Method for purifying protein using acid

Abstract

To provide a new method for efficiently purifying an exogenous protein expressed in a plant.SOLUTION: A method for purifying a foreign protein expressed in a plant, comprising the step of adding an acid to an extract of a tissue of a plant in which a foreign protein is expressed to degrade contaminating proteins derived from the plant.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for purifying a foreign protein expressed in a plant protein expression system using an acid. [Background technology]

[0002] Substance production in plants using genetic engineering technology has the potential to express proteins that are difficult to produce using exogenous protein expression systems using animal cells, and is expected to be utilized in fields such as the production of pharmaceuticals, foods, and healthcare products. However, compared to technologies for producing useful substances using animal cells, there are currently very few reports on technologies for producing useful substances using plants. Furthermore, most technologies for producing useful substances using plants are at the research protocol level, with few examples of commercial success. Furthermore, technologies that are considered to be commercially successful require large facilities, and there are still many challenges in terms of site area and implementation costs.

[0003] One process that has issues to be resolved in plant-based protein expression systems is the separation and purification of exogenous proteins expressed in plants. Most of the currently reported technologies for separating and purifying exogenous proteins expressed in plants require numerous reagents and multi-step operations, and issues have been pointed out in terms of efficiency and cost (Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6560495 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a novel method for efficiently purifying a foreign protein expressed in a plant. [Means for solving the problem]

[0006] As a result of intensive research into the above-mentioned problems, the inventors discovered that exogenous proteins can be purified extremely efficiently by adding an acid to an extract of a plant in which the exogenous protein has been expressed, thereby decomposing contaminating plant proteins. Based on this finding, the inventors continued their research and completed the present invention. That is, the present invention is as follows.

[0007] [1] A method for purifying a foreign protein expressed in a plant, comprising the step of adding an acid to an extract of plant tissue in which the foreign protein has been expressed, thereby decomposing contaminating plant proteins. [2] The method described in [1], wherein the extract is a crude extract. [3] The method described in [1], wherein the extract is a supernatant obtained by centrifuging a crude extract. [4] The method according to any one of [1] to [3], wherein the step is carried out at 1 to 25°C. [5] The method according to any one of [1] to [4], wherein the plant is a Nicotiana plant. [6] [5] The method described in [5], wherein the Nicotiana plant is Nicotiana benthamiana. [7] The method according to any one of [1] to [6], wherein the exogenous protein is a protein that is resistant to degradation by acid. [8] [7] The method described in [7], wherein the protein resistant to degradation by acid is at least one selected from the group consisting of collagen, elastin, keratin, and fibroin. [9] The method according to any one of [1] to [8], wherein the acid is at least one selected from the group consisting of acetic acid, hydrochloric acid, formic acid, propionic acid, butyric acid, lactic acid, glycolic acid, and citric acid.

[10] [9] The method according to claim 9, wherein the acid is acetic acid. [Effects of the Invention]

[0008] According to the present invention, foreign proteins expressed in plants can be purified with extremely high efficiency. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 shows that plant-derived proteins are decomposed by acetic acid. [Figure 2] FIG. 2 shows the effect of acetic acid concentration on collagen. [Figure 3] FIG. 3 shows the effect of treatment time on collagen in acetic acid treatment. [Figure 4] FIG. 4 shows that plant-derived proteins are decomposed by hydrochloric acid. [Figure 5] FIG. 5 shows the results when an extract obtained from tobacco in which collagen was overexpressed was applied to the purification method of the present invention.

[0010] The present invention will be described in detail below.

[0011] The present invention provides a method for purifying an exogenous protein expressed in a plant (hereinafter, sometimes referred to as the "purification method of the present invention"), which comprises the step of adding an acid to an extract of plant tissue in which the exogenous protein has been expressed, thereby decomposing contaminating plant-derived proteins.

[0012] In the purification method of the present invention, the plant in which an exogenous protein is expressed is not particularly limited, but is preferably a model plant for which cultivation techniques, breeding techniques, or gene transfer techniques have been established. Examples of such plants include, but are not limited to, 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., Brassica napus), the genus Solanum (e.g., Solanum lycopersicum), the genus Medicago (e.g., Medicago truncatula), and the genus Nicotiana (e.g., Nicotiana benthamiana). Such plants may be wild-type plants or genetically modified plants.

[0013] In the purification method of the present invention, the exogenous protein is not particularly limited as long as it is exogenous to the plant in which the protein is produced.

[0014] The purification method of the present invention is characterized by decomposing contaminating plant-derived proteins with acid. Thus, in one embodiment, the exogenous protein may be a protein that is resistant to acid degradation. "Decomposition by acid" refers to acid hydrolysis of the protein. Furthermore, "resistance to acid degradation" does not only mean that the protein is not hydrolyzed at all or substantially by acid, but also means that the protein is decomposed to some extent when the purification method of the present invention is applied, but that a certain amount or more of the protein can be recovered in an undecomposed state after completion of the purification method of the present invention. Such a "certain amount" also means, for example, when acetic acid is added to an extract (supernatant after centrifugation) of tobacco (N. benthamiana) leaves in which an exogenous protein has been expressed to a final concentration of 0.5 M and allowed to stand at 4°C for 2 hours to decompose the plant protein, at least 30% or more (preferably 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more) of the protein is not decomposed at the end of the decomposition process.

[0015] In one aspect, proteins resistant to acidic hydrolysis include, but are not limited to, proteins with tertiary or quaternary structures, proteins with structures that contribute to protein stability, such as β-sheet structures, proteins that are rich in specific amino acids that form strong peptide bonds (proline, hydroxyproline, etc.), and proteins that are rich in hydrophobic amino acids that stabilize the structure through hydrophobic interactions.

[0016] In one embodiment, proteins that are resistant to degradation by acid include, but are not limited to, collagen, elastin, keratin, and fibroin.

[0017] The expression of a foreign protein in a plant may be transient or constitutive. Means for expressing a foreign protein in a plant include, but are not limited to, a method of introducing an expression vector containing a nucleic acid sequence encoding the foreign protein into a plant cell, or a method of introducing a viral vector having a nucleic acid sequence encoding the foreign protein into a plant.

[0018] Introduction of an expression vector containing a nucleic acid sequence encoding a foreign protein into plant cells can be carried out by methods known per se. Introduction of the expression vector can be carried out in appropriate tissues (e.g., callus, roots, leaves, seeds, meristems, etc.) according to the type of plant using known methods (e.g., the Agrobacterium method, the PEG method, electroporation, particle gun method, direct whisker introduction method, etc.). Among these, the Agrobacterium method is particularly preferred because it is easy to scale up. Introduction of an expression vector containing a nucleic acid sequence encoding a foreign protein into plant cells can also be carried out using a viral vector carrying the nucleic acid sequence encoding the foreign protein. Introduction of the viral vector can be carried out in the above tissues according to the type of plant using the viral vector method.

[0019] To constitutively express an exogenous 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 into which the expression vector has been introduced is then cultured to select transgenic plant cells in which a nucleic acid sequence encoding the exogenous protein has been introduced into the genome. Callus is then induced from the cells, and rooting is induced from the callus to obtain a transgenic plant stably expressing the exogenous protein. Alternatively, Agrobacterium can be infected into the callus. Typically, Agrobacterium is used in which a nucleic acid sequence encoding the exogenous 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, or meristems present in the shoot apex or axillary buds using a particle gun. In the particle gun method and the Agrobacterium method, the introduced plants often become chimeric, so it is necessary to use sample cells for gene introduction that will allow the above nucleic acid sequence to be introduced into germline cells at a high frequency, such as embryos, hypocotyl segments, embryogenic callus, and isolated meristems.

[0020] 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.

[0021] 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.

[0022] For example, methods for transiently expressing exogenous 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 with 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 tissue.

[0023] The plant tissue used in the purification method of the present invention is not particularly limited as long as it is a tissue containing an exogenous protein. Examples of such tissue include, but are not limited to, callus, root, leaf, seed, or meristem. In a preferred embodiment, the plant tissue may be a leaf.

[0024] Plant tissue extracts can be prepared using methods known per se. For example, they can be prepared by grinding the plant tissue using a mortar and pestle and suspending it in an appropriate buffer. Any buffer can be used as long as it does not inactivate the desired exogenous protein, and examples of such buffers include Tris-HCl buffer, phosphate buffer, and HEPES buffer. The extract may also contain surfactants (e.g., Triton X-100 (trademark), Tween 20 (trademark), etc.) and protease inhibitors.

[0025] In one embodiment, the extract may be prepared by grinding plant tissue and suspending it in water, a buffer solution, or the like (i.e., a "crude extract"), or may be a supernatant obtained by centrifuging the crude extract (i.e., an extract obtained by removing cell debris and insoluble materials from the crude extract). In a preferred embodiment, the extract may be a supernatant obtained by centrifuging the crude extract.

[0026] The conditions for centrifuging the crude extract are not particularly limited, as long as cell debris and insoluble substances are precipitated, the desired exogenous protein is transferred to the supernatant, and the biological activity of the desired exogenous protein is not inactivated. For example, the centrifugal acceleration that can be used is usually 1,000 × g or higher, preferably 3,000 × g or higher, 5,000 × g or higher, 7,000 × g or higher, 9,000 × g or higher, 11,000 × g or higher, 13,000 × g or higher, 15,000 × g or higher, 17,000 × g or higher, or 19,000 × g or higher, but is not limited to these. The upper limit is usually 50,000×g or less, preferably 48,000×g or less, 46,000×g or less, 44,000×g or less, 42,000×g or less, 40,000×g or less, 38,000×g or less, 36,000×g or less, 34,000×g or less, or 32,000×g or less, but is not limited to these. In one embodiment, the centrifugal acceleration used when centrifuging the crude extract is typically 1,000 to 50,000 × g, and preferably 3,000 to 48,000 × g, 5,000 to 46,000 × g, 7,000 to 44,000 × g, 9,000 to 42,000 × g, 11,000 to 40,000 × g, 13,000 to 38,000 × g, 15,000 to 36,000 × g, 17,000 to 34,000 × g, or 19,000 to 32,000 × g, but is not limited to these.

[0027] The centrifugation time is not particularly limited, as long as cell debris and insoluble substances are precipitated, the desired exogenous protein is transferred to the supernatant, and the biological activity of the desired exogenous protein is not inactivated. The centrifugation time can typically be, but is not limited to, 1 minute or more, 2 minutes or more, 3 minutes or more, 4 minutes or more, 5 minutes or more, 6 minutes or more, 7 minutes or more, 8 minutes or more, or 9 minutes or more. The upper limit of the centrifugation time can typically be, but is not limited to, 1 hour or less, preferably 55 minutes or less, 50 minutes or less, 45 minutes or less, 40 minutes or less, 35 minutes or less, 30 minutes or less, 25 minutes or less, or 20 minutes or less. In one embodiment, the centrifugation time can typically be, but is not limited to, 1 minute to 1 hour, preferably 2 minutes to 55 minutes, 3 minutes to 50 minutes, 4 minutes to 45 minutes, 5 minutes to 40 minutes, 6 minutes to 35 minutes, 7 minutes to 30 minutes, 8 minutes to 25 minutes, or 9 minutes to 20 minutes.

[0028] The temperature during centrifugation is also not particularly limited, as long as cell debris and insoluble substances are precipitated, the desired exogenous protein is transferred to the supernatant, and the biological activity of the desired exogenous protein is not inactivated. The temperature during centrifugation can usually be 0°C or higher, preferably 1°C or higher, 2°C or higher, 3°C or higher, 4°C or higher, 5°C or higher, 6°C or higher, 7°C or higher, or 8°C or higher. The upper limit is usually 37°C or less, preferably 34°C or less, 33°C or less, 32°C or less, 31°C or less, 30°C or less, 29°C or less, 28°C or less, 27°C or less, 26°C or less, 25°C or less, 24°C or less, 23°C or less, 22°C or less, 21°C or less, 20°C or less, 19°C or less, 18°C ​​or less, 17°C or less, 16°C or less, 15°C or less, 14°C or less, 13°C or less, 12°C or less, 11°C or less, 10°C or less, 9°C or less, 8°C or less, 7°C or less, 6°C or less, 5°C or less, or 4°C or less. In one embodiment, the temperature during centrifugation may be generally 0 to 37°C, preferably 0 to 30°C, 0 to 25°C, 0 to 20°C, 0 to 15°C, 0 to 10°C, 0 to 9°C, 0 to 8°C, 0 to 7°C, 1 to 6°C, 1 to 5°C, 2 to 5°C, or 3 to 5°C.

[0029] If the exogenous protein is trapped in the precipitated cell debris or insoluble material after the centrifugation step is completed, the desired exogenous protein can be recovered by recovering the residue portion of the sample obtained after centrifugation and taking measures (e.g., filtration) to separate the desired exogenous protein trapped in the recovered residue portion from the residue.

[0030] In the purification method of the present invention, the step of adding an acid to the plant tissue extract to decompose impurity proteins derived from the plant (hereinafter, sometimes referred to as the "step of decomposing impurity proteins") may be carried out under any conditions as long as the majority of the desired exogenous protein is not inactivated or decomposed.

[0031] The conditions that can be used in the process of decomposing impurity proteins may be appropriately determined taking into consideration the type of plant in which the exogenous protein is expressed, the type of exogenous protein to be expressed, the type and / or concentration of the acid used, the treatment temperature, the treatment time, etc.

[0032] Any acid can be used in the step of decomposing impurity proteins as long as it can decompose plant-derived proteins and does not inactivate or decompose most of the desired exogenous proteins. Examples of acids that can be used include, but are not limited to, acetic acid, hydrochloric acid, formic acid, propionic acid, butyric acid, lactic acid, glycolic acid, and citric acid. Generally, strong acids tend to decompose not only plant-derived proteins but also exogenous proteins, and are dangerous to handle, so weak acids may be preferred. In a preferred embodiment, the acid is acetic acid.

[0033] Furthermore, the concentration of the acid that can be used in the step of decomposing impurity proteins may be any concentration as long as it is capable of decomposing plant-derived proteins and does not inactivate or decompose a large portion of the desired exogenous protein. When a weak acid (e.g., acetic acid) is used, the final concentration may be, but is not limited to, typically 0.01 M or higher, preferably 0.05 M or higher, 0.1 M or higher, 0.5 M or higher, or 1.0 M or higher. The upper limit of the concentration may be, but is not limited to, typically 5.0 M or lower, preferably 4.5 M or lower, 4.0 M or lower, 3.5 M or lower, or 3.0 M or lower. In one aspect, when a weak acid (e.g., acetic acid) is used, the concentration may be, but is not limited to, typically 0.01 to 5.0 M, preferably 0.05 to 4.5 M, 0.1 to 4.0 M, 0.5 to 3.5 M, or 1.0 to 3.0 M. When a strong acid (e.g., hydrochloric acid) is used, the final concentration may be, but is not limited to, typically 0.001 M or higher, preferably 0.05 M or higher, 0.1 M or higher, 0.5 M or higher, or 1.0 M or higher. Furthermore, the upper limit of the concentration may be, but is not limited to, typically 2.0 M or lower, preferably 1.8 M or lower, 1.6 M or lower, 1.4 M or lower, or 1.2 M or lower. In one embodiment, when a strong acid (e.g., hydrochloric acid) is used, the concentration may be, but is not limited to, typically 0.001 to 2.0 M, preferably 0.05 to 1.8 M, 0.1 to 1.6 M, 0.5 to 1.4 M, or 1.0 to 1.2 M. The concentration may be appropriately determined in consideration of the treatment time.

[0034] Furthermore, the treatment time that can be used in the step of decomposing impurity proteins may be any concentration as long as it is capable of decomposing plant-derived proteins and does not inactivate or decompose a large portion of the desired exogenous protein. The treatment time may be, for example, typically 5 minutes or more, preferably 10 minutes or more, 30 minutes or more, 45 minutes or more, or 1 hour or more, but is not limited to these. The upper limit of the treatment time may be typically 48 hours or less, preferably 24 hours or less, 12 hours or less, 6 hours or less, or 3 hours or less, but is not limited to these. In one embodiment, the treatment time may be typically 5 minutes to 48 hours, preferably 10 minutes to 12 hours, 30 minutes to 6 hours, or 1 hour to 3 hours, but is not limited to these. The treatment time may be determined appropriately depending on the type of acid used.

[0035] Furthermore, the treatment temperature that can be used in the step of decomposing impurity proteins may be any temperature as long as it is capable of decomposing plant-derived proteins and does not inactivate or decompose a large portion of the desired exogenous protein. The treatment temperature may be, for example, typically 0.5°C or higher, preferably 1.0°C or higher, 1.5°C or higher, 2.0°C or higher, 2.5°C or higher, or 3.0°C or higher, but is not limited to these. The upper limit may be typically 25°C or lower, preferably 20°C or lower, 17°C or lower, 14°C or lower, 11°C or lower, or 8°C or lower, but is not limited to these. In one aspect, the treatment temperature may be typically 0.5 to 25°C, preferably 1.0 to 20°C, 1.5 to 17°C, 1.5 to 14°C, 2.0 to 11°C, or 2.5 to 8°C, but is not limited to these.

[0036] The purification method of the present invention may additionally include a step for the purpose of further purifying or concentrating the exogenous protein. For example, the sample solution obtained through the step of decomposing impurity proteins may be subjected to centrifugation, affinity chromatography, ultrafiltration, dialysis, and / or salting out, etc., to further purify and / or concentrate the exogenous protein.

[0037] The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way. [Example]

[0038] Example 1. Decomposition of plant-derived proteins with acetic acid Wild-type N. benthamiana leaves were crushed and suspended in PBS to prepare a crude extract. Acetic acid was added to the crude extract to a final concentration of 0M, 0.05M, 0.5M, or 5M. The crude extract with added acetic acid was allowed to stand at 4°C for 1 hour. The resulting sample was then centrifuged (conditions: 4,000 × g, 10 minutes, 4°C) and the supernatant was collected. The collected supernatant was then subjected to SDS-PAGE and protein assay. SDS-PAGE was performed using a commercially available 4-15% acrylamide gel (BioRad). Protein Assay Dye Reagent (BioRad) was used for the protein assay. The results of SDS-PAGE (CBB staining) are shown in Figure 1. The results of the protein assay are shown below.

[0039] [Protein Assay] Acetic acid concentration (M) Total protein (μg / mL) 0 565.10 0.05 55.32 0.5 61.58 5 392.81

[0040] As shown in Figure 1, the supernatant obtained after acetic acid treatment and subsequent centrifugation showed a significant reduction in the amount of plant-derived proteins, including Rubisco. Similar results were also confirmed by protein assays. Furthermore, when the final concentration of acetic acid was 5 M, the total protein amount was higher than when the final concentration of acetic acid was 0.05 M or 0.5 M. Without wishing to be bound by theory, this is thought to be due to the migration of some of the plant-derived insoluble proteins into the supernatant due to the action of the high concentration of acetic acid. Based on these results, it was demonstrated that adding acetic acid to the supernatant obtained by centrifuging a crude plant extract to remove cell debris and insoluble materials is more efficient at removing plant-derived proteins than adding acetic acid to the crude plant extract itself.

[0041] Example 2. Effect of acetic acid on exogenous proteins The effect of acetic acid on exogenous proteins was investigated. Bovine type II collagen (COL2) was used as the exogenous protein. A crude extract of wild-type N. benthamiana was obtained using the same procedure as in Example 1. This crude extract was centrifuged (conditions: 3,000 × g, 5 minutes, 4°C) to obtain the supernatant. COL2 (600 μg / g-FW (fresh weight)) was added to the obtained supernatant, and acetic acid was further added to a final concentration of 0 M, 0.05 M, 0.5 M, 1 M, 2 M, 3 M, 4 M, or 5 M. After the addition of acetic acid, the sample was allowed to stand at 4°C for 2 hours. The obtained sample was then centrifuged (conditions: 12,000 × g, 20 minutes, 4°C) to obtain the supernatant. The obtained supernatant was subjected to SDS-PAGE. A 4-20% polyacrylamide gel (BioRad) was used for SDS-PAGE. The polyacrylamide gel after SDS-PAGE was stained with CBB to quantify the amount of COL2. The results of the CBB staining are shown in Figure 2. In the figure, samples with added COL2 are indicated by a "+" and samples without added COL2 are indicated by a "-". The intensity of the band estimated to be COL2 in Figure 2 was quantified relative to the intensity of the sample without added acetic acid (0 M), and the results are shown below.

[0042] [Quantification of band intensity] Acetic acid concentration (M) Band intensity (%) 0 100 0.05 101 0.5 88 1 75 2 66 3 68 4 59 5 49

[0043] As shown in Figure 2 and the results of quantifying band intensity, the amount of COL2 protein decreased as the concentration of added acetic acid increased, but even at the maximum acetic acid concentration (5 M), approximately 50% of COL2 could be recovered. Considering that the recovery rate in large-scale COL expression systems using animal cells is less than 50%, these results suggest that a purification process involving the degradation of plant-derived proteins with acetic acid is an efficient method.

[0044] Example 3. Effects of treatment time and temperature The effects of acetic acid treatment time and temperature on exogenous proteins were examined. Bovine type II collagen (COL2) was used as the exogenous protein. A crude extract of wild-type N. benthamiana was obtained using the same procedure as in Example 1. This crude extract was centrifuged (conditions: 3,000 × g, 10 minutes, 4°C) to obtain the supernatant. COL2 (600 μg / g-FW) was added to the resulting supernatant, and acetic acid was added to a final concentration of 0.5 M. After the addition of acetic acid, the sample was incubated at 4°C or 24°C. Aliquots were collected at 1 hour, 3 hours, 6 hours, and 24 hours. Each collected sample was subjected to SDS-PAGE. COL2 content was quantified by CBB staining and quantification of band intensity. Figure 3 shows the results of quantifying COL2 content at each time point, with the value before acetic acid treatment (0 h) set as the reference (100%).

[0045] As shown in Figure 3, the COL2 levels at 4°C were 61% at 1 hour, 65% at 3 hours, 65% at 6 hours, and 49% at 24 hours, and 53% at 1 hour, 52% at 3 hours, 49% at 6 hours, and 49% at 24°C. This means that nearly 50% or more of the COL2 was recovered at both 4°C and 24°C. Although the amount of COL2 recovered decreased slightly with increasing treatment time, approximately 50% of the COL2 was still recovered even after 24 hours.

[0046] Example 4. Decomposition of plant-derived proteins with hydrochloric acid We investigated whether hydrochloric acid could be used instead of acetic acid. A crude extract of wild-type N. benthamiana was obtained using the same procedure as in Example 1. This crude extract was centrifuged (conditions: 3,000 × g, 10 minutes, 4°C) to obtain the supernatant. Hydrochloric acid (final concentration: 0.01 M, 0.1 M, or 1 M) was added to the resulting supernatant, and the mixture was allowed to stand at 5°C for 2, 8, or 24 hours. After the hydrochloric acid treatment, the sample was subjected to ammonium sulfate precipitation, and the precipitate fraction was collected. The collected precipitate was suspended in SDS-PAGE sample buffer, boiled at 95°C for 5 minutes, and subjected to SDS-PAGE. A 4-20% criterion gel (BioRad) was used for SDS-PAGE. The results of CBB staining are shown in Figure 4.

[0047] As shown in Figure 4, plant-derived proteins could be decomposed using hydrochloric acid instead of acetic acid.

[0048] Example 5. Recovery of foreign proteins expressed in tobacco leaves We investigated whether a foreign protein expressed in tobacco could be purified by a method involving the decomposition of plant-derived proteins using acetic acid. Human COL2 was used as the foreign protein. Using the Agrobacterium method, N. benthamiana transiently expressing human COL2 was constructed. Two strains of N. benthamiana transiently expressing human COL2 were constructed (referred to as H strain / P19 and G strain / P19). Specifically, the procedure is as follows:

[0049] [H stock / P19] (1) Agrobacterium tumefaciens LBA4404 carrying the plasmid pBE2113, which contains a nucleic acid encoding a signal peptide derived from N. benthamiana, a nucleic acid encoding the human type II collagen α chain, a nucleic acid encoding a C-terminal propeptide, and a nucleic acid encoding a FLAG peptide (SEQ ID NO: 1), and (2) Agrobacterium tumefaciens LBA4404 carrying the plasmid pBI121 into which the P19 (RNA silencing suppressor) gene (SEQ ID NO: 3) has been inserted.

[0050] [G stock / P19] (1) Agrobacterium tumefaciens LBA4404 carrying the plasmid pBE2113, which contains a nucleic acid encoding a signal peptide derived from N. benthamiana, a nucleic acid encoding an N-terminal propeptide, a nucleic acid encoding a human type II collagen α chain, a nucleic acid encoding a C-terminal propeptide, and a nucleic acid encoding a FLAG peptide (SEQ ID NO: 2), and (2) Agrobacterium tumefaciens LBA4404 carrying the plasmid pBI121 into which the P19 (RNA silencing suppressor) gene (SEQ ID NO: 3) has been inserted.

[0051] Leaves from the two recombinant N. benthamiana plants were collected and ground using a pestle and mortar. Three volumes of PBS were added to prepare crude extracts (denoted "1" in Figure 5). A surfactant (0.1% Tween 20) was added to the crude extract from G strain / P19. The crude extracts were centrifuged (20,000 × g, 10 minutes, 4°C), and the supernatants were collected (in Figure 5, the supernatant fraction is designated "2S" and the precipitate fraction is designated "2P"). Acetic acid was added to the collected supernatant 2S to a final concentration of 0.5 M and the mixture was allowed to stand for 2 hours at 4°C. After acetic acid treatment, the sample was centrifuged (12,000 × g, 20 minutes, 4°C) to separate the precipitate (denoted "3P" in Figure 5) and the supernatant (denoted "3S" in Figure 5), which were then collected. The collected supernatant was subjected to ammonium sulfate precipitation (40% ammonium sulfate, 45 minutes, 4°C) and then centrifuged (20,000 × g, 20 minutes, 4°C) to separate it into a supernatant fraction (designated "4S" in Figure 5) and a precipitate fraction (designated "4P" in Figure 5). The resulting precipitate fraction (designated "4P" in Figure 5) was resuspended in PBS. Samples from each stage were subjected to Western blotting to analyze the amount of human COL2 contained in each fraction. The amount of COL2 recovered was quantified from the signal intensity of the COL2 band in the Western blot, and the COL2 recovery rate was calculated. The recovery rate was defined as the amount of COL2 contained in "4P" when the amount of COL2 in "2S" was set to 100%. The Western blot results are shown in Figure 5.

[0052] As shown in Figure 5, exogenous human COL2 expressed in tobacco leaves was found in large amounts in the 3S and 4P fractions after acetic acid treatment, demonstrating that efficient purification is possible using acetic acid treatment. Furthermore, the recovery rate of COL2 without the addition of a surfactant (i.e., H strain / P19) was approximately 70% to 90%, whereas the recovery rate with the addition of a surfactant (i.e., G strain / P19) was over 50%. This suggests that the addition of a surfactant is not essential for protein purification using acetic acid treatment. [Industrial Applicability]

[0053] According to the present invention, useful proteins produced in plants can be efficiently purified, and therefore the present invention is extremely useful in the field of bioindustry.

Claims

1. A method for purifying a foreign protein expressed in a plant, comprising the step of adding an acid to an extract of plant tissue in which the foreign protein has been expressed, thereby decomposing contaminating plant proteins.

2. 2. The method of claim 1, wherein the extract is a crude extract.

3. 2. The method according to claim 1, wherein the extract is a supernatant obtained by centrifuging a crude extract.

4. 2. The method of claim 1, wherein the step is carried out at 1 to 25°C.

5. The method according to claim 1, wherein the plant is a Nicotiana plant.

6. 6. The method according to claim 5, wherein the Nicotiana plant is Nicotiana benthamiana.

7. The method according to claim 1, wherein the exogenous protein is a protein that is resistant to degradation by acid.

8. The method according to claim 7, wherein the protein resistant to degradation by acid is at least one selected from the group consisting of collagen, elastin, keratin, and fibroin.

9. 2. The method of claim 1, wherein the acid is at least one selected from the group consisting of acetic acid, hydrochloric acid, formic acid, propionic acid, butyric acid, lactic acid, glycolic acid, and citric acid.

10. 10. The method of claim 9, wherein the acid is acetic acid.

Images