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

Abstract

To provide a method for removing abundant plant-derived soluble proteins from a plant-derived extract.SOLUTION: 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.SELECTED DRAWING: None

Description

[Technical field]

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

[0002] Technologies for producing products using living organisms such as plants, animals, or microorganisms are expected to revolutionize manufacturing, leading to the realization of a carbon-recycling society and sustainable economic growth, because they enable the production of substances from biomass that does not depend on fossil resources as raw materials. Among these, the production of substances from plants using recombinant DNA technology has the potential to express proteins that are difficult to produce due to their complex structure or high molecular weight, and is considered promising for use in the pharmaceutical, food, and healthcare fields, but there are still few commercial achievements, and technology for mass production has not been established. In particular, the separation and purification of expressed target proteins requires multiple reagents and multi-step operations (Patent Document 1), and there is 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 contained in large amounts in plant-derived extracts. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6560495 Summary of the Invention [Problem to be solved by the invention]

[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. [Means for solving the problem]

[0005] The present inventors centrifuged crude extracts of wild-type Nicotiana benthamiana and N. benthamiana introduced with a green fluorescent protein (GFP) gene, and separated them into a supernatant (S1) and a precipitate (P1). Furthermore, S1 was subjected to a freeze-thaw treatment, centrifuged, and separated into a supernatant (S2) and a precipitate (P2). Then, the soluble proteins contained in S1 and S2 were separated by SDS-PAGE, and the polyacrylamide gel was stained with Coomassie blue. As a result, it was confirmed that ribulose 1,5-bisphosphate carboxylase / oxygenase (RuBisCO), a plant-derived soluble protein confirmed to be contained in large amounts in S1, had almost disappeared in S2. At the same time, a new precipitate P2 was generated by freeze-thawing and centrifugation of S1, from which the precipitate had already been removed as P1 by centrifugation, and these were considered to be a new insoluble fraction containing RuBisCO. Furthermore, Western blot analysis of the amount of GFP in S1 and S2 revealed that freeze-thaw treatment did not affect the solubility of GFP, which continued to remain in the supernatant. In addition, the inventors found that a short freezing treatment of about 10 minutes can remove a small amount of plant-derived soluble proteins, and a freezing treatment of 60 minutes can remove a sufficient amount of RuBisCO, that the present invention can be carried out 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 freezing and thawing. From the above, it was revealed that freezing and thawing treatment insolubilizes plant-derived soluble proteins including RuBisCO, while not affecting the solubility of exogenous proteins such as GFP, GUS, or FGF1, and as a result, the two can be separated. In addition, the freezing time did not affect the above-mentioned effects. The inventors further studied based on these findings 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 steps of: (1) freezing and thawing a plant-derived extract to insolubilize plant-derived soluble proteins; and (2) removing insoluble proteins from the extract. [2] The method according to [1], wherein the plant-derived soluble protein comprises RuBisCO. [3] The method according to [1] or [2], wherein the plant is a plant belonging to the Nicotiana genus. [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 removal of insoluble proteins from the extract is carried out 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 prior to step (1).

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

[0007] According to the method for separating plant-derived soluble proteins including RuBisCO from a plant-derived extract described herein, while the conventional separation and purification of exogenous soluble proteins expressed in plants required multiple reagents and multiple steps, it is possible to remove plant-derived soluble proteins including RuBisCO, which is a dominant protein in plants, by insolubilizing them through a freeze-thaw treatment, and to increase the presence rate of the exogenous soluble proteins upstream of the purification process. As a result, it is possible to shorten the purification process of the exogenous soluble proteins, and the present invention may be useful in the bioindustry field. [Brief description of the drawings]

[0008] [Figure 1] Figure 1 shows a flow chart of the extraction process and the freeze-thaw process of N. benthamiana leaf samples. S1 indicates the supernatant obtained by centrifuging the unfrozen crude extract, S2 indicates the supernatant obtained by centrifuging S1 after thawing after freezing for 1 or 7 days, and S3 indicates the supernatant obtained by centrifuging the crude extract after thawing after freezing for 1 day. [Diagram 2] Figure 2 shows the results of freeze-thawing of crude extracts and supernatants from wild-type and GFP-transfected N. benthamiana leaves (n=3). Wild-type and GFP-transfected 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 molecular weight marker, RuBisCO L indicates RuBisCO large subunit, and RuBisCO S indicates RuBisCO small subunit. [Diagram 3]Figure 3 shows the relative amounts of proteins in each supernatant. (A) The relative amounts of recombinant GFP estimated from Western blot analysis. p values ​​(significance probability) are 2.96×10-3 for S2 and 2.42×10-3 for S3, respectively. (B) The relative amounts of RuBisCO were estimated from the band intensities of Coomassie Blue stained polyacrylamide gels. p values ​​were 3.01×10-4 for S2 and 6.46×10-3 for S3, respectively. (C) The relative amounts of total proteins were estimated from the band intensities of Coomassie Blue stained polyacrylamide gels. Error bars represent standard deviation (n=3). [Figure 4] 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 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 stained with Coomassie blue (top) or analyzed by Western blot (bottom). (D) The relative amounts of GFP in S1, S2 (frozen for 1 day) and S2 (frozen for 7 days) are shown. The p-values ​​(significance probability) were 0.294 for S2 (frozen for 1 day) and 0.456 for S2 (frozen for 7 days), respectively, indicating no significant difference. M indicates molecular weight marker, RuBisCO L indicates RuBisCO large subunit, and RuBisCO S indicates RuBisCO small subunit. [Diagram 5]Figure 5 shows the effect of short freeze-thaw treatments. Crude extracts from wild-type and three independent GFP-expressing leaves were frozen at -30°C for 10 or 60 min and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (bottom). M is molecular weight marker, RuBisCO L is RuBisCO large subunit, RuBisCO S is RuBisCO small subunit, S1 is unfrozen supernatant, and S3 is supernatant of crude extract frozen at -30°C for 10 or 60 min. (B) Total soluble proteins (TSP) and GFP concentrations in the corresponding supernatants. [Figure 6] Figure 6 shows the effect of short-term freeze-thaw treatment on GFP function. Relative GFP fluorescence intensity of S1 (unfrozen) and S3 (supernatant of crude sap frozen at -30°C for 10 or 60 min) from GFP-expressing plants is shown. [Figure 7] Figure 7 shows the effect of freeze-thaw treatment at -20°C as the freezing temperature. 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 is molecular weight marker, RuBisCO L is RuBisCO large subunit, RuBisCO S is RuBisCO small subunit, S1 is non-frozen supernatant, and S3 is the supernatant of crude extract frozen at -20°C for 1 day. (B) Concentrations of TSP and GFP in the corresponding supernatants. [Figure 8] Figure 8 shows the effect of freeze-thaw treatment on GFP function when the freezing temperature was -20°C. The relative GFP fluorescence intensity of S1 and S3 from GFP-expressing plants is shown. S1 is the unfrozen supernatant, and S3 is the supernatant of a crude extract frozen at -20°C for 1 day. [Figure 9]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 min and then thawed. (A) Proteins were separated by SDS-PAGE and detected by Coomassie blue staining (top) or Western blot (middle). Results of GUS staining are also shown (bottom). (B) TSP concentration in the corresponding supernatant. M is molecular weight marker, RuBisCO L is RuBisCO large subunit, RuBisCO S is RuBisCO small subunit, S1 is unfrozen supernatant, and S3 is supernatant of crude extract frozen at -30°C for 60 min. [Figure 10] 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 is molecular weight marker, RuBisCO L is RuBisCO large subunit, RuBisCO S is RuBisCO small subunit, S1 is non-frozen supernatant, and S3 is supernatant of crude extract frozen at -30°C for 1 day. (B) Concentrations of TSP and FGF1 in the corresponding supernatants. [Figure 11] Figure 11 shows the extraction and confirmation of expression of human type II collagen protein expressed by a transient expression system using N. benthamiana. Crude extracts obtained from leaves infected with Agrobacterium (D, D2, F, F2, H, H2 strains; n=3) encoding various designed human type II collagens were separated by non-reducing SDS-PAGE, and human type II collagen was detected by Western blotting. Negative controls are "WT", an extract obtained from uninfected plants, and "GFP", an extract obtained from leaves infected with Agrobacterium encoding GFP. Positive control was human type II collagen protein derived from human tissue (indicated as "human COL2 (1 μg)"). M indicates molecular weight marker. [Figure 12]Figure 12 shows the results of freeze-thawing of human type II collagen protein. The sample was made by freezing crude juice at -30℃ for 2 weeks, then thawing and centrifuging it, and analyzing the resulting supernatant. The figure shows the results of Western blotting to detect human type II collagen after separation by non-reducing SDS-PAGE. (Samples prepared under the same conditions as in Figure 11 were freeze-thawed; n=2) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention provides a method for removing a plant-derived soluble protein 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, step (1)).

[0011] In step (1), the plant from which the plant-derived extract is extracted is not particularly limited, but is assumed to be a model plant for which cultivation techniques, breeding techniques, or gene introduction 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), the genus Nicotiana (e.g., N. benthamiana), etc. In addition, the above-mentioned 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 and is not insolubilized by freezing and thawing in step (1) described below. Here, the soluble protein refers to a protein that has many basic amino acid residues that are positively charged near neutral pH, acidic amino acid residues that are negatively charged, or polar amino acid residues at the site that corresponds to the surface of the soluble protein, and is highly soluble in water because water molecules bind to the dissociable groups of the basic or acidic amino acid residues (ionic hydration) or hydrogen bonds are formed between polar groups (OH groups, etc.) and water molecules, thereby increasing the affinity with water molecules. Examples of basic amino acids include Lys, Arg, and His. Examples of acidic amino acids include Glu and Asp. Examples of polar amino acids include serine and threonine. In addition, the protein that is not insolubilized by freezing and thawing refers to a protein in which the higher-order structure of the soluble protein does not change and insolubilization due to aggregation does not occur due to freezing and thawing. Examples of such exogenous soluble proteins include antibodies, cytokines (IFN-α, IFN-β, FGF, etc.), various enzymes, antigen proteins of viruses, bacteria, and cancers, extracellular matrix proteins such as collagen, cell signaling-related proteins, and high-value-added proteins such as albumin. For example, by using a part of a viral protein as an antigen, it becomes possible to mass-produce antigens that are useful in developing vaccines and antibodies against viruses. Alternatively, it is also possible to mass-produce cancer antigens that are useful in developing anticancer drugs.

[0013] The expression of the exogenous soluble protein may be either transient or constant. Means for expressing the exogenous soluble protein in a plant include a method of introducing an expression vector containing DNA encoding the exogenous soluble protein into a plant cell, 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 cell by culturing the plant cell.

[0014] The introduction of an expression vector containing DNA encoding an exogenous soluble protein into a plant cell can be carried out by a method known per se, for example, in the form of an expression vector containing DNA encoding an exogenous soluble protein. The introduction of the expression vector can be carried out according to a known method (e.g., the Agrobacterium method, the PEG method, the electroporation method, the particle gun method, the whisker direct introduction method, etc.) into an appropriate tissue (e.g., callus, root, leaf, seed, growing point, etc.) according to the type of plant. Among them, the Agrobacterium method is particularly preferred because it is easy to scale up. The introduction of an expression vector containing DNA encoding an exogenous soluble protein into a plant cell can also be carried out by introducing it using a viral vector having a nucleic acid sequence encoding the exogenous soluble protein. The introduction of the viral vector can be carried out according to the viral vector method into the above-mentioned tissue according to the type of plant.

[0015] When a foreign soluble protein is constitutively expressed in a plant, for example, a part of tobacco tissue (e.g., tobacco leaf) or hypocotyl is cut off and infected with Agrobacterium, and the tissue or hypocotyl into which the expression vector has been introduced is cultured to select a genetically modified plant cell in which DNA encoding the foreign soluble protein has been introduced into the genome. Then, callus is induced from the cell, and roots are induced from the callus to obtain a genetically modified plant that stably expresses the foreign soluble protein. Alternatively, Agrobacterium may be infected into the callus. Typically, such Agrobacterium is one in which DNA encoding the foreign soluble protein has been introduced into a T-DNA fragment of an Agrobacterium expression vector. When the PEG method or electroporation method is used, a protoplast is prepared from an appropriate cell or tissue according to a conventional method, and an expression vector is introduced into the protoplast. When the particle gun method is used, an expression vector adsorbed on gold particles can be introduced into a callus, an immature embryo, a growing point present in a shoot apex, or an axillary bud, etc., using a particle gun. In the particle gun method and the Agrobacterium method, since the plants into which genes are introduced often become chimeras, it is necessary to use sample cells for gene introduction that will allow the above DNA to be introduced into germ line 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 a known method depending on the type of plant cells or tissues. The medium used for culture is preferably a solid medium (e.g., agar medium, agarose medium, gellan gum medium, etc.). The medium preferably contains a carbon source, a nitrogen source, inorganic substances, etc. necessary for the growth of the 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 appropriately selected usually within the range of about 20°C to about 35°C depending on the type of plant cells.

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

[0018] Examples of methods for transiently expressing an exogenous soluble protein in a plant include a method in which whole tobacco plants are immersed in a suspension of Agrobacterium, sealed, and subjected to a reduced pressure (e.g., 0.09 MPa) (vacuum infiltration method), or a method in which an Agrobacterium suspension is infiltrated into intercellular spaces of leaves using a needleless syringe (syringe infiltration method). After the treatment, tobacco tissues or whole tobacco plants are cultured for several days, making it possible to obtain plants that transiently express an exogenous soluble protein in the tobacco tissue infiltrated with Agrobacterium.

[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 a plant tissue disruption." For example, a plant extract can be prepared by disrupting all or a part of a plant (roots, leaves, stems, or flowers) and suspending it in water or a buffer (e.g., Tris-HCl buffer, phosphate buffer, HEPES buffer, etc.). The extract can further contain a surfactant (e.g., Triton X-100, TM , Tween 20 TM etc.) and protein degradation inhibitors may also be included.

[0020] The plant-derived extract obtained as described above contains insoluble substances such as fragments as well as soluble substances such as endogenous and exogenous soluble proteins. Therefore, the removal method of the present invention may further include a step of removing insoluble substances from the plant-derived extract before step (1). The step of removing insoluble substances from the plant-derived extract is not particularly limited as long as the insoluble substances can be removed, and a preferred example is a step of removing insoluble substances from the plant-derived extract by centrifugation. The conditions of the centrifugation are not particularly limited as long as the insoluble substances are precipitated, and 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 in that the recovery rate of soluble substances is good, the centrifuge is a general-purpose machine, the processing time is short, and the precipitate can be simply determined based on the molecular weight. Another step of removing insoluble substances from the plant-derived extract is a step of removing insoluble substances by filtering the plant-derived extract. The filtration 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 filtration membrane is not particularly limited as long as it can filter insoluble substances, and examples thereof include 10 μm to 700 μm, etc. 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 may be, for example, within the range of -30°C to -20°C, but are not limited thereto. The freezing period is not limited as long as the plant-derived extract is frozen, but may be, for example, within the 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 due to freezing, the loss of biological activity may be avoided by shortening the freezing period. When an exogenous soluble protein having such characteristics is used, the freezing period of the plant-derived extract is usually 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, but is not limited thereto.

[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, by freezing and thawing the plant-derived extract, it is possible to insolubilize the plant-derived soluble proteins contained in the plant-derived 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 in the photosynthetic reaction of plants, and catalyzes the reaction of fixing carbon dioxide to ribulose 1,5-bisphosphate to produce two molecules of 3-phosphoglyceric acid. RuBisCO is the protein most abundant in plants, and is the protein that is most removed in the process of purifying proteins other than RuBisCO from plants. RuBisCO usually exists as a heterohexamer consisting of eight large subunits and eight small subunits, and exists as a soluble protein in the plant-derived extract. It is believed that the freezing and thawing treatment in step (1) changes the higher-order structure of the large subunits and small subunits of RuBisCO, or the heterohexamer composed of them, causing insolubilization. In addition, when 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 is not insolubilized by the above-mentioned freezing and thawing, and therefore 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 above-mentioned frozen and thawed plant-derived extract (hereinafter, step (2)).

[0025] The step of removing the insolubilized protein from the frozen and thawed plant-derived extract is not particularly limited as long as the insolubilized protein can be removed, and a preferred example is a step of removing the insolubilized protein by centrifuging the frozen and thawed plant-derived extract. The conditions of the centrifugation are not particularly limited as long as the insolubilized protein is precipitated, and examples include about 10,000 to about 30,000×g, about 2 to about 20 minutes, and about 0 to about 4° C. By removing the resulting precipitate, it is possible to separate the insolubilized protein from the plant-derived extract. Another step of removing the insolubilized protein from the frozen and thawed plant-derived extract is a step of 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. When the plant expresses an exogenous soluble protein that is not insolubilized by the freezing and thawing in step (1), it is possible to separate the insolubilized protein from the plant-derived extract and increase the proportion of the exogenous soluble protein in the soluble proteins contained in 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. 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 carrying a plant expression vector (GFP_pBE2113) expressing GFP was cultured in 3 ml of YEP medium (containing kanamycin (50 μg / mL), streptomycin (300 μg / mL), and rifampicin (100 μg / mL)) at 28°C overnight with shaking at 220 rpm. The culture was centrifuged at 5,000×g for 10 minutes at room temperature of 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 (add immediately before use)) and further incubated at OD 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 to 3 hours, and then grown in a chamber for 4 days (23°C, 16 hours light, 8 hours dark).

[0028] Leaves (100 mg) of wild-type N. benthamiana and leaves (100 mg) of N. benthamiana transfected with the GFP gene as described above were crushed using a multi-beads shocker (1,700 rpm, 30 sec), and the crushed material was suspended in 500 μl of phosphate buffer (pH 7.2, 0.1% (v / v) Tween 20, 1% (v / v) protease inhibitor cocktail (Sigma-Aldrich #P9599)) to obtain crude extracts of each plant body. The resulting crude extracts were centrifuged (23,000×g, 10 min, 2°C) to obtain the supernatant (S1) and precipitate (P1). Furthermore, a separately prepared supernatant (S1) was left at -30°C (1 day), thawed in a refrigerator (4°C), and centrifuged (23,000×g, 10 min, 2°C) to obtain a supernatant (S2) and a precipitate (P2). A separately prepared crude extract was left at -30°C (1 day), 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 flow chart in Figure 1 was followed.

[0029] In both wild-type and GFP-transfected plants, the crude extract was centrifuged to remove the insoluble fraction P1, and the S1 was freeze-thawed and centrifuged again, resulting in the formation of a new precipitate P2 (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] In addition, when the colors of the supernatants were compared under a blue LED, none of the wild-type S1 to S3 showed any fluorescence, but GFP fluorescence was observed in all S1 to S3 obtained from plants into which the GFP gene had been introduced, and the fluorescence intensities of S1 (before freeze-thaw treatment) and S2 (after freeze-thaw treatment) were comparable (Figure 2B). This indicates that the GFP that remained in the supernatant without being precipitated by centrifugation before freeze-thaw treatment continued to remain in the supernatant without being insolubilized by freeze-thaw treatment, and as a result, it was possible to separate GFP from the material insolubilized by freeze-thaw treatment.

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

[0032] Furthermore, the results of Western blot analysis showed that the amount of GFP contained in S1 to S3 of the GFP gene-introduced type was relatively compared. As a result, when the amount of GFP in S1 was set to 1, the amount of GFP in S2 was more than 0.9 (Figure 3A). On the other hand, the results of Coomassie blue staining showed that the amount of RuBisCO contained in S1 to S3 of the GFP gene-introduced type was relatively compared. When the amount of RuBisCO in S1 was set to 1, the amount in S2 was less than 0.1 (Figure 3B). Furthermore, the results of Coomassie blue staining showed that the amount of total protein contained in S1 to S3 of the GFP gene-introduced type was relatively compared. As a result, when the amount of total protein in S1 was set to 1, the amount in S2 was less than 0.3 (Figure 3C). From these results, it was found that more than 70% of the total protein was removed from the crude extract derived from N. benthamiana leaves by the freeze-thaw treatment, and more than 90% of RuBisCO was removed. Furthermore, it was found that GFP, a soluble protein that was transiently expressed, was hardly affected by the freeze-thaw treatment and remained in the supernatant. Therefore, the freeze-thaw treatment can simply and quickly insolubilize plant-derived soluble proteins from crude plant extracts, which can be easily precipitated and removed by centrifugation. In addition, when an exogenous soluble protein is expressed, the freeze-thaw treatment can easily and dramatically improve the purification rate of the target soluble protein. In addition, the same results were obtained without the procedure of centrifuging the crude plant extract and removing the insoluble fraction before performing the freeze-thaw treatment.

[0033] Example 2 Verification of the effect of freezing period on the separation of plant-derived soluble proteins and exogenous soluble proteins 1 As in Example 1, wild-type N. benthamiana leaves (100 mg) and leaves (100 mg) obtained from N. benthamiana introduced with the GFP gene were ground (1,700 rpm, 30 sec) using a multi-beads shocker, and the ground material was suspended in 500 μl of phosphate buffer (containing 0.1% (v / v) Tween 20 and protease inhibitors) to obtain crude extracts of each plant body. The obtained crude extracts were immediately centrifuged (23,000×g, 10 min, 2° C.) to obtain supernatant S1 and precipitate P1. In addition, the supernatant S1 was allowed 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 the supernatant S2 (frozen for 1 day), the supernatant S2 (frozen for 7 days), and the precipitate P2 (frozen for 1 day) and precipitate P2 (frozen for 7 days).

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

[0035] In addition, when the color of the supernatant was compared under a blue LED, none of the wild-type S1, S2 (frozen for 1 day), and S2 (frozen for 7 days) showed GFP fluorescence, but the S1, S2 (frozen for 1 day), and S2 (frozen for 7 days) obtained from the plant body into which the GFP gene had been introduced all showed fluorescence, and the fluorescence intensity was comparable (Figure 4B). This suggests that the freezing period does not affect the state of GFP.

[0036] Proteins in wild-type and GFP gene-introduced S1, S2 (frozen for 1 day), and S2 (frozen for 7 days) were separated by SDS-PAGE and then subjected to Coomassie blue staining and Western blot analysis. As a result, a RuBisCO (L / S) band was detected in S1, but not in S2 (frozen for 1 day) or S2 (frozen for 7 days) (Fig. 4C). In addition, no GFP band was detected in wild-type S1, S2 (frozen for 1 day), or S2 (frozen for 7 days), but GFP bands were detected in all S1, S2 (frozen for 1 day), and S2 (frozen for 7 days) obtained from plants with the GFP gene introduced, and the band intensities were the same among S1, S2 (frozen for 1 day), and S2 (frozen for 7 days). In fact, the results of Western blot analysis showed that the relative amounts of GFP in GFP gene-introduced S1, S2 (frozen for 1 day), and S2 (frozen for 7 days) showed no difference in the amounts of GFP. These results suggest that the freezing period does not affect the precipitation or the state of GFP. It was shown that freezing treatment can insolubilize proteins including RuBisCO from crude plant extracts, and they can be easily removed by centrifugation. It was also shown that when an exogenous soluble protein is expressed, freezing treatment can easily and dramatically improve the purification rate of the target soluble protein.

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

[0038] As shown in Figure 5, freezing for 10 minutes was effective in removing RuBisCO, although it was weak. Freezing for 60 minutes was effective in removing RuBisCO. Furthermore, no effect was observed on GFP during the freeze-thaw treatment under these conditions (Figure 5(B) and Figure 6).

[0039] Example 4 Verification of the effect of freezing temperature on the separation of plant-derived soluble proteins from exogenous soluble proteins The same experiment as in Example 3 was carried out, 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 FIG. 7 and FIG. 8.

[0040] As shown in Figures 7 and 8, when the freezing temperature was set to -20°C, the same results were obtained as 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 were reduced, but no effect was observed on GFP.

[0041] Example 5 Examination of the effect of freeze-thaw treatment on proteins other than GFP 1 (GUS) The effect of freeze-thaw treatment on transiently expressed GUS (β-glucuronidase) was evaluated. Crude extracts from GUS-expressing leaves were frozen at -30°C for 60 min and thawed at 4°C. Western blot analysis confirmed GUS expression in all samples from S1 and S3, and GUS activity was detected in the supernatant (Figure 9(a)). TSP was sufficiently reduced even after 60 min 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 Examination of the effect of freeze-thaw treatment on proteins other than GFP 2 (human FGF1) The effect of freeze-thaw treatment 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 the amount of FGF1 after freeze-thaw treatment (Figure 10(B)).

[0043] Example 7 Examination of the effect of freeze-thaw treatment on proteins other than GFP 3 (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] A crude extract from leaves expressing human Collagen II was 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, similar to the sample before freezing.

[0045] Example 8 Proteomic analysis To investigate the effects of freeze-thaw treatment, proteome analysis was performed on the supernatants before and after freeze-thawing of wild-type plants. As a result, 695 proteins were identified in the test supernatants. The average abundance ratio of three proteins was 1 or more, and the abundance ratio of 692 proteins was less than 1. Table 1 shows the top 20 proteins whose amounts decreased after freeze-thawing. As shown in Table 1, the amounts of many endogenous proteins in the supernatant after freeze-thaw treatment were decreased compared to 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] [Table 1-1]

[0047] [Table 1-2]

[0048] SDS-PAGE analysis showed that the amounts of the large and small subunits of RuBisCO in the freeze-thawed supernatants were significantly reduced compared to the unfrozen supernatants: in the frozen samples, the average abundance ratios of the large and small subunits of RuBisCO were reduced to approximately 10% and 20%, respectively, of the unfrozen supernatants (Table 2).

[0049] [Table 2]

[0050] These values ​​were higher than those estimated from the band intensities observed in SDS-PAGE. In contrast, only 15 proteins were hardly affected by the freeze-thaw treatment (their average abundance was 90% or more) (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] [Table 3-1]

[0052] [Table 3-2] [Industrial Applicability]

[0053] Conventionally, the separation and purification of exogenous soluble proteins expressed in plants required multiple reagents and multiple steps, but according to the present invention, it is possible to remove plant-derived soluble proteins, including RuBisCO, a dominant protein in plants, by insolubilizing them through a freeze-thaw treatment, and it is possible to increase the proportion of the exogenous soluble proteins upstream in the purification process. 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.

Claims

1. A method for removing plant-derived soluble proteins from a plant-derived extract, comprising the steps of: (1) freezing and thawing a plant-derived extract to insolubilize plant-derived soluble proteins; and (2) Removing insoluble 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. 3. The method of claim 1 or 2, wherein the plant expresses an exogenous soluble protein that is not insolubilized by freezing and thawing.

6. 3. 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 -20°C or lower.

8. 3. 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 of 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 the removal of insoluble matter from the plant-derived extract is carried out by centrifugation or filtration.

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