JP4267621B2 - Cell-free protein synthesis method - Google Patents

Description

  The present invention relates to a protein synthesis method using a cell-free system.

  With the completion of the genome project coming soon, the focus of research is rapidly expanding from gene structure analysis to gene function analysis. A protein in a cell is not functioning alone, but is functionally expressed by interacting with a wide variety of protein factors, nucleic acids, low molecular species, cell membrane components, etc. It is thought that the biological function is carried out as the sum of

  One of the main tasks of the post-genome project is to analyze the relationship between the structure and function of various protein factor complexes. The results obtained from this research range from basic biology including structural biology and biochemistry to elucidation of the relationship between gene translation products and pathogenesis in the medical field as an application, and further to the development of pharmaceuticals. It is expected to provide extremely important knowledge.

  As a method of performing protein synthesis reaction that proceeds efficiently in cells in vitro, components such as ribosome, which is a protein translation apparatus provided in cells, have been extracted from living organisms, and a translation template, Research has been actively conducted on so-called cell-free protein synthesis methods in which proteins are synthesized in vitro by adding amino acids serving as substrates, energy sources, various ions, buffer solutions, and other effective factors (Patent Documents). 1-5).

  E. coli, wheat germ, or rabbit reticulocytes are used to prepare a reaction system for cell-free protein synthesis, that is, a cell extract or a tissue extract for protein synthesis used in a cell-free protein synthesis system. ing. The cell-free protein synthesis system has the advantages of maintaining performance comparable to that of living cells in terms of peptide synthesis rate and translation reaction accuracy, and does not require complicated chemical reaction steps or complicated cell culture steps. Therefore, practical systems have been developed so far. However, cell extracts extracted from cells of organisms generally have low protein synthesis efficiency due to their extremely unstable protein synthesis ability, and the quality of cell extracts during storage has been markedly degraded. The amount of the synthesized product obtained by the cell protein synthesis system is a small amount that can be detected by radioisotope labeling or the like, and could not be used as a practical protein production means.

The present inventors have previously described (1) a cell extract preparation for cell-free protein synthesis and a cell-free protein synthesis method as a method for solving the disadvantages of the conventional cell-free protein synthesis system, and (2) versatility and high performance. A template molecule having an efficiency function and a cell-free protein synthesis method using the template molecule are provided (Patent Documents 6-7).
JP-A-6-98790 JP-A-6-225783 JP-A-7-194 JP-A-9-291 JP 7-147992 A WO00 / 68412 WO01 / 27260

An apparatus for continuously performing cell-free protein synthesis for improving the efficiency of protein synthesis has been reported. Conventional continuous cell-free protein synthesizers include ultrafiltration membrane methods, dialysis membrane methods, column chromatography methods in which a translation template is immobilized on a resin (Spirin, A., et al., (1993) Methods in Enzymology). , 217, 123-142). Among these, the ultrafiltration membrane method and the dialysis membrane method are widely used because they are easy to handle. However, in the continuous method using these membranes, <1> the material strength of the membrane used is low; <2> the membrane function is degraded due to clogging; <3> the operation is complicated and complicated. Issues that need to be solved, such as the need for technology;
In addition, the continuous cell-free protein synthesis method, which is performed manually using the ultrafiltration membrane method, dialysis membrane method, etc., can be used for protein synthesis from a small number of genes, but increases the protein production from a large number of genes. It was difficult to do with efficiency. Therefore, the shortcomings of the conventional continuous cell-free protein synthesis method were solved for the development of a high-throughput multi-sample fully automated protein synthesis system that enables highly efficient protein production from a large number of genes. There is an urgent need to develop new technologies.

In the present invention, in order to solve the above problems, the following cell-free protein synthesis method is used.
One aspect of the present invention is to directly contact a synthetic reaction solution (reaction phase) containing a biological extract with a substrate and an energy source supply solution (supply phase), and by free diffusion through the contact interface of both phases, The supply phase substrate and energy source molecules are continuously supplied to the reaction phase translation reaction system, and by-products generated in the reaction phase are eliminated, thereby extending the duration of the synthesis reaction and improving the efficiency of the synthesis reaction. It is a cell-free protein synthesis method by a diffusion continuous batch method characterized by enhancing the pH.
The present invention may also be a cell-free protein synthesis method characterized in that, in the cell-free protein synthesis method, a wheat germ extract is used as a biological extract.
Furthermore, the present invention may be a cell-free protein synthesis method characterized by using an E. coli extract as a biological extract in the cell-free protein synthesis method.
Further, in the cell-free protein synthesis method, a cell-free protein synthesis method characterized by diluting out by-products generated in the reaction phase to the supply phase is also included in the scope of the present invention.
Furthermore, in the cell-free protein synthesis method, the cell-free protein synthesis method is also included in the scope of the present invention, wherein the direct contact interface formed between the reaction phase and the supply phase is a vertical plane. It is.
In one embodiment of the present invention, a cell-free protein synthesis reaction solution containing a wheat germ extract is pre-incubated (pre-incubation), and then a substrate and an energy source supply solution are added to the cell-free protein synthesis containing the wheat germ extract. A cell-free protein synthesis method comprising diluting a reaction solution.
Furthermore, one embodiment of the present invention is an amino acid, ATP, GTP, or creatine phosphate necessary for protein synthesis using a gel filtration column or a semipermeable membrane in the reaction solution after stopping the synthesis reaction in the batch cell-free protein synthesis method. The cell-free protein synthesis method is characterized in that the efficiency of the synthesis reaction is enhanced by re-supplying the raw materials and energy sources, etc., and simultaneously removing the by-products generated in the reaction from the reaction solution.

In the present invention, the following cell-free protein synthesis method is used in order to solve the above problems.
“1. A cell-free protein synthesis method comprising the following steps in a batch-type cell-free protein synthesis method:
1) Step of re-supplying one or more of amino acids, ATP, GTP and creatine phosphate to the reaction solution after stopping the synthesis reaction 2) Excluding by-products generated in the reaction solution after stopping the synthesis reaction from the reaction solution Step 2 2. The cell-free protein synthesis method according to item 1, wherein the refeeding step and the removing step are performed using a gel filtration column or a semipermeable membrane.
3. 3. The cell-free protein synthesis method according to item 1 or 2, wherein a step of restarting a protein synthesis reaction is performed after the resupplying step and the eliminating step.
4). 4. The cell-free protein synthesis method according to item 3 above, wherein the step of refeeding, the step of eliminating, and the step of restarting the synthesis reaction are repeated. "

  One embodiment of the present invention is a general cell-free protein synthesis method in which a synthetic reaction solution (reaction phase) containing a biological extract and a substrate and energy source supply solution (supply phase) are semipermeable membranes or ultrafiltration membranes. By-diffusion generated in the reaction phase at the same time as the substrate and energy source molecules of the supply phase are continuously supplied to the reaction phase by free diffusion through the contact interface of both phases, with direct contact without separation by a barrier such as It is a diffusion continuous batch cell-free protein synthesis method characterized in that the reaction duration is extended by excluding substances to the feed phase, thereby increasing the efficiency of the synthesis reaction.

  In the diffusion continuous batch cell-free protein synthesis method, the interface between both phases may be formed as a horizontal plane or a vertical plane. In order to form the interface as a horizontal plane, for example, a reaction phase is first added to a reaction vessel to form a lower layer, and then a supply phase is gently overlaid on the reaction phase so as not to disturb the interface between both phases. Good (see FIG. 1). The reaction vessel may have any shape and size as long as it provides a sufficient diffusion rate of the solute between the two phases. For example, a test tube or a multiwell microtiter plate can be used, but is not limited thereto. It is also possible to form an interface between both phases in a vertical plane by layering the synthesis reaction solution (reaction phase) and the supply solution (supply phase) and then adding a centrifugal operation to the reaction vessel containing them. is there.

  The larger the contact interface area of both phases, the higher the mass exchange rate by diffusion and the higher the protein synthesis efficiency. Accordingly, the optimum volume ratio of the supply phase to the reaction phase varies depending on the interface area between the two phases. The volume ratio of the supply phase to the reaction phase is not particularly limited. For example, when the interface is circular and the diameter is 7 mm, the ratio is preferably 1: 4 to 1: 8, more preferably 1: 5.

The synthesis reaction solution forming the reaction phase contains a biological extract necessary for a cell-free protein synthesis reaction and a desired mRNA as a template for protein synthesis, and is a composition used in a conventionally known batch-type cell-free protein synthesis system. Consists of. As the biological extract, a known biological extract used in a conventional cell-free protein synthesis method such as a wheat germ extract, an E. coli extract, or a rabbit reticulocyte extract can be used. These extracts can be prepared according to a method known per se. Wheat germ extract has been reported [Madin K. et al. et al. , Proc. Natl. Acad. Sci. USA (2000), 97, 559-564] (WO00 / 68412) is preferably used. Specifically, for example, when a wheat germ extract is used as a biological extract, the synthesis reaction solution contains 48% of the total volume (concentration is 200 A ₂₆₀ nm units / ml). Composition of concentration [1,000 units / ml ribonuclease inhibitor (RNAsin), 30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 0.5 mg / ml creatine kinase 1.2 mM adenosine triphosphate (ATP), 0.25 mM guanosine triphosphate (GTP), 16 mM creatine phosphate, 0.380 mM spermidine, 20 L-type amino acids (each 0.3 mM), 0.05% NP-40, 600 μg / ml mRNA]. The composition of the synthesis reaction solution is not limited to the above composition, and any composition that allows a cell-free protein synthesis reaction to proceed efficiently can be used. For example, after synthesizing mRNA with a transcription reaction solution containing a plasmid (encoding the target gene), RNA polymerase, nucleotides, etc. instead of the above mRNA, the composition of the transcription reaction solution is subsequently determined by gel filtration or dialysis. You may convert into the solution which consists of a composition suitable for translation reaction, and you may use the obtained solution as a synthesis reaction solution (refer Example 6 below).

  The composition of the synthesis reaction solution can be appropriately changed depending on the type of biological extract to be used. When Escherichia coli is used as a biological extract, a report [Pratt, J. et al. M.M. Transcription and Translation (1984), 179-209, Hames, B .; D. & Higgins, S .; J. et al. , Eds, IRL Press, Oxford], a protein synthesis reaction solution having a composition according to the same method may be prepared using an E. coli extract prepared according to the same method. For example, the Escherichia coli extract contains 50% of the total volume, and has the following final composition (57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mM ammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothrei Toll, 0.3 U / ml pileate kinase, 0.17 mg / ml E. coli tRNA mixed solution, 34 mg / ml L-5-formyl-5,6,7,8-tetrahydrophoric acid, 6.7 μg / ml plasmid (objective) Gene), 33 μg / ml T7 RNA polymerase, 1.2 mM ATP, 0.85 mM GTP and UTP and CTP, 56 mM phosphoenolpyruvate, 20 L-type amino acids (0.2 mM each)] Prepare a transcription reaction solution, first synthesize mRNA. Thereafter, the composition of the transcription reaction solution may be converted into a solution having a composition suitable for translation reaction by gel filtration or dialysis, and the resulting solution may be used as a synthesis reaction solution (Example 6 below). See). In addition, in the case of a cell-free protein synthesis system using an Escherichia coli extract, after first synthesizing mRNA with a transcription reaction solution as described above, the supply solution is overlaid on the transcription reaction solution to carry out a translation reaction under static conditions. The protein synthesis reaction may be performed at a suitable temperature. The composition of the synthesis reaction solution is not limited to the above composition, and any composition that allows a cell-free protein synthesis reaction to proceed efficiently can be used. For example, instead of the plasmid (encoding the target gene), T7 RNA polymerase, UTP, and CTP in the above transcription reaction solution, a method known per se (Gurevic, VV, (1996) Methods in Enzymology, 275, The mRNA encoding the target gene prepared in 383-397) may be added as appropriate to prepare a synthesis reaction solution having a composition suitable for the translation reaction.

  Furthermore, by adding sugar alcohols such as inositol, xylitol and / or ficoll to the above synthetic reaction liquid to increase the viscosity and density of the synthetic reaction liquid, and by controlling the mixing speed between the two phases of the reaction phase and the supply phase Further, the protein synthesis reaction can be further stabilized.

  The supply solution forming the supply phase contains a substrate and an energy source, for example, amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for protein synthesis reaction. Specifically, for example, when the above protein synthesis reaction solution containing wheat germ extract is used in the reaction phase, 30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithio A feed solution consisting of a composition of threitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine and 20 L-type amino acids (each 0.3 mM) can be used. When the above protein synthesis reaction solution containing an E. coli extract is used for the reaction phase, for example, 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mM ammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol. 34 mg / ml L-5-formyl-5,6,7,8-tetrahydrophoric acid, 1.2 mM ATP, 0.85 mM GTP and UTP and CTP, 56 mM phosphoenolpyruvate, 20 L-type amino acids A feed solution having a composition (each 0.2 mM) can be used.

  The protein synthesis reaction is carried out under static conditions, and the reaction temperature is the optimum temperature usually used in various cell-free protein synthesis methods. When a wheat germ extract is used as the biological extract, the temperature is 20 ° C. to 30 ° C., preferably 26 ° C., and when an E. coli extract is used, the temperature is 30 ° C. to 37 ° C., preferably 30 ° C.

  One embodiment of the present invention is a cell-free protein synthesis method using a wheat germ extract, by pre-incubating the reaction solution (pre-incubation) and then adding the diluted solution to dilute the synthesized reaction solution, It is a diluted batch cell-free protein synthesis method that extends the duration of the synthesis reaction and thereby increases protein synthesis efficiency.

  In the diluted batch type cell-free protein synthesis method, a conventional batch type cell-free protein synthesis reaction solution, for example, a synthesis reaction solution having the above-described composition, is first pre-incubated for 15 to 30 minutes to carry out protein synthesis. Thereafter, supply in the diffusion continuous batch protein synthesis method including a substrate and an energy source, a substrate and an energy source, such as amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for protein synthesis reaction. A solution having the same composition as the solution is added, and the wheat germ extract contained in the reaction solution is diluted from 7% to about 12%, and the synthesis reaction is performed. The optimum temperature for the protein synthesis reaction is 20 ° C. to 30 ° C., preferably 26 ° C. when a wheat germ extract is used. Enzymes and translated protein factors are generally known to be less stable at low concentrations. Therefore, by adding a known stabilizer such as inositol, xylitol, ficoll, etc. to the synthesis reaction solution in advance, It is possible to further improve efficiency.

  The dilution batch type protein synthesis method was very effective in a cell-free protein synthesis system using a wheat germ extract, but its effect could not be confirmed in a system using an E. coli extract. This may be due to the characteristics of the wheat germ extract.

  Also, in this method, preincubation is a very important step, and if this operation is omitted, the efficiency of the protein synthesis reaction will decrease, so that a stable translation initiation complex is formed during this preincubation. Conceivable. However, the molecular mechanism related to this unique phenomenon is for further study.

  Furthermore, one embodiment of the present invention is a batch-type cell-free protein synthesis method, in which a substrate or an energy source necessary for protein synthesis using a gel filtration column or a semipermeable membrane as a reaction solution after termination of the synthesis reaction, such as amino acids, ATP, A cell-free protein synthesis method characterized in that the efficiency of the synthesis reaction is improved by re-supplying raw materials such as GTP and creatine phosphate and at the same time eliminating by-products generated in the reaction from the reaction solution. . This method is a batch method in which the protein synthesis reaction, the supply of the substrate and energy source to the reaction solution, and the removal of by-products are a discontinuous process, and basically a continuous cell-free protein of Spirin et al. It is different from the synthesis method.

  In this discontinuous batch-type cell-free protein synthesis method, a conventional batch-type cell-free protein synthesis reaction is started using a reaction vessel such as a test tube, and the temperature of the reaction solution is set to 0 ° C. to 4 ° C. after the synthesis reaction is stopped. A low molecular weight obtained by completely stopping the protein synthesis reaction by lowering to 0 ° C., and equilibrating the reaction solution after the termination of the reaction in advance with a solution containing a substrate or an energy source such as amino acid, ATP, GTP, creatine phosphate, etc. It is subjected to chromatography using gel filtration particles for compound separation, such as Sephadex G-25. As the solution used for equilibration, a solution having the same composition as the supply solution in the diffusion continuous batch protein synthesis method can be used.

  By-products are excluded in the Sephadex particles by the gel filtration operation, and a cell-free protein synthesis solution exchanged with fresh amino acids, ATP, GTP, creatine phosphate and the like can be recovered in the void fraction. When the collected solution is kept warm again, the translation reaction is started and the protein synthesis reaction proceeds for several hours. When the synthesis reaction stops again, the above gel filtration operation is repeated again. By repeating this method, the synthesis reaction that stops in a short time in a normal batch method can be continued for a long time, and as a result, the protein synthesis yield increases.

  In the discontinuous batch type cell-free protein synthesis method, the dialysis method may be used in place of the gel filtration method for the purpose of resupplying substrates and energy sources and eliminating reaction byproducts. The above effects can be obtained.

  As described above, <1> a reaction phase comprising a synthetic reaction solution containing a cell extract according to the present invention and a supply phase comprising a substrate containing amino acids, ATP, GTP, creatine phosphate, etc., and an energy source supply solution are directly used. A diffusion continuous batch method in which the substrate and energy source of the feed phase are continuously fed to the reaction phase by free diffusion through the contact interface of both phases, and by-products generated in the reaction phase are excluded to the feed phase. <2> In a cell-free protein synthesis system using wheat germ, a dilution batch method by reducing the cell extract concentration contained in the synthesis reaction solution, and further <3> gel filtration method or dialysis after the protein synthesis reaction is stopped Using this method, the amino acid, ATP, GTP, creatine phosphate, and other substrates and energy sources necessary for protein synthesis are resupplied to the synthesis reaction solution. At the same time, the discontinuous batch method that eliminates the by-products generated by the reaction discontinuously, unlike conventional continuous batch cell-free protein synthesis method is very effective as a cell-free protein synthesis method. Each of these methods may be performed alone or in combination. For example, for the purpose of improving protein synthesis efficiency, a diffusion continuous batch method and a discontinuous batch method, or a combination of a dilution batch method and a discontinuous batch method may be performed. In addition, the concentration of the cell extract or tissue extract added first may be increased, and the above three methods may be combined.

  In addition, the present invention allows the duration of the cell-free protein synthesis reaction to be extended, so that the protein synthesis efficiency is significantly improved as compared to the conventional batch method, and the continuous cell-free protein utilizing the semipermeable membrane established by Spirin et al. Cell Protein Synthesis [Spirin, A. , Et al. , (1993) Methods in Enzymology, 217, 123-142], a cell-free protein synthesis method could be established.

  Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples.

As an example of a diffusion continuous batch type cell-free protein synthesis method, protein synthesis was performed using a wheat germ extract by the multi-layer diffusion continuous batch method shown in FIG.
Wheat germ extract has been reported [Madin K. et al. et al. , Proc. Natl. Acad. Sci. USA (2000), 97, 559-564] (WO 00/68412).
In addition, a versatile plasmid pEU1 (FIG. 6) (WO01 / 27260) constructed by Endo was used to synthesize mRNA as a translation template in the wheat germ cell-free protein synthesis reaction. A jellyfish green fluorescent protein (GFP) gene (gfp gene) was used as a gene encoding the target protein, and the gene was inserted into the plasmid according to a conventional method. The obtained plasmid was cleaved with HindIII to obtain a linear form, and mRNA was synthesized by a conventional method using this as a transcription template. The synthesized mRNA does not have CAP at the 5 ′ end, has an AMV-Ω sequence at the 5 ′ end as an untranslated sequence, and 500 bases derived from the plasmid at the 3 ′ end. The AMV-Ω sequence refers to a base sequence in which the 5 ′ terminal leader structure of alfalfa mosaic virus mRNA (AMV-mRNA) and the 5 ′ terminal Ω sequence of tobacco mosaic virus mRNA (TMV-mRNA) are connected in series ( WO 01/27260). The addition of these untranslated sequences enhances the stability of the RNA, and as a result, the use of this mRNA increases the efficiency of cell-free protein synthesis. The same results as below were obtained even when mRNA having CAP at the 5 ′ end was used.

Next, the composition of the following final concentration containing the wheat germ extract (concentration is 200A ₂₆₀ nm units / ml) [48 units of the whole volume: 1,000 units / ml ribonuclease inhibitor (RNAsin) (TAKARA) Manufactured), 30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 0.5 mg / ml creatine kinase, 1.2 mM adenosine triphosphate (ATP), 0 .25 mM guanosine triphosphate (GTP), 16 mM creatine phosphate, 0.380 mM spermidine, 20 types of L-type amino acids (each 0.3 mM), 0.05% NP-40, 600 μg / ml mRNA] A reaction solution was prepared. In order to measure the protein synthesis amount, 4 μCi of ¹⁴ C-leucine (300 mCi / mmol) was added to 1 ml of the protein synthesis reaction solution [Proc. Natl. Acad. Sci. USA (2000), 97, 559-564].
This protein synthesis reaction solution is added to reaction vessels of 7 mm, 5 mm, and 3 mm in diameter (a microtiter plate, a 1.5 mL capacity test tube, and a 0.2 mL capacity test tube, respectively), and a 5-fold volume thereon. [30 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.65 mM magnesium acetate, 2.85 mM dithiothreitol, 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.380 mM spermidine And 20 kinds of L-type amino acids (0.3 mM)] are gently layered so that the interface is not disturbed, and incubated at 26 ° C. for 3, 6, 9, and 17 hours under static conditions to conduct a protein synthesis reaction It was. The amount of the synthesized protein was measured using the incorporation of a radioisotope into the trichloroacetic acid insoluble fraction as an index according to a conventional method, and the synthesized protein was confirmed by autoradiography as usual [Endo, Y. et al. et al. (1992) J. Am. Biotech. , 25, 221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564].

The results are shown in FIG. 2 (A) and FIG. 2 (B).
As a control, a conventional batch cell-free protein synthesis method was performed. In this method, mRNA, wheat germ extract, and a protein synthesis reaction solution containing these are the same as those used in the diffusion continuous batch cell-free protein synthesis method, except that the feed solution is not added.
As is clear from (A) of FIG. 2, the protein synthesis reaction was stopped in 1 hour after the start of the reaction in the conventional batch system (O-O). This result is the same as the previously reported result [Endo, Y. et al. (1992) J. Am. Biotech. , 25, 221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564].
On the other hand, in the multi-layer method (large □-□) using a reaction vessel with a 7 mm aperture (interface area is 0.385 cm ² ), the synthesis reaction continues even after 17 hours from the start of the reaction. More than 9 times the law. Furthermore, as a result of investigating the influence of the interface area between the reaction phase and the supply phase on the synthesis reaction using reaction vessels of different sizes, the synthesis efficiency 9 hours after the start of the reaction is compared with that when using a reaction vessel with a diameter of 7 mm. In a reaction vessel having a diameter of 5 mm (interface area is 0.196 cm ² ) (medium □-□), the reaction vessel having a diameter of 3 mm (interface area is 0.071 cm ² ) (small □ ---) was 75%.
In addition, the autoradiography shown in FIG. 2B shows both the synthesis reaction time lapse of the protein synthesized by the conventional batch method and the diffusion continuous batch method, and the molecular weight and synthesis amount of the synthesized product ( This completely supported the experimental results obtained by examining the amount of protein synthesis by measuring ¹⁴ C-leucine uptake shown in A). In FIG. 2B, the diffusion continuous batch method is indicated as a multi-layer method. Moreover, the result of protein synthesis by the diffusion continuous batch method showed only the result in a reaction vessel having a diameter of 7 mm.

Also, in this method, after synthesizing mRNA with a transcription reaction solution by the transcription / translation integrated protein synthesis method shown in Example 6 below, the composition of the transcription reaction solution is subsequently translated by gel filtration or dialysis. The same results were obtained when the protein was synthesized in the same manner as described above by converting the solution into a solution having a composition suitable for the reaction and using the resulting solution as a synthesis reaction solution.
From the above results, <1> diffusion continuous batch protein synthesis method using wheat germ extract has significantly higher synthesis efficiency than conventional batch methods, and <2> the synthesis efficiency is the interface area between the reaction phase and the supply phase. It was found that the larger the is, the better. It was also found that the increase in synthetic yield by this method was due to the duration of the synthesis reaction time.

  As an example of a diluted batch-type cell-free protein synthesis method, a pre-incubation at 26 ° C. for 15 minutes in a conventional batch method using the protein synthesis reaction solution containing the wheat germ extract prepared in Example 1 and mRNA encoding GFP After that, after adding a 5-fold volume of the diluted solution, the protein synthesis reaction was performed by further incubation at 26 ° C. for 3, 6, and 9 hours under static conditions. As the diluting solution, a solution having the same composition as the feed solution prepared in Example 1 was used. The amount of the synthesized protein was measured in the same manner as in Example 1, and the results are shown in FIG. 2 (A) (■-■) and FIG. 2 (B).

As apparent from FIG. 2 (A), when cell-free protein synthesis is performed in a diluted batch type compared to the conventional batch type (○ ―― ○) in which the synthesis reaction stops in 1 hour, the reaction starts 6 The synthesis reaction continued linearly until time (■ ―― ■).
The autoradiography shown in FIG. 2 (B) fully supports the experimental results obtained by examining the amount of protein synthesis by measuring ¹⁴ C-leucine incorporation shown in FIG. 2 (A). there were.
Although this diluted batch cell-free protein synthesis method is inferior in synthesis efficiency to the diffusion continuous batch method shown in Example 1, the protein synthesis amount is about 3 times that of the conventional batch method, and the synthesis is significantly higher. Showed efficiency.

Also in this method, after synthesizing mRNA with a transcription reaction solution by the transcription / translation integrated protein synthesis method shown in Example 6 below, the composition of the transcription reaction solution is subsequently determined by gel filtration or dialysis. The same results were obtained when the protein was synthesized in the same manner as described above using the solution obtained as a synthesis reaction solution after conversion to a solution having a composition suitable for the translation reaction.
Further, in the above diluted batch type cell-free protein synthesis method, when the pre-incubation reaction operation was omitted, the remarkable phenomenon of the synthesis reaction as described above was not observed. Furthermore, in a cell-free protein synthesis system using an Escherichia coli extract, a dilution batch type effect was not observed.
As described above, in the cell-free protein synthesis system using the wheat germ extract, it has been demonstrated that the diluted batch cell-free protein synthesis method is also an effective protein synthesis means.

  In the diffusion continuous batch cell-free protein synthesis method, in addition to the GFP synthesis performed in Example 1, it was demonstrated that E. coli-derived dihydrofolate reductase (DHFR) can be synthesized, and this method is used for the synthesis of general protein molecular species. It was confirmed that it was effective.

  Protein synthesis was performed in the same manner as in Example 1 using a protein synthesis reaction solution having the same composition as in Example 1 except that the mRNA was a DHFR-encoding mRNA. The results are shown in FIG. The amount of synthesized protein was measured using the incorporation of a radioisotope into the trichloroacetic acid insoluble fraction as an index according to a conventional method, and the synthesized protein was confirmed by separation by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue ( CBB) staining [Endo, Y. et al. et al. (1992) J. Am. Biotech. , 25, 221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564].

  As shown in Fig. 3 (A), in the DHFR synthesis, the synthesis reaction time of the diffusion continuous batch cell-free protein synthesis method (■ ―― ■) is also compared with the conventional batch method (○ ―― ○). Significantly persisted. The analysis result of the time course of the synthesis reaction and the amount of the synthesized product by SDS-polyacrylamide gel electrophoresis [(B) in FIG. 3, the arrow indicates a stained band with DHFR Coomassie Brilliant Blue] is shown in FIG. 3 (A). The results shown were fully supported. From the measurement of the band staining intensity of the synthetic DHFR, it was found that 0.9 mg of DHFR per ml of reaction volume could be synthesized by the reaction for 8 hours by the diffusion continuous batch cell-free protein synthesis method.

  Demonstration that the diffusion continuous batch cell-free protein synthesis method is universally effective for cell-free protein synthesis systems using cell extracts prepared from any species using E. coli extract did.

  The Escherichia coli extract has been reported previously [Pratt, J. M.M. Transcription and Translation (1984), 179-209, Hames, B .; D. & Higgins, S .; J. et al. , Eds, IRL Press, Oxford]. In this example, mRNA is first synthesized by a transcription / translation integrated cell-free protein synthesis system (see Example 6), and the transcription reaction solution containing the mRNA is a reaction solution suitable for translation reaction by gel filtration. After conversion into the composition, in addition to the reaction vessel, the supply solution was overlaid in the same manner as in Example 1, and a protein synthesis reaction was performed at 30 ° C. under stationary conditions.

First, 50% of the total volume of the E. coli extract was contained, and the final concentration composition was as follows: 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mM ammonium acetate, 16 mM magnesium acetate; 7 mM dithiothreitol, 0.3 U / ml pile beet kinase, 0.17 mg / ml E. coli tRNA mixed solution, 34 mg / ml L-5 formyl-5,6,7,8-tetrahydrophoric acid, 6.7 μg / m plasmid (Encoding GFP gene), E. coli consisting of 33 μg / ml T7 RNA polymerase, 1.2 mM ATP, 0.85 mM GTP and UTP and CTP, 56 mM phosphoenolpyruvate, 20 L-type amino acids (each 0.2 mM) Prepare cell-free protein synthesis reaction solution at 30 ℃ Was synthesized mRNA is incubated for 30 minutes. Next, the reaction solution composition is converted into a composition suitable for translation reaction by gel filtration, and 25 μl of this solution is transferred to a reaction vessel (7 mm diameter microtiter plate), and a supply solution having the following composition is gently overlaid. Protein synthesis was performed at 30 ° C. When protein synthesis was measured using amino acid uptake as an index, ¹⁴ C-leucine (300 mCi / mmol) was added to the reaction solution at 4 μCi per 1 ml.
A plasmid serving as a transcription template for mRNA is pK7-RAS having a T7-phage promoter sequence reported by Kikawa et al. [Kigawa, T. et al. , Et al. (1995) J. MoI. Biomol. Based on NMR, 6,129-134], the RAS gene was replaced with the jellyfish GFP gene.

The composition of the supply solution used in the E. coli cell-free protein synthesis system was as follows: final concentration 57 mM HEPES-KOH (pH 8.2), 75 mM potassium acetate, 36 mM ammonium acetate, 16 mM magnesium acetate, 1.7 mM dithiothreitol, 34 mg / ml L- 5 formyl-5,6,7,8-tetrahydrophoric acid, 1.2 mM ATP, 0.85 mM GTP and UTP and CTP, 56 mM phosphoenolpyruvate, 20 L-type amino acids (0.2 mM each) Become. When protein synthesis was measured using amino acid uptake as an index, ¹⁴ μCi of ¹⁴ C-leucine was added to 1 ml of the reaction solution.

FIG. 4A shows the results of measuring protein synthesis by incorporation of ¹⁴ C-leucine. The GFP synthesis reaction was completely stopped in 3 hours after the start of the reaction in the conventional batch method (○ ―― ○), but until 17 hours after the start of the reaction in the diffusion continuous batch cell-free protein synthesis method (■ ―― ■). Lasted. When the amount of synthetic protein was calculated from the amount of amino acid incorporated, the amount of synthesis by the diffusion continuous batch cell-free protein synthesis method reached more than four times that by the batch method. The time course of the synthesis reaction by autoradiogram shown in FIG. 4B and the analysis results of the molecular weight and synthesis amount of the synthesized product completely supported the result of FIG. On the other hand, in the control experiment in which both phases were mixed with a vortex mixer immediately after the feed solution (feed phase) was overlaid on the protein synthesis reaction solution (reaction phase), protein synthesis was significantly reduced compared to the conventional batch method. .

This fact is consistent with the findings reported by Kim [Kim, D. et al. M.M. (1996) Eur. J. et al. Biochem. 239, 881-886], that is, in the E. coli cell-free protein synthesis system, it is completely consistent with the result that it is important to increase the concentration of the cell extract in the reaction solution for the efficiency of synthesis. This result is due to the decrease in the reaction rate due to the decrease in the concentration of the components required for protein synthesis in the reaction solution, such as ribosomes, due to the extended phenomenon of the protein synthesis reaction observed in the diffusion continuous batch method. It clearly shows the characteristics of the diffusion continuous batch cell-free protein synthesis method.
Furthermore, using the E. coli extract, mRNA was first synthesized in a transcription / translation integrated cell-free protein synthesis system (see Example 6), and then the supply solution was overlaid on the reaction solution in the same manner as in Example 1. Even when the protein synthesis reaction was carried out at 30 ° C. under static conditions, the same results as above were obtained.

  Below, the example of the cell-free protein synthesis method using the wheat germ by the discontinuous batch type using the gel filtration method is demonstrated. First, the same protein synthesis solution as prepared in Example 1 was added to a normal small test tube or a 96-well titer plate, and allowed to react as usual at 26 ° C. under static conditions. Under this reaction condition, protein synthesis is stopped for several hours, for example, in the case of a reaction solution containing 48% of the volume of wheat germ extract, the synthesis reaction is completely stopped in 1 hour from the start of the reaction. This can be confirmed from the measurement of amino acid incorporation into protein and polyribosome analysis by sucrose density gradient centrifugation [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564]. The Sephadex G- that has been equilibrated with a supply solution containing a substrate or an energy source, for example, amino acids, ATP, GTP, other ions necessary for the protein synthesis reaction or a buffer solution, is prepared by stopping the reaction solution from which the synthesis reaction has stopped. After gel filtration using 25 columns, protein synthesis was further performed at 26 ° C. The feed solution has the same composition as the feed solution used in Example 1.

As shown by the results of ¹⁴ C-leucine incorporation in FIG. 5, in the conventional batch method, the synthesis reaction was completely stopped in 1 hour after the start of the reaction (■-■). However, after 3 hours from the start of the reaction, the gel filtration operation was performed as described above (indicated by an arrow in FIG. 5), and further incubation was carried out to start the protein synthesis reaction again (□-□). Furthermore, since the rate gradient of ¹⁴ C-leucine incorporation was almost the same as that at the beginning of the reaction, it was found that the protein synthesis efficiency after gel filtration did not decrease compared to that at the beginning of the reaction.
Also in this method, after synthesizing mRNA with a transcription reaction solution by the transcription / translation integrated protein synthesis method shown in Example 6 below, the composition of the transcription reaction solution is subsequently determined by gel filtration or dialysis. The same results were obtained when the protein was synthesized in the same manner as described above using the solution obtained as a synthesis reaction solution after conversion to a solution having a composition suitable for the translation reaction.

  The cell-free protein synthesis system using wheat germ is extremely stable [Endo, Y. et al. et al. (1992) J. Am. Biotech. , 25, 221-230] [Proc. Natl. Acad. Sci. USA (2000) 97, 559-564], it is possible to maintain the reaction time for a long time by repeating this gel filtration operation. Therefore, the discontinuous batch cell-free protein synthesis method is useful as a highly efficient protein synthesis method in a cell-free protein synthesis system using, for example, wheat germ.

  After synthesizing mRNA in a transcription reaction solution by a transcription / translation integrated protein synthesis method, the composition of the transcription reaction solution is subsequently converted to a solution composed of a composition suitable for translation reaction by gel filtration or dialysis. The obtained solution can be used as a synthesis reaction solution.

  First, using a spin column equipped with a gel filtration filter as a reaction container, template DNA, four types of substrate ribonucleoside-5′-3 phosphate, and optionally CAP molecules, RNA polymerase, spermidine, Transcription reaction solution consisting of magnesium ions and an appropriate buffer [80 mM HEPES-KOH (pH 7.6), 16 mM magnesium acetate, 2 mM spermidine, 10 mM dithiothreitol, 2.5 mM ATP, 2.5 mM GTP, 2.5 mM CTP 2.5 mM UTP, 1 U / μL ribonuclease inhibitor, 3 U / μL SP6 RNA polymerase (manufactured by TAKARA)]. A solution obtained by removing template DNA, RNA polymerase, and ribonuclease inhibitor from the above transcription reaction solution is separately prepared and used as an external dialysis solution, and dialysis-type continuous synthesis of mRNA is performed.

  After the mRNA synthesis, the spin column is centrifuged at a low speed, and the above-mentioned transcription reaction solution is converted into a protein synthesis solution suitable for translation reaction by performing gel filtration using the protein synthesis solution (without mRNA) shown in Example 1. .

The cell-free protein synthesis method according to the present invention includes an ultrafiltration membrane method and a dialysis membrane method using a semipermeable membrane, and a column chromatography method in which a translation template is immobilized on a resin [Spirin, A. et al. , Et al. , (1993) Methods in Enzymology, 217, 123-142], by introducing the efficiency of the three types of synthesis reactions into the conventional batch system, respectively, by any means. It was shown that protein synthesis in a cell-free system using tissue / cell extracts can be performed with high efficiency.
The cell-free protein synthesis method according to the present invention is a low membrane material strength, a membrane function deterioration due to clogging, and a complicated operation, as found in a conventional continuous cell-free protein synthesis method using a membrane. Therefore, protein synthesis can be carried out with much higher efficiency than conventional methods. Therefore, the technology according to the present invention will be a basic element technology for automating the production of gene products (proteins), which will be the basis for functional analysis and structural analysis of a huge number of genes that will be brought forward with the completion of future genome projects. . In particular, it can be said that it is indispensable as an elemental technology for automating cell-free protein synthesis systems, such as the development of fully automated cell-free protein synthesis robots for multiple specimens.

It is a figure explaining the spreading | diffusion continuous batch type cell-free protein synthesis method taking a multilayer system as an example. In the drawing, the portion surrounded by the leader line is a sectional view of the hole of the titer plate. The green fluorescent protein (GFP) synthesis | combination by the diffusion continuous batch type cell-free protein synthesis method using a wheat germ extract is shown. FIG. 2A shows the results of measuring protein synthesis by incorporation of ¹⁴ C-leucine. The vertical axis represents the amount of protein synthesis, and the horizontal axis represents the incubation time. Diffusion using a conventional batch type (○ ―― ○) and reaction vessel of 7 mm (large □ ―― □), 5 mm (in □ ―― □), or 3 mm (small □ ―― □) The protein synthesis by a continuous batch system (multilayer system) is shown. ■ ―― ■■ indicates protein synthesis by dilution batch method. The radioactivity count indicating the amount of protein synthesis on the vertical axis was expressed per equivalent amount of embryo extract. FIG. 2B is an autoradiogram of the synthesized product. Fig. 2 shows dihydrofolate reductase (DHFR) synthesis by a diffusion continuous batch cell-free protein synthesis method using wheat germ extract. FIG. 3A shows the result of measuring protein synthesis by incorporation of ¹⁴ C-leucine. Protein synthesis by the conventional batch method (○ ―― ○) and diffusion continuous batch method (multilayer method) using a reaction vessel with a diameter of 7 mm is shown (■-■). The radioactivity count indicating the amount of protein synthesis on the vertical axis was expressed per equivalent amount of embryo extract. FIG. 3 (B) is an SDS-polyacrylamide gel electrophoresis diagram of the synthesized product stained with Coomassie brilliant blue. The GFP synthesis | combination by the diffusion continuous batch type cell-free protein synthesis method using E. coli extract is shown. FIG. 4A shows the result of measuring protein synthesis by incorporation of ¹⁴ C-leucine. Protein synthesis by the conventional batch method (○ ―― ○) and diffusion continuous batch method (multilayer method) using a reaction vessel with a diameter of 7 mm is shown (■-■). □ ―― □ indicates protein synthesis by a dilution batch type cell-free protein synthesis method. The radioactivity count indicating the amount of protein synthesis on the vertical axis was expressed per equivalent amount of E. coli extract. FIG. 4B is an autoradiogram of the synthesized product. GFP synthesis by discontinuous gel filtration batch cell-free protein synthesis method using wheat germ extract is shown (□-□). The arrow indicates the time point when the reaction solution was subjected to gel filtration. ■ ―― ■ shows the results when gel filtration was not performed. The structure of the versatile plasmid pEU1 is shown.

Claims (2)

A batch-type cell-free protein synthesis method comprising the following steps:
1) A step of resupplying one or more of amino acids, ATP, GTP and creatine phosphate to the reaction solution after the synthesis reaction is stopped,
2) a step of removing by-products generated in the reaction solution from the reaction solution after the synthesis reaction is stopped;
3) The step of resuming the protein synthesis reaction after the step of resupplying in 1) and the step of eliminating in 2) above,
4) A step of repeating the step of resupplying in 1) above, the step of eliminating in 2) above and the step of restarting the synthesis reaction in 3) above .   2. The cell-free protein synthesis method according to claim 1, wherein the refeeding step and the removing step are performed using a gel filtration column or a semipermeable membrane.

Images