JP2005087208A - Method for producing cell extract for synthesizing acellular protein - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for industrially efficiently producing cell extracts for synthesizing acellular proteins having a high synthesizing efficiency. <P>SOLUTION: The method for producing cell extracts for synthesizing acellular proteins comprises a step for subdividing the cells, a step for providing the cell extract by mixing the subdivided cells with an extractant and a step for separating/purifying a liquid containing the cell extracts, wherein the subdivided products of the cells are treated with an activator in or after one of these steps. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for producing a cell extract for cell-free protein synthesis, and more specifically, a method for industrially efficiently producing a cell extract for cell-free protein synthesis with high synthesis efficiency, and a cell-free translation system To a protein synthesis method, a protein synthesis solution, and a cell-free protein synthesis reagent kit.

  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 a method of synthesizing a protein in a test tube (cell-free system) using ribosomes extracted from an organism and using them. It is actively performed (see, for example, Patent Documents 1 to 5). In these methods, Escherichia coli (see Non-Patent Document 1), plant germ, rabbit reticulocyte (see Non-Patent Document 2), and the like have been used as ribosome raw materials.

  As a method for obtaining a cell 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, followed by washing to obtain endosperm After removing the components, a method of pulverizing, extracting and purifying is performed.

  As a conventional method for finely pulverizing embryos, it has been considered that, for example, it is 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. After the extraction solvent was added to the resulting germ and stirred, a solution containing the germ extract collected and purified by centrifugation or the like was used as an enzyme stock solution for cell-free protein synthesis reaction. This enzyme stock solution contains many unnecessary components not involved in the protein synthesis reaction, and there may be a protein synthesis inhibitory effect.

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.
JP-A-6-98790 JP-A-6-225783 JP-A-7-194 Japanese Patent Laid-Open No. 9-291 JP 7-147992 A Methods in Enzymol. 1983; 101: 674-90. Prokaryotic coupled transcription-translation. Chen HZ, Zubay G. Pratt, JM Eur J Biochem. 1976 AUG 1; 67 (1): 247-56. An efficient mRNA-dependent translation system from reticulocyte lysates.

  The cell-free protein synthesis system is a useful method that can maintain the performance comparable to that of living cells in the peptide synthesis reaction rate and the accuracy of the translation reaction, and can obtain the target protein without carrying out complicated purification steps. It is. Therefore, several inventions related to the improvement of synthesis efficiency have been disclosed in order to use the synthesis system more effectively for industrial use. However, in order to improve industrial utility, it is necessary to supply not only the synthesis efficiency but also a large amount of various substances used in the synthesis system with stable and high quality.

  In order to solve the above problems, the inventors have increased the synthesis efficiency of a cell extract used in a cell-free protein synthesis system, and efficiently produced the extract to an industrially feasible level, A method for extracting factors necessary for protein synthesis was examined. As a result, it has been found that when a cell fragment is treated with a certain active agent, the protein synthesis activity is dramatically improved. The present invention has been accomplished based on these findings.

  That is, according to the present invention, (1) a step of subdividing cells, a step of obtaining a cell extract by mixing the subdivided cells and an extraction solvent, and a step of separating and purifying a cell extract-containing solution. There is provided a method for producing a cell extract for cell-free protein synthesis comprising treating a cell subdivision with an activator during or after any of these steps.

  According to a preferred embodiment of the present invention, the group (2) wherein the activator comprises aluminum oxide, active aluminum oxide, silica gel, zeolite, activated carbon, titanium oxide, zirconium oxide, gel chromatography carrier, and column carrier to which various ligands are bound. (3) The method according to (1) above, which is at least one activator selected from the following: (3) The method according to (1) or (2) above, wherein the cell is a plant germ cell, (4) The method according to (3) above, which is a wheat germ, (5) the plant embryo is obtained by selecting a plant germ from a pulverized product obtained by pulverizing plant seeds and then washing the germ. The method according to any one of (1) to (5), wherein the method according to (3) or (4) and (6) the fragmentation is performed by impact or cutting are provided.

  According to another aspect of the present invention, there is provided (7) a cell-free protein synthesis method comprising contacting a cell extract produced by the method according to any one of (1) to (6) above with a translation template. Is done.

  According to another aspect of the present invention, (8) a cell extract produced by the method according to any one of (1) to (6), ATP, GTP, creatine phosphate, creatine kinase, L-type amino acid, potassium ion And a solution for cell-free protein synthesis characterized by containing magnesium ions.

  According to another aspect of the present invention, (9) the cell extract produced by the method according to any one of (1) to (6) above or the protein synthesis solution according to (8) above is included. A reagent kit for cell-free protein synthesis is provided.

  According to the present invention, a cell extract for cell-free protein synthesis that is stable and highly active can be obtained by efficiently extracting a factor necessary for protein synthesis from the cytoplasm and further treating with an activator. In addition, since the cell extract obtained by the method of the present invention has high purity and excellent protein synthesis activity, it is possible to prepare a large amount of protein in a cell-free system, for example, a large amount of a new enzyme or antibody in the field of evolutionary molecular engineering. Very useful.

  The reason why a cell extract-containing solution having excellent characteristics can be obtained according to the present invention is not necessarily clear, but there is a possibility that a protein synthetic substance in a cell is activated by treatment with an activator. .

Hereinafter, the present invention will be described in more detail.
1. Method for producing cell extract
1) Raw material cell and its preparation The cell used as a raw material in the present invention may be any cell as long as it has a protein synthesis ability, such as a microbial cell, a plant cell, and an animal cell. Examples of microbial cells include E. coli (Methods in Enzymol. 1983; 101: 674-90. Prokaryotic coupled transcription-translation. Chen HZ, Zubay G. Pratt, JM). Examples of animal cells include rabbit reticulocytes ( Eur J Biochem. 1976 AUG 1; 67 (1): 247-56. An efficient mRNA-dependent translation system from reticulocyte lysates. Pelham HR, Jackson RJ.). Examples of plant cells include cells of gramineous plants such as wheat, barley, rice, and corn, and germ cells of these gramineous plant seeds (hereinafter sometimes simply referred to as “plant germ”) are preferred. Used. Plant germs that can be used include the above wheat, barley, rice or corn, with wheat being particularly preferred. These raw material cells can be easily prepared by a commonly used method known per se.

  Hereinafter, the case where plant germs that are particularly preferably used, for example, wheat germs, are used as raw material cells will be described in more detail. In addition, when using microbial cells or animal cells as a raw material, since a cell extract can be obtained by a commonly used method described in the above document, detailed description thereof is omitted.

  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 the 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 generally known classifier, 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.

  A crude embryo fraction can also be obtained by a method using the difference in specific gravity between embryo, seed coat, and endosperm, for example, heavy liquid sorting. A crude embryo fraction can also be obtained by using a method utilizing the difference in rolling friction coefficient between embryo, seed coat, and endosperm, for example, using a vibration sorter. In order to obtain a crude germ fraction containing more germs, a plurality of the above methods may be combined. Furthermore, the germ is selected 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 at a concentration of about 0.5%, for example.

  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.

2) Step of subdividing cells The cells having the ability to synthesize proteins obtained as described above are subdivided. The subdividing method is not particularly limited, and may be performed by a conventionally used method of pulverizing by grinding or crushing with a mortar, stamp mill, ball mill or the like. Plant germs are small and strong, so they cannot be pulverized easily. Therefore, it is preferable to finely pulverize the embryo after washing it in advance with liquid nitrogen or the like.

  In the present invention, when cells are subdivided, it is more preferable to subdivide them by impact or cutting rather than by grinding or crushing. 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. This means that the cells are destroyed under conditions that can be minimized.

  The apparatus and method that can be used when subdividing are not particularly limited as long as the above conditions are satisfied. For example, it is preferable to use an apparatus having a blade-like object that rotates at high speed, such as a Waring blender. 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.

  By subdividing cells, especially embryos, by impact or cutting as in the present invention, cell organelles such as cell nuclei, cell membranes and cell walls are not destroyed, but at least part of them are not destroyed. Remain. That is, especially when this subdivision method is applied to embryos, cell organelles such as embryonic cell nuclei, cell membranes and cell walls are not destroyed more than necessary, so that impurities such as DNA and lipid contained therein are mixed. Therefore, RNA and ribosome necessary for protein synthesis localized in the cytoplasm can be efficiently extracted from the germ with high purity.

3) Step of obtaining a cell extract by mixing the subdivided cells and the extraction solvent Next, after adding the extraction solvent to the subdivided cells and stirring, the cell extract-containing liquid is recovered by centrifugation or the like. A cell extract can be obtained by purification by gel filtration or the like.

  Also, the step of subdividing the germ by mixing the subdivided embryo with the extraction solvent by causing the extraction solvent to be present when subdividing the cells, for example, the embryo by impact or cutting, to obtain a germ extract Can be 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. In addition, 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 a non-frozen one.

  As an extraction solvent, an aqueous solution containing a buffer, potassium ion, magnesium ion and / or 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 a 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.

4) Step of separating and purifying the cell extract-containing solution The cell extract obtained above is separated and purified by gel filtration or the like. The gel filtration can be performed, for example, using a gel filtration device that has been equilibrated in advance with a solution (an aqueous solution containing HEPES-KOH, potassium acetate, magnesium acetate, dithiothreitol, or 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 production method of cell extracts for cell-free protein synthesis, such as embryo extract, may be employed.

The cell extract-containing solution after gel filtration may contain microorganisms, particularly spores such as filamentous fungi, and it is preferable to exclude 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 case of wheat germ extract, those obtained as described above are substances that suppress or suppress the protein synthesis function contained in or retained by the source cells themselves (such as tritin, thionine, ribonuclease, mRNA, tRNA, translated protein factor, ribosome, etc. Endosperm containing substances that act on and inhibit its function) is almost completely removed and purified. Here, the term “endosperm is almost completely removed and purified” 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 substantially deadeninated. The term “not converted to a degree” means that the deadenination rate of ribosome is less than 7%, preferably 1% or less.

5) Treatment with activating agent The method for producing a cell extract for cell-free protein synthesis of the present invention comprises a step of subdividing cells, a step of obtaining a cell extract by mixing the subdivided cells and an extraction solvent, It has one feature in that the subdivided cells (cell subdivisions) are treated with an activator during or after any one of the steps of separating and purifying the cell extract-containing liquid. . As described above, the treatment time with the activator is not particularly limited as long as it is in these steps, but it is particularly preferable to carry out the treatment after separating and purifying the cell extract-containing solution.

  The activator is not particularly limited as long as it is a water-insoluble solid carrier, and the protein synthesis ability of the finely divided product can be improved by bringing the solid carrier into contact with the cell finely divided product. Examples thereof include aluminum, activated aluminum oxide, silica gel, zeolite, activated carbon, titanium oxide, zirconium oxide, a carrier for gel chromatography, and a column carrier to which various ligands are bonded. Titanium dioxide is preferred as the titanium oxide, and zirconium dioxide is preferred as the zirconium oxide. Examples of the carrier for gel chromatography include an aluminum oxide carrier, a polyvinyl alcohol carrier, an agarose carrier, a cellulose carrier, a dextran carrier and the like. Among these, an aluminum oxide carrier is preferable. Examples of the column carrier to which various ligands are bound include, for example, a Quaternary ammonia binding carrier, which is a strong anion exchanger binding carrier, a Quaternary aminoethyl binding carrier, a dimethylaminoethyl binding carrier, which is a weak anion exchanger binding carrier, Examples include a methyl sulfonate binding carrier, which is a cation exchanger binding carrier, a sulfopropyl binding carrier, a carboxymethyl binding carrier which is a weak cation exchanger binding carrier, an active aluminum oxide whose surface is acidic, basic or neutral. Among them, methyl sulfonate binding carrier, sulphopropyl binding carrier, carboxymethyl binding carrier, basic Active aluminum oxide is preferred.

  These activators can also be used in combination of two or more. Of these activators, aluminum oxide is particularly preferred. Further, these activators are commonly used and known per se and can be easily obtained.

  The treatment method using the activator is not particularly limited, and an appropriate treatment method is used according to the characteristics of each step, for example, the activator and the cell extract-containing liquid are mixed and contacted, and then the activator is separated. A method or the like may be adopted. Alternatively, the cell extract can be passed through a column packed with an activator for treatment.

  The usage amount of the activator is not particularly limited, and the usage amount may be appropriately determined using the protein synthesis activity of the obtained cell extract as an index. Specifically, in the case of aluminum oxide, 3 to 1000 mg, preferably about 15 to 640 mg, per 1 g of cells (for example, embryo) is appropriate. In the step of obtaining the cell extract and the subsequent steps, the amount is usually about 10 to 1000 mg, preferably about 50 to 640 mg per 1 ml of the cell extract.

  There is no particular limitation on the treatment time (the contact time between the fragmented product and the activator) of the cell fragmented product and the activator, and it is usually 5 to 60 minutes, preferably 10 to 20 minutes.

  There is no particular limitation on the treatment temperature (contact time between the fragmented product and the activator) of the cell fragmented product and the activator, and it is usually 4 to 50 ° C, preferably about 20 to 25 ° C.

  By performing the treatment with the activator, a cell extract for cell-free protein synthesis with further excellent activity can be obtained.

2. Cell-free protein synthesis method Protein synthesis by a cell-free protein synthesis system can be carried out in the same manner as in the prior art except that the cell 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 Japan Molecular Biology Society, It may be a continuous supply system of amino acids and energy sources such as the 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 an ultrafiltration membrane dialysis system in which the cell extract of the present invention is used as an internal dialysis solution and a mixed solution containing an energy source and an amino acid is used as an external dialysis solution, a large amount of protein can be continuously prepared. . 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.

  Since the embryo extract of the present invention contains an amount of tRNA required for the protein synthesis reaction, it is not essential to add a separately prepared tRNA as in the prior art.

3. Protein synthesis solution and protein synthesis reagent kit In addition, the cell extract of the present invention obtained as described above, for example, embryo extract, and ATP, GTP, creatine phosphate, creatine which are essential components for protein synthesis An aqueous solution containing a kinase, L-type amino acid, potassium ion, and magnesium ion 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. The cell-free protein synthesis solution of the present invention does not require the preparation of a reaction solution as in the prior art for protein synthesis, and simply and efficiently adds a large amount of protein simply by adding the target translation template (mRNA). Can be synthesized. The protein synthesis solution can be prepared by using the extraction solvent as a solvent and adding the components as necessary.

  The reagent kit of the present invention includes at least the above-mentioned cell extract, for example, embryo extract or the above-mentioned protein synthesis solution. As optional elements, a diluent (buffer solution), a dialysate, an energy supply solution, an expression vector, and a control use An expression vector, a restriction enzyme and the like may be contained. The reagent kit may contain a reagent used for a transcription system such as RNA polymerase.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, the scope of the present invention is not limited at all by the following example.

In the following examples, l represents liter, ml represents milliliter, M represents mol / liter, mM represents mmol / liter, and μg represents microgram.
[Example 1]

Finely ground extraction by Waring blender Using Chihoku wheat (unsanitized) from Hokkaido, according to the method described in the examples of WO02 / 095377, the germ was separated and the purity of the germ (included per gram of any sample) Selection was performed until the weight ratio of the embryos reached 98% or more. 50 g of the obtained wheat germ was suspended in distilled water at 4 ° C., and washed with 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 wheat germ extract-containing liquid. First, the washed wheat germ was mixed 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), and a Waring blender. And ground for 30 seconds at a rotational speed of 5000 to 20000 rpm. 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 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. This is a Sephadex G-25 column that has been 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 the supernatant, and concentrated using an ultrafiltration membrane. This was used as a wheat germ extract-containing liquid. The concentration of the sample was adjusted so that the optical density (OD) (A ₂₆₀ ) at ₂₆₀ nm was 180 to 250 (A ₂₆₀ / A ₂₈₀ = 1.5 or so).
[Example 2]

Aluminum oxide treatment of wheat germ extract
1) Preparation of aluminum oxide suspension 10 g of aluminum oxide (for cell disruption, purity 95% or more, particle size 12 μm, manufactured by Nacalai Tesque) Buffer-1 (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2 Suspended in 10 ml of 65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM)) did. Next, centrifugation was performed at 15000 rpm, 5 minutes, and 4 degrees Celsius, and the supernatant was removed. The same operation was repeated twice to wash the aluminum oxide. The washed aluminum oxide was suspended in 10 ml of Buffer-1 to prepare a suspension under the conditions of 1 g aluminum oxide / 1 ml Buffer-1.

2) Treatment of wheat germ extract using aluminum oxide suspension For the aluminum oxide suspension prepared in 1) above, the wheat germ extract prepared in Example 1 was treated with 0: 100, 1: 100, 2 : 100, 4: 100, 8: 100, 16: 100, 32: 100, 64: 100, 100: 100, and suspended, and slowly stirred at room temperature for 15 minutes so as not to precipitate aluminum oxide. The suspension after stirring was centrifuged at 15000 rpm for 5 minutes at 4 degrees Celsius, and the supernatant was collected. The supernatant was again centrifuged at 15000 rpm for 5 minutes at 4 degrees Celsius, and the supernatant was recovered to completely remove aluminum oxide.

3) Preparation of wheat germ extract after aluminum oxide treatment The liquid composition of the wheat germ extract treated under the above conditions was adjusted to a final concentration of 30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM) Prepared. Thereafter, the wheat germ extract prepared with the liquid composition was buffer-2 (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.65 mM magnesium acetate, 2.5 mM dithiothrei so that the absorbance at 260 nm was 75 Abs. Toll, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM)) were used for adjustment.

Hereinafter, the treatment amount of aluminum oxide was aluminum oxide suspension: germ extract = 0: 100, 1: 100, 2: 100, 4: 100, 8: 100, 16: 100, 32: 100, 64: 100 and Samples of 100: 100 were respectively 0 mg / WGEml-1, 10 mg / WGEml-1, 20 mg / WGEml-1, 40 mg / WGEml-1, 80 mg / WGEml-1, 160 mg / WGEml-1, 320 mg / WGEml-1. 640 mg / WGEml-1 and 1000 mg / WGEml-1.
[Example 3]

Evaluation of wheat germ extract after aluminum oxide treatment (batch method)
A solution (30 mM HEPES-KOH, pH 7.8, containing 16 μl of wheat germ extract (various samples) prepared in Example 2 at a concentration of 2.5 mg / ml of mRNA encoding the green fluorescent protein (GFP) gene. 100 mM potassium acetate, 2.65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-forms Amino acid (0.3 mM each) 8 μl was mixed to prepare a protein synthesis solution. This protein synthesis solution was subjected to a protein synthesis reaction at 26 degrees Celsius for 3 hours.

  The mRNA serving as a translation template is a plasmid GFP / pEU containing the omega (Ω) sequence of tobacco mosaic virus (TMV), which is a translation initiation reaction sequence linked to the SP6 promoter sequence, and further the GFP gene DNA linked 3 ′ downstream. (WO01 / 27260) was used as a template for transcription using SP6 RNA polymerase (manufactured by Promega) according to a conventional method, and the resulting RNA was purified by ethanol precipitation and used according to a conventional method.

The protein synthesis solution after the reaction was diluted 5-fold, and the fluorescence intensity was measured. These results are shown in FIG. 1 (the fluorescence intensity is five times the measured value and the value of the stock solution is displayed). As shown in FIG. 1, it was confirmed that the protein synthesis ability of the wheat germ extract was increased by the batch method by treatment with aluminum oxide under the optimum conditions.
[Example 4]

Evaluation of wheat germ extract after aluminum oxide treatment (multilayer method)
A solution (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate) containing 16.5 μl of wheat germ extract (various samples) prepared in Example 2 at a concentration of 2.5 mg / ml of mRNA encoding the GFP gene. 2.65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM)) 8.4 μl was mixed to prepare a protein synthesis solution.

  This protein synthesis solution was added to 125 μl of Buffer-1 (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphorus. An acid, 0.380 mM spermidine, 20 types of L-type amino acids (each 0.3 mM) were overlaid, and a protein synthesis reaction was performed at 26 degrees Celsius for 17 hours.

  The mRNA serving as a translation template is a plasmid GFP / pEU containing the omega (Ω) sequence of tobacco mosaic virus (TMV), which is a translation initiation reaction sequence linked to the SP6 promoter sequence, and further the GFP gene DNA linked 3 ′ downstream. (WO01 / 27260) was used as a template for transcription using SP6 RNA polymerase (manufactured by Promega) according to a conventional method, and the resulting RNA was purified by ethanol precipitation and used according to a conventional method.

The protein synthesis solution after the reaction was diluted 5-fold, and the fluorescence intensity was measured. These results are shown in FIG. 2 (the fluorescence intensity is five times the measured value and the value of the stock solution is displayed). As shown in FIG. 2, it was confirmed that the protein synthesis ability of the wheat germ extract was significantly increased by the multi-layer method by treatment with aluminum oxide under the optimum conditions.
[Example 5]

Evaluation of wheat germ extract after aluminum oxide treatment (dialysis method)
A solution (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2) containing 40 μl of wheat germ extract (various samples) prepared in Example 2 at a concentration of 2.5 mg / ml of mRNA encoding the GFP gene. 65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mg / ml creatine kinase, 0.380 mM spermidine, 20 L-type amino acids 3 mM)) 20 μl was mixed to prepare a protein synthesis solution.

  This protein synthesis solution was put into a dialysis membrane, and Buffer-1 (30 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 2.65 mM magnesium acetate, 2.5 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, A protein synthesis reaction was performed at 26 degrees Celsius using 2.5 ml of 16 mM creatine phosphate, 0.380 mM spermidine, 20 types of L-type amino acids (each 0.3 mM) as a dialysate.

  The mRNA serving as a translation template is a plasmid GFP / pEU containing the omega (Ω) sequence of tobacco mosaic virus (TMV), which is a translation initiation reaction sequence linked to the SP6 promoter sequence, and further the GFP gene DNA linked 3 ′ downstream. (WO01 / 27260) was used as a template for transcription using SP6 RNA polymerase (manufactured by Promega) according to a conventional method, and the resulting RNA was purified by ethanol precipitation and used according to a conventional method.

  The protein synthesis solution after the reaction was diluted 100 times, and the fluorescence intensity was measured. These results are shown in FIG. 3 (fluorescence intensity is multiplied by 100 and the value of the stock solution is displayed).

  As shown in FIG. 3, it was confirmed that the protein synthesis ability of the wheat germ extract was significantly increased by the dialysis method by treatment with aluminum oxide under the optimum conditions.

It is a figure which shows the evaluation result by the batch method of the wheat germ extract after an aluminum oxide process. In the figure, the vertical axis indicates the fluorescence intensity (protein synthesis amount) of GFP, and the horizontal axis indicates the type of sample. It is a figure which shows the evaluation result by the multilayer method of the wheat germ extract after an aluminum oxide process. In the figure, the vertical axis indicates the fluorescence intensity (protein synthesis amount) of GFP, and the horizontal axis indicates the type of sample. It is a figure which shows the evaluation result by the dialysis method of the wheat germ extract after an aluminum oxide process. In the figure, the vertical axis indicates the fluorescence intensity (protein synthesis amount) of GFP, and the horizontal axis indicates the type of sample.

Claims (9)

  Manufacture of a cell extract for cell-free protein synthesis comprising a step of subdividing cells, a step of obtaining a cell extract by mixing the subdivided cells and an extraction solvent, and a step of separating and purifying a cell extract-containing solution A method comprising treating a cell fragment with an activator during or after any of these steps.   The activator is at least one activator selected from the group consisting of aluminum oxide, active aluminum oxide, silica gel, zeolite, activated carbon, titanium oxide, zirconium oxide, gel chromatography carrier, and column carrier to which various ligands are bound. The method of claim 1, wherein   The method according to claim 1 or 2, wherein the cell is a plant germ cell.   The method according to claim 3, wherein the plant germ is wheat germ.   The method according to claim 3 or 4, wherein the plant germ is obtained by washing the plant germ after selecting the plant germ from a pulverized product obtained by pulverizing plant seeds.   The method according to claim 1, wherein the fragmentation is performed by impact or cutting.   A cell-free protein synthesis method comprising contacting the cell extract produced by the method according to claim 1 with a translation template.   A cell-free protein comprising the cell extract, ATP, GTP, creatine phosphate, creatine kinase, L-type amino acid, potassium ion and magnesium ion produced by the method according to any one of claims 1 to 6. Synthesis solution.   A reagent kit for cell-free protein synthesis comprising the cell extract produced by the method according to any one of claims 1 to 6 or the protein synthesis solution according to claim 8.