JP4148962B2 - Germ extract for cell-free protein synthesis, method for producing the same, and method for protein synthesis using the same - Google Patents

Description

  The present invention relates to a germ extract for cell-free protein synthesis, a method for preparing the same, and a method for synthesizing a protein using the same, and a self-protein synthesis reaction inhibition mechanism that is activated when a biological tissue or cell is damaged, that is, physiologically equipped. A germ-free solution for cell-free protein synthesis with high synthesis efficiency by removing the self-protein synthesis reaction destruction mechanism as a defense mechanism against pathogens and neutralizing the protein synthesis reaction inhibition activity induced by crushing It relates to a method of preparation.

  In general, the cell-free protein synthesis system has performance comparable to that of living cells in terms of peptide synthesis reaction rate and translation reaction accuracy, but the synthesis efficiency is 0.1% to 1% of that of living cells. There is a disadvantage that it is low. The cell-free protein synthesis systems used today are mainly E. coli, wheat germ, and rabbit reticulocyte-derived systems that are also commercially available. In combination with labeling and immunological methods, it is limited to use as a means of analyzing gene translation products, and is rarely used as a means of preparing proteins (inTranscription and Translation: A practical approach; BD Hames, SJHiggins, Eds .; IRL Press: NewYork, 1984).

  The cause of the decrease in the efficiency of protein synthesis in a cell-free system can be divided into the following three possibilities. (1) Decreased activity of protein synthesis factors associated with cell fluid extraction from organisms, (2) Decreased activity of various factors involved in protein synthesis and substrate concentration during in vitro synthesis reaction, (3) The results produced in 1 and 2 are combined to reduce translation activity. So far, many studies have been made on the efficiency of cell-free protein synthesis systems, but Spirin et al. Limited amino acids, ATP, and GTP as raw materials to cell-free systems prepared by conventional methods. By supplying continuously through an outer filtration membrane (continuous cell-free system), in any of the cell-free systems described above, the reaction time has been sustained for 20 hours or more, and the protein exceeds 20 times the conventional protein. Synthetic yields were achieved (AS Spirin et al. (1988), Science, 242, 1162-1164). In general, the concentration of protein synthesis factors such as ribosomes in cell-free reaction solutions is as low as around 10% compared to that in living cells. However, Yokoyama et al. Used continuous dialysis membranes for reaction solutions containing concentrated E. coli extracts. Have succeeded in synthesizing relatively small molecules of proteins such as CAT and Ras in a high yield of 3-5 mg per ml of reaction system (Kikawa et al., 21st Japan Molecular Biology Society, WID6). These results indicate that, among the possibilities of (2) above, a decrease in substrate concentration contributes to the phenomenon of protein synthesis efficiency decrease in a cell-free system. In other words, the efficiency of the continuous system prevents the decrease of amino acids and energy sources (it is considered that the metabolic enzyme group of those substrates coexist in the decrease in the substrate concentration during the reaction), and at the same time, metabolism such as AMP and GMP. It can be explained that the protein synthesis efficiency is increased by eliminating the accumulation of the product.

  On the other hand, from the study of the ribosome inactivation mechanism of lysin, a cytotoxin protein contained in castor seeds, a group of "antiviral proteins" produced by plants are RNA N-glycosidases identical to lysin A chain. (Y. Endo et al. (1988) Biochim. Biophys. Acta, 954, 224-226). In other words, this enzyme acts on the ribosome and catalyzes the hydrolysis of one N-glycoside bond at a specific site to which the peptide chain elongation factor of its large RNA (23S rRNA in E. coli and 28S rRNA in eukaryotes) binds. As a result of this reaction, the ribosome loses its peptide chain elongation function by dissociation of one molecule of adenine (Y. Endo et al. (1992), TIBS, 17, 266-269). ). Wheat seed endosperm contains a large amount of RNA N-glycosidase termed tritin (W. K. Roberts et al. (1989) Biochemistry, 18, 2615-2621). On the other hand, a protein called thionine, which was isolated as an antibacterial substance, was known to be widely distributed in the plant kingdom, including wheat. Recently, wheat thionine is localized in the endosperm, and this protein initiates a translation initiation reaction. Inhibiting it was shown to strongly inhibit protein synthesis (J. Brummer et al. (1994) Eur. J. Biochem., 219, 425-433, P. Hughes et al. (1997) Plant Physiol., 114, 1568).

  Therefore, as an improvement means for the above (1), JP-A-7-203984 discloses an activity inhibitory factor induced by cell destruction in a cell-free protein synthesis system, which comprises nucleic acid synthesis and / or protein synthesis. Describes a method for synthesizing a protein in which the reaction efficiency is increased by suppressing the activation of an activity inhibitor that is a factor that inhibits the activity of the protein. In this publication, as a method for suppressing the activation of an activity inhibitor, a means for removing it from the protein synthesis system and a means for inhibiting the activation in the protein synthesis system are exemplified. Since it is difficult to selectively remove the activity inhibitor, means using a specific inhibitor is recommended. Specifically, an inhibitor called tritin present in wheat germ is suppressed by a tritin antibody.

  Conventionally, the preparation of wheat germ for cell-free protein synthesis has been performed by the method of A. H. Erikson and G. Blobel (1983), Methods in Enzymol., 96, 38-50. However, as is clear from the reference examples described later, in the germ isolation by the conventional method, it was inevitable to mix endosperm containing tritin, which is an inhibitory substance. For this reason, the ribosome was already 7% in the germ extract immediately after preparation and 25% after 4 hours of incubation, and the activity was reduced. The object of the present invention is to prepare a highly efficient cell extract for cell-free protein synthesis in which endogenous specific inhibitors (such as tritin) that inhibit the protein synthesis reaction are eliminated by a very practical and efficient method. Develop technology.

  As a result of intensive studies to solve the above problems, the present inventors have found that a wheat seed specific inhibitor (ribosome inactivating substance) is localized in the endosperm and can be eliminated by mechanical or chemical treatment. As a result, the present invention has been completed. That is, the present invention is characterized in that an embryo extract for cell-free protein synthesis characterized by not containing plant seed endosperm and an embryo free of endosperm by sonication in an aqueous solution The production method and the protein synthesis method using the germ extract are present.

  The embryo extract of the present invention is characterized by using an embryo from which the endosperm portion has been almost completely removed. As described above, the so-called “embryonic extract” in the related art is in a state in which the endosperm portion is not sufficiently removed and the endogenous specific inhibitor remains contained. In order to prepare the germ extract of the present invention, it is necessary to remove the endosperm containing the endogenous specific inhibitory substance almost completely and purify the germ. In the present invention, the embryo extract containing no endosperm is an embryo extract obtained by removing the endosperm part to such an extent that the ribosome is not substantially deadeninated. In addition, the extent to which ribosomes are not substantially deadenylated means that the ribosome deadenination rate is less than 7%, preferably 1% or less.

  Plant seeds that can be used in the present invention include seeds of plants that are usually selected from wheat, barley, rice, corn and spinach. Among these, wheat, barley or rice is mentioned as a plant seed suitable for the present invention, and wheat is particularly preferred. Furthermore, when wheat germ particles are separated into three types: small grains (0.71 to 0.85 mm), medium grains (greater than 0.85 mm and up to 1 mm), and light grains (medium grains and light weight) Small embryos are preferred because they have the highest protein synthesis activity. In order to prepare the germ extract of the present invention, a crude embryo fraction is first obtained. In order to select this crude germ fraction, it is possible to use a method that has been used in the conventional preparation of germ extract, for example, mechanically crushing plant seeds, collecting germs, flotation, removal of seed coat by adsorption By performing the above, a crude embryo fraction can be obtained.

  In the present invention, this crude embryo fraction is further processed in order to purify the germ. Examples of this treatment method include a method of ultrasonicating an aqueous solution in which a crude germ fraction is dispersed. In addition, high-concentration germ extraction is performed by preparing in the presence of high-concentration amino acids, surfactants, formycin 5′-phosphate (hereinafter referred to as 5′FMP), etc., during germ purification. When a liquid-soluble fraction is obtained and protein synthesis is performed using this fraction, protein synthesis can be performed with higher efficiency.

  The embryo extract of the present invention can be used in a so-called cell-free protein synthesis system. Protein synthesis by the cell-free protein synthesis system of the present invention can be performed in the same manner as in the prior art except that an embryo extract containing no endosperm is used. This method may be a known batch method or a continuous supply system of amino acids and energy sources such as the continuous cell-free protein synthesis system of Spirin et al. 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 ATP, GTP, creatine phosphate, and the like, and examples of the amino acid include 20 L-type amino acids.

  Hereinafter, the present invention will be described in more detail by taking a wheat germ system as an example. The present inventors have studied a method for washing a crude germ fraction separated from wheat seeds using a mill for the purpose of removing contaminating endosperm-derived substances. As a result, it became possible to almost completely exclude contamination of the endosperm-derived protein into the germ fraction by sonicating the crude germ fraction dispersed in an aqueous solution containing 5′FMP and the like. In FIG. 2A, the degree of contamination of endosperm protein before and after ultrasonic washing was tested by immunoblotting using an anti-tritin antibody, using tritin as an index. You can see that In the wheat germ cell-free protein synthesis system prepared using this washed germ as a material, deadenination of the ribosome is not observed (FIG. 2B). Although the results are not shown, the contamination level of thionine, which is known to be localized in the endosperm, was examined by the same method as in the case of tritin. As a result, this ultrasonic cleaning method almost completely removed the germ fraction. It became clear that

  Using such purified embryos, we attempted batch-type cell-free protein synthesis using mRNA encoding dihydrofolate reductase (DHFR) as a model template. Compared with the cell line, the synthesis reaction duration was remarkably prolonged and the synthesis efficiency increased (FIG. 2C). This result can be explained as a result of eliminating contamination of the embryo fraction with endogenous protein synthesis inhibitors such as tritin and thionine by washing.

  The wheat germ fraction thus obtained contains seed coat fragments and various germ particle sizes. Therefore, it is thought that there is a difference in cell-free protein synthesis activity depending on the germ particle size and the like, and as shown in Example 2 described later, using a sieve and an electrostatically charged body, small particles, medium particles, and light particles 3 Fractions were fractionated and each activity was measured. As shown in FIG. 3, it was found that the small germ had the highest cell-free protein synthesis activity. For the following experiment, a cell-free system was prepared from this small germ. In this system, as shown by polyribosome pattern analysis, 60% of ribosomes form polyribosomes 1 hour after the start of the reaction, and at least 67% of the ribosomes in the reaction system form polyribosomes after 2 hours. This is about 10 times that of the conventional method (FIG. 4D). Low concentrations of cycloheximide are known to specifically inhibit the elongation reaction rather than the translation initiation reaction. However, when a protein synthesis reaction is carried out in the presence of 0.2 mM cycloheximide, 78% of the ribosomes are polymorphic. Ribosomes are formed (FIG. 4C). These results indicate that both the translation initiation reaction activity and the peptide chain elongation activity are remarkably increased, and furthermore, the ribonuclease activity is extremely low in this system. FIG. 5 shows the reaction solution separated by SDS-polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue, and the arrow in the figure indicates the synthesized DHFR. From the densitometric quantification of the band, the synthesized protein is estimated to be 0.2 mg in a reaction of 2 hours per ml of reaction volume, and it has achieved a significantly higher translation efficiency compared to conventional commercial kits. I understood.

  Next, the method for preparing the embryo extract was examined. Many types of aminoacyl-tRNA synthetases are considered to have functional structures as supramolecular complexes in cells together with tRNA and peptide chain elongation factors (BS Negrutskiiet al. (1991), Proc. Natl. Acad. Sci. USA, 88, 4991-4995). Therefore, the composition of the germ extract using various surfactants such as NP-40 and a solution containing 20 types of L-type amino acids with high concentrations was examined for the relationship with protein synthesis ability. As shown in FIG. 6, both showed significantly enhanced efficiency of protein synthesis when added alone, but it was found that a more remarkable effect was observed when used together.

  As mentioned above, taking wheat germ as an example, the method for preparing a cell extract for high-efficiency cell-free protein synthesis has been shown, but the technology based on the idea of preventing the operation of protein synthesis inhibition mechanism of this living organism accompanying cell disruption is It can also be applied to the development of a method for preparing a cell extract for highly efficient cell-free protein synthesis from other plant seeds.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the following examples should be regarded as an aid for obtaining specific recognition of the present invention, and the scope of the present invention is limited by the following examples. Is not to be done.

  In order to elucidate the involvement of endogenous protein synthesis inhibitors in the phenomenon of reduced efficiency of protein synthesis in cell-free systems, in relation to the possibility of decreased protein synthesis factor activity associated with cell fluid extraction from organisms The following experiment was conducted. First, using a wheat germ cell-free protein synthesis system as a research material, an embryo extract prepared by isolating and preparing germs by a conventional method (Erickson, AH et al. (1996) Meth. In Enzymol., 96, 38-50) The detachment method by aniline treatment under acidic conditions (Y. Endo et al. (1987) J. Biol. Chem., 262,5908-5912, S. Yoshinari et al. (1996) Eur. J. Biochem., 242, 585-591). As a result, as shown in FIG. 1A, 7% of the ribosome contained in the extract immediately after preparation is already deadenated by tritin. Furthermore, it has been clarified that deadenination further proceeds with the incubation time. Intracellular protein synthesis proceeds efficiently on so-called polyribosomes, in which 10 to 20 molecules of ribosomes are bound at an average mRNA size. Taking into account that ribosomes are completely frozen (freeze) by mRNA de-adenination by RNA N-glycosidase, 10% of the ribosome population in the synthetic system is depleted in the case of mRNAs of average size in higher animals. It is considered that the protein synthesis reaction is completely stopped by adenination. Formycin 5'-phosphate (hereinafter referred to as 5'FMP) is a competitive inhibitor of RNA N-glycosidase (J. Ren et al. (1994) Structure, 2, 7-16). When prepared in the presence and added to the reaction solution, it was found that this deadenination reaction of ribosome by tritin can be prevented (FIG. 1B).

Isolation of wheat germ The method of isolating intact (with germination ability) from seeds using mill, flotation and sieving is the method of Johnston et al. (Johnston, FB et al. (1957) Nature, 179, 160-161). Improved and used. The seeds from Hokkaido (undisinfected) are added to the mill (Fritsch Rotor Speed Mill pulverisette 14) at a rate of 100 grams per minute, and the seeds are gently crushed at a rotation speed of 8000 rpm. This was crushed again at 6000 rpm, and a coarse germ fraction (mesh size 0.71 mm-1.00 mm) was obtained by sieving, and then a mixture of carbon tetrachloride and cyclohexane (carbon tetrachloride: cyclohexane = 2.5: 1) was added. By the flotation used, germs having germination ability were collected from the floating fraction, and the organic solvent was removed by drying at room temperature. Impurities such as seed coat mixed in the embryo fraction were adsorbed and removed using an electrostatically charged body such as a polyethylene plate. Furthermore, the germ particles are separated into three fractions: small particles (0.71 mm to 0.85 mm), medium particles (0.85 mm to 1 mm), and light particles (0.85 mm to 1 mm and light weight) using a sieve and an electrostatically charged body. did. The small particle fraction showed the highest protein synthesis activity (FIG. 3). The light grains are presumed to be those in which the small wounded germs produced in the germ during seed crushing progressed during the flotation operation. Next, in order to completely remove the wheat endosperm components from this sample, it is suspended in a 0.5% solution of NP40, which is a nonionic surfactant, and washed using an ultrasonic cleaner until the cleaning solution does not become cloudy. Was repeated. Ultrasonic cleaning was performed once again in the presence of distilled water to purify the wheat germ.

Preparation and storage of wheat germ extract The following wheat germ extract and protein synthesis solution were prepared in accordance with conventional methods (Erickson, AH et al. (1996) Meth. In Enzymol., 96, 38-50). . The following operation is carried out at 4 degrees Celsius. Purified wheat germ frozen in liquid nitrogen is ground in a mortar, and 1 ml of the extracted solution (80 mM HEPES-KOH, partially modified from Patterson et al.) Per 1 g of the obtained powder. , pH 7.8, 200 mM potassium acetate, 2 mM magnesium acetate, 4 mM calcium chloride, 8 mM dithiothreitol, each containing 1 mM (microM) protease inhibitor FUT, E-64, and PMSF) The mixture was stirred while being careful not to generate bubbles. The supernatant obtained by centrifugation at 30k xg for 15 minutes is recovered as an embryo extract and equilibrated in advance with a solution (40 mM HEPES-KOH, pH 7.8, 100 mM potassium acetate, 5 mM magnesium acetate, 4 mM dithiothreitol). Gel filtration was performed using a Sephadex G-25 column (Coarse), and the sample concentration was adjusted to 170 to 250 A260 nm (A260 / A280 = 1.5), and if necessary, polyethylene glycol and dehydrating agent were used. After concentrating by the dialysis method described above, it was aliquoted and frozen and stored in liquid nitrogen. A solution containing 0.1% NP-40 and 20 types of 0.6 mM L-type amino acids was used.

Preparation of Batch-type Wheat Germ Cell-Free Protein Synthesis Solution The reaction solution contains 20-60% of the germ extract and has the following composition according to the method of Erickson et al. 1000 units / ml Ribonuclease inhibitor (RNasin), 30 mM HEPES-KOH, pH 7.6, 95 mM potassium acetate, 2.65 mM magnesiumacetate, 2.85 mM dithiothreitol, 0.5 mg / ml creatine kinase, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine, 20 types of L-amino acids (each 0.3 mM), 0.05% NP-40, and other methods already reported (Endo, Y. et al. (1992) J. Biotech., 25, 221 -230) dihydrofolatereductase mRNA with CAP (80 mg / ml reaction volume) and 50 mCi (per ml reaction volume) [U- ^14C ] leucine (166 mCi / mmol). In addition, potassium, magnesium, and spermidine in the reaction solution were reacted at each optimum concentration according to the template mRNA species to be used. The reaction was performed at 20 to 26 degrees Celsius.

Preparation of continuous wheat germ cell-free protein synthesis system by dialysis The method of continuous wheat germ cell-free protein synthesis has been reported (Endo, Y. et al., (1992) J. Biotech., 25, 221-230) The reaction solution of Example 2 was placed in a despodializer (Spectra / PorRCE, MWCO: 25k, volume: 0.5 ml), and the dialysis solution (20 mM HEPES-KOH, pH 7.6, 95 mM potassium acetate) 10 times the volume of the reaction solution. , 2.65 mM magnesium acetate, 4 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatinephosphate, 0.380 mM spermidine, 20 L-amino acids (each 0.3 mM), 0.005% NaN3, 0.05% NP-40, E The reaction was performed at 20 degrees Celsius in a dialysis system against -64, PMSF 1 mM each).

Protein Synthesis Under the conditions obtained by the above method, wheat germ continuous cell-free synthesis was attempted. As shown in FIG. 7, various proteins having a molecular weight of 5,000 to 130,000 were efficiently synthesized. The synthesis efficiency was 0.3-1.8 mg / ml reaction volume (Table 1).

In the examples, the method for analyzing protein synthesis activity was performed according to the above-mentioned description by Endo et al. In addition, ^14C- leucine incorporation measurement method, separation of synthetic protein by SDS-polyacrylamide gel electrophoresis, staining with Coomassie brilliant blue, and polyribosome pattern analysis by sucrose density gradient centrifugation are described in Endo et al. (Endo, Y. et al. (1992) J. Biotech., 25, 221-230, Endo, Y. et al. (1975) Biochim. Biophys. Acta, 383, 305-315). It was.

  The germ extract for cell-free protein synthesis of the present invention is extremely useful for mass-production of proteins in a cell-free system, for example, mass production of new enzymes and antibodies in the field of evolutionary molecular engineering. In combination with protein labeling methods, specific and efficient labeling of proteins in cell-free systems is possible, and protein-protein interactions and protein-nucleic acid interactions are examined in genome function analysis etc. Useful for.

(A) Deadenination in a wheat germ cell-free system. A cell-free protein synthesis system was prepared from wheat germ prepared by a conventional method, and kept warm at 26 degrees Celsius. The reaction solution was taken out over time, RNA extracted and purified by the phenol method was treated with aniline, rRNA was separated by 4% polyacrylamide gel electrophoresis, and stained with ethidium bromide. The arrow in the gel photograph is a fragment on the 3 ′ side of 28S rRNA resulting from cleavage of the phosphodiester at the deadenine site by the elimination reaction with aniline, and the number at the top of the gel indicates the reaction time (h). From the fluorescence intensity ratio of this RNA fragment and 5.8S rRNA, the deadenine rate was calculated based on a standard graph prepared in advance. The vertical axis of the graph represents the deadenination rate (%), and the horizontal axis represents the reaction time (hr). (B) Inhibition of RNA N-glycosidase by 5'FMP (blocking deadenination reaction). Each reaction time is 5 hours. Removal of tritin from the germ by ultrasonic cleaning. (A) The tritin content was determined by separating the protein from the embryo extract using SDS-polyacrylamide gel electrophoresis, and then staining the tritin with tritin antibody (兎) and goat anti-goat IgG antibody-horseradish peroxidase. Lanes 1 to 4 are embryos isolated by a conventional method, Lane 5 is a novel method, and the left end of the lane is a molecular weight marker. (B) RNA N-glycosidase activity in washed germ extract. A protein synthesis system was prepared using various extracts, and a sample after 3 hours of reaction was examined for deadenine activity in the same manner as in FIG. 1A. Separately purified tritin was added as a lane 1 positive control. Lanes 2 to 5 were prepared by conventional methods, and lanes 6 to 9 were prepared from embryos isolated by a novel method. The arrow indicates the 3'-side fragment of 28S rRNA resulting from cleavage of the phosphodiester at the deadenine site by b elimination reaction with aniline. (C) Protein synthesis activity in batch system using washed germ extract. As the translation template, mRNA encoding DHFR with 5'CAP was used. Germ particle size and cell-free protein synthesis activity. As shown in Example 2, three fractions of small grains (0.71 mm to 0.85 mm), medium grains (0.85 mm to 1 mm), and light grains (0.85 mm to 1 mm and sucked by an electrostatically charged polyethylene plate) Each cell-free protein synthesis activity was measured using DHFR-mRNA with 5′CAP as a template. Polyribosome pattern analysis using sucrose density gradient centrifugation. Ribosomes were separated by 10-40% linear sucrose density gradient centrifugation from the reaction solution at the start of protein synthesis, 0 hours (A), 1 hour (B), and 2 hours (C, D). In the figure (C), protein synthesis reaction was performed in the presence of 0.2 mM cycloheximide (■ --- ■). (D) is a cell-free system using an extract prepared from a conventional unwashed germ. Analysis of translation products by SDS-polyacrylamide gel electrophoresis. (A) After electrophoresis, stained with CBB (Coomassie Brilliant Blue) (B) Autoradiogram. The protein synthesis reaction was carried out at 26 ° C in the presence of 14C-leucine at 0 ° (lane 1), 1 hour (lane 3), and 2 hours (lanes 2 and 4) using DHFR-mRNA with 5'CAP as a template. . Lane 2: Commercially available kit; Lane 3, 4: Embryo isolated by the novel method. Arrows indicate translation products. Examination of embryo extract preparation method. In order to examine the method of preparing the embryo extract from the viewpoint of the composition of the extract preparation solution, the conventional composition solution (X --- X) and a solution with a final concentration of 0.05% NP-40 (- -), Prepare a germ extract using a solution containing 300 mM each of 20 L-type amino acids (●--●) and a solution containing NP-40 and amino acids at the same concentration (● --- ●). A cell-free system was constructed. All the protein synthesis reaction systems were reacted in the same manner as in FIG. 4 except that NP-40 was contained (final concentration 0.05%). An example of a protein synthesized using a wheat germ continuous cell-free synthesis system. A continuous cell-free synthesis system was constructed using an extract prepared from a small grain germ in the presence of a mixture of NP-40 and amino acids, and protein synthesis was performed. The reaction was incubated at 20 degrees for 18 hours and then analyzed by autoradiography as in FIG. The template RNAs are lane 1: bromo mosaic virus (BMV); lane 2: DHFR; lane 3: human mitochondrial met-tRNA synthetase; lanes 4-6; proteasome activating enzymes a, b, g. BMV lanes 1 to 4 and arrowheads indicate the respective translation products.

Claims (3)

Plant seed for cell-free protein synthesis in which the deadenination rate of ribosome is less than 7% by sonication in an aqueous solution containing a surfactant and / or formycin 5'-phosphate (Formycin 5'-phosphate) A method for producing a plant seed germ extract for cell-free protein synthesis, comprising obtaining a germ extract.   The method for producing a plant seed germ extract for cell-free protein synthesis according to claim 1, wherein the plant seed is selected from any one of wheat, barley, rice, corn and spinach. A cell-free protein synthesis method using the plant seed germ extract for cell-free protein synthesis obtained by the production method according to claim 1 or 2 .