JP4643444B2 - High-throughput synthesis system and synthesis apparatus for automatically performing the system - Google Patents

Description

  This application claims the priority from Japanese patent application numbers 2003-122930, 2003-281500, which is incorporated herein by reference.

  The present invention relates to a cell-free synthesis system from a template material. More specifically, the present invention relates to a system for maximizing the efficiency of a synthesis reaction when a material is synthesized using a template material as a raw material. The present invention also relates to a synthesis apparatus for automatically performing the system.

As a means of synthesizing biopolymers such as proteins and RNA in vitro, so-called batches are prepared by reacting the substrates, ions, buffers, cell extracts, and synthetic enzymes necessary for these reactions in a test tube. The method has been used for a long time. However, this batch reaction method is based on the reaction principle, and the synthesis reaction can be performed in a short time due to the decrease in the substrate concentration accompanying the progress of the reaction and the increase in the concentration of by-products having a synthesis inhibitory action. As a result, there was a major limitation that the amount of the desired product was low. Former Soviet Union Spirin et al., Using an ultrafiltration membrane, a dialysis membrane, or a column chromatography method in which a translation template is immobilized on a resin, etc. enables simultaneous supply of energy sources such as amino acids, ATP, GTP, and substrates. A continuous cell-free protein synthesis method that enables continuous removal of target products and by-products from the reaction system has been published (Non-patent Document 1). However, the continuous method using these semipermeable membranes and ultrafiltration columns requires the assembly of a complicated device, and further complicated processing, which requires sufficient experience. At the same time, this method has been a major difficulty in the automation of the reaction system.
Endo et al. Have invented a contact interface diffusion method capable of continuously supplying a substrate and removing a byproduct without using a semipermeable membrane as a method for solving these problems (Patent Document 1). ). This method made it possible to synthesize a large amount of protein without using a special reaction apparatus. However, since this method is based on the principle of molecular diffusion, there is a disadvantage that it takes a long time to synthesize a necessary amount of protein, and the solution has been struggled.
Furthermore, the problems found in the cell-free synthesis method of proteins remain in the RNA synthesis method in vitro, especially in the high-throughput synthesis method of mRNA, which is a translation template essential for the cell-free protein synthesis reaction. Endo et al. Have developed a continuous synthesis method using a dialysis membrane as a study of RNA synthesis methods, and have found that this method is an RNA synthesis method with extremely high synthesis yield that greatly improves the performance of the batch method. (Patent Document 2). However, this continuous RNA synthesis method using a semipermeable membrane or the like requires assembly of a complicated apparatus and complicated processing, and sufficient work experience is also required. Furthermore, this method has been a major difficulty in the automation of reaction systems.
The requirements for high-throughput synthesis of biopolymers such as proteins and RNA in the post-genome era can be considered as follows. 1) Mass synthesis is possible within a short time, 2) Many types of molecules can be synthesized within a short time, and 3) It can be an elemental technology for automation of reactors based on simple principles. And so on. In particular, the requirements of 1) and 2) are extremely important not only from the viewpoint of reducing the cost of synthesis, but also for preparing a protein having relatively unstable physical properties.
WO02 / 24939 WO01 / 27260 Spirin, A.M. et al. (1993) Meth. in Enzymol. , 217, 123-142

The subject of the present invention is a system technology for a high-throughput in vitro synthesis reaction method of biopolymers such as proteins and RNA, which is completely different from the principle of the continuous synthesis method using a complex apparatus such as a semipermeable membrane. And an automatic synthesizer using the system. This makes it possible to easily and efficiently perform comprehensive preparation and mass production for analysis of structures and functions of gene products such as RNA and proteins.
In order to solve the above problems, the present invention has been conducted in various ways for the purpose of maintaining a high reaction rate for a long time in a synthesis system using a template substance as a raw material. The solution to the problem has been achieved by completing a cell-free system including a combination and an automatic synthesizer using the system.
That is, the present invention is as follows.
1. A cell-free synthesis system comprising a template material as a raw material and comprising the following means, control means, or a combination thereof:
1) A template substance, a substrate, and a reaction solution are brought into contact with each other to lead to a synthesis reaction.
2) The reaction solution is diluted before and after substantially decreasing the synthesis rate, approximately before and after the termination of the synthesis reaction, or in the middle thereof.
3) A concentration process is performed following the dilution process.
4) Perform the synthesis reaction using the concentrated reaction system.
Or 1) A template substance, a substrate, and a reaction solution are brought into contact with each other to lead to a synthesis reaction.
2) Concentrate the reaction solution before or after substantially lowering the synthesis rate, before or after substantially stopping the synthesis reaction, or on the way thereof.
3) A dilution process is performed following the concentration process.
4) Perform the synthesis reaction using the diluted reaction system.
2. The system according to item 1 above, wherein by-products are removed from the reaction system by concentration treatment.
3. 3. The system according to item 1 or 2, wherein the substrate, energy source and / or template material are replenished into the reaction system in the dilution process.
4.1) The system according to item 1 above, wherein the means of 4) is repeated a plurality of times.
5. 5. The system according to any one of items 1 to 4, wherein the template substance is a transfer template.
6). 5. The system according to any one of items 1 to 4, wherein the template substance is a translation template.
7). 7. The system according to item 6 above, wherein the reaction solution contains a cell extract.
8). A cell-free synthesis system that combines the transcription product production system obtained by the system of the previous item 5 and the translation product production system obtained by the system of the previous item 6 or 7.
9. A synthesis kit comprising at least one reagent used in a cell-free synthesis method from a template substance using the system according to any one of 1 to 8 above.
10. 9. A cell-free synthesis method from a template substance using the system according to any one of 1 to 8 above.
11. A cell-free synthesizer from a template substance using the system according to any one of 1 to 8 above.
12 9. An apparatus for automatically performing the system according to any one of 1 to 8 above, wherein the following control means is used to perform a synthesis system using a synthesis reaction initial phase having a high protein synthesis reaction rate a plurality of times. A cell-free synthesizer comprising:
(1) means for initiating synthesis;
(2) means for concentrating the reaction solution;
(3) means for diluting the reaction solution;
(4) means for reactivating the synthesis reaction;
(5) Means for repeating the above means (2) to (4);
Or (1) means for initiating synthesis;
(2) means for diluting the reaction solution;
(3) means for concentrating the reaction solution;
(4) means for reactivating the synthesis reaction;
(5) Means for repeating the means (2) to (4) above.
13. The means for concentrating the reaction solution uses an ultrafiltration membrane, and when the reaction solution is removed from the reaction system through the ultrafiltration membrane, a substance that cannot pass through the ultrafiltration membrane in the reaction solution, 13. The synthesizer according to item 12, which is concentrated in the reaction system.
14 14. The synthesizer according to item 13 above, wherein in the means for concentrating the reaction solution, a centrifuge and / or a suction pump is used to remove the reaction solution to the outside of the reaction system.
15. 13. The synthesizer according to item 12, wherein the means for diluting the reaction solution is to add a diluted solution or a substrate solution to the reaction solution.
16. In the cell-free protein synthesis step, in order to automatically perform the steps from the transcription template to the production of the protein encoded by the translation template, the synthesizer according to any one of the preceding items 11-15, comprising at least one of the following control means:
(1) means for variably controlling the temperature in the reaction vessel;
(2) Means for dispensing a sample or reagent into a reaction vessel;
(3) means for transporting the reaction vessel;
(4) Precipitation and concentration filtration means 17. A program for implementing the system according to any one of the preceding items 1 to 8, wherein the following information processing means is used to implement a synthesis system using a synthesis reaction initial phase having a high protein synthesis reaction rate multiple times: Including programs.
(1) Information processing means for setting a synthesis time within the initial phase range of the synthesis reaction just before the amount of synthesis per unit time tends to decrease based on information on the volume of the reaction vessel and the concentration of the reaction solution,
(2) Information processing means for adding a dilute solution to the reaction vessel when the synthesis time within the synthesis reaction initial phase range of (1) is reached, and setting the reaction solution outside a concentration range that can substantially undergo a synthesis reaction,
(3) Information processing means for concentrating the reaction liquid in the reaction vessel after (2) and setting the volume of the reaction liquid increased by addition of the diluent to return to the original liquid volume,
(4) After (3), an information processing means for setting the reaction optimal temperature in order to restart the synthesis reaction,
(5) Information processing means for repeating (1) to (4) a plurality of times,
Or
(1) Information processing means for setting a synthesis time within the initial phase range of the synthesis reaction just before the amount of synthesis per unit time tends to decrease based on information on the volume of the reaction vessel and the concentration of the reaction solution,
(2) Information processing means for concentrating the reaction solution in the reaction vessel when the synthesis time in the synthesis reaction initial phase range of (1) is reached, and setting the reaction solution outside the concentration range that can substantially undergo the synthesis reaction,
(3) After (2), an information processing means for adding a dilute solution to the reaction vessel and setting the volume of the reaction liquid reduced by the concentration back to the original liquid volume,
(4) After (3), an information processing means for setting the reaction optimal temperature in order to restart the synthesis reaction,
(5) Information processing means for repeating (1) to (4) a plurality of times.
18. A cell-free synthesizer in which the program according to the preceding item 17 is incorporated, and information processing of the program is performed in cooperation with the apparatus, so that synthesis is started, reaction solution is diluted and concentrated, A cell-free synthesizer characterized in that a reactivation means is executed and a synthesis system using a synthesis reaction initial phase having a high protein synthesis reaction rate can be repeatedly executed a plurality of times.
19. 19. A protein synthesized by the synthesizer according to the above items 11 to 16, 18.

FIG. 1 is a graph showing differences in experimental results between a cell-free protein synthesis method of the present invention and a conventional cell-free protein method using a batch method.
FIG. 2 is a graph showing the results of an experiment in which cell-free protein synthesis was performed in the same manner as in Example 1 except that the concentration of the wheat germ extract was changed.
FIG. 3A: Cell-free protein synthesis was carried out in the same manner as in Example 1 except that the concentration of the wheat germ extract was 80 OD, the concentration of GFP mRNA was 640 μg / ml, and the interval for performing the repeated repetition of dilution and concentration was changed. It is a graph which shows the experimental result performed.
FIG. 3B is a polyacrylamide gel electrophoresis result showing a quantitative difference of synthesized GFP due to a difference in intervals at which discontinuous repetition of dilution and concentration is performed.
FIG. 4 is a diagram schematically showing an example of a configuration in which a by-product can be removed in the cell-free protein synthesis method of the present invention by combining a concentrator having a filtration membrane and a liquid feed pump. .
FIG. 5 is a graph showing the results of experiments in which protein synthesis was performed by the cell-free protein synthesis method of the present invention using the configuration using the filtration membrane and the liquid feed pump shown in FIG. .
FIG. 6A shows a discontinuous repetition of dilution and concentration using a translation reaction solution similar to that in Example 1 except that 60 OD wheat germ extract and 450 μg / ml dihydrofolate eductase (DHFR) mRNA were used as translation templates. It is a figure which shows the experimental result at the time of performing a cell-free protein synthesis | combination like Example 1 except having repeated operation twice at 1 hour intervals.
B is an electrophoretogram showing the experimental results of synthesizing a GFP fusion protein in which streptavidin is fused to the N-terminus by the method of the present invention and isolating the GFP fusion protein outside the system.
FIG. 7 is a diagram showing the difference in experimental results between the RNA synthesis method of the present invention and the conventional method for synthesizing RNA in vitro by the batch method.
FIG. 8 is a graph showing the experimental results of performing the cell-free protein synthesis method of the present invention using mRNA purified by ethanol precipitation and unpurified mRNA as translation templates.
FIG. 9 is a graph showing the results of an experiment in which the cell-free protein synthesis method of the present invention was performed using a translation reaction solution prepared using the transcription reaction solution after the transcription reaction of Experimental Example 7 as it was.
FIG. 10 is a schematic diagram of an automatic synthesizer.
FIG. 11 is a diagram of SDS-PAGE showing the results of an apparatus example.

Explanation of symbols

1: Filtration (filter) superconcentrator 2: Liquid feed pump 3: Reaction tank 4: Container containing (RNA) substrate solution 5: Liquid feed switching valve 6: Container T1: Tube T1
T2: Tube T2
T3: Tube T3
T4: Tube T4
T5: Tube T5
(1): Template plate (2): Reagent tank 1 (solution for transcription reaction)
(3): Reagent tank 2 (Translation reaction solution)
(4): Reagent tank 3 (diluted solution)
(5): 300 μl tip (6): 20 μl tip (7): Robot arm (8): Dispenser 1
(9) Dispenser 2
(10): Transfer / filtrate receiving plate (11): Chip disposal port (12): Thermostatic chamber 1
(13): MTP (multi titer plate) stage (14): Translation plate (15): Lift platform (16): Centrifuge (17): Waste port

The present invention is a synthesis method characterized by maximally utilizing the characteristics of an initial phase of a synthesis reaction having a high reaction rate, which is a general batch type or diffusion continuous batch type using a template material as a raw material. Further, the present invention is a synthesizer characterized in that a synthesizer that makes maximum use of the characteristics can be automatically made available.
That means is that the reaction solution is diluted or concentrated before or after substantially decreasing the synthesis rate, before or after the synthesis reaction is substantially stopped, or in the middle thereof. In this dilution process or concentration process, components such as substrates, energy sources, ions, buffers, and template substances required for synthesis using the template substance as a raw material are supplemented or concentrated.
The diluted or concentrated reaction solution is then subjected to a concentration treatment when diluted, or a dilution treatment when concentrated. By this treatment, the reaction solution is returned to the original optimum reaction concentration. In the concentration treatment, components in the reaction solution are removed or separated, and reaction products and / or reaction byproducts are collected and / or removed. By the concentration or dilution, each element of the synthesis reaction is adjusted again to the optimum concentration. This dilution and concentration treatment is repeated discontinuously, so that a large amount of protein can be synthesized.
The principle of the present invention is a synthesis method in which the synthesis reaction using the template substance is repeatedly diluted and concentrated repeatedly.
The automatic synthesizing apparatus of the present invention is based on the principle of automating a synthesis method that is repeatedly performed discontinuously.
In the present invention, one of the synthesis systems using a template material as a raw material is a protein translation synthesis system using mRNA as a template, and in particular, cell extraction for protein synthesis that uses a ribosome, which is a protein translation apparatus provided in a cell, from a cell. A cell-free protein synthesis system that uses liquid. The other means an RNA synthesis system by in vitro transcription reaction using a transcription template using RNA polymerase as an enzyme.
(Cell-free protein synthesis system)
Among cell-free protein synthesis systems, those using wheat germ extract have particularly low nucleolytic enzyme activity and stable and high translation activity compared to cell-free protein synthesis systems using E. coli extract. (Madin K. et al., (2000) Proc. Natl. Acad. Sci. USA 97, 559-564), which enables highly efficient protein synthesis in a transcription / translation system using a plasmid as a template. (PCT / JP99 / 04088). Hereinafter, in the present invention, a wheat germ cell-free protein synthesis system and a method for synthesizing a transcription template capable of functioning in the synthesis system will be described as examples. The basic principle of the present invention is cell extraction from other microorganisms or animal cells. It can also be applied to a cell-free protein synthesis system using a product and a transcription template synthesis system used therefor.
(Cell extract)
Commercially available cell extracts include E. coli-derived E. coli. E. coli S30 extract system (Promega), RTS 500 Rapid translation System (Roche), Rabbit Reticulocyte Lysate System (Promega) derived from rabbit reticulocyte, Proteos derived from wheat germ (Promega) ^TM ) (TOYOBO). Among these, it is particularly preferable to use a germ extract derived from plant seeds, and examples include wheat, barley, rice, corn, spinach seeds, etc., and a system using a wheat germ extract is optimal.
(Translation product production system)
Here, the translation product production system means that a component containing ribosome, which is a protein translation apparatus provided in a cell, is extracted from an organism, a translation template, a nucleic acid serving as a substrate, an amino acid, an energy source, and various ions. , Buffer solution, and other effective factors are added in a test tube.
(Cell-free protein synthesis system)
Here, the cell-free protein synthesis system means that a component including ribosome, which is a protein translation apparatus provided in a cell, is extracted from an organism and transferred to this extract, or a translation template, a substrate nucleic acid, amino acid, energy Source, various ions, buffer, and other effective factors are added in a test tube.
(Wheat germ extract)
Among these, the cell extract-containing solution of the present invention is preferably one using a wheat germ extract.
As a method for preparing a wheat germ extract, as a method for isolating wheat germ, for example, Johnston, F. et al. B. et al. , Nature, 179, 160-161 (1957), and the like. As a method for extracting a cell extract-containing liquid from an isolated embryo, for example, Erickson, A. et al. H. et al. (1996) Meth. In Enzymol. 96, 38-50 etc. can be used. In addition, the methods of Japanese Patent Application Nos. 2002-23139 and 2002-231340 are exemplified.
The germ extract suitably used in the present invention is a substance that suppresses the protein synthesis function contained in or retained by the raw wheat itself (tritin, thionine, ribonuclease, mRNA, tRNA, translated protein factor, ribosome, etc. The substance that acts on and suppresses its function) is almost completely removed. That is, the endosperm where these inhibitors are localized is almost completely removed and purified. The degree of endosperm removal can be evaluated by monitoring the activity of tritin contaminated in the wheat germ extract, that is, the activity of deadenating the ribosome. If the ribosome is not substantially adenylated, it is judged that there is no contaminating endosperm-derived component in the embryo extract, that is, the endosperm is almost completely removed and purified. The extent to which the ribosome is not substantially deadeninated means that the deadenination rate of the ribosome is less than 7%, preferably 1% or less.
The embryo extract contains a protein derived from a cell extract-containing solution and separately added as necessary. The content is not particularly limited, but from the viewpoint of storage stability in a lyophilized state, ease of use, etc., in the composition before lyophilization, preferably 1 to 10% by weight of the whole composition, more It is preferably 2.5 to 5% by weight, and in the lyophilized composition after lyophilization, it is preferably 10 to 90% by weight, more preferably 25 to 70% by weight of the entire lyophilized composition. The protein content here is calculated by measuring the absorbance (260, 280, 320 nm).
(Removal of microorganisms)
The cell extract-containing solution may contain microorganisms, particularly spores such as filamentous fungi, and it is preferable to remove 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 it is preferable to use a filter sterilization filter. The pore size of the filter is not particularly limited as long as microorganisms that may be mixed are removable, but usually 0.1 to 1 micrometer, preferably 0.2 to 0.5 micrometer is appropriate. .
Furthermore, in order to perform cell-free protein synthesis having a specific effect by adding a low molecular weight synthesis inhibitor removing step and / or a reducing agent concentration reducing step at any stage of the cell extract-containing liquid preparation step Cell extract-containing liquid.
(Method for removing low molecular weight synthesis inhibitors from cell extract-containing solutions)
The cell extract-containing solution contains a low-molecular synthesis inhibitor having protein synthesis inhibitory activity (hereinafter sometimes referred to as “low-molecular synthesis inhibitor”), and by removing these, protein synthesis A highly active cell extract-containing solution can be obtained. Specifically, the low molecular weight synthesis inhibitor is fractionated and removed from the constituents of the cell extract-containing solution based on the difference in molecular weight. The low molecular weight synthesis inhibiting substance can be fractionated as a molecule having a molecular weight equal to or smaller than the smallest factor necessary for protein synthesis contained in the cell extract-containing solution. Specifically, it can be fractionated and removed with a molecular weight of 50,000 to 14,000 or less, preferably 14,000 or less.
As a method for removing a low-molecular-weight synthesis inhibitor from a cell extract-containing solution, a commonly used method known per se is used. Specifically, a method by dialysis through a dialysis membrane, a gel filtration method, or Examples thereof include an ultrafiltration method. Among these, the method by dialysis is preferable in terms of ease of supplying the substance to the dialysis internal solution.
Examples of the dialysis membrane used for the removal operation of the low molecular weight synthesis inhibitor by dialysis include those having a removal molecular weight of 50,000 to 12,000, specifically, regenerated cellulose having a removal molecular weight of 12,000 to 14,000. A membrane (manufactured by Viscose Sales, Chicago) or Spectra / pore 6 (manufactured by SPECTRUM LABORTORIES INC., CA, USA) having a removal molecular weight of 50,000 is preferably used. An appropriate amount of a cell extract-containing solution or the like is placed in such a dialysis membrane and dialyzed using a conventional method. The time for dialysis is preferably about 30 minutes to 24 hours.
(Stabilization of cell extract-containing solution)
When an insoluble component is generated in the cell extract-containing solution when removing the low-molecular-weight synthesis inhibitor, this generation is inhibited (hereinafter referred to as “stabilization of the cell extract-containing solution”). The protein synthesis activity of the cell extract-containing solution or translation reaction solution finally obtained can be increased. As a specific method for stabilizing the cell extract-containing solution or translation reaction solution, the cell extract-containing solution or translation reaction solution must be at least high-energy when removing the low-molecular synthesis inhibitor described above. Examples thereof include a method of carrying out as a solution containing a phosphoric acid compound such as ATP or GTP (hereinafter sometimes referred to as “stabilizing component”). ATP is preferably used as the high energy phosphoric acid compound. Further, it is preferably performed in a solution containing ATP and GTP, more preferably ATP, GTP, and 20 kinds of amino acids.
These components may be preliminarily added with a stabilizing component, incubated, and then subjected to a step of removing a low molecular weight inhibitor. If a dialysis method is used to remove a low molecular weight inhibitor, dialysis is used. It is also possible to remove the low-molecular-weight synthesis inhibitor by adding a stabilizing component to the external liquid and performing dialysis. It is more preferable to add a stabilizing component to the external dialysis solution because a new stabilizing component is always supplied even if the stabilizing component is decomposed during dialysis. This can also be applied when using a gel filtration method or an ultrafiltration method, and after each carrier is equilibrated with a filtration buffer containing a stabilizing component, a cell extract-containing solution containing a stabilizing component or The same effect can be obtained by providing a translation reaction solution and performing filtration while adding the above buffer solution.
The addition amount of the stabilizing component and the stabilization treatment time can be appropriately selected depending on the type of cell extract-containing liquid and the preparation method. These selection methods include the addition of a stabilizing component, which is experimentally used in various amounts and types, to the cell extract-containing solution, followed by a removal step of the low-molecular inhibitor after an appropriate time, and after the obtained treatment. Examples include a method of separating a cell extract-containing solution into a solubilized component and an insolubilized component by a method such as centrifugation, and selecting one having a small amount of insoluble components. Furthermore, a method of performing cell-free protein synthesis using the obtained post-treatment cell extract-containing solution and selecting one having a high protein synthesis activity is also preferred. In addition, in the above selection method, when using a cell extract-containing solution and a dialysis method, an appropriate stabilizing component is also added to the external dialysis solution, and after dialysis is performed for an appropriate time using these, the obtained cells Examples of the method include selection based on the amount of insoluble components in the extract-containing liquid, the protein synthesis activity of the obtained cell extract-containing liquid, and the like.
As an example of the stabilization conditions of the cell extract-containing solution selected in this way, specifically, in the case of performing the step of removing the low-molecular synthesis inhibitor by dialysis, the wheat germ extract-containing solution, In addition, in the external dialysis solution, ATP is 100 μM to 0.5 mM, GTP is 25 μM to 1 mM, 20 amino acids are each 25 μM to 5 mM, and dialysis is performed for 30 minutes to 1 hour or more. . The temperature for dialysis may be any temperature as long as the protein synthesis activity of the cell extract-containing solution is not lost and dialysis is possible. Specifically, the minimum temperature is a temperature at which the solution does not freeze, usually −10 ° C., preferably −5 ° C., and the maximum temperature is a temperature limit of 40 ° C. that does not adversely affect the solution used for dialysis, Preferably it is 38 degreeC.
Further, if the removal of the low-molecular synthesis inhibitor is performed after preparing the cell extract-containing solution, it is not necessary to further add the stabilizing component to the cell extract-containing solution.
(Preparation of mRNA)
The preparation method of mRNA as a template substance is not limited as long as it encodes the target protein, and any method known per se can be used. Of course, it is particularly preferable to use one prepared from DNA as a template substance by the means of the present invention, and further to use it continuously in a series of systems.
(Preparation of translation reaction solution)
For protein synthesis from mRNA, amino acids (20 kinds of essential amino acids or analogs or mutant amino acids depending on the purpose) or energy source (ATP and / or GTP) are used as a substrate in a cell extract for protein synthesis containing ribosomes. ), Various ions (calcium ion, magnesium ion, ammonium ion, etc.), buffer solution (HEPES-KOH, Tris-acetic acid, etc.), ATP regeneration system (combination of phosphoenolpyruvate and pyruvate kinase, creatine phosphorus as necessary) Combination of acid and creatine kinase, etc.), nuclease inhibitor (ribonuclease inhibitor, nuclease inhibitor, etc.), tRNA, reducing agent (dithiothreitol, etc.), polyethylene glycol, 3 ′, 5′-cAMP, folic acid, antibacterial agent ( Sodium azide, Add a solution (translation reaction solution) containing a component suitable for translation reaction (such as Npicillin) to a concentration suitable for cell-free protein synthesis means, add the mRNA for the translation template to this, and add the translation reaction solution. Prepare. This translation reaction solution is prepared at a temperature suitable for the translation reaction, the translation reaction is performed, and the target protein is synthesized.
(Transcription reaction)
A transcription template may be prepared according to a general-purpose template molecule construction method (WO01 / 27260, 02/18586) already reported by the present inventors. For example, a desired structural gene is introduced into a plasmid vector pEU and SP6 RNA is introduced. A method of performing transcription by polymerase treatment and the like are preferably exemplified, but of course, the method for preparing mRNA is not limited thereto. Mix the transcription template with a solution (also called a transcription reaction solution) containing components necessary for the transcription reaction, such as RNA polymerase compatible with the promoter in the transcription template and a substrate for RNA synthesis (four types of ribonucleoside triphosphates). Then, this is incubated at about 20 to about 60 ° C., preferably about 30 to about 42 ° C. for about 30 minutes to about 16 hours, more preferably about 2 to 5 hours to carry out the transcription reaction.
(Synthetic reaction)
In the cell-free synthesis system of the present invention, a system including the following means, control of means, or a combination thereof is performed.
One of the essential means is to bring the template substance, the substrate, and the reaction solution into contact with each other and guide them to the synthesis reaction system. The template substance, substrate, and reaction solution (transcription reaction solution or translation reaction solution) described above are prepared at an optimal concentration and temperature, and an optimal synthesis reaction is performed. The synthesis reaction is usually performed for about 10 minutes to 2 hours, more preferably 20 minutes to 1 hour. This time can be changed according to each system, and the optimum time can be prepared by experimental repetition. In the discontinuous repetitive synthesis method of the present invention, it is particularly preferable that this one-cool processing time is relatively short.
(Dilution or concentration)
The essential means 2 is to dilute or concentrate the reaction system as a reaction time before or after substantially lowering the synthesis rate, before or after substantially stopping the synthesis reaction, or during the reaction. The term “approximately before and after the decrease in the synthesis rate” means the timing at which the amount of mRNA or protein synthesized per hour tends to decrease from the maximum amount over time, and is generally understood as a line rather than a point. Further, “before and after substantially stopping the synthesis reaction” refers to a level at which the amount of synthesis has dropped to a level where it cannot be substantially detected. In this case as well, it is generally understood as a line rather than a point. In the process, the synthesis rate starts to decrease and the synthesis reaction stops. In order to obtain more preferable synthesis efficiency, the reaction system is diluted or concentrated before and after substantially decreasing the synthesis rate. This time is optimally 10 minutes to 1 hour.
Dilution or concentration of the reaction system is performed as follows.
Dilution is performed by adding about 1 to 20 times, preferably about 2 to 10 times the volume of aqueous solution to the reaction system. The aqueous solution contains a template substance, a substrate, and a reaction solution as desired. Particularly preferably, a solution containing a template substance, a substrate, an energy source and the like is used. The concentration of each component added is selected to be adjusted to the optimum concentration after the concentration of the next treatment. By this dilution, the reaction system is remarkably reduced in its synthesis ability. With this state, the present invention is called discontinuous.
Concentration can use any means known per se that can concentrate substances that cannot pass through the reaction solution (eg, synthetic proteins, ribosomes, etc.) into the reaction system when the reaction solution is removed from the reaction system. It is. Preferable examples include filtration using an ultrafiltration membrane, treatment with a centrifugal separator, gel filtration, a suction pump, a method of generating a pressure difference in the liquid phase or gas phase, and the like. In this process, by adjusting the passage diameter of the membrane, the reaction product and reaction by-product are separated and removed by centrifugal separation or molecular sieve. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a film having a molecular weight cut of 10,000 to 100,000 Da is preferably used.
By this concentration treatment, the reaction solution is concentrated to 1/5 to 2/3 volume of the original volume, and as a result, the optimum synthesis concentration of each synthesis factor is greatly shifted. By this concentration, the reaction system is remarkably reduced in its synthesis ability. With this state, the present invention is called discontinuous.
(Reactivation of synthesis reaction)
The essential means 3 is reactivation of the synthesis reaction. Those diluted in the previous stage are concentrated, and those that have been concentrated are diluted.
In the former case, the concentration process is performed following the dilution process. Here, the concentration means returning the liquid volume of the reaction system increased by dilution to the original liquid volume. The concentration means is not particularly limited, and any concentration means known per se can be used. Preferable examples include filtration using an ultrafiltration membrane, treatment using a centrifugal separator, gel filtration, a method of generating a pressure difference in the liquid phase or gas phase, and the like. In this process, by adjusting the passage diameter of the membrane, the reaction product and reaction by-product are separated and removed by centrifugal separation or molecular sieve. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a film having a molecular weight cut of 10,000 to 100,000 Da is preferably used.
In the latter case, a dilution process is performed following the concentration process. Here, “dilution” means returning the liquid volume of the reaction system reduced by concentration to the original liquid volume. The diluted solution contains a template substance, a substrate, and a reaction solution as desired. Particularly preferably, a solution containing a template substance, a substrate, an energy source and the like is used. The concentration of each component added is selected to be adjusted to the optimum concentration after dilution.
Thus, the temperature of the reaction system restored to the optimum concentration of the reaction system is adjusted again to the optimum reaction temperature, and the reaction system is reactivated. The optimum temperature is 15 to 25 ° C.
In addition, for separation / recovery / removal of reaction products and / or reaction by-products, treatment based on affinity with the treatment target can be suitably performed. Based on affinity, a method of immobilizing an affinity substance, bringing it into contact with the target substance, binding it, and then eluting and collecting the target substance is exemplified. Examples of the affinity substance include an antibody against a protein, a ligand for a receptor, and a nucleic acid for a transcription factor if the recovered substance is a protein. The target product is modified with an appropriate tag marker (for example, streptavidin, histidine tag, GST, maltose binding protein, etc.), and a substance that can specifically bind to the modified substance (for example, biotin, divalent metal ion, etc.) , Glutathione, maltose, etc.).
In the present invention, it is possible to repeat the dilution and concentration processes a plurality of times in a discontinuous manner, and by this repetition, the regeneration of the cell-free synthesis system is achieved a plurality of times. This regeneration achieves mass protein synthesis.
In the present invention, the dilution and concentration processes can be performed in combination with the control means as a series of steps. For this control, the drive source (motor, pneumatic / hydraulic equipment, other operation controllable actuators, etc.) and computer controlled control circuit, sequence control circuit, etc., turn the operation on / off, the degree of operation, The operation speed and operation interval are adjusted. Further, a signal transfer driver, an operation confirmation sensor, an operation control switch, a timer, and the like can be appropriately provided as desired.
As described above, the present invention achieves regeneration of a cell-free synthesis system by repeating dilution and concentration a plurality of times in a discontinuous manner. The effect will be described more specifically below.
(Dilution-concentrated batch cell-free protein synthesis method)
In this synthesis method, the synthesis reaction vessel can start a synthesis reaction using a batch reaction vessel as a vessel that enables concentration using a conventionally used ultrafiltration membrane or dialysis membrane. In order to reactivate the reaction system after the protein synthesis reaction under optimal conditions using cell-free protein synthesis means, for example, before and after the synthesis reaction is stopped, other buffers such as amino acids serving as substrates and energy sources, ATP and GTP Components required for protein synthesis such as dialysis and ions (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556) The solution containing the template substance mRNA is added to 1 to 20 times the volume of the reaction solution at a time and diluted (dilution treatment). Thereafter, the reaction vessel is centrifuged, for example, by a centrifuge to return to the original reaction solution volume even if the increased solution in the vessel is discharged through the filtration membrane (concentration treatment). This concentration treatment can also be performed by generating a pressure difference in the liquid phase or gas phase regardless of the centrifugal force. By this dilution and concentration treatment, a fresh substrate solution is supplied to the reaction system, and at the same time, a by-product substance that inhibits the translation reaction generated in the synthesis reaction can be eliminated. In this way, for example, by repetitively diluting and concentrating the reaction solution with a substrate-containing solution before and after the synthesis reaction is stopped, reactivation of the reaction system is achieved, and protein synthesis is possible over a long period of time. Become. This method is different from the so-called continuous method, which uses a semipermeable membrane developed by Spirin et al. To continuously replenish the substrate and energy source and discard the metabolites. It can reach several thousand times (in the method of the present invention, the production amount obtained over about 30 hours by the method using a semipermeable membrane can be obtained in about 2 to 3 hours) and has a great difference in quality. In the protein synthesis method using this principle, it is possible to selectively isolate the synthesized protein from the reaction system simultaneously with the elimination of by-products by selecting the fractional molecular weight size of the filtration membrane to be used. It becomes. That is, by using this principle, it is possible to simultaneously synthesize proteins and purify synthesized products. The same effect can be achieved when the order of dilution after the concentration is changed is changed.
(Dilution-concentrated batch in vitro RNA synthesis method: Transcript production system)
The composition of general RNA synthesis reaction solution using phage RNA synthase, the reaction method and the analysis method of synthetic RNA were in accordance with the methods described in WO01 / 27260 and WO02 / 18586. In the synthesis method of the present invention, it is possible to start a synthesis reaction using a batch reaction vessel as a container capable of concentration equipped with a conventional ultrafiltration membrane or dialysis membrane. The size of the ultrafiltration membrane to be used is selected so that RNA does not pass through. Before and after the termination of the synthesis reaction, a solution containing components required for RNA synthesis such as four types of ribonucleotide triphosphates, ions and buffers as substrates (in the method described in WO01 / 27260 and WO02 / 18586) The dialyzed externally prepared solution is abbreviated as RNA substrate solution hereinafter) and diluted by adding 1 to 20 times the volume of the reaction solution. By centrifuging the reaction vessel through a centrifuge, the increased solution in the vessel is discharged through the filtration membrane and concentrated to the original reaction solution volume. The process in this process can also be performed by generating a pressure difference in the liquid phase or gas phase regardless of the centrifugal force. By these treatments, a fresh substrate solution is supplied to the reaction system, and at the same time, the mononucleotide and pyrophosphate generated in the batch RNA synthesis reaction are excluded from the system, so that the RNA synthesis reaction that has been virtually stopped by the principle of chemical equilibrium. Is resumed by shifting the equilibrium. By repeatedly diluting the reaction solution with the substrate and filtering and concentrating before and after stopping the reaction, it becomes possible to synthesize RNA over a long period of time. This method is different in principle from the continuous RNA synthesis method by dialysis method (WO00 / 68412) previously invented by Endo et al. In protein synthesis means using this principle, RNA containing no nucleotides, buffer components or ions is obtained by repeatedly diluting the synthesized reaction solution with water (or a desired solution) and concentrating it through a filtration membrane. Can be recovered as water (or a solution containing the target component). That is, in this method, by selecting a DNA molecular species as a template, high-speed, high-yield and low-cost (the amount of expensive RNA synthase used is only the amount required in ordinary batch systems). The desired mRNA can be prepared as a solution.
The same effect can be achieved when the order of dilution after the concentration is changed is changed.
(Automatic synthesizer)
The automatic synthesizer of the present invention provides a method for automatically performing a series of reaction operations of a synthetic method in which at least a synthesis reaction (protein synthesis) using a translation template is repeatedly diluted and concentrated repeatedly. Furthermore, the present invention provides a method for automatically performing a reaction operation from the transcription template synthesized in the transcription product production system until the protein encoded by the template is produced. Here, “automatically perform the operation” means that the experimenter does not directly apply a manual operation to the reaction system (reaction vessel) during a series of steps. Therefore, when each step is executed, if the experimenter manually operates predetermined operation buttons and switches provided in the automatic synthesizing apparatus of the present invention to be used, the “automatic” requirement in the present invention is impaired. It is not a thing.
In the present invention, an apparatus for automatically performing a reaction operation from a transcription template synthesized in a transcription product production system to production of a protein encoded by the template comprises at least the following means (1) to (5): It is characterized by having.
Hereinafter, although each embodiment will be described in detail with specific embodiments, the method of the present invention is not limited thereto as long as it has the characteristics of the translation template purification step.
(1) Transfer template manufacturing process
In the automatic synthesizer of the present invention, this step does not necessarily need to be performed automatically, and a transfer template obtained manually can be used in the following automated steps. More preferably, a series of steps until the production of the protein encoded by the template is automatically performed.
As used herein, “transcription template” refers to DNA that can be used as a template molecule for in vitro transcription reaction, and has at least a base sequence encoding a target protein downstream of an appropriate promoter sequence. An appropriate promoter sequence refers to a promoter sequence that can be recognized by RNA polymerase used in a transcription reaction, and examples thereof include SP6 promoter and T7 promoter. Any DNA encoding the target protein may be used.
The transcription template preferably has a base sequence having an activity of controlling translation efficiency between the promoter sequence and the base sequence encoding the target protein, and is derived from an RNA virus such as an Ω sequence derived from tobacco mosaic virus. A 5 ′ untranslated region and / or a Kozak sequence or the like can be used. Furthermore, the transcription template preferably includes a 3 ′ untranslated region including a transcription termination region and the like downstream of the base sequence encoding the target protein. As the 3 ′ untranslated region, about 1.0 to about 3.0 kilobases downstream from the stop codon is preferably used. These 3 ′ untranslated regions are not necessarily those of the gene encoding the target protein.
The transfer template can be produced by a method known per se. For example, a host cell such as Escherichia coli having a plasmid into which a DNA containing the same base sequence as the desired transcription template is inserted is cultured, and a large amount of the plasmid is prepared using a known purification method. The transcription template DNA is excised from the plasmid, the restriction enzyme is removed by phenol treatment and chloroform treatment, and alcohol precipitation with ethanol or isopropanol (if necessary, an appropriate amount of salt such as ammonium acetate, sodium acetate, sodium chloride, etc. And the like. The obtained DNA precipitate can be dissolved in ultrapure water or a transcription reaction solution described later and used for the following transcription reaction.
An apparatus for carrying out such a series of operations automatically or semi-automatically (referring to an embodiment in which an experimenter directly adds a manual operation to a reaction system in a part of the process) is known, and this is described in this book. By incorporating it into the automatic synthesizer of the invention, it is possible to automatically perform from the production of the transcription template to the production of the target protein. However, in consideration of the object of the present invention to provide a high-throughput synthesis system according to the present invention for high-throughput analysis, simplification of the apparatus, reduction of required time, etc., the following polymerase chain reaction (PCR) It is more preferable to use a method of producing a transfer template by the method.
In a preferred embodiment of the synthesizer of the present invention, a method is used in which a host in which a DNA encoding a target protein is cloned (for example, Escherichia coli having a plasmid containing the DNA) is directly subjected to PCR to amplify a transcription template. . For example, an oligonucleotide containing a suitable promoter sequence, a 5 ′ untranslated sequence having an activity to control translation efficiency, and a part of the 5 ′ end region of DNA encoding the target protein is publicly known by using an automatic DNA synthesizer. Using this method as a sense primer, and using an oligonucleotide having a 3 'end region sequence of the 3' untranslated sequence as an antisense primer, DNA encoding the target protein as a template and the 3 'untranslated downstream A desired transcription template can be obtained by directly adding a host such as Escherichia coli having a plasmid containing a sequence to a PCR reaction solution and performing an amplification reaction under normal conditions. In order to prevent the generation of short-chain DNA (resulting in yield loss of the target product and low-molecular translation product noise) generated by non-specific amplification, the promoter-splitting primer described in WO 02/18586 is used. It can also be used.
The amplification reaction can be carried out in a commercially available 96-well plate for PCR using a commercially available PCR thermal cycler, or a similar temperature variable control device can be linked with the synthesizer of the present invention, or the synthesis of the present invention. Each means for performing the transcription / translation reaction of the apparatus can be applied to PCR as it is.
The transcription template DNA obtained as described above may be subjected to a transcription reaction after being purified by chloroform extraction or alcohol precipitation. However, in order to simplify the apparatus and shorten the required time, the PCR reaction solution can be used as it is. It is preferable to use it as a solution. In preparation of a transcription template, by using direct PCR from the above-mentioned host, the steps can be greatly omitted as compared with a method in which a large amount of a plasmid is once prepared and subjected to restriction enzyme treatment to obtain a transcription template. A large amount of transcription template can be synthesized in a short time with a small number of steps. That is, since a step of cultivating E. coli having a plasmid incorporating the target gene and maximally preparing the plasmid is not required, the time required for ultracentrifugation for culture and plasmid purification can be shortened. In addition, a restriction enzyme treatment for cutting out a transcription template from a plasmid, a phenol treatment for removing restriction enzymes, chloroform treatment, alcohol precipitation for purification of transcription template, and dissolution of DNA as a transcription template Therefore, there is no inhibition of transcription reaction due to residual phenol / chloroform and loss of transcription template due to multi-step purification operation. Further, since the number of steps required for the reaction can be reduced, there is a further advantage that the number of chips used can be reduced.
(2) Transcription reaction process
The synthesizer of the present invention includes a step of generating mRNA, which is a translation template, by in vitro transcription reaction from transcription template DNA encoding a target protein prepared using a method known per se. In this step, a solution containing a transcription template provided in a reaction system (for example, a commercially available reaction vessel such as a 96-well titer plate), preferably the PCR reaction solution, and an RNA polymerase (for example, compatible with the promoter in the transcription template). , SP6 RNA polymerase, etc.) and a solution containing components necessary for transcription reaction such as RNA synthesis substrate (4 types of ribonucleoside triphosphates) (also referred to as “transcription reaction solution”), It is carried out by incubating the mixture at 20 ° C. to about 60 ° C., preferably about 30 ° C. to about 42 ° C. for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours. Dispensing means for transfer template solution and transcription reaction solution into reaction container, mixing, etc., are performed by dispensing means of an automatic synthesizer described later (for example, pipetter (when using a commercially available 96-well titer plate as reaction container, It is preferable to use one having 8 or 12 dispensing tips adapted to the well interval). Incubation for the transcription reaction can be performed while controlling the temperature at a constant temperature by a temperature control means of a synthesizer described later.
(3) Translation template purification process
The transcription product generated as described above (ie, “translation template”) optionally includes a 5 ′ untranslated sequence and / or a 3 ′ untranslated sequence having an activity of controlling the translation efficiency inserted into the transcription template. An RNA molecule having a base sequence encoding a target protein. In the reaction solution after the transcription reaction, in addition to the translation template RNA, unreacted ribonucleoside triphosphate, reaction by-product pyrophosphate, and other salts contained in the transcription reaction solution are mixed. Since these substances are known to inhibit the subsequent translation reaction, these substances are removed by exchanging the reaction solution. Examples of such solution exchange means include a method in which a reaction solution is placed in a container with a filter, a solution containing a substance that inhibits translation reaction is removed by centrifugation, a solution such as a suitable buffer is newly added, and this is repeated. However, it is not limited to this. If the newly added solution is used as a translation reaction solution, the next translation reaction can proceed without purifying the template. Further, as a means for removing these substances, there is a method in which a translation template is selectively precipitated to separate and remove unreacted substrates and the like. Examples of such precipitation means include salting out and the like, and an alcohol precipitation method is preferably exemplified, but is not limited thereto. When the alcohol precipitation method is used, the alcohol to be used is not particularly limited as long as it can selectively precipitate RNA. For example, ethanol, isopropanol and the like are preferable, and ethanol is more preferable. In the case of ethanol, it is preferable to use about 2 to about 3 times the amount of the transcription reaction solution, and in the case of isopropanol, about 0.6 to about 1 amount of the transcription reaction solution. Moreover, the yield of precipitation can be increased by making a suitable salt coexist. Examples of such salts include ammonium acetate, sodium acetate, sodium chloride, lithium chloride and the like. For example, when ammonium acetate is used, it is desirable to add it so that the final concentration is about 0.5M to about 3M. The alcohol precipitation may be performed at room temperature.
(4) Translation reaction process
The translation reaction can be performed by incubating the translation reaction solution containing the translation template obtained as described above at a temperature suitable for the translation reaction for an appropriate time. The translation reaction is usually performed for about 10 minutes to 2 hours, more preferably 20 minutes to 1 hour. This time can be changed according to each system, and the optimum time can be prepared by experimental repetition. In the discontinuous repetitive synthesis method of the present invention, it is particularly preferable that this one-cool processing time is relatively short.
As described above, in the above reaction, dilution or concentration of the reaction system is performed before or after substantially lowering the synthesis rate, before or after substantially stopping the synthesis reaction, or in the course thereof. The term “approximately before and after the decrease in the synthesis rate” means the timing at which the amount of protein synthesized over time tends to decrease from the maximum amount, and is generally understood as a line rather than a point. Further, “before and after substantially stopping the synthesis reaction” refers to a level at which the amount of synthesis has dropped to a level where it cannot be substantially detected. In this case as well, it is generally understood as a line rather than a point. In the process, the synthesis rate starts to decrease and the synthesis reaction stops. In order to obtain more preferable synthesis efficiency, the reaction system is diluted or concentrated before and after substantially decreasing the synthesis rate. This time is optimally 10 minutes to 1 hour.
Dilution or concentration of the reaction system is performed as follows.
Dilution is performed by adding about 1 to 20 times, preferably about 2 to 10 times the volume of aqueous solution to the reaction system. The aqueous solution contains a template substance, a substrate, and a reaction solution as desired. Particularly preferably, a solution containing a template substance, a substrate, an energy source and the like is used. The concentration of each component added is selected to be adjusted to the optimum concentration after the concentration of the next treatment. By this dilution, the reaction system is remarkably reduced in its synthesis ability. With this state, the present invention is called discontinuous.
Concentration can use any means known per se that can concentrate substances that cannot pass through the reaction solution (eg, synthetic proteins, ribosomes, etc.) into the reaction system when the reaction solution is removed from the reaction system. It is. Preferable examples include filtration using an ultrafiltration membrane, treatment with a centrifugal separator, gel filtration, a suction pump, a method of generating a pressure difference in the liquid phase or gas phase, and the like. In this process, by adjusting the passage diameter of the membrane, the reaction product and reaction by-product are separated and removed by centrifugal separation or molecular sieve. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a film having a molecular weight cut of 10,000 to 100,000 Da is preferably used.
By this concentration treatment, the reaction solution is concentrated to 1/5 to 2/3 volume of the original volume, and as a result, the optimum synthesis concentration of each synthesis factor is greatly shifted. By this concentration, the reaction system is remarkably reduced in its synthesis ability. With this state, the present invention is called discontinuous.
Subsequently, the synthesis reaction is reactivated. Those diluted in the previous stage are concentrated, and those that have been concentrated are diluted.
In the former case, the concentration process is performed following the dilution process. Here, the concentration means returning the liquid volume of the reaction system increased by dilution to the original liquid volume. The concentration means is not particularly limited, and any concentration means known per se can be used. Preferable examples include filtration using an ultrafiltration membrane, treatment using a centrifugal separator, gel filtration, a method of generating a pressure difference in the liquid phase or gas phase, and the like. In this treatment, by adjusting the passage diameter of the membrane, the reaction product and reaction by-product are separated and removed by centrifugal separation or molecular sieve. The molecular weight cut size of the membrane, the centrifugal speed, and the gel filtration conditions can be optimally adjusted depending on the physical properties of the product to be processed known per se. In the present invention, a membrane having a molecular weight cut of 10,000 to 100,000 Da is preferably used.
In the latter case, a dilution process is performed following the concentration process. Here, “dilution” means returning the liquid volume of the reaction system reduced by concentration to the original liquid volume. The diluted solution contains a template substance, a substrate, and a reaction solution as desired. Particularly preferably, a solution containing a template substance, a substrate, an energy source and the like is used. The concentration of each component added is selected to be adjusted to the optimum concentration after dilution.
Thus, the temperature of the reaction system restored to the optimum concentration of the reaction system is adjusted again to the optimum reaction temperature, and the reaction system is reactivated. The optimum temperature is 15 to 25 ° C.
In the present invention, it is possible to repeat the dilution and concentration processes a plurality of times discontinuously, and by this repetition, the regeneration of the cell-free synthesis system is achieved a plurality of times. The number of repetitions is 2 to 20 times, preferably 5 to 10 times. This regeneration achieves mass protein synthesis.
Other steps may be carried out in accordance with any conventionally known procedures and conditions that can be applied for automation, and are not particularly limited.
In the present invention, an apparatus for performing the above operation is characterized by having at least the following means (a) to (e).
(A) means for variably controlling the temperature in the reaction vessel;
(B) means for dispensing a sample or reagent into the reaction vessel;
(C) means for transporting the reaction vessel;
(D) precipitation and concentration filtration means;
as well as
(E) Control means for controlling the means (a) to (d) to operate in accordance with the method of the present invention described above.
By using an apparatus having at least the configurations of (a) to (e), the reaction operation until the protein encoded by the template is generated from the transcription template synthesized in the transcription product production system of the present invention described above. It can be performed automatically. Hereinafter, each configuration will be specifically described in detail.
(A) Means for variably controlling the temperature in the reaction vessel
Means for variably controlling the temperature in the reaction vessel include a transcription reaction, a translation reaction incubation and a translation reaction stop, a translation template precipitation, or a transcription template preparation process by PCR using the automatic synthesizer of the present invention. In the case of carrying out automatically, it is a means for adjusting the liquid temperature in the reaction vessel to an appropriate temperature condition in the amplification reaction of the PCR method. The temperature range to be variably controlled is not particularly limited, but a temperature range normally required in a series of reaction operations for cell-free protein synthesis including preparation of a transcription template (for example, about 4 ° C. to about 100 ° C., preferably about As long as the temperature of the liquid in the reaction vessel can be variably controlled within a range of 15 ° C. to about 60 ° C., the means for realizing this is not particularly limited. For example, conventionally known Takara PCR thermal cycler MP (manufactured by Takara Bio Inc.), Gene Amp PCR System 9700 (manufactured by Applied Biosystems Inc.), and the like can be mentioned. Specifically, in addition to the work stage for placing the reaction vessel, which is a place where pipetting is performed, a plurality of stages for placing the reaction vessel are provided in the apparatus, and the temperature of the entire space on the stage is adjusted. As a result, the temperature in the reaction vessel is variably controlled.
(B) Means for dispensing sample or reagent into reaction vessel
The means for dispensing a sample or reagent into a reaction container is to dispense a sample or reagent into a reaction container in order to perform a series of cell-free protein synthesis reactions such as transcription reaction, translation reaction, and PCR in the reaction container. Means. Here, “sample” refers to transcription template, translation template, PCR template plasmid (or host (eg, E. coli) having the plasmid), etc., and “reagent” refers to transcription reaction solution, translation reaction solution, translation reaction. It refers to a solution, a diluted solution, an alcohol, a salt solution, a PCR reaction solution and the like. As such a dispensing means, a pipette arm (dispensing machine) that can be dispensed automatically in a conventionally known manner is particularly suitable as long as it can dispense by adjusting the amount of sample and reagent according to the process. It can be realized without limitation. In addition, the pipette arm can have a function of discarding a used tip at a tip disposal port of a synthesizer and a function of discharging a sucked filtrate or the like to a waste solution port.
In addition to the above functions, the dispensing means more preferably has a mixing function (eg, pipetting, stirring, etc.) for homogenizing two or more types of solutions and precipitating dissolution.
Further, by adding a substrate solution or a diluting solution into each cell of the reaction vessel with a pipette arm, a diluting process for the reaction solution can be performed.
(C) Means for transporting the reaction vessel
The means for transporting the reaction container is a means for moving the reaction container to each stage, centrifuge, elevator, and thermostat. The means for transporting the reaction container is not particularly limited as long as the reaction container can be transported to a target place, and can be realized by any conventionally known appropriate means. For example, it can be realized by using a robot arm used in a conventional synthesis apparatus.
(D) Means for precipitation and concentration filtration
Precipitation and concentration filtration means are means for concentrating and filtering the reaction solution during the operation of repetitive translation reaction or the operation of precipitating cloudy matter generated during the transcription reaction. Such means is particularly limited as long as it can precipitate white turbid matter generated during the reaction and can be separated into solid and liquid, and can concentrate the reaction liquid during repeated translation reactions. It can implement | achieve by the conventionally well-known appropriate means, without. For example, the means can be realized by using a conventionally known centrifuge and other appropriate devices conventionally used for filtration and freeze-drying.
(E) Control means
In the control means, the operation of the drive source (motor, pneumatic / hydraulic equipment, other actuators that can be controlled in operation, etc.) used for each means in order to operate the means (a) to (d) is turned on / off. A control device for controlling the degree and state of operation is included. The configuration of the control is such that the operations of the means (a) to (d) described above are performed automatically from the transcription template synthesized in the transcription product production system until the protein encoded by the template is generated. Can be achieved.
The control device may be configured by combining control devices necessary for controlling the operation of each means, such as a control circuit including a computer having a control program, a sequence control circuit, etc., in the order according to the purpose. The control configuration is such that signals, power, pneumatic pressure, hydraulic pressure, and the like can be supplied to each means so that each means operates. Further, a driver necessary for directly sending a drive signal to the drive source of each means, various sensors and switches necessary for detecting an operation state of the drive source of each means may be added as appropriate.
The reaction vessel applicable to the synthesizer of the present invention is not particularly limited, and various conventionally known reaction vessels that have been used for cell-free protein synthesis reaction can be used. For example, a 96-well PCR plate , 96-well titer plates, 8-strip tubes and tubes (1.5 mL, 15 mL, 50 mL, etc.). For example, when a batch method or multi-layer method is used as a translation reaction system, a small reaction system such as a 96-well plate is used. A translation reaction can be performed, and according to the synthesizer of the present invention, a transcription reaction can also be performed in a small reaction system, so that a transcription reaction, a purification of a translation template, a translation reaction, and a transcription for further a transcription reaction if desired A series of cell-free protein synthesis methods including PCR for template production can be performed simultaneously on multiple types of proteins in multiple reaction systems. Many proteins can be synthesized during.
Furthermore, the present invention provides an apparatus for performing a synthesis method in which dilution and concentration are repeated discontinuously in a synthesis reaction using a translation template. The apparatus of the present invention is characterized by having at least the following means (1) to (5).
(1) means for initiating synthesis;
(2) means for concentrating the reaction solution;
(3) means for diluting the reaction solution;
(4) means for reactivating the synthesis reaction;
(5) Means for repeating the above means (2) to (4);
Or
(1) means for initiating synthesis;
(2) means for diluting the reaction solution;
(3) means for concentrating the reaction solution;
(4) means for reactivating the synthesis reaction;
(5) Means for repeating the above means (2) to (4);
By using an apparatus having at least the configurations of (1) to (5), the above-described high-throughput synthesis system of the present invention can be automatically performed. Hereinafter, each means will be described in detail.
(1) means for initiating synthesis;
(1) Move the dispenser to the reagent tank containing the translation reaction solution and aspirate the translation reaction solution.
(2) Move the dispenser onto the translation plate (the bottom of the cell is a filter), and discharge the translation reaction solution into each cell of the translation plate containing the transcription product.
Following (3) and (2), the dispenser pipettes each cell.
(4) The robot arm covers the translation plate, transports the plate to the thermostat and sets it in the thermostat.
(5) Keep the translation plate warm to start synthesis.
(6) After completion of the set synthesis time, the robot arm conveys the translation plate to the MTP stage.
(2) means for diluting the reaction solution;
(1) Move the dispenser to the reagent tank containing the diluted solution and aspirate the diluted solution.
(2) Move the dispenser onto the translation plate and discharge the diluted solution into each cell of the translation plate containing the translation product.
(3) Means for concentrating the reaction solution
(1) The robot arm puts the translation plate on the transfer / filtrate receiving plate.
(2) The robot arm conveys the translated translation plate and the transfer / filtrate receiving plate to the lifting platform.
(3) The elevating table on which the translated translation plate and the transfer / filtrate receiving plate are placed is lowered so as to match the height of the centrifuge.
(4) The robot arm sets the superimposed translation plate and transfer / filtrate receiving plate in the centrifuge. At this time, when the translation plate and the transfer / filtrate receiving plate are a set, a dummy plate is set on the diagonal line. In the case of two sets, the plates are set diagonally.
(5) Start centrifugation with a centrifuge. At this time, since the bottom of the translation plate is a filter, the filtrate is removed in the cells of the transfer / filtrate receiving plate, and each cell in the translation plate is concentrated.
(6) The robot arm conveys the translated translation plate and the transfer / filtrate receiving plate from the centrifuge to the lifting platform.
(7) Raise the lifting platform on which the overlaid translation plate and the transfer / filtrate receiving plate are placed.
(8) The robot arm transports the superimposed translation plate and transfer / filtrate receiving plate to the MTP stage, and separates and sets the translation plate and the transfer / filtrate receiving plate.
(4) Means for reactivating the synthesis reaction
When the dilution process is performed first in the previous stage, the concentration process is performed subsequently. When the process is performed previously, the dilution process is performed.
(1) The robot arm transports the translation plate after concentration or the translation plate after dilution to a thermostat and sets it in the thermostat.
(2) Keep the translation plate warm to start synthesis.
(5) Means for repeating the above means (2) to (4)
(1) After the set time is over, the temperature of the thermostatic bath is lowered to stop the substantial synthesis.
(2) The robot arm transports the translation plate to the MTP stage.
(3) The means described in (2) to (4) is repeated a plurality of times.
The means of the synthesizer described above is an example of an apparatus for automatically performing a high-throughput synthesis system, and may further include a waste liquid treatment means by a concentration process, a tip disposal process means of a dispenser, and the like.
In addition, a program including the following information processing means is also a subject of the present invention in order to execute a synthesis system using a synthesis reaction initial phase having a high protein synthesis reaction rate for implementing a high-throughput synthesis system a plurality of times.
(1) Information processing means for setting a synthesis time within the initial phase range of the synthesis reaction just before the amount of synthesis per unit time tends to decrease based on information on the volume of the reaction vessel and the concentration of the reaction solution,
(2) Information processing means for adding a dilute solution to the reaction vessel when the synthesis time within the synthesis reaction initial phase range of (1) is reached, and setting the reaction solution outside a concentration range that can substantially undergo a synthesis reaction,
(3) Information processing means for concentrating the reaction liquid in the reaction vessel after (2) and setting so that the volume of the reaction liquid increased by addition of the diluted solution is returned to the original liquid volume,
(4) After (3), an information processing means for setting the reaction optimal temperature in order to restart the synthesis reaction,
(5) Information processing means for repeating (1) to (4) a plurality of times,
Or
(1) Information processing means for setting a synthesis time within the initial phase range of the synthesis reaction just before the amount of synthesis per unit time tends to decrease based on information on the volume of the reaction vessel and the concentration of the reaction solution,
(2) Information processing means for concentrating the reaction solution in the reaction vessel when the synthesis time in the synthesis reaction initial phase range of (1) is reached, and setting the reaction solution outside the concentration range that can substantially undergo the synthesis reaction,
(3) After (2), an information processing means for adding a dilute solution to the reaction vessel and setting the volume of the reaction liquid reduced by the concentration back to the original liquid volume,
(4) After (3), an information processing means for setting the reaction optimal temperature in order to restart the synthesis reaction,
(5) Information processing means for repeating (1) to (4) a plurality of times.
Furthermore, in a conventionally known synthesis apparatus in which the above program is incorporated, the information processing of the program is performed in cooperation with the synthesis apparatus to start synthesis, dilute / concentrate the reaction solution, and reactivate the synthesis reaction. An object of the present invention is also a synthesizer characterized in that a synthesizing system is executed and a synthesis system using a synthesis reaction initial phase having a high protein synthesis reaction rate can be repeatedly executed a plurality of times.
In addition, the transcription reaction and the translation reaction are preferably performed in a sealed reaction vessel. From such a viewpoint, a reaction vessel with a lid is used, and the device further opens and closes the lid of the reaction vessel. It is preferable to have a means. For example, when a 96-well plate is used as the reaction vessel, the lid is exemplified by a rubber lid that can seal each hole individually. Since it is preferable that the lid can be brought into close contact with the reaction vessel in a closed state, a lid having a certain weight (for example, about 500 g) is used, or a lid or the like is sandwiched between the lid and the reaction vessel. It is conceivable to close the lid. The means for opening and closing the lid of the reaction vessel can be realized using, for example, a mechanism in which a conventionally known chucking mechanism / suction mechanism and a robot arm are combined.
The synthesizer of the present invention may have means for stocking the reaction reagent as necessary in addition to the means described above.
As described above, the high-throughput system of the present invention and the apparatus that automatically performs the system can simultaneously and easily synthesize a plurality of types of proteins. For example, multiple transcription templates and translation templates encoding various mutant proteins can be prepared, and multiple mutant proteins can be synthesized simultaneously for analysis without requiring detailed design of the mutants. Is useful.
The high-throughput system of the present invention and an apparatus that automatically performs the system can be suitably used for high-throughput functional analysis of various proteins. For example, as a result of homology search, a gene group encoding a protein containing a conserved common domain (for example, a kinase domain) is used as a template, and the protein is simultaneously synthesized by the method of the present invention using the apparatus of the present invention. On the other hand, a group of proteins that can be phosphorylation targets (for example, transcription factors) are synthesized in the same manner, and both are mixed in various combinations. ³² It is possible to identify which protein kinase phosphorylates which protein using the uptake of P-labeled ATP as an index.
Alternatively, a gene group encoding a protein containing a motif specific to a transcription factor (eg, Zn finger, leucine zipper, etc.) is used as a template, and the protein is simultaneously synthesized by the method of the present invention using the apparatus of the present invention. By investigating the ability to bind to cis-element sequences, heterodimer formation with other transcriptional regulatory factors, and the ability to bind to the transcriptional regulatory region of specific gene promoters, information for elucidating crosstalk woven by transcription factors Obtainable.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples.
(Cell-free protein synthesis method)
1) Reaction of DNA to mRNA (transcription reaction)
Transcription reaction solution [final concentration, 80 mM HEPES-KOH pH 7.8, 16 mM magnesium acetate, 10 mM dithiothreitol, 2 mM spermidine, 2.5 mM 4NTPs (4 types of nucleotide triphosphates), 0.8 U / μl RNase inhibitor, 0.1 μg / μl DNA [plasmid GFP (Green fluorescein protein)], 1.6 U / μl SP6 RNA polymerase] was prepared and reacted at 37 ° C. for 3 hours. After the reaction, the mixture was centrifuged at 12,000 rpm for 1 hour at 4 ° C. After centrifugation, the supernatant was collected and added to ethanol at a final concentration of about 70% and ammonium acetate to a final concentration of about 0.27M. And then centrifuged at 3,000 rpm for 30 minutes (ethanol precipitation). After this ethanol precipitation was performed three times, an appropriate amount of dialysis buffer (final concentration, 35 mM HEPES-KOH pH 7.8, 3.1 mM magnesium acetate, 103 mM potassium acetate, 16 mM creatine, 1.2 mM ATP, 0.26 mM GTP, 2 (5 mM DTT, 0.43 mM spermidine, 0.3 mM of various amino acids) to adjust the RNA concentration to about 5 to 10 mg / ml.
Further, mRNA synthesis according to the present invention by discontinuous repetition of dilution and concentration was performed according to Experimental Example 7.
2) Preparation of translation reaction solution
The embryo extract was used as a translation reaction solution (final concentration 35 mM HEPES-KOH pH 7.8, 103 mM potassium acetate (KOAc), 3.1 mM magnesium acetate (Mg (OAc) 2), 16 mM creatine phosphate, 1.2 mM ATP, 0 .26 mM GTP, 2.5 mM dithiothreitol (DTT), 0.43 mM spermidine, 0.3 mM AAs (mixture of 20 L-type amino acids), 1.03 mg / ml creatine kinase] The extract was prepared so that the concentration (OD 260 nm) was 40 to 120.
3) Translation reaction
The prepared mRNA solution was added to the translation reaction solution containing the embryo extract according to the concentration of the embryo extract during the translation reaction, and the reaction was performed. In other words, the O.D. D. When 260 nm was 100, the amount of mRNA to be added was 0.8 mg / ml, and the amount of mRNA to be added was prepared by proportional calculation.
The reaction solution prepared above was reacted at 20 ° C. After a certain period from the start of the reaction (10 minutes to 3 hours), the reaction solution is diluted with 2 to 3 volumes of the above-mentioned translation reaction solution, and then concentrated with an ultrafiltration membrane having a molecular weight cut of 30,000 Da to obtain a reaction solution. Was returned to the same amount as before dilution. This operation was repeated several times. Alternatively, it was first concentrated to about 1/2 to 1/3 volume, and then diluted to the original volume using the translation reaction solution.
When additional mRNA is added to the translation reaction solution by batch operation, the transcription reaction solution prepared in 1) (before ethanol precipitation) or the mRNA solution prepared through ethanol precipitation operation is adjusted to the concentration of the wheat germ solution. Added accordingly.
4) Confirmation of protein synthesis effect
The amount of synthetic protein was measured according to the method described in the report of Madin K et al. (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556). went.
5) Form of wheat germ extract
The wheat germ extract, cell-free protein synthesis reaction solution composition, mRNA preparation method, and batch-type cell-free protein synthesis method used in this study are described in Madin K et al. (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556), Japanese Patent No. 3255784, Japanese Patent Laid-Open No. 2000-236896, WO 00/68412, Sawasaki. T. T. et al. et al. (Proc. Natl. Acad. Sci. USA (2002), 99, 14652-14657).
(Example of equipment)
FIG. 4 shows a schematic diagram of a system for allowing the dilution and concentration operations of the present invention to be performed a plurality of times and performing the synthesis reaction discontinuously. When the dilution and concentration operations are realized by a configuration having a filtration membrane and a liquid feed pump, for example, use of a mechanism schematically shown in FIG. 4 is exemplified. By using a mechanism in which the filtration concentrator 1 having a filtration membrane and the liquid feed pump 2 are combined, the (RNA) substrate solution (transcription or) translation reaction before and after the completion of the translation reaction in the reaction tank 3 After diluting the liquid, the by-product is filtered by the filter concentrator 1 and discharged into the container 6. On the other hand, the concentrated (transcription or translation) reaction solution can be returned to the reaction vessel 3 to resume the reaction.
In the system shown in FIG. 4, the container 4 containing the (RNA) substrate solution and the reaction vessel 3 are connected via a tube T1, a liquid feed switching valve 5 and a tube T2, and this liquid feed switching valve 5 is connected to the tube T3. The reaction tank 3 is connected via the liquid feed pump 2, the tube T4, the filtration concentrator 1, and the tube T5. In this way, the liquid feed switching valve 5 accommodates the (RNA) substrate solution by (a) the state in which no liquid is fed into the reaction tank 3 (the valve is closed), and (b) the liquid feed pump 2 is operated. The (RNA) substrate solution can be pumped up from the vessel 4 that has been prepared, and can be directly fed into the reaction tank 3, and (c) the translation reaction solution in the reaction tank 3 is transferred by the operation of the liquid feed pump 2. A state in which the translation reaction solution can be circulated so as to be pumped up and passed through the filtration concentrator 1 and then returned to the reaction vessel 3 (d) The vessel 4 containing the substrate solution by the operation of the liquid feed pump 2 Then, the state in which the (RNA) substrate solution is pumped up, passed through the filtration concentrator 1 and fed into the reaction vessel 3 can be switched appropriately.
Using this configuration, the operation is performed according to the following procedure.
1. In the reaction tank 3, a batch reaction (transcription or translation) is performed. During this time, the liquid feed switching valve 5 is closed [state (a) above], the liquid feed pump 3 is also stopped, and the liquid feed into the reaction tank 3 is stopped.
2. At any time before or after the (transcription or) translation reaction is stopped, the liquid feed switching valve 5 is switched [state (b) above], the liquid feed pump 2 is operated, and the (RNA) substrate solution is put into the reaction tank 3. Feed and mix in.
3. By switching the liquid feed switching valve 5 (state (c) above), the liquid feed pump 2 is operated to pump up the (transcription or) translation reaction liquid in the reaction tank 3 and pass through the filtration concentrator 1 to produce by-products. The product is discharged into the container 6 and circulated so that the concentrated reaction liquid is returned to the reaction tank 3.
4). The liquid feed switching valve 5 is switched [state (d) above], the liquid feed pump 2 is operated to pump up the (RNA) substrate solution in the container 4, and the liquid is fed into the reaction tank 3 through the filter concentrator 1. Then, the translation reaction solution partially remaining in the filter concentrator 1 is washed out into the reaction vessel 3.
5. The liquid feed switching valve 5 is switched [state (b) above], the liquid feed pump 2 is operated, the (RNA) substrate solution in the container 4 is pumped up, and the liquid is fed into the reaction tank 3 to be fed into the reaction tank. After adjusting the amount of the reaction solution (translation or translation) in 3 to the original volume, the solution feed switching valve 5 is switched [state (a)] to stop the solution feed.
Return to 6.1 and repeat the above operations.
In the case of realizing an apparatus using such a system, as a liquid feeding pump to be used, a conventionally known appropriate liquid feeding pump can be used without particular limitation. For example, a peristaltic pump [for example, LKB-Pump-p1 ( Pharmacia, etc.) and the like. Moreover, a conventionally well-known thing can also be used as a filtration concentrator without a restriction | limiting in particular. Specifically, a cross flow filtration device, VF05C2 type (molecular weight 30,000 cut, manufactured by Sartorius) and the like are exemplified.
According to the system in which the dilution and concentration means is realized with a configuration having a filtration membrane and a liquid feed pump, the protein synthesis yield is improved as compared with the case of using a centrifuge (see Experimental Example 4). In addition, a device capable of synthesizing a large-scale cell-free protein can be constructed. Furthermore, it is possible to construct a small-capacity apparatus by combining a small reaction vessel and a filtration concentrator, which is different from the configuration having a filtration membrane and a centrifuge as a by-product removing means described above. The device is not necessarily a big one. In addition, such a configuration having a filtration membrane and a liquid feed pump facilitates precise control of the dilution / concentration process, and is extremely useful for automating general cell-free protein synthesis technology for various purposes. It can be expected to become an important core technology.
Experimental example 1
FIG. 1 shows the experimental results using a wheat germ extract as an example (Example 1) of a batch-type cell-free protein synthesis method in which the dilution and concentration treatments of the present invention are repeated discontinuously. A comparative example is the result by the conventional cell-free protein synthesis method by a batch method. The vertical axis represents the amount of synthetic protein (mg / ml), and the horizontal axis represents the reaction time (hours). In FIG. 1, the line connecting black circles (●-●) is the result of the method of the present invention, and the line connecting white circles (◯ -O) is the result of the conventional batch method. For the synthesis reaction, a wheat germ extract having a concentration of 40A 260 nm / ml (absorbance of wheat germ extract at a wavelength of 260 nm) and 320 μg / ml of green fluorescein protein (GFP) -encoding mRNA (translation template) are mixed, Cell-free protein synthesis was performed at 20 ° C.
The synthetic reaction of the comparative experiment was carried out using a concentrated container with an ultrafiltration membrane having a molecular weight cut off of 30,000 (cut molecular weight: 30,000 kDa). In the batch reaction method of the conventional method, the change over time in the amount of protein produced using fluorescence activity as an index shows a typical hyperbolic type. That is, the reaction rate decreases through the maximum rate phase at the beginning of the reaction, and after 3 hours when the reaction is stopped, no increase in the amount of synthetic protein is observed even with long-term incubation (◯-○). This result is in good agreement with previous reports (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556). Although the cause of this reaction stop has not been completely elucidated, the decrease in the concentration of ATP and GTP (energy depletion), which is an energy source, and the accumulation of AMP and GMP, which are byproducts that cannot be regenerated, are It is thought that protein synthesis is suppressed by the mechanism.
The synthesis reaction of the present invention is carried out in a container equipped with an ultrafiltration membrane, and the protein synthesis substrate solution (substrate and energy source is 3 times the volume of the reaction solution 2 hours after the start of the reaction, which has a high reaction rate and before stopping the reaction). Add) and stir (dilution treatment). Next, the solution was diluted with a centrifuge, and the solution diluted with this substrate solution was concentrated to the volume of the reaction solution before dilution (at the time indicated by the arrow) (concentration treatment). By repeating this series of dilution and concentration processes a plurality of times in a discontinuous manner, low-molecular substances including by-products are eliminated and reduced in concentration through the ultrafiltration membrane, while ATP consumed in the synthesis reaction, GTP and amino acid were recovered to near their respective concentrations at the start of the reaction. Simultaneously with this concentration treatment, ribosomes derived from wheat germ, tRNA and all other translation factors are concentrated to the original concentration at the start of the reaction. By such treatment, it can be confirmed that the protein synthesis reaction once reduced is resumed, and after reaching the original high initial rate, the reaction again decreases (●-●). By repeating such a discontinuous dilution / concentration treatment, it becomes possible to utilize the characteristics of the time domain with a high initial reaction rate, and achieve highly efficient cell-free protein synthesis. As shown in FIG. 1, by repeating the dilution / concentration treatment four times, the synthesis amount, which was 0.072 mg per 1 ml reaction volume in the batch system of GFP, can be increased to 0.41 mg. It was. The amount of synthetic protein was calculated according to a GFP fluorescence intensity measurement technique (fluorescence maximum wavelength 508 nm), assuming that 1 μg / ml of GFP has a fluorescence intensity of 2.0.
Although sufficient synthesis efficiency was achieved by this treatment, a phenomenon was observed in which the protein synthesis rate of the system gradually decreased with repeated dilution and concentration treatments. This can be caused by leakage of the translation factor (s) derived from the germ into the filtrate, loss from the reaction system due to adsorption on the ultrafiltration membrane, degradation of mRNA, or adsorption on the ultrafiltration membrane. It was estimated that the concentration decreased due to the like.
Experimental example 2
FIG. 2 is a graph showing the results of an experiment in which cell-free protein synthesis was performed in the same manner as in Example 1 except that the concentration of the wheat germ extract was changed, and the vertical axis represents the amount of protein synthesized (mg / ml). The horizontal axis indicates the reaction time (hours). In FIG. 2, a line connecting small black circles (· − ·) indicates experimental results when the concentration of wheat germ extract is 60A260 nm / ml and the GFP mRNA concentration is 480 μg / ml (Example 2). The line (●-●) connecting indicates the experimental results when the concentration of the wheat germ extract is 80 A 260 nm / ml and the GFP mRNA concentration is 640 μg / ml (Example 3). In FIG. 2, arrowheads indicate the time point when the above operation is performed.
As shown in FIG. 2, the protein synthesized in a cell-free system by the discontinuous repeated operation of dilution and concentration four times in Example 2 was 0.62 mg per ml of the reaction solution, and four times dilution and concentration in Example 3 The protein synthesized in a cell-free system by the discontinuous operation was 1.13 mg per 1 ml of the reaction solution. Thus, the amount of protein synthesis by the cell-free protein synthesis method of the present invention increases as the concentration of the translation factor in the reaction system increases, and is significantly improved as compared with the experimental results of the conventional batch method shown in FIG.
Experimental example 3
Since the time for reducing the synthesis rate in the usual batch method depends on the decrease in the substrate concentration in the reaction solution and the accumulated concentration of by-products, it is inversely related to the translation factor concentration of the system. That is, the higher the translation factor concentration (wheat germ extract concentration) in the system, the shorter the time to stop the reaction (Madin K et al., Proc. Natl. Acad. Sci. USA (2000), 97, 556-559). Thus, when translation factors are used at high concentrations, it is considered possible to maintain a high synthesis rate by shortening the time interval for discontinuous repeated dilution and concentration operations. It can be expected to increase production efficiency within a unit time.
FIG. 3A shows a cell-free manner in the same manner as in Example 1 except that the concentration of the wheat germ extract was 80 A 260 nm / ml, the concentration of GFP mRNA was 640 μg / ml, and the interval for performing the repeated repetition of dilution and concentration was changed. It is a graph which shows the experimental result which performed protein synthesis, FIG. 3: B is the polyacrylamide gel electrophoresis result which shows the quantitative difference of the synthesized GFP by the difference in the interval which performs the discontinuous repetition operation of dilution and concentration.
In the graph of FIG. 3A, the vertical axis represents the amount of protein synthesized (mg / ml), the horizontal axis represents the reaction time (hours), and the arrowheads represent the time points at which discontinuous repetition of dilution and concentration was performed. In FIG. 3A, the black circled line (●-●) shows the experimental results when the above-mentioned discontinuous repetition of dilution and concentration was performed at 0.5 hour intervals from the start of the reaction (Example 4). The line (○-○) connecting, indicates the experimental results when the above-mentioned discontinuous repetition of dilution and concentration was performed at 1 hour intervals from the start of the reaction (Example 5). In FIG. 3A, arrowheads indicate the time points at which the above dilution and concentration discontinuous repetition operations were performed.
FIG. 3B shows Example 4 in which the above-described dilution and concentration discontinuous repetition operation was performed at intervals of 0.5 hours, and Example 4 was performed except that the above-described dilution and concentration discontinuous repetition operation was performed at intervals of 2 hours. It compares and shows the case (Example 6) performed similarly. In addition, the arrowhead in FIG. 3B has shown the dyeing | staining band of GFP. Polyacrylamide gel electrophoresis is performed at each reaction time (in Example 4, 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5 hours have elapsed from the start of the reaction, in Example 6, 1 μl of the reaction solution at 0, 2, 4, 6, 8, 10, 12, 14 hours after the start of the reaction) was separated by polyacrylamide native gel electrophoresis, and the protein was stained with Coomassie brilliant blue.
As shown in FIGS. 3A and 3B, Example 4 in which the above dilution and concentration discontinuous repetition operation was performed at intervals of 0.5 hours was performed by performing the above dilution and concentration discontinuous repetition operation at intervals of 1 hour. Example 5 The yield per unit time is higher than that of Example 6 in which the above dilution and concentration discontinuous operation was repeated at intervals of 2 hours. In Example 4, a synthesis amount of 1.12 mg / ml is obtained in a reaction of 2.5 hours. This is the same concentration of the reaction solution, and the above dilution and concentration are repeated repeatedly at intervals of 2 hours for 10 hours. It exceeds the yield obtained by the reaction. This result shows that the higher the translation factor concentration in the reaction system (wheat germ extract concentration), the lower the concentration that accompanies consumption of energy sources and the like, and at the same time, the accumulated concentration of by-products increases. It directly indicates that the time to decrease and stop the reaction is shortened. Therefore, in setting the time for the discontinuous repeated operation of dilution and concentration, the maximum synthetic yield can be achieved by selecting the optimal timing depending on the concentration of the wheat germ extract contained in the reaction system.
Proteins include instability (high reactivity) as a general physical property. Therefore, in order to obtain a high-quality protein, it is one of the essential conditions to construct a technique for increasing the synthesis yield in a short time, and the principle of the present invention proved in FIG. 3 can be said to be extremely effective.
Experimental Example 4
FIG. 5 shows an experiment in which protein synthesis was carried out by the cell-free protein synthesis method of the present invention using the configuration in which the filtration membrane and the liquid feed pump shown in FIG. 4 were used for removal of by-products (Example 7) It is a graph which shows a result. The vertical axis represents the amount of protein synthesized (mg / ml), and the horizontal axis represents the reaction time (hours). In FIG. 5, a line connecting black circles (●-●) shows the experimental results of Example 7, and a line connecting white circles (◯-○) shows the experimental results of the conventional batch method. Moreover, in FIG. 5, arrowheads show the time of performing the above-mentioned discontinuous repetition operation of dilution and concentration.
In Example 7, a cross flow filtration apparatus (VF05C2 type, manufactured by Sartorius, molecular weight 30,000 cut) was used as a filtration concentrator, and LKB-Pump-P1 (Pharmacia) was used as a liquid feed pump. In addition, a translation reaction solution similar to that in Example 1 was used except that 80A 260 nm / ml wheat germ extract and 640 μg / ml concentration GFP mRNA were used. Done at intervals. As a reaction container, a graduated plastic tube (Falcon tube) was used, the reaction volume was 15 ml, and dilution in a discontinuous repeated operation of dilution and concentration was performed using a translation reaction solution having a volume of 45 ml.
As shown in FIG. 5, when a configuration having a filtration membrane and a liquid feed pump is used as a means for removing by-products, the ultrafiltration membrane and the centrifuge in Experimental Examples 1 to 3 are used. It was confirmed that the present invention can be suitably used in the cell-free protein synthesis method of the present invention, as in the case of the apparatus using the. In Example 7, the protein was synthesized with a synthetic yield of 2.1 mg per ml of reaction volume by reaction for 4 hours, which was about twice as high as when using the above-mentioned apparatus using a centrifuge and an ultrafiltration membrane.
Experimental Example 5
FIG. 6A shows a discontinuous dilution and concentration using the same translation reaction solution as in Example 1 except that 60A 260 nm / ml wheat germ extract and 450 μg / ml dihydrofolate reductase (DHFR) mRNA were used as translation templates. It is a figure which shows the experimental result at the time of performing a cell-free protein synthesis | combination like Example 1 (Example 8) except having repeated the repetition operation twice at 1 hour intervals. As a comparative experiment, cell-free protein synthesis was carried out under the same conditions except for the conventional batch method (no discontinuous repetition of dilution and concentration). In Example 8, a reaction vessel equipped with an ultrafiltration membrane having a cut molecular weight of 30,000 Da (manufactured by Millipore) was used for the synthesis of DHFR having a molecular weight of about 20,000 KDa in a cell-free system.
In FIG. 6A, lanes 1, 2, and 3 are the discharged solution (filtrate) after the reaction by the discontinuous repeated operation of Example 1, 0, 1, and 2 dilution and concentration, and the dilution and concentration discontinuities, respectively. 1 μl of each reaction solution in the conventional batch method at the same time as the repetitive operation was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the result of Coomassie brilliant blue staining is shown (the arrowhead in the figure is: DHFR which is a synthetic product is shown.). As can be seen from the staining intensity of the DHFR band indicated by the arrowhead in FIG. 6A, the experimental results of the conventional batch method (the “reaction solution” on the left side of each lane) confirmed the synthesis of a trace amount of DHFR after 1 hour of reaction. However, in the experimental results of Example 8 (the “filtrate” on the right side of each lane), the DHFR produced in the reaction system was filtered by the discontinuous operation of dilution and concentration once and twice. It was confirmed that the liquid could be taken out efficiently.
Experimental Example 6
FIG. 6B shows the results of an experiment in which a GFP fusion protein in which streptavidin is fused to the N-terminus by the cell-free protein synthesis method of the present invention (Example 9) and the GFP fusion protein is isolated outside the system. FIG.
The cell-free protein synthesis itself in this experiment was the same as in Example 1 except that mRNA encoding GFP fusion protein was used as a translation template, and the discontinuous repetition of dilution and concentration was performed 4 times at 1 hour intervals. went. In order to isolate the GFP fusion protein outside the system, first, magnetic beads immobilized with 1 biotin are added to the reaction solution, and the resulting fusion product is used for the magnetic beads using the affinity between biotin and streptavidin. The beads were bonded to the two fusion proteins that were selectively collected in the reaction vessel, and the procedure was such that the beads were selectively removed from the reaction vessel using a magnet. Electrophoresis and staining were performed in the same manner as in Experimental Example 5.
As shown in FIG. 6B, the product detected in the reaction solution before the isolation operation disappears from the reaction solution by this operation (an arrowhead in the reaction solution lane after isolation), and the system is efficiently passed through the magnetic beads. It can be seen that it is isolated outside (synthetic product shown in the isolation product lane).
Experimental Example 7
FIG. 7 is a diagram showing the difference in experimental results between the RNA synthesis method of the present invention (Example 10) and the conventional method for synthesizing RNA in vitro by the batch method. In FIG. 7, the left of FIG. 7 shows the result of the conventional method by the batch method, the right of FIG. 7 shows the result of the RNA synthesis method of the present invention, and the arrow shows the mRNA product encoding GFP.
In each case, a transcription reaction solution [final concentration, 80 mM HEPES-KOH pH 7.8, 16 mM magnesium acetate, 10 mM dithiothreitol, using a GFP gene of jellyfish inserted into a wheat germ cell-free dedicated plasmid vector pEU as a transcription template. 2 mM spermidine, 2.5 mM 4NTPs (4 types of nucleotide triphosphates), 0.8 U / μl RNase inhibitor, 0.1 μg / μl DNA [plasmid GFP (Green Fluorescent protein)], 1.6 U / μl SP6 RNA Polymerase] was prepared and reacted at 37 ° C. for 3 hours.
The RNA synthesis reaction in Example 10 was carried out 3 hours after the start of the reaction, diluted by adding 3 times the amount of the above transcription reaction solution to the transcription reaction solution, and then using a centrifuge to remove low molecules through a filtration membrane. By excluding the reaction solution from the reaction solution system, the transcription reaction solution was concentrated to the original concentration. Thereafter, the synthesis reaction was allowed to proceed by discontinuously repeating dilution and concentration at intervals of 3 hours. At the time of discontinuous repeated operation of each dilution and concentration, the transcription reaction solution was collected, and the transcription product (mRNA) separated by agarose gel electrophoresis was visualized by ethidium bromide staining.
As shown in FIG. 7, in the method of synthesizing RNA in vitro by the conventional batch method, the RNA synthesis reaction is stopped in about 3 hours. In Example 10 in which repeated operations were performed, mRNA could be synthesized in a yield 2.4 times that of the conventional method.
Experimental Example 8
FIG. 8 is a graph showing the results of an experiment in which the cell-free protein synthesis method of the present invention was performed using mRNA purified by ethanol precipitation and unpurified mRNA as translation templates, and the vertical axis represents the synthesized protein. Amount (mg / ml), horizontal axis indicates reaction time (hours). Purified mRNA was prepared according to the method described in Example (cell-free protein synthesis method) 1) mRNA reaction (transcription reaction) from DNA. Unpurified mRNA was prepared by the method described in Example 10 and centrifuged at 12,000 rpm for 1 hour, and the supernatant was recovered and prepared. In FIG. 8, the black circled line (●-●) indicates that the transcription reaction solution containing the mRNA synthesized in Example 10 was added to the translation reaction solution at a ratio of 1: 2, and after mixing, the same amount as the mixture solution Dialysis buffer [35 mM HEPES-KOH pH 7.8, 103 mM potassium acetate (KOAc), 3.1 mM magnesium acetate (Mg (OAc) 2), 16 mM creatine phosphate, 1.2 mM ATP, 0.26 mM GTP, 2. 5 mM dithiothreitol (DTT), 0.43 mM spermidine, 0.3 mM AAs (mixture of 20 L-type amino acids)], diluted, concentrated to the original mixture volume, and again dialyzed with the same volume Add buffer, repeat the same procedure, and after concentration, add creatine kinase to a final concentration of about 1 mg / ml to perform the protein synthesis reaction. The results of the experiment in Example 11 (Example 11) are shown, and the white circled line (○-○) is a translation reaction solution prepared using the ethanol synthesized in Experiment Example 7 and purified by ethanol precipitation. The experimental result at the time of performing protein synthesis reaction (Example 12) is shown. In Examples 11 and 12, the above mRNA was used at a concentration of 480 μg / ml, and a wheat germ extract having a concentration of 60A / 260 nm / ml was used.
As shown in FIG. 8, when the protein synthesis rates were compared for Examples 11 and 12, no significant difference was observed. From this result, according to the cell-free protein synthesis method of the present invention, a simple cell-free protein synthesis method that uses a translation reaction solution prepared using a transcription reaction solution after transcription without purifying mRNA is realized. I found it possible.
Experimental Example 9
FIG. 9 is a graph showing the results of an experiment in which the cell-free protein synthesis method of the present invention was performed using a translation reaction solution prepared using the transcription reaction solution after the transcription reaction in Experimental Example 7, and the vertical axis was synthesized. The amount of protein (mg / ml), and the horizontal axis represents the reaction time (hours). In FIG. 9, the upward arrowhead indicates the time point at which the dilution and concentration discontinuous repetition operations were performed. The downward arrow indicates the time when the transcription reaction solution (including mRNA) after the transcription reaction is added.
As a translation reaction solution, 80A 260 nm / ml wheat germ extract and a transcription reaction solution after transcription reaction (including 9.6 mg of GFP mRNA) were added to 15 ml of the translation reaction solution similar to Example 1 (final concentration of GFP mRNA: 640 μg). / Ml), this was concentrated to a liquid volume of 8 ml, and then the translation reaction solution was prepared by dilution with the above translation reaction solution and subsequent concentration operation, and the translation reaction was started. As a means for removing the by-product, a configuration having a filtration membrane and a liquid feed pump is used in the same manner as shown in FIG. 4, and GFP mRNA (translation template) is added every 4 hours from the start of the reaction. Then, dilution / concentration with a solution containing a substrate and an energy source was performed at 30-minute intervals by discontinuous repeated operation.
As the experimental results show, it can be confirmed that the translation reaction that was almost stopped is restarted by the addition of mRNA. Furthermore, it can be seen that protein synthesis can be sustained for a long time by combining this dilution / concentration method with the addition of mRNA.
Example of apparatus
Examples of the automatic synthesis apparatus according to the present invention will be described below. However, the automatic synthesizing apparatus according to the present invention is not limited to the apparatus of the following embodiment as long as the high-throughput synthesis system can be automatically implemented.
In order to implement the automatic synthesizer of the present invention, the following were set in the automatic synthesizer, and GFP protein synthesis was performed using the synthesizer.
Transcription mold
A pEU plasmid vector in which GFP was incorporated was used as a transcription template, using a sense primer and an antisense primer, and DNA obtained by PCR in a 30 μl system. This DNA was housed in a 96-well PCR plate (template plate: (1)).
Solution for transcription reaction
4.95 ml of a solution containing a final concentration of 80 mM HEPES-KOH, 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTPs, 1 U / μl SP6 RNA polymerase, 1 U / μl RNasin was prepared, and reagent tank 1 (▲ 2 ▼).
Translation reaction solution
Final concentrations of 30 mM HEPES-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM magnesium acetate , 100 mM potassium acetate, 0.005% sodium azide, 40 ng / μl creatine kinase, and 5.5 ml of a solution containing wheat germ extract with an optical density (OD) at 260 nm of 80 or 40 units. It put into the reagent tank 2 ((3)). In addition, the protein synthesis | combination using the solution for translation reactions containing an 80 unit and 40 unit wheat germ extract was performed separately.
Diluted solution
Final concentrations of 30 mM HEPES-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM magnesium acetate 100 ml of a solution containing 100 mM potassium acetate and 0.005% sodium azide was prepared and placed in the reagent tank 3 (4) in the apparatus.
Synthetic container
Plate for template (PCR 96-hole with lid (vertical 8 x horizontal 12) plate (with template DNA): (1)), Transfer / filtrate receiving plate (96-hole with lid (vertical 8 x horizontal 12) titer Plate: (10)), translation plate (PCR 96-hole with lid (vertical 8 x horizontal 12) plate (cell bottom is filter): (14)), dummy plate (balance during centrifugation)
Chip
300 μl chip (for transcription reaction solution, translation reaction solution, diluted solution: (5)) (280), 20 μl chip (for template product: (6)) (96)
Dispenser 1: Number of pipetters 8 (for mounting 300 μl tips: (8))
Dispenser 2: 8 pipetters (for mounting 20 μl tips: (9))
GFP protein synthesis was performed using the synthesizer according to the present invention in the following steps.
<Step 1: Transfer>
(1) The robot arm (7) removed the cover of the transfer / filtrate receiving plate (10).
(2) Dispenser 1 (8) was mounted to 300 μl tip (5) and moved to reagent tank 1 (2) containing the transfer reaction solution.
(3) The dispenser 1 (8) sucked 95 μl (45 μl × 2 times + 5 μl) of the transcription reaction solution from the reagent tank 1 (2).
(4) The dispenser 1 (8) moves to the position of the transfer / filtrate receiving plate (10), and 45 μl of the transfer reaction solution is placed in each vertical cell (eight) of the plate. Discharged twice.
(5) The steps (2) to (4) were further performed on each of the 7 vertical columns of cells in which the transcription reaction solution was not added.
(6) Dispenser 1 (8) moved to the tip disposal port (11) position and the tip was discarded.
(7) The robot arm (7) removed the lid of the mold plate (1).
(8) Dispenser 2 (9) was equipped with a 20 μl tip (6).
(9) Each tip of the dispenser 2 (9) sucked 5 μl of air (performed to increase the efficiency of suction).
(10) Move the dispenser 2 (9) to the position of the mold plate (1), and aspirate 5 μl of the mold sample in each of the vertical cells (8) of the mold plate (1). did.
(11) The dispenser 2 (9) was moved to the transfer / filtrate receiving plate (10) position, and the entire amount of the mold sample was discharged to each of the vertical cells (8 pieces) of the plate.
(12) The vertical cells (eight) of the transfer / filtrate receiving plate (10) were mixed by pipetting with the tips of the dispenser 2 (9).
(13) Dispenser 2 (9) moved to the tip disposal port (11) position, and the tip was discarded.
(14) The steps (8) to (13) were further performed on each of the 7 vertical columns of cells in which the mold sample was not yet placed.
(15) The robot arm (7) covered the mold plate (1) and the transfer / filtrate receiving plate (10).
(16) The robot arm (7) transported and set the transfer / filtrate receiving plate (10) to the thermostat 1 (12) set to 37 ° C. in advance.
(17) The transcription reaction was carried out at 37 ° C. for 4 hours in the thermostatic chamber 1 (12).
<Step 2: Removal of precipitate after transfer>
(1) The robot arm (7) transported the transfer / filtrate receiving plate (10) from the thermostat 1 (12) to the MTP stage (13).
(2) The robot arm (7) removed the transfer / filtrate receiving plate (10).
(3) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(4) Each tip of dispenser 1 (8) sucked 5 μl of air.
(5) The dispenser 1 (8) is moved to the reagent tank 3 (4) containing the diluted solution, and the diluted solution is aspirated (the amount of suction is the amount of liquid after concentration centrifugation by the row on the plate) Because of the change, it was finely adjusted between 80 μl and 120 μl for each column.)
(6) The dispenser 1 (8) was moved to the transfer / filtrate receiving plate (10) position, and the entire diluted solution was discharged into each of the vertical cells (8) of the plate.
(7) The steps (4) to (6) were further performed on each of the 7 columns of cells not containing the diluted solution.
(8) The dispenser 1 (8) moved to the tip disposal port (11) and discarded the tip.
(9) The robot arm (7) covers the translation plate (5) and puts it on the transfer / filtrate receiving plate (10). To take).
(10) The robot arm (7) transported the overlaid translation plate (14) and the transfer / filtrate receiving plate plate (10) to the lifting platform (15).
(11) The lifting platform on which the translated translation plate ((14)) and the transfer / filtrate receiving plate ((10)) are placed is lowered to match the height of the centrifuge ((16)). .
(12) The robot arm (7) opened the door of the centrifuge (16).
(13) The centrifuge plate (16) was positioned so that the plate could be set.
(14) The robot arm (7) transported and set the superimposed translation plate (14) and the transfer / filtrate receiving plate (10) to the centrifuge.
(15) Positioning was performed in the same manner as in (13), and the dummy plate was set as a balance in a centrifuge.
(16) Centrifugation was performed at 3100 g for 15 minutes with a centrifuge ((16)).
<Step 3: First transfer buffer buffer exchange>
(1) After the centrifuge ((16)) was stopped, the robot arm opened the centrifuge door, and then the centrifuge performed alignment (alignment of the robot arm and centrifuge plate installation site). .
(2) The robot arm (7) transported the translated translation plate (14) and the transfer / filtrate receiving plate (10) from the centrifuge (16) to the lifting platform.
(3) Positioning was performed in the same manner as in (1), and the robot arm (7) transported the dummy plate to the lifting platform.
(4) The robot arm (7) transports the translated translation plate (14) and the transfer / filtrate receiving plate (10) to the MTP stage (13), and the translation plate (▲). 14 ▼) and the transfer / filtrate receiving plate (10) were set separately.
(5) The robot arm (7) removed the lid of the translation plate (14).
(6) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(7) Each tip of dispenser 1 (8) sucked 5 μl of air.
(8) The dispenser 1 (8) moves to the transfer / filtrate receiving plate (10) position, and each vertical cell (8) of the transfer / filtrate receiving plate (10). The whole amount was aspirated.
(9) The dispenser 1 (8) moved to the translation plate (14) position, and the entire amount was discharged to each vertical cell (eight) of the translation plate (14).
(10) The processes of (6) to (9) were further performed on each cell in the other seven vertical columns.
(11) The dispenser 1 (8) covered the translation plate (14).
(12) Dispenser 1 (8) placed translation plate (14) on transfer / filtrate receiving plate (10) (upper: translation plate, lower: transfer / filtrate receiver) Plate).
(13) Centrifugation was performed in the same manner as in Steps 2 (10) to (16).
<Step 4: Second transfer of the transfer buffer>
(1) In the same manner as in Steps 3 (1) to (5), the transfer / filtrate receiving plate and the translation plate were taken out from the centrifuge, set on the MTP stage, and the lid of the translation plate was removed.
(2) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(3) Each tip of dispenser 1 (8) sucked 5 μl of air.
(4) Dispenser 1 (8) moved to reagent tank 3 (4) containing the diluted solution.
(5) Each cell of dispenser 1 (8) sucked the diluted solution (the amount of suction was finely adjusted between 80 μl and 120 μl for each row).
(6) The dispenser 1 (8) moved to the translation plate (14) position, and the entire amount was discharged to each vertical cell (eight) of the translation plate (14).
(7) The processes of (3) to (6) were further performed on each cell in the other seven vertical columns.
(8) The dispenser 1 (9) moved to the tip disposal port (11) position, and the tip was discarded.
(9) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(10) Each tip of dispenser 1 (8) sucked 5 μl of air.
(11) Dispenser 1 (8) moved to the translation plate (14) position.
(12) The vertical cells (8) of the translation plate (14) were mixed by pipetting with the tips of the dispenser 1 (8).
(13) The processes of (8) to (12) were further performed on each cell in another seven vertical columns (the chip may be replaced in each cell).
(14) The dispenser 1 (9) moved to the tip disposal port (11) position, and the tip was discarded.
(15) The robot arm (7) covered the translation plate (14).
(16) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(17) Each tip of dispenser 1 (8) sucked 5 μl of air.
(18) Dispenser 1 (8) moved to the transfer / filtrate receiving plate (10) position.
(19) The dispenser 1 (8) sucked the entire amount in each of the vertical cells (eight) of the transfer / filtrate receiving plate (10).
(20) Dispenser 1 (8) moved to the waste liquid port (17) position.
(21) Dispenser 1 (8) discharged the entire amount to the waste liquid port (17).
(22) The processes of (17) to (21) were performed on each of the cells in the other seven vertical columns.
(23) The dispenser 1 (9) moved to the tip disposal port (11) position, and the tip was discarded.
(24) The translation plate (14) was centrifuged in the same manner as in Steps 2 (9) to (16).
<Step 5: Translation reaction solution dispensing>
(1) In the same manner as in steps 3 (1) to (5), take out the transfer / filtrate receiving plate and translation plate from the centrifuge, set them on the MTP stage, and put the lid of the translation plate (14) on. I removed it.
(2) Dispenser 1 (8) was equipped with 300 μl tips (5), and each tip sucked 5 μl of air.
(3) Dispenser 1 (8) moved to reagent tank 2 (3) containing the translation reaction solution.
(4) The dispenser 1 (8) sucked 50 μl of the translation reaction solution from the reagent tank 2 (3).
(5) Dispenser 1 (8) moved to the position of translation plate (14) and discharged the entire amount in each vertical cell (8) of translation plate (14). .
(6) The vertical cells (8) of the translation plate (14) were mixed by pipetting with the tips of the dispenser 1 (8).
(7) The processes of (2) to (6) were performed on each of the cells in the other seven vertical columns.
(8) The dispenser 1 (9) moved to the tip disposal port (11) position, and the tip was discarded.
(9) The robot arm (7) covered the translation plate (14).
(10) The robot arm (7) transported the translation plate (14) to the thermostat 1 (12).
(11) The translation plate (14) was kept warm at 26 ° C. for 1 hour.
(12) During the heat retention step of (11) (hereinafter, the step of discarding the filtrate), the dispenser 1 (8) is equipped with 300 μl tips (5), and each tip is air Was aspirated.
(13) Dispenser 1 (8) moved to transfer / filtrate receiving plate (10) position.
(14) The dispenser 1 (8) sucked the entire amount in each of the vertical cells (eight) of the transfer / filtrate receiving plate (10).
(15) Dispenser 1 (8) moved to the waste liquid port (17) and discharged the entire amount.
(16) The processes of (12) to (15) were performed on each of the cells in the other seven vertical columns.
(17) The dispenser 1 (9) moved to the tip disposal port (11) position, and the tip was discarded.
<Step 6: Repeat translation>
The following steps were repeated 6 times.
(1) The robot arm (7) transported the translation plate (14) from the thermostat 1 (12) to the MTP stage (13).
(2) The robot arm (7) removed the lid of the translation plate (14).
(3) Dispenser 1 (8) was equipped with a 300 μl tip (5).
(4) Each tip of dispenser 1 (8) sucked 5 μl of air.
(5) The dispenser 1 (8) is moved to the reagent tank 3 (4) containing the diluted solution, and the diluted solution is aspirated (the aspirated amount depends on the row on the plate. Because of the change, it was fine-tuned between 80 μl and 120 μl for each row).
(6) The dispenser 1 (8) was moved to the position of the translation plate (14), and the entire diluted solution was discharged into each vertical cell (eight) of the plate.
(7) The steps (4) to (6) were further performed on each of the 7 columns of cells not containing the diluted solution.
(8) Centrifugation was performed in the same manner as in Steps 2 (9) to (16) (centrifugation time was 18 minutes).
(9) In the same manner as in Steps 3 (1) to (5), the transfer / filtrate receiving plate and the translation plate were taken out of the centrifuge, set on the MTP stage, and the translation plate lid was removed.
(10) Dispenser 1 (8) was equipped with 300 μl tips (5), and each tip sucked 5 μl of air.
(11) Dispenser 1 (8) moved to the translation plate (14) position.
(12) Using the tips of the dispenser 1 (8), the vertical cells (8) of the translation plate (14) were mixed by pipetting.
(13) The dispenser 1 (9) moved to the tip disposal port (11) position and discarded the tip (the tip may not be replaced in some cases).
(14) The processes of (10) to (13) were performed on each of the other seven vertical cells.
(15) The robot arm (7) covered the translation plate (14).
(16) The robot arm (7) transported the translation plate (14) to the thermostat 1 (12).
(17) The translation plate (14) was kept warm at 26 ° C. for 1 hour.
(18) During the heat retention step of (17) (hereinafter, the step of discarding the filtrate), dispenser 1 (8) is equipped with a 300 μl tip (5) and sucked 5 μl of air. did.
(19) Dispenser 1 (8) moved to the transfer / filtrate receiving plate (10) position.
(20) The dispenser 1 (8) sucked the entire amount in each of the vertical cells (eight) of the transfer / filtrate receiving plate (10).
(21) Dispenser 1 (8) moved to the waste liquid port (17) position.
(22) Dispenser 1 (8) discharged the entire amount.
(23) The processes of (18) to (22) were performed on each of the cells in the other seven vertical columns.
<Step 7: End (after step 6 is repeated six times)>
(1) After the heat retention in step 6 (18), the temperature of the thermostatic bath was set to 4 ° C.
(2) The translation plate was removed from the synthesizer.
The measurement of the amount of the synthesized GFP synthesized by the above steps is reported by Madin K et al. (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556). And analyzed by SDS-PAGE (FIG. 11).
In FIG. 11, the protein was synthesized at the position of the GFP band in all 1-8 lanes of 40 units and 1-4 lanes of 80 units. Further, by comparing the band concentration of BSA (125, 250, 500 ng) and the band concentration of GFP in each lane, it can be estimated that at least 250 ng of GFP is synthesized in all lanes.
As is clear from the above explanation, the high-throughput synthesis system of the present invention comprising a discontinuous repeated operation of dilution and concentration by selecting a reaction initial time with a high reaction rate, and an apparatus for automatically performing the system, have high quality. It turns out that it is possible to synthesize | combine protein efficiently in a short time, and it is very effective also in isolation of the target protein. This principle is also useful for efficient synthesis of RNA in vitro.
Furthermore, protein and RNA synthesis methods applying this principle have various disadvantages seen in the continuous method of Spirin et al., Ie, the complexity of the device, the low strength of the membrane, clogging of the membrane during operation, It was shown that the reactor caused by the complexity can be solved. In addition, the disadvantage of the continuous method, which requires a particularly long synthesis reaction time, is not only a waste of time, but also remains as a major problem that must be solved from the aspect of ensuring the quality of the protein produced. However, this drawback was an unsolved problem even in the multilayer method.

  The technology invented here will provide basic elemental technologies for protein research in the post-genomic era. In particular, it can be said that it is indispensable as an elemental technology that enables simple and efficient comprehensive preparation and mass production for analysis of structures and functions of RNA and proteins.

Claims (5)

A cell-free protein synthesis system using a translation template as a raw material, comprising the following means, its control means, or a combination thereof:
1) Contacting a reaction solution containing a translation template, a substrate, and a wheat germ extract leads to a synthesis reaction.
2) Diluting the reaction solution before and after substantially lowering the synthesis rate, before and after substantially stopping the synthesis reaction, or in the middle thereof,
3) Concentrate after dilution.
4) Perform the synthesis reaction using the concentrated reaction system.
5) Repeat the means 2) to 4) a plurality of times,
Or 1) contacting a reaction solution containing a translation template, a substrate, and a wheat germ extract to lead to a synthesis reaction,
2) Concentrate the reaction solution before and after substantially lowering the synthesis rate, before and after substantially stopping the synthesis reaction, or in the middle thereof,
3) A dilution process is performed following the concentration process.
4) Perform the synthesis reaction with the diluted reaction system.
5) Repeat the means 2) to 4) a plurality of times,
Here, the wheat germ extract has a ribosome deadenification rate of less than 1%.
Cell-free protein synthesis method.   2. The method according to claim 1, wherein the wheat germ extract has a low molecular weight synthesis inhibitor having a molecular weight of 14,000 or less removed.   The method according to claim 1 or 2, wherein by-products are removed from the reaction system by concentration treatment.   The method according to any one of claims 1 to 3, wherein the substrate, the energy source and / or the template substance are replenished into the reaction system in the dilution process.   The method according to any one of claims 1 to 4, wherein the number of repetitions is 2 to 20 times.

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