JP3956314B2 - Embryo extract for cell-free protein synthesis and method for producing the same - Google Patents

Description

  The present invention relates to an embryo extract for cell-free protein synthesis and a method for producing the same. More specifically, the present invention relates to a cell-free protein synthesis embryo extract having high synthesis efficiency, a method for producing the same efficiently industrially, and the like.

  The protein synthesis reaction that takes place in the cell proceeds by a process in which the information is first transcribed from mRNA with genetic information into mRNA, and the ribosome translates the mRNA information to synthesize the protein. Yes. Currently, as a method of performing protein synthesis in a cell in vitro, such as in a test tube, for example, research on cell-free protein synthesis, in which ribosomes are extracted from organisms and used to conduct a protein synthesis reaction in a test tube. It is actively carried out (Japanese Patent Laid-Open No. 6-98790, Japanese Patent Laid-Open No. 6-225783, Japanese Patent Laid-Open No. 7-194, Japanese Patent Laid-Open No. 9-291 and Japanese Patent Laid-Open No. 7-147992). In this method, Escherichia coli, plant embryos, rabbit reticulocytes and the like have been used as raw materials for ribosomes.

As a method for obtaining an embryo extract for cell-free protein synthesis containing ribosomes from plant embryos, usually, after seeding plant seeds, seed coats and endosperm fractions are removed to obtain crude embryo fractions, and further, endosperm is washed. After removing the components, a method of pulverizing, extracting and purifying is performed.
As a conventional method for finely pulverizing germs, it was considered necessary to pulverize germs as finely as possible in order to improve the efficiency of extraction of water-soluble components including protein synthesis components such as ribosomes and tRNAs. However, since the germ is small and strong, it cannot be easily pulverized. Accordingly, conventionally, a method of finely pulverizing the washed embryo with liquid nitrogen or the like and then grinding or crushing with a mortar, stamp mill, ball mill or the like has been widely used. Extracted solvent is added to the resulting germ and stirred, and then the germ extract-containing solution (embryo extract) is collected and purified by centrifugation, etc., and used as an “enzyme stock solution” for cell-free protein synthesis reaction It was. This “enzyme stock solution” contains a lot of unnecessary components not involved in the protein synthesis reaction, and there may be a protein synthesis inhibitory action.
Moreover, since the extract obtained by the above conventional method does not contain the amount of tRNA required for the protein synthesis reaction, it was essential to add a separately prepared tRNA. Furthermore, it is difficult to obtain a large amount of the extract in a short time by the conventional method.

In order to solve the above-mentioned problems, the present inventors have increased the protein synthesis efficiency of an embryo extract used in a cell-free protein synthesis system and produced the extract efficiently to such an extent that it can be industrially implemented. As a result of studying a method for efficiently extracting a factor necessary for protein synthesis from plant germ, the present invention has been achieved.
That is, the present invention is as follows.
“(1) Plant germ extract with reduced ribonuclease activity.
(2) The embryo extract according to (1) above, wherein the ribonuclease activity is 10 pg / μl or less in terms of RNase A.
(3) The ribonuclease activity is 80% or less compared to a germ extract obtained by a method (hereinafter simply referred to as a conventional method) comprising a step of grinding or crushing plant germs in the absence of an extraction solvent and subdividing them The embryo extract according to (1) or (2), wherein the germ extract is reduced to a low level.
(4) A plant embryo extract characterized by having a DNA content of 230 μg / ml or less when the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm is 90.
(5) Plant germ having a total content of total fatty acids (palmitic acid, oleic acid, and linoleic acid) when the optical density (OD) (A ₂₆₀ ) at 90 nm is 90 is 0.03 g / 100 g or less Extract liquid.
(6) Plant germ extract having a DNA content of 230 μg / milliliter or less and a total fatty acid content of 0.03 g / 100 g or less when the optical density (OD) at 260 nm (A ₂₆₀ ) is 90 liquid.
(7) The germ extract according to any one of (1) to (6) above, wherein the plant germ is a wheat, barley, rice or corn germ.
(8) A method for producing a plant embryo extract, comprising a step of subdividing plant germ by impact or cutting.
(9) A method for producing a plant embryo extract, comprising a step of subdividing plant germ in the presence of an extraction solvent.
(10) The production method according to the above (9), wherein the extraction solvent contains one or more selected from the group consisting of a buffer solution, potassium ions, magnesium ions and a thiol group antioxidant.
(11) The production method according to (9) above, wherein the subdivision is performed by impact or cutting.
(12) The production method according to any one of (8) to (11), wherein the plant germ is one in which an endosperm component is not substantially contaminated.
(13) The production method according to any one of (8) to (12), wherein the plant germ is wheat, barley, rice or corn germ.
(14) A plant germ extract obtained by the production method according to any one of (8) to (13) above.
(15) A cell-free protein synthesis method characterized by using the germ extract according to any one of (1) to (7) and (14) above.
(16) The embryo extract according to any one of (1) to (7) and (14) above, containing ATP, GTP, creatine phosphate, creatine kinase, L-type amino acid, potassium ion and magnesium ion A solution for cell-free protein synthesis characterized by the above.
(17) A solution for cell-free protein synthesis containing the embryo extract according to any one of (1) to (7) and (14) above, and sufficient protein without adding tRNA A cell-free protein synthesis solution characterized by having a synthetic activity.
(18) A kit for performing cell-free protein synthesis, comprising at least the embryo extract according to any one of (1) to (7) and (14) above.
(19) A kit for performing cell-free protein synthesis, comprising at least the cell-free protein synthesis solution according to (16) or (17). "
More details
“(A) A plant germ extract characterized by having a DNA content of 230 μg / milliliter or less when the optical density (OD) (A ₂₆₀ ) at 90 nm is 90”.
(B) Plant germs having a total content of total fatty acids (palmitic acid, oleic acid and linoleic acid) when the optical density (OD) (A ₂₆₀ ) at 90 nm is 90 is 0.03 g / 100 g or less Extract liquid.
(C) Plant germ extract having a DNA content of 230 μg / milliliter or less and a total fatty acid content of 0.03 g / 100 g or less when the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm is 90 liquid.
(D) The plant germ extract according to any one of (a) to (c) above, wherein the plant germ is wheat, barley, rice or corn germ.
(E) The method for producing a plant embryo extract according to any one of the above (a) to (d), comprising a step of fragmenting plant embryos by impact or cutting.
(F) The manufacturing method of the plant embryo extract of any one of said (a)-(d) including the process of subdividing a plant embryo in presence of an extraction solvent.
(G) The production method of the above (f), wherein the extraction solvent contains one or more selected from the group consisting of a buffer solution, potassium ions, magnesium ions and a thiol group antioxidant.
(H) The production method of (f) above, wherein the subdivision is performed by impact or cutting.
(I) The production method according to any one of (e) to (h), wherein the plant germ is one in which an endosperm component is not substantially contaminated.
(J) The production method according to any one of (e) to (i) above, wherein the plant germ is wheat, barley, rice, or corn germ.
(K) A plant germ extract obtained by the production method according to any one of (e) to (j) above.
(L) A cell-free protein synthesis method comprising using the plant germ extract according to any one of (a) to (d) and (k) above.
(M) The plant germ extract according to any one of the above (a) to (d) and (k), ATP, GTP, creatine phosphate, creatine kinase, L-type amino acid, potassium ion and magnesium ion A solution for cell-free protein synthesis characterized by the above.
(N) A kit for carrying out cell-free protein synthesis, comprising the plant germ extract according to any one of (a) to (d) and (k) above.
(O) A kit for carrying out cell-free protein synthesis comprising the solution for cell-free protein synthesis of (m) above. Is.

Hereinafter, the present invention will be described in more detail.
The plant germ used in the present invention can be obtained from plant seeds. Examples of plant seeds that can be used include seeds of plants selected from gramineous plants such as wheat, barley, rice and corn. Among these, examples of the plant seed suitable for the present invention include the above-mentioned wheat, barley, rice or corn, and particularly preferred is wheat.

  Since the amount of germs contained in plant seeds is small, it is preferable to remove portions other than germs as much as possible in order to obtain germs efficiently. Usually, first, a mechanical force is applied to plant seeds to obtain a mixture containing embryos, endosperm crushed material, and seed coat crushed material, and the endosperm crushed material, seed coat crushed material, etc. are removed from the mixture to obtain a crude embryo fraction. (A mixture containing germ as a main component and containing an endosperm crushed material and a seed coat crushed material). The force applied to the plant seeds only needs to be strong enough to separate the germ from the plant seeds.

Usually, a mixture containing embryos, endosperm crushed material, and seed coat crushed material is obtained by pulverizing plant seeds using a known pulverizing apparatus.
Plant seeds can be pulverized using a generally known pulverizer, but it is preferable to use a pulverizer of a type that applies impact force to an object to be pulverized, such as a pin mill or a hammer mill. The degree of pulverization may be appropriately selected according to the size of the plant seed germ to be used. For example, in the case of wheat seed, the maximum length is usually 4 mm or less, and preferably the maximum length is 2 mm or less. To do. The pulverization is preferably performed by a dry method.

  Next, a crude germ fraction is obtained from the pulverized plant seeds obtained using a classifier known per se, for example, a sieve. For example, in the case of wheat seeds, a crude germ fraction having a mesh size of 0.5 mm to 2.0 mm, preferably 0.7 mm to 1.4 mm is usually obtained. Furthermore, if necessary, seed coat, endosperm, dust, etc. contained in the obtained crude germ fraction may be removed using wind power or electrostatic force.

In addition, a crude embryo fraction can be obtained by a method using the difference in specific gravity between embryo, seed coat, and endosperm, for example, heavy liquid sorting. In order to obtain a crude germ fraction containing more germs, a plurality of the above methods may be combined. Furthermore, the germ is sorted from the obtained crude germ fraction using, for example, visual observation or a color sorter.
Since the embryo fraction obtained in this manner may have an endosperm component attached thereto, it is usually preferable to further perform a washing treatment for germ purification.

  As the washing treatment, it is preferable to disperse and suspend the embryo fraction in water or an aqueous solution usually cooled to 10 ° C. or less, preferably 4 ° C. or less, and wash until the washing solution does not become cloudy. Moreover, it is more preferable to disperse and suspend the embryo fraction in an aqueous solution containing a surfactant, usually at 10 ° C. or lower, preferably at 4 ° C. or lower, and wash until the washing solution does not become cloudy. The surfactant is preferably a nonionic surfactant, and can be widely used as long as it is a nonionic surfactant. Specifically, for example, bridges (Brij), Triton, Nonidet P40, Tween, etc., which are polyoxyethylene derivatives, are exemplified as preferable examples. Of these, Nonidet P40 is most suitable. These nonionic surfactants can be used, for example, at a concentration of 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. Further, these cleaning processes may be performed in combination with ultrasonic processing.
After selecting the plant germ from the pulverized product obtained by pulverizing the plant seed as described above, the intact (having germination ability) germ obtained by washing is subdivided in the presence or absence of the extraction solvent. In the latter case, a germ extract is obtained by further extracting the subdivided plant germ with an extraction solvent, and a germ extract-containing solution (hereinafter simply referred to as germ extract) is separated and purified.

  In the present invention, the fragmentation can be performed using a conventionally known method as a method for pulverizing the embryo such as grinding or crushing, but it is more preferable to divide the embryo by impact or cutting. In particular, when the embryo is subdivided in the absence of the extraction solvent, it is desirable to use impact or cutting. Here, “subdivide by impact or cutting” means the destruction of cell organelles such as cell nuclei, mitochondria, chloroplasts, cell membranes and cell walls of plant embryos compared to conventional grinding or crushing. It means to destroy the plant germ under conditions that can be minimized.

  An apparatus or method that can be used when subdividing the embryo by impact or cutting is not particularly limited as long as the above conditions are satisfied. For example, an apparatus having a blade that rotates at high speed, such as a Waring blender. Is preferably used. The rotation speed of the blade is usually 1000 rpm or more, preferably 5000 rpm or more, and usually 30000 rpm or less, preferably 25000 rpm or less. The rotation time of the blade is usually 5 seconds or longer, preferably 10 seconds or longer. The upper limit of the rotation time is not particularly limited, but is usually 10 minutes or less, preferably 5 minutes or less. The temperature at the time of subdivision is not particularly limited as long as the protein synthesis ability of the embryo is not lost, but it is preferably within a temperature range where subdivision operations can be performed at 10 ° C. or less, particularly preferably about 4 ° C. It is.

On the other hand, when the germ is subdivided by grinding or crushing, it should be performed by grinding or crushing the germ in the presence of an extraction solvent using a known device such as a mortar and pestle, stamp mill, ball mill or the like. Can do. In this case, the same solvent as described above is preferable.
Particularly suitable subdivision methods include subdivision by impact or cutting as described above. However, by using such a method, cell organelles such as embryonic cell nuclei, cell membranes and cell walls are all destroyed. Rather, at least a part of it can be left without being destroyed. In other words, since organelles such as embryonic cell nuclei, cell membranes and cell walls are not destroyed more than necessary, they contain less impurities such as DNA and lipids contained in them, and are necessary for the synthesis of proteins localized in the cytoplasm. RNA, ribosome, etc. can be extracted from embryos with high purity and efficiency.

Germination of the embryo can be performed in the presence or absence of an extraction solvent.
When embryo subdivision is performed in the presence of an extraction solvent, the procedure is specifically as follows.
By substituting an extraction solvent when the embryo is subdivided (preferably by impact or cutting), the step of subdividing the plant germ and the step of extracting the subdivided plant embryo with the extraction solvent are performed simultaneously. In this case, the germ and an extraction solvent in an amount necessary for extraction are mixed, and the germ is subdivided in the presence of the extraction solvent. The amount of the extraction solvent is usually 0.1 ml or more, preferably 0.5 ml or more, more preferably 1 ml or more with respect to 1 g of germ before washing. The upper limit of the amount of extraction solvent is not particularly limited, but is usually 10 ml or less, preferably 5 ml or less, per 1 g of germ before washing. The mixture is separated by centrifugation or the like, and the resulting supernatant is recovered as an embryo extract. Furthermore, you may attach | subject to purification processes, such as gel filtration.

If subdivision and solvent extraction are performed simultaneously as one step, an embryo extract can be obtained efficiently, and cell organelles such as embryonic cell nuclei, cell membranes and cell walls can be obtained more efficiently than when extraction is performed after subdivision. Factors necessary for protein synthesis localized in the cytoplasm can be sufficiently extracted even in a state in which the destruction of the cells is suppressed.
In addition, when the embryo subdivision is performed in the absence of the extraction solvent, the procedure is specifically as follows. First, an extractant is added to the subdivided embryo, preferably by impact or cutting, so that cell organelles such as cell nuclei, cell membranes and cell walls are not destroyed more than necessary. The supernatant obtained is recovered as an embryo extract. Furthermore, you may attach | subject to purification processes, such as gel filtration. The amount of the extraction solvent used and the upper limit thereof are set to the same values as in the case of subdividing the embryo in the presence of the extraction solvent.
The embryo to be subdivided may be a frozen one as in the prior art or an unfrozen one, but it is preferable to use an unfrozen one.

  As the extraction solvent, an aqueous solution containing at least one of a buffer solution, potassium ions, magnesium ions and a thiol group antioxidant can be used. Moreover, you may further add a calcium ion, an L-type amino acid, etc. as needed. For example, a solution containing N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) -KOH, potassium acetate, magnesium acetate, L-type amino acid and / or dithiothreitol, or the method of Patterson et al. A partially modified solution (a solution containing HEPES-KOH, potassium acetate, magnesium acetate, calcium chloride, L-type amino acid and / or dithiothreitol) can be used as an extraction solvent. The composition and concentration of each component in the extraction solvent are known per se, and those usually used in the method for producing an embryo extract for cell-free protein synthesis may be employed.

  The gel filtration can be performed, for example, using a gel filtration device that has been equilibrated in advance with a solution (a solvent containing HEPES-KOH, potassium acetate, magnesium acetate, dithiothreitol, or an L-type amino acid). The composition and concentration of each component in the gel filtration solution are also known per se, and those usually used in the method for producing an embryo extract for cell-free protein synthesis may be employed.

The germ extract after gel filtration may contain microorganisms, particularly spores such as filamentous fungi, and it is preferable to eliminate these microorganisms. In particular, it is important to prevent the growth of microorganisms during long-term (one day or longer) cell-free protein synthesis reactions. The means for removing microorganisms is not particularly limited, but a filter sterilization filter is preferably used. The pore size of the filter is not particularly limited as long as microorganisms that may be mixed can be excluded, but usually 0.1 to 1 micrometer, preferably 0.2 to 0.5 micrometer is appropriate. . Incidentally, since the spore size of Bacillus subtilis of a small class is 0.5 μm × 1 μm, it is effective to remove the spore by using a 0.20 micrometer filter (for example, Minisart ^™ manufactured by Sartorius). In the filtration, it is preferable to first use a filter having a larger pore size, and then use a filter having a pore size capable of eliminating microorganisms that may be mixed.

In the present invention, the embryo is preferably subdivided in the presence of an extraction solvent, and particularly preferably the embryo is subdivided by impact or cutting in the presence of the extraction solvent.
The embryo extract obtained in this manner is used for substances (such as tritin, thionine, ribonuclease, and phosphatase, such as mRNA, tRNA, translation factor protein, and ribosome) that suppress or inhibit the protein synthesis function contained or retained by the source cells themselves. Endosperm containing a substance that acts and suppresses its function) is substantially not contaminated and purified. Here, “endosperm is not substantially contaminated” means an embryo extract obtained by removing the endosperm part to such an extent that the ribosome is not substantially deadeninated, and the ribosome is not substantially deadeninated. The degree means that the ribosome deadenification rate is less than 7%, preferably 1% or less.

  In addition, by performing a step of eliminating a substance that suppresses a low molecular weight protein synthesis function that can be mixed into the germ extract by dialysis or the like, a germ extract having a more excellent protein synthesis ability can be obtained. Such an exclusion step is preferably carried out in the presence of stabilizing components such as ATP, GTP and amino acids.

Furthermore, the germ extract of the present invention, for example, the germ extract obtained by the above-described procedure is characterized in that the ribonuclease activity is reduced. Here, “reduced” is intended to mean that the activity is lower when compared with a plant embryo extract for cell-free protein synthesis obtained by a conventionally known method for preparing an embryo extract. The ribonuclease activity is 85% or less in terms of RNase A compared to a germ extract obtained by a method in which plant germ fragmentation is performed by grinding or crushing in the absence of an extraction solvent (ie, conventional method), An embryo extract that is preferably 80% or less, more preferably 75% or less is applicable. For example, the germ extract with reduced ribonuclease of the present invention has a ribonuclease activity of 10 pg / μl or less in terms of RNase A.
The ribonuclease activity can be measured by a method known per se. Specifically, mRNA as a substrate for ribonuclease (the type of the mRNA is not particularly limited, and a germ extract solution as a control with the germ extract solution of the present invention ( It is obvious to those skilled in the art that the same kind can be selected as long as the same type is used in a comparison experiment with a germ extract obtained by a conventional extraction method), and the mRNA was treated with each germ extract. By examining the degree of RNA degradation at this time, the ribonuclease activity mixed in the germ extract of the present invention can be relatively known, and at that time, ribonuclease having a known concentration as a standard product, for example, RNA by RNase A Knowing the amount of ribonuclease mixed in the germ extract of the present invention by comparing with the degree of degradation It can be. In this specification, “in terms of RNase A” indicates that the value is obtained when RNase A is used as a ribonuclease of known concentration used as a standard product. A specific method will be described later.

In addition, the germ extract of the present invention, for example, the germ extract obtained by the above-described procedure, has a DNA content of not more than 230 μg / ml when the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm is 90, preferably 200 μg / ml or less, more preferably 180 μg / ml or less. In the present invention, the DNA content is a value measured by the following method.

Method for measuring DNA content
PicoGreen dsDNA quantitation reagent (manufactured by Molecular Probe) is used, and Calf Thymus DNA Standard (manufactured by Pharmacia Biotech) is used as a standard sample, and measurement is performed using a microplate fluorometer (SPECTRAmax GEMINI XS, manufactured by Molecular Devices).
First, 10 microliters of proteinase K (10 mg / milliliter) is added to 200 microliters of sample, and it reacts at 55 degreeC overnight. Extract the reaction solution with an equal amount of phenol, extract with an equal amount of phenol / chloroform (1: 1) to deproteinize, add 1/10 volume of 3 mol / liter sodium acetate and 2 volumes of ethanol. Ethanol precipitation. After washing with 70% ethanol, it is dissolved in 10 mmol / liter Tris-HCl buffer (pH 8.0) (TE) containing 1 mmol / liter sodium ethylenediaminetetraacetate. To this solution, 5 microliters of RNaseA (10 mg / milliliter) is added and reacted overnight at 37 ° C., and RNA is removed by extraction with phenol and phenol / chloroform as described above. The sample is ethanol precipitated, washed and dissolved in 200 microliters of TE. Mix the PicoGreen dsDNA quantitation reagent diluted 200 times with TE and the sample diluted to the appropriate concentration with TE at a ratio of 1: 1, measure the fluorescence at an excitation / fluorescence wavelength of 485/530 nm, and determine the DNA content from the standard curve. Is calculated.

Moreover, embryo extract of the present invention, the optical density _(O.D..) In 260nm _{(A 260)} is optical density at 260nm when the 90 _(O.D..) Total fatty acid when the _{(A 260)} is 90 The content is 0.03 g / 100 g or less, preferably 0.02 g / 100 g or less, more preferably 0.018 g / 100 g or less. In the present invention, the total fatty acid content is a value measured by the following method (gas chromatographic method). In the present invention, the total fatty acid content is the total amount of palmitic acid, oleic acid and linoleic acid contents.

Measurement method for total fatty acid content To 5.0 g of sample, 2 mg of heptadecanoic acid (internal standard), 4 ml of ethanol, 6.7 ml of ion-exchanged water, and 8.3 ml of 12 mol / liter hydrochloric acid are added for acid decomposition. . Next, 16 ml of ethanol is added, 100 ml of a diethyl ether-petroleum ether mixture (volume ratio 1: 1) is added for extraction treatment, and the extraction treatment with 60 ml of the mixture is performed twice. After washing with water and evaporating the extraction solvent, saponification and methyl esterification are performed according to ACOS Official Method Ce 1b-89 (1997), and then measured by gas chromatography under the following operating conditions.

<Gas chromatograph operating conditions>
Measurement model: GC-17A manufactured by Shimadzu Corporation
Detector: FID
Column: DB-23 manufactured by J & W, φ0.25 mm × 30 mm, df. 0.25μm
Column temperature: 70 ° C. (1 minute hold) → 170 ° C. (10 ° C./minute temperature increase) → 210 ° C.
(1.2 ℃ / min temperature rise)
Inlet temperature: 250 ° C
Detector temperature: 250 ° C
Gas flow rate: Helium 1.5 ml / min Gas pressure: Hydrogen 60 kPa, Air 50 kPa
Introduction system: Splitless

The content of each fatty acid (palmitic acid, oleic acid, and linoleic acid) is calculated from the obtained measured value according to the following formula, and the total fatty acid content is calculated from these.
Each fatty acid content (g / 100 g) = (E × F × H / D × G) × 0.1
(In the above formula, D is the peak area of heptadecanoic acid, E is the peak area of each fatty acid, F is the amount of heptadecanoic acid added (mg), G is the amount of sample collected, H is the sensitivity correction factor (each of the heptadecanoic acid measured in advance) Represents the sensitivity of fatty acids).

  Furthermore, the germ extract of the present invention, for example, the germ extract obtained by the above-mentioned procedure, when performing cell-free protein synthesis using it, compared to the case of using the germ extract obtained by the conventional extraction method, It is possible to suppress consumption of energy sources such as ATP. This suggests that the phosphatase activity of the germ extract of the present invention may be reduced.

The present invention also provides a method for synthesizing a cell-free protein using the embryo extract of the present invention.
The cell-free protein synthesis method can be performed in the same manner as in the prior art except that the embryo extract obtained as described above is used. This method may be a known batch method, Spirin et al. (AS Spirin et al. (1988), Science, 242, 1162-1164) and Yokoyama et al. (Kikawa et al., 21st Annual Meeting of the Molecular Biology Society of Japan). A continuous supply system of amino acids and energy sources such as a continuous cell-free protein synthesis system of WID6). In the batch method, if the protein synthesis is carried out for a long time, the reaction may stop. By using the latter amino acid and energy source continuous supply system, the reaction can be maintained for a long time, and further efficiency can be improved. It becomes possible. Moreover, when protein is synthesize | combined by a continuous supply system, a dialysis method can also be used. For example, in the ultrafiltration membrane dialysis system using the germ extract of the present invention as the dialysis internal solution and the mixed solution containing the energy source and amino acid as the dialysis external solution, it is possible to prepare a large amount of protein continuously. . Here, examples of the energy source include adenosine triphosphate (ATP), guanosine triphosphate (GTP), creatine phosphate, and the like, and examples of amino acids include 20 kinds of L-type amino acids.

  Embryo extract of the present invention (embryo extract obtained by a method characterized by performing embryo subdivision in the presence of an extraction solvent, or subdivided by impact or cutting in the absence of an extraction solvent, and then extracted In the embryo extract obtained by a method characterized by extraction with a solvent), since the organelles such as cell nuclei of embryos, cell membranes and cell walls are prevented from being destroyed more than necessary during the preparation, For example, ribonuclease contamination is greatly reduced. Thus, since the tRNA required for protein synthesis is rarely degraded by ribonuclease, the embryo extract of the present invention usually contains an amount of tRNA required for the protein synthesis reaction. Thus, it is not an essential requirement to add a separately prepared tRNA. Such an embryo extract functions as a solution for cell-free protein synthesis containing at least a plant embryo extract and is an essential or appropriate component for protein synthesis such as ATP, GTP, creatine phosphate, creatine kinase, L If there is a type amino acid, potassium ion, magnesium ion, etc., it has sufficient protein synthesis activity without adding tRNA. Here, the cell-free protein synthesis solution “having sufficient protein synthesis activity without adding tRNA” means that a conventional plant germ is previously added with liquid nitrogen or the like without adding tRNA, as shown in the Examples below. It has protein synthesis activity at least equivalent to that obtained by adding a sufficient amount of tRNA to the embryo extract prepared by freezing, grinding or crushing in a mortar, etc. and then solvent extraction (amount of protein synthesis reaches a plateau) It means a solution for cell-free protein synthesis, preferably a solution in which the protein synthesis activity when tRNA is not added is equal to or more than the protein synthesis activity when tRNA is added.

  Furthermore, the embryo extract of the present invention having sufficient protein synthesis activity without adding the above-mentioned tRNA, and proteins such as ATP, GTP, creatine phosphate, creatine kinase, L-type amino acid, potassium ion and magnesium ion An aqueous solution containing components essential or appropriate for synthesis can be conveniently used as a ready-made cell-free protein synthesis solution. Content of the said component is not specifically limited, What is necessary is just the density | concentration which can perform a cell-free protein synthesis reaction. Such a ready-made cell-free protein synthesis solution does not require the preparation of a reaction solution as in the prior art for protein synthesis, and simply and efficiently simply by adding the target translation template (mRNA). Proteins can be synthesized in large quantities. The protein synthesis solution can be prepared by using the extraction solvent as a solvent and adding the components as necessary.

  The kit for carrying out cell-free protein synthesis of the present invention contains at least the above-mentioned embryo extract or cell-free protein synthesis solution and, as an optional element, other reagents and reaction vessels necessary for cell-free protein synthesis, for example, Diluent (buffer), dialysate, energy source, expression vector, expression vector for positive control, amino acid serving as a substrate, dialysis tube and the like may be included. The reagent kit may contain a reagent used for a transcription system such as RNA polymerase.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples should be considered as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is not limited in any way by the following examples. It is not something.
In the following examples, l represents liter, ml represents milliliter, M represents mol / liter, mM represents mmol / liter, and μg represents microgram.

Fine grinding extraction with Waring blender (1)
Chihoku wheat from Hokkaido (unsanitized) was added to the mill (Fritsch Rotor Speed Mill pulverisette type 14) at a rate of 100 g per minute, and the seeds were gently crushed at a rotational speed of 7000 rpm. This crushing process was repeated 4 times. After collecting a fraction containing germs having germination ability with a sieve (mesh size 0.71 mm to 1.00 mm), a mixture of carbon tetrachloride and cyclohexane [carbon tetrachloride: cyclohexane = 2.4: 1 (volume ratio) )] To collect floating fractions containing germinating germs, remove organic solvent by drying at room temperature, remove impurities such as seed coat mixed by room temperature ventilation, and then remove crude embryos Got the minute.

  Next, using a belt-type color sorter BLM-300K (manufacturer: Anzai Manufacturing Co., Ltd., distributor: Anzai Sogyo Co., Ltd.), germs were selected from the crude germ fraction using the color difference as follows. . This color sorter has means for irradiating light to the crude germ fraction, means for detecting reflected light and / or transmitted light from the crude germ fraction, means for comparing the detected value with the reference value, and deviating from the reference value. It is an apparatus having a means for selecting and removing the thing within the reference value.

The crude germ fraction was supplied to the color sorter belt at 1000 to 5000 grains / m ² and the reflected light was detected by irradiating the crude germ fraction on the belt with a fluorescent lamp. The belt conveyance speed was 50 m / min. A monochrome CCD line sensor (2048 pixels) was used as the light receiving sensor.
First, in order to eliminate black components (seed coat, etc.) from the germ, attach a beige belt, set a reference value between the brightness of the germ and the seed coat, and remove anything that deviates from the reference value by suction. It was. Next, in order to sort the endosperm, the belt was replaced with a dark green belt, a reference value was set between the brightness of the germ and the brightness of the endosperm, and those that deviated from the reference value were removed by suction. Suction was performed using 30 suction nozzles (one suction nozzle arranged per 1 cm in length) installed approximately 1 cm above the conveyor belt.

By repeating this method, germs were selected until the purity of the germs (the weight ratio of germs contained in 1 g of any sample) reached 98% or more.
50 g of the wheat germ obtained as described above was suspended in distilled water at 4 ° C., and washed using an ultrasonic cleaner until the cleaning solution became cloudy. Next, it was suspended in a 0.5% by volume solution of Nonidet P40, and washed with an ultrasonic cleaner until the cleaning solution did not become cloudy to obtain a wheat germ from which the endosperm was removed.

  Subsequently, the following operation was performed at 4 ° C. to obtain a germ extract (wheat germ extract-containing solution). First, washing wheat germ with 100 ml of extraction solvent (HEPES-KOH (pH 7.6) 80 mM, potassium acetate 200 mM, magnesium acetate 10 mM, calcium chloride 4 mM, 20 L-type amino acids 0.6 mM and dithiothreitol 8 mM) The mixture was placed in a blender and pulverized at a rotational speed of 5000 to 20000 rpm for 30 seconds. After scraping off germs and the like adhering to the inner wall of the blender, the work of grinding again at 5000 to 20000 rpm for 30 seconds was performed twice. The particle size distribution of the obtained germ pulverized product was measured using a laser scattering type particle size distribution device (LA-920 manufactured by Horiba, Ltd.). The result is shown in FIG.

The resulting mixture of the extract and crushed germ was transferred to a centrifuge tube and centrifuged at 30000 g for 30 minutes to collect the supernatant. This was further centrifuged at 30 000 g for 30 minutes and the supernatant was collected 5 times to obtain a non-turbid supernatant. A Sephadex G-25 column in which this was equilibrated in advance with a solution (HEPES-KOH (pH 7.6) 40 mM, potassium acetate 100 mM, magnesium acetate 5 mM, 20 L-type amino acids each 0.3 mM and dithiothreitol 4 mM). Gel filtration was performed. The obtained liquid was centrifuged at 30000 g for 12 minutes to collect a supernatant, which was used as a wheat germ extract-containing liquid. The concentration of the sample was adjusted with the extraction solvent so that the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm was 80 to 150 (A ₂₆₀ / A ₂₈₀ = 1.5).

Preparation of wheat germ cell-free protein synthesis system by dialysis method Concentration adjustment of the wheat germ extract-containing solution obtained in Example 1 with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm is 90 Then, green fluorescent protein (GFP) was synthesized according to the method described in “Endo, Y. et al., PNAS, January 18, 2000 / vol.97 / no.2 / 559-564”. The GFP activity was quantified from the fluorescence intensity of 510 nm at an excitation wavelength of 490 nm using a TD-360 Mini-Fluorometer manufactured by Turner Designs. As shown in FIG. 3 (indicated as blender method in FIG. 3), the fluorescence intensity was observed to be about 350,000 after 24 hours and 480000 after 48 hours, confirming the synthesis of GFP.

Analysis of liquid containing wheat germ extract (1)
A wheat germ extract-containing liquid was prepared in the same manner as in Example 1 using Chihoku wheat (unsanitized) from Hokkaido. The concentration of the sample was adjusted with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 90, and the DNA and RNA contents of each sample were measured. The results are shown below.

RNA (μg / ml) DNA (μg / ml)
Sample a1 1411 141
Sample a2 1554 142
In addition, the measuring method of content of DNA and RNA is as follows.

Method for measuring DNA content
PicoGreen dsDNA quantitation reagent (manufactured by Molecular Probe) was used, and Calf Thymus DNA Standard (manufactured by Pharmacia Biotech) was used as a standard sample, and measurement was performed using a microplate fluorometer (SPECTRAmax GEMINI XS, manufactured by Molecular Devices).

  First, 10 μl of proteinase K (10 mg / ml) was added to 200 μl of the sample, and reacted at 55 ° C. overnight. Extract the reaction solution with an equal amount of phenol, extract with an equal amount of phenol / chloroform (1: 1), deproteinize, add 1/10 volume of 3M sodium acetate and 2 volumes of ethanol to precipitate ethanol. Went. After washing with 70% ethanol, it was dissolved in 10 mM Tris-HCl buffer (pH 8.0) (TE) containing 1 mM sodium ethylenediaminetetraacetate. 5 μl of RNase A (10 mg / ml) was added to this solution and reacted overnight at 37 ° C., and RNA was removed by extraction with phenol and phenol / chloroform in the same manner as described above. The sample was ethanol precipitated, washed and dissolved in 200 μl TE. Mix the PicoGreen dsDNA quantitation reagent diluted 200 times with TE and the sample diluted 100 times with TE at a ratio of 1: 1, measure the fluorescence at an excitation / fluorescence wavelength of 485/530 nm, and determine the DNA content from the standard curve. Calculated.

Measuring method of RNA content Using BioGreen RNA Quantitation reagent (Molecular Probes) as a fluorescent reagent, Ribosomal RNA standard (16S and 23S rRNA from E.coil) (Molecular probe) as standard RNA, and microplate fluorescence It was measured using a meter (SPECTRAmax GEMINI XS, manufactured by Molecular Devices).

  First, 3 μl of DNase I (RNase free, 1 U / μl) was added to 100 μl of the extract, and reacted at 37 ° C. for 30 minutes. 300 μl of purified water was added to the reaction solution, extracted twice with 400 μl of water-saturated phenol, and then extracted with 400 μl of chloroform to remove DNA. 1/10 amount of 3M sodium acetate and 2 times amount of ethanol were added thereto, ethanol precipitation was carried out and dissolved in 100 μl of TE. Furthermore, 100 μl of a 200-fold diluted solution of BioGreen RNA Quantitation reagent was added to 100 μl of a sample diluted 1500 times with TE, and the fluorescence content was measured at an excitation / fluorescence wavelength of 480/520 nm, and the RNA content was calculated from the standard curve.

Analysis of liquid containing wheat germ extract (2)
A wheat germ extract-containing liquid was prepared in the same manner as in Example 1 using Chihoku wheat (unsanitized) from Hokkaido. The concentration of the sample was adjusted with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 90, and the content of lipid (acid decomposition method) and total fatty acid (gas chromatographic method) in each sample Was measured. The results are shown below.

Lipid (g / 100g) Total fatty acid (g / 100g)
Sample a3 Below detection limit 0.03
Sample a4 Below detection limit 0.03
In addition, the measuring method of content of a lipid and a fatty acid is as follows.

Method for measuring lipid content (acid decomposition method)
5.0 g of sample (S) and 10 ml of concentrated hydrochloric acid were placed in a container, and acid decomposition was performed by heating in a hot water bath at 80 ° C. for 40 minutes. The resulting decomposition product was transferred to a Majoronia tube with ethyl ether, and extracted with shaking with a diethyl ether-petroleum ether mixture (volume ratio 1: 1). Washing with ion-exchanged water until the ether layer no longer showed acidity. The ether layer was recovered and transferred to a weighing container (W ₁ g). After the ether was distilled off, it was further dried at 105 ° C. for 1 hour and weighed (W ₂ g). The lipid content (g / 100 g) was calculated according to the following formula.
Lipid content (g / 100 g) = (W ₂ −W ₁ ) ÷ S × 100

Method for measuring total fatty acid content (gas chromatographic method)
To 5.0 g of the sample, 2 mg of heptadecanoic acid (internal standard), 4 ml of ethanol, 6.7 ml of ion-exchanged water, and 8.3 ml of 12 mol / liter hydrochloric acid were added for acid decomposition. Next, 16 ml of ethanol was added, 100 ml of diethyl ether-petroleum ether mixture (volume ratio 1: 1) was added for extraction treatment, and extraction treatment with 60 ml of the mixture was performed twice. After washing with water and evaporating the extraction solvent, saponification and methyl esterification were performed according to ACOS Official Method Ce 1b-89 (1997), and then measured by gas chromatography under the following operating conditions.

<Gas chromatograph operating conditions>
Measurement model: GC-17A manufactured by Shimadzu Corporation
Detector: FID
Column: DB-23 manufactured by J & W, φ0.25 mm × 30 mm, df. 0.25μm
Column temperature: 70 ° C. (1 minute hold) → 170 ° C. (10 ° C./minute temperature increase) → 210 ° C.
(1.2 ℃ / min temperature rise)
Inlet temperature: 250 ° C
Detector temperature: 250 ° C
Gas flow rate: Helium 1.5 ml / min Gas pressure: Hydrogen 60 kPa, Air 50 kPa
Introduction system: Splitless

The content of each fatty acid (palmitic acid, oleic acid, and linoleic acid) was calculated from the obtained measured values according to the following formula, and the total fatty acid content was calculated therefrom.
Each fatty acid content (g / 100 g) = (E × F × H / D × G) × 0.1
(In the above formula, D is the peak area of heptadecanoic acid, E is the peak area of each fatty acid, F is the amount of heptadecanoic acid added (mg), G is the amount of sample collected, H is the sensitivity correction factor (each of the heptadecanoic acid measured in advance) Represents the sensitivity of fatty acids).

Analysis of liquid containing wheat germ extract (3)
A wheat germ extract-containing liquid was prepared in the same manner as in Example 1 using white heron wheat (unsanitized) from Okayama Prefecture, and the sample had an optical density (OD) (A ₂₆₀ ) at 90 nm of 90. The DNA and RNA contents were measured in the same manner as in Example 3 with the extraction buffer solution. The results are shown below.

RNA (μg / ml) DNA (μg / ml)
Sample a5 1401 123

Comparative Example 1 Frozen finely ground extraction with mortar (1)
Chihuk wheat from Hokkaido (undisinfected) was crushed and selected in the same manner as in Example 1 to obtain a crude germ fraction, and the germ was selected with a color sorter until the purity of the embryo reached 98% or more. . Furthermore, the germ was washed by the same method as in Example 1 to obtain a wheat germ from which the endosperm was removed.

Subsequently, the following operation was performed at 4 ° C. to obtain a wheat germ extract-containing liquid. First, the washed wheat germ was frozen with liquid nitrogen and pulverized in a mortar. The particle size distribution of the obtained germ pulverized product is shown in FIG.
10 ml of an extraction solvent (HEPES-KOH (pH 7.6) 80 mM, potassium acetate 200 mM, magnesium acetate 10 mM, calcium chloride 4 mM, 20 L-type amino acids 0.6 mM and dithiothreitol 8 mM) was added to a mortar and mixed. The resulting mixture of the extract and crushed germ was transferred to a centrifuge tube and centrifuged at 30000 g for 30 minutes to collect the supernatant. This was further centrifuged under the same conditions as in Example 1 to obtain a non-turbid supernatant. After gel filtration, the supernatant was centrifuged, and the resulting supernatant was used as a wheat germ extract-containing solution. The concentration of the sample was adjusted with the extraction solvent so that the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm was 170 to 250 (A ₂₆₀ / A ₂₈₀ = 1.5).

Comparative Example 2 Preparation of Wheat Germ Cell-Free Protein Synthesis System by Dialysis Method The wheat germ extract-containing solution obtained in Comparative Example 1 was extracted so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 90. The concentration was adjusted with a solvent, and the synthesis of Green fluorescent protein (GFP) was performed according to the method described in "Endo, Y. et al., PNAS, January 18, 2000 / vol.97 / no.2 / 559-564". went. The GFP activity was quantified from the fluorescence intensity of 510 nm at an excitation wavelength of 490 nm using a TD-360 Mini-Fluorometer manufactured by Turner Designs. As shown in FIG. 3 (indicated as mortar method in FIG. 3), the fluorescence intensity was observed to be about 200000 after 24 hours and 260000 after 48 hours, and it was confirmed that GFP was synthesized. It was a low value.

Comparative Example 3 Analysis of liquid containing wheat germ extract (1 ')
A wheat germ extract-containing liquid was prepared in the same manner as in Comparative Example 1 using Chihoku wheat (unsanitized) from Hokkaido. The concentration of the sample was adjusted with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 90, and the DNA and RNA contents of each sample were measured in the same manner as in Example 3. . The results are shown below.

RNA (μg / ml) DNA (μg / ml)
Sample b1 1567 283
Sample b2 1574 283
Sample b3 1578 232
As is clear from the above results, the RNA content is almost the same as the value of the embryo extract-containing liquid obtained in Example 3 (about 1.1 times compared with the sample a1 and the sample b1), and the DNA content Was about twice.

Comparative Example 4 Analysis of liquid containing wheat germ extract (2 ')
A wheat germ extract-containing liquid was prepared in the same manner as in Comparative Example 1 using Chihoku wheat (unsanitized) from Hokkaido. The concentration of the sample was adjusted with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 90, and the lipid (acid decomposition method) and total fatty acid (gas chromatograph) of each sample were the same as in Example 4. Method) The content was measured. The results are shown below.

Lipid (g / 100g) Total fatty acid (g / 100g)
Sample b4 0.1 0.06
Sample b5 0.05 0.05
As is clear from the above results, the lipid and total fatty acid contents are both higher than the content of the embryo extract-containing liquid obtained in Example 4, and the total fatty acid contents are as follows: Sample a3 and Sample b4 In the comparison of 2 times.

Fine grinding extraction with Waring blender (2)
Using Chihoku wheat from Hokkaido (undisinfected), an extraction solvent that does not contain calcium chloride as a germ extraction solvent (HEPES-KOH (pH 7.8) 80 mM, potassium acetate 200 mM, magnesium acetate 10 mM, 20 types of L-type amino acids 0 each A solution containing wheat germ extract was obtained in the same manner as in Example 1 except that 6 mM and 8 mM dithiothreitol were used. The concentration of the sample was adjusted with the extraction solvent so that the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm was 80 to 150 (A ₂₆₀ / A ₂₈₀ = 1.5).

Batch-type wheat germ cell-free protein synthesis reaction (1)
The concentration of the wheat germ extract-containing liquid obtained in Example 6 was adjusted with an extraction solvent so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 110, and the preparation of the reaction solution and the reaction method were already reported. (Madin, K. et al., Proc. Natl. Acad. Sci. USA 2000, 97, 559-564). That is, a reaction solution having the composition shown in (* 1) below was prepared.

As a model translation template (WO01 / 27260), DHFR mRNA (without cap structure) prepared by transcription from a dihydrofolate dehydrogenase (DHFR) gene incorporated into a wheat germ cell-free protein synthesis system-specific expression vector (pEU) 60 μg / ml was added, and the synthesis reaction was carried out at 26 ° C. The synthetic activity was measured from [ ¹⁴ C] -leucine incorporated into the protein in 5 μl reaction solution at 26 ° C. for 2 hours.

  Here, in order to confirm whether or not tRNA should be added, the synthesis was performed in 4 cases of 0 μg / ml, 50 μg / ml, 100 μg / ml, and 150 μg / ml as a result of tRNA addition. Synthetic activity was obtained (indicated as blender method in FIG. 4). Moreover, it became clear from this result that a germ extract-containing solution having sufficient protein synthesis activity can be obtained even when calcium is not present in the extraction solvent.

(* 1) Composition of wheat germ cell-free protein synthesis solution The reaction solution is a wheat germ extract having an optical density at 260 nm (OD) (A ₂₆₀ ) of 200%, which is 24% of the reaction volume (accordingly, In the case where the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm is 110, it is 43.5%). 30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 1.2 mM adenosine triphosphate (ATP), 0.25 mM guanosine triphosphate (GTP), 16 mM Creatine phosphate, 0.5 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM), 1000 units / ml ribonuclease inhibitor (RNase inhibitor).

Comparative Example 5 Frozen finely ground extraction with mortar (2)
Using Chihoku wheat from Hokkaido (undisinfected), an extraction solvent that does not contain calcium chloride as a germ extraction solvent (HEPES-KOH (pH 7.8) 80 mM, potassium acetate 200 mM, magnesium acetate 10 mM, 20 types of L-type amino acids 0 each A solution containing wheat germ extract was obtained in the same manner as in Example 1 except that 6 mM and 8 mM dithiothreitol were used. The concentration of the sample was adjusted with the extraction solvent so that the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm was 170 to 250 (A ₂₆₀ / A ₂₈₀ = 1.5).

Comparative Example 6 Batch Wheat Germ Cell-Free Protein Synthesis Reaction The concentration of the wheat germ extract-containing liquid obtained in Comparative Example 5 was adjusted so that the optical density (OD) (A ₂₆₀ ) at 260 nm was 210. In the same manner as in Example 7, a reaction solution was prepared, protein synthesis was performed, and [ ¹⁴ C] -leucine incorporated into the protein was measured. The reaction solution was a wheat germ extract having an optical density (OD) at 260 nm (A ₂₆₀ ) of 200, which was 24% of the reaction volume (hence, the optical density (OD) at 260 nm). In the case of A ₂₆₀ ) of 210, the one containing 22.8%) was used.

  As is clear from FIG. 4, the synthesis was performed in 4 cases of tRNA addition amounts of 0 μg / ml, 50 μg / ml, 100 μg / ml, and 150 μg / ml in the same manner as in Example 7. As a result, it was sufficient when tRNA was not added. No synthetic activity was obtained (indicated as mortar method in FIG. 4).

Measurement of Ribonuclease Activity (1) Germ Extract Solution As a wheat germ extract solution for measuring ribonuclease activity, the germ extract solution of the present invention (Example 1) and an extract solution obtained by extraction by a conventional method (Comparative Example 1) Was used. For the activity measurement, the conventional germ extract is 30 mM Hepes-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0.3 mM spermidine, 0.3 mM. 20 amino acids, 2.5 mM magnesium acetate, 100 mM potassium acetate, 0.005% sodium azide (hereinafter referred to as “DB”) exchanged with MicroSpin G-25 Column were used. The extract of the present invention comprises B. Is included. Based on the OD ₂₆₀ measurement value, the concentrations of both embryo extracts (embryo extract of the present invention and embryo extract obtained by extraction by a conventional method) were aligned.

(2) Preparation of reaction solution As a ribonuclease substrate, pEU-DHFR (see WO01 / 27260) was subjected to PCR using a sense primer and an antisense primer, and mRNA obtained by transferring it to a template was used. . For transcription, prepare 400 μl of the reaction system so that it becomes 80 mM Hepes-KOH, 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTP, 1 U / μl SP6 RNA polymerase, 1 U / μl RNase inhibitor (RNasin), 10% PCR product. And kept at 37 ° C. for 3 hours. When labeling mRNA with radioisotope ³² P, 8 μl of [α- ³² P] UTP was added to 400 μl of a reaction system with a UTP concentration of 1.2 mM. After incubation, after centrifugation at 10,000 g for 2 minutes, 53 μl of 7.5M ammonium acetate and 1 ml of ethanol were added to the supernatant, followed by centrifugation at 20,000 g for 15 minutes. After washing the precipitate with 1 ml of 70% ethanol, 80 μl of D.P. B. RNA dissolved in was used. Each composition is shown below.

MRNA without radioisotopes

5xTB 80μl
48 mM of 25 mM NTP
SP6 RNA polymerase 5 μl
RNase inhibitor 5 μl
DNA 40 μl
Milli Q water 222μl
400 μl

MRNA with radioisotope
5xTB 80μl
25 mM ATP CTP GTP 10 mM UTP 48 μl
[Α- ³² P] UTP 8 μl
5 μl of SP6 RNA polymerase
RNase inhibitor 5 μl
DNA 40 μl
Milli Q water 214μl
400 μl

(3) RNA degradation control (assay with RNase A standard)
As controls for RNA degradation, RNaseA was added to 2 μl of mRNA (without radioisotope) and 1 μl of mRNA (with radioisotope), respectively, so that a reaction system of 30 μl was obtained by adding 30 pg, 300 pg, 3 ng, and 30 ng. B. And kept at 26 ° C. for 30 minutes. Each composition is shown below.

mRNA
(No radioisotope) 2 μl 2 μl 2 μl 2 μl 2 μl
mRNA
(With radioisotope) 1μl 1μl 1μl 1μl 1μl
RNase A-30 pg 300 pg 3 ng 30 ng
D. B.
Total 30μl 30μl 30μl 30μl 30μl

The activity of ribonuclease contained in the germ extract of the present invention and the germ extract by the conventional method is 2 μl of mRNA (without radioisotope), 1 μl of mRNA (with radioisotope), and final concentration of 40 OD ₂₆₀ / ml. Add 8 μl or 12 μl respectively. B. To 30 μl. Four of each were prepared and kept at 26 ° C.

Germ extract of the present invention Conventional germ extract mRNA
(Without radioisotope) 2μl 2μl
mRNA
(With radioisotope) 1μl 1μl
Embryo extract 8μl 12μl
D. B. 19 μl 15 μl
Total 30μl 30μl

  After 10 and 30 minutes of incubation, the reaction is stopped by freezing with liquid nitrogen. Milli Q water 255 μl, 10% SDS 15 μl, phenol / milli Q water 300 μl was added, vortexed for 10 minutes, and centrifuged at 20,000 g for 10 minutes. 300 μl of the aqueous phase was transferred to a new tube, 300 μl of phenol / milli Q was added, vortexed for 10 minutes, and centrifuged at 20,000 g for 10 minutes. 300 μl of the aqueous phase was transferred to a new tube, 300 μl of chloroform was added, vortexed for 10 minutes, and centrifuged at 20,000 g for 1 minute. 300 μl of the aqueous phase was transferred to a new tube, 30 μl of 5M sodium chloride and 750 μl of ethanol were added, and centrifuged at 20,000 g for 15 minutes. The precipitate was washed with 1 ml of 70% ethanol, dissolved in 54 μl of milli Q water, and 6 μl of 10 × Sample buffer (0.25% bromophenol blue, 50 mM Tris-HCl pH 7.6, 6% glycerol) was added, and 5 μl. The sample was electrophoresed in 1.2% agarose. The presence or absence of RNA degradation was confirmed by autoradiogram (FUJIFILM: BAS-2500). The results are shown in FIG. M represents a marker. (A) is the figure which showed the mode of RNA decomposition | disassembly at the time of processing mRNA which is a substrate with the RNaseA containing solution prepared by changing the density | concentration in 4 steps. (B) is a view showing the degradation of RNA when mRNA as a substrate is treated with the germ extract of the present invention or the germ extract obtained by extraction by a conventional method.

  In the RNase A treatment, mRNA was almost completely degraded with 100 pg / ml RNAase A. The band intensities of the germ extract of the present invention and the conventional germ extract were calculated from the PLS of ImageGauge (FUJIFILM: BAS2500) with RNase A untreated as 100%. The results are shown in FIG. It turns out that the ribonuclease per extract of this invention is 10 pg / microliter or less in conversion of RNaseA.

The reason why the embryo extract having the above-described excellent characteristics can be obtained by the present invention is not necessarily clear, but is considered as follows.
Protein synthesis systems in plant embryos (ribosomes, tRNAs, aminoacyl-tRNA enzymes, translation factors, etc.) are used for active protein synthesis that starts in response to the supply of mRNA transferred to the cytoplasm following transcription in the cell nucleus at germination. All the factors are localized only in the cytoplasm where the translation reaction takes place. That is, factors involved in protein synthesis are not present in parts other than the cytoplasm (cell organelles such as embryonic cell nuclei, cell membranes and cell walls). Cell organelles such as cell nuclei, cell membranes and cell walls contain unnecessary DNA and lipids that are not involved in protein synthesis, so that frozen embryos can be ground or crushed with a mortar, stamp mill, ball mill, etc. The germ extract-containing liquid obtained by the method of pulverizing by crushing and then extracting with an extraction solvent contains a lot of unnecessary components not involved in these protein synthesis reactions.

Components that are not involved in the protein synthesis reaction include those that inhibit the protein synthesis reaction, form a higher-order structure of the synthesized product, and exhibit an interfering action in functional analysis. For example, DNA is an acidic polymer that can bind to basic proteins such as ribosomal proteins to inhibit translation reactions, and binds strongly to embryo-derived DNA fragments during DNA-binding protein synthesis. There may be one having a base sequence. In general, the fat component is often an interfering substance in the protein purification process, and usually needs to be removed by a pretreatment operation. In addition, some low molecular weight substances may have a protein synthesis inhibitory effect.
In addition, contamination with ribonuclease leads to degradation of template mRNA, and contamination with phosphatase leads to degradation of energy sources such as ATP.

  The present invention provides an embryo extract (useful for a cell-free protein synthesis system) in which subdivision is performed in the presence of an extraction solvent, or the embryo is subdivided by impact or cutting and then extracted by adding an extraction solvent. . By utilizing a new technique that has not been used in the past, such as impact or cutting, when subdividing, cell organelles such as cell nuclei, cell membranes, and cell walls are not destroyed more than necessary, and therefore, there is a problem with conventional methods. It is possible to prevent extraction of unnecessary components that are not involved in the protein synthesis reaction or components that have adverse effects. In addition, even when preparing an embryo extract using conventional methods such as grinding or crushing, it is possible to extract more rapidly by carrying out the fragmentation in the presence of an extraction solvent. Therefore, it is possible to prevent unnecessary components that are not involved in the protein synthesis reaction from being extracted.

  According to the present invention, it is possible to efficiently extract factors necessary for protein synthesis from the cytoplasm, and to obtain an embryo extract with less impurities such as DNA and lipid contained in organelles such as cell nuclei, cell membranes and cell walls. Therefore, a stable and highly efficient embryo extract for cell-free protein synthesis and a solution for cell-free protein synthesis can be provided. By using the embryo extract for protein synthesis or the protein synthesis solution of the present invention, stable and highly efficient cell-free protein synthesis can be achieved. In addition, because it can produce a large amount of high-purity embryo extract in a short time, it is extremely useful for mass-production of proteins in cell-free systems, for example, mass production of new enzymes and antibodies in the field of evolutionary molecular engineering. It is.

  This application is based on patent application Nos. 2002-23138, 2002-23139 and 2002-23140 filed in Japan, the contents of which are incorporated in full herein.

FIG. 1 is a graph showing the particle size distribution of the crushed germ obtained in Example 1. FIG. FIG. 2 is a diagram showing the particle size distribution of the crushed germ obtained in Comparative Example 1. FIG. 3 is a graph showing the fluorescence intensity of GFP synthesized in Example 2 and Comparative Example 2. FIG. 4 is a diagram showing the results of DHFR synthesis in Example 7 and Comparative Example 6. The vertical axis represents the radioactivity incorporated into the protein ([ ¹⁴ C] -leucine incorporation: dpm / 5 μl), and the horizontal axis represents the amount of tRNA added. FIG. 5 is an electrophoresis image showing the results of measuring the ribonuclease activity of the germ extract of the present invention and the germ extract obtained by extraction by the conventional method. (A) is the figure which showed the mode of RNA decomposition | disassembly at the time of processing mRNA which is a substrate with the RNaseA containing solution prepared by changing the density | concentration in 4 steps. (B) It is a figure which shows decomposition | disassembly of RNA when mRNA which is a substrate is each processed with the germ extract of this invention, and the germ extract obtained by the extraction by a conventional method. M represents a marker. FIG. 6 is a graph showing the amount of remaining RNA by measuring the density of the band in the electrophoretic image obtained in FIG. 5 and assuming that RNase A untreated is 100% (RNA is not degraded).

Claims (5)

When the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm of the embryo extract is 90, the DNA content is 230 μg / ml or less, and the total fatty acid content is 0.03 g / 100 g or less. A plant embryo extract containing ribosome, tRNA, DNA, total fatty acids, and a solution for cell-free protein synthesis containing magnesium ions.   The solution for cell-free protein synthesis according to claim 1, wherein the plant germ is wheat, barley, rice or corn germ.   The solution for cell-free protein synthesis according to claim 1 or 2, wherein the endosperm is not substantially contaminated.   A cell-free protein synthesis method comprising using the cell-free protein synthesis solution according to any one of claims 1 to 3.   A kit for cell-free protein synthesis, comprising the cell-free protein synthesis solution according to any one of claims 1 to 3.

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