JPWO2004070047A1 - Automatic protein synthesis method and apparatus for performing the same - Google Patents

Abstract

Provided are a method for automatically performing a series of steps from a transcription template to production of a protein encoded by the template, and an automatic protein synthesizer capable of executing the method. Specifically, in the cell protein synthesis, a reaction is automatically performed until a protein encoded by a transcription template is generated.

Description

  This application claims the priority from Japanese Patent Application No. 2003-033009, which is incorporated herein by reference.

  The present invention relates to a method for automatically performing cell-free protein synthesis and an automatic protein synthesizer therefor.

Today, with the rapid progress of genomics and the completion of genome projects for various organisms, the focus of research has shifted greatly from gene structure analysis to gene function analysis. Transgenic technologies, including knockout, have produced tremendous results in the production of mutants to which the target function has been added by introducing or destroying genes whose functions have already been elucidated. Is currently limited to data interpretation based on the phenotype obtained through the black box of cells. This is apparent from the fact that only a few genes have been identified for function by these techniques. On the other hand, genes whose functions have been elucidated are accompanied by biochemical support. Therefore, it can be said that biochemical analysis of gene products is essential for functional analysis of unknown genes obtained from genomic information.
Genetic engineering techniques for expressing cloned genes in living cells are widely used for protein synthesis, but foreign proteins that can be obtained by this method are limited to molecular species that can evade the life-sustaining mechanism of the host. It will be. On the other hand, with the progress of organic synthesis technology, automatic peptide synthesizers have become widespread, and it has become routine to synthesize peptides consisting of tens of amino acids, but chemically synthesizing proteins with larger molecular weights Even now, it is extremely difficult due to limitations of yield and side reactions. Furthermore, in Europe and the United States, there are strong ethical criticisms regarding protein production and novel molecular search methods that use conventional living organisms as they are, and there is a concern that international regulations will become even more strict.
As a new protein synthesis method that overcomes such problems, there can be mentioned a cell-free protein synthesis method that incorporates biochemical techniques and makes the best use of the superior properties of organisms. In this method, a transcription system and translation system for genetic information of living organisms are prepared in an artificial container, and a system that can take in desired amino acids including non-natural types from a designed and synthesized transcription template is reconstructed. It is. Since this system is not subject to the constraints of living organisms, it can be expected that the number of protein molecular species that can be synthesized will be expanded to almost infinite. Such cell-free protein synthesis methods have been developed 40 years ago to report that protein synthesis ability remains in ground cell fluid, and cells derived from Escherichia coli, wheat germ, rabbit reticulocytes have been developed. The extract is still widely used for protein synthesis. Among them, cell extracts derived from eukaryotes such as cell extracts derived from wheat germ are attracting attention because of their high protein synthesis ability and accuracy of protein folding.
In order to analyze the function of a gene obtained from a vast amount of genomic information, it is desired to develop a high-throughput synthesis system that can synthesize multiple samples of proteins easily in a short time. In the cell-free protein synthesis method, (1) an apparatus for automatically performing each step of a transcription reaction for obtaining mRNA as a translation template from a transcription template and (2) a translation reaction for synthesizing a protein using mRNA as a template. Has already been developed. In addition, when a cell extract derived from E. coli, a prokaryote, is used, a transcription reaction and a translation reaction can be carried out in one solution, so that a protein is automatically extracted from a transcription template through the transcription reaction and the translation reaction. An apparatus for synthesizing with is already developed. However, for eukaryotic cell extracts such as wheat germ having excellent protein synthesis ability, manual operation is unavoidable until the translation template obtained by the transcription reaction is used for the translation reaction as described above. Therefore, a series of operations until the protein encoded by the transcription template is generated cannot be automatically performed.
That is, in the conventional cell-free protein synthesis method using the above eukaryotic cell extract, unreacted substrates and by-products in the transcription reaction inhibit the subsequent translation reaction, so it is essential to remove them. The removal operation by alcohol precipitation or a gel filtration column was performed (for example, see the right column steps b12 to b16 in FIG. 1 as a typical protocol of the conventional method). At this time, since the salt contained in the buffer used for gel filtration inhibits the subsequent translation reaction, at least a part of the protein has little or no protein unless reprecipitation and / or washing operations are performed. Can only be obtained. However, when such reprecipitation / washing operation is performed, the translational template precipitate held on the bottom surface of the reaction vessel by the surface tension peels off, and in a normal centrifuge that can be used as a configuration of an automatic synthesizer, In order to remove the supernatant without losing the translation template even after centrifugation, the experimenter had to carefully and carefully perform the procedure manually using a pipette or the like.
In addition, the purified translation template is once dissolved in a solution (translation reaction solution) containing ultrapure water, a substrate for translation reaction, an energy source, etc., and then a cell containing a protein synthesizer such as ribosome. Although it was mixed with the extract and used for the translation reaction (right column steps b18 and b19 in FIG. 1), the translation template mRNA is extremely difficult to dissolve in water or the solution for translation reaction. It had to be dissolved by stirring with a tip or the like.

Accordingly, an object of the present invention is to solve the above-mentioned problems in the automation of cell-free protein synthesis, and thus, from a series of steps from the transcription template to the production of the protein encoded by the template, preferably from the production of the transcription template. It is to provide a method for automatically carrying out the steps up to protein production, and to provide an automatic protein synthesizer capable of executing the method, particularly an automatic protein synthesizer capable of simultaneously synthesizing various types of proteins. .
As a result of intensive studies to solve the above problems, the present inventors have (1) the step of removing the unreacted substrate after precipitation of the translation template obtained after the transcription reaction and the accompanying buffer exchange. That there is no effect on the protein translation efficiency even if the washing step and the precipitation step are omitted, and (2) the cell for protein synthesis is not dissolved in water or the solution for translation reaction once. Surprisingly, it was found that the precipitate dissolves very easily when the extract is added directly. The present inventor has developed a novel cell-free protein synthesis protocol based on these findings, and succeeded in constructing an automatic protein synthesizer capable of executing the protocol, thereby completing the present invention. .
That is, the present invention is as follows.
1. In the cell-free protein synthesis step, in the reaction step from the transcription template to the production of the protein encoded by the template, the translation template in the reaction solution after the transcription reaction is precipitated, the supernatant is removed, and the translation template is dried. A cell-free protein synthesis method comprising, after the step, a step of dissolving the precipitate by directly adding a cell extract for protein synthesis to the precipitate.
2. 2. The cell-free protein synthesis method according to item 1, further comprising a step of amplifying and preparing a transcription template encoding the target protein.
3. 3. A cell-free protein synthesis method according to item 1 or 2, characterized in that a host cell containing a gene encoding a target protein is directly subjected to a polymerase chain reaction to amplify a transcription template.
4). 4. The cell-free protein synthesis method according to any one of items 1 to 3, wherein the reaction from the preparation of the transcription template to the production of the protein encoded by the transcription template is automatically performed.
5). 5. The cell-free protein synthesis method according to any one of items 1 to 4, wherein the translation reaction solution is layered on a protein synthesis cell extract containing a translation template to cause a translation reaction.
6). 5. The cell-free protein synthesis method according to any one of items 1 to 4, wherein the translation reaction solution is added to and mixed with a protein synthesis cell extract containing a translation template to cause a translation reaction.
7). 7. The cell-free protein synthesis method according to any one of 1 to 6 above, which comprises any one of the following steps.
(1) A step of amplifying a transcription template by PCR amplification;
(2) A step of mixing the transcription reaction solution and the transcription template;
(3) Incubating the mixture of the transcription reaction solution and the transcription template for a certain period of time;
(4) a step of precipitating the translation template;
(5) removing the supernatant in the reaction vessel;
(6) a step of drying the translation template precipitate; and (7) a step of adding the cell extract for protein synthesis to the dried translation template and dissolving the translation template.
8). 8. The cell-free protein synthesis method according to any one of items 1 to 7, wherein all reaction steps are performed in the same reaction vessel.
9. 9. The cell-free protein synthesis method according to any one of items 1 to 8, wherein a plurality of types of proteins are reacted and synthesized in a plurality of reaction vessels at the same time.
10. 10. The cell-free protein synthesis method according to any one of 1 to 9 above, wherein the cell extract for protein synthesis is a plant seed germ extract.
11. 11. The cell-free protein synthesis method according to 10 above, wherein the plant seed germ extract is a wheat seed germ extract.
12 12. The cell-free protein synthesis method according to 11 above, wherein the wheat seed germ extract is an extract from which the endosperm components of germ and the low-molecular protein synthesis inhibitor substance are substantially removed.
13. 13. A cell-free protein synthesizer comprising at least one of the following control means for carrying out the cell-free protein synthesis method according to any one of 1 to 12 above.
(A) means for variably controlling the temperature in the reaction vessel;
(B) means for dispensing a sample or reagent into the reaction vessel;
(C) precipitation means;
(D) means for removing the supernatant;
(E) Drying means; and (f) Control means for operating the means (a) to (e) according to the process described in 1 above in any one of the preceding items 1 to 12. 14. The cell-free protein synthesizer according to item 13 above, wherein the means for removing the supernatant is a means for causing the reaction container to turn over.
15. 15. The cell-free protein synthesizer according to item 13 or 14, further comprising means for opening and closing the lid of the reaction vessel.

FIG. 1 is a diagram showing the time required for a process comparing a specific example of the method of the present invention with a conventional method.
FIG. 2 is a graph showing the experimental results of Experimental Example 2.
FIG. 3 is an SDS-PAGE photograph showing the experimental results of Experimental Example 3.

The present invention provides a method (hereinafter sometimes simply referred to as “the method of the present invention”) in which a reaction operation is automatically performed from at least a transcription template provided to a reaction system until a protein encoded by the template is generated. provide. Here, “automatically perform the operation” means that the experimenter does not directly apply a manual operation to the reaction system (reaction vessel) during a series of steps. Therefore, when performing each step, it is necessary for an experimenter to manually operate predetermined operation buttons and switches provided in the automatic protein synthesizer of the present invention to be used. There is no loss. The reaction vessel to be used may be different in each step, but the series of steps is the same for the purpose of simplifying the structure of the apparatus for carrying out the method of the present invention and preventing loss of samples and products. It is preferably designed to be carried out in a reaction vessel.
The method of the present invention is divided into a transcription reaction step, a translation template purification step, and a translation reaction step. Preferably, the method of the present invention is characterized in that the steps from preparation of a transcription template to generation of a protein encoded by the template are automatically performed. Therefore, in a preferred embodiment, the method of the present invention further includes a step of preparing a transcription template prior to the transcription reaction step. Hereinafter, each step will be described in detail with specific embodiments, but the method of the present invention is not limited thereto as long as it has at least one of the above two characteristics in the translation template purification step. .
(1) Transfer template manufacturing process
In the method of the present invention, this step does not necessarily need to be performed automatically, and a transfer template obtained manually can be used in the following automated steps. More preferably, the series of steps up to the generation of the encoded protein is performed automatically.
As used herein, “transcription template” refers to DNA that can be used as a template molecule for in vitro transcription reaction, and has at least a base sequence encoding a target protein downstream of an appropriate promoter sequence. An appropriate promoter sequence refers to a promoter sequence that can be recognized by RNA polymerase used in a transcription reaction, and examples thereof include SP6 promoter and T7 promoter. Any DNA encoding the target protein may be used.
The transcription template preferably has a base sequence having an activity of controlling translation efficiency between the promoter sequence and the base sequence encoding the target protein. For example, the transcription template can be derived from RNA virus-derived 5 such as Ω sequence derived from tobacco mosaic virus. 'Untranslated regions and / or Kozak sequences can be used. Furthermore, the transcription template preferably includes a 3 ′ untranslated region including a transcription termination region and the like downstream of the base sequence encoding the target protein. As the 3 ′ untranslated region, about 1.0 to about 3.0 kilobases downstream from the stop codon is preferably used. These 3 ′ untranslated regions are not necessarily those of the gene encoding the target protein.
The transfer template can be produced by a method known per se. For example, a host cell such as Escherichia coli having a plasmid into which a DNA containing the same base sequence as the desired transcription template is inserted is cultured, and a large amount of the plasmid is prepared using a known purification method. The transcription template DNA is excised from the plasmid, the restriction enzyme is removed by phenol treatment and chloroform treatment, and alcohol precipitation with ethanol or isopropanol (if necessary, an appropriate amount of salt such as ammonium acetate, sodium acetate, sodium chloride, etc. And the like. The obtained DNA precipitate can be dissolved in ultrapure water or a transcription reaction solution described later and used for the following transcription reaction.
An apparatus for carrying out such a series of operations automatically or semi-automatically (referring to an embodiment in which an experimenter directly adds a manual operation to a reaction system in a part of the process) is known, and this is described in this book. By incorporating it into the automatic protein synthesizer of the invention, it is possible to automatically perform from the preparation of the transcription template to the production of the target protein. However, the present invention provides an automatic cell-free protein synthesis system for high-throughput analysis. In view of the above objectives, simplification of the apparatus, shortening of the required time, etc., it is more preferable to use the following method for preparing a transcription template by the polymerase chain reaction (PCR) method.
In a preferred embodiment of the method of the present invention, a method is used in which a host in which a DNA encoding a target protein has been cloned (for example, E. coli having a plasmid containing the DNA) is directly subjected to PCR to amplify a transcription template. For example, an oligonucleotide containing a suitable promoter sequence, a 5 ′ untranslated sequence having an activity to control translation efficiency, and a part of the 5 ′ end region of DNA encoding the target protein is publicly known by using an automatic DNA synthesizer. Using this method as a sense primer, and using an oligonucleotide having a 3 'end region sequence of the 3' untranslated sequence as an antisense primer, DNA encoding the target protein as a template and the 3 'untranslated downstream A desired transcription template can be obtained by directly adding a host such as Escherichia coli having a plasmid containing a sequence to a PCR reaction solution and performing an amplification reaction under normal conditions. In order to prevent the generation of short-chain DNA (resulting in yield loss of the target product and low-molecular translation product noise) generated by non-specific amplification, the promoter-splitting primer described in WO 02/18586 is used. It can also be used.
The amplification reaction can be carried out in a commercially available 96-well plate for PCR using a commercially available PCR thermal cycler, or a similar temperature variable control device is linked with the automatic protein synthesizer of the present invention, or the present invention. Each means for performing the transcription / translation reaction of the automatic protein synthesizer can be directly applied to PCR.
The transcription template DNA obtained as described above may be subjected to a transcription reaction after being purified by chloroform extraction or alcohol precipitation. However, in order to simplify the apparatus and shorten the required time, the PCR reaction solution can be used as it is. It is preferable to use it as a solution. In preparation of a transcription template, by using direct PCR from the above-mentioned host, the steps can be greatly omitted as compared with a method in which a large amount of a plasmid is once prepared and subjected to restriction enzyme treatment to obtain a transcription template. A large amount of transcription template can be synthesized in a short time with a small number of steps (see FIG. 1). In other words, the time required for ultracentrifugation for culture and plasmid purification is reduced because the step of culturing Escherichia coli having a plasmid incorporating the target gene and preparing a large amount of plasmid (right column step b1 in FIG. 1) is not required. (E.g., it can be significantly reduced from 72 hours to 6 hours as shown in FIG. 1). Furthermore, restriction enzyme treatment (step b2) for cutting out the transcription template from the plasmid, phenol treatment for removing the restriction enzyme and the like, chloroform treatment (steps b3 and b4), and alcohol precipitation (step for purification of the transcription template) b5) Since the step of dissolving the DNA precipitate as the transcription template (step b6) can be omitted, there is no inhibition of transcription reaction due to residual phenol / chloroform, and no loss of transcription template due to multi-step purification operation. . Further, since the number of steps required for the reaction can be reduced, there is a further advantage that the number of chips used can be reduced.
(2) Transcription reaction process
The method of the present invention includes a step of generating mRNA as a translation template by in vitro transcription reaction from transcription template DNA encoding a target protein prepared using a method known per se. The step includes a solution containing a transcription template provided in a reaction system (for example, a commercially available container such as a 96-well titer plate), preferably the above-mentioned PCR reaction solution, and an RNA polymerase (for example, suitable for the promoter in the transcription template). After mixing with a solution (also referred to as “transcription reaction solution”) containing components necessary for a transcription reaction such as SP6 RNA polymerase) and a substrate for RNA synthesis (four types of ribonucleoside triphosphates), about 20 ° C. It is carried out by incubating the mixture at about 60 ° C., preferably about 30 ° C. to about 42 ° C. for about 30 minutes to about 16 hours, preferably about 2 hours to about 5 hours. The operation of dispensing and mixing the transcription template solution and the transcription reaction solution into the reaction vessel is performed by dispensing means of an automatic protein synthesizer described later (for example, a pipetter (when using a commercially available 96-well titer plate as the reaction vessel). , And the like having a dispensing tip of 8 or 12 series adapted to the well interval are preferably used). Incubation for the transcription reaction can be performed while the temperature is controlled at a constant temperature by a temperature control means of an automatic protein synthesizer described later.
(3) Translation template purification process
The transcription product generated as described above (ie, “translation template”) optionally includes a 5 ′ untranslated sequence and / or a 3 ′ untranslated sequence having an activity of controlling the translation efficiency inserted into the transcription template. An RNA molecule having a base sequence encoding a target protein. In the reaction solution after the transcription reaction, in addition to the translation template RNA, unreacted ribonucleoside triphosphate, reaction by-product pyrophosphate, and other salts contained in the transcription reaction solution are mixed. Since these substances are known to inhibit the subsequent translation reaction, the translation template is selectively precipitated to separate and remove unreacted substrates and the like. Examples of such precipitation means include salting out and the like, and an alcohol precipitation method is preferably exemplified, but is not limited thereto. When the alcohol precipitation method is used, the alcohol to be used is not particularly limited as long as it can selectively precipitate RNA. For example, ethanol, isopropanol and the like are preferable, and ethanol is more preferable. In the case of ethanol, it is preferable to use about 2 to about 3 times the amount of the transcription reaction solution, and in the case of isopropanol, about 0.6 to about 1 amount of the transcription reaction solution. Moreover, the yield of precipitation can be increased by making a suitable salt coexist. Examples of such salts include ammonium acetate, sodium acetate, sodium chloride, lithium chloride and the like. For example, when ammonium acetate is used, it is desirable to add it so that the final concentration is about 0.5M to about 3M. The alcohol precipitation may be performed at room temperature.
Alcohol and salt solution can be added using a dispensing means of an automatic protein synthesizer described later, and translation template RNA is precipitated at a constant temperature using a temperature control means of an automatic protein synthesizer described later. Under the conditions.
Although transcription template DNA is also present in the transcription reaction solution, it has been reported that the transcription template inhibits the subsequent translation reaction. Therefore, DNase treatment is conventionally performed after the transcription reaction, followed by phenol treatment and chloroform treatment. To denature and remove DNase (see right column steps b9 to b11 in FIG. 1). The present inventors have found that in the method of the present invention, even if a transcription template coexists in the translation reaction solution, it does not cause a decrease in translation efficiency, and simplifies the automatic protein synthesizer and shortens the time required. In addition, DNase treatment was omitted in order to inhibit translation due to residual phenol / chloroform and to prevent loss of translation template due to multi-step purification operation. Therefore, the method of the present invention is further characterized in that the step of disassembling and removing the transcription template is not performed after the transcription reaction.
The translation template RNA precipitated as described above can be precipitated on the bottom surface of the reaction vessel by any means known per se. Examples of such means include centrifugation, filtration, standing, lyophilization, etc., preferably centrifugation. When utilizing centrifugation, it can be carried out, for example, at room temperature or lower, preferably about 25 ° C. or lower, more preferably from about 4 ° C. to about 15 ° C., and from about 400 × g to about 22000 × g. Such a centrifugation operation can be carried out by incorporating a centrifugal separator used in a known tabletop centrifuge or the like into a below-described automatic protein synthesizer as a precipitation means.
Since the translation template precipitated on the bottom surface of the reaction vessel by the first precipitation operation is held on the bottom surface by surface tension, it is not necessary for the experimenter to manually remove the supernatant after the precipitation operation. It can also be performed using a known apparatus (eg, BioRobot 8000 (Qiagen)), or simply by turning the reaction vessel upside down. Considering simplification of the apparatus, reduction of required time, reduction of the number of chips to be used, the latter method is advantageous for automation.
After removing the supernatant, the translation template RNA precipitate is dried. Drying is more than the time necessary for removing components (eg, alcohol) that can inhibit the translation reaction in the remaining supernatant, and does not reduce translation efficiency by causing insolubilization by complete drying. The method and time are not particularly limited as long as it is carried out within a certain amount of time, but it is preferably about 10 minutes or less, more preferably about 3 minutes to about 3 minutes, by a method known per se such as natural drying, air drying, reduced pressure drying and the like. Can be done for 8 minutes.
The dried translation template precipitate is dissolved in a protein synthesis cell extract for use in the following translation reaction step. The cell extract for protein synthesis used herein may be any cell extract that can translate a translation template to produce a protein encoded by the template. Specifically, Escherichia coli, Plant seed germs, cell extracts such as rabbit reticulocytes, and the like are used. These may be commercially available, or a method known per se, specifically, in the case of an Escherichia coli extract, Pratt, J. et al. M.M. et al. Transcription and Translation, Hames, 179-209, B .; D. & Higgins, S .; J. et al. , Eds), IRL Press, Oxford (1984).
Examples of commercially available cell extracts for protein synthesis include E. coli-derived cell extracts. Examples include those attached to E. coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche). Rabbit Reticulocyte Lysate System (Promega) and Rabbit Reticulocyte Lysate System (Promega) PROTEIOS is derived from wheat germ ^TM Examples include those attached to TOYOBO. Among them, it is preferable to use a plant seed germ extract system, and plant seeds are preferably gramineous plants such as wheat, barley, rice and corn, and seeds such as spinach, and in particular, wheat seed germ extract. Those using are suitable. Furthermore, a wheat seed germ extract extract from which the endosperm components of germ and the low-molecular protein synthesis inhibitor substance are substantially removed is more preferable. This is because the components and substances involved in protein synthesis inhibition in the extract are reduced as compared with the conventional wheat seed germ extract.
As a method for producing a wheat germ extract, for example, Johnston, F. et al. B. et al. , Nature, 179, 160-161 (1957), or Erickson, A. et al. H. et al. (1996) Meth. In Enzymol. 96, 38-50 etc. can be used. Furthermore, treatments such as removal of endosperm containing translation inhibitory factors such as tritin, thionine, and nucleolytic enzyme contained in the extract (JP 2000-236896 A, etc.), and activation of translation inhibitory factors are suppressed. It is also preferable to perform processing (Japanese Patent Laid-Open No. 7-203984).
When using an extract of plant seed germ, such as wheat germ, the translation efficiency is increased by preincubating the extract at, for example, about 15 ° C. to about 26 ° C. for about 3 minutes or more before adding it to the precipitation of the translation template. There are cases where it can be improved.
The cell extract for protein synthesis may be added as it is to the precipitation of the translation template, or other components necessary or suitable for the translation reaction, that is, amino acids serving as substrates, energy sources, various ions, buffers, ATP regeneration One or more of a system, nucleolytic enzyme inhibitor, tRNA, reducing agent, polyethylene glycol, 3 ′, 5′-cAMP, folate, antibacterial agent, etc. are mixed with the cell extract beforehand and then added to the precipitate May be.
By adding the cell extract for protein synthesis directly to the precipitate of the translation template, the precipitate dissolves very easily. For example, the cell extract is used as a template for the translation template by using a dispensing means of an automatic protein synthesizer described later. If the dispensing means is designed to be used as a liquid mixing means (eg, pipetting, stirring, etc.) after being added to the precipitate, the mixing operation can be performed for a short time. The following translation reaction process can be started.
(4) Translation reaction process
The cell extract for protein synthesis containing the translation template obtained as described above is added to the substrate amino acid, energy source, various ions, buffer solution, ATP regeneration system, nucleolytic enzyme inhibitor, tRNA, reducing agent, polyethylene glycol. Add a solution (also referred to as a “translation reaction solution”) that contains necessary or suitable components for translation reaction, such as 3 ′, 5′-cAMP, folate, antibacterial agent, etc., and temperature suitable for translation reaction The translation reaction can be carried out by incubating for an appropriate time. The amino acid serving as a substrate is usually 20 kinds of natural amino acids constituting a protein, but analogs and isomers thereof can be used depending on the purpose. Moreover, ATP and / or GTP are mentioned as an energy source. Examples of various ions include acetates such as potassium acetate, magnesium acetate, and ammonium acetate, and glutamates. As the buffer, Hepes-KOH, Tris-acetic acid or the like is used. Examples of the ATP regeneration system include a combination of phosphoenolpyruvate and pyruvate kinase, or a combination of creatine phosphate (creatine phosphate) and creatine kinase. Examples of the nuclease inhibitor include ribonuclease inhibitors and nuclease inhibitors. Among these, as a specific example of the ribonuclease inhibitor, RNase inhibitor (manufactured by TOYOBO, etc.) derived from human placenta is used. tRNA is described in Monitor, R .; , Et al. , Biochim. Biophys. Acta. , 43, 1 (1960), etc., or a commercially available product can be used. Examples of the reducing agent include dithiothreitol. Antibacterial agents include sodium azide, ampicillin and the like. These addition amounts can be appropriately selected within a range that can be usually used in cell-free protein synthesis.
The mode of addition of the translation reaction solution can be appropriately selected according to the translation reaction system to be used. The translation reaction system used in the method of the present invention may be any system known per se that can be applied to the automatic protein synthesizer of the present invention, for example, a batch method (Pratt, JM et al.,). Transcribation and Translation, Hames, 179-209, BD & Higgins, SJ, eds), IRL Press, Oxford (1984)), amino acids, energy sources, etc. are continuously supplied to the reaction system. Cell-free protein synthesis method (Spirin, AS et al., Science, 242, 1162-1164 (1988)), dialysis method (Kikawa et al., 21st Japan Molecular Biology Society, WID6), or layered method ( International Publication No. 02/24939 pamphlet) and the like. Furthermore, a discontinuous gel filtration method (Japanese Patent Laid-Open No. 2000-333673) for supplying template RNA, amino acid, energy source and the like to the synthesis reaction system when necessary, and discharging the synthesized product and degradation product when necessary, and a synthesis reaction tank Is prepared with a carrier capable of molecular sieving, and the above synthetic material or the like is developed using the carrier as a mobile phase, a synthetic reaction is performed during the development, and the synthesized protein can be recovered as a result (Japanese Patent Laid-Open No. 2000-316595). Publication) etc. can be used. However, from the viewpoint of simplifying the structure of an automated protein synthesizer, saving space, low cost, and providing a multi-sample simultaneous synthesis system applicable to high-throughput analysis, the batch method or the multi-layer method is preferable, and a relatively large amount of protein can be obtained. The multi-layer method is particularly preferable in that it can be obtained.
When performing the translation reaction by the batch method, the translation reaction solution may be added to and mixed with the protein synthesis cell extract containing the translation template. Alternatively, when the components contained in the translation reaction solution are previously mixed with the protein synthesis cell extract, the addition of the translation reaction solution can be omitted. As a “translation reaction solution” obtained by mixing a cell synthesis solution for protein synthesis containing a translation template and a solution for translation reaction, for example, when a wheat germ extract is used as the cell extract for protein synthesis, 10 to 50 mM. HEPES-KOH (pH 7.8), 55-120 mM potassium acetate, 1-5 mM magnesium acetate, 0.1-0.6 mM spermidine, each 0.025-1 mM L-amino acid, 20-70 μM, preferably 30-50 μM DTT, 1-1.5 mM ATP, 0.2-0.5 mM GTP, 10-20 mM creatine phosphate, 0.5-1. Those containing OU / μl ribonuclease inhibitor, 0.01-10 μM protein disulfide isomerase, and 24-75% wheat germ extract are used. When such a translation reaction solution is used, the preincubation is about 10 to about 40 ° C. for about 5 to about 10 minutes, and the incubation in this reaction (translation reaction) is also about 10 to about 40 ° C., preferably about 18 to In the batch method, the reaction is usually carried out at a temperature of about 30 ° C., more preferably about 20 to about 26 ° C., until the reaction is stopped.
When the translation reaction is performed by the multilayer method, the protein synthesis is performed by layering the translation reaction solution on the cell synthesis solution for protein synthesis containing the translation template so as not to disturb the interface. Specifically, for example, if necessary, a cell extract for protein synthesis that has been preincubated for an appropriate period of time is added to the precipitation of the translation template to dissolve it, thereby forming a reaction phase. The reaction is carried out by layering the solution for translation reaction (supply phase) on the upper layer of the reaction phase so as not to disturb the interface. The interface between both phases does not necessarily have to be formed in a horizontal plane by means of multiple layers, and it is also possible to form a horizontal plane by centrifuging the mixed solution containing both phases. When the diameter of the circular interface between the two phases is 7 mm, the volume ratio of the reaction phase to the supply phase is suitably 1: 4 to 1: 8, but 1: 5 is preferred. The larger the interfacial area consisting of both phases, the higher the mass exchange rate by diffusion and the higher the protein synthesis efficiency. Accordingly, the capacity ratio of both phases varies depending on the interface area between both phases. For example, in a system using a wheat germ extract, the translation reaction is performed at about 10 to about 40 ° C., preferably about 18 to about 30 ° C., more preferably about 20 to about 26 ° C. It can be performed for 10 to about 20 hours. In addition, when an E. coli extract is used, the reaction temperature is suitably about 30 ° C to about 37 ° C.
The cell-free protein synthesis method of the present invention comprises the following steps: (1) a step of precipitating the translation template in the reaction solution after the transcription reaction, a step of removing the unreacted substrate and a 1 or By omitting multiple washing steps and precipitation steps, and subjecting to translation reaction; and (2) dissolving the precipitate by directly adding the cell extract for protein synthesis to the resulting translation template precipitate, This is the first time that a series of operations from a transcription template to production of a protein encoded by the template is automatically performed. However, the features (1) and (2) are each independently new and have advantageous effects. Therefore, as long as it has one of these characteristics, a cell-free protein synthesis method that does not have the other characteristic (that is, an automatic cell-free protein synthesis method that solves only the problem solved by the other characteristic part by other means, or Regarding the problem solved by the other characteristic part, a “semi-automated cell-free protein synthesis method” manually performed by an experimenter as usual is also included in the present invention.
As described above, as long as the method of the present invention has the characteristics of (1) and / or (2) above, the other steps are performed according to any conventionally known procedures and conditions that can be applied for automation. Although it does not restrict | limit in particular, In addition to the characteristic of said (1) and / or (2), it is preferable to have at least 1 process of the following.
・ Process to amplify and prepare transcription template by PCR
・ Process of mixing transcription reaction solution and transfer template
・ Incubating a mixture of a transcription reaction solution and a transcription template for a certain period of time
-Precipitation of transcription product (ie translation template)
・ Removing the supernatant in the reaction vessel
・ Drying the translation template precipitate
A step of adding a cell extract for protein synthesis to a dried translation template and dissolving the translation template
-Layering the translation reaction solution on the protein synthesis cell extract containing the translation template
-Layering the translation reaction solution on the protein synthesis cell extract containing the translation template and incubating it for a certain period of time
Or
・ Process to amplify and prepare transcription template by PCR
・ Process of mixing transcription reaction solution and transfer template
・ Incubating a mixture of a transcription reaction solution and a transcription template for a certain period of time
-Precipitation of transcription product (ie translation template)
・ Removing the supernatant in the reaction vessel
・ Drying the translation template precipitate
A step of adding a cell extract for protein synthesis to a dried translation template and dissolving the translation template
・ Process to perform protein synthesis reaction by batch method
According to the cell-free protein synthesis method of the present invention, a cell-free protein synthesis reaction can be automatically performed without an operator performing a manual operation on the way. In addition, according to the method of the present invention, the steps required for the entire reaction can be reduced without changing the protein synthesis efficiency from the conventional one. (From the viewpoint of automation, reducing the number of steps causes a mistake. It is also important in the sense of making it difficult to reduce the time required for protein synthesis, and it is possible to reduce loss of translation templates due to degradation. In addition, there are further advantages such that the transcription reaction system can be reduced and the amount of DNA to be used can be reduced.
In addition, according to the present invention, it is possible to perform the entire cell-free protein synthesis reaction in the same reaction vessel, and the design of the apparatus for performing the method of the present invention is not a complicated structure as will be described later. There is also an advantage that space can be saved. Furthermore, according to the method of the present invention, a plurality of types (especially many types) of proteins can be easily synthesized simultaneously by using, for example, a 96-well plate as a reaction vessel for performing a transcription reaction or a translation reaction. And can be suitably used for applications such as high-throughput functional analysis of proteins as described below.
In this invention, the apparatus (namely, automatic protein synthesizer) for performing the said method is also provided. The apparatus of the present invention is characterized by having at least the following means (a) to (f).
(A) means for variably controlling the temperature in the reaction vessel;
(B) means for dispensing a sample or reagent into the reaction vessel;
(C) precipitation means;
(D) means for removing the supernatant;
(E) drying means; and
(F) Control means for controlling the means (a) to (e) to operate in accordance with the method of the present invention described above.
By using an apparatus having at least the configurations of (a) to (f), the above-described cell-free protein synthesis reaction of the present invention can be automatically performed. Hereinafter, each configuration will be specifically described in detail.
(A) Means for variably controlling the temperature in the reaction vessel
Means for variably controlling the temperature in the reaction vessel include a transcription reaction, a translation reaction incubation, a translation template precipitation, or an automated protein synthesizer according to the present invention that automatically performs a transcription template preparation process by PCR. In some cases, it is a means for adjusting the liquid temperature in the reaction vessel to an appropriate temperature condition in the amplification reaction of the PCR method. The temperature range to be variably controlled is not particularly limited, but a temperature range normally required in a series of reaction operations for cell-free protein synthesis including preparation of a transcription template (for example, about 4 ° C. to about 100 ° C., preferably about Any means that can variably control the liquid temperature in the reaction vessel within a range of 26 ° C. to about 99 ° C. is not particularly limited. For example, conventionally known Takara PCR thermal cycler MP (manufactured by Takara Bio Inc.), Gene Amp PCR System 9700 (manufactured by Applied Biosystems Inc., etc.) and the like can be mentioned. Specifically, in addition to the work stage for placing the reaction vessel, which is a place where pipetting is performed, a plurality of stages for placing the reaction vessel are provided in the apparatus, and the temperature of the entire space on the stage is adjusted. As a result, the temperature in the reaction vessel is variably controlled.
(B) Means for dispensing sample or reagent into reaction vessel
The means for dispensing a sample or reagent into a reaction container is to dispense a sample or reagent into a reaction container in order to perform a series of cell-free protein synthesis reactions such as transcription reaction, translation reaction, and PCR in the reaction container. Means. Here, “sample” refers to a transcription template, translation template, PCR template plasmid (or host having the plasmid (eg, E. coli)), etc., and “reagent” refers to a transcription reaction solution, a cell extract for protein synthesis, It refers to translation reaction solution, alcohol, salt solution, PCR reaction solution and the like. As such a dispensing means, a pipette arm or the like that can be dispensed by a conventionally known appropriate automatic method can be used without particular limitation as long as the amount of the sample and reagent can be adjusted according to the process. Can be realized. In addition to the above functions, the dispensing means more preferably has a mixing function (eg, pipetting, stirring, etc.) for homogenizing two or more types of solutions and precipitating dissolution.
(C) Means for precipitation
The precipitation means is means for precipitating an appropriate target (for example, a translation template after the transcription reaction) in the reaction container. Such precipitation means is not particularly limited and can be realized by any conventionally known appropriate means as long as it can precipitate the target in the reaction vessel and enables solid-liquid separation. For example, the precipitation means can be realized by using a conventionally known centrifuge used for ethanol precipitation or the like, or any other appropriate device conventionally used for filtration or freeze-drying.
(D) Means for removing supernatant
The means for removing the supernatant is means for removing the supernatant to separate the precipitate from the supernatant after the appropriate target in the reaction vessel is precipitated by the precipitation means. In the apparatus of the present invention, the means for removing the supernatant can be realized by means for causing the reaction container to turn over, means for sucking only the supernatant in the reaction container, and the like. That is, as described above, according to the cell-free protein synthesis method of the present invention, after the step of precipitating the translation template in the reaction solution after the transcription reaction, the step of removing the unreacted substrate and one or more times associated therewith. Since the washing step and the precipitation step are omitted and used for the translation reaction, the translation template (mRNA) can be used for the drying step while attached to the bottom of the reaction vessel. Therefore, the supernatant can be automatically separated from the precipitate without losing the precipitation of the translation template by the means for removing the supernatant as described above. Specifically, in the case where the means for removing the supernatant is a means for performing an operation of turning the reaction container upside down, for example, the reaction container is fixed on it using a rotational power of a motor or the like. It can be realized by a mechanism using a plate or the like that can be turned over, or a mechanism using a robot arm that can be gripped and reversed. Further, in the case of means for aspirating only the supernatant in the reaction vessel, it can be realized by using a conventionally known appropriate one such as BioRobot 8000 (manufactured by Qiagen).
(E) Drying means
The drying means can be realized by, for example, a centrifugal dryer, an evaporator or the like when drying by a method other than natural drying. However, in the automatic protein synthesis method apparatus according to the present invention, it is also possible to use a conventionally known drying apparatus that is incorporated inside or can be connected to the outside.
(F) Control means
In the control means, the operation of the drive source (motor, pneumatic / hydraulic equipment, other operation controllable actuator, etc.) used for each means in order to operate the means (a) to (e) is turned on / off. A control device for controlling the degree and state of operation is included. The control configuration is such that the steps of the method are performed in accordance with the method of the present invention (for example, a cell-free protein synthesis reaction according to the procedure shown in FIG. 1), and the object of the method is achieved. The operation of the means (a) to (e) can be controlled.
The control device may be configured by combining control devices necessary for controlling the operation of each means, such as a control circuit including a computer having a control program, a sequence control circuit, and the like. The control configuration is such that signals, power, pneumatic pressure, hydraulic pressure, and the like can be supplied to each means so that each means operates in the order along. Further, a driver necessary for directly sending a drive signal to the drive source of each means, various sensors and switches necessary for detecting an operation state of the drive source of each means may be added as appropriate.
The reaction vessel applicable to the apparatus of the present invention is not particularly limited, and various conventionally known reaction vessels that have been used for cell-free protein synthesis reaction can be used. For example, a 96-well PCR plate, 96 Hole titer plates, 8-strip tubes and tubes (1.5 mL, 15 mL, 50 mL, etc.) can be used. For example, when batch or multi-layer methods are used as translation reaction systems, translation reactions can be performed in small reaction systems such as 96-well plates. Furthermore, according to the method of the present invention, the transcription reaction can also be performed in a small reaction system, so that the transcription reaction / translation template purification, translation reaction, and preparation of a transcription template for further transcription reaction if desired. A series of cell-free protein synthesis methods, including PCR, can be performed simultaneously on multiple types of proteins in multiple reaction systems. It is possible to synthesize the protein.
In addition, the transcription reaction and the translation reaction are preferably performed in a sealed reaction vessel. From such a viewpoint, a reaction vessel with a lid is used, and the device further opens and closes the lid of the reaction vessel. It is preferable to have a means. For example, when a 96-well plate is used as the reaction vessel, the lid is exemplified by a rubber lid that can seal each hole individually. Since it is preferable that the lid can be brought into close contact with the reaction vessel in a closed state, a lid having a certain weight (for example, about 500 g) is used, or a lid or the like is sandwiched between the lid and the reaction vessel. It is conceivable to close the lid. The means for opening and closing the lid of the reaction vessel can be realized using, for example, a mechanism in which a conventionally known chucking mechanism / suction mechanism and a robot arm are combined.
The automatic protein synthesizer of the present invention may have means for stocking the reaction reagent, if necessary, in addition to the means described above.
As described above, the method and apparatus of the present invention can synthesize a plurality of types of proteins easily and automatically at the same time. For example, multiple transcription templates and translation templates encoding various mutant proteins can be prepared, and multiple mutant proteins can be synthesized simultaneously for analysis without requiring detailed design of the mutants. Is useful.
The method and apparatus of the present invention can be suitably used for high-throughput functional analysis of various proteins. For example, as a result of homology search, a gene group encoding a protein containing a conserved common domain (for example, a kinase domain) is used as a template, and the protein is simultaneously synthesized by the method of the present invention using the apparatus of the present invention. On the other hand, a group of proteins that can be phosphorylation targets (for example, transcription factors) are synthesized in the same manner, and both are mixed in various combinations. ³² It is possible to identify which protein kinase phosphorylates which protein using the uptake of P-labeled ATP as an index.
Alternatively, a gene group encoding a protein containing a motif unique to a transcription factor (for example, Zn finger, leucine zipper, etc.) is used as a template, and the protein is simultaneously synthesized by the method of the present invention using the apparatus of the present invention. By investigating the ability to bind to cis-element sequences, heterodimer formation with other transcriptional regulatory factors, and the ability to bind to the transcriptional regulatory region of specific gene promoters, information for elucidating crosstalk woven by transcription factors Obtainable.
Experimental example
The present invention will be specifically described below with reference to experimental examples, but the present invention is not limited to the following examples.
Experimental Example 1: Examination of effect on translation efficiency of washing with 70% alcohol in mRNA precipitation caused by ethanol precipitation
(1) Preparation of translation template mRNA
About GFP gene of jellyfish and DHFR gene of Escherichia coli, a transcription reaction solution (80 mM Hepes-KOH) containing SP6 RNA polymerase (manufactured by Promega) using a gene inserted into wheat germ cell-free plasmid vector pEU as a transcription template. 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTP, 1 U / μl SP6 RNA polymerase, 1 U / μl Rnasin). The obtained mRNA was subjected to ethanol precipitation using ammonium acetate, and then divided into those that were washed with 70% alcohol and those that were not washed, and each was used as a translation template.
(2) Protein synthesis by wheat germ cell-free protein synthesis system (multilayer method)
Translation reaction solution containing 5.8 μl of wheat germ extract (each final concentration of 29 mM Hepes-KOH (pH 7.8), 95 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nacalai Tectonics) 20 types of 0.23 mM L-type amino acids, 2.9 mM dithiothreitol, 1.2 mM ATP (manufactured by Wako Pure Chemical Industries), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries), 15 mM creatine phosphate (Wako Pure Chemical Industries, Ltd.) 0.9U / μl RNase inhibitor (manufactured by TAKARA), 50 ng / μl tRNA (Moniter, R., et al., Biochim. Biophys. Acta., 43, 1- (1960)), 0.46 μg / L creatine kinase (Roche), 2 nCi / μl ¹⁴ 25 μl of C-leu (manufactured by Moravec) was prepared. A protein synthesis reaction was separately carried out by the multilayer method for each of the mRNA from which the precipitate was washed and the mRNA from which the precipitate was not washed. 8 μg / μl of translation template is added to the above translation reaction solution to form a lower phase, and an upper phase (each final concentration of 29 mM Hepes-KOH (pH 7.8), 95 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM) Spermidine (manufactured by Nacalai Tectonics), 20 types of 0.23 mM L-type amino acids, 2.9 mM dithiothreitol, 1.2 mM ATP (manufactured by Wako Pure Chemical Industries), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 2nCi / μl ¹⁴ C-leu (manufactured by Moravec)) 125 μl was overlaid and incubated at 26 ° C. for 16 hours.
After starting the reaction, the solution for translation reaction after 16 hours was spotted on a 5 μl filter paper, and using a liquid scintillation counter (LS6000IC: manufactured by Beckman Coulter, Inc.) by a solid support method, ¹⁴ C-leu uptake was measured. In both DHFR and GFP genes, there was no difference in the synthesis amount of the target protein between the mRNA precipitate produced by ethanol precipitation and the one that was washed with 70% alcohol and the one that was not treated. This indicates that in the protein synthesis system (multilayer method), washing treatment with 70% alcohol in mRNA precipitation caused by ethanol precipitation has no effect on translation efficiency. The above results can be used not only for the layered method but also for all other synthetic methods in wheat germ cell-free protein.
Thus, it has been found that the translation efficiency is not affected at all even if the conventional process of washing mRNA precipitation with 70% alcohol is excluded. By excluding this step, which may cause the loss or reduction of translation template mRNA, not only the reproducibility of the synthesis amount of the target protein can be improved, but also the working time can be reduced. These are particularly effective for fully automatic protein synthesizers that process samples in large quantities.
Experimental Example 2: Solubilization rate of mRNA pellet with Milli Q water and cell extract for protein synthesis
pEU-DHFR was subjected to PCR using a sense primer and an anti-primer, and mRNA obtained by transferring it to a template was used. For transcription, prepare 400 μl of a reaction system so that it becomes 80 mM Hepes-KOH, 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTP, 1 U / μl SP6 RNA polymerase, 1 U / μl Rnasin, 10% PCR product, and 37 ° C. Incubated for 3 hours. mRNA radioisotope ³² When labeling with P, the UTP concentration is 1.2 mM [α- ³² 8 μl of P] UTP was added to make the reaction system 400 μl. ³² To the mRNA labeled with P, 53 μl of 7.5M ammonium acetate and 1 ml of ethanol were added and stirred well, followed by centrifugation at 20,000 × g for 15 minutes. The pellet was dissolved in 100 μl of Milli Q water, applied to NICK Column (Amersham Bioscience) previously equilibrated with 3 ml of 100 mM sodium chloride, 10 mM Tris-HCl (pH 7.6), 0.5 mM EDTA, and 400 μl of 100 mM sodium chloride, The product was washed with 10 mM Tris-HCl (pH 7.6) and 0.5 mM EDTA, and purified by elution with 400 μl of 100 mM sodium chloride, 10 mM Tris-HCl (pH 7.6) and 0.5 mM EDTA.
³² 23 μl of mRNA not labeled with P ³² Milli Q water (25 μl), 7.5 mM ammonium acetate (7.7 μl) and ethanol (144 μl) were added to P-labeled mRNA (2 μl), and the mixture was stirred well and centrifuged at 20,000 × g for 15 minutes. The supernatant was removed and the pellet was air-dried. 6.25 μl of Milli Q water, 30 mM Hepes-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0 .3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM magnesium acetate, 100 mM potassium acetate, 0.005% sodium azide, 400 ng / μl creatine kinase, optical density (OD) at 260 nm (A ₂₆₀ 25 μl of a translation reaction solution prepared so as to be 60 wheat germ extract. After 5 minutes, 5 μl of the solution was measured by the Cherenkov method.
The experimental results are shown in FIG. The value before ethanol precipitation was 66143 for the extract and 66741 for Milli Q water. The cpm values after dissolution were 22,285 for the extract and 13,828 for Milli Q water. This shows that the extract is more soluble than Milli Q water.
Experimental Example 3: Protein synthesis method using automatic protein synthesizer
Using an automatic protein synthesizer for carrying out the method of the present invention, the following were set, respectively, and cell-free protein synthesis was performed.
・ Transfer mold
DNA obtained by PCR using a pEU plasmid vector incorporating DHFR and GFP in a 30 μL system using a sense primer and an antisense primer (contained in a 96-well PCR plate)
・ Transcription reaction solution
A solution containing a final concentration of 80 mM Hepes-KOH, 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTP, 1 U / ml SP6 RNA polymerase, 1 U / ml Rnasin
・ Ethanol precipitation mixture
Milli Q water 3.54ml, 7.5M ammonium acetate 1.32ml, ethanol 24.66ml mixture
・ Translation solution
Final concentrations of 30 mM Hepes-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM magnesium acetate, 100 mM potassium acetate, 0.005% sodium azide, 400 ng / μl creatine kinase, optical density (OD) at 260 nm (A ₂₆₀ ) Contains 60 units of wheat germ extract
・ Multilayer liquid
Final concentrations of 30 mM Hepes-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM dithiothreitol, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM magnesium acetate, Overlay solution prepared to be 10 mM potassium acetate, 0.005% sodium azide
・ Reaction vessel
Two round-bottom 96-well microplates that can withstand centrifugation
・ Tip for dispensing
200 boxes of 200 μl chips (5 boxes for transcription reaction solution, 1 box for translation reaction solution, 1 box for ethanol precipitation x 2 microplates, 1 box for multi-layers x 2 microplates), 2 boxes of 96 μl chips (1 for PCR products) Box x 2 microplate)
・ Microplate for collecting ethanol precipitation supernatant
Cell-free protein synthesis was performed according to the following procedure.
(1) Transfer the PCR plate from the cooling stage to the work stage.
(2) Move the microplate from the cooling stage to the work stage.
(3) Remove the PCR plate lid.
(4) Remove the microplate lid.
(5) Dispense 22.5 μl of the transcription reaction solution to the microplate.
(6) Transfer 2.5 μl of PCR product from the PCR plate to a microplate and pipette 10 times.
(7) Cover the PCR plate.
(8) Cover the microplate.
(9) Restore the PCR plate.
(10) Incubate the microplate at 37 ° C. for 3 hours.
(11) Remove the microplate lid and hold it.
(12) Dispense 147.6 μl of ethanol precipitation mixture into a microplate. Pipetting 10 times.
(13) Centrifuge at 1,000 × g for 30 minutes without closing the microplate.
(14) Place the absorbent cotton-coated plate on the microplate and turn it over to remove the supernatant.
(15) Allow to air dry for 5 minutes.
(16) Dispense 25 μl of the translation reaction solution onto the microplate.
(17) Slowly overlay 125 μl of the overlay solution on top of 25 μl of the translation reaction solution on the microplate.
(18) Cover the microplate.
(19) Incubate the microplate at 26 ° C. for 20 hours.
Protein synthesis was performed as described above. For comparison, protein synthesis was performed manually.
FIG. 3 is an SDS-PAGE photograph showing the experimental results. The amount of protein synthesis was confirmed by SDS-PAGE. 10 μl of well-pipetted DHFR and GFP synthesized, 3 × SDS sample buffer [150 mM Tris-HCl pH 6.8, 6% SDS, 0.2% (W / W) bromophenol blue, 30% (V / V ) Glycerol, 3% (V / V) β-mercaptoethanol] 20 μl, Milli Q water 15 μl was mixed and boiled at 98 ° C. for 5 minutes, and 9 μl was electrophoresed on a 12.5% polyacrylamide gel.
As a result of the above experiments, the amount of protein synthesized by the automatic protein synthesizer and the manual was similar.

  As is apparent from the above description, according to the present invention, it is possible to provide a method for automatically performing a cell-free protein synthesis reaction and a protein synthesizer that can be used in the method.

Claims (15)

In the cell-free protein synthesis step, in the reaction step from the transcription template to the production of the protein encoded by the template, the translation template in the reaction solution after the transcription reaction is precipitated, the supernatant is removed, and the translation template is dried. A cell-free protein synthesis method comprising, after the step, a step of dissolving the precipitate by directly adding a cell extract for protein synthesis to the precipitate. The cell-free protein synthesis method according to claim 1, further comprising a step of amplifying and preparing a transcription template encoding a target protein. 3. The cell-free protein synthesis method according to claim 1 or 2, wherein a host cell containing a gene encoding a target protein is directly subjected to a polymerase chain reaction to amplify a transcription template. The cell-free protein synthesis method according to any one of claims 1 to 3, wherein the reaction from the creation of a transcription template to the production of a protein encoded by the transcription template is automatically performed. The cell-free protein synthesis method according to any one of claims 1 to 4, wherein the translation reaction solution is layered on a protein synthesis cell extract containing a translation template to cause a translation reaction. The cell-free protein synthesis method according to any one of claims 1 to 4, wherein the translation reaction solution is added to and mixed with a cell extract for protein synthesis containing a translation template to cause a translation reaction. . The cell-free protein synthesis method according to any one of claims 1 to 6, comprising any one of the following steps.
(1) A step of amplifying a transcription template by PCR amplification;
(2) A step of mixing the transcription reaction solution and the transcription template;
(3) Incubating the mixture of the transcription reaction solution and the transcription template for a certain period of time;
(4) a step of precipitating the translation template;
(5) removing the supernatant in the reaction vessel (6) drying the translation template precipitate; and (7) adding the protein synthesis cell extract to the dried translation template, The process of dissolving. The cell-free protein synthesis method according to any one of claims 1 to 7, wherein all reaction steps are carried out in the same reaction vessel. The cell-free protein synthesis method according to any one of claims 1 to 8, wherein a plurality of types of proteins are simultaneously synthesized in a plurality of reaction vessels. The cell-free protein synthesis method according to any one of claims 1 to 9, wherein the protein extract for protein synthesis is a plant seed germ extract. The cell-free protein synthesis method according to claim 10, wherein the plant seed germ extract is a wheat seed germ extract. 12. The cell-free protein synthesis method according to claim 11, wherein the wheat seed germ extract is an extract from which the endosperm components of germ and the low-molecular protein synthesis inhibitor substance are substantially removed. A cell-free protein synthesizing apparatus for carrying out the cell-free protein synthesizing method according to any one of claims 1 to 12, comprising at least one of the following control means.
(A) means for variably controlling the temperature in the reaction vessel;
(B) means for dispensing a sample or reagent into the reaction vessel;
(C) precipitation means;
(D) means for removing the supernatant;
(E) Drying means; and (f) Control means for operating the means (a) to (e) according to the process according to any one of claims 1 to 12. The cell-free protein synthesizer according to claim 13, wherein the means for removing the supernatant is a means for performing an operation of turning the reaction container upside down. The cell-free protein synthesizer according to claim 13 or 14, further comprising means for opening and closing the lid of the reaction vessel.

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