomata, water pore, or cross section of the plant body. In this case, the plunger may be moved manually or by applying other mechanical external force.

[0054] An example of the method of extraction using pressurization is a method of extracting the buffer solution from the inside of the plant body through the stomata, water pores, or cross section by pressurizing the inside of a container that contains a plant body into which a buffer solution has been injected. Pressurization in the container can be performed, for example, by injecting gas into the container, or by compressing the gas in the container. When the container is a syringe, for example, it may be performed by sealing one side of the syringe and moving the plunger from the other side toward the tip of the syringe, or by injecting gas from the other side. The pressure in the pressurized container is not particularly limited as long as it is higher than atmospheric pressure and can extract the buffer solution from the inside of the plant body through the stomata, water pores, or cross section, and may be, for example, 0.01 to 5 MPa, 0.01 to 1 MPa, 0.01 to 0.5 MPa, or 0.01 to 0.1 MPa. The pressurization time is not particularly limited, and may be, for example, 0 to 5 minutes, 0 to 1 minute, 0 to 0.5 minutes, 1 second to 5 minutes, 1 second to 1 minute, or 1 second to 0.5 minutes.

[0055] An example of the method of extraction using reduced pressure is a method of extracting the buffer solution from the inside of the plant body through the stomata, water holes, or cross section by reducing the pressure inside a container that contains a plant body injected with a buffer solution. The pressure reduction may be performed while the plant body injected with the buffer solution is immersed in the buffer solution. When the pressure reduction is performed inside a container while the plant body injected with the buffer solution is immersed in the buffer solution, the buffer solution inside the plant body is extracted from the stomata, water holes, or cross section, and is contained in the buffer solution in which the plant body is immersed. The pressure reduction inside the container can be performed, for example, by removing the air from the container. When the container is a syringe, the pressure reduction can be performed, for example, by sealing one side of the syringe and moving the plunger from the other side in the direction opposite to the tip of the syringe, or by removing the air from the plunger from the other side. The pressure in the reduced pressure container is not particularly limited as long as it is lower than atmospheric pressure and the buffer solution inside the plant body can be extracted from the stomata, water holes, or cross section, but may be, for example, -0.01 to -0.1 MPa or -0.01 to -0.05 MPa. The decompression time is not particularly limited, and may be, for example, 0 to 5 minutes, 0 to 1 minute, 0 to 0.5 minutes, 1 second to 5 minutes, 1 second to 1 minute, or 1 second to 0.5 minutes.

[0056] The extraction of the buffer solution from inside the plant body by the above-mentioned pressurization or decompression may include pressurization or decompression using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container (e.g., a pressure-resistant tank). When air pressure is used, for example, a pressure-resistant container containing a plant immersed in a buffer solution can be pressurized by adding a gas, and decompressed by removing the gas (FIG. 3(a)). When a liquid is used, for example, a pressure-resistant container containing a plant immersed in a buffer solution can be pressurized by adding a liquid, and decompressed by removing the liquid (FIG. 3(b)). When a press is used, for example, a sealed space containing the plant immersed in the buffer solution can be narrowed by the press in a pressure-resistant container containing the plant immersed in the buffer solution, thereby compressing the gas and / or liquid, and conversely, the sealed space can be widened or a pressure reducing valve can be used to decompress the space (FIG. 3(c)).

[0057] An example of the method of extraction using osmotic pressure is immersing a plant body in a buffer solution having an osmotic pressure higher than that of the buffer solution (buffer solution contained in the apoplast) injected into the inside of the plant body through the stomata, water pores, or cross section. For example, a plant body can be immersed in a buffer solution having a higher concentration than that of the buffer solution injected into the inside of the plant body through the stomata, water pores, or cross section. For example, when the buffer solution injected into the inside of the plant body through the stomata, water pores, or cross section is the first buffer solution, and the second buffer solution used in this step is the second buffer solution having a higher concentration than the first buffer solution, the buffer solution inside the plant body can be extracted by immersing the plant body in the second buffer solution having a higher concentration than the first buffer solution. The concentration of the buffer solution may be, for example, a salt concentration or a sugar concentration.

[0058] As the buffer, for example, a general buffer such as a Tris buffer, a phosphate buffer, or a citrate buffer can be used. In the present invention, the buffer is not particularly limited, but can be appropriately set by a person skilled in the art while considering the influence on each protein characteristic such as hydration, stability, activity, etc. that may be given to the target protein. The concentration of the Tris buffer may be, for example, 10 to 300 mM, or 10 to 100 mM. The concentration of the phosphate buffer may be, for example, 10 to 200 mM. The concentration of the citrate buffer may be, for example, 10 to 200 mM. The pH of the buffer may be, for example, pH 3 to 11, pH 5 to 9, or pH 7 to 9.

[0059] The time for immersing the plant body in the second buffer solution may be, for example, 1 to 120 minutes, 5 to 60 minutes, or 10 to 30 minutes.

[0060] The buffer solution used may be the same as the buffer solution injected into the plant body through the stomata, water pores or cross section, or may be different. When the buffer solution inside the plant body is extracted using osmotic pressure, a buffer solution having at least an osmotic pressure different from that of the buffer solution injected into the plant body through the stomata, water pores or cross section is used.

[0061] The buffer may contain a salt. Examples of salts contained in the buffer include common salts such as sodium chloride, magnesium chloride, and sodium phosphate. The type and concentration of inorganic salts can be appropriately determined by those skilled in the art, taking into consideration the effects on each protein characteristic, such as hydration, stability, and activity that can be imparted to the target protein. The salt concentration of the buffer may be, for example, 1 to 3000 mM or 1 to 1000 mM. When the buffer solution inside the plant body is extracted from the stomata, water pores, or cross section using osmotic pressure, the salt concentration of the second buffer solution in which the plant body is immersed is preferably higher than the salt concentration of the first buffer solution (buffer solution contained in the apoplast) injected into the plant body from the stomata, water pores, or cross section, and may be, for example, 10 to 3000 mM or 10 to 1000 mM. In addition, for example, the salt concentration of the second buffer solution may be 1 time or more, 2 times or more, 1 time to 1000 times, or 2 times to 100 times the salt concentration of the first buffer solution. By immersing the plant body in a second buffer solution having a higher salt concentration than the first buffer solution injected inside the plant body, the first buffer solution containing the target protein is extracted into the second buffer solution by osmotic pressure.

[0062] The buffer may contain sugar. Examples of sugars contained in the buffer include common sugars such as sucrose, trehalose, and maltose. The type and concentration of sugar can be appropriately determined by a person skilled in the art, taking into consideration the effect on each protein characteristic, such as hydration, stability, and activity that can be imparted to the target protein. The sugar concentration of the buffer may be, for example, 0.1 to 50% or 0.1 to 10%. When the buffer solution inside the plant body is extracted from the stomata, water pores, or cross section using osmotic pressure, the sugar concentration of the second buffer solution in which the plant body is immersed is preferably higher than the sugar concentration of the first buffer solution (buffer solution contained in the apoplast) injected into the plant body from the stomata, water pores, or cross section, and may be, for example, 0.1 to 50% or 0.1 to 10%. In addition, for example, the sugar concentration of the second buffer solution may be 1 time or more, 2 times or more, 1 time to 1000 times, or 2 times to 100 times the sugar concentration of the first buffer solution. By immersing the plant body in a second buffer solution having a higher sugar concentration than the sugar concentration of the first buffer solution injected inside the plant body, the first buffer solution containing the target protein is extracted into the second buffer solution by osmotic pressure.

[0063] The buffer solution may contain additives, such as chelating agents such as EDTA, NTA, and DTPA, antioxidants such as α-mercaptoethanol, ascorbate, sodium metabisulfate, and dithiothreitol, surfactants such as SDS, CTAB, CHAPS, and TritonX-100, denaturants such as urea and guanidine hydrochloride, amino acids such as aspartic acid and glutamic acid, alcohols such as glycerol and isopropanol, enzymatic proteins such as cellulase, protease, and pectinase, and nonenzymatic proteins such as expansin.

[0064] The buffer may be one that does not contain an additive. By adding an additive such as a chelating agent, a step of removing the additive using a column or the like may be required after the buffer is extracted from the pores, water pores, or cross section. The present invention has the advantage that a step of removing the additive is not required because the target protein can be extracted using a buffer that does not contain an additive.

[0065] The temperature at which this step is carried out is not particularly limited, and may be, for example, 1 to 50° C., 1 to 30° C., 1 to 15° C., or 4 to 50° C. In order to prevent denaturation of the target protein, the temperature is preferably 30° C. or lower. The temperature may be the same as or different from the temperature in the step of injecting a buffer solution into the plant body through the stomata, water pores, or cross section.

[0066] An example of a method of extraction using centrifugal force is to place a plant body into which a buffer solution has been injected through the stomata, water pores, or cross section, in a container, and centrifuge the plant body. The buffer solution inside the plant body is released into the container through the stomata, water pores, or cross section by centrifugal force. The conditions for centrifugation are not particularly limited, but examples include centrifugation at 100 rpm to 20,000 rpm for 10 to 30 minutes.

[0067] By extracting the buffer solution containing the target protein, the target protein can be recovered in a state contained in the buffer solution. Depending on the use of the target protein, the method may further include a step of separating and purifying the target protein from the buffer solution. The step of purifying the target protein from the buffer solution can be performed according to a method well known to those skilled in the art, for example, salting out, ethanol precipitation, ultrafiltration, dialysis, filter filtration, aqueous two-phase separation, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, medium-high pressure liquid chromatography, reversed phase chromatography, hydrophobic chromatography, etc., or a combination thereof.

[0068] Furthermore, since the method according to this embodiment does not require crushing of the plant body, it does not require a purification step for removing unnecessary plant body components contained in the extract obtained by crushing the plant body. However, this does not preclude the method according to this embodiment from including an additional purification step for removing impurities and the like contained in the buffer solution extracted from the stomata, water pores, or cross section in order to further increase the purity of the target protein.

[0069] The method according to the present embodiment may also include a step of confirming the presence and / or expression level of the target protein. This step may be performed after extraction of a buffer solution containing the target protein, or after separation and purification. The presence and / or expression level of the target protein may be confirmed by a method well known to those skilled in the art, such as Western blotting and amino acid sequence analysis.

[0070] From the viewpoint of improving the extraction efficiency of the target protein, the method according to the present embodiment preferably includes a step of injecting a buffer solution into the plant body through a stomata, water pores or cross section by pressurizing or depressurizing the buffer solution, and a step of extracting the buffer solution from the plant body through a stomata, water pores or cross section by pressurizing or depressurizing the buffer solution. In this case, for example, the buffer solution may be injected into the plant body through pressurizing or depressurizing the buffer solution from the plant body by ... The upper limit of the number of repetitions of the above sets is not particularly limited, and may be, for example, 100 sets or less, 50 sets or less, or 20 sets or less. It is preferable that the plant body remains immersed in the buffer solution during all the repetitions of the sets.

[0071] Furthermore, from the viewpoint of improving the extraction efficiency of the target protein, in the method of this embodiment, the step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section preferably includes injecting the buffer solution by pressurization or depressurization, and the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section preferably includes extraction using osmotic pressure.

[0072] [Method for producing a target protein] A method for producing a target protein according to one embodiment of the present invention includes the steps of preparing a plant body that expresses a target protein in the apoplast, injecting a buffer solution into the plant body through the stomata, water pores or cross-section of the plant body, and extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section.

[0073] In the step of preparing a plant that expresses a target protein in the apoplast, the method for preparing the plant can be carried out by the method described above in relation to the method for recovering the target protein.

[0074] Specific aspects of the method according to this embodiment, including a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-sections of the leaves of the plant body, and a step of extracting the buffer solution from the inside of the plant body through the stomata, water pores or cross-sections, can be applied without limitation to the specific aspects of the above-mentioned method for recovering a target protein. EXAMPLES

[0075] [Example 1] Production of a plant expressing a target protein in the apoplast The gene for phosphodiesterase (target protein) was cloned into a tobacco mosaic virus (TMV) vector (Icon Genetics), transformed into Agrobacterium, and then cultured. The culture mixture was then infiltrated into Nicotiana benthamiana leaves, and the infected leaves were harvested after about a week.

[0076] [Example 2 Extraction and recovery of target protein] The target protein was extracted and recovered by the following three methods. Note that MgCl2 was added to the buffer composition used in each extraction because it is essential for stabilizing the enzyme of interest in this example.

[0077] (Injection and extraction of buffer solution by pressure / vacuum) The infected leaves were immersed in a 5-fold volume of buffer (50 mM Tris) containing 2 mM MgCl2, stored in a sealed pressure-resistant syringe, and pressurized (<0.1 MPa) for a few seconds with a piston to return to normal pressure, and then depressurized (>-0.1 MPa) for a few seconds to return to normal pressure. This pressurization and depressurization process was repeated five times to recover the buffer solution. Hereinafter, this recovered buffer solution will also be referred to as the pressurized and depressurized extract.

[0078] (Injection of buffer solution under reduced pressure and extraction by immersion in hypertonic buffer solution) The harvested infected leaves were immersed in a buffer solution (50 mM Tris) containing 2 mM MgCl2, placed in a pressure-resistant desiccator, and the pressure was reduced (~0.1 MPa) using a vacuum pump to return to normal pressure. The infected leaves were collected, the droplets were wiped off, and the leaves were immersed in a 5-fold amount by weight of a high-salt buffer solution (20 mM PB pH 7.4, 0.5 M NaCl, 5 mM MgCl2) at room temperature for 10 to 30 minutes to collect the buffer solution. Hereinafter, this collected buffer solution is also referred to as the osmotic extract.

[0079] (Extraction by physical destruction) The infected leaves were frozen in liquid nitrogen and crushed in a mortar and pestle, and suspended in a 4-fold volume of 50 mM Tris buffer containing 2 mM MgCl2 for 10 minutes at room temperature. The mixture was then centrifuged (4°C, 10 minutes, 12,000×g) to recover the supernatant. Hereinafter, this supernatant is also referred to as the crushed extract.

[0080] [Example 3: Analysis of each extract] Each of the extracts collected in Example 2 was analyzed by SDS-PAGE, Western blot analysis, measurement of total protein content, measurement of phosphodiesterase content, and measurement of phosphodiesterase activity.

[0081] (SDS-PAGE and Western blot analysis) The samples were treated with non-reducing denaturing sample loading buffer, and proteins were separated by SDS-PAGE. The gels were stained with CBB staining solution (Bio-Rad Laboratories). The gels were also subjected to Western blot analysis with HRP-labeled anti-phosphodiesterase rabbit polyclonal antibody to identify phosphodiesterase bands.

[0082] (Total protein content measurement) Total protein content was measured by the TCA-BCA method (MicroBCA Protein Assay Kit, Thermo Fisher Scientific) using bovine serum albumin as the reference standard.

[0083] (Phosphodiesterase activity measurement) Phosphodiesterase activity was measured using a Sphingomyelinase Assay Kit (Abcam).

[0084] (Phosphodiesterase content measurement (ELISA)) A 96-well microplate was coated with anti-phosphodiesterase rabbit polyclonal antibody at 5°C for 15-18 hours. The plate was washed three times with PBST (0.01M PBS (phosphate buffered saline) (pH 7.4) containing 0.1% Tween-20) and blocked with 2% bovine serum albumin in PBST at 25°C for 1 hour. Purified phosphodiesterase was diluted in PBST as a standard, and samples were diluted in PBST. The plate was washed three times with PBST, and the standard and samples were added and incubated at 25°C for 2 hours. The plate was washed three times with PBST, and HRP-labeled anti-phosphodiesterase rabbit polyclonal antibody was added and incubated at 25°C for 1 hour. The plate was washed three times with PBST, and a color-developing substrate (1-StepML tra TMB-ELISA, Thermo Fisher Scientific) was added and incubated at 25°C for 20 minutes, and 0.313M sulfuric acid was added to stop the reaction. The absorbance at a wavelength of 450 nm was measured using a plate reader (EnSpire, PerkinElmer).

[0085] [Example 4 Comparison of extraction effects (1)] The components extracted from the extract obtained by general physical disruption (the disruption extract of Example 2) and the extract obtained by pressurization and decompression (the pressurized and decompressed extract of Example 2) were compared.

[0086] The results of SDS-PAGE and Western blot analysis of the crushed extract and the pressurized / reduced extract are shown in Figure 1. Compared to the crushed extract (lane 2 in Figure 1), the pressurized / reduced extract (lane 1 in Figure 1) contained equal or greater amounts of phosphodiesterase. Furthermore, no plant-derived components other than phosphodiesterase were detected in the pressurized / reduced extract.

[0087] Table 1 shows the results of measurements of total protein content, phosphodiesterase content, and phosphodiesterase activity, as well as the amount extracted by pressure and vacuum when the amount extracted by physical crushing per infected leaf is set as 100%.

[0088] [Table 1]

[0089] Although the total protein extraction amount of the pressurized / decompressed extract was reduced to one-eighth compared to the crushed extract, the amount of phosphodiesterase extracted was increased by more than two-fold. This shows that pressurized / decompressed extraction is a method capable of extracting expressed proteins with high efficiency while significantly suppressing the extraction of unnecessary plant components.

[0090] [Example 5 Comparison of extraction effects (2)] The components extracted from the extract obtained by general physical disruption (disruption extract in Example 2) were compared with those extracted from the extract obtained by immersion in a high osmotic pressure buffer (osmotic pressure extract in Example 2).

[0091] The results of SDS-PAGE and Western blot analysis of the disruption extract and osmotic extract are shown in Figure 2. The osmotic extract (lane 2 in Figure 2) contained the same amount of phosphodiesterase as the disruption extract (lane 1 in Figure 2). Furthermore, no plant-derived components other than phosphodiesterase were detected in the osmotic extract.

[0092] Table 2 shows the results of measurements of total protein content, phosphodiesterase content, and phosphodiesterase activity, as well as the amount extracted by immersion in a hypertonic buffer solution when the amount extracted by physical crushing per infected leaf is set to 100%.

[0093] [Table 2]

[0094] Although the total protein extraction amount of the osmotic extract was reduced to one-eighth compared to the crushed extract, the amount of phosphodiesterase extracted was comparable. This suggests that extraction by immersion in a high osmotic buffer is an efficient method for extracting expressed proteins while significantly suppressing the extraction of unnecessary plant components.

Claims

1. Injecting a buffer solution into the plant body through the stomata, water pores or cross section of the plant body expressing the target protein in the apoplast; and A step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross section. A method for recovering a target protein expressed in the apoplast, comprising: A recovery method, wherein the step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section includes injecting the buffer solution by applying pressure, or the step of extracting the buffer solution from the plant body through the stomata, water pores or cross-section includes extracting the buffer solution by applying pressure (excluding pressure applied by centrifugation) or by applying reduced pressure.

2. The method according to claim 1, comprising repeating two or more sets of a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section and a step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section.

3. The method according to claim 1, wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the plant body through the stomata, water pores or cross-section.

4. A step of injecting a buffer solution into a plant body through a stomata, a water stomata or a cross section of a plant body expressing a target protein in an apoplast; and A step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross section. A method for recovering a target protein expressed in the apoplast, comprising: A recovery method, wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution contained in the apoplast.

5. The method according to claim 4, wherein the step of injecting the buffer solution into the plant body through the stomata, water pores or cross-sections comprises injecting the buffer solution by applying pressure or vacuum.

6. The method according to claim 4 or 5, wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section comprises extracting the buffer solution by applying pressure or vacuum.

7. The step of injecting a buffer solution into the plant body through a stomata, a water hole or a cross section includes injecting the buffer solution by applying pressure or pressure; The step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section includes extracting the buffer solution by applying pressure or pressure; The method according to claim 4, comprising a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section, and a step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section, which are defined as one set, and which comprises repeating this set of two or more sets.

8. The method according to any one of claims 1 to 7, wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the inside of the plant body through the stomata, water pores or cross-section.

9. The method according to any one of claims 1 to 3, 5 and 7, wherein injecting the buffer solution into the plant body by pressurization or depressurization comprises pressurizing or depressurizing using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container.

10. The method according to any one of claims 1 to 3, 6 and 7, wherein extracting the buffer solution inside the plant body by pressurization or decompression comprises pressurizing or decompressing using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container.

11. Providing a plant that expresses a protein of interest in the apoplast; Injecting a buffer solution into the plant body through the stomata, water holes or cross-section of the plant body; and Extracting a buffer solution from inside the plant body through the stomata, water pores or cross section; A method for producing a target protein, comprising: A manufacturing method in which the step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section includes injecting the buffer solution by applying pressure, or the step of extracting the buffer solution from the plant body through the stomata, water pores or cross-section includes extracting the buffer solution by applying pressure (excluding pressurization by centrifugation) or by applying reduced pressure.

12. The method described in claim 11, comprising a set of steps of injecting a buffer solution into the plant body through the stomata, water pores or cross-sections and extracting the buffer solution from inside the plant body through the stomata, water pores or cross-sections, and repeating this set of steps two or more times.

13. The method according to claim 11, wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution injected into the plant body through the stomata, water pores or cross-section.

14. A step of preparing a plant body expressing a target protein in the apoplast; Injecting a buffer solution into the plant body through the stomata, water holes or cross-section of the plant body; and Extracting a buffer solution from inside the plant body through the stomata, water pores or cross section; A method for producing a target protein, comprising: A production method, wherein the step of extracting a buffer solution from inside the plant body through the stomata, water pores or cross section comprises immersing the plant body in a buffer solution having a higher osmotic pressure than the buffer solution contained in the apoplast.

15. The method according to claim 14, wherein the step of injecting the buffer solution into the plant body through the stomata, water pores or cross-sections comprises injecting the buffer solution by applying pressure or vacuum.

16. The method according to claim 14, wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-sections comprises extracting the buffer solution by applying pressure or vacuum.

17. The step of injecting a buffer solution into the plant body through a stomata, a water hole or a cross section includes injecting the buffer solution by applying pressure or pressure; The step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section includes extracting the buffer solution by applying pressure or pressure; The method according to claim 14, comprising a step of injecting a buffer solution into the plant body through the stomata, water pores or cross-section, and a step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross-section, which are defined as one set, and repeating this set of two or more sets.

18. The method according to any one of claims 11 to 17, wherein the step of extracting the buffer solution from inside the plant body through the stomata, water pores or cross section comprises immersing the plant body in a buffer solution having a higher salt concentration than the buffer solution injected into the inside of the plant body through the stomata, water pores or cross section.

19. The method according to any one of claims 11 to 13, 15 and 17, wherein injecting the buffer solution into the plant body by pressurization or depressurization comprises pressurizing or depressurizing using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container.

20. The method according to any one of claims 11 to 13, 16 and 17, wherein extracting the buffer solution inside the plant body by pressurization or decompression comprises pressurizing or decompressing using a gas, a liquid, a press, or a combination thereof in a pressure-resistant container.

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