CN1777674B - High-flux protein synthesis system and synthesis device for automatically operating same - Google Patents

Abstract

A systematic technique for in vitro synthesis reaction of high-throughput biopolymers such as proteins and RNA is developed. More specifically, the present invention relates to a cell-free synthesis system using a template material as a raw material, comprising the following steps, control steps thereof, or a combination thereof: 1) the template substance, the substrate and the reaction solution are brought into contact with each other and introduced into the synthesis reaction system. 2) Before or after the synthesis rate is slightly decreased, before or after the synthesis reaction is stopped, or during these conditions, the reaction system is placed outside the synthesis reaction system, and the solution is diluted. 3) The concentration treatment is performed after the dilution treatment. 4) And returning the reaction system to the synthesis reaction system. Or 1) bringing a template substance, a substrate and a reaction solution into contact with each other and introducing them into a synthesis reaction system. 2) Before or after the synthesis rate is slightly decreased, before or after the synthesis reaction is stopped, or during these conditions, the reaction system is placed outside the synthesis reaction system, and the solution is concentrated. 3) The concentration treatment is followed by a dilution treatment. 4) And returning the diluted reaction system to the synthesis reaction system.

Description

High-throughput protein synthesis system and synthesis device for automatic operation of the system

This application claims the priority of Japanese Patent Application Nos. 2003-122930 and 2003-281500, which are hereby incorporated by reference.

technical field

The present invention relates to a cell-free synthesis system starting from a template substance. More specifically, it relates to a system for maximizing synthesis reaction efficiency when synthesizing substances using template substances as raw materials. At the same time, it relates to a synthesis device for automatic operation of the system.

Background technique

As a method for synthesizing biological macromolecules such as proteins and RNA outside the body, the so-called batch method is used to prepare substrates, ions, buffers, cell extracts, and synthetic enzymes necessary for those reactions in test tubes. It has been used since ancient times. However, this batch reaction method, due to its reaction principle, causes the concentration of the substrate to decline with the progress of the reaction, and the concentration of by-products that inhibit the synthesis rises, etc., so that the synthesis reaction is stopped in a short time. As a result, It is the reduction of the yield of the target product, which has a great limitation. A.Spirin et al. of the former Soviet Union reported that by using ultrafiltration membranes, dialysis membranes, or column chromatography of immobilizing translation templates on resins, amino acids, ATP, GTP, etc. energy sources and substrates can be continuously supplied, and at the same time, energy sources such as ATP and GTP can be continuously supplied. A continuous cell-free protein synthesis method that removes target products and by-products from a reaction system (Non-Patent Document 1). However, the continuous method using these semi-permeable membranes and ultrafiltration columns requires complicated device assembly and cumbersome handling, requiring sufficient experience. In addition, this method also has great difficulties in the automation of the reaction system.

Endo et al. invented a contact interface diffusion method (Patent Document 1) that can continuously supply a substrate and remove by-products without using a semipermeable membrane as a method to solve these problems. According to this method, a large amount of protein can be synthesized without using a special reaction apparatus. However, this method is based on the principle of molecular diffusion and has the disadvantage that it takes a long time to synthesize a necessary amount of protein, which needs to be solved.

Furthermore, RNA synthesis methods in test tubes, especially cell-free synthesis methods in which proteins are present even in high-throughput synthesis methods of translational template mRNA necessary for cell-free protein synthesis reactions Problems, as subjects to be solved. Endo et al. studied the RNA synthesis method and developed a continuous synthesis method using a dialysis membrane. This method greatly improves the performance of the batch method to synthesize RNA with a very high yield (Patent Document 2). However, the continuous RNA synthesis method using such a semipermeable membrane or the like requires assembly of a complicated device, and the handling is cumbersome and requires sufficient work experience. Furthermore, this method has a great difficulty in automating the reaction system.

In today's post-genome era, the following points can be considered as necessary conditions for high-throughput synthesis of biological macromolecules such as proteins and RNA. 1) It is possible to synthesize a large amount in a short period of time; 2) It is possible to synthesize various types of molecules in a short period of time; and 3) Based on a simple principle, it is possible to obtain technical elements for automating a reaction device, etc. In particular, the requirements of 1) and 2) are extremely important not only from the viewpoint of low cost for simple synthesis, but also in order to maintain the active state of the protein with relatively unstable physical properties.

(Patent Document 1) WO02/24939

(Patent Document 2) WO01/27260

(Non-Patent Document 1) Spirin, A.et al. (1993) Meth.in Enzymol., 217, 123-142

Contents of the invention

The object of the present invention is to provide a technical system for high-throughput in-vitro synthesis of biopolymers such as proteins and RNA, and to provide a technical system using this system, which is completely different from the principle of continuous synthesis using complex devices such as semi-permeable membranes. Automatic synthesis device. This enables simple and efficient overall preparation and mass production for the purpose of analyzing the structure and function of gene products such as RNA and protein.

In order to solve the above-mentioned problems, the present invention has carried out various researches aimed at maintaining its high reaction rate for a long time in a synthesis system using a template substance as a raw material. As a result, a cell-free The completion of the system and the automatic synthesis device using the system realized the solution of the problem.

That is, the present invention is as follows.

1. A cell-free synthesis system, which is a synthesis system using a template substance as a raw material, and is characterized in that it contains the following steps, its control steps, or a combination thereof:

1) Bringing the template substance, substrate and reaction solution into contact and introducing them into the synthesis reaction,

2) Dilute the reaction solution before and after the synthesis rate is slightly reduced, before and after the synthesis reaction is about to stop, or during these states,

3) Concentration treatment is carried out after dilution treatment,

4) Carrying out the synthesis reaction through the concentrated reaction system;

or

1) Bringing the template substance, substrate and reaction solution into contact and introducing them into the synthesis reaction,

2) before and after the synthesis rate is slightly reduced, before and after the synthesis reaction is about to stop, or in the process of these states, the reaction solution is concentrated,

3) performing dilution treatment after concentration treatment,

4) Carry out the synthesis reaction through 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. The system according to item 1 or 2 above, wherein the substrate, energy source and/or template substance are added to the reaction system by dilution treatment.

4. The system of item 1 above, wherein the steps 1) to 4) are repeated multiple times.

5. The system according to any one of items 1 to 4 above, wherein the template substance is a transcription template.

6. The system according to any one of items 1 to 4 above, wherein the template substance is a translation template.

7. The system of item 6 above, wherein the reaction solution includes a cell extract.

8. A cell-free synthesis system comprising a combination of a transcription product producing system obtained by the system of the preceding item 5 and a translation product producing system obtained by the system of the preceding item 6 or 7.

9. A synthesis kit comprising at least one reagent used in the cell-free synthesis method starting from a template substance using the system described in any one of items 1 to 8 above.

10. A cell-free synthesis method starting from a template substance, using the system described in any one of 1 to 8 above.

11. A device for cell-free synthesis starting from a template substance, using the system described in any one of 1 to 8 above.

12. A cell-free synthesis device for automatically implementing the system according to any one of items 1 to 8 above, wherein the synthesis of the initial phase of the synthesis reaction using a high protein synthesis reaction rate is performed multiple times system, equipped with at least the following control mechanisms:

(1) The body that starts the synthesis,

(2) A mechanism for concentrating the reaction solution,

(3) A mechanism for diluting the reaction solution,

(4) A mechanism for reactivating a synthetic reaction,

(5) Organizations that make the above-mentioned (2)-(4) organizations operate repeatedly;

or

(1) The body that starts the synthesis,

(2) A mechanism for diluting the reaction solution,

(3) A mechanism for concentrating the reaction solution,

(4) A mechanism for reactivating a synthetic reaction,

(5) A mechanism that repeatedly operates the above-mentioned mechanisms (2) to (4).

13. The synthesis device described in the preceding item 12, wherein the mechanism for concentrating the reaction solution is a mechanism using an ultrafiltration membrane, so that when the reaction solution is removed to the outside of the reaction system through the ultrafiltration membrane, the reaction solution cannot pass through the ultrafiltration membrane. The substance is concentrated in the reaction system.

14. The synthesis apparatus described in item 13 above, wherein, in the mechanism for concentrating the reaction liquid, a centrifuge and/or a suction pump are used to remove the reaction liquid from the reaction system.

15. The synthesis device described in item 12 above, wherein the means for diluting the reaction solution is to add a dilution solution or a matrix solution to the reaction solution.

16. The synthesis device described in the preceding paragraphs 11 to 15, wherein, in the cell-free protein synthesis process, in order to automatically implement the process from the transcription template to the production of the protein encoded on the translation template, at least one of the following control mechanisms is provided:

(1) A mechanism for variable control of the temperature in the reaction vessel;

(2) A mechanism for dispensing samples or reagents into the reaction vessel;

(3) The mechanism for transporting the reaction vessel;

(4) Mechanisms for precipitation and concentration filtration.

17. A program for implementing the system described in any one of items 1 to 8 above, wherein the synthesis system using the initial phase of the synthesis reaction with a high protein synthesis reaction rate is implemented multiple times, including the following information processing means:

(1) Based on the information on the volume of the reaction vessel and the concentration of the reaction solution, an information processing mechanism that sets the synthesis time within the range of the initial phase of the synthesis reaction before the synthesis amount per unit time becomes a decreasing tendency process,

(2) An information processing mechanism that adds a dilute solution to the reaction container when the synthesis time within the range of the initial phase of the synthesis reaction in (1) is reached, and sets the reaction solution to a concentration range outside the range where a substantial synthesis reaction can be performed,

(3) After (2), the reaction liquid in the reaction container is concentrated, and the information processing mechanism is set to return the liquid volume of the reaction liquid increased due to the addition of the diluent to the original liquid volume,

(4) After (3), an information processing mechanism for setting the optimum reaction temperature for restarting the synthesis reaction,

(5) An information processing mechanism for repeating (1) to (4) multiple times;

or

(1) Based on the information on the volume of the reaction vessel and the concentration of the reaction solution, an information processing mechanism that sets the synthesis time of the synthesis amount per unit time within the range of the initial phase of the synthesis reaction before it becomes a decrease tendency process,

(2) An information processing mechanism that concentrates the reaction solution in the reaction vessel and sets the reaction solution at a concentration range outside the concentration range where a substantial synthesis reaction can be performed when the synthesis time in the initial phase of the synthesis reaction of (1) is reached,

(3) After (2), add a dilute solution to the reaction container, and an information processing mechanism for returning the liquid amount of the reaction liquid reduced by concentration to the original liquid amount,

(4) After (3), an information processing mechanism for setting the optimum reaction temperature for restarting the synthesis reaction,

(5) An information processing mechanism for repeating (1) to (4) multiple times.

18. A cell-free system synthesis device incorporating the program described in the preceding paragraph 17, wherein the data processing of the program cooperates with the device to start the synthesis, dilute and concentrate the reaction solution, and perform the synthesis reaction. The mechanism of reactivation can be repeated multiple times using a synthesis system that uses the initial phase of the synthesis reaction with a high protein synthesis reaction rate.

19. A protein synthesized by the synthesis device described in 11-16, 18 above.

Description of drawings

FIG. 1 : A graph showing the difference in experimental results between the cell-free protein synthesis method of the present invention and the past cell-free protein synthesis method by batch method.

Fig. 2 is a graph showing the results of experiments for cell-free protein synthesis performed in the same manner as in Example 1, except that the concentration of the wheat germ extract was changed.

Fig. 3A: It is an experiment of cell-free protein synthesis in the same manner as in Example 1, except that the concentration of wheat germ extract was 800D, the concentration of GFPmRNA was 640 μg/ml, and the intervals of discontinuous repeated operations of dilution and concentration were changed. Result graph.

FIG. 3B : A polyacrylamide gel electrophoresis result graph showing the difference in the amount of synthesized GFP due to the different intervals of the discrete repeated operations of dilution and concentration.

Fig. 4: A schematic diagram showing a structure in which by-products generated in the cell-free protein synthesis method of the present invention can be removed by a combination of a concentrator having a filter membrane and a liquid delivery pump.

Fig. 5: Using the structure shown in Fig. 4 to remove by-products by means of a filter membrane and a liquid delivery pump, an experimental result diagram of protein synthesis by the cell-free protein synthesis method of the present invention.

Fig. 6: A shows except adopting the mRNA of the wheat germ extract of 600D, the dihydrofolate reductase (DHFR) of 450g/ml as the translation template, using the same translation reaction solution as in Example 1, except implementing dilution and It is a graph showing experimental results when cell-free protein synthesis was carried out in the same manner as in Example 1, except that the concentrated discontinuous operation was repeated twice.

B is an electrophoresis graph showing the experimental results of synthesizing a GFP fusion protein fused with streptavidin at the N-terminus by the method of the present invention and separately isolating the GFP fusion protein outside the system.

Fig. 7 is a graph showing the difference in experimental results between the RNA synthesis method of the present invention and a conventional in vitro RNA synthesis method using a batch method.

Fig. 8 is a graph showing the experimental results of implementing the cell-free protein synthesis method of the present invention using ethanol-precipitated purified mRNA and unpurified mRNA respectively as translation templates.

Fig. 9 is a graph showing the experimental results of the cell-free protein synthesis method of the present invention using the translation reaction solution prepared by using the transcription reaction solution after the transcription reaction of Experimental Example 7 as it is.

Figure 10: Schematic diagram of the automatic synthesis device

Figure 11: SDS-PAGE graph showing device embodiment results

(Symbol Description)

1: filter concentrator

2: liquid delivery pump

3: Reaction tank

4: Container with (RNA) matrix solution

5: liquid delivery switching valve

6: container

T1: Tube T1

T2: Tube T2

T3: Tube T3

T4: Tube T4

T5: Tube T5

①: Template board

②: Reagent tank 1 (solution for transcription reaction)

③: Reagent tank 2 (solution for translation reaction)

④: Reagent tank 3 (diluted solution)

⑤: 300μl interface tube (チツプ) ⑥: 20μl interface tube

⑦: Mechanical arm

⑧: Dispensing machine 1

⑨: Dispensing machine 2

⑩: Plate for receiving transcription and filtrate

：接口管废弃口

: Interface tube waste port

：恒温槽1

: constant temperature bath 1

: MTP (multi titer plate) console

: Board for translation

：升降台

:Lifts

: centrifuge waste port

Detailed ways

The synthesis method of the present invention is characterized in that the characteristics of the initial phase of the synthesis reaction with a high reaction rate in the general batch method or the continuous diffusion batch method using the template substance as a raw material are utilized to the maximum extent. Furthermore, the synthesizing apparatus of the present invention is characterized in that it can automate a synthesizing method that makes the most of this characteristic.

Its method is to dilute or concentrate the reaction solution before and after the synthesis rate slightly decreases, or before and after the synthesis reaction is about to stop, or during these states. Components such as substrates, energy sources, ions, buffers, and template substances necessary for synthesis using template substances as raw materials are supplemented or concentrated by these dilution treatments or concentration treatments.

These diluted or concentrated reaction solutions are then subjected to a concentration treatment when diluted or a dilution treatment when concentrated. Through this treatment, the reaction solution was returned to the original optimum concentration for the reaction. Through this concentration treatment, components in the reaction solution can be removed or separated, and reaction products and/or reaction by-products can be recovered and/or removed. By such concentration or dilution, the various elements of the synthesis reaction can be brought to the optimum concentration again. Furthermore, by repeating such dilution and concentration treatments discontinuously, a large amount of protein can be synthesized.

The principle of the present invention is a synthesis method of discontinuously repeating dilution and concentration for a synthesis reaction using a template substance.

In addition, the principle of the automatic synthesis apparatus of the present invention is to automate a discontinuously repeated synthesis method.

In the present invention, one of the synthesis systems using template substances as raw materials is a protein translation synthesis system using mRNA as a template, especially a cell-free protein synthesis system using a cell extract for protein synthesis. The ribosome, which is a protein translation device, is taken out of the cell and used. The other refers to an RNA synthesis system that uses RNA polymerase as an enzyme to perform an in vitro transcription reaction through a transcription template.

(cell-free protein synthesis system)

In the cell-free protein synthesis system, compared with the cell-free protein synthesis system using Escherichia coli extract, the synthesis system using wheat germ extract has lower nucleolytic enzyme activity, and has the characteristics of stable and high translation activity (Madin K.et al., (2000) Proc.Natl.Acad.Sci.USA 97,559-564), can synthesize protein (PCT/JP99/04088 ). Hereinafter, in the present invention, the synthesis method of the transcription template that can play a role in the wheat germ cell-free protein synthesis system and the synthesis system is used as an example to illustrate, but the basic principle of the present invention can also be applied to adopt other A cell-free protein synthesis system derived from cell extracts of microorganisms and animal cells and a synthesis system of transcription templates for this system.

(cell extract)

Commercially available cell extracts include Escherichia coli-derived E.coli S30 extractsystem (Promega), RTS 500 Rapid Translation System (Roche), rabbit reticulocyte-derived Rabbit Reticulocyte Lysate System (Promega), derived from Proteios( ^TM ) of wheat germ (TOYOBO) and the like. Among them, it is particularly preferable to use germ extracts derived from plant seeds, examples of which include seeds of wheat, barley, rice, corn, and spinach, and a system using wheat germ extracts is most suitable.

(Translation product production system)

Here, the so-called translation product production system is to extract components such as ribosomes, which are protein translation devices in cells, from organisms, and convert translation templates, nucleic acids used as substrates, amino acids, energy sources, various ions, buffers, and other effective factors A method of adding to the extract and reacting it in a test tube.

(cell-free protein synthesis system)

Here, the so-called cell-free protein synthesis system is to extract components including ribosomes, which are intracellular protein translation devices, from living organisms, and convert transcription or translation templates, nucleic acids as substrates, amino acids, energy sources, various ions, buffers, and A method in which other effective factors are added to the extract to make it react in a test tube.

(wheat germ extract)

As the cell extract-containing solution of the present invention, a solution containing a wheat germ extract is preferably used.

With regard to the preparation method of wheat germ extract, as the method for isolating wheat germ, for example can adopt the method etc. described in Johnston, F.B.et al., Nature, 179,160-161 (1957); The method for extracting the cell extract-containing liquid from the germ, for example: the method described in Erickson, A.H.et al., (1996) Meth.In Enzymol., 96, 38-50, etc. can be used. In addition, methods of Japanese Patent Application No. 2002-23139 and Japanese Patent Application No. 2002-231340 can be cited.

The germ extract that can be suitably used in the present invention has almost completely removed the substances that inhibit the protein synthesis function (trichin, thionine, ribonuclease, etc.) Factors and ribosomes, etc., and inhibit their function). That is, the endosperm in which these inhibitory substances are locally present is almost completely removed and purified. The degree of endosperm removal can be assessed by examining the activity of tricoflavones, ie ribosomal deadenylation activity, entrapped in wheat germ extracts. If the ribosomes are not substantially adenylated, no endosperm-derived components are included in the germ extract, that is, it is judged that the endosperm is almost completely removed and purified. The degree to which ribosomes are not substantially deadenylated means that the deadenylation rate of ribosomes is less than 7%, preferably 1% or less.

The above-mentioned germ extract contains protein derived from the cell extract-containing solution and optionally added. Its content is not particularly limited, but from the perspective of storage stability in a freeze-dried state, ease of use, etc., in the composition before freeze-drying, the composition as a whole is preferably 1 to 10% by weight, more preferably 1 to 10% by weight. 2.5 to 5% by weight; in addition, in the freeze-dried composition after freeze-drying, the composition as a whole is preferably 10 to 90% by weight, more preferably 25 to 70% by weight. In addition, the protein content mentioned here was calculated by measuring the absorbance (260, 280, 320 nm).

(to remove microorganisms)

Microorganisms, especially spores of filamentous fungi (mold) are often mixed in the cell extract-containing liquid, and it is preferable to remove these microorganisms in advance. In particular, it is important to inhibit the proliferation of microorganisms as they are commonly seen in long-term (1 day or longer) cell-free protein synthesis reactions. The means for removing microorganisms is not particularly limited, but a filter sterilization filter is preferably used. The pore diameter of the filter is not particularly limited as long as microorganisms that may be mixed in can be removed, but it is usually 0.1 to 1 μm, preferably 0.2 to 0.5 μm.

Furthermore, at a certain stage of the cell extract-containing solution preparation process, by adding a process of removing low-molecular synthesis inhibitors and/or reducing the concentration of a reducing agent, it can be used as a method for the purpose of cell-free protein synthesis with a specific effect. The cell extract contains liquid.

(Method for Removing Low Molecular Synthesis Inhibiting Substances from Cell Extract Containing Liquid)

The cell extract-containing liquid contains low-molecular synthesis inhibitors having protein synthesis inhibitory activity (hereinafter often referred to as "low-molecular synthesis inhibitors"), and by removing these substances, a cell extract-containing liquid with high protein synthesis activity can be obtained. Specifically, low-molecular-weight synthesis inhibitors are respectively removed from the constituent components of the cell extract-containing liquid according to the difference in molecular weight. The low-molecular-weight synthesis inhibitor is the smallest substance among the factors necessary for protein synthesis contained in the cell extract-containing solution, and can be distinguished as molecules having the following molecular weights. Specifically, it can be distinguished and removed as a molecule having a molecular weight of 50,000 to 14,000 or less, preferably 14,000 or less.

As a method for removing low-molecular-weight synthesis inhibitors from the cell extract-containing liquid, known common methods can be used, and specifically, methods such as dialysis through a dialysis membrane, gel filtration, or ultrafiltration, etc., can be used. . Among them, the method of using dialysis is preferable in view of easiness of material supply to the dialyzed internal fluid.

As the dialysis membrane used in the operation of removing low-molecular synthesis inhibitors by dialysis, a dialysis membrane having a removal molecular weight of 50,000 to 12,000 can be exemplified, and specifically, a regenerated cellulose membrane having a removal molecular weight of 12,000 to 14,000 is preferably used ( Viskase Sales, manufactured in Chicago) and SPECTRA/aperture 6 (manufactured by SPECTRUM LABOTRATORIES NC., CA, USA) with a molecular weight of 50,000 for removal, etc. An appropriate amount of a cell extract-containing solution or the like is added to this dialysis membrane, and dialysis is performed by a usual method. The time for dialysis is preferably about 30 minutes to 24 hours.

(stabilization of cell extract containing liquid)

When removing low-molecular synthesis inhibitors, when insoluble components are generated in the cell extract-containing liquid, by inhibiting its production (hereinafter, often referred to as "stabilization of the cell extract-containing liquid"), the final obtained The protein synthesis activity of the cell extract containing solution or the solution for translation reaction. As a specific method for stabilizing the cell extract-containing solution or the translation reaction solution, for example, when removing the above-mentioned low-molecular synthesis inhibitors, the cell extract-containing solution or the translation reaction solution is made to contain at least a high-energy phosphoric acid compound , such as ATP or GTP (hereinafter often referred to as "stabilizing components") solution. Preferably ATP is used as the high energy phosphate compound. Furthermore, it is preferably carried out in a solution containing ATP and GTP, more preferably in a solution containing ATP, GTP and 20 kinds of amino acids.

These components can be added in advance with stabilizing components, and after incubation, they can be provided to the removal process of low-molecular-weight inhibitory substances. When dialysis is used to remove low-molecular-weight synthetic inhibitory substances, the stabilizing components can also be added to the external dialysis fluid for dialysis. to remove low molecular weight synthesis inhibitors. If the stabilizing component is added to the external dialysis fluid in advance, even if the stabilizing component is decomposed during dialysis, a new stabilizing component can be continuously supplied, which is preferable. This also applies to the case of gel filtration and ultrafiltration. After equilibrating various carriers with a filtration buffer containing a stabilizing component, a cell extract-containing solution containing a stabilizing component or a solution for translation reaction is provided, and then The same effect can be obtained by performing filtration while adding the above-mentioned buffer solution.

The addition amount of the stabilizing component and the stabilization treatment time can be appropriately selected according to the type and preparation method of the cell extract-containing liquid. As these selection methods, for example, adding stabilizing components divided into experimental amounts and types into the cell extract-containing solution, performing a process of removing low-molecular inhibitory substances after an appropriate period of time, centrifuging, etc. A method of separating the obtained treated cell extract into a soluble component and an insoluble component, and selecting a stabilized component containing less insoluble components. Furthermore, it is preferable to perform cell-free protein synthesis using the obtained treated cell extract-containing solution, and to select a stabilizing component with high protein synthesis activity. In addition, in the above-mentioned selection method, in the case of using the cell extract-containing solution and the dialysis method, it can be mentioned that an appropriate stabilizing component is also added to the dialysis fluid, and after dialysis is performed for an appropriate time with them, according to the obtained The selection method is based on the amount of insoluble components in the cell extract-containing liquid and the protein synthesis activity of the obtained cell extract-containing liquid.

In this way, as an example of the stabilization conditions of the selected cell extract-containing liquid, specifically, in the case of performing the removal step of low-molecular-weight synthesis inhibitors by dialysis, it can be mentioned that in its wheat germ extract-containing liquid And the method of adding ATP 100 μM to 0.5 mM, GTP 25 μM to 1 mM, and 25 μM to 5 mM each of the 20 kinds of amino acids to the external fluid for dialysis, and performing dialysis for 30 minutes to 1 hour or longer. The temperature at the time of dialysis may be any temperature at which dialysis can be performed without losing the protein synthesis activity of the cell extract-containing solution. Specifically, the lowest temperature is the temperature at which the solution does not freeze, usually -10°C, preferably -5°C; the highest temperature is 40°C, preferably 38°C, within the limit of not adversely affecting the solution used in dialysis.

Furthermore, if the low-molecular synthesis inhibitors are removed after preparation of the cell extract-containing liquid, it is not necessary to further add the aforementioned stabilizing components to the cell extract-containing liquid.

(Preparation of mRNA)

The preparation method of mRNA as a template substance is not limited as long as it is a substance encoding a target protein, and any known method can be used. Of course, it is particularly preferable to prepare DNA as a template substance by the method of the present invention, and to use it continuously in a series of systems.

(Preparation of solution for translation reaction)

The synthesis of protein from mRNA is carried out by adding a solution (translation reaction solution) containing a component suitable for translation reaction to a ribosome-containing cell extract for protein synthesis at a concentration suitable for the cell-free protein synthesis method. Contains amino acids used as substrates (using 20 essential amino acids or similar or modified amino acids according to the purpose), energy sources (ATP and/or GTP), and various ions as needed (calcium ions, magnesium ions, ammonium ions, etc.) , buffer (HEPES-KOH, Tris-acetic acid, etc.), ATP regeneration system (combination of phosphoenolpyruvate and pyruvate kinase, combination of phosphocreatine and creatine kinase, etc.), nucleolytic enzyme inhibitor (ribose Nuclease inhibitors, nuclease inhibitors, etc.), tRNA, reducing agents (dithiothreitol, etc.), polyethylene glycol, 3', 5'-cAMP, folic acid, antibacterial agents (sodium azide, ampicillin etc.), etc., and then add translation template mRNA therein to prepare a solution for translation reaction. This translation reaction solution is adjusted to a temperature suitable for the translation reaction, and the translation reaction is carried out to synthesize the target protein.

(transcriptional reaction)

The transcription template can be prepared according to the widely applicable template molecular construction method (WO01/27260, 02/18586) reported by the present inventors. A suitable example is: for example, introducing the desired structural gene into the plasmid vector pEU, A method of transcription by treating with SP6 RNA polymerase, etc., of course, the method of preparing mRNA is not limited thereto. The transcription template is mixed with a solution (also called a transcription reaction solution) containing components necessary for the transcription reaction, such as an RNA polymerase suitable for the promoter in the transcription template and a substrate for RNA synthesis (four kinds of ribonucleoside triphosphates). , the mixture is incubated at about 20 to about 60°C, preferably at 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.

(synthesis reaction)

In the cell-free synthesis system of the present invention, a system including the following steps, control of the steps, or a combination thereof can be realized.

One of the necessary steps is bringing a template substance, a substrate, and a reaction solution into contact with and introducing into a synthesis reaction system. The template substance, substrate and reaction solution (transcription reaction solution or translation reaction solution) are adjusted to the optimum concentration and temperature to perform the optimum synthesis reaction. The synthesis reaction is usually carried out for about 10 minutes to 2 hours, more preferably 20 minutes to 1 hour. This time can be changed depending on each system, and the optimum time can be adjusted by repeated experiments. In the discontinuously repeated synthesis method of the present invention, it is particularly preferable that the treatment time in one cycle is relatively short.

(diluted or concentrated)

The second necessary step is to dilute or concentrate the reaction system before and after the synthesis rate slightly decreases, before and after the synthesis reaction is about to stop, or during these states as the above-mentioned reaction time. The so-called before and after the slight decrease in the synthesis rate refers to the timing when the synthesis amount of mRNA or protein per unit time tends to decrease from the maximum amount over time, which can generally be understood as a line rather than a point. In addition, before and after the stop of the synthesis reaction refers to a level at which the amount of synthesis drops to a substantially undetectable level, and such a situation can generally be understood as a line rather than a point. The term "in progress" in these states refers to a region in which the synthesis rate starts to decrease and the synthesis reaction stops. Furthermore, in order to obtain better synthesis efficiency, the reaction system should be diluted or concentrated before and after the synthesis rate is slightly reduced. This time is most preferably from 10 minutes to 1 hour.

The dilution or concentration of the reaction system was carried out as follows.

Dilution is performed by adding about 1 to 20 times, preferably about 2 to 10 times the volume of an aqueous solution to the reaction system. The aqueous solution contains a template substance, a substrate, and a reaction solution as necessary. It is particularly preferred to use a solution containing a template substance, a substrate, an energy source, and the like. The addition concentration of each component is selected so that the concentration most suitable for synthesis can be prepared after the subsequent concentration treatment. By this dilution, the reaction system is in a state where the synthesis ability is significantly reduced. The present invention is therefore in a state of discontinuity.

Concentration means that when the reaction solution is removed from the reaction system, the impassable substances (such as synthetic proteins, ribosomes, etc.) in the reaction solution can be concentrated into the reaction system, and any well-known concentration method can be used. Preferable methods include filtration treatment with an ultrafiltration membrane, treatment with a centrifuge, gel filtration treatment, suction pump, and a method of generating a pressure difference in a liquid phase or a gas phase. In this treatment, the reaction product and reaction by-products are separated and removed by centrifugation or molecular sieves by adjusting the passing pore size of the membrane. The molecular weight cut-off size of the membrane, centrifugal speed, and gel filtration conditions can be adjusted to the optimum according to the well-known physical properties of the product to be processed. In the present invention, it is preferable to use a molecular weight separation membrane of 10,000 to 100,000 Da.

Through this concentration treatment, the reaction solution is concentrated to 1/5 to 2/3 of its original capacity, and as a result, the optimum concentration of each synthesis factor is greatly deviated. By such concentration, the reaction system is in a state where the synthesis ability is significantly lowered. The present invention is therefore in a state of discontinuity.

(reactivation of synthesis reaction)

The third necessary procedure is the reactivation of the synthesis reaction. The reaction solution diluted in the previous stage is concentrated, and the concentrated reaction solution is subjected to dilution treatment.

In the former case, the concentration treatment is performed after the dilution treatment. Concentration here refers to returning the liquid volume of the reaction system increased by dilution to the original liquid volume. The concentration method is not particularly limited, and any known concentration method can be used. Preferable methods include filtration treatment using an ultrafiltration membrane, treatment with a centrifuge, gel filtration treatment, a method of generating a pressure difference in a liquid phase or a gas phase, and the like. In this treatment, the reaction product and reaction by-products are separated and removed by centrifugation or molecular sieves by adjusting the passing pore size of the membrane. The molecular weight cut-off size of the membrane, centrifugal speed, and gel filtration conditions can be adjusted to the optimum according to the well-known physical properties of the product to be treated. In the present invention, it is preferable to use a molecular weight separation membrane of 10,000 to 100,000 Da.

In the latter case, the dilution treatment is performed after the concentration treatment. The term "dilution" here refers to returning the liquid volume of the reaction system reduced by concentration to the original liquid volume. A template substance, a substrate, and a reaction solution are contained in the diluted solution as necessary. It is particularly preferable to use a solution containing a template substance, a substrate, an energy source, and the like. The added concentration of each component should be selected according to the most suitable concentration that can be prepared after dilution.

In this way, the reaction system is restored to the optimum concentration of the reaction system, the temperature can be adjusted to the optimum reaction temperature again, and the reaction system can be activated again. The optimum temperature is 15-25°C.

In addition, for the separation, recovery, and removal of reaction products and/or reaction by-products, it is preferable to perform a treatment based on the affinity with the object to be treated. The term based on affinity can be exemplified as a method in which an affinity substance is first immobilized, brought into contact with the target substance to bind it, and then the target substance is dissolved and recovered. The so-called affinity substance can be exemplified, for example, if the recovered substance is a protein, it is an antibody corresponding to the protein, a ligand corresponding to a receptor, a nucleic acid corresponding to a transcription factor, and the like. In addition, the product of interest is modified with an appropriate label (for example, streptavidin, histidine tag, GST, maltose-binding protein, etc.), and a substance that can specifically bind to these modified substances (for example, biotin , divalent metal ions, glutathione, maltose, etc.) to purify.

In the present invention, the treatment of dilution and concentration can be repeated multiple times discontinuously, and multiple regenerations of the cell-free synthesis system can be achieved through this repetition. A large amount of protein synthesis can be achieved through this regeneration.

In the present invention, the treatment of dilution and concentration can be completed as a series of processes in combination with a control mechanism. In order to achieve this kind of control, it is possible to adjust the on/off of the operation, the degree of operation, the operation of speed, operating interval. In addition, a drive device for signal transmission, a sensor for operation confirmation, a switch for operation control, a timer, etc. may be appropriately equipped as necessary.

As described above, the present invention realizes the regeneration of the cell-free synthesis system by repeated dilution and concentration discontinuously, and its effect is described more specifically as follows.

(dilution concentration batch method cell-free protein synthesis method)

In this synthesis method, as a batch reaction vessel, a synthesis reaction vessel equipped with an ultrafiltration membrane and a dialysis membrane that has been used in the past and capable of concentration can be used as a batch reaction vessel to start the synthesis reaction. After the protein synthesis reaction is carried out under the optimal conditions by the cell-free protein synthesis method, for example, before and after the synthesis reaction is stopped, in order to reactivate the reaction system, other buffers and ions containing substrates and amino acids as energy sources, ATP and GTP (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000) 97, 559-556 described in the dialyzed external fluid, hereinafter referred to as matrix solution) and template substances The solution of the mRNA is added and diluted at one time according to the volume of 1 to 20 times of the reaction solution (dilution treatment). Thereafter, by centrifuging the reaction container on, for example, a centrifuge, the solution increased in the container is discharged through a filter membrane, and the original reaction liquid capacity is restored (concentration treatment). This concentration treatment can be achieved by creating a pressure difference in the liquid phase or the gas phase instead of relying on centrifugal force. Through this dilution and concentration treatment, while providing fresh substrate solution to the reaction system, by-products generated by the synthesis reaction that inhibit the translation reaction can be eliminated. In this way, for example, by discontinuously repeating dilution and concentration of the reaction solution with a solution containing a substrate or the like before and after the stop of the synthesis reaction, the reactivation of the reaction system is completed, and a protein can be synthesized for a long time. This method is different from the principle of the so-called continuous method that uses semi-permeable membranes developed by Spirin et al. to continuously carry out supplementation of substrates, energy sources, and waste of metabolites. Its synthetic effect reaches tens to thousands of times (in the present invention) In the method, it takes 2 to 3 hours to obtain the yield obtained by the method using a semipermeable membrane in about 30 hours), which essentially has a large difference. Furthermore, in the protein synthesis method adopting this principle, by selecting the cut molecular weight of the filter membrane, it is possible to selectively separate the synthesized protein from the reaction system while excluding by-products. That is, by utilizing this principle, protein synthesis and synthesis product purification can be performed simultaneously. In addition, the same effect can be obtained also when changing the order of diluting after performing concentration.

(Dilution Concentration Batch RNA Synthesis in Test Tube: Transcript Production System)

The general RNA synthesis reaction composition, reaction method and analysis method of synthesized RNA using phage RNA synthetase are in accordance with the methods described in WO01/27260 and WO02/18586. In the synthesis method of the present invention, a conventional container capable of concentration equipped with an ultrafiltration membrane and a dialysis membrane can be used as a batch reaction vessel to start the synthesis reaction. The cutting molecular weight of the ultrafiltration membrane employed is selected to be of a size through which RNA cannot pass. Before and after the stop of the synthesis reaction, a solution (prepared according to the method described in WO01/27260 and WO02/18586) containing four kinds of ribonucleotide triphosphates, ions, and buffer as substrates, etc. Dialyzed external fluid, hereinafter referred to as RNA matrix solution), was added and diluted according to 1 to 20 times the volume of the reaction solution. Centrifuge the reaction container on a centrifuge, drain the increased solution in the container through the filter membrane, and concentrate to the original capacity of the reaction solution. This process can be handled not by centrifugal force but by creating a pressure difference in the liquid or gas phase. Through these treatments, while supplying fresh substrate solution to the reaction system, mononucleotides and pyrophosphates produced in the batch RNA synthesis reaction are excluded from the reaction system, so that the chemical equilibrium principle that has actually stopped The RNA synthesis reaction starts anew by shifting the equilibrium. By repeating the operations of diluting the reaction liquid with the substrate and concentrating by filtration before and after stopping the reaction, long-term RNA synthesis is possible. This method is different from the principle of the continuous RNA synthesis method through dialysis (WO00/68412) invented by Endo et al. In the protein synthesis method using this principle, by repeating the operation of diluting with water (or a desired solution) and concentrating with a filter membrane, RNA that does not contain nucleotides, buffer components, and ions can be used as water. (or a solution containing the target component) recovery. That is, with this method, desired mRNA can be prepared at high speed, high yield, and low cost (the amount of expensive RNA synthetase used is only necessary for the usual batch method) by selecting the DNA molecule species to be used as a template. solution.

In addition, in the case of changing the order of performing concentration and then dilution, the same effect can be achieved.

(automatic synthesis device)

The automatic synthesis apparatus of the present invention provides a method for automatically performing a series of reaction operations of a synthesis method in which at least a synthesis reaction (protein synthesis) is performed by translating a template by repeating dilution and concentration discontinuously. Furthermore, the present invention provides a method for automatically performing a reaction operation from a transcription template synthesized by a transcription product production system to production of a protein encoded by the template. The so-called "automatically performing the operation" here means that in a series of procedures, the experimenter does not need to manually operate the reaction system (reaction vessel) directly. Therefore, when carrying out each process, the experimenter manually uses the predetermined operation buttons and switches set in the automatic synthesis device of the present invention, without impairing the requirement of "automatic" in the present invention.

In the present invention, the device for automating the reaction operation from the transcription template synthesized by the transcription product production system to the production of the protein encoded by the template is characterized by having at least the following steps (1) to (5).

Hereinafter, specific embodiments related to each step will be described in detail, but the method of the present invention is not limited as long as it has the characteristics of a translation template purification step.

(1) Production process of transcription template

In the automatic synthesis device of the present invention, this step does not necessarily have to be automatically performed, and transcription templates obtained manually can also be used in the following automated steps. A series of steps up to the template-encoded protein are automatically performed.

The term "transcription template" in this specification refers to a DNA that can be used as a template molecule for an in vitro transcription reaction, and has at least a nucleotide sequence encoding a desired protein downstream of an appropriate promoter sequence. The appropriate promoter sequence refers to a promoter sequence that can be recognized by RNA polymerase used in the transcription reaction, and examples thereof include SP6 promoter, T7 promoter, and the like. The DNA encoding the protein of interest may be any gene.

The transcription template preferably has a base sequence that controls translation efficiency between the promoter sequence and the base sequence encoding the target protein. For example, the 5' untranslated region derived from RNA viruses such as the Ω sequence derived from tobacco mosaic virus can be used , and/or Cozak's sequence (Kozak's sequence), etc. Furthermore, the transcription template preferably includes a 3' untranslated region including a transcription termination region downstream of the base sequence encoding the protein of interest. The so-called 3' untranslated region is preferably about 1.0 to about 3.0 kilobases downstream of the stop codon. These 3' untranslated regions do not have to be the original sequence of the gene encoding the protein of interest.

Preparation of transcription templates can be carried out by well-known techniques. Examples include culturing host cells such as Escherichia coli containing a plasmid inserted with a DNA having the same nucleotide sequence as a desired transcription template. Cut out the transcription template DNA from the plasmid, remove restriction enzymes by phenol treatment and chloroform treatment, and further alcohol precipitation with ethanol or isopropanol (if necessary, add an appropriate amount of ammonium acetate, sodium acetate, sodium chloride and other salts), etc. Method for purifying transcription templates. The obtained DNA precipitate is dissolved in ultrapure water or the solution for transcription reaction described below, and can be used in the following transcription reaction.

Devices that are implemented automatically or semi-automatically (referring to the state in which the experimenter directly manually operates the reaction system in a part of the process) for such a series of operations are known, and by assembling these devices into the automatic synthesis device of the present invention, it is possible to automatically perform from From the production of transcription templates to the production of proteins. However, in consideration of the purpose of providing the high-throughput synthesis system involved in the present invention for high-throughput analysis, simplification of the device, and shortening the time required, it is preferable to use the following polymerase chain reaction (PCR). A method for making transcription templates.

In a preferred embodiment aspect of the synthesis device of the present invention, a method in which a transcription template is amplified by direct PCR is used in a host (for example: Escherichia coli having a plasmid containing the DNA) in which the DNA encoding the protein of interest is cloned. For example, an oligonucleotide containing a suitable promoter sequence, a 5' non-translated sequence having the activity of controlling translation efficiency, and a partial region of the 5' end of DNA encoding a protein of interest is synthesized by a well-known method using an automatic DNA synthesis machine, and this As a sense primer, an oligonucleotide containing the sequence of the 3' end region of the 3' untranslated sequence was used as an antisense primer, and the DNA encoding the target protein and its downstream plasmid containing the 3' untranslated sequence in the large intestine A host such as bacteria can be added as a template into the direct PCR reaction solution, and the desired transcription template can be obtained by performing amplification reaction under normal conditions. In addition, in order to prevent the generation of short-strand DNA generated by non-specific amplification (resulting in low recovery of the product of interest and interference of low-molecular translation products), the promoter described in International Publication No. 02/18586 pamphlet can also be used Split primers.

The amplification reaction can also be carried out in a 96-well plate for commercially available PCR using a commercially available thermal cycler for PCR, and the same temperature variable control device can be linked with the synthesis device of the present invention, or the Various methods of transcription and translation reactions of synthetic devices are directly applicable to PCR.

The transcription template DNA obtained above can also be used for transcription reaction after being purified by chloroform extraction and alcohol precipitation. However, in order to simplify the equipment and shorten the required time, it is preferable to use the PCR reaction solution directly as the transcription template solution. In the production of transcription templates, compared with the method of preparing a large number of plasmids at one time and treating the plasmids with restriction enzymes to obtain transcription templates, by using the above-mentioned direct PCR from the host, the process can be particularly saved, and the number of processes can be reduced in a short time. time to synthesize transcription templates in large numbers. That is, since there is no need to culture Escherichia coli containing recombinant target gene plasmids to prepare large quantities of plasmids, the time required for ultracentrifugation for culturing and purifying plasmids can be shortened. In addition, it is possible to omit the steps of restriction enzyme treatment for excising the transcription template from the plasmid, phenol treatment for removing restriction enzymes, chloroform treatment, alcohol precipitation for purification of the transcription template, and precipitation for dissolving the transcription template DNA. , Therefore, there is no inhibition of the transcription reaction caused by the residue of phenol/chloroform and the loss of the transcription template caused by the multi-step purification operation. In addition, since the number of steps required in the reaction can be reduced, there is further an advantage that only a small number of mouthpieces and the like can be used.

(2) Transcription reaction process

The synthesis device of the present invention includes a step of producing translation template mRNA by an in vitro transcription reaction from transcription template DNA encoding a protein of interest prepared by a known method. The solution containing the transcription template provided in the reaction system (for example: a commercially available reaction container such as a 96-well titer plate), preferably the above-mentioned PCR reaction solution, and an RNA polymerase (for example: SP6 RNA polymerase) containing a promoter suitable for the transcription template enzymes, etc.) and a solution (also referred to as "transcription reaction solution") of essential components in transcription reactions such as substrates for RNA synthesis (four nucleoside triphosphates) This transcription reaction step is carried out by incubating at about 30°C to about 42°C for 30 minutes to 16 hours, preferably about 2 hours to about 5 hours. The dispensing and mixing of the transcription template solution and the transcription reaction solution into the reaction container can be performed using the following dispensing device of the automatic synthesis device (for example: pipette (when using a commercially available 96-well titer plate as the reaction container) , it is preferred to use a device with 8- or 12-piece dispensing mouthpieces suitable for the hole spacing) etc.). On the other hand, the incubation for the transcription reaction can be carried out while controlling a constant temperature by the temperature control mechanism of the synthesis apparatus described below.

(3) Purification process of translation template

As described above, the resulting transcript (i.e. "translation template") is an RNA molecule comprising 5' untranslated sequences and/or 3' untranslated sequences active in controlling translation efficiency inserted into the transcription template as desired , having the base sequence encoding the protein of interest. The reaction solution after the transcription reaction is mixed with unreacted nucleoside triphosphates other than the translation template RNA, pyrophosphate by-products of the reaction, salts contained in other transcription reaction solutions, etc., because these substances are known to inhibit subsequent translation reactions , so these substances are removed by exchanging the reaction solution. As such a solution exchange method, a method of adding a reaction solution to a container equipped with a filter, centrifuging to remove a solution containing a substance that inhibits the translation reaction, adding a new solution such as a suitable buffer, and repeating the operation , but not limited to this. If the newly added solution is used as the solution for the translation reaction, the next step of the translation reaction can be carried out without purification of the template. Furthermore, methods for removing these substances include selectively precipitating translation templates, separating and removing unreacted substrates, and the like. As such a precipitation method, salting-out etc. are mentioned, For example, an alcohol precipitation method is preferable, but it is not limited to this. In the case of the alcohol precipitation method, the alcohol used is not particularly limited as long as it selectively precipitates RNA. For example, ethanol, isopropanol, etc. are preferable, and ethanol is more preferable. In the case of ethanol, it is preferable to use about 2 times to about 3 times the amount of the transcription reaction solution; in the case of isopropanol, it is preferable to use about 0.6 times to about 1 times the amount of the transcription reaction solution. Also, by co-existing with appropriate salts, precipitation yields can be increased. 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 preferably added so that the final concentration becomes about 0.5M to about 3M. Alternatively, alcohol precipitation can be performed at room temperature.

(4) Translation reaction process

As described above, the translation reaction can be carried out by incubating the translation reaction solution containing the obtained translation template at an appropriate temperature for an appropriate time. The translation reaction is usually carried out for about 10 minutes to 2 hours, more preferably 20 minutes to 1 hour. This time can be changed according to each reaction system, and can be adjusted to the optimum time through repeated experiments. In the discontinuously repeated synthesis method of the present invention, the treatment time of this one cycle is particularly preferably shorter.

As mentioned above, in the above reaction, the dilution or concentration of the reaction system is carried out before and after the synthesis rate slightly decreases, before and after the synthesis reaction is about to stop, or during these states. The so-called before and after a slight decrease in the synthesis rate means that the amount of protein synthesized per unit time changes with time and has a tendency to decrease from the maximum amount, which can generally be understood as a line rather than a point. Also, before and after the synthesis reaction is about to stop means that the amount of synthesis has decreased to a level where it is substantially undetectable, and this situation can generally be understood as a line rather than a point. The term "during these states" refers to the period from when the synthesis rate starts to decrease to when the synthesis reaction stops. Here, in order to obtain better synthesis efficiency, the reaction system is diluted or concentrated before and after the synthesis rate is slightly reduced. This time is most suitable for 10 minutes to 1 hour.

Dilute or concentrate the reaction system in the following manner.

Dilution is performed by adding about 1 to 20 times, preferably about 2 to 10 times, the volume of an aqueous solution to the reaction system. The aqueous solution contains a template substance, a substrate, and a reaction solution as desired. It is particularly preferable to use a solution containing a template substance, a substrate, an energy source, and the like. The addition concentration of each component should be selected according to the standard which can prepare the synthesis|combination optimum concentration after concentrating in the following processing. By this dilution, the reaction system is in a state where the synthesis ability is significantly reduced. Hence the state, which the present invention refers to as discontinuous.

Concentration can utilize any well-known concentration method that can concentrate impassable substances in the reaction solution (for example, synthetic proteins, ribosomes, etc.) in the reaction system when the reaction solution is removed from the reaction system. Preferable examples include filtration treatment using an ultrafiltration membrane, treatment with a centrifuge, gel filtration treatment, a suction pump, a method of generating a pressure difference in a liquid phase or a gas phase, and the like. In this treatment, reaction products and reaction by-products are separated and removed by adjusting the passing pore size of the membrane, centrifuging, or passing through molecular sieves. The molecular weight cut-off size of the membrane, centrifugation speed, and gel filtration conditions can be adjusted to the optimum according to the well-known physical properties of the target product. In the present invention, it is preferable to use a separation membrane with a molecular weight cut off of 10,000 to 100,000 Da.

Through this concentration treatment, the reaction solution is concentrated to 1/5 to 2/3 of its original capacity, and as a result, each synthesis factor is greatly deviated from the optimum concentration. Due to such concentration, the reaction system is brought into a state where the synthesis ability is remarkably lowered. Hence the state, which the present invention refers to as discontinuous.

This is followed by reactivation of the synthesis reaction. The reaction solution diluted in the previous stage is concentrated, and the concentrated reaction solution is diluted.

In the former case, the concentration treatment is performed after the dilution treatment. Concentration here means returning the liquid volume of the reaction system increased by dilution to the original liquid volume. The concentration method is not particularly limited, and all known concentration methods can be used. Preferable examples include filtration treatment using an ultrafiltration membrane, treatment with a centrifuge, gel filtration treatment, a method of generating a pressure difference in a liquid phase or a gas phase, and the like. In this treatment, reaction products and reaction by-products are separated and removed by adjusting the passing pore size of the membrane, centrifuging, or passing through molecular sieves. The molecular weight cut-off size of the membrane, centrifugation speed, and gel filtration conditions can be adjusted to the optimum according to the well-known physical properties of the target product. In the present invention, it is preferable to use a molecular weight cut-off separation membrane of 10,000 to 100,000 Da.

In the latter case, the dilution treatment is performed after the concentration treatment. The term "dilution" here means returning the liquid volume of the reaction system reduced by concentration to the original liquid volume. The dilute solution contains the template substance, the substrate, and the reaction solution as desired. It is particularly preferable to use a solution containing a template substance, a substrate, an energy source, and the like. The added concentration of each component should be selected according to the optimal concentration that can be adjusted after dilution.

In this way, the temperature of the reaction system restored to the optimum concentration of the reaction system can be adjusted to the optimum reaction temperature again, and the reaction system can be activated again. The optimum temperature is 15-25°C.

In the present invention, the treatment of dilution and concentration can be repeated multiple times discontinuously, and multiple regenerations of the cell-free synthesis system can be achieved through this repetition. The number of repetitions is from 2 to 20, preferably from 5 to 10. Through this regeneration, a large amount of protein synthesis can be completed.

The other steps can be performed in any known order and conditions applicable to automation, and are not particularly limited.

In the present invention, the device for performing the above-mentioned operations is characterized by having at least the following mechanisms (a) to (e).

(a) Mechanism for variably controlling the temperature in the reaction vessel

(b) Mechanism for dispensing samples or reagents into reaction vessels

(c) Mechanism for transporting reaction vessels

(d) device for precipitation and concentration filtration

and (e) a control mechanism for controlling the above-mentioned mechanisms (a) to (d) so as to operate according to the above-mentioned method of the present invention.

By using such an apparatus equipped with at least the structures (a) to (e), the reaction operation from the transcription template synthesized by the above-mentioned transcription product production system of the present invention to the production of the protein encoded by the template can be automatically carried out. Each structure will be described in detail below.

(a) Mechanism for variably controlling the temperature in the reaction vessel

The mechanism for variable control of the temperature in the reaction container refers to the incubation of the transcription reaction, the translation reaction, the stop of the translation reaction, the precipitation of the translation template, or the automatic synthesis of the transcription template by the PCR method using the automatic synthesis device of the present invention. It is a mechanism to adjust the temperature of the liquid in the reaction container to an appropriate temperature condition in the amplification reaction of the PCR method, etc. during the production process. The controllably variable temperature range is not particularly limited as long as it is a temperature range usually necessary in a series of reaction operations including transcription template production and cell-free protein synthesis (for example: about 4°C to about 100°C, preferably about 15°C A mechanism capable of controlling and changing the temperature in the reaction vessel within a temperature range of about 60° C. can be used as the means for realizing this without any particular limitation. For example, the well-known Takara PCR thermal cycler MP (manufactured by Takara Biological Co., Ltd.), (Gene Amp PCR System 9700 (manufactured by Applied Biosystems Inc., manufactured) and the like can be mentioned. Specifically, it is related to the place and setting where the pipetting operation and the like are performed. The operating table for placing the reaction vessels is different. A plurality of operating tables for placing the reaction containers are designed in the device, and the temperature of the entire space on the operating table is controlled and changed, so as to realize the control and change of the temperature in the reaction containers.

(b) Devices for dispensing samples or reagents into reaction vessels

The so-called mechanism for dispensing samples or reagents into the reaction container refers to a mechanism for dispensing samples or reagents into the 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 . Here, "sample" refers to a transcription template, a translation template, a template plasmid for PCR (or a host (such as Escherichia coli) containing the plasmid), etc.; Dilution solution, alcohol, salt solution, solution for PCR reaction, etc. As such a dispensing mechanism, as long as it is a mechanism that can adjust the amount of dispensing samples and reagents according to the process and perform dispensing, any well-known and appropriate automatic dispensing pipetting arm (dispensing machine) can be used without particular limitation. ) and so on to achieve. Furthermore, the pipetting arm may have a function of discarding the used mouthpiece at the mouthpiece disposal port of the synthesizer and a function of discharging the sucked filtrate to the waste liquid port.

In addition, the dispensing mechanism preferably has the above functions plus a mixing function (such as pipetting, stirring, etc.) for homogenizing two or more solutions and dissolving precipitates.

In addition, the dilution treatment of the reaction liquid can be performed by adding a substrate solution or a diluent solution to each unit of the reaction vessel via the pipetting arm.

(c) Mechanism for transporting reaction vessels

The so-called mechanism for transporting the reaction container refers to a mechanism for moving the reaction container to each operation table, centrifuge, lifting table, and constant temperature bath. Such a mechanism for transporting the reaction container can be implemented by any known and appropriate mechanism without particular limitation as long as the reaction container can be transported to the destination. For example, it can be done using a robot arm used in a conventional synthesis device.

(d) mechanism for sedimentation and concentration filtration

The precipitation and concentration filtration mechanism refers to the operation of precipitating the white suspension generated in the transcription reaction or the mechanism of repeatedly concentrating and filtering the reaction solution in the translation reaction. Such a mechanism is capable of precipitating the white suspension generated during the reaction and separating the solid, and there is no particular limitation as long as the reaction solution during the translation reaction can be repeatedly concentrated, and a well-known suitable mechanism can be used. This mechanism can be accomplished, for example, using well known centrifuges, as well as suitable devices previously used in filtration and freeze-drying.

(e) Control agencies

The control mechanism includes the operation of the drive source (engine, air compressor, oil compressor, other transmission devices that can control the operation, etc.) used in each mechanism to operate the mechanisms (a) to (d) above. A control device that controls start and stop, degree of operation, and status. Its control structure is to automate the operation of the above-mentioned mechanism (a) to (d) and the reaction operation from the transcription template synthesized in the transcription product production system to the production of the protein encoded by the template.

The above-mentioned control device, for example, can be composed of a control circuit including a computer with a control program, a program-controlled circuit, etc., and control devices necessary for controlling the operation of the above-mentioned mechanisms. Signals can be provided to each mechanism, and control structures such as electric power, air pressure, and oil pressure can be provided as needed. In addition, necessary drivers for sending direct drive signals to the driving sources of the above-mentioned mechanisms, and necessary sensors and switches for detecting the operating states of the driving sources of the above-mentioned mechanisms may also be appropriately added.

In addition, there are no particular limitations on the reaction vessel that can be applied to the synthesis device of the present invention, and various well-known reaction vessels that have been used in cell-free protein synthesis reactions can be used, and examples include 96-well PCR plates, 96-well titration plates, 8-tube test tubes, and test tubes (1.5ml, 15ml, 50ml, etc.), but for example, when the translation reaction system adopts the batch method and the multilayer method, the translation reaction can be performed in a small reaction system such as a 96-well plate, and , due to the use of the synthesis device of the present invention, the transcription reaction can also be carried out in a small reaction system, including the purification of the transcription reaction and translation template, the translation reaction, and PCR for making a transcription template that is further provided to the transcription reaction as desired. A series of reaction operations of the cell-free protein synthesis method can be performed simultaneously on various proteins in various reaction systems, and various proteins can be synthesized in a short time.

Furthermore, the present invention provides an apparatus for carrying out a synthesis reaction by translating a template by a synthesis method in which dilution and concentration are repeated in a discrete manner. The device of the present invention is characterized by having at least the following (1) to (5) mechanisms.

(1) The body that starts the synthesis,

(2) A mechanism for concentrating the reaction solution,

(3) A mechanism for diluting the reaction solution,

(4) mechanism of reactivation of synthesis reaction,

(5) A mechanism that makes the above-mentioned mechanisms (2) to (4) operate repeatedly;

or

(1) The body that starts the synthesis,

(2) A mechanism for diluting the reaction solution,

(3) A mechanism for concentrating the reaction solution,

(4) mechanism of reactivation of synthesis reaction,

(5) A mechanism for repeatedly operating the above-mentioned mechanisms (2) to (4).

The above-mentioned high-throughput synthesis system of the present invention can be automated by using an apparatus having at least such structures (1) to (5). Each institution is described in detail below.

(1) The organization that starts the synthesis

①Move the dispensing machine to the reagent tank where the translation reaction solution is added, and attract the translation reaction solution.

②Move the dispensing machine above the plate for translation (the bottom of the unit is the filter), and spit out the translation reaction solution into each unit of the plate for translation where the transcription product is added.

③Next to ②, the dispensing machine performs the pipetting operation of each unit.

④The robot arm will cover the cover of the plate for translation, and transport the plate to the constant temperature bath and install it in the constant temperature bath.

⑤Insulate the translation plate and start the synthesis.

⑥After the set synthesis time is over, the robotic arm transports the plate for translation to the MTP console.

(2) Mechanism for diluting the reaction solution

①Move the dispensing machine to the reagent tank where the diluted solution is added, and attract the diluted solution.

②Move the dispensing machine above the translation plate, and spit out the diluted solution into each unit of the translation plate where the translation product is added.

(3) Mechanism for concentrating the reaction solution

①The robotic arm superimposes the plate for translation on the plate for receiving transcription and filtrate.

②The robotic arm transports the stacked plates for translation and plates for receiving transcription and filtrate to the lift table.

③ Lower the lifting table on which the stacked plates for translation and plates for receiving transcription and filtrate are placed to match the height of the centrifuge.

④The robotic arm installs the stacked plates for translation and plates for receiving transcription and filtrate into the centrifuge. At this time, when the plate for translation and the plate for receiving transcription and filtrate are set as a set, partitions are installed on the diagonal. In addition, in the case of two sets, mutual boards are installed on diagonal lines.

⑤ Start centrifugation through the centrifuge. At this time, since the bottom of the plate for translation is a filter, the filtrate can be removed to the units of the plate for transcription and filtrate, and each unit in the plate for translation is concentrated.

⑥The robotic arm transports the stacked plates for translation and plates for receiving transcription and filtrate from the centrifuge to the lift table.

⑦ Raise the lifting platform on which the stacked plates for translation and plates for receiving transcription and filtrate are loaded.

⑧The robotic arm transports the overlapping plate for translation and the plate for receiving transcription/filtrate to the MTP operation table, and installs the plate for translation and the plate for receiving transcription/filtrate separately.

(4) Mechanism to reactivate the synthesis reaction

Concentration treatment is continued in the case of dilution treatment in the previous stage, and dilution treatment is continued in the case of concentration treatment in the previous stage.

①The robotic arm transports the concentrated translation plate or the diluted translation plate to the constant temperature tank and installs it in the constant temperature tank.

②Insulate the translation plate and start the synthesis.

(5) A mechanism that repeatedly operates the above-mentioned mechanisms (2) to (4)

① After the set time has elapsed, lower the temperature of the constant temperature bath to stop substantial synthesis.

②The robotic arm transports the translation plate to the MTP console.

③The mechanism described in (2)-(4) is repeated several times.

In addition, the mechanism of the synthesis apparatus described above is an example of automatic operation of a high-throughput synthesis system, and may further include a mechanism for treating waste liquid by concentration treatment, a mechanism for disposing of a mouthpiece of a dispensing machine, and the like.

In addition, a program including the following information processing means is also an object of the present invention in order to perform a synthesis system using the initial phase of the synthesis reaction at which the protein synthesis reaction rate is high for implementing a high-throughput synthesis system multiple times.

(1) Based on the information on the volume of the reaction vessel and the concentration of the reaction solution, an information processing mechanism that sets the synthesis time within the scope of the initial phase of the synthesis reaction before the synthesis amount per unit time appears to decrease;

(2) An information processing mechanism that adds a dilute solution to the reaction vessel and sets the reaction solution at a concentration range beyond the possible concentration range for a substantial synthesis reaction when the synthesis time within the range of the initial phase of the synthesis reaction in (1) is reached;

(3) After (2), concentrate the reaction liquid in the reaction vessel, and set the information processing mechanism so that the liquid volume of the reaction liquid increased due to the addition of the dilute solution returns to the original liquid volume;

(4) After (3), an information processing mechanism for setting the optimum reaction temperature in order to restart the synthesis reaction;

(5) An information processing mechanism for repeating (1) to (4) multiple times.

or

(1) Based on the information on the volume of the reaction vessel and the concentration of the reaction solution, an information processing mechanism that sets the synthesis time within the scope of the initial phase of the synthesis reaction before the synthesis amount per unit time appears to decrease;

(2) An information processing mechanism that concentrates the reaction liquid in the reaction vessel and sets the reaction liquid outside the concentration range capable of carrying out the substantial synthesis reaction when the synthesis time within the range of the initial phase of the synthesis reaction in (1) is reached;

(3) After (2), add a diluting solution into the reaction container, and set the information processing mechanism so that the liquid volume of the reaction liquid reduced by concentration returns to the original liquid volume;

(4) After (3), an information processing mechanism for setting the optimum reaction temperature in order to restart the synthesis reaction;

(5) An information processing mechanism for repeating (1) to (4) multiple times.

Furthermore, in the case of a well-known synthesis device incorporating the above-mentioned program, the information processing of the program cooperates with the synthesis device to perform the steps of starting synthesis, diluting and concentrating the reaction solution, and reactivating the synthesis reaction, which can be repeated many times. The object of the present invention is also a synthesis apparatus characterized by performing a synthesis system using a synthesis reaction initial stage in which the protein synthesis reaction rate is high.

In addition, when performing transcription reaction and translation reaction, it is preferable to carry out the reaction container in an airtight manner. From this point of view, it is preferable to use a reaction container with a lid, and it is preferable that the device has a mechanism for opening and closing the lid of the reaction container. As the above-mentioned lid, when a 96-well plate is used as the reaction vessel, a rubber lid that can seal each well can be exemplified. In the closed state, it is preferable that the lid can tightly cover the reaction vessel, so it can be considered to adopt a lid with a certain weight (for example, about 500G), or clamp the lid and the reaction vessel with a clamp to close the lid. In addition, the mechanism for opening and closing the lid of the reaction container can be realized by, for example, a combination of a well-known chuck structure, suction structure, and robot arm.

The synthesis apparatus of the present invention may have a mechanism for storing reaction reagents as necessary, in addition to the above-mentioned mechanisms.

As described above, the high-throughput system of the present invention and the device for automating the system can easily and automatically synthesize a variety of proteins at the same time. For example, it is useful to prepare multiple transcription templates and translation templates for proteins encoding various variants, simultaneously synthesize a large number of variant proteins, and provide them for analysis without detailed design of the variants.

And the high-throughput system of the present invention and the device for automatically operating the system can be well used for high-throughput functional analysis of various proteins. For example, as a result of the homology search, the gene group encoding a protein comprising a preserved common functional region (such as a protein kinase functional region, etc.) is used as a template to simultaneously synthesize the protein by using the device of the present invention through the method of the present invention. Similarly, target protein groups that may become phosphorylated (such as transcription factors, etc.) are synthesized, and the two are mixed in various combinations. For example, by taking up ³² P-labeled ATP as an indicator, it is possible to simultaneously identify which protein kinase and which protein Phosphorylation.

Alternatively, using the gene group encoding a protein containing a unique motif (Zn finger structure, leucine zipper, etc.) The combination of known cis-component sequences, the ability to form heterodimers with other transcriptional control factors, and the ability to bind to the transcriptional control region of a specific gene promoter, etc., can be used to elucidate the interaction of transcription factor interweaving Information.

Example

The following examples are given to illustrate the present invention in detail, but the present invention is not limited to the examples described below.

(cell-free protein synthesis method)

1) Reaction from DNA to mRNA (transcription reaction)

Prepare transcription reaction solution [final concentration is 80mM HEPES-KOH pH 7.8, 16mM magnesium acetate, 10mM dithiothreitol, 2mM spermidine, 2.5mM 4NTPs (4 nucleotide triphosphates), 0.8U/μl RNase inhibitor, 0.1μg/μl DNA [plasmid GFP (Green Fluorescent Protein)], 1.6U/μl SP6 RNA polymerase], react at 37°C for 3 hours. After the reaction, centrifuge at 12000rmp for 1 hour at 4°C, collect the supernatant after centrifugation, add ethanol to a final concentration of about 70%, and ammonium acetate to a final concentration of about 0.27M, let stand on ice at 4°C for 10 minutes, and then centrifuge at 3000rmp for 30 minutes (ethanol precipitation). After the ethanol precipitation was carried out 3 times, an appropriate amount of dialysis buffer (final concentration of 35mM HEPES-KOH pH7.8, 3.1mM magnesium acetate, 103mM potassium acetate, 16mM creatine, 1.2mM ATP, 0.26mM GTP, 2.5mM DTT , 0.43mM spermidine, 0.3mM of various amino acids) to adjust the RNA concentration to about 5-10mg/ml.

In addition, according to the present invention, mRNA was synthesized by repeating discontinuous operations of dilution and concentration in accordance with Experimental Example 7.

2) Preparation of solution for translation reaction

Use the translation reaction solution [final concentration of 35mM HEPES-KOH pH7.8, 103mM potassium acetate (KOAc), 3.1mM magnesium acetate (Mg(OAc)2), 16mM phosphocreatine, 1.2mM ATP, 0.26mMGTP, 2.5mM Thiothreitol (DTT), 0.43mM spermidine, 0.3mM AAs (a mixture of 20 kinds of L-type amino acids), 1.03mg/ml creatine kinase], modulate the germ extract, so that the germ extract during translation reaction The concentration (O.D.260nm) is 40 to 120 to use.

3) Translation response

The above-prepared mRNA solution was added to the translation reaction solution containing the germ extract to react according to the concentration of the germ extract during the translation reaction. That is, the amount of mRNA added when the O.D.260nm in the translation reaction of the germ extract was 100 was defined as 0.8 mg/ml, and the amount of mRNA added was calculated according to the ratio.

The reaction solution prepared above was reacted at 20°C. After a certain period of time (10 minutes to 3 hours) from the start of the reaction, dilute the reaction solution with 2 to 3 times the volume of the above-mentioned translation reaction solution, then concentrate it with an ultrafiltration membrane with a cut-off molecular weight of 30,000 Da, and return the reaction solution to the same level as before dilution. quantity status. Repeat this operation several times. Alternatively, concentrate to about 1/2 to 1/3 volume in advance, and then dilute to the original volume with the above-mentioned translation reaction solution.

In the case of adding mRNA to the translation reaction solution in a batch method, add the transcription reaction solution prepared in 1) (the solution before ethanol precipitation) according to the concentration of the above-mentioned wheat germ liquid, or prepare through the ethanol precipitation operation The mRNA solution was carried out.

4) Determination of protein synthesis effect

The quality of the synthesized protein was determined 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).

5) Morphology of wheat germ extract

In addition, the wheat germ extract, cell-free protein synthesis reaction solution composition, mRNA preparation method, and batch cell-free protein synthesis method used in this study are based on the report of Madin K et al. (Madin K. et al., Proc. .Natl.Acad.Sci.USA(2000), 97,559-556), Patent No. 3255784, Japanese Patent Application Laid-Open No. 2000-236896, WO00/68412, Sawasaki T.etal. Reports such as (Proc.Natl.Acad .Sci.USA (2002), 99, 14652-14657).

(Example of device)

Fig. 4 shows a schematic diagram of a system in which the dilution and concentration operations of the present invention can be performed multiple times to discontinuously perform synthesis reactions. In the case where dilution and concentration operations are performed by a structure having a filter membrane and a liquid-feeding pump, a structure as schematically shown in FIG. 4 can be exemplified. By utilizing the combined structure of the filter concentrator 1 with a filter membrane and the liquid delivery pump 2, after the translation reaction liquid is diluted (transcription or) with the (RNA) substrate solution before and after the termination of the translation reaction in the reaction tank 3, the liquid is passed through the filter concentrator 1 The by-products are filtered and discharged into vessel 6. On the other hand, the reaction can be restarted by returning the concentrated (transcription or) translation reaction solution to the reaction tank 3 again.

In the system shown in Figure 4, the container 4 for receiving (RNA) matrix solution and the reaction tank 3 are connected through the tube T1, the liquid delivery switching valve 5 and the tube T2, and the liquid delivery switching valve 5 is connected through the tube T3, the liquid delivery pump 2 , tube T4, filter concentrator 1, and tube T5 are connected to reaction tank 3. In this way, the liquid-sending switching valve 5 is a structure capable of appropriately switching the following states: (a) a state in which liquid is not sent to the reaction tank 3 (valve closed state); The container 4 with the (RNA) matrix solution absorbs the (RNA) matrix solution and can directly send the liquid to the reaction tank 3; (c) absorb the (transcription or) translation in the reaction tank 3 through the operation of the liquid delivery pump 2 The reaction solution is returned to the reaction container 3 after passing through the filter concentrator 1, so that the (transcription or) translation reaction solution can be circulated; (d) is sucked from the container 4 containing the matrix solution by the operation of the liquid delivery pump 2 The (RNA) substrate solution is allowed to pass through the filter concentrator 1 and be in a state where it can be fed into the reaction tank 3 .

With such a structure, operations are performed in the following order.

1. In the reaction tank 3, a (transcription or) translation reaction is performed by a batch reaction. During this period, the liquid feeding switching valve 5 is closed [state (a) above], the liquid feeding pump 3 is also stopped, and the liquid feeding into the reaction tank 3 is stopped.

2. At any time before or after the stop of the (transcription or) translation reaction, switch the liquid delivery switching valve 5 [the state of (b) above], operate the liquid delivery pump 2, and supply the (RNA) substrate solution to the reaction tank 3 for mixing.

3. Change the liquid delivery switching valve 5 [the state of (c) above], make the liquid delivery pump 2 operate, absorb the (transcription or) translation reaction solution in the reaction tank 3, pass it through the filter concentrator 1, and discharge the by-products to the container 6, and circulate the concentrated reaction liquid back to the reaction tank 3.

4. Switch the liquid delivery switching valve 5 [the state of (d) above], make the liquid delivery pump 2 operate to absorb the (RNA) substrate solution in the container 4, and send it to the reaction tank 3 through the filter concentrator 1, and put Part of the (transcription or) translation reaction solution remaining in the filter concentrator 1 is washed out and flowed into the reaction tank 3 .

5. Switch the liquid delivery switching valve 5 [the state of (b) above] to operate the liquid delivery pump 2, absorb the (RNA) substrate solution in the container 4, send the solution to the reaction tank 3, and transfer the (RNA) substrate solution in the reaction tank 3 to the reaction tank 3. After the volume of the transcription or) translation reaction solution is adjusted to the original volume, the liquid supply switching valve 5 is switched [the state of (a) above] to stop the liquid supply.

6. Go back to 1 and repeat the above operation.

When implementing a device utilizing such a system, a well-known and suitable liquid-feeding pump can be used as the liquid-feeding pump used, and is not particularly limited, and examples thereof include peristaltic pumps [for example, LKB-Pump-P1 (manufactured by Phamacia Co., Ltd.), etc.] and the like. Also, well-known ones can be used as the filter concentrator without particular limitation. Specifically, a cross flow filter device, VF05C2 type (cut molecular weight: 30,000, manufactured by Sartorius Co., Ltd.) and the like can be exemplified.

A system that realizes the above-mentioned dilution and concentration mechanism by a structure having a filter membrane and a liquid-feeding pump can increase the yield of protein synthesis compared with the case of using a centrifuge (see Experimental Example 4), and can be constructed to perform large-scale capacity. A device for cell-free protein synthesis. Furthermore, by combining a small reaction vessel and a filter concentrator, a small-capacity device can be constructed. As a mechanism for removing the above-mentioned by-products, it is not necessary to have a large-scale device, unlike a structure having a filter membrane or a centrifuge. In addition, with such a filter membrane and liquid-feeding pump-like structure, precise control of the dilution and concentration processes is easy, and it is expected to be extremely important for the automation of general cell-free protein synthesis techniques suitable for various purposes. backbone technology.

Experimental example 1

As an example (Experimental Example 1) of the batch-type cell-free protein synthesis method in which the dilution and concentration treatments of the present invention are repeated discontinuously, FIG. 1 shows the experimental results using a wheat germ extract. The comparative example is the result obtained by the conventional cell-free protein synthesis method of the batch method. The vertical axis is the amount of synthesized protein (mg/ml), and the horizontal axis shows the reaction time (hour). In FIG. 1 , the lines (●-●) connecting black circles are the method of the present invention, and the lines (○-○) connecting white circles are the results of the conventional batch method. In the synthesis reaction, the wheat germ extract at a concentration of 40A260nm/ml (absorbance of the wheat germ extract at 260nm wavelength) was mixed with 320 μg/ml of mRNA (translation template) encoding Green Fluorescent Protein (GFP), and carried out at 20°C without Cellular protein synthesis.

The synthesis reaction of the comparative experiment is carried out by using a concentration vessel (separation molecular weight 30,000KDa) with an ultrafiltration membrane with a cut-off molecular weight of 30,000. The temporal change of the amount of protein produced with fluorescence activity as an indicator exhibits a typical hyperbolic shape in the conventional batch method. That is, the reaction rate decreased after the maximum rate at the initial stage of the reaction, and the amount of synthesized protein did not increase after the reaction was nearly stopped for 3 hours even after a long time of incubation (○-○). This result is quite consistent with the reports so far (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 559-556). The reason for the cessation of this reaction has not been fully elucidated. It is mainly considered that the concentration of ATP and GTP as energy sources decreases (energy depletion) and the accumulation of non-renewable by-products AMP and GMP inhibits protein synthesis through a certain mechanism.

Synthetic reaction of the present invention is carried out in the container that ultrafiltration membrane is housed, and reaction speed is higher, before reaction stops, is equivalent to after 2 hours of reaction beginning, adds the protein synthesis matrix solution (containing matrix and energy of 3 times of capacity of reaction solution volume) source) and stir (dilution treatment). Then, the solution diluted with this substrate solution was concentrated by centrifugation to the volume of the reaction solution before dilution (at the time indicated by the arrow) (concentration treatment). By discontinuously repeating this dilution and concentration series of treatments several times, the low-molecular substances containing by-products are eliminated through the ultrafiltration membrane, and their concentration is reduced. On the other hand, the ATP, GTP, and amino acids consumed by the synthesis reaction are separated Return to the respective concentrations close to the start of the reaction. Through this concentration process, ribosomes, tRNA and all other translation factors derived from wheat germ were concentrated to the original concentration at the beginning of the reaction. It was confirmed that by this treatment, once the low protein synthesis reaction started again, the initial high rate was maintained, and then the reaction decreased again (●-●). By repeating the dilution and concentration treatments discontinuously, high-efficiency cell-free protein synthesis can be achieved by utilizing the characteristics of the time zone where the initial reaction rate is high. As shown in Figure 1, the synthetic amount of 0.072 mg synthesized in 1 ml reaction capacity in the GFP batch method can be increased to 0.41 mg by repeated dilution and concentration treatment 4 times. Here, the amount of synthesized protein was calculated based on the fluorescence intensity measurement technique of GFP (the maximum fluorescence wavelength is 508 nm), and the fluorescence intensity of 1 μg/ml of GFP was calculated as 2.0.

Sufficient synthesis efficiency was achieved by this treatment, but the rate of protein synthesis in the system gradually decreased as the dilution and concentration treatments were repeated. The reason for this is presumed to be that the germ-derived translation factor (group) leaks into the filtrate or adsorbs on the ultrafiltration membrane to cause loss inside the reaction system, decomposes mRNA, or decreases the concentration due to adsorption on the ultrafiltration membrane.

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. The vertical axis is the amount of protein synthesized (mg/ml), and the horizontal axis shows the reaction time (hours). In FIG. 2 , the lines connecting small black circles (·-·) show the experimental results when the concentration of the wheat germ extract was 60A260nm/ml and the concentration of GFPmRNA was 480μg/ml (Example 2). The big black circle lines (●-●) show the experimental results when the concentration of wheat germ extract is 80A260nm/ml and the concentration of GFPmRNA is 640μg/ml (Example 3). And in FIG. 2 , the arrows indicate the timing at which the above-mentioned operations are performed.

As shown in Figure 2, in Example 2, through 4 times of dilution and concentrated discontinuous repeated operations, the protein synthesized by the cell-free system was 0.62 mg per 1 ml of reaction solution, and in Example 3 through 4 times of dilution and The protein synthesized by the concentrated discontinuous repeated operation cell-free system was 1.13 mg per 1 ml of reaction liquid. In this way, the higher the concentration of translation factors in the reaction system, the higher the amount of protein synthesis obtained by the cell-free protein synthesis method of the present invention, which is significantly higher than the experimental results of the previous batch method shown in FIG. 1 .

Experimental example 3

In the usual batch method, the time for the reduction of the synthesis rate depends on the reduction of the substrate concentration in the reaction solution and the accumulation concentration of by-products, which is inversely proportional to the concentration of the translation factor in the system. That is, the higher the translation factor concentration (wheat germ extract concentration) of the system, the shorter the time to stop the reaction (Madin K. et al., Proc. Natl. Acad. Sci. USA (2000), 97, 556-559) . Therefore, in the case of using high-concentration translation factors, it is considered that by shortening the time interval between discontinuous repeated operations of dilution and concentration, a high synthesis rate can be maintained, and thus the production efficiency of protein per unit time can be expected to increase.

Fig. 3A shows that when the concentration of the wheat germ extract is 80A260nm/ml and the concentration of GFPmRNA is 640 μg/ml, the cell-free protein synthesis is carried out in the same manner as in Example 1, except that the interval of the discontinuous repeated operation of dilution and concentration is changed. Figure 3B is the result of polyacrylamide gel electrophoresis showing the difference in the amount of GFP synthesized due to the difference in the interval of the discrete repeated operation of dilution and concentration.

In the graph of FIG. 3A , the vertical axis is the amount of synthesized protein (mg/ml), and the horizontal axis is the reaction time (hour). Arrows indicate when the discrete repetition of dilution and concentration was performed. Furthermore, in FIG. 3A, the lines (●-●) connecting black circles show the experimental results when the above-mentioned dilution and concentration were repeated discontinuously at intervals of 0.5 hours from the start of the reaction (Example 4), and the lines connecting white circles (○-○) shows the experimental results when the above-mentioned dilution and concentration were repeated discontinuously at intervals of 1 hour from the start of the reaction (Example 5). In FIG. 3A , arrows indicate the timing at which the above-mentioned discontinuous repetition of dilution and concentration is performed.

Figure 3B shows Example 4, which performs the above-mentioned discontinuous repetition of dilution and concentration at intervals of 0.5 hours, and performs the same operation as in Example 4 except that the above-mentioned discontinuous repetition of dilution and concentration is performed at intervals of 2 hours. When (embodiment 6) compares. Here, the arrow in Fig. 3B shows the stained band of GFP. Polyacrylamide gel electrophoresis at each reaction time (in embodiment 4, pass through the moment of 0,0.5,1,1.5,2,2.5,3,3.5 hours from the beginning of reaction; in embodiment 6, pass through from the beginning of reaction 0, 2, 4, 6, 8, 10, 12, and 14 hours) of the reaction solution 1 μl was separated by polyacrylamide unmodified gel electrophoresis, and the protein was stained with Coomassie brilliant blue.

As shown in Figure 3A and B, Example 4 in which the above-mentioned discontinuous repeated operations of dilution and concentration were performed at intervals of 0.5 hours, and Example 5 in which the above-mentioned discontinuous repeated operations of dilution and concentration were performed at intervals of 1 hour , The output per unit time is higher than that of Example 6 in which the above-mentioned dilution and concentration are repeated discontinuously at intervals of 2 hours. In Example 4, the synthesis amount of 1.12 mg can be obtained per 1 ml of the reaction in 2.5 hours, but this is more than the reaction liquid of the same concentration, which is repeated discontinuously by diluting and concentrating at intervals of 2 hours, and allowed to react for 10 hours. The yield obtained is high. This result directly shows that when the concentration of translation factors in the reaction system (concentration of wheat germ extract) is higher, the consumption concentration of energy sources and the like becomes lower in a short period of time, and at the same time, the accumulation concentration of by-products becomes higher, and the reaction speed decreases and reaches The time to stop the reaction is shortened. Therefore, the maximum synthetic yield can be obtained by selecting the optimum timing depending on the concentration of the wheat germ extract contained in the reaction system in setting the timing of the discontinuous repeated operations of dilution and concentration.

Instability (high reactivity) of protein is mentioned as its general physical property. Therefore, in order to obtain high-quality protein, it becomes one of the necessary conditions to construct a technique to increase the synthesis yield in a short time. It can be said that the principle of the present invention demonstrated in FIG. 3 is extremely effective.

Experimental example 4

Fig. 5 is a graph showing the experimental results of protein synthesis (Example 7) using the cell-free protein synthesis method of the present invention using the structure shown in Fig. 4 using a filter membrane and a liquid delivery pump to remove by-products. The vertical axis is the amount of protein synthesized (mg/ml), and the horizontal axis shows the reaction time (hours). In FIG. 5 , the lines (●-●) connecting black circles show the experimental results of Example 7, and the lines (○-○) connecting white circles are the experimental results by the conventional batch method. In FIG. 5 , the arrows indicate the timing at which the above-mentioned discontinuous repetition of dilution and concentration is performed.

In Example 7, a cross-flow filter (VF05C2, manufactured by Sartorius, cut molecular weight: 30,000) was used as the filter concentrator, and LKB-Pump-P1 (Pharmacia) was used as the liquid delivery pump. Except containing wheat germ extract at 80A260nm/ml concentration and GFPmRNA at 640μg/ml concentration, using the same translation reaction solution as in Example 1, diluting and concentrating at 0.5 hour intervals were repeated discontinuously. A graduated plastic tube (Falcon tube) was used as the reaction vessel, and the reaction capacity was 15 ml. Dilution during the discontinuous repeated operation of dilution and concentration was performed using a solution for translation reaction with a capacity of 45 ml.

As shown in FIG. 5, when a structure having a filter membrane and a liquid-feeding pump is used as a mechanism for removing byproducts, it has been confirmed that the cell-free solution of the present invention can well utilized in protein synthesis methods. In Example 7, 2.1 mg per 1 ml of reaction capacity can be synthesized through a 4-hour reaction, which can synthesize a protein with a yield about 2 times higher than that of the above-mentioned device using a centrifuge and an ultrafiltration membrane.

Experimental example 5

Fig. 6A is to show except adopting the mRNA of the wheat germ extract of 60A260nm/ml, the dihydrofolate reductase (DHFR) of 450 μ g/ml as translation template, use the same translation reaction solution as Example 1; It is a schematic diagram of the experimental results when the same cell-free protein synthesis as in Example 1 was performed (Example 8) except for the discontinuous repetition of dilution and concentration. As a comparative experiment, cell-free protein synthesis was performed under the same conditions except for the conventional batch method (discontinuous repeated operation without dilution and concentration). In Example 8, for the synthesis of a cell-free system of DHFR with a molecular weight of about 20,000 KDa, a reaction vessel equipped with an ultrafiltration membrane with a cut-off molecular weight of 30,000 Da (manufactured by Millipore Corporation) was used.

In Fig. 6A, swimming lanes 1, 2, and 3 are the discharge solution (filtrate) after the discontinuous repeated operation reaction of embodiment 8 by 0, 1, and 2 times of dilution and concentration respectively. Continuously repeat the operation at the same time using 1 μl of the reaction solution of the previous batch method for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), showing the results of Coomassie brilliant blue staining (the arrow in the figure shows the synthetic product DHFR). Judging from the staining intensity of the DHFR band indicated by the arrow in Fig. 6A, in the experimental results of the batch method in the past ("reaction solution" on the left side of each lane), only a trace amount of DHFR synthesis was confirmed after 1 hour of reaction , and in the experimental results of Example 8 ("filtrate" on the right side of each swimming lane), through the discontinuous repeated operation of 1, 2 dilutions and concentration, it can be confirmed that the DHFR generated by the reaction system is efficiently expressed in the filtrate .

Example 6

Fig. 6B is an electrophoresis graph showing the experimental results of synthesizing a GFP fusion protein fused with streptavidin at the N-terminus (Example 9) and separating the GFP fusion protein out of the system using the cell-free protein synthesis method of the present invention.

The cell-free protein synthesis itself in this experiment was carried out in the same manner as in Example 1, except that the mRNA encoding the GFP fusion protein was used as a translation template, except that dilution and concentration were performed 4 times at intervals of 1 hour. The operation of isolating the GFP fusion protein out of the system is to add biotin-immobilized magnetic beads to the reaction solution according to 1, and use the affinity between biotin and streptavidin to select the resulting fusion product. 2. The magnetic beads bound to the fusion protein are selectively taken out of the reaction vessel by a magnet. In addition, electrophoresis and staining were carried out in the same manner as in Experimental Example 5.

As shown in Figure 6B, the products that could be detected in the reaction solution before the separation operation disappeared from the reaction solution after this operation (the arrow of the swimming lane of the reaction solution after separation), and it can be seen that the magnetic beads can be effectively separated out of the system ( The synthesized product is shown in the isolated product lane).

Experimental example 7

Fig. 7 is a graph showing the difference between the experimental results of the RNA synthesis method of the present invention (Example 10) and the conventional in vitro RNA synthesis method of the batch method. In FIG. 7 , the left side of FIG. 7 shows the results of the past method of the batch method, and the right side of FIG. 7 shows the results of the RNA synthesis method of the present invention, and arrows show mRNA products encoding GFP.

The GFP genes of jellyfish were respectively inserted into the plasmid vector pEU dedicated to the wheat germ cell-free system as a transcription template, and the transcription reaction solution was prepared [the final concentration was 80mM HEPES-KOH pH7.8, 16mM magnesium acetate, 10mM dithiothreitol, 2mM Spermidine, 2.5mM 4NTPs (4 nucleotide triphosphates), 0.8U/μl RNase inhibitor, 0.1μg/μl DNA [plasmid GFP (Green Fluorescent Protein)], 1.6U/μl SP6 RNA polymerase], react at 37°C 3 hours.

In the RNA synthesis reaction of Example 10, 3 hours after the start of the reaction, 3 times the amount of the above-mentioned transcription reaction solution was added to the transcription reaction solution for dilution, and the low molecules were excluded from the reaction solution system by using a centrifuge through a filter membrane. , Concentrate the transcription reaction solution to the original concentration. Thereafter, the operation of diluting and concentrating was repeated discontinuously at intervals of 3 hours to proceed the synthesis reaction. When each dilution and concentration were repeated discretely, the transcription reaction solution was aspirated, stained with ethidium bromide, and the transcription product (mRNA) separated by agarose gel electrophoresis was visualized.

As shown in Fig. 7, in the conventional in vitro RNA synthesis method of the batch method, the RNA synthesis reaction was stopped in about 3 hours, but the above-mentioned dilution and concentration operations were repeated discontinuously after 3 hours of reaction at a high synthesis rate. In Example 10, it was possible to synthesize mRNA at a yield 2.4 times that of the conventional method.

Experimental example 8

Figure 8 is a graph showing the experimental results of the cell-free protein synthesis method of the present invention using ethanol precipitation purified mRNA and unpurified mRNA as translation templates, the vertical axis is the amount of protein synthesized (mg/ml), and the horizontal axis shows Reaction time (hours). Purified mRNA was prepared according to the method described in Example (Cell-free protein synthesis method) 1) Reaction from DNA to mRNA (transcription reaction). After the unpurified mRNA was prepared by the method described in Example 10, it was centrifuged at 12,000 rpm for 1 hour, and the supernatant was collected for preparation. In Figure 8, the lines (●-●) connecting the black circles show 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 mixture was added The same amount of dialysis buffer [35mM HEPES-KOH pH7.8, 103mM potassium acetate (KOAc), 3.1mM magnesium acetate (Mg(0Ac)2), 16mM creatine phosphate, 1.2mM ATP, 0.26mM GTP, 2.5mM disulfide Threitol (DTT), 0.43mM spermidine, 0.3mM AAs (a mixture of 20 kinds of L-type amino acids)] diluted, then concentrated to the capacity of the original mixture, added the same amount of dialysis buffer again, and repeated the same The operation, the experimental results when creatine kinase was added after concentration to make the final concentration about 1 mg/ml for protein synthesis (Example 11). The lines connecting the white circles (○-○) show the experimental results when the protein synthesis reaction (Example 12) was performed using the translation reaction solution prepared from the mRNA synthesized in Experimental Example 7 by ethanol precipitation and purification. . In Examples 11 and 12, it was carried out in the same manner as in Example 4, except that the above-mentioned mRNA was used at a concentration of 480 μg/ml, and the wheat germ extract at a concentration of 60A260 nm/ml was used.

As shown in FIG. 8 , when comparing the protein synthesis rates of Examples 11 and 12, no significant difference was observed. From this result, it can be seen that by the cell-free protein synthesis method of the present invention, a simple cell-free protein synthesis method can be realized by using a transcription reaction solution after transcription of unpurified mRNA to prepare a translation reaction solution.

Experimental example 9

9 is a schematic diagram showing the experimental results of the cell-free protein synthesis method of the present invention using the transcription reaction solution after the transcription reaction of Experimental Example 7 to prepare the translation reaction solution. The vertical axis is the amount of synthesized protein (mg/ml) , and the horizontal axis shows the reaction time (hours). In FIG. 9 , upward arrows indicate the timing at which the dilution and concentration operations are repeated in a discrete manner. The downward arrow indicates the time when the transcription reaction solution (including mRNA) after the transcription reaction was added.

The translation reaction solution is to add the 80A260nm/ml wheat germ extract and the transcription reaction solution (containing GFPmRNA 9.6 mg) after the transcription reaction to 15 ml of the same translation reaction solution as in Example 1 (the final concentration of GFPmRNA is 640 μg/ml) , after concentrating the solution to a liquid volume of 8 ml, the translation reaction solution was prepared by diluting the solution for translation reaction described above and the subsequent concentration operation, and the translation reaction was started. As a mechanism for removing by-products, the structure with a filter membrane and a liquid delivery pump is used in the same manner as shown in Figure 4, and GFPmRNA (translation template) is added every 4 hours from the start of the reaction, and then the matrix and energy source are passed through at intervals of 30 minutes. Repeated discontinuous dilution and concentration of the solution.

As shown by the experimental results, by adding mRNA, it was confirmed that the translation reaction, which was almost stopped, started again. Furthermore, it was found that protein synthesis can be sustained for a long time by combining this dilution-concentration method with additional addition of mRNA.

Embodiment of the device

Embodiments of the automatic synthesis apparatus of the present invention will be described below. However, the automatic synthesis apparatus of the present invention is not limited to the apparatus of the examples described below as long as it can automatically implement a high-throughput synthesis system.

In order to implement the automatic synthesis device of the present invention, the following items were assembled in the automatic synthesis device, and GFP protein synthesis was performed using the automatic synthesis device.

transcription template

Using pEU plasmid vector incorporated with GFP, sense primers and antisense primers, PCR was carried out in a 30 μl system, and the obtained DNA was used as a transcription template. The DNA was packed in a 96-well PCR plate (plate for template ①).

Transcription reaction solution

Prepare 4.95ml of a solution containing a final concentration of 80mM HEPES-KOH, 16mM magnesium acetate, 2mM spermidine, 10mMDTT, 3mM NTPs, 1U/μl SP6 RNA polymerase, 1U/μl RNasin, and put it into the reagent tank 1 (②) in the device.

Solution for translation reaction

Prepared with final concentrations of 30mM HEPES-KOH (pH7.8), 1.2mM ATP, 0.25mM GTP, 16mM creatine phosphate, 2mM dithiothreitol, 0.3mM spermidine, 0.3mM 20 kinds of amino acids, 2.7mM magnesium acetate , 100mM potassium acetate, 0.005% sodium azide, 40ng/μl creatine kinase, 5.5ml of a solution of wheat germ extract with an optical density (OD) at 260nm of 80 or 40 units (unit), the reagents in the device Inside slot 2(③). In addition, protein synthesis was performed using a solution for translation reaction containing 80 units or 40 units of the wheat germ extract, respectively.

dilute solution

Prepared with final concentrations of 30mM HEPES-KOH (pH7.8), 1.2mM ATP, 0.25mMGTP, 16mM creatine phosphate, 2mM dithiothreitol, 0.3mM spermidine, 0.3mM 20 kinds of amino acids, 2.7mM magnesium acetate , 100 ml of a solution of 100 mM potassium acetate and 0.005% sodium azide, and put it into the reagent tank 3 (④) in the device.

synthesis container

、隔板(离心时平衡用)接口管300μl接口管(转录反应用溶液用、翻译反应用溶液用、稀释溶液用：⑤)(280支)、20μl接口管(模板产物用：⑥)(96支)Plate for template (PCR 96-well (vertical 8 x horizontal 12) plate with lid (plate containing template DNA): ①), plate for receiving transcription and filtrate (96-well (vertical 8 x horizontal 12) titer plate with lid: ⑩ ), plate for translation (PCR 96-well (vertical 8 x horizontal 12) plate with cover (the bottom of the well is a filter):

, Separator (for balance during centrifugation) mouthpiece 300μl mouthpiece (for transcription reaction solution, translation reaction solution, dilution solution: ⑤) (280 pieces), 20μl mouthpiece (for template product: ⑥) (96 branch)

Dispensing machine 1: 8 suction tubes (for connecting 300μl interface tube: ⑧)

Dispensing machine 2: 8 suction tubes (for connecting 20μl interface tube: ⑨)

GFP protein synthesis was performed using the synthesis apparatus of the present invention in the following steps.

<Process 1: Transcription>

(1) The robotic arm (⑦) opens the cover of the plate (⑩) for receiving transcription and filtrate.

(2) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧), and move to the reagent tank 1 (②) containing the transcription reaction solution.

(3) The dispensing machine 1 (⑧) draws 95 µl of the transcription reaction solution from the reagent tank 1 (②) (45 µl x 2 times + 5 µl).

(4) The dispensing machine 1 (⑧) moves to the position (⑩) for receiving the plate for transcription and filtrate, and discharges 45 μl of the transcription reaction solution twice into each vertical well (8 holes) of the plate.

(5) Steps (2)-(4) were further carried out in each of the wells in the 7 vertical rows to which the transcription reaction solution had not been added.

)的位置，废弃接口管。(6) The dispensing machine 1 (⑧) moves to the discard port of the mouthpiece (

), discard the mouthpiece.

(7) The mechanical arm (⑦) opens the cover of the template plate (①).

(8) Install the 20μl mouthpiece (⑥) on the dispensing machine 2 (⑨).

(9) Each mouthpiece of the dispensing machine 2 (⑨) sucks 5 μl of air (implemented to improve suction efficiency).

(10) Move the dispensing machine 2 (⑨) to the position of the template plate (①), and suck out 5 μl of the template sample from each vertical well (8) of the template plate (①).

(11) Move the dispensing machine 2 (⑨) to the position (⑩) for receiving the plate for transcription and filtrate, and discharge the template sample in full amount into each vertical well (8 holes) of the plate.

(12) Pass each mouthpiece of the dispensing machine 2 (⑨) and mix with a pipette each vertical well (8) of the plate for receiving transcription and filtrate (⑩).

(13) Move the dispensing machine 2 (⑨) to the discard port of the mouthpiece ( ), discard the mouthpiece.

(14) The steps (8)-(13) are further carried out in the wells in the vertical 7 rows to which the template sample has not been added.

(15) The manipulator (⑦) covers the template plate (①) and the transcription and filtrate receiving plate (⑩).

)中安装。(16) The robot arm (⑦) transports the plate for receiving transcription and filtrate (⑩) to the constant temperature tank 1 (

) installed.

)中，进行37℃4小时的转录反应。(17) in the constant temperature bath 1 (

), a transcription reaction was performed at 37° C. for 4 hours.

<Step 2: Precipitation removal after transcription>

)运送至MTP操作台(

)。(1) The robotic arm (⑦) receives the plate for transcription and filtrate (⑩) from the thermostat 1 (

) to the MTP console (

).

(2) The robotic arm (⑦) opens the cover of the plate (⑩) for receiving transcription and filtrate.

(3) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(4) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

(5) Move the dispensing machine 1(⑧) to the reagent tank 3(④) where the dilute solution is added, and suck the dilute solution (the liquid volume after concentrating and centrifuging changes due to the columns on the board, so the suction volume is in each column Make fine adjustments between 80 μl and 120 μl.).

(6) Move the dispensing machine 1 (⑧) to the position for receiving the plate for transcription and filtrate (⑩), and discharge the whole amount of the diluted solution into each vertical well (8 holes) of the plate.

(7) The steps (4)-(6) are further carried out in each of the wells in the 7 rows in the vertical direction to which the dilution solution has not been added.

)位置，废弃接口管。(8) The dispensing machine 1 (⑧) moves to the discard port of the mouthpiece (

) position, discard the mouthpiece.

(9) Mechanical arm (⑦) is covered with translation plate ( ) overlay on the plate (⑩) for transferring and receiving transcription and filtrate (to balance with the separator during centrifugation).

)以及接受转录·滤液用板(⑩)运送至升降台()上。(10) The mechanical arm (⑦) transfers the overlapping translation plate (

) and the plate for receiving transcription and filtrate (⑩) is transported to the lifting platform ( )superior.

)以及接受转录·滤液用板(⑩)的升降台下降至与离心机(

)的高度吻合。(11) Place the overlapping translation plates (

) and the lifting platform for receiving transcription and filtrate plates (⑩) descends to the centrifuge (

) are highly consistent.

(12) The mechanical arm (⑦) opens the centrifuge ( ) door.

(13) Centrifuge ( ), in order to allow the plate to be installed, adjust the position of the plate setting part of the centrifuge.

(14) The mechanical arm (⑦) will overlap the translation plate ( ) and the plate for receiving transcription and filtrate (⑩) is transported to the centrifuge for placement.

(15) Adjust the position in the same way as (13), and install the separator on the centrifuge for balance.

)上，进行3100g、15分钟的离心。(16) in the centrifuge (

) was centrifuged at 3100 g for 15 minutes.

<Step 3: The first exchange of the buffer of the transcription solution>

(1) Centrifuge ( ) stops, the mechanical arm opens the door of the centrifuge), and then the centrifuge performs alignment (the position of the mechanical arm coincides with the position of the plate setting part of the centrifuge).

(2) The robotic arm (⑦) transfers the overlapping translation plate ( ) and plate for receiving transcription filtrate (⑩) from the centrifuge ( ) to the lifting platform.

(3) Perform the same alignment as (1), and the mechanical arm (⑦) transports the partition to the lifting platform.

)，分开安装翻译用板()和接受转录·滤液用板(⑩)。(4) The robotic arm (⑦) transfers the overlapping translation plate ( ) and the plate for receiving transcription and filtrate (⑩) is transported to the MTP operation station (

), install the translation board separately ( ) and a plate for receiving transcription and filtrate (⑩).

(5) Robotic arm (⑦) opens translation plate ( ) cover.

(6) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(7) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

(8) The dispensing machine 1 (⑧) moves to the position for receiving the plate for transcription and filtrate (⑩), and sucks all the solution from each vertical well (8) of the plate for receiving transcription and filtrate (⑩).

)的纵向各孔(8个)内吐出全部溶液。(9) Move the dispensing machine 1 (⑧) to the translation board ( ) to the translation board (

) in the longitudinal holes (8) to spit out all the solutions.

(10) The steps (6)-(9) are further carried out in each of the other 7 longitudinal rows of holes.

(11) Dispensing machine 1 (⑧) covers the plate for translation ( ) cover.

(12) Dispensing machine 1 (⑧) will translate the plate ( ) is superimposed on the plate for receiving transcription and filtrate (⑩) (top: plate for translation; bottom: plate for receiving transcription and filtrate).

(13) Centrifuge in the same manner as steps (10) to (16).

<Process 4: The second exchange of transcription buffer>

As in steps 3 (1) to (5), take out the plate for receiving transcription/filtrate and the plate for translation from the centrifuge, install it on the MTP console, and open the cover of the plate for translation.

(2) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(3) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

(4) Move the dispensing machine 1 (⑧) to the reagent tank 3 (④) filled with the diluted solution.

(5) Each hole of the dispensing machine 1 (⑧) sucks the diluted solution (the amount of suction is finely adjusted between 80 μl and 120 μl for each column.).

)的位置，向翻译板()的纵向各孔(8个)内吐出全部溶液。(6) Dispensing machine 1 (⑧) moves to translation plate (

) to the translation board ( ) in the longitudinal holes (8) to spit out all the solutions.

(7) Steps (3)-(6) are further carried out in each hole of the other 7 longitudinal rows.

)的位置，废弃接口管。(8) The dispensing machine 1 (⑧) moves to the discard port of the mouthpiece (

), discard the mouthpiece.

(9) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(10) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

)的位置。(11) The dispensing machine 1 (⑧) moves to the plate for translation (

)s position.

(12) Mix the translation plate ( ) longitudinal holes (8).

(13) Steps (8)-(12) are further carried out in each of the other 7 longitudinal rows of holes (in some cases, mouthpieces are exchanged in each hole).

(14) The dispensing machine 1 (⑧) moves to the discard port of the mouthpiece ( ), discard the mouthpiece.

(15) The mechanical arm (⑦) covers the translation board ( ) cover.

(16) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(17) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

(18) The dispensing machine 1 (⑧) moves to the position (⑩) for receiving the plate for transcription and filtrate.

(19) The dispensing machine 1 (⑧) sucks the entire solution from each vertical well (8) of the plate for receiving transcription and filtrate (⑩).

)位置。(20) The dispensing machine 1 (⑧) moves to the waste liquid port (

)Location.

)吐出全量。(21) Dispensing machine 1 (⑧) to the waste liquid port (

) spit out the full amount.

(22) The steps (17)-(21) are further carried out in each of the other 7 longitudinal rows of holes.

(23) The dispensing machine 1 (⑨) moves to the discard port of the mouthpiece ( ) position, discard the mouthpiece.

(24) Carry out translation plate ( ) centrifugal.

<Step 5: Dispense the solution for translation reaction>

)的盖子。(1) Same as step 3 (1) to (5), take out the plate for receiving transcription and filtrate and the plate for translation from the centrifuge, install it on the MTP console, and open the plate for translation (

) cover.

(2) Install the 300 μl mouthpiece (⑤) on the dispensing machine 1 (⑧), and each mouthpiece sucks 5 μl of air.

(3) The dispensing machine 1 (⑧) moves to the reagent tank 2 (③) containing the solution for translation reaction.

(4) Dispenser 1 (⑧) draws 50 μl of translation reaction solution from reagent tank 2 (③).

)的纵向各孔(8个)中吐出全部溶液。(5) Dispensing machine 1 (⑧) moves to translation plate ( ) to the translation board (

) in the longitudinal wells (8) spit out all the solution.

(6) Mix the plate for translation ( ) longitudinal holes (8).

(7) The steps (2)-(6) are further carried out in each of the other 7 longitudinal rows of holes.

(8) The dispensing machine 1 (⑨) moves to the discard port of the mouthpiece ( ) position, discard the mouthpiece.

(9) Mechanical arm (⑦) is covered with translation plate ( ) cover.

)。(10) The mechanical arm (⑦) will translate the plate ( ) to the constant temperature bath 1 (

).

)在26℃保温1小时。(11) Put the translation plate (

) at 26°C for 1 hour.

(12) During the heat preservation process of (11) (the process of discarding the filtrate thereafter), the dispensing machine 1 (⑧) is equipped with 300 μl mouthpieces (⑤), and each mouthpiece sucks 5 μl of air.

(13) The dispensing machine 1 (⑧) moves to the position (⑩) for receiving the plate for transcription and filtrate.

(14) The dispensing machine 1 (⑧) sucks the entire solution from each vertical well (8) of the plate for receiving transcription and filtrate (⑩).

(15) The dispensing machine 1 (⑧) moves to the waste liquid port ( ) position, spit out the full amount.

(16) The steps (12) to (15) are further carried out in each of the other 7 longitudinal rows of holes.

)位置，废弃接口管。(17) The dispensing machine 1 (⑨) moves to the discard port of the mouthpiece (

) position, discard the mouthpiece.

<Process 6: Repeat translation>

Repeat the following procedure 6 times.

)运送至MTP操作台(

)。(1) The robot arm (⑦) will translate the plate ( ) from the constant temperature bath 1 (

) to the MTP console (

).

)的盖子。(2) The mechanical arm (⑦) opens the translation plate (

) cover.

(3) Install the 300μl mouthpiece (⑤) on the dispensing machine 1 (⑧).

(4) Each mouthpiece of the dispensing machine 1 (⑧) sucks 5 μl of air.

(5) Move the dispensing machine 1 (⑧) to the reagent tank 3 (④) where the diluted solution is added, and suck the diluted solution (the amount of liquid after centrifugation changes due to the columns on the plate, so the suction volume for each column is 80μl～ Make fine adjustments between 120μl.).

(6) The dispensing machine 1 (⑧) moves to the plate for translation ( ), and spit out all the diluted solutions into each vertical well (8) of the plate.

(7) Steps (4)-(6) were further carried out in each of the 7 longitudinal rows of wells to which the dilution solution had not been added.

(8) Perform centrifugation in the same manner as in steps 2 (9) to (16) (centrifugation time: 18 minutes).

(9) In the same manner as in steps 3 (1) to (5), the plate for receiving transcription/filtrate and the plate for translation are taken out from the centrifuge, installed on the MTP console, and the cover of the plate for translation is opened.

(10) Install the 300 μl mouthpiece (⑤) on the dispensing machine 1 (⑧), and each mouthpiece sucks 5 μl of air.

(11) The dispensing machine 1 (⑧) moves to the plate for translation ( )s position.

)的纵向各孔(8个)。(12) Mix the translation plate (

) longitudinal holes (8).

(13) The dispensing machine 1 (⑨) moves to the discard port of the mouthpiece ( ), discard the mouthpiece (there is also a case of replacing the mouthpiece).

(14) The steps of (10)-(13) are further carried out in each of the other 7 longitudinal rows of holes.

(15) The mechanical arm (⑦) covers the translation board ( ) cover.

(16) The mechanical arm (⑦) will translate the plate ( ) to the constant temperature bath 1 ( ).

)在26℃保温1小时。(17) plate for translation (

) at 26°C for 1 hour.

(18) During the heat preservation process of (17) (the process of discarding the filtrate later), the dispensing machine 1 (⑧) is equipped with a 300 μl mouthpiece (⑤) to suck 5 μl of air.

(19) The dispensing machine 1 (⑧) moves to the position for receiving the plate for transcription and filtrate (⑩).

(20) The dispensing machine 1 (⑧) sucks out all the solutions in the vertical wells (8) of the plate for receiving transcription and filtrate (⑩).

)的位置。(21) The dispensing machine 1 (⑧) moves to the waste liquid port (

)s position.

(22) The dispensing machine 1 (⑧) spits out all the solutions.

(23) The steps (18) to (22) are further carried out in each of the other 7 vertical rows of holes.

<Process 7: End (after repeating process 6 six times)>

(1) After completion of the heat preservation in step 6 (18), the temperature of the thermostat was set to 4°C.

(2) Take out the plate for translation from the synthesizer.

For the GFP synthesized by the above procedure, according to the report of Madin K. et al. et al. (Madin K. et al., (2000) Proc. Natl. Acad. Sci. USA 97, 559-564) described Methods The amount of synthesized protein was measured and analyzed by SDS-PAGE ( FIG. 11 ).

In FIG. 11 , in lanes 1-8 of 40 units and lanes 1-4 of 80 units, proteins were synthesized at the position of the GFP band. Furthermore, when the band concentration of BSA (125, 250, 500 ng) was compared with the concentration of the GFP band in each lane, it can be estimated that at least 250 ng of GFP was synthesized in all the lanes.

As can be clearly seen from the above description, the high-throughput synthesis system of the present invention formed by the discontinuous operation of repeated dilution and concentration and the device for automatically performing the system can achieve a high-throughput reaction in a short time by selecting a relatively high reaction initial time. High-quality proteins can be efficiently synthesized within the system, and the target protein can be separated extremely effectively. In addition, it has been clarified that this principle is also useful for the efficient synthesis of RNA in vitro.

Furthermore, protein and RNA synthesis methods using this principle have been shown to be able to solve various disadvantages seen in the continuous method of Spirin et al., that is, the complexity of the device, the low strength of the membrane, the clogging of the membrane during operation, and the complexity of the operation. Problems in the reaction device caused by etc. Coupled with the disadvantage that the continuous method requires a particularly long synthesis reaction time, it not only wastes time, but also leaves a major problem that must be solved in terms of ensuring the quality of the produced protein, and this disadvantage has not been solved in the overlapping method.

Possibility of industrial use

The technology invented here can provide the basic backbone technology for protein research in the post-genome era. In particular, for the analysis of the structure and function of RNA and proteins, it can be said that it is indispensable as a key technology that can be easily and efficiently comprehensively prepared and mass-produced.

Claims (3)

1. A protein synthesis method, which uses a translation template as a raw material, and is characterized in that it comprises the following steps: 1) Bringing the translation template, amino acid, and a solution for translation reaction including a ribosome-containing wheat germ cell extract into contact with each other, and introducing them into the synthesis reaction, 2) Dilute the translation reaction solution before and after the synthesis rate is slightly reduced, before and after the synthesis reaction is about to stop, or during these states, 3) Concentration treatment is performed after dilution treatment, 4) carry out the synthesis reaction through the concentrated reaction system, 5) Repeat the process of 2) to 4) multiple times; or 1) Bringing the translation template, amino acid, and a solution for translation reaction including a ribosome-containing wheat germ cell extract into contact with each other, and introducing them into the synthesis reaction, 2) Concentrating the solution for translation reaction before and after the synthesis rate is slightly reduced, before and after the synthesis reaction is about to stop, or during these states, 3) Dilution treatment is performed after concentration treatment, 4) Carry out the synthesis reaction through the diluted reaction system, 5) Repeat the process of 2) to 4) multiple times; The translation reaction solution contains amino acids and ribosomes, the concentration is concentrated to 1/5-2/3 volume by filtration or suction pump treatment, and the dilution is 1-20 times by adding an aqueous solution. 2. The method according to claim 1, characterized in that, the by-products are removed outside the reaction system by concentration treatment. 3. The method according to claim 1, characterized in that amino acids, ATP and/or GTP and translation templates are added to the reaction system through dilution treatment.

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