Method for removing plant-derived soluble protein from plant-derived extract

Abstract

The present invention provides a method for removing a plant-derived soluble protein from a plant-derived extract, the method comprising the following steps: (1) a step for freezing and thawing a plant-derived extract to cause the insolubilization of a plant-derived soluble protein; and (2) a step for removing the insolubilized protein from the extract.

Description

Method for removing plant-derived soluble proteins from plant-derived extract

[0001] The present invention relates to a method for removing plant-derived soluble proteins from a plant-derived extract, and the like.

[0002] Technologies for producing products using living organisms, such as plants, animals, or microorganisms, enable the production of materials from biomass without relying on fossil fuels as raw materials, and are therefore expected to revolutionize manufacturing, leading to the realization of a carbon-recycling society and sustainable economic growth. Among these technologies, plant-based production using genetic engineering has the potential to express proteins that are difficult to produce due to their complex structure or high molecular weight, making it promising for use in the pharmaceutical, food, and healthcare fields. However, commercial success remains limited, and mass-production technologies have yet to be established. In particular, the separation and purification of expressed target proteins requires multiple reagents and multi-step procedures (Patent Document 1), leaving a great demand for efficient purification methods. In particular, there is a great demand for purification methods that can efficiently remove plant-derived soluble proteins, such as RuBisCO, which are present in large quantities in plant-derived extracts.

[0003] Patent No. 6560495

[0004] An object of the present invention is to provide a method for removing plant-derived soluble proteins present in large amounts from a plant-derived extract.

[0005] We centrifuged crude extracts of wild-type Nicotiana benthamiana and N. benthamiana transfected with a green fluorescent protein (GFP) gene and separated them into supernatant (S1) and precipitate (P1). S1 was then freeze-thawed, centrifuged, and separated into supernatant (S2) and precipitate (P2). The soluble proteins contained in S1 and S2 were then separated by SDS-PAGE, and the polyacrylamide gel was stained with Coomassie blue. Ribulose 1,5-bisphosphate carboxylase / oxygenase (RuBisCO), a plant-derived soluble protein previously found to be abundant in S1, was found to be almost completely absent in S2. Furthermore, freeze-thawing and centrifugation of S1, which had already been centrifugally removed as P1, generated a new precipitate, P2, which we believe represents a new insoluble fraction containing RuBisCO. Furthermore, Western blot analysis of the amount of GFP contained in S1 and S2 revealed that freeze-thawing did not affect the solubility of GFP, and that it remained in the supernatant. Furthermore, the inventors discovered that a short freezing period of approximately 10 minutes was sufficient to remove a small amount of plant-derived soluble proteins, while a 60-minute freezing period removed a sufficient amount of RuBisCO. They also found that the present invention can be implemented even when GUS protein or human FGF1 is used as an exogenous protein, and that various plant-derived soluble proteins other than RuBisCO can be removed by freeze-thawing. These findings demonstrate that freeze-thawing insolubilizes plant-derived soluble proteins, including RuBisCO, while not affecting the solubility of exogenous proteins such as GFP, GUS, or FGF1, thereby enabling their separation. Furthermore, the freezing time did not affect the above effects. Based on these findings, the inventors conducted further research and completed the present invention.

[0006] That is, the present invention is as follows: [1] A method for removing plant-derived soluble proteins from a plant-derived extract, comprising the following steps: (1) freezing and thawing the plant-derived extract to insolubilize the plant-derived soluble proteins, and (2) removing the insolubilized proteins from the extract. [2] The method according to [1], wherein the plant-derived soluble proteins include RuBisCO. [3] The method according to [1] or [2], wherein the plant is a plant of the genus Nicotiana. [4] The method according to [3], wherein the Nicotiana plant is N. benthamiana. [5] The method according to any one of [1] to [4], wherein the plant expresses an exogenous soluble protein that is not insolubilized by freezing and thawing. [6] The method according to any one of [1] to [5], wherein the freezing period is one day or more. [7] The method according to any one of [1] to [6], wherein the freezing temperature is −20° C. or lower. [8] The method according to any one of [1] to [7], wherein the insolubilized proteins are removed from the extract by centrifugation or filtration. [9] The method according to any one of [1] to [8], further comprising a step of removing insoluble substances from the plant-derived extract before step (1).

[10] The method according to [9], wherein the removal of insoluble substances from the plant-derived extract is carried out by centrifugation or filtration.

[0007] According to the method for separating plant-derived soluble proteins, including RuBisCO, from a plant-derived extract described herein, whereas conventional separation and purification of exogenous soluble proteins expressed in plants required multiple reagents and multi-step procedures, by performing a freeze-thaw treatment, plant-derived soluble proteins, including RuBisCO, which is a dominant protein in plants, can be insolubilized and removed, thereby increasing the abundance of the exogenous soluble proteins upstream of the purification process. As a result, the purification process for exogenous soluble proteins can be shortened, and the present invention may be useful in the bioindustry field.

[0008] Figure 1 shows a flowchart of the extraction and freeze-thaw treatments for N. benthamiana leaf samples. S1 represents the supernatant obtained by centrifugation of unfrozen crude extract, S2 represents the supernatant obtained by centrifugation of S1 after 1 or 7 days of freezing and thawing, and S3 represents the supernatant obtained by centrifugation of crude extract thawed after 1 day of freezing. Figure 2 shows the results of freeze-thawing of crude extracts and supernatants from wild-type N. benthamiana leaves and GFP-transfected N. benthamiana leaves (n = 3). Wild-type and GFP-transfected leaves S1–S3 and P1–P3 were photographed under visible light (A) and blue LED (B). (C) Proteins in S1–S3 were separated by SDS-PAGE and stained with Coomassie blue (top) or analyzed by Western blot (bottom). M indicates a molecular weight marker, RuBisCO L indicates the RuBisCO large subunit, and RuBisCO S indicates the RuBisCO small subunit. Figure 3 shows the relative amounts of proteins contained in each supernatant. (A) The relative amount of recombinant GFP estimated from Western blot analysis is shown. The p-value (significance probability) for S2 is 2.96 × 10 -3 , 2.42 × 10 for S3 -3 (B) The relative amounts of RuBisCO were estimated from the band intensities on a Coomassie blue-stained polyacrylamide gel. The p values ​​were 3.01 × 10 for S2 and 3.01 × 10 for S3, respectively. -4 , 6.46 x 10 for S3 -3(C) The relative amounts of total protein were estimated from the band intensities on a Coomassie Blue-stained polyacrylamide gel. Error bars represent standard deviations (n ​​= 3). Figure 4 shows the results of freeze-thawing the supernatants of extracts from wild-type and GFP-transgenic N. benthamiana leaves (n = 3). Wild-type and GFP-transgenic N. benthamiana leaves (S1 and S2) were photographed under visible light (A) and blue LED (B). (C) Proteins in S1 and S2 were separated by SDS-PAGE and then stained with Coomassie Blue (top) or analyzed by Western blot (bottom). (D) The relative amounts of GFP in S1, S2 (1 day frozen), and S2 (7 days frozen) are shown. The p-values ​​(significance probability) for S2 (1 day frozen) and S2 (7 days frozen) were 0.294 and 0.456, respectively, indicating no significant difference. M represents a molecular weight marker, RuBisCO L represents the RuBisCO large subunit, and RuBisCO S represents the RuBisCO small subunit. Figure 5 shows the effects of short-term freeze-thaw treatment. Crude extracts from wild-type and three independent GFP-expressing leaves were frozen at -30°C for 10 or 60 minutes and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (bottom). M represents a molecular weight marker, RuBisCO L represents the RuBisCO large subunit, and RuBisCO S represents the RuBisCO small subunit. S1 represents the unfrozen supernatant, and S3 represents the supernatant of crude extracts frozen at -30°C for 10 or 60 minutes. (B) Concentrations of total soluble proteins (TSP) and GFP in the corresponding supernatants. Figure 6 shows the effect of short-term freeze-thaw treatment on GFP function. Figure 7 shows the relative GFP fluorescence intensity of S1 (unfrozen) and S3 (supernatants of crude sap frozen at -30°C for 10 or 60 minutes) from GFP-expressing plants. Figure 7 shows the effect of freeze-thaw treatments at -20°C. Crude extracts from wild-type and three independent GFP-expressing leaves were frozen at -20°C for 1 day and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (bottom).M denotes molecular weight marker, RuBisCO L denotes RuBisCO large subunit, RuBisCO S denotes RuBisCO small subunit, S1 denotes unfrozen supernatant, and S3 denotes supernatant of crude extract frozen at -20°C for 1 day. (B) Concentrations of TSP and GFP in the corresponding supernatants. Figure 8 shows the effect of freeze-thaw treatment on GFP function at -20°C. The relative GFP fluorescence intensity of S1 and S3 from GFP-expressing plants is shown. S1 denotes unfrozen supernatant, and S3 denotes supernatant of crude extract frozen at -20°C for 1 day. Figure 9 shows the effect of freeze-thaw treatment on GUS (β-glucuronidase) protein. Crude extracts from wild-type and three independent GUS-expressing leaves were frozen at -30°C for 60 minutes and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (middle). GUS staining results are also shown (bottom). (B) TSP concentration in the corresponding supernatant. M: molecular weight marker; RuBisCO L: RuBisCO large subunit; RuBisCO S: RuBisCO small subunit; S1: unfrozen supernatant; S3: supernatant of crude extract frozen at -30°C for 60 minutes. Figure 10 shows the effect of freeze-thaw treatment on human FGF1 protein. Crude extracts from wild-type and three independent FGF1-expressing leaves were frozen at -30°C for 1 day and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (bottom). M: molecular weight marker; RuBisCO L: RuBisCO large subunit; RuBisCO S: RuBisCO small subunit; S1: unfrozen supernatant; S3: supernatant of crude extract frozen at -30°C for 1 day. (B) TSP and FGF1 concentrations in the corresponding supernatant. FIG. 11 shows the results of extracting human type II collagen protein expressed in a transient expression system using N. benthamiana, and confirming its expression.Crude extracts from leaves infected with Agrobacterium strains (D, D2, F, F2, H, and H2; n = 3) encoding various engineered human type II collagen proteins were separated by non-reducing SDS-PAGE and subjected to Western blot analysis to detect human type II collagen. Negative controls included "WT," an extract from uninfected plants, and "GFP," an extract from leaves infected with Agrobacterium strains encoding GFP. The positive control was human type II collagen protein derived from human tissue (denoted "human COL2 (1 μg)"). M indicates a molecular weight marker. Figure 12 shows the results of freeze-thaw treatment of human type II collagen protein. Crude sap samples were frozen at -30°C for 2 weeks, then thawed and centrifuged. The resulting supernatant was analyzed. The figure shows the results of Western blot analysis to detect human type II collagen after separation by non-reducing SDS-PAGE. (Samples prepared under the same conditions as in Figure 11 were subjected to freeze-thaw treatment; n=2).

[0009] The present invention provides a method for removing plant-derived soluble proteins from a plant-derived extract (hereinafter referred to as the removal method of the present invention).

[0010] The removal method of the present invention includes a step of insolubilizing plant-derived soluble proteins by freezing and thawing a plant-derived extract (hereinafter, referred to as step (1)).

[0011] In step (1), the plant from which the plant-derived extract is extracted is not particularly limited, but is expected to be a model plant for which cultivation techniques, breeding techniques, or gene transfer techniques have been established, and examples of such plants include plants belonging to the genus Arabidopsis (e.g., Arabidopsis thaliana), the genus Oryza (e.g., Oryza sativa), the genus Triticum (e.g., Triticum aestivum), the genus Brachypodium (e.g., Brachypodium distachyon), and the genus Nicotiana (e.g., N. benthamiana). Furthermore, the above plants may be wild-type or genetically modified plants capable of expressing an exogenous soluble protein that is not insolubilized by the freezing and thawing in step (1).

[0012] The exogenous soluble protein that is not insolubilized by freezing and thawing is not limited as long as it is a soluble protein that is not insolubilized by the freezing and thawing process described below in step (1). Here, a soluble protein refers to a protein that has many positively charged basic amino acid residues, negatively charged acidic amino acid residues, or polar amino acid residues at its surface at near-neutral pH, and that is highly soluble in water due to increased affinity for water molecules caused by binding of water molecules to the dissociative groups of basic or acidic amino acid residues (ionic hydration) or by hydrogen bonds formed between polar groups (e.g., OH groups) and water molecules. Basic amino acids include Lys, Arg, and His. Acidic amino acids include Glu and Asp. Polar amino acids include serine and threonine. A protein that is not insolubilized by freezing and thawing refers to a protein whose higher-order structure is not changed by freezing and thawing, and which does not become insolubilized due to aggregation. Examples of such exogenous soluble proteins include antibodies, cytokines (IFN-α, IFN-β, FGF, etc.), various enzymes, viral, bacterial, and cancer antigen proteins, extracellular matrix proteins such as collagen, cell signaling-related proteins, and high-value-added proteins such as albumin. Using parts of viral proteins as antigens, for example, enables the mass production of antigens useful for the development of vaccines and antibodies against viruses. Alternatively, it is also possible to mass-produce cancer antigens useful for the development of anticancer drugs.

[0013] The expression of an exogenous soluble protein may be transient or constitutive. Means for expressing an exogenous soluble protein in a plant include a method of introducing an expression vector containing DNA encoding the exogenous soluble protein into plant cells, and a method of introducing the exogenous soluble protein using a viral vector having a nucleic acid sequence encoding the exogenous soluble protein. Any known method may be used as long as it allows the exogenous soluble protein to be expressed in the plant cells by culturing the plant cells.

[0014] Introduction of an expression vector containing DNA encoding an exogenous soluble protein into plant cells can be carried out by known methods, for example, in the form of an expression vector containing DNA encoding the exogenous soluble protein. Introduction of the expression vector can be carried out using known methods (e.g., the Agrobacterium method, PEG method, electroporation, particle gun method, direct whisker introduction method, etc.) into appropriate tissues (e.g., callus, roots, leaves, seeds, meristem, etc.) depending on the type of plant. Among these, the Agrobacterium method is particularly preferred because it is easy to scale up. Introduction of an expression vector containing DNA encoding an exogenous soluble protein into plant cells can also be carried out using a viral vector carrying a nucleic acid sequence encoding the exogenous soluble protein. Introduction of the viral vector can be carried out into the above tissues depending on the type of plant using the viral vector method.

[0015] To constitutively express an exogenous soluble protein in a plant, for example, a portion of tobacco tissue (e.g., tobacco leaf) or hypocotyl is excised and infected with Agrobacterium. The tissue or hypocotyl containing the expression vector is then cultured to select transgenic plant cells in which DNA encoding the exogenous soluble protein has been introduced into the genome. Callus is then induced from the cells, and rooting is induced from the callus to obtain transgenic plants stably expressing the exogenous soluble protein. Alternatively, Agrobacterium can be infected into the callus. Typically, Agrobacterium is used in which DNA encoding the exogenous soluble protein has been introduced into a T-DNA fragment of an Agrobacterium expression vector. When using the PEG method or electroporation method, protoplasts are prepared from appropriate cells or tissues according to standard procedures, and the expression vector is then introduced into them. When using the particle gun method, an expression vector adsorbed to gold particles can be introduced into callus, immature embryos, shoot tips, or meristems present in axillary buds using a particle gun. In the particle gun method and the Agrobacterium method, the transgenic plants often become chimeric, so it is necessary to use sample cells that will allow the DNA to be introduced into germline cells at a high frequency, such as embryos, hypocotyl segments, embryogenic callus, and isolated meristems.

[0016] Plant cells or tissues into which an expression vector has been introduced can be cultured according to known methods, depending on the type of plant cell or tissue. The medium used for culture is preferably a solid medium (e.g., agar medium, agarose medium, gellan gum medium, etc.). Furthermore, the medium preferably contains carbon sources, nitrogen sources, minerals, etc. necessary for the growth of genetically modified cells. For example, N6 medium, MS medium, MSR medium, LS medium, B5 medium, etc. are used as basal media. Plant growth substances (e.g., auxins, cytokinins, etc.) may be added to the medium as appropriate. The pH of the medium is preferably about 5 to about 8. The culture temperature can be selected appropriately, usually within the range of about 20°C to about 35°C, depending on the type of plant cell.

[0017] Those skilled in the art can select genetically modified plant cells by appropriate publicly known methods depending on the type of marker gene used. For example, when a drug resistance gene (e.g., a kanamycin resistance gene (nptII) or a hygromycin resistance gene (hpt)) is used, genetically modified plant cells can be selected by culturing them in the presence of the corresponding drug.

[0018] For example, methods for transiently expressing exogenous soluble proteins in plants include immersing whole tobacco plants in a suspension of Agrobacterium, sealing them, and applying a reduced pressure (e.g., 0.09 MPa) (vacuum infiltration), or infiltrating the Agrobacterium suspension into the intercellular spaces of leaves using a needleless syringe (syringe infiltration). After treatment, tobacco tissues or whole tobacco plants are cultured for several days, allowing plants to be obtained that transiently express exogenous soluble proteins in the Agrobacterium-infiltrated tobacco tissues.

[0019] In step (1), the plant-derived extract may be obtained by known means. In the present invention, the plant-derived extract refers to a suspension obtained by adding water or a buffer to crushed plant tissue. For example, a plant extract can be prepared by crushing all or part of a plant (roots, leaves, stems, or flowers) and suspending the crushed plant tissue in water or a buffer (e.g., Tris-HCl buffer, phosphate buffer, HEPES buffer, etc.). The extract may further contain a surfactant (e.g., Triton X-100). TM , Tween 20 TM ) and protein degradation inhibitors may also be included.

[0020] The plant-derived extract obtained as described above contains soluble substances such as endogenous and exogenous soluble proteins as well as insoluble substances such as debris. Therefore, the removal method of the present invention may further include a step of removing insoluble substances from the plant-derived extract prior to step (1). The step of removing insoluble substances from the plant-derived extract is not particularly limited as long as it can remove the insoluble substances, but a preferred example is a step of removing insoluble substances from the plant-derived extract by centrifugation. The centrifugation conditions are not particularly limited as long as they result in precipitation of the insoluble substances, but examples include about 10,000 to about 30,000 × g, about 2 to about 20 minutes, and about 0 to about 4°C. Centrifugation is a simple and rapid method due to its high recovery rate of soluble substances, the fact that the centrifuge is a general-purpose machine, the processing time is short, and the precipitate can be determined simply based on molecular weight. Another step of removing insoluble substances from the plant-derived extract is to remove the insoluble substances by filtering the plant-derived extract. The filter membrane used for filtration is not particularly limited as long as it can filter insoluble substances, and examples thereof include gauze, filter paper, etc. The pore size of the filter membrane is not particularly limited as long as it can filter insoluble substances, and examples thereof include 10 μm to 700 μm. By removing the resulting precipitate, it is possible to remove insoluble substances from the plant-derived extract in advance.

[0021] In step (1), the plant-derived extract is frozen. The freezing conditions are not particularly limited as long as the extract is frozen, but examples include, but are not limited to, a range of -30°C to -20°C. The freezing period is also not limited as long as the plant-derived extract is frozen, but examples include a range of 10 minutes to 14 days. Alternatively, the extract may be frozen instantaneously using liquid nitrogen. In one embodiment, when an exogenous soluble protein loses some or all of its biological activity upon freezing, the loss of biological activity may be avoided by shortening the freezing period. When an exogenous soluble protein having such properties is used, the freezing period of the plant-derived extract can usually be, but is not limited to, 10 minutes to 12 hours, preferably 10 minutes to 11 hours, 10 minutes to 10 hours, 10 minutes to 9 hours, 10 minutes to 8 hours, 10 minutes to 7 hours, 10 minutes to 6 hours, 10 minutes to 5 hours, 10 minutes to 4 hours, 10 minutes to 3 hours, 10 minutes to 2 hours, or 10 minutes to 1 hour.

[0022] In step (1), the frozen plant-derived extract is thawed. The thawing conditions are not particularly limited as long as the frozen extract is thawed. The thawing temperature is, for example, 4°C.

[0023] As described above, freezing and thawing a plant-derived extract can insolubilize plant-derived soluble proteins contained in the extract. Examples of plant-derived soluble proteins include RuBisCO. RuBisCO is the only enzyme involved in the carbon dioxide fixation reaction in the Calvin cycle during plant photosynthesis. It catalyzes the fixation of carbon dioxide to ribulose 1,5-bisphosphate to produce two molecules of 3-phosphoglycerate. RuBisCO is the most abundant protein in plants and is the protein most removed during the purification process of proteins other than RuBisCO from plants. RuBisCO typically exists as a heterohexadecameric complex consisting of eight large and eight small subunits, and is present as a soluble protein in plant-derived extracts. The freezing and thawing process in step (1) is thought to alter the higher-order structure of the large and small subunits of RuBisCO, or the heterohexadecameric complex composed of them, resulting in insolubilization. In addition, if a plant expresses an exogenous soluble protein that is not insolubilized by freezing and thawing, the exogenous soluble protein contained in the plant-derived extract will not be insolubilized by the above-mentioned freezing and thawing, and the plant-derived soluble protein can be separated from the exogenous soluble protein.

[0024] The separation method of the present invention includes a step of removing insolubilized proteins from the frozen and thawed plant-derived extract (hereinafter, step (2)).

[0025] The process for removing insolubilized protein from frozen and thawed plant-derived extract is not particularly limited as long as it can remove the insolubilized protein. A preferred example is a process for removing the insolubilized protein by centrifuging the frozen and thawed plant-derived extract. The centrifugation conditions are not particularly limited as long as they result in precipitation of the insolubilized protein. Examples include a temperature of about 10,000 to about 30,000 × g, about 2 to about 20 minutes, and about 0 to about 4°C. Removal of the resulting precipitate allows the insolubilized protein to be separated from the plant-derived extract. Another process for removing insolubilized protein from frozen and thawed plant-derived extract is a process for removing the insolubilized protein by filtering the frozen and thawed plant-derived extract. The filter membrane used for filtration is not particularly limited as long as it can filter the insolubilized protein, and examples include gauze and filter paper. The pore size of the filter membrane is not particularly limited as long as it can filter the insolubilized protein, and examples include 10 μm to 700 μm. If the plant expresses an exogenous soluble protein that is not insolubilized by the freezing and thawing in step (1), the proportion of the exogenous soluble protein in the soluble proteins contained in the plant-derived extract can be increased by separating the insolubilized protein from the plant-derived extract.

[0026] The present invention will be described below with reference to examples, although the present invention is not limited to these examples.

[0027] Example 1: Verification of the effect of freezing and thawing on the separation of plant-derived soluble proteins from exogenous soluble proteins. Agrobacterium, into which a plant expression vector (GFP_pBE2113) expressing GFP had been introduced, was cultured overnight at 28°C with shaking at 220 rpm in 3 ml of YEP medium (containing kanamycin (50 μg / mL), streptomycin (300 μg / mL), and rifampicin (100 μg / mL)). The culture was centrifuged at 5,000 × g for 10 minutes at room temperature (23°C) to recover the Agrobacterium. The recovered Agrobacterium was suspended in approximately 5-10 ml of suspension buffer (10 mM MES-KOH (pH 5.7), 10 mM MgCl2, 150 μM acetosyringone (added immediately before use)) and further analyzed by OD analysis. 600 The suspension was diluted with the same suspension buffer so that the pH was 0.5. The suspension was left to stand for about 2 hours, and then infiltrated into the underside of N. benthamiana leaves using a 1 mL syringe without a needle (syringe infiltration method). After roughly removing the suspension from the leaves, the leaves were left to stand for about 2-3 hours and then grown in a chamber for 4 days (23°C, 16 hours light, 8 hours dark).

[0028] Wild-type N. benthamiana leaves (100 mg) and GFP-transfected N. benthamiana leaves (100 mg) were crushed using a multi-bead shocker (1,700 rpm, 30 sec). The crushed material was suspended in 500 μl of pH 7.2 phosphate buffer (0.1% (v / v) Tween 20, 1% (v / v) protease inhibitor cocktail (Sigma-Aldrich #P9599)) to prepare crude extracts of each plant. The resulting crude extracts were centrifuged (23,000 × g, 10 min, 2°C) to obtain supernatant (S1) and precipitate (P1). Furthermore, a separately prepared supernatant (S1) was left at -30°C for 1 day, then thawed in a refrigerator (4°C), and centrifuged (23,000 × g, 10 min, 2°C) to obtain a supernatant (S2) and a precipitate (P2). Separately, a separately prepared crude extract was left at -30°C for 1 day, then thawed in a refrigerator (4°C), and centrifuged (23,000 × g, 10 min, 2°C) to obtain a supernatant (S3) and a precipitate (P3). Specifically, the procedure was as shown in the flowchart in Figure 1.

[0029] In both wild-type and GFP-transfected plants, the crude extract was centrifuged to remove the insoluble fraction P1. When the extract S1 was freeze-thawed and centrifuged again, a new precipitate, P2, was formed (Fig. 2A). This suggests that some of the soluble material remaining in the supernatant before the freeze-thaw treatment was insolubilized by the freeze-thaw treatment.

[0030] Furthermore, when the supernatant colors were compared under a blue LED, none of the wild-type S1 to S3 showed fluorescence, whereas all S1 to S3 obtained from plants transfected with the GFP gene showed GFP fluorescence, and the fluorescence intensities of S1 (before freeze-thawing) and S2 (after freeze-thawing) were comparable (Fig. 2B). This indicates that the GFP that remained in the supernatant without being precipitated by centrifugation before freeze-thawing remained in the supernatant without being insolubilized by freeze-thawing, and as a result, GFP could be separated from the material insolubilized by freeze-thawing.

[0031] Proteins contained in S1–S3 from wild-type and GFP-transfected plants were separated by SDS-PAGE, and the polyacrylamide gel was stained with Coomassie blue. The plant-derived soluble protein RuBisCO (L / S) bands (approximately 10 kDa (RuBisCO S) and 50 kDa (RuBisCO L)) were detected in S1, but not in S2 or S3 (Fig. 2C). Furthermore, when the amount of GFP contained in S1–S3, which contain GFP, was analyzed by Western blot analysis, no GFP band was detected in any of the wild-type S1–S3, but GFP bands were detected in all of the GFP-transfected S1–S3, and the band intensities were similar among S1–S3. This also indicates that RuBisCO(L / S), which remained in the supernatant before freeze-thawing, was insolubilized by the freeze-thaw treatment, whereas GFP remained in the supernatant without being insolubilized by the freeze-thaw treatment. As a result, it was shown that RuBisCO(L / S) and GFP could be separated by the freeze-thaw treatment.

[0032] Furthermore, Western blot analysis was performed to compare the relative amounts of GFP in the GFP-transfected strains S1–S3. The results showed that, when the amount of GFP in S1 was set to 1, the amount of GFP in S2 exceeded 0.9 (Fig. 3A). Coomassie blue staining was used to compare the relative amounts of RuBisCO in the GFP-transfected strains S1–S3. When the amount of RuBisCO in S1 was set to 1, the amount in S2 was less than 0.1 (Fig. 3B). Coomassie blue staining was also used to compare the relative amounts of total protein in the GFP-transfected strains S1–S3. When the amount of total protein in S1 was set to 1, the amount in S2 was less than 0.3 (Fig. 3C). These results indicate that freeze-thawing removes more than 70% of the total protein from crude extracts of N. benthamiana leaves, and more than 90% of RuBisCO is removed. Furthermore, we found that transiently expressed soluble GFP proteins were largely unaffected by freeze-thaw treatment and remained in the supernatant. Therefore, freeze-thaw treatment is a simple and rapid way to insolubilize plant-derived soluble proteins from crude plant extracts, which can then be easily precipitated and removed by centrifugation. Furthermore, when an exogenous soluble protein is expressed, freeze-thaw treatment can easily and dramatically improve the purification rate of the target soluble protein. Furthermore, similar results were obtained even without the prior procedure of centrifuging the crude plant extract and removing the insoluble fraction.

[0033] Example 2: Effect of Freezing Period on the Separation Effect of Plant-Derived Soluble Proteins and Exogenous Soluble Proteins 1. As in Example 1, wild-type N. benthamiana leaves (100 mg) and GFP-transfected N. benthamiana leaves (100 mg) were ground using a multi-bead shocker (1,700 rpm, 30 sec), and the ground material was suspended in 500 μl of phosphate buffer (0.1% (v / v) Tween 20, containing protease inhibitors) to obtain crude extracts of each plant. The resulting crude extracts were immediately centrifuged (23,000 × g, 10 min, 2°C) to obtain supernatant S1 and precipitate P1. In addition, supernatant S1 was left to stand at -30°C (for 1 or 7 days), thawed in a refrigerator (4°C), and centrifuged (23,000 × g, 10 min, 2°C) to obtain supernatant S2 (frozen for 1 day), supernatant S2 (frozen for 7 days), and precipitate P2 (frozen for 1 day) and precipitate P2 (frozen for 7 days).

[0034] When the color of the precipitates was compared under visible light, P1, obtained by centrifugation of crude extracts, was green for both wild-type and GFP-transfected cells, whereas P2 (frozen for 1 day) and P2 (frozen for 7 days) obtained by centrifugation of S1 were both white, showing no difference (Fig. 4A). This suggests that the freezing period does not affect the precipitates.

[0035] Furthermore, when the color of the supernatant was compared under blue LED, none of wild-type S1, S2 (1 day frozen), and S2 (7 days frozen) showed GFP fluorescence, whereas S1, S2 (1 day frozen), and S2 (7 days frozen) obtained from GFP-transfected plants all showed fluorescence, and the fluorescence intensity was comparable (Fig. 4B). This suggests that the freezing period does not affect the state of GFP.

[0036] Proteins in wild-type and GFP-transfected plants S1, S2 (1 day frozen), and S2 (7 days frozen) were separated by SDS-PAGE and subjected to Coomassie blue staining and Western blot analysis. RuBisCO (L / S) bands were detected in S1, but not in S2 (1 day frozen) or S2 (7 days frozen) (Fig. 4C). Furthermore, no GFP bands were detected in wild-type plants S1, S2 (1 day frozen), or S2 (7 days frozen). However, GFP bands were detected in all plants S1, S2 (1 day frozen), and S2 (7 days frozen) derived from GFP-transfected plants, and the band intensities were similar among S1, S2 (1 day frozen), and S2 (7 days frozen). In fact, Western blot analysis revealed no differences in the relative amounts of GFP in the GFP-transfected plants S1, S2 (1 day frozen), and S2 (7 days frozen). These results suggest that the freezing period does not affect the precipitation or the state of GFP. It was shown that freezing insolubilizes proteins, including RuBisCO, from crude plant extracts, allowing them to be easily removed by centrifugation. Furthermore, when an exogenous soluble protein is expressed, freezing can dramatically improve the purification rate of the target soluble protein.

[0037] Example 3: Verification of the effect of freezing period on the separation effect of plant-derived soluble proteins and exogenous soluble proteins 2 The same experiment as in Example 2 was conducted except that the crude extract was frozen for 10 minutes or 60 minutes to evaluate the effect of short-term freezing. The results are shown in Figure 5.

[0038] As shown in Figure 5, freezing for 10 minutes was effective in removing RuBisCO, albeit weakly. Freezing for 60 minutes was effective in removing RuBisCO. Furthermore, GFP was not affected by the freeze-thaw treatment under these conditions (Figures 5(B) and 6).

[0039] Example 4: Verification of the effect of freezing temperature on the separation effect of plant-derived soluble proteins and exogenous soluble proteins The same experiment as in Example 3 was conducted, except that the crude extract was frozen for 1 day at a freezing temperature of -20°C, and the effect of using a freezing temperature of -20°C was evaluated. The results are shown in Figures 7 and 8.

[0040] As shown in Figures 7 and 8, the results obtained when the freezing temperature was set to -20°C were similar to those obtained when the freezing temperature was set to -30°C. Specifically, the amounts of total soluble proteins (sometimes referred to as "TSP") and RuBisCO in the supernatant decreased, but GFP was not affected.

[0041] Example 5: Study of the effect of freeze-thawing on proteins other than GFP (GUS). The effect of freeze-thawing on transiently expressed GUS (β-glucuronidase) was evaluated. Crude extracts from GUS-expressing leaves were frozen at -30°C for 60 minutes and thawed at 4°C. Western blot analysis confirmed GUS expression in all samples S1 and S3, and GUS activity was detected in the supernatant (Figure 9(a)). TSP was significantly reduced even after 60 minutes of freezing (Figure 9(b)). This suggests that RuBisCO can be removed by a shorter freezing time without inactivating freeze-sensitive proteins.

[0042] Example 6: Study of the effect of freeze-thawing on proteins other than GFP (Human FGF1) The effect of freeze-thawing on transiently expressed human FGF1 was evaluated. Crude extracts from FGF1-expressing leaves were frozen at -30°C for 1 day and thawed at 4°C. FGF1 was detected by SDS-PAGE and Western blot analysis (Figure 10(A)). ELISA analysis showed little change in FGF1 levels after freeze-thawing (Figure 10(B)).

[0043] Example 7: Study of the effect of freeze-thaw treatment on proteins other than GFP (Human Collagen II) The effect of freeze-thaw treatment on transiently expressed human Collagen II was evaluated. Crude extracts from leaves expressing human Collagen II were detected by Western blot analysis (Figure 11). As a result, a band specific to human Collagen II was detected.

[0044] Crude extracts from leaves expressing human Collagen II were frozen at -30°C for 2 weeks and then thawed at 4°C, and human Collagen II was detected by Western blot analysis (Figure 12). As a result, a band specific to human Collagen II was detected even after freezing and thawing, just as in the unfrozen sample.

[0045] Example 8 Proteome Analysis To examine the effects of freeze-thaw treatment, proteome analysis was performed on the supernatant of wild-type plants before and after freeze-thawing. As a result, 695 proteins were identified in the test supernatant. The average abundance ratio of three proteins was 1 or greater, and the average abundance ratio of 692 proteins was less than 1. Table 1 shows the top 20 proteins whose abundance decreased after freeze-thawing. As shown in Table 1, the abundance of many endogenous proteins in the supernatant after freeze-thaw treatment was reduced compared to that in the unfrozen supernatant. In particular, ribosomal proteins were effectively removed from the supernatant after freeze-thaw treatment. In the table, abundance ratio means (abundance after freeze-thaw treatment) / (abundance before freeze-thaw treatment).

[0046]

[0047]

[0048] SDS-PAGE analysis revealed that the amounts of the large and small subunits of RuBisCO in the supernatant after freeze-thawing were significantly reduced compared with the unfrozen supernatant. In the frozen samples, the average abundance ratios of the large and small subunits of RuBisCO were approximately 10% and 20%, respectively, of the unfrozen supernatant (Table 2).

[0049]

[0050] These values ​​were higher than those estimated from the band intensities observed by SDS-PAGE. In contrast, only 15 proteins were significantly unaffected by the freeze-thaw treatment (their average abundances were over 90%) (Table 3). Interestingly, most of the identified proteins were chloroplast-type: A0A1U7YAD0 (inactive purple acid phosphatase 29 isoform X2) and A0A1S3XW44 (GDSL esterase / lipase APG-like) (Table 3).

[0051]

[0052]

[0053] While conventional separation and purification of exogenous soluble proteins expressed in plants required multiple reagents and multi-step procedures, the present invention makes it possible to insolubilize and remove plant-derived soluble proteins, including RuBisCO, a dominant protein in plants, by performing a freeze-thaw treatment, thereby increasing the abundance of the exogenous soluble proteins upstream in the process of purifying the exogenous soluble proteins. As a result, it is possible to shorten the purification process of exogenous soluble proteins, and the present invention may be useful in the bioindustry field.

[0054] This application is based on Japanese Patent Application No. 2023-106995 (filing date: June 29, 2023) and Japanese Patent Application No. 2023-200214 (filing date: November 27, 2023), the contents of which are incorporated in their entirety herein.

Claims

1. A method for removing plant-derived soluble proteins from a plant-derived extract, comprising the steps of: (1) insolubilizing the plant-derived soluble proteins by freezing and thawing the plant-derived extract; and (2) removing the insolubilized proteins from the extract.

2. The method of claim 1, wherein the plant-derived soluble protein comprises RuBisCO.

3. The method according to claim 1 or 2, wherein the plant is a plant belonging to the genus Nicotiana.

4. The method according to claim 3, wherein the Nicotiana plant is Nicotiana benthamiana.

5. The method of claim 1 or 2, wherein the plant expresses an exogenous soluble protein that is not insolubilized by freezing and thawing.

6. The method of claim 1 or 2, wherein the freezing period is one day or more.

7. The method according to claim 1 or 2, wherein the freezing temperature is below -20°C.

8. The method according to claim 1 or 2, wherein the removal of insoluble proteins from the extract is carried out by centrifugation or filtration.

9. The method according to claim 1, further comprising the step of removing insoluble matter from the plant-derived extract prior to step (1).

10. The method according to claim 9, wherein removal of insoluble matter from the plant-derived extract is carried out by centrifugation or filtration.

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