Plant germ for cell-free protein synthesis

Abstract

Plant germ of the invention has gibberellin added thereto for cell-free protein synthesis. A plant germ extract of the invention for cell-free protein synthesis is obtained from plant germ having gibberellin added thereto. A reaction liquid of the invention for cell-free protein synthesis contains an extract from plant germ having gibberellin added thereto. A method of the invention for cell-free protein synthesis includes using the extract or reaction liquid for cell-free protein synthesis. A method of the invention for improving protein synthesis capability includes adding gibberellin to plant germ.

Description

Plant germ for cell-free protein synthesis

[0001] The present invention relates to a plant embryo for cell-free protein synthesis, an extract and reaction solution for cell-free protein synthesis obtained using the same, and a method for cell-free protein synthesis and a method for improving protein synthesis ability using the same.

[0002] The artificial synthesis of proteins has become increasingly necessary in various fields in recent years, beyond academic applications. Ideally, the artificial synthesis of proteins would require a method that can produce the desired protein inexpensively, stably, and in large quantities, and that can be easily carried out without requiring specialized equipment or complicated processes.

[0003] Methods for artificially synthesizing proteins include those that synthesize the required protein by introducing a gene of interest into cells derived from various organisms, such as mammals, insects, plants, and Escherichia coli. Other known methods include cell-free protein synthesis systems that use wheat or rabbit reticulocyte-derived materials (Patent Document 1). In these systems, genetic information is transcribed from DNA into mRNA, and ribosomes translate the mRNA information to synthesize proteins outside of a living organism, such as a test tube. While cell-free protein synthesis systems offer advantages such as accurate translation, they suffer from drawbacks such as poor synthesis efficiency and yield. Various studies have been conducted to overcome these drawbacks. For example, in cell-free protein synthesis systems using wheat germ, methods have been reported in which a cell extract-containing solution is formulated by freeze-drying (Patent Document 2), a polyhydric alcohol is added to stabilize the solution under frozen storage (Patent Document 3), animal- and microbial-derived impurities are removed (Patent Document 4), an initial substrate concentration is maintained (Patent Document 1), and a protein concentration is concentrated in an extract solution used for protein synthesis to maintain a certain level (Patent Document 5).

[0004] Japanese Patent Laid-Open No. 1-503119 Japanese Patent Laid-Open No. 2000-316594 US2003 / 0199076 A1 Japanese Patent Laid-Open No. 2007-97438 Japanese Patent Laid-Open No. 6-225783

[0005] When wheat is used as a material for cell-free protein synthesis, it is expected that the material can be obtained inexpensively and that it will be possible to synthesize target proteins in large quantities at low cost. However, the above-mentioned conventional methods for cell-free protein synthesis have not been able to improve yields as much as expected, and they have problems such as requiring special equipment and limiting the uses of the synthesized proteins when additives or other substances are used.

[0006] Therefore, the present invention relates to providing a plant embryo or the like for cell-free protein synthesis, which enables synthesis of a target protein at low cost and in high yield in a cell-free protein synthesis system without requiring complicated devices or steps.

[0007] As a result of extensive research, the present inventors have found that by adding gibberellin, a plant hormone, to the germ of a plant such as wheat, the protein synthesis capacity in a cell-free protein synthesis system can be improved without the need for complicated equipment or processes, and have completed the present invention based on this finding.

[0008] The present invention provides a plant embryo for cell-free protein synthesis containing added gibberellin. The present invention also provides an extract for cell-free protein synthesis extracted from the plant embryo. The present invention also provides a reaction solution for cell-free protein synthesis containing the extract for cell-free protein synthesis. The present invention also provides a method for cell-free protein synthesis using the extract for cell-free protein synthesis or the reaction solution for cell-free protein synthesis.

[0009] The present invention also provides a method for improving protein synthesis ability, which comprises adding gibberellin to plant embryos, allowing the embryos to stand at low temperatures, and then extracting the embryos, thereby enhancing the protein synthesis ability of the embryo extract obtained by the extraction in cell-free protein synthesis.

[0010] The present invention also provides a method for improving protein synthesis ability in cell-free protein synthesis, comprising the following steps (1) to (5): (1) adding a plant hormone to a plant embryo; (2) allowing the embryo to stand under low-temperature conditions; (3) crushing and homogenizing the embryo after allowing the embryo to stand; (4) adding a solution for extracting embryo components to dissolve the embryo; and (5) purifying the dissolved embryo.

[0011] FIG. 1 is a graph showing the evaluation results of the examples and comparative examples.

[0012] According to the present invention (plant embryo for cell-free protein synthesis, extract for cell-free protein synthesis, reaction solution for cell-free protein synthesis, cell-free protein synthesis method, or method for improving protein synthesis ability), it becomes possible to synthesize a target protein inexpensively and in high yield in a cell-free protein synthesis system without the need for complicated equipment or steps. According to the present invention, it becomes possible to inexpensively and easily supply a variety of proteins for a wide range of uses, such as not only protein synthesis for research purposes but also as medical drugs, diagnostic medicines, research reagents, and alternative proteins for animal feed or human food, thereby promoting industrial use.

[0013] The present invention is described below based on preferred embodiments. The present invention relates to a cell-free protein synthesis system using plant germ. In the present invention, the term "cell-free protein synthesis system" refers to both a cell-free translation system that synthesizes proteins or polypeptides by reading information from mRNA, and a system that includes both a cell-free transcription system and a cell-free translation system that synthesizes RNA using DNA as a template. The plant germ is derived from a plant, and can be from various plants such as wheat, barley, rice, corn, and spinach. From the viewpoints of improving protein synthesis ability and / or ease of availability, germs derived from the seeds of grasses are preferred, and wheat germ is particularly preferred. In the present invention, proteins produced by cell-free protein synthesis methods generally include polypeptides and proteins with molecular weights that can be synthesized by cell-free protein synthesis. For example, polypeptides or proteins with molecular weights of 10 to 200 kD may be synthesized. Furthermore, although the following description may use wheat germ as an example, the method is equally applicable to the germs of plants other than wheat germ.

[0014] Generally known methods can be used to prepare plant embryos from plant seeds that contain ribosomes and can be used for cell-free protein synthesis. Examples of methods for preparing plant embryos include a step of preparing a plant embryo to which gibberellin is to be added by crushing the plant seeds, sieving, heavy medium sorting, removing the seed coat and endosperm fraction by visual inspection, etc., and removing the endosperm components by washing, and a method of crushing, extracting, and purifying the prepared plant embryo. In the present invention, it is preferable to add gibberellin to the plant embryo after the preparation step.

[0015] The plant embryos to which gibberellin is added are preferably those obtained through, for example, (A1) the plant seed crushing step and (A2) the embryo sorting step, and more preferably those obtained through the following (A3) moisture content adjustment step.

[0016] (A1) Plant Seed Crushing Process Because plant seeds contain a small amount of germ, in order to efficiently obtain the germ, it is preferable to remove as many parts of the plant seeds as possible other than the germ before crushing them. In the crushing process, typically, a mechanical force is first applied to the plant seeds to obtain a crushed plant seed product containing the germ, crushed endosperm, and crushed seed coat. This crushing process can be carried out using a known crushing device. As the crushing device, a crushing device that applies an impact force to the material to be crushed, such as a pin mill or hammer mill, may be used. The degree of crushing may be appropriately selected depending on the size of the germ of the plant seed used. For example, in the case of wheat seeds, the seeds are crushed to a maximum length of preferably 4 mm or less, more preferably 2 mm or less. Crushing may be carried out in a dry or wet manner.

[0017] (A2) Germ Sorting Step In the germ sorting step, it is preferable to sort a crude germ fraction. After sorting the crude germ fraction, it is preferable to further perform a purification treatment to increase the proportion of germ-derived components. In sorting the crude germ fraction, crushed endosperm, crushed seed coat, etc. are removed from the crushed plant seed product obtained in the crushing step to obtain a crude germ fraction. The crude germ fraction is mainly composed of germ and contains crushed endosperm and crushed seed coat. The crude germ fraction is obtained from the crushed plant seed product using a known classification device, such as a sieve. For example, in the case of wheat seeds, a crude germ fraction with a mesh size of 0.5 mm to 2.0 mm, preferably 0.7 mm to 1.4 mm, can usually be obtained. Furthermore, if necessary, seed coats, endosperm, debris, etc. contained in the obtained crude germ fraction may be removed using wind power or electrostatic force. Thereafter, or instead of the aforementioned classification using a sieve or the like, a crude germ fraction can also be obtained by a method utilizing the difference in specific gravity between the germ, the seed coat, and the endosperm, for example, by heavy medium sorting, in which the crushed plant germ is placed in a liquid, and the seed coat and endosperm fractions are removed by utilizing the difference in specific gravity to obtain a crude germ fraction. The liquid used in this case is usually a mixed solution of carbon tetrachloride and cyclohexane, but organic chlorine solvents such as carbon tetrachloride are particularly preferred because they can be selected depending on the intended use of the synthetic protein, taking into consideration environmental and health aspects. To obtain a crude germ fraction containing a larger amount of germ, multiple methods described above may be combined.

[0018] The germ fraction may be obtained by sorting the germ from the crude germ fraction obtained in this manner, for example, visually or using a color sorter. Furthermore, since endosperm components may adhere to the germ obtained in this manner, a further washing treatment is preferably performed to purify the germ. For example, the washing treatment is preferably performed by dispersing or suspending the germ fraction in water or an aqueous solution cooled to typically 10°C or below, preferably 4°C or below, and washing until the washing solution becomes clear. Furthermore, it is more preferable to disperse or suspend the germ fraction in an aqueous solution containing a surfactant, typically at 10°C or below, preferably 4°C or below, and washing until the washing solution becomes clear. Nonionic surfactants are preferred. A wide range of nonionic surfactants are available. Specific examples of suitable surfactants include polyoxyethylene derivatives such as Brij, Triton, Nonidet (registered trademark) P40, and Tween. Among these, Nonidet (registered trademark) P40 is the most suitable. These nonionic surfactants can be used at a concentration of, for example, about 0.5%. Either or both of the cleaning treatment with water or an aqueous solution and the cleaning treatment with a surfactant may be performed. These cleaning treatments may also be performed in combination with ultrasonic treatment.

[0019] (A3) Water Content Adjustment Step In the present invention, the water content of the germ fraction may be adjusted to improve the protein yield when the germ obtained using the above-mentioned method is used for cell-free protein synthesis. The water content (% by mass) of the germ after adjustment is preferably 30% or less, more preferably 28.6% or less, and even more preferably 24.8% or less. There is no particular lower limit for the water content (% by mass), but it is, for example, 1.6% or more and preferably 18.8% or less. The water content of the germ can be adjusted, for example, as follows. As an example of adjusting the water content, 1.6 to 8.0 mL of water is added to 20 g of wheat germ, and the water content of the germ is measured after refrigeration at 4°C for one day.

[0020] The method for adjusting the moisture content of plant embryos is not particularly limited as long as it achieves the desired final moisture content (e.g., 30% or less), and both the amount of water added and the hydration process (conditions) may be appropriately selected. Hydration methods may be appropriately selected, such as submerging the resulting plant embryos in water or spraying with a sprayer. After hydration, a refrigeration step of approximately one day may be performed to homogenize the moisture content. A freeze-drying step may also be performed as needed. In a preferred embodiment of the present invention, since too much or too little moisture affects protein synthesis, it is preferable to adjust the moisture content by adjusting the amount of water added, and also to appropriately adjust the moisture content by freeze-drying. When freeze-drying is used, the moisture content after freeze-drying may be used as the moisture content of the embryo, or a predetermined amount of water may be added to the freeze-dried embryo to achieve the desired moisture content. The moisture content of the embryo after moisture adjustment is calculated using the following formula: Moisture content of wheat germ after adjustment (mass %) = [(mass after moisture adjustment - dry mass before moisture adjustment) / mass after moisture adjustment] x 100 The dry mass was calculated by weighing 2 g of wheat germ into a heat-resistant container, heating it at 135°C for 1 hour, and then weighing it again, and subtracting the lost mass from the mass of the wheat germ before heating. The weight was measured to the nearest 0.001 g.

[0021] When the moisture content is adjusted by adding water, the water added to the germ may be, but is not limited to, pure water, ion-exchanged water, etc. To adjust the moisture content, a solution containing a plant hormone (such as gibberellin), which will be described later, may be used alone or in combination with other water as described above. The amount of plant germ used can be adjusted depending on the dry matter amount before adding water, and then supplied to the cell-free protein synthesis system.

[0022] (Gibberellin Treatment) In the present invention, the plant hormone gibberellin is used to improve the cell-free protein synthesis capacity using plant embryos. Gibberellins are known as compounds endogenous to plants or produced by microorganisms. They are broadly divided into two types: 20-carbon gibberellins (e.g., A12, A13, A14, etc.) and 19-carbon gibberellins (e.g., A1, A3, A4, A7, A9, etc.). Any gibberellin or gibberellin analogue may be used, and those skilled in the art can appropriately select and implement the treatment. In other words, the gibberellin used in the present invention also includes gibberellin analogues. The amount of gibberellin added to plant embryos can be, for example, 0.5 to 500 ppm (μg / mL: mass equivalent) based on the dry weight of the embryo. Gibberellin is preferably added as a gibberellin solution. For example, gibberellin A3 can be adjusted to 100 μg / mL with pure water to prepare a gibberellin solution, and about 80 μL of the gibberellin solution is added per 1 g of dry mass of wheat germ.

[0023] The adjustment of the moisture content of wheat germ and the gibberellin treatment may be carried out separately or simultaneously. One example of a method for carrying out these treatments separately and simultaneously is, for example, adding 400 μL of a gibberellin solution (e.g., 100 μg / mL) with an adjusted concentration to 5 g of germ, as described below. Furthermore, when adjusting the moisture content of wheat germ and adding gibberellin are carried out together, for example, if 800 μL of water is to be added, this can be carried out by adding a gibberellin solution (e.g., 50 μg / mL) with an adjusted concentration to 5 g of germ. Those skilled in the art can make appropriate adjustments depending on the concentration of the gibberellin solution and the amount of water to be added.

[0024] (Infiltration treatment in a low-temperature environment) After the addition of gibberellin, the plant embryo may be subjected to infiltration treatment by leaving it in a low-temperature environment for a predetermined period of time. The leaving is preferably static. This infiltration treatment allows gibberellin to penetrate into the plant embryo, thereby improving the cell-free protein synthesis ability. This infiltration treatment is performed, for example, by adding a gibberellin solution and then leaving it statically at 4°C for 24 hours.

[0025] The plant embryos to which gibberellin has been added in this manner are a preferred embodiment of the plant embryos for cell-free protein synthesis according to the present invention, and by using them in protein synthesis by a cell-free protein synthesis system, the protein synthesis ability can be improved compared to when gibberellin is not added. It is also preferable that the plant embryos after gibberellin treatment are subjected to protein synthesis by a cell-free protein method as an extract for cell-free protein synthesis extracted from the plant embryos. For example, an extract can be prepared from the plant embryos after gibberellin addition in the same manner as when gibberellin treatment is not performed, and used for cell-free protein synthesis. Whether or not a plant embryo has had gibberellin added can be determined by LC-MS or the like of the embryo extract.

[0026] An embryo extract is obtained from the plant embryos treated with gibberellin obtained as described above. The method for obtaining the embryo extract can be a conventionally known method. For example, embryos frozen in liquid nitrogen are finely pulverized, an extraction solvent is added and stirred, and then the embryo extract-containing liquid is recovered by centrifugation or the like. Thereafter, the extract may be purified by gel filtration or the like, as necessary. The plant embryos before or after the gibberellin treatment may be subjected to a finely pulverized step, and the finely pulverized embryos may be used.

[0027] A preferred example of a method for obtaining an extract for cell-free protein synthesis or a reaction solution for cell-free protein synthesis of the present invention from plant embryos treated with gibberellin will be described below. The method for obtaining an extract from plant embryos preferably includes (B1) a grinding step and (B2) an extraction and purification step. (B1) Grinding Step: The grinding step is preferably a grinding and homogenization step in which homogenization of the ground material (crushed material) is achieved by stirring during grinding. In the grinding and homogenization step, the plant embryos used are ground using a grinding device such as a mill. Preferably, the plant embryos after addition of the gibberellin solution are frozen using liquid nitrogen or the like, and the frozen embryos are ground using a grinding device. Homogenization can also be achieved by a grinding method involving stirring. Homogenization by stirring during grinding or by separate stirring after grinding is preferred from the perspective of reliably improving protein synthesis ability in the cell-free protein synthesis method. Grinding the embryos in a frozen state is also preferred from the perspective of suppressing gibberellin degradation. When storing plant embryos after gibberellin treatment until extraction, the storage temperature is preferably 25°C or below, more preferably 10°C or below, to prevent gibberellin decomposition. It is also preferable to store plant embryos in a frozen state in a centrifuge tube, as described below. (B2) Extraction and Purification Steps In the extraction step, for example, an appropriate amount of ground embryos is weighed and placed in a container such as a centrifuge tube or flask, and a germ component extraction solution is added and shaken. After shaking, the mixture is centrifuged either directly in the centrifuge tube or transferred to a centrifuge tube, and the supernatant is recovered. The recovered supernatant is preferably sterilized and / or gel filtration is performed to obtain the desired germ extract. An extraction buffer can be used as the germ component extraction solution. Examples of extraction buffers include extraction buffers 1 and 2 described in the Examples below. The germ component extraction solution preferably contains at least potassium acetate, magnesium acetate, and a reducing agent (e.g., dithiothreitol), and preferably further contains calcium chloride or the like. The sterilization treatment is preferably a treatment that can be carried out at low temperatures in order to prevent denaturation of the components contained in the extracted wheat germ extract. The purpose of gel filtration is to purify the plant germ dissolved in the germ component extraction solution.

[0028] The embryo extract thus obtained from plant embryos usually contains at least ribosomes and translation factors. Examples of translation factors include initiation factors, elongation factors, and translation termination factors. An extract obtained from plant embryos to which gibberellin has been added, or a liquid or solid obtained by subjecting the embryo extract to lyophilization, spray drying, concentration, or the like, is a preferred embodiment of the extract for cell-free protein synthesis of the present invention, and is preferably used in a cell-free protein synthesis system. Synthesis of a target protein in a cell-free protein synthesis system using the extract for cell-free protein synthesis of the present invention can be carried out by employing known methods, except for using the obtained embryo extract. A commercially available synthesis kit may be used for the cell-free protein synthesis reaction. Known methods can be used for the cell-free protein synthesis reaction. A commercially available synthesis reaction kit may be used.

[0029] In the case of a cell-free translation system that synthesizes proteins or polypeptides by reading information from mRNA, for example, by adding mRNA containing information for a target protein to an extract obtained from plant embryos to which gibberellin has been added and containing ribosomes, translation factors, etc., and allowing the reaction to proceed, the target protein can be efficiently produced and the yield of the target protein can also be improved. Furthermore, when the cell-free protein synthesis system includes a cell-free transcription system that synthesizes RNA using DNA as a template and a cell-free translation system, for example, a synthesis reaction kit preferably contains various components of the cell-free transcription system, such as RNA polymerase, deoxynucleotide triphosphates, an amino acid solution, creatine kinase, etc., as well as various proteins involved in transcription, such as DNA polymerase and magnesium sulfate. By adding an extract obtained from plant embryos to which gibberellin has been added and containing ribosomes, translation factors, etc., and DNA containing information for a target protein to such a synthesis reaction kit and allowing the reaction to proceed, the target protein can be efficiently produced and the yield of the target protein can also be improved.

[0030] The efficiency of protein synthesis by a cell-free protein synthesis method using the extract according to the present invention can be determined, for example, as follows. By carrying out such a determination step, the yield of the protein can be estimated before synthesizing the target protein. The "extract according to the present invention" referred to here is preferably an embryo extract obtained by the above-mentioned method from plant embryos to which gibberellin has been added, but there are no particular limitations on the extraction method, properties, etc., as long as it is obtained from plant embryos to which gibberellin has been added.

[0031] Using a cell-free protein synthesis kit, an mRNA sequence is synthesized from the DNA sequence of a protein similar in properties to the target protein. The protein used to assess synthesis efficiency can be, for example, a fluorescent protein such as GFP, but is not limited to this; any protein can be used to evaluate synthesis efficiency. Fluorescent proteins such as GFP are preferred because they are irradiated with specific excitation light, allowing for easy determination of the protein amount from the fluorescence intensity. Synthesis efficiency can be assessed, for example, by synthesizing protein using the synthesized mRNA as a template and measuring the yield over a predetermined period of time. For example, after a certain time has elapsed since the start of translation, the translation reaction solution placed in a plate reader is irradiated with 485 nm excitation light, and the fluorescence intensity is measured.

[0032] The cell-free protein synthesis method of the present invention broadly includes, for example, methods for synthesizing proteins in a cell-free protein synthesis system using the above-mentioned plant embryo for cell-free protein synthesis or extract for cell-free protein synthesis, or a cell-free protein synthesis reaction solution containing the extract for cell-free protein synthesis, and is not particularly limited in the type of protein to be synthesized, the type of template DNA used, the production method, etc. Specific examples of the cell-free protein synthesis reaction solution of the present invention include a mixed solution containing an embryo extract obtained by extraction from a plant embryo to which gibberellin has been added using the above-mentioned method, which normally contains ribosomes, translation factors, etc., and other components necessary for cell-free protein synthesis, such as an amino acid solution, creatine kinase, and an mRNA solution obtained by transcription of the above-mentioned template DNA.

[0033] The method for improving protein synthesis ability (Method 1) of the present invention is a method for improving protein synthesis ability by adding gibberellin to plant embryos, allowing them to stand at low temperature, and then performing extraction, thereby improving the protein synthesis ability of the embryo extract obtained by extraction in cell-free protein synthesis. In a preferred embodiment, the method for adding gibberellin to plant embryos is the same as the above-mentioned gibberellin treatment, and the method for allowing them to stand at low temperature and then performing extraction is the same as the above-mentioned method for permeation treatment in a low-temperature environment. According to the method for improving protein synthesis ability (Method 1) of the present invention, protein synthesis ability in a cell-free protein synthesis system can be easily improved without the need for complicated equipment or steps.

[0034] The method for improving protein synthesis ability (second method) of the present invention is a method for increasing protein synthesis ability in cell-free protein synthesis, and includes the following steps (1) to (5): (1) adding gibberellin to plant embryos, (2) allowing the embryos to stand under low-temperature conditions, (3) crushing and homogenizing the plant embryos after allowing the embryos to stand, (4) adding a solution for extracting embryo components to dissolve the plant embryos, and (5) purifying the dissolved plant embryos.

[0035] In a preferred embodiment, step (1) is the gibberellin treatment described above, step (2) is the permeation treatment in a low-temperature environment described above, step (3) is the (B1) crushing step described above, and steps (4) and (5) are the (B2) extraction and purification steps described above, with the respective descriptions of each step being applied as appropriate. In step (2), the incubation time under low-temperature conditions is preferably, for example, 1 hour or more, more preferably 6 hours or more, with no particular upper limit, and may be, for example, 3 months. In step (4), lysis of the plant germ refers to the elution of ribosomes, translation factors, and the like, required for translating mRNA into protein, into a solution for extracting germ components. In step (5), purification of the lysed plant germ refers to the purification and recovery of the components eluted in step (3). According to the method for improving protein synthesis ability (method 2) of the present invention, protein synthesis ability in a cell-free protein synthesis system can be easily improved without the need for complicated equipment or processes.

[0036] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. [Comparative Example 1] A wheat germ extract was prepared from wheat germ obtained from Nisshin Flour Milling Co., Ltd. The wheat germ extract was prepared by partially modifying a previously published method (Methods in Enzymology (1983), Vol. 96, pp. 38-50) and following the steps (1) to (8) below. (1) Wheat germ was pulverized and homogenized using a mill (Silent Mill: manufactured by Iwatani Corporation). (2) Based on the moisture value measured by the method described below, wheat germ was weighed so that the dry weight was 3 g and placed in a 50 mL centrifuge tube. 10 mL of "Extraction Buffer 1" prepared with the composition shown in Table 1 below was added. [Method for measuring moisture content] 2 g of wheat germ was weighed and placed in a heat-resistant container, heated at 135°C for 1 hour, and then reweighed. The mass loss was calculated as a mass ratio to the mass of the wheat germ before heating, and this was used as the moisture content. The weight was measured to the nearest 0.001 g. (3) The sample was shaken for 5 minutes using a shaker (Utility Shaker US-S100: AS ONE Corporation) (set speed: 200 rpm). (4) The sample was centrifuged using a centrifuge (Avanti JXN-30: Beckman Coulter) (centrifugation conditions: 23,000 G, 10 minutes). (5) After centrifugation, the inner aqueous layer of the supernatant was collected and sterilized using a 0.22 μm filter. (6) After removing the storage solution from the PD-10 column (manufactured by Cytiva), "Extraction Buffer 1" shown in Table 1 below was added to the column and the gel was homogenized. (7) 2.5 mL of the solution recovered in (5) above after removing impurities was added to the PD-10 column, and the solution that passed through the gel was discarded. (8) 3.5 mL of "Extraction Buffer 2" prepared with the composition shown in Table 2 below was added to the column, and the recovered solution was used in the test as wheat germ extract.

[0037]

[0038]

[0039] Example 1: Preparation of Wheat Germ for Cell-Free Protein Synthesis Gibberellin A3 (Tokyo Chemical Industry Co., Ltd.) was adjusted to 100 μg / mL with purified water to prepare a gibberellin solution. 400 μL of the prepared gibberellin solution was added to 5 g of wheat germ similar to that used in Comparative Example 1, and the mixture was allowed to stand at 4°C for 24 hours. Example 2: Preparation of Extract for Cell-Free Protein Synthesis Wheat germ extract was prepared from gibberellin-treated wheat germ using the same method (1) to (8) as in Comparative Example 1. Example 3: Cell-Free Protein Synthesis Method Using a cell-free protein synthesis kit (NUProtein Co., Ltd.), mRNA was synthesized from plasmid DNA containing the GFP-derived base sequence listed in Table 3 below, following the kit protocol. GFP protein translation was performed using the synthesized mRNA, the kit's Amino Acid Mix, and the wheat germ extract prepared in Comparative Example 1 or Example 2, following the kit protocol. Sixteen hours after the start of translation, the translation reaction solution was irradiated with 485 nm excitation light using a plate reader (ARVO-X4: manufactured by PerkinElmer) and the GFP fluorescence intensity was measured (measurement wavelength: 535 nm). The results are shown in Figure 1 below.

[0040]

[0041] 1 , the wheat germ extract obtained from gibberellin-treated plant embryos (Example 2) had significantly improved GFP fluorescence intensity compared to the wheat germ extract obtained from non-gibberellin-treated plant embryos (Comparative Example 1). This demonstrates that the use of gibberellin-treated plant embryos can improve the protein synthesis ability and protein yield in a cell-free protein synthesis system without requiring complex equipment and processes.

[0042] According to the present invention, materials necessary for cell-free protein synthesis using plant-derived embryos can be efficiently extracted, and an embryo extract with few impurities can be obtained, thereby providing a stable and highly efficient embryo extract for cell-free protein synthesis and a solution for cell-free protein synthesis.The use of the embryo extract for protein synthesis or the solution for protein synthesis of the present invention enables stable and highly efficient cell-free protein synthesis, which can contribute to the mass production of proteins not only for research purposes but also in various fields such as pharmaceuticals, foods, and industrial products.

Claims

1. Gibberellin-supplemented plant germ for cell-free protein synthesis.

2. An extract for cell-free protein synthesis extracted from the plant embryo according to claim 1.

3. A reaction solution for cell-free protein synthesis, comprising the extract for cell-free protein synthesis according to claim 2.

4. A method for cell-free protein synthesis, which uses the extract for cell-free protein synthesis according to claim 2 or the reaction solution for cell-free protein synthesis according to claim 3.

5. A method for improving protein synthesis ability, which comprises adding gibberellin to plant embryos, allowing them to stand at low temperatures, and then extracting them, thereby increasing the protein synthesis ability of the embryo extract obtained by extraction in cell-free protein synthesis.

6. A method for improving protein synthesis ability in cell-free protein synthesis, comprising the following steps (1) to (5): (1) adding gibberellin to plant embryos, (2) allowing the embryos to stand under low-temperature conditions, (3) crushing and homogenizing the embryos after allowing the embryos to stand, (4) adding a solution for extracting embryo components to dissolve the embryos, and (5) purifying the dissolved embryos.