TW200526786A - Automatic process for protein synthesis and apparatus therefor - Google Patents

Abstract

The present invention provides an automatic process for protein synthesis and apparatus therefore. According to the invention, a non-cellular protein synthesis process provides a sequence of operations from transcription template to formation of protein encoded by the template so that the translation template in the reaction solution is precipitated after the transcription reactions. After the procedures of removing the supernatant liquid and drying the translation template, the precipitation is directly added with a cell extract for protein synthesis. Subsequently, a procedure for dissolving the precipitation is performed. Also, an automatic protein synthesis apparatus capable of implementing the above-mentioned method is provided. In particular, an automatic protein synthesis apparatus capable of synthesizing a plurality of types of protein simultaneously is provided.

Description

200526786 发明 Description of the invention: [Technical field to which the invention belongs] The present invention relates to a method for automatically performing cell-free protein synthesis, and an automatic protein synthesis device for implementing the same. [Previous technology] With the rapid development of gene mix (Genomix) and the completion of chromosomal plans of various organisms, the focus of research topics has largely changed from the analysis of genetic factor structure to the analysis of genetic factor function. With the introduction and destruction of genetic factors whose functions have been clarified, Knock-out-based transgenic technology has brought great results in the manufacture of mutants with additional functions, but unknown genetic factors In terms of functional analysis, it is still based on the interpretation of information based on the expression form obtained through the black box of cells. This situation can be understood from the fact that only a few genetic factors can be identified by these techniques. In addition, genetic factors whose functions have been elucidated can almost be said to be evidence that they must be biochemical. Therefore, for the functional analysis of unknown genetic factors obtained from the chromosome, the biochemical analysis of genetic factor products can be said to be necessary. In terms of protein synthesis, genetic engineering methods that make Coulomb-like genetic factors present in living cells are widely used, but foreign proteins that can be obtained from this method are limited to sneaking into the host Molecular species of life support institutions. In addition, with the advancement of organic synthesis technology, automatic peptide synthesizers have become popular. It is common practice to synthesize peptides formed from dozens of amino acids, but chemically synthesize large-molecular-weight proteins. However, due to the limits of yield, side reactions, etc., it is extremely serious even at present. Moreover, in Europe and the United States, 200526786 directly uses the living form of living protein production and the ethical critique of the new molecular exploration method is extremely strict, and it is also worrying that international regulations will be more severe. To solve this problem, as a new type of protein synthesis method, a cell-free protein synthesis method can be adopted. This method uses a biochemical method to maximize the excellent characteristics of the organism. In this method, a replication system and a translation system for living genetic information are placed in an artificial container, and a system containing a non-natural form of amino acids that can be taken in from a designed and synthesized replication model is reconstructed. Since such a system is not restricted to living organisms, it is expected that the protein that can be synthesized can be expanded to almost infinity. This type of cell-free protein synthesis method has been reported 40 years ago that residual protein synthesis energy in ground cell fluid has been developed. Various methods have been developed, including cell extracts from coliforms, wheat germ, and rabbit reticulocytes. It is still widely used for protein synthesis. In particular, cell extracts from wheat germs and other cell extracts from eukaryotes are highly valued for their high protein synthesis energy and their precise protein retention. In order to clarify the genetic factors obtained from huge genetic information, it is expected that a High through VII synthesis system capable of synthesizing multi-sample proteins in a short time and easily will be presented. In the cell-free protein synthesis method, j can obtain the transcription reaction of the translation mode mRNA from the replication mode, k, and other projects that synthesize proteins by using the mRNA as a mold. Equipment for automatically performing these tasks has been developed. In addition, if a cell extract from a coliform belonging to a prokaryote is used, in order to enable it to perform a transcription reaction and a translation reaction in a valley fluid, the equipment for automatically synthesizing proteins from the transcription mode after the transcription reaction and translation reaction has been performed. Was developed. However, eukaryotic cell extracts such as wheat germs with excellent protein synthesis ability, as mentioned above, the translation model obtained from the replication reaction must unavoidably be operated manually before it can be used as a translation reaction. The series of operations before the protein f encoded by the 200526786 transcription board cannot be automated. That is, 'the past cell-free synthesis method using cell extracts from the eukaryotes mentioned above', because non-anti-filamental organisms that replicate reflections hinder subsequent translation reactions, 'it was considered necessary to remove them and perform alcohol Removal operations such as precipitation and gel filtration columns (such as the typical original protocol in the previous method, please refer to the projects bl2 to bl6, including: • ethanol precipitation using acetic acid (bl2), column treatment (bl3), Ethanol precipitation (bl4) using sodium chloride, ethanol precipitation (W5) using steel acetate, 70% ethanol precipitation (W6)). At this time, the salt contained in the buffer used for gel filtration will hinder subsequent translation reactions. Therefore, without reprecipitation and / or washing operations, at least a part of the protein will only be used. Get a little or no protein at all. However, if the re-sinking and washing operations are performed, the precipitation of the translation mold maintained by the surface tension at the bottom of the reactor will be peeled off. Using a centrifugal separator that can be used as an automatic synthesis equipment structure, the centrifugation will only become a liquid. In the floating state, in order to remove the supernatant without error, the experimenter must carefully carry out 0 with a pipette. In addition, in the past, once the refined translation mold was dissolved in ultrapure water and After the solution of the translation reaction, the solution of Kibe, energy source, etc. (for translation reaction) is mixed with a cell extract containing protein synthesis equipment such as ribonucleosomes, etc., and then provided to the translation reaction (Fig. 1, Project bl8, bl9) 'However, it is extremely difficult to dissolve the niRNA in translation nucleus for translation reaction> Grain fluid' The experimenter must stir each sample with the front end of a pipette to dissolve it. [Summary of the Invention] 200526786 Therefore, the object of the present invention is to solve the above-mentioned problems in the automation of cell-free protein synthesis, and to provide a series of projects from copy mode to protein generation encoded by the mold, from the production of copy mode to eggs The self-producing engineering makes it a secret method, and provides automatic protein synthesis equipment that can put this method into practice, especially automatic protein synthesis equipment that can synthesize multiple types of proteins at the same time. In order to solve the above-mentioned problems, the inventor of this case, after careful research, found that (1) Lai obtained after the replication reaction, after it was precipitated, the non-anti-filamentary sanitary process and the buffer from the domain were replaced, and the cleaning process was replaced. Even if Shen Dian Engineering is omitted, it will not affect the translation efficiency of the protein, and (2) the precipitation of the translation mold is not once dissolved in water or the solution for translation reaction, and when the cell extract for protein synthesis is directly added, the precipitation unexpectedly Dissolves extremely easily. Based on these ideas, the inventors have developed a new original proposal for cell-free protein synthesis (Protto), and successfully constructed an automatic protein synthesis device that can implement the original proposal, and even completed the present invention. That is, the present invention is as follows: 1. In the cell-free protein synthesis project, from the replication model to the reaction engineering of the protein encoded by the mold, the translation model in the reaction solution after the replication reaction is precipitated, and the supernatant is removed. After the liquid and the drying process of the turning mold, the process of immersing the plate by adding protein and fine cell extract directly to the precipitate, the cell-free protein synthesis method including the above method 〇 2 · Purpose The process of enlarging and then modulating the replication model of a protein protein is also included in the cell-free protein synthesis method described in item 1 above. 3- The host cell containing the genetic factor that symbolizes the protein of interest is directly supplied as a polymerase. 200526786 Chain reaction “Amplification of the remodeling process” is characterized by the cell-free protein synthesis method described in the preceding item 丨 or 2. 4. The method for synthesizing the cell-free protein described in any one of the preceding paragraphs 1 to 3, which is characterized by automatic progress, from the production of a replica model to produce a protein material encoded by the replica model. 5. Superimpose the translation inverse test on the egg containing the clams and extract the liquid from f. Then take the translation responder as the characteristic of the cell-free protein synthesis method described in any one of items 1 to 4 above. 6. A method for synthesizing a cell-free protein by adding a solution for translation reaction and mixing it with a cell extract for protein synthesis containing a translation mold to make it oily. 7. A method for synthesizing cell-free protein as described in any one of the items 1 to 6 below in accordance with any of the following items. (1) A process of modulating a replica mode by PCR amplification. (2) A process in which a solution for copying reaction is mixed with a copying mold. (3) A process of cultivating a mixture of the transcription reaction bribe and the replication mold—the cultivation process for a fixed time. (4) The process of precipitating translation dies. (5) The process of drying the deposit of the translation mold. (6) The process of removing the supernatant of the reaction solution, and the process of removing the eggs from their own quality and taking the remaining 6 jobs, and dissolving the positions. 8. Let all reaction projects enter the same reaction vessel. ^ ^ ^ 疋 仃 No. 200526786 Cell protein synthesis method described in any one of items 1 to 7. 9. A method of synthesizing a plurality of proteins simultaneously in a plurality of reaction vessels and synthesizing the cell-free protein according to any one of items 1 to 8. 10. The cell extract for protein synthesis described above is a cell-free protein synthesis method according to any one of the preceding items 1 to 9 of the plant seed germ extract. 11. The aforementioned plant seed germ extract is a method for synthesizing a cell-free protein as described in item i 0 of the wheat seed germ extract. 12. The aforementioned wheat seed germ extract, the endosperm component of the germ and the low-molecular-weight protein synthesis, and the extract from which the inhibitor material has been substantially removed. The cell-free protein synthesis method according to item 11 above. System A is a device for carrying out the cell-free protein synthesis method described in any one of the preceding paragraphs 1 to 12, and has a cell-free protein synthesis device having at least one of the following control means. (a) Means for variably controlling the temperature in the reaction vessel. (b) Means of separately injecting a sample or a reagent into a reaction container. (c) Shen Dianshou 丨}. (d) Remove the supernatant. (e) Drying means and (0 means the means (a) to (e) for controlling the engineering operation according to any of the preceding paragraphs 1 to 12 The means for performing the action is the cell-free protein synthesis device according to item 13 above. 200526786 15 · Furthermore, the cell-free protein synthesis device according to item 13 or μ is characterized by including means for opening and closing the lid of the reaction container. [Implementation method The present invention provides a method for automatically performing a reaction operation from at least a replication mode provided to a reaction system to the production of a protein symbolized by the mode (hereinafter sometimes referred to simply as "the method of the invention"). Here, the “making operation to be performed automatically” means that the experimenter does not directly perform manual operation on the reaction system (reaction valley) in a series of projects. Therefore, when each project is implemented, the automatic protein of the present invention is adopted The predetermined operation set on the synthesizing device is operated according to the description and the switch, and it is performed by the experimenter manually, without damaging the "automatic" sound piece in the present invention. The reaction capacity can be different in each of the X-paths. However, under the simplification of the equipment structure for carrying out the method of the present invention, and for the purpose of preventing staleness and product loss of scale, it is best to design a series of projects in the same process. The method is preferably carried out in a reactor. The method of the present invention is divided into a copying reaction process, a refined jade process of a translation mode, and the best translation reaction engineering. The method of the present invention is from the production of a copy mode to the encoding of the mode. The process up to the generation of proteins is characterized by an automatic performer. Therefore, in the best mode, the method of the present invention leads the replication reaction engineering, and also includes the production process of replication molds. Hereinafter, specific implementation of the sample for each process_ As mentioned above, but the method of the present invention is to refine the Wangcai of the Lai Lai, as long as it has the two characteristics mentioned above to the state, it is not allowed to be among them. 'This course is not necessary-it must be done automatically. The 11 200526786 copy core that can be obtained by Yang can also be used in the following automatic cylinder process, but it includes the process from the creation of the copy model to the protein encoded by this model. -The series of guarding processes is preferably performed automatically. The "replication mode" in the book of the present invention refers to the legs of the mold molecules that can be used for the in vitro replication reaction, at least in the order of the appropriate promoter. The downstream has the sequence of encoding the target protein (code). The so-called proper sequence of the promoter refers to the sequence of the promoter of the polymerase A used in the replication reaction, such as the SP6 promoter, T7 co-agent, etc. The DNA encoding the target protein may be any type. The replication mode preferably has an active base sequence that controls translation efficiency between the sequence of the co-agent and the mechanical base sequence encoding the target protein, For example, the Ω sequence of the cigarette-like virus can be derived from RNA virus 5, non-translated field, and / or Kusak sequence, etc. Furthermore, it is better to include the replication model in the mechanical base of the protein encoding standard. Downstream covers 3 areas of replication terminal, non-translated areas. The 3 ′ non-translated field is best to be about 3.0 kilobases from the downstream of the stop codon (CQdQn). These 3 ’untranslated fields do not necessarily have to be the original fields of the genetic factors encoding the target protein. The production of the replica mold can be performed according to a well-known method. For example, a host cell containing a DNA having the same salty sequence as the desired replica, a coliform having a cytoplasmic genetic body incorporated with the DNA, the host cell is cultured, and the cytoplasm is largely prepared by a well-known purification method. After the heredity (bl), use appropriate restriction enzymes to remove the copy model from the cytoplasmic heredity (say, followers), and then remove the restriction enzymes after phenol treatment (b3) and three-gas methane treatment (b4). A method of refining a replica using alcohol precipitation (b5) of ethanol and isopropanol (if necessary, adding an appropriate amount of ammonium acetate, sodium acetate, sodium vaporization salt, etc.). The precipitate of the obtained DNA is dissolved in ultrapure water (b6) and a solution for an overwrite reaction described later in 2005 2005786 to provide the following overwrite reactions. In order to integrate the required series of operations automatically or semi-automatically (in the case of a part of the project where the experimenter directly adds a manual operation to the reaction system), the existing equipment is incorporated into the automatic protein synthesis device of the present invention. After that, it can be performed automatically from the creation of the replica to the production of the target protein. It is considered that the purpose of the present invention is to provide an automatic cell-free protein synthesis system in order to analyze High through put, and the simplification of the equipment and shorten the time required. For example, it is preferable to use the following method for making a replica of a polymerase chain reaction (PCR) method. In the method of the present invention, the aspect of best practice is to use a host (for example, a coliform having a cytoplasmic heredity containing the DNA) in which the DNA encoded by the protein of interest is coulombized, and use the host directly for PCR. , Enlarge the copy mode. For example, a 5 untranslated sequence with controlled, appropriate aggregator sequence, translation efficiency activity, and the ancestor that encodes the protein of interest, and will contain oligonucleotides that are part of the 5 'end domain of the DNA, using DNA automatically After the synthesizer synthesizes it by a known method, it applies a sense primer, and then applies an anti-sense primer to a 3, untranslated sequence of 3, terminal domain sequence oligonucleotides, and uses it as a model to encode the DNA of the target protein. And coliform bacteria containing the 3 'untranslated cytoplasmic heredity downstream are directly added to the pcR reaction solution.' Under normal conditions, the amplified copy can be obtained to obtain the desired replica (al) . In addition, in order to prevent the generation of short-stranded DNA due to non-specific amplification due to non-specific amplification (as a result, the yield of the target product is reduced and the low-molecular translation product murmurs) are generated, the International Publication No. 02/18586 can also be used. The recorded co-agent break type primer. The amplification reaction can also be performed using a commercially available thermal cycler for PCR on a commercially available 96-well plate for PCR. The same temperature-variable control device and the protein of the invention since 13 200526786 can also be used. The synthesizing equipment is linked, or various methods are directly applied to the PCR, so as to perform the replication and translation reactions of the automatic protein synthesis equipment of the present invention. As described above, the replication mode DM is subjected to trichloride? After fresh extraction and alcohol Shen Dian refinement, it is also available for copying reaction. However, in order to simplify the equipment and shorten the required time, it is best to use the pcR reaction solution as a copying mold. In the production of replication models, by using direct PCR from the above-mentioned host, the cytoplasmic genetic body is modulated in a large amount and then subjected to restriction enzyme treatment to obtain a replication model. With this financial method, she can omit the ball. Lai Shao's number of balls synthesized a large number of duplication modules in a short time (see Figure 2). That is, there is no need to cultivate the large intestine g of the cytoplasmic heredity with the genetic factor of the edited target, and a large number of feminine hereditary organisms can be used. Therefore, the ultracentrifugation facility necessary for the cultivation and purification of the cytoplasmic hereditary can be shortened. It takes time (for example, as shown in Figures 1 and 2, ㈣ 办 敎 _ 转 办). χ, the key for the hereditary body to remove the replica model_: treatment (work hungry), benzene removal process to remove the yeast scales, three-gas torrefaction treatment (Weicheng b3 and b4), replica model refining Lai Shen Dian (Engineering Project, Shen Dian Project (Solution b6) that dissolves replica DNA, the above-mentioned projects can be omitted, so there is no obstacle to Lai-Wen's reaction due to phenanthrene / trichloromethyl domain retention, and Meida Wei复 The copy mode error from π in the refining operation, and the benefits of reducing the number of steps required for the reaction can be reduced. (2) Copy reaction engineering The method of the present invention includes modulating a method known per se. The copy DNA of the target protein is coded to generate a translation model 111 in vitro copy reaction. This project is a copy model containing a reaction system (such as a commercially available container such as a 96-well titer plate). Solution 14 200526786 'It is better to copy the above PCR reaction solution with RNA polymerase (such as SP6 RNA polymerase) suitable for the polymerization aid in replication mode, and the substrate for RNA synthesis (4 ribonucleoside 3 phosphates), etc. Ingredients required for reaction After mixing the solution (also referred to as a "solution for overwriting reaction") (a2, b7), the mixture is preferably cultured at about 20 ° C to 60 < > C 'about 3 (TC to 42 ° C) for about 30 minutes to 16 hours, preferably about 2 hours to 5 hours (a3, b8). Dispensing and copying the replica mold solution, the replication reaction solution, and the reaction container can be performed by the automatic protein synthesis equipment described below. Means (such as 'pipettes' (e.g. when using commercially available 96-well titer plates as reaction vessels, it is best to use wafers with 8- or 12-well dispensing intervals suitable for the Well interval). The heat preservation can be performed by a temperature control method of the automatic protein synthesis equipment described below, while controlling at a certain temperature. (3) The refining product of the translation mold refining process (ie, the "translation mold") generated by the 5, untranslated sequence and / or 3, untranslated sequence RNA molecules that encode the protein of the target protein in the transcription solution required to have the activity of controlling the translation efficiency inserted in the transcription mode. In the reaction solution after the transcription reaction 'Except for scaly face A, it's all over Anti-lying chicks 3 and anti-biochar filling acids, salts contained in other replication reaction solutions, etc., but these substances are known to hinder subsequent translation reactions, so unreacted substrates are separated after selective precipitation of the translation mold Removal. The method used in Shen Dian can be, for example, a method of equalization, and it is best to use the method of precipitation, but it is not limited. For example, when using alcohol precipitation method, if the alcohol used can selectively precipitate RNA, that is, There is no particular limitation. For example, ethanol, reversal, etc., and ethanol is the best. If it is ethanol, it is best to duplicate the reaction solution by about 2 to 3 times. If it is isopropanol, use the replication reaction solution. 6 times and 1 time. Also, the coexistence method of adding appropriate salt can increase the harvest of precipitation. This kind of salt can be 15 200526786 疋 Ammonium acetate, sodium acetate, sodium chloride, lithium chloride, etc. For example, if ammonium acetate is used, it is best to add the final concentration to about 0.5M to 3M. Alternatively, alcohol precipitation can be performed at room temperature. The addition of alcohol and salt solution can be performed using a dispensing method of an automatic protein synthesis device described later, and the precipitation of translation mold RNA can be performed under a certain temperature condition by using a temperature control method of an automatic protein synthesis device described later. In addition, transcription DNA also exists in the transcription reaction solution, but it has been reported that it will hinder the subsequent translation reaction. Therefore, DNase treatment (b9) and phenol treatment (bio), The operation of denaturing and removing DNase after three gas methane treatment (bii) (refer to the projects b9 to bll in the right column of FIG. 1). The present inventors found that in the method of the present invention, even if the replication mode coexists in the translation reaction liquid, the translation efficiency will not be reduced. In order to simplify the purification of the automatic protein synthesis equipment and shorten the time required, and to prevent the phenol / trichloride Residual methane hinders translation and due to errors in the translation model caused by the refining operations of many projects, DNase treatment is omitted. Therefore, the method of the present invention is characterized by a process of decomposing and removing the copy mode without performing the copy reaction. The translation-mode RNA thus precipitated can be deposited on the bottom of the reaction container (a4, bl2) by any method known per se. Such means may be, for example, centrifugation, filtration, standing, binding drying and the like, but centrifugation is preferred. If centrifugation is used, it can be carried out at a temperature below room temperature, preferably about 25C or less, more preferably about 4 ° C to 15 ° C, and about 4000xg to 22000xg. The centrifugation operation used can be performed as a precipitation means by combining a centrifugation method used in a well-known tabletop centrifuge or the like into an automatic protein synthesis apparatus described later. The translation mold that settled at the bottom of the reaction vessel under the initial sinking operation was maintained at the bottom due to surface tension. Therefore, the supernatant removal operation after the precipitation operation does not require the experimenter to manually enter 16 200526786 lines, for example, also It can be performed using well-known equipment (for example: BioRobot 8000 (manufactured by Kiagen)) or simply inverting the reaction vessel up and down. For example, considering the simplification of the equipment, shortening the required time, reducing the number of wafers used The latter method is more advantageous for automation. After removing the supernatant, the precipitation of the translation mold RNA is dried (a5, bl7). Drying is, if it exceeds the residual supernatant, it may become a component that hinders the translation reaction (such as alcohol). ) Is removed within the time required to cause insolubilization due to complete drying and does not reduce translation efficiency, the method and time are not particularly limited, but for example, natural drying, air drying, and reduced pressure drying By a method known per se, it can be carried out preferably for about 10 minutes or less, and more preferably for about 3 to 8 minutes. For the following translation reaction engineering, the cell extract for protein synthesis is used to dissolve it (a6). The cell extract for protein synthesis used here can be used to generate the protein encoded by the mold after translating the translation mold. Any extraction solution may be used, but specifically, cell extracts of coliform, plant seed germ, rabbit reticulocytes, etc. may be used. These may also be commercially available extracts, and the methods known per se, specifically, It can be modulated according to the method described in Pratt, JM, etal, Transcription and Tranlntion, Hames, 179-209, BD & Higgins, SJ, eds), IRL Press, Oxford (1984). Commercially available cell extracts for protein synthesis may be those derived from coliforms such as E. coli i S30 extract system (promega) and RTS 500 Rapid Tranlation.

System (manufactured by Roche), those from rabbit reticulocytes such as Rabbit Reticulocyte Lysate Sytem (promega), and wheat germ 17 200526786 buds (such as PR0TEI0STMCT0Y0B0). Among them, it is best to use a plant seed bud extract liquid, and the first plant seeds are preferably wheat, barley, rice, corn and other rice plants and spinach seeds, which are the most suitable for wheat seed material. In addition, wheat seed germ extracts from which the endosperm of the germ is wounded and eggs with low 77 seeds are substantially removed from the substance that inhibits the synthesis of the substance are more suitable. Because these are related to the extract of overcut wheat seed germ, the components and substances related to the protein synthesis hindrance in the extract are reduced. Wheat germ extract can be prepared by methods such as Johnston, F.B. etal, Nature, 179, 160 ^ 161 (1957), ilErickson, AH etal., (1996) Meth. In Enzymol., 96, 38 -50 and other methods. In addition, removal of inhibitors of translation factors contained in the extract, such as endosperm containing trisulfazine, sulfur, nuclease, etc. (Japanese Patent Laid-Open No. 2000-236896, etc.), and suppression of activation of inhibitors of translation factors (Japanese Patent Application Laid-Open No. 7 2 03984) and the like are preferably performed. If an extraction solution of plant germ such as wheat germ is used, the extraction solution may be added before the precipitation of the translation mold, for example, at a temperature of about 15 ° C ~ 26 ° C for more than 3 minutes to increase the translation efficiency. The cell extraction solution for protein synthesis can also be directly added to the precipitation of the translation mold, or other components required or extremely suitable for the translation reaction, that is, amino acids, energy sources, various ions, buffers, ATP used as a substrate One or more of a regeneration system, a nucleic acid degrading enzyme inhibitor, a vα, a reducing agent, a polyethylene glycol, 3,5, a cAMP, a folate, an antibacterial agent, etc. are mixed with the cell extract before adding the precipitate. The cell extract for protein synthesis is directly added to the precipitation of the translation mold, which is easily soluble 18 200526786. Therefore, for example, 'the dispensing method of the automatic protein synthesis device described later is used, the cell extract is added to the precipitation of the translation mold, and then it is left to stand or the dispensing method is designed to be a liquid mixing method (e.g., pipetting) , Stirring, etc.), under this mixing operation, the following translation reaction process can be started in a short time. (4) Translation reaction engineering is added to a cell extract for protein synthesis containing a translation mold obtained by the above method, and contains amino acids, energy sources, various ions, buffers, ATP regeneration systems, nucleic acid degrading enzyme inhibitors, and tRNAs that serve as substrates. , Reducing agent, polyethylene glycol, 3,5, cAMP, folate, antibacterial agent, etc., the translation reaction must be or the most suitable solution (also known as "translation reaction solution") to suit the translation reaction After incubation at the appropriate temperature, the translation reaction can proceed. The amino acids that form the matrix are generally the 20 natural amino acids that make up proteins, but analogies and heterosexuals can also be used depending on the purpose. As the energy source, ATP and / or GTP can be used. Each ion includes acetate, magnesium acetate, ammonium acetate, and other acetic acid salts, and glutamine salts. The buffer solution was Hepes-K0Η, Tris ~ acetic acid, etc. The APT regeneration system is a combination of phosphoenol acetone and acetone kinase, or a combination of creatine (creatine phosphate) and creatine kinase. Nucleolytic enzyme inhibitors include ribonuclease inhibitors and nucleic acid S # inhibitors. A specific example of the ribonucleic acid inhibitor is RNase inhibitor (manufactured by TOZO 0B0, etc.) from a human placenta. The tRNA can be obtained by the method described in Moniter, R., et al., Biochim. Biophys. Acta, 43, 1 (1960), or a commercially available finished product. Reducing agents such as DTT (Dithiothreito) and so on. Antibacterial agents include sodium azide and ampicillin. The amount of these additives can be appropriately selected within a range generally available for synthesis of cytoproteins. The addition of the solution for translation reaction may be appropriately selected depending on the translation reaction system 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 synthesis device of the present invention, such as a program group method (BatchXPratt, J. M et al.

Transcription and Tranlation, Hames, 179-209, Beta D & Higgins, J., eds), IRLPress, Oxford (1984)), and continuous cell-free protein synthesis method in which amino acids and energy sources are continuously supplied to the reaction system (Spirin, AS etal., Science, 1162-1164 (1988)), dialysis method (Kikawa et al., 21st Japanese Molecular Biology Society wiD6), or multi-layer overlap method (International Publication No. 02/24939). In addition, if necessary, mold RNA, amino acid, energy source, etc. are supplied to a synthetic reaction system, and a discontinuous gel filtration method in which synthetic and decomposed substances are discharged when necessary (Japanese Patent Application Laid-Open No. 2000-333673) and a synthetic reaction The tank is prepared with a molecular sieve-capable support, and the synthetic material and the like are developed using the support as a mobile phase, and a synthetic reaction is performed during the development to recover the synthesized protein (Japanese Patent Application Laid-Open No. 2000-316595). . However, from the point of view of the provision of a multi-sample simultaneous synthesis system applicable to the structure of an automatic protein synthesis device, which is simplistic, space-saving, low-cost, and high-throughput analysis, a program group method or a multi-layer overlapping method may be preferred. From the standpoint of obtaining a larger amount of protein, the multi-layer overlap method is particularly useful. When a translation reaction is performed by a program-level method, the solution for translation reaction is added to a cell extract for protein synthesis containing a translation mold and mixed. Alternatively, when the components contained in the solution for translation reaction are mixed with the cell extract for protein synthesis in advance, the addition of the solution for translation reaction may be omitted. 20 200526786 "translation reaction solution" obtained by mixing a cell death solution for protein synthesis containing a transdermal mold and a solution for translation reaction. For example, when wheat germ extract is used as a cell extraction solution for protein synthesis, 10 can be used. ~ SOmMHEPES-K0Η (PΗ7 · 8), 5 5 ~ 1 20mM potassium acetate '1 ~ 5mM magnesium acetate, 0 · 6mM spermidine, each 0.025 ~ ImM L-amino acid, 2 ~ 7〇μM, most Fortunately, DTT of 3 ～ 5 ～ _, 1 ～ 1 · 5πιΜ ΑΡΡ, 0.2 ～ 0 · 5πιΜ GTP, 1Q ~ 2QmM creatine, 0.5 ~ 1 · OU / μ 1 ribonuclease inhibitor, 〇 〇1 ～ 10μM protein disulfide isomerase, and those containing 24 to 7 5% wheat germ extract. When using this translation reaction solution, 'pre-cultivate at about 10 to about 40 ° C for about 5 to 10 minutes. The culture of this reaction (translation reaction) is the same at about 10 to 40 ° C, preferably about 丄8 ~ 3 (rC, more preferably about 20 ~ 2 6 ° C, until the reaction is stopped, it is generally performed by the program method χ 〇 minutes ~ 7 hours 〇 When the translation reaction is performed by the multi-layer overlapping method, when the translation reaction is included In the cell extract solution for protein synthesis of the mold, the solution for translation reaction is multilayered to avoid disturbing the interface, and then the protein is synthesized (a7, b20). Specifically, for example, if necessary, a protein for protein synthesis is precultured for an appropriate time. The cell extract is added to the precipitation of the translation mold to dissolve it to form a reaction phase. The reaction reaction solution (supply phase) is layered on the upper layer of the reaction phase to avoid disturbing the interface, and then the reaction is performed. The interface of the two phases It is not necessary to use multiple layers to form a horizontal plane, and the mixed liquid containing two phases can be centrifuged to form a horizontal plane. If the circular interface diameter of the two phases is 7ππη, the capacity ratio of the reaction phase to the supply phase is 1: 4 ~ 1: 8 for Appropriate, but 1: 5 is the most appropriate. The larger the interface area formed by the two phases, the higher the material exchange rate under diffusion, and the higher the protein synthesis efficiency. Therefore, the capacity ratio of the two phases will depend on the interface between the two phases. The area varies. Translation reaction, for example, when using a wheat germ extract 21 200526786 liquid extraction system, under static conditions, at about 10 ~ 40 ° C, preferably about 18 ~ 30 ° C, more preferably It is about 20 ~ 2 6 ° C, and usually can be carried out for about 10 ~ 20 hours (a8, b21). In addition, when using coliform extract, the reaction temperature is about 30 ° C ~ 3 7 ° C is appropriate The process of cell-free protein synthesis of the present invention involves the following steps: (1) the process of precipitating the translation mold in the reaction solution after the replication reaction, the process of removing the unreacted substrate, and the subsequent 1 or Several washing processes and precipitation processes were omitted to provide translation reactions, and (2) by adding the cell extract for protein synthesis directly to the precipitation of the obtained translation mold, the method of dissolving the precipitation was completed for the first time by automation Copy mode to protein protein encoded by this mode Become a stop-series operation. However, each of the above-mentioned features of ⑴ and 各自 are independent new methods and obtain beneficial effects. Therefore, as long as one of these methods has the characteristics of the other and has the characteristics of the other cell-free Protein synthesis method (that is, the problem that is only covered by the characteristic part of the other party 'is solved by other means The "cell-free protein synthesis method" performed manually is also included in the present invention. Other Γ㈣, this method _, only _ and ⑵ and 7 or __ above, and only need to be combined, as long as _ can be turned over A well-known engineering order and conditions, such as one engineer Satoshi / 1 'But in addition to the above-mentioned ⑴ and __, there are at least the following processes to modulate the replication mode by PCR amplification: • Mixing the solution for the replication reaction with the replication mode Engineering • Project in which the mixture of the solution-producing mold for replication reaction is cultivated for a certain period of time 22 200526786 的 Engineering for precipitating the replication product (that is, the translation mold) Synthetic cell extract is added to the dried translation mold to dissolve the translation model. • The cell extract for protein synthesis containing the translation mold is multi-layered and overlapped with the reaction solution for translation.% Project • The protein synthesis cell containing the translation mold is used. Extraction solution is multi-layered and the reaction solution is cultured for a certain period of time, or • the replication mode is amplified by PCR, and the process is prepared by mixing the replication reaction solution with the replication mode. The process of cultivating the mixture of replication molds over a certain period of time ♦ The process of precipitating the replication products (that is, the translation molds) _ The process of drying the precipitation of the translation molds Add the cell extract for protein synthesis to the dried translation molds, Engineering of translation model dissolution ♦ Engineering of protein synthesis reaction by program method If the cell-free protein synthesis method of the present invention is used, the experimenter can perform the cell-free protein synthesis reaction automatically without having to perform manual operations in the middle. In addition, if the method of the present invention is adopted, the protein synthesis efficiency is not different from that in the past, and the process required for the entire reaction can be reduced (from an automation point of view, reducing the number of processes means that errors are less likely to occur, and it is also important) It can greatly reduce the time required for protein synthesis. At the same time, it can also reduce the loss under decomposition. 23 200526786 Translation mode can reduce the amount of DNA used and so on. Model, and it can reduce the replication reaction system.

The reaction container used for the reaction and translation reaction, and if the method of the present invention is used, for copying, for example, a 96-well plate can be used at the same time to synthesize the surface (especially most) protein f, which can be used as a protein as described later. Functional analysis of through put and other uses.

(a) means for variable control of the temperature in the reaction chamber (b) means for separately injecting samples or reagents into the reaction container (c) means for precipitation (d) means for removing the supernatant (e) means for drying; and (f) Means for controlling the above (a) to (e), and the means for controlling the operation of the method according to the present invention using the equipment having at least (a) to (f) can synthesize the above-mentioned cell-free protein of the present invention The reaction proceeds automatically. Each component will be described in detail below. (a) Means for variable control of the temperature in the reaction container The means for variable control of the temperature in the reaction container is a replication reaction, culture of translation reaction, precipitation of translation mold, or automatic protein of the present invention When synthesizing equipment to automate the process of making replicas of the PCR method, when the amplification reaction of the PCR method is performed, etc., the reaction method of the container _ liquid temperature adjustment _ when the temperature conditions are used. Although the temperature range of the variable control is not particularly limited, in the cell-free protein synthesis f_ series reaction operation including the production of replica molds, if the temperature range generally regarded as necessary (for example, about 4 ° C ~ 100 ° c, preferably within 26 ° c ~ 99 °, the reaction volume 11_ liquid temperature can be controlled by means of variable control, if used as a means to achieve it, there is no limit. For example, TA_A is known in the past pGR_Tantan (manufactured by Tak Shipbuilding Co., Ltd.), Gene Amp PCR System 9700 (manufactured by Applied Biosystems Inc., etc.), etc. Specifically, for example, in a place where suction is performed, and a reaction vessel is placed there. Separate the stations, set several workbenches for reaction vessels in the equipment, and control the temperature of the entire space on the workbenches variably, and finally you can achieve the variable control of the temperature in the reaction vessel. (B) In The method of injecting samples or reagents into the reaction container each year is a method of injecting samples or reagents into the reaction container separately. In the reaction container, a series of cell-free protein synthesis reactions, such as replication reactions, translation reactions, and PCR, are performed. Means of injecting a sample or a reagent into a reaction container respectively. Here, "sample" refers to a copy model, a translation model, a PCR model cytoplasmic heredity (or a host having a cytoplasmic heredity (such as coliform)), etc. `` Reagent '' refers to the solution for copying reaction, cell extract for protein synthesis, solution for translation reaction, alcohol, salt solution, solution for PCR reaction, etc. If the dispensing method used is different according to the engineering, the amount of reagent can be divided. Dispensers after adjustment can be achieved without special restrictions by using pipette arms, etc., which are known to be suitable for automatic dispensing in the past. In addition to the above-mentioned functions, the dispensation method can be used to homogenize two or more solutions and dissolve Shen Dian. Other mixing functions (such as pipetting, stirring, etc.) are better. (C) Precipitation method 25 200526786 The so-called Shen Dian method system allows appropriate objects in the reaction volume n (such as copying the translation model after the reaction) to be laudable. Gu The Shen Xuan section of the Shen Xuan section can be achieved by appropriate means known to the public, such as the above-mentioned object Shen Dian, and can be separated. Precipitation can be accomplished by centrifugal separation known in the past, and other appropriate equipment used in filtration and freeze drying. (D) Means of removing the supernatant The method of removing the supernatant is by ± Hearing means, after the reaction capacity is appropriate for Shen Dian, the method of removing the supernatant should be separated from the Shen Dian and the supernatant. In the device of the present invention, the means for removing the supernatant can be used, for example, to perform a flip reaction The means of the container only attracts the hands of the upper pi & etc. in the reaction trough. That is, as described above, if the cell-free protein synthesis method of the present invention is used, the translation in the reaction solution after the replication reaction is performed After the mold precipitation process is completed, the process of removing the unreacted substrate and the subsequent cleaning process or precipitation process are omitted. To provide translation reactions, the translation mold (mRNA) can be attached to the bottom of the reaction container. In the state for dry engineering. Therefore, the above-mentioned means for removing the supernatant can be used to automatically separate the supernatant from the precipitate without losing the translation mold. The means for removing the supernatant is specifically, for example, in order to reverse the reaction container. For example, a rotating power such as a motor can be used to directly fix the reaction container on the plate and the device can be reversed. The above-mentioned devices can be achieved by turning the robot arm device and the like. In addition, the means for attracting only the supernatant in the reaction container can be achieved by using a suitable tool known in the past such as BiROBot8000 (manufactured by KiAGen). (e) Drying means 26 200526786 The so-called drying means is, for example, when drying by a method other than natural drying. Dry, touch and finish. However, the automatic egg self-synthesizing method and equipment of the present invention can also use an overly unknown drying device that can be combined with a fairy part or connected to the outside. (f) Control means The control means includes operation switches and operations of a drive source (motor, hydraulic, hydraulic equipment, other actuators that can control operation) that can control each of the means for causing the operations of ⑷ to ⑷. The extent and shape of the fresh ㈣ equipment. The result is that according to the method of the present invention (for example, the cell-free protein synthesis reaction in the order shown in FIG. 2), the actions of the means of commenting (a) to ⑹ can be controlled to make the X-path of the method Performed to achieve the purpose of this method. The aforementioned control system includes, for example, a computer with an age-controlling computer, a frequency control circuit, and the like, which control the age necessary for the operations of the above-mentioned means, and can also be combined to form a method according to the method of the present invention. In the sequence, the above-mentioned means are operated, and each means can provide power, air pressure, oil pressure and other control structures according to the signal and needs. In addition, a driver necessary for sending a direct driving signal to a driving source of each of the above-mentioned means, and various sensors, switches, etc. necessary for detecting the state of the driving source of each of the above-mentioned means may be appropriately added. There are no particular restrictions on the reaction vessels applicable to the apparatus of the present invention, and various conventionally known reaction vessels that can be used for cell-free protein synthesis reactions, such as 96-well pCR plates, 96-well titer plates, 8 hoses and hoses (a 5m L, 15m L, 50m L, etc.), but when using the program group method and multi-layer overlapping method as a translation reaction system, you can use a smaller reaction system such as a 96-well plate for translation In addition, if the method of the present invention is adopted, a smaller reaction system can also be used for the replication reaction. Therefore, several reaction systems can be used for simultaneous replication of several proteins. 27 200526786 Reaction 'refining of translation model, translation reaction, and as required A large number of proteins can be synthesized in a short time by performing a series of reaction operations including a cell-free protein synthesis method including PCR for making a replication model for a replication reaction. In addition, when the copying reaction and the translation reaction are performed, it is preferable to carry out the reaction container in a closed state. From this viewpoint, it is preferable to use a covered reaction container, and the device has a means for controlling the opening and closing of the lid of the reaction container. Those are better. In the case of the above-mentioned lids, for example, when a 96-well plate is used for a reaction container, a rubber lid can be used to seal each well separately. In the closed state, it is better to close the lid to the reaction container, so a lid with a certain weight (for example, about 500 g) can be used, or the lid and the reaction container can be clamped with something like a clip Close the lid again. In addition, the method of performing the opening and closing of the descent of the descent can be achieved by, for example, using a conventionally known device that combines a chuck, a suction device, and a robot arm. The automatic protein synthesis device of the present invention may have a means for storing a reaction reagent, in addition to the above-mentioned means, if necessary. In addition, in the case of mRNA drying engineering, if it is dried by a method other than natural drying, it may also have a means for drying (for example, a centrifugal dryer or an evaporator). As described above, the method and apparatus of the present invention can synthesize several kinds of proteins easily and automatically at the same time. For example, it is very useful to have several copy models and translation models that encode the proteins of various variants and synthesize several proteins of several variants at the same time, which can be used for analysis without detailed design of the variants, which is extremely useful. In addition, the method and equipment of the present invention can be extremely suitable for providing functional analysis of high thrugh put of various proteins. For example, the results of a human logic search, using the apparatus of the present invention to synthesize the __subgroup # containing the encoded common 28 200526786 domain (such as kinase collar scale) protein as a model, using the device of the present invention, In addition, the protein group (such as replication factor) that can be the target of acidification is synthesized in the same way, and the two are mixed in various combinations. For example, 32P-labeled ATP is taken as an index, and what kind of protein can be identified. What proteins are phosphorylated by protein kinases. Or 疋, contains specific topics in the transcription factor, such as & shirt, leucine

Zipper, etc.), using its encoded genetic factor group as a model, turn the device of the present invention, and synthesize the protein at the same time according to the method of the present invention, which can be combined with the known ciselement arrangement and other replication control Factors such as the formation of heterodimers of factors and the binding force with the replication control field of specific genetic factor promoters can obtain crosstalk information unraveling the replication @ 子 所 woven. Examples The present invention will be specifically described below by way of examples, but the present invention is not limited to the following examples. Experimental Example 1 · The effect of translation efficiency after washing with 70% alcohol in the mRNA precipitation produced by ethanol precipitation. (1) Modulation of translation mode mRNA The GFP genetic factor for the jellyfish and the DHFR genetic factor of E. coli were inserted into the wheat germ cell line-specific cytoplasmic heredity vector pEU, respectively, and used as replication models. SP 6 RNA was used for polymerization. Enzyme (promega) overwrite reaction solution (8 OmM Hepes-K0Η, 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 3 mM NTP, ΐυ / μ1 SP6 RNA polymerase, 1U / μΙ Rnasin) performed a copy 29 200526786 response. The obtained mRNA was divided into two parts, and after being precipitated with ethanol using ammonium acetate, the precipitated mRNA was washed with 70% alcohol and the unwashed one was used as a translation mold, respectively. (2) Using wheat germ cell-free protein synthesis system (multi-layer overlapping method) to synthesize proteins to prepare a translation reaction solution containing wheat germ extract 5.8 μl 25 μl (individual final concentration is 2 9 mM Hepes-KOΗ (ρΗ7 · 8), 9 5 mM potassium acetate, 2.7 mM magnesium acetate, 0.4 mM spermidine (manufactured by Nakley), 20 kinds of each 0.23 mM L-amino acid, 2.9 mM DTT, 1.2 mM ATP (Wako Pure Chemical Industries, Ltd. Manufactured), 0.25 mM GTP (manufactured by Wako Pure Chemical Industries, Ltd.), 15 mM creatine phosphonate (manufactured by Wako Pure Chemical Industries, Ltd.), 0.9 υ / μΙ RNa seinhibit οr (manufactured by TAKARA), 50 η g / μΙ t RNA (Moniter, R., et al., Biochim. Biophys. Acta., 43, 1-(1960)), 0.46 μθ / 1 creatine kinase (manufactured by Roche), 2nCi / pll4C-leu (Morra (Made by Baker)) 25 μl. The above-mentioned Shendian mRNA was washed, and the unwashed mRNA was subjected to a protein synthesis reaction by a multi-layer overlapping method. In the above translation reaction solution, a translation mold of 8 Mg / μ 1 was added as the lower phase, and the multiple layers overlapped the upper phase (individual final concentrations were 2 9 mM Hepes-K0Η (Ph7 · 8), 9 5 mM potassium acetate, and 2 · 7ιηΜ acetic acid). Jin, 0.4mM spermidine (manufactured by Naklai Co.), 0.23m each of 20 types of 熥 1 type amino acids, 2.9111] ^ 017, 1.21111 ^ octabutane? (Manufactured by Wako Pure Chemical Industries, Ltd.), 0.25mM GTP (Wako Pure Chemical Industries, Ltd.) (Manufactured by the company), 2nCi / M14C_leu (manufactured by Morabeck Co.), and cultivated at 25 ° C for 16 hours at 26 ° C. After the reaction started, 5 μl of the translation reaction solution after 16 hours was dripped on the filter paper, and the solid support method was adopted to use a liquid flash counting tube (LS6000IC · Beckmencott Co., Ltd.) to measure 14C- leu. Into. Among the two genetic factors, DHFR and GFP, there was no difference in the amount of target protein synthesis between those treated with ethanol and those who were not treated with ethanol. This phenomenon indicates that in a protein synthesis system (multilayer overlapping method), the washing treatment with 7⑽ alcohol in the wish-like precipitate produced by ethanol precipitation has no effect on translation efficiency. The above results are not limited to the overlap method, and can be applied to all other synthesis methods of wheat germ cell-free protein. From the above, it can be seen that even if the 70% alcohol washing process of mRNA precipitation performed in the past is deleted ', it will not have any effect on the translation efficiency. This project, which may cause the disappearance and reduction of translation molds, will not only improve the reproducibility of the target protein synthesis amount, but also reduce the operation time after deletion. These are particularly effective for fully automatic protein synthesizers that process a large number of samples. Experimental Example 2: Solubility of Milli Q water and protein extraction solution for _A particles (Pellet). PCR was performed on PEE-DHFR using a Sense primer and an anti primer, and an mRNA that can be copied onto the mold was used. The reaction system was prepared by 400μ1, and the replication was changed to 80mM Hepes-K0Η, 16mM acetate, 2mM spermidine, 10InMDΠ, 3InMNTP, 1U / μl SP6 RNase polymerase.

Synthase, 1 U / μ 1 Rnasin, 10% P CR product, cultured at 37 ° C for 3 hours. When the mRNA is labeled with the radioisotope 32P, adjust the UTP concentration to 1.2 mM, add [α-32P] UTP 8μ1, and adjust the reaction system to 400μ1. The mRNA labeled with 32P was added with 53 μl of 7.5 M acetic acid and 1 ml of ethanol, followed by careful stirring, and centrifuged at 20,000 × g for 15 minutes. Pellet was dissolved in 100 μ 1 of Milli Q water, and NICK 31 200526786 equilibrated with 100 mM sodium gas, 10 mM Tris-HCl (pH 7.6), and 0.5 mM EDTA 3 m 1 was added in advance.

Column (Amacia Biosience), 400 μl of 100 mjy [sodium chloride, 10 mM Tris-HCl (pH 7.6), 0.5 mM EDTA, and washed with 100 μm of 100 mM sodium chloride, i. m M Tris-HCl (pH 7 · 6) and 0.5 mM EDTA were dissolved and purified. MRNA 2 3μ1 not labeled with 32P and mRNA 2μ1 labeled with 32P are added to Milli Q water 2 5μ1, 7. 5mM ammonium acetate 7. 7μ1, ethanol 1 4 4μ1, carefully stir, and perform 20,000xg for 15 minutes Centrifuge. Remove the supernatant air-dried particles (peiiet), plus

Milli Q water 6.25μ 1, or 30 mM Hepes-KOH (pH 7.8), 1.2 mM ATP, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM DTT, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM acetic acid, 100 mM potassium acetate, 0.005% sodium disulfide, 4.0 ng / μ 1 creatine kinase, adjusted to an optical density of 260 nm (0. d.) (A260) Translation reaction solution of wheat germ extract of 60, 25 μ1. The solution 5 μ 1 after 5 minutes was measured by the Cherenkov method. The above experimental results are shown in Figure 3. The values before ethanol precipitation were 66143 for the extraction solution and 66741 for the Mi 11 i Q water. The cpm value after dissolution is 22,285 for the extraction solution, and 13,828 for the Milli Q water. It can be seen that the extract is easier to dissolve than Milli Q water. Experimental Example 3: Protein synthesis method using automatic protein synthesis equipment Using the automatic protein synthesis equipment developed to implement the method of the present invention, the following subjects were selected for cell-free protein synthesis, respectively. • Copy mode DH U and GFP composed of pe U cytoplasmic genetic media using Sense primer and Anti sense primer to perform D CR from 3 Ομί system P CR (containment 32 200526786 on 9 6Well P CR board) _ for copy reaction The solution contains a final concentration of 8 OmM Hepes-K0Η, 16 mM magnesium acetate, 2 mM spermidine, 10 mMDTT, 3 mM NTP, 1 U / ml SP6 RNA polymerase, 1 U / m 1 Rnasin, and a solution for ethanol precipitation 1111 ( } Water 3.541111, 7.5] ^ A mixed solution of acetic acid 1.3 21111 and ethanol 24.66111 1_The solution for translation reaction contains a final concentration of 30 mM Hepes-KOH (pH 7.8), L 2mM ATP, 0.25 • ♦

*. mM GTP, 16 mM cranial acid, 2 mM DTT, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2. 7 mM acetate, 100 mM potassium acetate, 0.005% sodium disulfide, 400 η g / μΐ Creatine kinase, a solution of wheat germ extract with an optical density (OD) (A260) of 60 units at 260 nm_Multi-layer overlay solution prepared to a final concentration of 3 OmM Hepes-KOH (pH 7.8 ), 1.2 mM AT P, 0.25 mM GTP, 16 mM phosphocreatine, 2 mM DTT, 0.3 mM spermidine, 0.3 mM 20 amino acids, 2.7 mM acetate, 10 mM ethyl acetate, 0.005% Sodium disulfide multi-layer superimposed liquid reaction container can withstand centrifugation, 2 pieces of round bottom 96-well microplates 33 200526786 _Dispensing wafers 2 0 0μ 1 day piece 9 6 pieces 51 mesh (replicate reaction liquid Dedicated, special for translation retrospective solution 1 phase, x2 microplate for ethanol Shendian, χ2 microplate for multilayer overlap), 2 wafers 9 6 pieces 2 boxes (1 # χ 2 microplate for PCR products) ethanol Shendian supernatant recovered Microplate and cell-free protein synthesis is performed in the following order: (1) Move the PCR plate from the cooling stage to the workbench

(2) Move the microplate from the cooling stage to the workbench. (3) Remove the cover of the PCR plate. (4) Remove the cover of the microplate. (5) Dispense 22.5 μl of the reaction solution on the microplate. (6) Transfer the PCR product of the PCR plate 2.5 μb and transfer it to the microplate 10 times. (7) Cover the PCR plate. (8) Cover the microplate.

(9) Return the PCR plate to its original position. (10) Incubate the microplate at 37 ° C for 3 hours. (11) Open the microplate cover and place. (12) Dispense the ethanol precipitation mixed solution 147.6 μl in a microplate. Pipet 10 times. (13) The microplate was centrifuged at 1,000 × g without a lid for 30 minutes. (14) After the plate covered with absorbent cotton is turned over on the microplate, the supernatant is removed. (15) Allow to dry for 5 minutes. (16) Dispense 25 μ1 of the translation reaction solution on the microplate. 34 200526786 (17) On the microplate, place the superimposed / overlap solution on the upper layer of the translation reaction solution 25μ1 slowly and print 2 ^ 2 (18) Close the lid on the microplate. (19) Move the microplate to 26. 〇 Incubate for 20 hours. Protein synthesis was performed as described above, and for comparison, manual protein synthesis was performed.

FIG. 4 is an SDS-PAGE photograph of the above-mentioned test results. The amount of protein synthesis was confirmed from SDS-PAGE. Pipette the synthesized DHFR and GFP carefully 10μ1, 3xSDS Sample buffer [150mM

Tns-HCl pH 6.8, 6% SDS, 0.2% (W / W) indigo blue, 30% (V / V) glycerol, 3% (v / V) β-mercaptoethanol) 20 μ 1. Mix 15 μl of Milli Q water, boil 9 μl at 98 ° C for 5 minutes, and perform electrophoresis on a 12 · 5% polyacrylamide gel. The results of the above-mentioned test were the same as those of the automatic protein synthesis equipment and the amount of protein synthesis performed manually. Industrial feasibility As can be seen from the above description, if the present invention is adopted, a method for automatically performing a cell-free protein synthesis reaction 'and a protein synthesis device that can be used for the method can be provided. [Schematic description] Schematic description: Figure 1: Schematic diagram of the engineering process and required time. Figure 2: Schematic diagram of the engineering process and the time required for the method of the present invention. Figure 3: A graph of the experimental results of Experimental Example 2. Figure 4 is a photograph of the SDS-page of the experimental result of Experimental Example 3. Explanation of drawing number: (bl) Mass modulation of this cytoplasmic heredity 35 200526786 (b2) ------ Restriction enzyme treatment of cytosolic heredity DNA (b3) ------ Phenol treatment

Cb4) ------ Trichloromethane treatment (b5) ------ Ethanol precipitation using sodium chloride (b6) ------ Dissolve DNA in ultrapure water (al)- ---- Copying coliform directly after PCR treatment

(a2, b7)-Mixed with the solution for replication reaction (a3, b8)-37 ° C, 3 hours incubation (b9) ------ DNase treatment (blO) ----- phenol treatment (bll ) ----- Trichloromethane treatment (a4, bl2)-ethanol precipitation using ammonium acetate (M3) ----- split column treatment

(M4) ----- ethanol precipitation using sodium chloride (bl5) ----- ethanol precipitation using sodium acetate (M6) ----- 70% ethanol precipitation (a5, bl7)-drying the mRNA (bl8) ----- dissolving mRNA in ultrapure water (a6, bl9)-mixed with cell extract for protein synthesis (a7, b20)-multi-layer overlapping translation reaction solution (a8, b21) --- --26 ° C, 20 hours incubation 36

Claims (1)

200526786 Scope of patent application: 1-An automatic protein synthesis method, which is used in the cell-free protein synthesis engineering, from the replication model to the reaction engineering of the protein generated by the model, so that the replication reaction limb should be in the fluid. After the translation model is precipitated, after removing the impurities, you can directly add protein to the cell and extract it in the extraction test. It contains a method of protein synthesis that dissolves the Shen Chengwei process. 2. The cell-free egg self-synthesizing method according to item 1 of the patent application, which also includes a process of enlarging the mode of the target protein protein by copying the mode and then modulating the mode. 3 The cell-free protein synthesis method according to item 2 of the scope of the patent application, wherein the host cell containing the genetic factor encoding the target protein is directly used as a polymerase chain reaction to enlarge the copy mode and modulate the project. 4. The cell-free protein synthesis method according to any one of claims 1 to 3 in the scope of the patent application, wherein the reaction from the creation of the replication model to the generation of the protein encoded by the model is performed automatically. B. The cell-free protein synthesis method according to any one of claims 1 to 3, wherein a translation reaction solution is 'multilayered on a cell extraction solution for protein synthesis containing a translation mold' to perform a translation reaction By. 6. The cell-free protein synthesis method according to any one of claims 1 to 3, wherein a translation reaction solution is added and mixed in a cell extract for protein synthesis containing a translation mold to perform translation. reaction. 7. The cell-free protein synthesis method according to any one of items 1 to 3 of the scope of application for a patent, wherein 37 200526786, the cell-free protein synthesis method includes any one of the following projects: (1) Amplify the copy mode PCR and modulate it Project; (2) a process of mixing a replication reaction solution with a replication mold; (3) a process of cultivating a mixture of a replication reaction solution and a replication replica at a fixed time; (4) a process of precipitating a translation mold; (5) making The process of drying the precipitate of the translation mold; (6) the process of removing the supernatant of the reaction solution; and the process of adding the cell extract for protein synthesis to the dried translation mold and dissolving the translation mold. 8 The method for synthesizing cell-free proteins according to item 7 of Shen Qing's patent scope, wherein all reaction processes can be performed in the same reaction vessel. 9 The method for synthesizing cell-free proteins according to item 7 in the scope of the patent claim, wherein several types of proteins can be reacted simultaneously in several reaction vessels for synthesis. 10. The cell-free protein synthesis method according to any one of claims 1 to 3 in the scope of the patent application, wherein 'the aforementioned cell extract for protein synthesis is a plant seed germ extract. 11 The method for synthesizing cell-free protein according to item 10 of the scope of the patent application, wherein the plant seed germ extract is wheat seed germ extract. 12. The cell-free protein synthesis method according to item 11 of the scope of the patent application, wherein the wheat seed germ extract is an extract in which the endosperm component of the germ and the low molecular weight protein synthesis inhibitor have been substantially removed. 13 The cell-free protein synthesis method described in any one of the items 1 to 3 of the scope of the applied patent '38 200526786, wherein, for the purpose of inputting and reading from the machine track, cell-free protein synthesis which is one of the control methods under M operation X Equipment; (a) means for variable control of the temperature in the reaction vessel; (b) means for dispensing samples or reagents within the reaction valley; (c) means for precipitation; (d) means for removing the supernatant; (e) means for drying; and (f) means for controlling the operation of the means (a) to (e) described above by means of the processes described above. 14 Cell-free protein synthesis as described in item 13 of the scope of patent application In the device, the above-mentioned means for removing the supernatant is a means for inverting the reaction container. 15 The cell-free protein synthesis device according to item 14 of the scope of patent application, further comprising means for opening and closing the lid of the reaction vessel.