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WO2006051901A1 - Procede de synthese de proteines sans cellules - Google Patents

Procede de synthese de proteines sans cellules Download PDF

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Publication number
WO2006051901A1
WO2006051901A1 PCT/JP2005/020715 JP2005020715W WO2006051901A1 WO 2006051901 A1 WO2006051901 A1 WO 2006051901A1 JP 2005020715 W JP2005020715 W JP 2005020715W WO 2006051901 A1 WO2006051901 A1 WO 2006051901A1
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Prior art keywords
reaction
transcription
cell
protein synthesis
solution
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English (en)
Japanese (ja)
Inventor
Yaeta Endo
Tatsuya Sawasaki
Tomio Ogasawara
Ryo Morishita
Mihoro Saeki
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CellFree Sciences Co Ltd
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CellFree Sciences Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins

Definitions

  • the present invention relates to a cell-free protein synthesis method and a cell-free protein synthesis apparatus using the method. Specifically, in the cell-free protein synthesis process, the reaction process from the transcription template to the production of the protein encoded by the template is performed.
  • the present invention relates to a cell-free protein synthesis method including a step of directly adding a transcription solution containing RNA to a cell extract for protein synthesis, and a cell-free protein synthesizer in which the method is automated.
  • Non-patent Document 1 Patent Documents 1 to 5.
  • Cell extract or protein for protein synthesis used in this cell-free protein synthesis system
  • the tissue extract is prepared using E. coli, wheat germ, rabbit reticulocytes, or the like as a raw material.
  • the cell-free protein synthesis system retains performance comparable to that of living cells in two respects: the rate of peptide synthesis and the accuracy of the translation reaction, and this system is capable of complex chemical reaction processes and complicated processes. Because it has this advantage, this system has been developed as a practical system, but generally, a cell extract from which biological cells are also extracted. Has a low protein synthesis efficiency due to its extremely unstable protein synthesis ability, and a significant decrease in the quality of the cell extract during storage. The amount of the synthesized product was so small that it could be detected by radioisotope labeling, etc. As a result, this system was not available as a practical protein production means.
  • cell-free protein synthesis requires the following steps: 1. a transcription reaction to obtain a translational type mRNA from a transcriptional type 2. a translation reaction that synthesizes a protein using mRNA as the type. .
  • the purified translation mold is once dissolved in a solution (translation reaction solution) containing ultrapure water, a substrate for translation reaction, an energy source, etc., and then a protein synthesis apparatus such as ribosome.
  • a solution translation reaction solution
  • the sample was mixed with a cell extract containing sucrose and subjected to a translation reaction! / But, but the translation-type mRNA was extremely difficult to dissolve in water or a solution for translation reaction. A force that had to be dissolved by stirring with the tip of a nut.
  • Non-Patent Document 1 Spirin, A., et al., (1993) Methods in Enzymology, 217, 123- 142
  • Patent Document 1 JP-A-6-98790
  • Patent Document 2 JP-A-6-225783
  • Patent Document 3 JP-A-7-194
  • Patent Document 4 JP-A-9 291
  • Patent Document 5 JP-A-7-147992
  • the present inventors previously obtained the following knowledge as a method for solving the drawbacks of the conventional cell-free protein synthesis system.
  • the step of removing unreacted substrate and the accompanying washing step for buffer exchange It has been found that the precipitate dissolves unexpectedly very easily when the cell extract for protein synthesis is added directly, rather than once dissolving the translational precipitate in water or the translation reaction application solution. It was.
  • a cell-free protein synthesis method was prepared by dissolving the product to obtain a translation reaction solution (WO2004Z70047).
  • the above cell-free protein synthesis method requires the following steps, and the above cell-free protein synthesizer requires the following equipment, which makes it possible to easily synthesize multiple samples in a short time. Further problems remain as a synthesis apparatus for carrying out a cell-free protein synthesis method and automated protein synthesis that is compact and energy-saving.
  • the object of the present invention is to provide a cell-free protein synthesis method and a cell-free protein synthesizer without requiring the essential steps of the above conventional cell-free protein synthesis method.
  • the present inventor has examined the means for preparing a substrate in a transcription mold and a transcription reaction solution as an important factor for solving the above-mentioned problems. As a result, it was found that the transcription solution containing the mRNA of the transcription reaction product can be directly introduced into the protein synthesis system without performing the purification step of the mRNA in the transcription reaction solution after the transcription reaction, and the present invention has been completed.
  • the present invention comprises:
  • a transcription solution containing mRNA of a transcription reaction product is added to a cell for protein synthesis in the reaction process from the transcription mold to the production of a protein encoded by the mold.
  • a cell-free protein synthesis method comprising a step of directly adding to an extract.
  • a cell-free protein synthesis method comprising a step of directly transcribing a transcriptional form as a transcriptional form without purifying the form, and directly adding a transcription solution containing the transcription reaction product mRNA to the cell extract for protein synthesis.
  • the synthesis means consists of two layers, upper and lower, through the interface.
  • the upper layer is the supply phase
  • the lower layer is the reaction phase
  • the substrate and energy source molecules of the supply phase are continuously supplied to the reaction phase by natural expansion (diffusion) ( 5.
  • the method according to any one of items 1 to 4, which is a multi-layer method in which a by-product generated in a reaction phase is diluted and eliminated to a feed phase.
  • a synthesis kit comprising at least one of the reagents used in the method according to any one of 1 to 11 in the preceding paragraph.
  • Control means for operating the means 1) to 3) according to the process described in any one of 1 to 11 above.
  • the apparatus according to item 13 above which does not include means for precipitating the translation mold, means for removing the supernatant of the transcription solution, and means for drying the precipitated translation mold.
  • the cell-free protein synthesis means of the present invention has achieved the provision of a cell-free protein synthesis method and an automated protein synthesizer capable of easily synthesizing multiple samples in an unprecedented short time.
  • transcription type refers to DNA that can be used as a type 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 the transcription reaction, and examples thereof include one SP6 promoter and one T7 promoter. Any DNA encoding the target protein may be used.
  • the transcription type has a base sequence having an activity for controlling the translation efficiency between the promoter sequence and the base sequence encoding the target protein.
  • RNA viruses such as ⁇ sequences derived from tobacco mosaic virus 5 'untranslated region and / or Kozak sequence etc. derived from can be used.
  • the transcription type includes a 3 ′ untranslated region including a transcription termination region and the like downstream of the base sequence encoding the target protein.
  • the 3 ′ untranslated region about 1.0 to about 3.0 kilobases downstream from the stop codon is preferably used. These 3 'untranslated regions do not necessarily have to be those of the gene encoding the target protein.
  • DNA encoding the target protein is used.
  • the reaction product amplified and synthesized by the PCR method is directly converted into a transcription cage without purification.
  • the promoter-splitting primer described in International Publication No. WO02Z18586 can also be used. .
  • the transcription type DNA obtained as described above may be subjected to transcription reaction after purification by chloroform extraction or alcohol precipitation, but in the protein synthesis method of the present invention, the reaction solution after the PCR reaction is used as it is. It can be used as a transfer mold solution.
  • the reaction solution after the PCR reaction is used as it is. It can be used as a transfer mold solution.
  • an E. coli transformation step and a selection step for a clone into which the type DNA has been introduced are not required.
  • the number of steps can be greatly reduced, and the number of steps can be reduced in a short time. Mass production of molds is possible.
  • the method of the present invention was prepared without purifying the transcription type DNA encoding the target protein prepared using a method known per se, or the DNA amplified and synthesized by the PCR method described in (1).
  • the process involves a reaction system (eg For example, a solution containing a transcription cage provided in a 96-well titer plate), preferably a PCR reaction solution, and an RNA polymerase (for example, SP6 RNA polymerase) compatible with the promoter in the transcription cage
  • a reaction system eg For example, a solution containing a transcription cage provided in a 96-well titer plate
  • an RNA polymerase for example, SP6 RNA polymerase
  • the mRNA that is the translation type used in the examples of the present invention is GFP gene DNA (Chiu, W. – L., et al, Curr. Biol. 6, 325-330 (1996)).
  • the ⁇ sequence portion is the base of SEQ ID NO: 136 described in WOO 3Z056009. Transcription was performed using SP6 RNApolymerase (Promega) using the circular plasmid DNA replaced with the sequence as a cage.
  • the translation cage in the reaction solution after the transcription reaction is precipitated and contained in unreacted ribonucleoside triphosphate, pyrophosphate which is a reaction byproduct, and other transcription reaction application solutions in addition to the translation cage RNA.
  • the supernatant liquid containing the salt was removed, and further, components (for example, alcohol) that could inhibit the translation reaction in the remaining supernatant were removed to such an extent that they could be removed.
  • the transcription reaction step in the protein synthesis method of the present invention includes (1) a translational trapezoidal precipitation step in the reaction solution after the transcription reaction, (2) a supernatant removal step in the reaction solution after the transcription reaction, (3 ) It was found that even if the drying step after the removal step (2) (the steps (1) and (3) are referred to as the mRNA purification step is S) is not reduced in translation efficiency.
  • the transcription solution in the protein synthesis method of the present invention means a transcription solution containing unpurified mRNA after the transcription reaction. [0011]
  • the transcription solution of the present invention is added directly to the cell extract for protein synthesis.
  • the direct addition means addition to a cell synthesis solution for protein synthesis without adding any purification step to the transcription solution containing the transcription reaction product mRNA.
  • the cell extract for protein synthesis used here may be any one as long as it can translate the translation template and produce a protein encoded by the template.
  • Cell extracts such as Escherichia coli, plant seed germs, and rabbit reticulocytes are used. These may be commercially available, or a method known per se, such as Pratt, JM et al., Transcription and Translation, Hames, 179-209, BD & Higgins. , SJ, eds), IRL Press, Oxford (1988), and the like.
  • Examples of commercially available cell extracts for protein synthesis include those attached to E.coli S30 extract system (Promega) and RTS 500 Rapid Translation System (Roche) derived from E. coli. Examples include those attached to Rabbit Reticulocyte Lysate System (Promega), and those derived from wheat germ, such as those attached to PROTEIOS TM (TOYOBO).
  • plant seeds that are preferably used in the plant seed germ extract system are grasses such as wheat, barley, rice, and corn, and seeds such as spinach are especially preferred. Those using embryo extract are preferred.
  • a wheat seed germ extract from which the endosperm components and low-molecular protein synthesis inhibitors mixed in the extract preparation step are substantially removed is more preferred. This is because components and substances involved in protein synthesis inhibition in the extract are reduced as compared with the conventional wheat seed germ extract.
  • the best cell extract of the present invention is an extract derived from wheat germ, and further metabolites such as glucose that cause protein synthesis inhibition in the mixed endosperm components and germ tissue cells have been substantially removed. Since this is an extract, the method for preparing the raw material will be described below using this as an example.
  • a mechanical force is first applied to wheat seeds to obtain a mixture containing germ, endosperm crushed material, and seed coat crushed material. From the product, a crude germ fraction (a mixture containing germ as a main component and containing a crushed endosperm and a seed coat) is obtained. The force applied to the seeds only needs to be strong enough to separate the seed force germ. Specifically, a seed mixture is pulverized using a known pulverizer to obtain a mixture containing germ, endosperm crushed material, and seed coat crushed material.
  • the seeds can be pulverized using a generally known pulverizer, but it is preferable to use a pulverizer of a type that applies an impact force to the object to be pulverized, such as a pin mill or a hammer mill.
  • a pulverizer of a type that applies an impact force to the object to be pulverized such as a pin mill or a hammer mill.
  • the degree of grinding may be appropriately selected according to the size of the seed germ used.
  • the pulverization is preferably performed by a dry method.
  • a crude germ fraction is obtained from the obtained ground seed product using a generally known classifier, for example, a sieve.
  • a sieve for example, in the case of wheat, the mesh size is usually 0.5 mm to 2.
  • a crude embryo fraction can also be obtained by a method utilizing the difference in specific gravity between embryo, seed coat, and endosperm, for example, heavy liquid sorting.
  • a plurality of the above methods may be combined. Sarakuko, the embryos are selected from the obtained crude germ fraction using, for example, visual inspection or a color sorter.
  • the embryo fraction obtained in this way may have an endosperm component attached thereto, it is usually preferable to further perform a washing treatment for germ pure.
  • the washing treatment the embryo fraction is dispersed and suspended in water cooled to 10 ° C or lower, preferably 4 ° C or lower, or an aqueous solution or an aqueous solution containing a surfactant, and washed until the washing solution does not become cloudy. It is preferable to do. Further, it is more preferable to disperse and suspend the embryo fraction in an aqueous solution containing a surfactant, usually at 10 ° C. or less, preferably at 4 ° C. or less, and wash until the washing solution does not become cloudy.
  • the surfactant can be widely used as long as it is a nonionic surfactant that is preferably a nonionic surfactant.
  • a nonionic surfactant that is preferably a nonionic surfactant.
  • bridges Brij
  • Triton Triton
  • Nodet P40 Nonidet
  • Tween etc.
  • Nodette P40 Nonidet
  • These nonionic surfactants can be used at concentrations that are sufficient to remove endosperm components and can be used at concentrations that do not adversely affect protein synthesis activity of the germ component, for example, at a concentration of 0.5%.
  • Either or both of the washing treatment with water or an aqueous solution and the washing treatment with a surfactant may be performed. These cleaning treatments may be performed in combination with ultrasonic treatment.
  • germs having germination ability obtained by sorting and washing seed pulverized products as described above are preferably subdivided in the presence of an extraction solvent, The obtained embryo extract is separated and further purified to obtain an extract for cell-free protein synthesis.
  • an aqueous solution containing a buffer solution, potassium ions, magnesium ions and a Z- or thiol-based anti-oxidation agent can be used. If necessary, force ions, L-type amino acids and the like may be further added.
  • HEPES N-2-hydroxyethylpiperazine N'-2-ethanesulfonic acid
  • a partially modified solution (HEPES—a solution containing KOH, potassium acetate, magnesium acetate, calcium chloride salt, L-type amino acid and Z or dithiothreitol) can be used as an extraction solvent.
  • the composition and concentration of each component in the extraction solvent are known per se, and those used for the production of cell extracts for cell-free protein synthesis may be employed.
  • the required amount of extraction solvent is added to the germ and the embryo is subdivided in the presence of the extraction solvent.
  • the amount of the extraction solvent is usually 0.1 milliliters or more, preferably 0.5 milliliters or more, more preferably 1 milliliter or more, relative to the germ lg before washing.
  • the upper limit of the amount of the extraction solvent is not particularly limited, but is usually 10 milliliters or less, preferably 5 milliliters or less with respect to the germ lg before washing.
  • the embryos to be subdivided can be frozen as in the past, but it is more preferable to use frozen ones!
  • subdividing wrinkles As a method of subdividing wrinkles, a conventionally known crushing method such as grinding or crushing can be used.
  • a method of subdividing embryos by impact or cutting developed by the present inventors (WO03 / 06 4671).
  • “subdivide by impact or cutting” means destruction of plant germ cell nuclei, mitochondria, organelles such as chloroplasts, cell membranes and cell walls. Is to destroy the plant germ under conditions that can be minimized compared to conventional grinding or crushing.
  • the apparatus and method that can be used for subdividing are not particularly limited as long as the above conditions are satisfied.
  • an apparatus having a blade that rotates at high speed such as a Warinda blender.
  • the rotational speed of the blade is usually lOOOOrpm or more, preferably ⁇ 5000rpm or more, and usually 30000rpm or less, preferably ⁇ 25000rpm or less.
  • the rotation time of the blade is usually 5 seconds or longer, preferably 10 seconds or longer.
  • the upper limit of the rotation time is not particularly limited, but is usually 10 minutes or less, preferably 5 minutes or less.
  • the temperature at the time of fragmentation is preferably within a range where the operation can be carried out at 10 ° C or less, particularly preferably about 4 ° C.
  • the cell nucleus and cell wall of the embryo are not completely destroyed, and at least a part of them remains without being destroyed.
  • organelles such as embryonic cell nuclei, cell membranes and cell walls are not destroyed more than necessary, the synthesis of proteins localized in the cytoplasm is less contaminated with impurities such as DNA and lipids contained in them.
  • Necessary RNA and ribosomes can be extracted with high purity and efficiency efficiently.
  • the extraction solvent can be added after subdivision. More preferably, it is carried out in the presence of.
  • the conventional step of pulverizing the germ and the step of adding the extraction solvent to the pulverized embryo to obtain the germ extract can be performed simultaneously as a single step.
  • An embryo extract can be obtained.
  • the above method may be referred to as a “pender method”.
  • the embryo extract is centrifuged at 20,000 to 40,000 G, preferably 2.5 to 350,000 G, more preferably 30,000 G, and a centrifugal supernatant is obtained.
  • an inorganic carrier as a precipitation aid to separate the precipitate from the supernatant.
  • This precipitate contains a complex of calcium and an enzyme such as glycosidase. Preliminarily removing glycosidase helps to minimize glucose production as well as starch power.
  • suitable inorganic carriers include bentonite, activated carbon, silica gel, sea sand and the like. It is. By introducing this inorganic carrier, it is possible to almost completely prevent the precipitate from being mixed into the supernatant.
  • the wheat germ extract can be further purified by gel filtration or the like.
  • Gel filtration can be performed, for example, by equilibrating with an appropriate solution in advance and using a gel filtration apparatus.
  • the composition and concentration of each component in the gel filtration solution are known per se, and are used for the production of cell germ extracts for cell-free protein synthesis (eg, HEPES-KOH, potassium acetate, magnesium acetate, dithio).
  • a solvent containing thritol or L-type amino acid may be employed.
  • the germ cell extract thus obtained has extremely reduced RNase activity and phosphatase activity.
  • the germ extract-containing solution after gel filtration may contain spore such as microorganisms or filamentous fungi (breast), it is preferable to exclude these microorganisms. ,. In particular, it is important to prevent the growth of microorganisms during the long-term (one day or longer) cell-free protein synthesis reaction.
  • the means for eliminating microorganisms is not particularly limited, but it is preferable to use a filter sterilization filter.
  • the pore size of the filter is not particularly limited as long as microorganisms that may be mixed can be removed, but usually 0.1 to 1 micrometer, preferably 0.2 to 0.5 micrometers. Is appropriate.
  • the cell extract thus obtained is a substance that suppresses the protein synthesis function contained or retained by the wheat itself as a raw material (acts on the function by acting on various RNAs, translated protein factors, ribosomes, etc.) Inhibiting substances such as various ribonucleases and various protears Ase, tritin, thionine, etc.) are almost completely removed. That is, the endosperm where these inhibitors are localized has been almost completely removed and purified. The degree of endosperm removal can be evaluated by monitoring the activity of tritin contaminated in the wheat germ extract, that is, the activity of deadenating ribosomes.
  • the ribosome is not substantially deadenated, it is judged that there is no contaminating endosperm-derived component in the germ extract, that is, the endosperm is almost completely removed and purified.
  • the extent that the ribosome is not substantially deadenylated means that the ribosome deadenification rate is 7% or less, preferably 1% or less.
  • This fractionation treatment is preferably performed a plurality of times, and in particular, it is preferable to substantially remove substances having a molecular weight of 1 kDa or less.
  • the specific number of times is 1 to: LO times, preferably 2 to 9 times, more preferably 3 to 8 times, and most preferably 4 to 7 times.
  • sugar and phosphate sucrose are substantially reduced to 10 mM or less, preferably 6 mM or less (glucose in the extract having an absorbance of 200 OD / ml at 260 nm). As concentration).
  • the extract with reduced glucose concentration obtained by force possesses an unprecedented high cell-free protein synthesis ability.
  • the cell extract for protein synthesis that the transfer solution was added directly obtained as described above, the amino acid as a substrate, energy source, various ions, buffers, ATP regenerating system, nuclease inhibitor, t RNA, a reducing agent , Polyethylene glycol, 3 ', 5'- cAMP, folate, antibacterial and other solutions that contain necessary or suitable components for translation reaction (also called “translation reaction solution”) By incubating at an appropriate temperature for an appropriate time. Translation reaction.
  • the amino acid used as a substrate is usually 20 kinds of natural amino acids constituting a protein, and its analog or isomer can be used depending on the purpose.
  • Energy sources include ATP and Z or GTP.
  • Examples of various ions include acetates such as potassium acetate, magnesium acetate, and ammonium acetate, and glutamates.
  • As the buffer solution 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, specific examples of ribonuclease inhibitors include human placenta-derived RNase inhibitors (TOYOBO, etc.).
  • tRNA can be obtained by the method described in Moniter, R "et al, Biochim. Biophys. Acta., 43, 1 (1960), etc., or a commercially available one can be used.
  • antibacterial agent include sodium azide, ampicillin, etc.
  • the amount of these additives can be appropriately selected within a range that can be normally used in cell-free protein synthesis.
  • the mode of addition of the translation reaction solution can be appropriately selected depending on 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 cell-free protein synthesis method of the present invention.
  • the Notch method Pratt, JM et al., Transcription and Translation, Hames, 179-209, BD & Higgins, SJ, eds), IRL Press, Oxford (1984)
  • continuous cell-free protein synthesis that continuously supplies amino acids, energy sources, etc. to the reaction system.
  • a method in which a synthesis reaction tank is prepared by a carrier capable of molecular sieving, the above-described synthetic material is developed using the carrier as a mobile phase, a synthesis reaction is performed during the development, and the synthesized protein can be recovered as a result 200 0-316595) can be used. While simplifying the structure of the synthetic system, The multi-layer method is particularly advantageous in that it can provide a relatively large amount of protein that is preferred by the notch method or multi-layer method in terms of providing a multi-sample simultaneous synthesis system applicable to space-saving, low-cost, high-throughput analysis. I like it.
  • the solution for translation reaction may be added to the cell extract for protein synthesis to which the transcription solution is directly added and mixed.
  • the addition of the translation reaction application solution can be omitted.
  • a “translation reaction solution” obtained by mixing a cell extract solution for protein synthesis to which a transcription solution is directly added and a solution for translation reaction
  • a wheat germ extract for example, when a wheat germ extract is used as a cell extract solution for protein synthesis, 10 to 50 mM HEPES-KOH (pH 7.8), 55 to 120 mM potassium acetate, 1 to 5 mM magnesium acetate, 0.1 to 0.6 mM spermidine, 0.025 to each: LmM L—amino acid, 20 to 70 ⁇ m ⁇ , preferably 30-50 D DTT, 1-1.5 mM ATP, 0.2-0.5 mM GTP, 10-2 OmM creatine phosphate, 0.5-1.
  • Ounits / 1 ribonuclease baboon, , 0.01 to 10 ⁇ protein disulfide isomerase, and 24 to 75% wheat germ extract are used.
  • the pre-incubation 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. It is preferably about 18 to about 30 ° C., more preferably about 20 to about 26 ° C., and the batch method is usually performed for about 10 minutes to 7 hours until the reaction is stopped.
  • the substrate and the energy source supply solution are continuously or discontinuously supplied to the translation reaction system of the reaction phase by a free fall or a liquid feed pump, and the transfer solution derived from the transcription solution
  • a cell-free protein synthesis method using a fed-batch method characterized by extending the duration of the synthesis reaction and increasing the efficiency of the synthesis reaction by diluting high magnesium ions, nucleotides, and by-products generated in the reaction phase (Hereinafter, the supply batch method) is also targeted.
  • the size and form of the reaction vessel are not limited by feeding from the feed phase, and the mixing rate of both solutions, which is an important rate-limiting parameter for protein synthesis rate, can be controlled freely. Synthetic reactions can be controlled, enabling large-scale production of highly efficient tanks. Sarasako, by adding a supply phase with added mRNA, The response efficiency can be further increased.
  • the specific feed liquid addition rate is within the range in which the same amount of feed liquid as the reaction phase at the start of the reaction can be continuously or discontinuously supplied to the reaction phase in 5 minutes to 15 hours, preferably 10 minutes to 10 hours.
  • the flow rate is
  • the protein synthesis is performed by layering the translation reaction solution on the cell synthesis solution for protein synthesis to which the transcription solution is directly added so as not to disturb the interface.
  • a cell extract for protein synthesis that has been pre-incubated for an appropriate time as necessary is added to a translation-type precipitate and dissolved to form a reaction phase.
  • the reaction is carried out by layering the translation reaction solution (feed phase) on top of this reaction phase without disturbing the interface.
  • the interface between the two phases does not necessarily have to be formed in a horizontal plane by means of multiple layers. It is also possible to form a horizontal plane by centrifuging the mixed solution containing both phases.
  • the volume ratio of the reaction phase to the supply phase is suitably from 1: 4 to 1: 8, but 1: 5 is preferred.
  • the translation reaction is about 10 to about 40 ° C., preferably about 18 to about 30 ° C., more preferably about 20 to about 26 ° C. under static conditions. And usually about 10 to about 20 hours.
  • the reaction temperature is suitably about 30 ° C to about 37 ° C.
  • Sarako 1. Process of mixing the supply phase and reaction phase before and after the protein synthesis rate is substantially reduced, before and after the synthesis reaction is almost stopped, or in the process of the synthesis reaction. Including the step of centrifugal concentration to about the initial liquid volume, 3. the process of resuming synthesis by overlaying the supply liquid as the supply phase on the reaction phase, and 4. the process of repeating steps 1-3. There is also a characteristic cell-free protein synthesis method (hereinafter referred to as “repetitive multi-layer method”).
  • the cell-free protein synthesizer of the present invention comprises a transcription reaction product in a cell-free protein synthesis step from the transcription cocoon to the production of a protein encoded by the cocoon.
  • This is an automated cell-free protein synthesis method that includes adding a transcription solution containing mRNA directly to a cell extract for protein synthesis. Specifically, one of the following 1) and 5) steps 1 or 2 The above is automated.
  • the cell-free protein synthesizer of the present invention includes a step of precipitating the translation mold, a step of removing the supernatant, and a process of drying the precipitated translation mold in the protein synthesis process. Not required as an essential process. As a result, the cell-free protein synthesis step can be greatly omitted, and the synthesis time can be shortened.
  • “automation” means that the experimenter does not directly perform a manual operation on the reaction system (reaction vessel) during a series of cell-free protein synthesis steps. Therefore, when performing each step, it is necessary for the experimenter to manually operate the predetermined operation buttons and switches provided in the automatic synthesizer of the present invention to be used. This is not a damage.
  • an apparatus for automatically performing the reaction operation from the synthesized transcriptional vase to the production of the protein encoded by the vase is at least one of the following means (1) to (3).
  • the cell-free protein synthesizer of the present invention is used in the reaction process from the transcription cocoon to the production of the protein encoded in the cocoon.
  • the transcription solution containing the transcription reaction product mRNA is not limited thereto as long as it has a feature capable of automating the process of directly adding the transcription solution to the cell extract for protein synthesis.
  • this step does not necessarily need to be performed automatically.
  • a transcription mold obtained manually can be used for the following automated process. It is more preferable that the production process is automatically performed through a series of steps up to the production of the protein encoded in the saddle type! /.
  • An apparatus for carrying out such a series of operations automatically or semi-automatically (which means that an experimenter directly adds a manual operation to a reaction system as part of a process) is known.
  • the object of the present invention is to provide a high-throughput synthesis system for high-throughput analysis and the simplicity of the apparatus.
  • PCR polymerase chain reaction
  • Dispensing and mixing of the transfer cage solution and the transcription reaction solution into the reaction vessel are performed by the automatic synthesizer described later (for example, using a pipetter (commercially available 96-well titer plate as the reaction vessel). In some cases, it is preferable to use one having 8 or 12 dispensing tips adapted to the well interval). Incubation for the transcription reaction can be performed while the temperature is controlled at a constant temperature by the temperature control means of the synthesizer described later.
  • Operations such as dispensing of unpurified mRNA-containing transfer solution, supply solution, and reaction solution into the reaction vessel are performed by dispensing means of an automatic synthesizer described later (for example, pipetter (96-well titer commercially available as reaction vessel).
  • an automatic synthesizer for example, pipetter (96-well titer commercially available as reaction vessel).
  • pipetter 96-well titer commercially available as reaction vessel.
  • the above-described cell-free protein synthesizer of the present invention is characterized by having at least one of the following means (a) to (c).
  • the configuration of the cell-free protein synthesizer of the present invention differs from the configuration of the conventional synthesizer in that it requires means for precipitating the translation mold, means for removing the supernatant, and means for drying the precipitated translation mold. It is not necessary as a means of. This eliminates the need for a centrifuge in the configuration of the cell-free protein synthesizer. As a result, the cell-free protein synthesizer of the present invention achieved a reduction in cost of the synthesizer, a “compact”, an energy saving, and a shortened synthesis time. In addition, the influence of the centrifuge vibration on other processes could be eliminated.
  • Control means for controlling the means (a) to (c) to operate in accordance with the method of the present invention described above.
  • the means for variably controlling the temperature in the reaction vessel is the transcription reaction, the incubation of the translation reaction and the termination of the translation reaction, or the transcription slab-type preparation process by the PCR method using the automatic synthesizer of the present invention.
  • 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 the temperature range usually required in a series of reaction operations for cell-free protein synthesis including preparation of a transcription cage (for example, about 4 ° C to about 100 ° C).
  • any means capable of variably controlling the temperature of the liquid in the reaction vessel within the range of ° C is not particularly limited.
  • conventionally known Takara PCR thermal cycler MP manufactured by Takara Bio Inc.
  • Gene Amp PCR System 9700 manufactured by Applied Biosystems Inc.
  • 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 set. As a result, the temperature in the reaction vessel is variably controlled.
  • the means for dispensing a sample or reagent into a reaction container is to distribute the 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. It is a means of pouring.
  • sample refers to transcription type and translation type
  • reagent refers to transcription reaction solution, translation reaction solution, translation reaction solution, dilution solution, alcohol, salt solution, PCR reaction solution, supply Liquid, reaction liquid, etc.
  • a pipette arm (dispensing machine) that can be dispensed automatically in a conventionally known manner is used as long as it can be dispensed by adjusting the amount of sample and reagent according to the process. It can be realized without limitation.
  • the position of the chip can be preliminarily set in order to perform an appropriate multi-layer.
  • the pipette arm can have a function of discarding used tips at the tip disposal port of the synthesizer and a function of discharging the sucked filtrate to the waste solution port.
  • the dispensing means more preferably has a mixing function (eg, pipetting, stirring, etc.) for homogenizing two or more types of solutions.
  • a mixing function eg, pipetting, stirring, etc.
  • the means for transporting the reaction container is a means for moving the reaction container to each stage, the lifting platform, and the thermostat.
  • the means for transporting the strong reaction vessel is not particularly limited as long as the reaction vessel can be transported to the target place, and can be realized by any conventionally known appropriate means. For example, it can be realized by using a robot arm used in a conventional synthesis apparatus.
  • control means In the control means, the operation of the drive sources (motors, pneumatic equipment, other hydraulic actuators, etc. that can be controlled for operation) used for each means in order to operate the means (a) to (c) above is switched on / off.
  • a control device for controlling the degree and state of operation is included.
  • the configuration of the control can achieve the purpose of automatically performing the reaction operation from the synthesized transcription cage to the production of the protein encoded in the cage by the operation of the means (to (c) above. It is.
  • 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 power, air pressure, hydraulic pressure, and the like can be supplied to each means so that each means operates in order.
  • a driver necessary for directly sending a drive signal to the drive source of each of the above means, various sensors and switches necessary for detecting the operation state of the drive source of each of the above means may be added as appropriate.
  • the reaction vessel applicable to the synthesizer of the present invention may be any of various conventionally known reaction vessels that have been used for cell-free protein synthesis reactions without particular limitations. Yes, for example, 6-well plate, 24-well plate, 96-well plate, 384-well plate, 96-well PCR plate, 96-well titer plate, 8-strip tube or tube (1.5 mL, 15 mL, 50 mL, etc.)
  • a translation reaction can be carried out in a small reaction system such as a 96-well plate or a 384-well plate, and the synthesis reaction of the present invention also enables a transcription reaction.
  • a series of cell-free protein synthesis methods including transcription reaction 'translation reaction, PCR for preparation of transcription cage for further transcription reaction, if desired, can be performed in multiple reaction systems. It is possible to perform several types of proteins at the same time and synthesize many proteins in a short time.
  • the cell-free protein synthesis method of the present invention is characterized in that a transcription solution containing mRNA obtained by transcription can be directly introduced into a protein synthesis system without performing the mRNA purification step in the transcription reaction solution after the transcription reaction. It is what. Further, the present invention is a cell-free protein synthesizer characterized by automating the cell-free protein synthesis method of the present invention.
  • the target gene can be amplified and synthesized by the PCR method to obtain a DNA template, which can be directly transcribed as a transcription template without purification and introduction into the plasmid.
  • a synthesis kit containing at least one of the reagents used in the cell-free protein synthesis method of the present invention is useful because it can easily provide the cell-free protein synthesis of the present invention.
  • Hokkaido chihoku wheat seeds or Ehime chikugoizumi seeds were added to the mill (Fritsch: Rotor Speed Mill pulverisettel type 4) at a rate of lOOg per minute, and the seeds were gently ground at a rotation speed of 8, OOOrpm.
  • Flotation using cyclohexane 2.4: 1) collects the floating fraction containing germinating embryos, removes the organic solvent by room temperature drying, and then blows at room temperature. Thus, impurities such as the seed coat mixed together were removed to obtain a crude germ fraction.
  • This color sorter includes means for irradiating light to the crude germ fraction, means for detecting reflected light and Z or transmitted light from the crude germ fraction, means for comparing the detected value with the reference value, Supply the crude germ fraction to 1000 to 5000 grains / cm 2 on the beige belt of the color sorter, which is a device that has a means to sort out and remove those that fall outside or within the reference value.
  • the reflected light was detected by irradiating the upper crude germ fraction with a fluorescent lamp.
  • the belt conveyance speed was 50 mZ.
  • a monochrome CCD line sensor (2048 pixels) was used as the light receiving sensor.
  • a reference value was set between the brightness of the germ and the brightness of the seed coat, and those that deviated from the reference value were removed by suction.
  • a reference value was set between the brightness of the germ and the brightness of the endosperm, and anything that deviated from the reference value was removed by suction.
  • Suction was performed using 30 suction nozzles (one suction nozzle per 1 cm length) installed approximately lcm above the conveyor belt. By repeating this method, the germs were selected until the purity of the germs (the weight ratio of the germs contained in any lg sample) reached 98% or more.
  • the obtained wheat germ fraction was suspended in distilled water at 4 ° C, and washed with an ultrasonic washing machine until the washing solution did not become cloudy. Next, it is suspended in a 0.5% by volume solution of Nodette P40 (manufactured by Nakarai Tectus Co., Ltd.), and washed with an ultrasonic cleaner until the washing solution does not become cloudy. Obtained.
  • the homogenate (crushed material) obtained above was mixed with 20% weight of sea sand or swollen cef. Adex G25 particles were added and mixed. Sea sand was subjected to the following treatment prior to addition of homogenate: water washing ⁇ 5 volumes of 0.1N NaOH or KOH washing ⁇ water washing ⁇ 0.1N HC1 washing ⁇ water washing ⁇ 100-120 ° RNase inactivation treatment by heating C, followed by drying treatment.
  • the translucent supernatant was obtained by centrifuging the homogenate mixed with sea sand twice at 30,000 xg for 30 minutes followed by one centrifugation for 12 minutes (S-30 fraction).
  • the transcription reaction was incubated at 37 degrees for 2.5 hours.
  • the transcription reaction solution is as follows.
  • Transcription reaction liquid yarn 300 ⁇ 1 5 ⁇ transcription buffer (400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT), 150 ⁇ ⁇ 25 mM 4NTPs, 18.75 ⁇ 1 RNAsin (80 Units), 200 ⁇ 1 PCR product (GFP transcription type: pEU-E01-GFP (SP6 promoter-E01-GFP) is a vertical type with two primers on the 5 'and 3' sides. A transcription mold was constructed by one-step PCR (30 cycles)), and water 300.125 1 was added to 18.75 ⁇ 1 SP6 polymerase (80 units) to make a total volume of 1 ml.
  • GFP transcription type pEU-E01-GFP (SP6 promoter-E01-GFP)
  • Transcription reaction liquid yarn 200 ⁇ 1 5 ⁇ transcription buffer (400 mM HEPES, pH 7.6; 80 mM Magnesium acetate; 10 mM Spermidine; 50 mM DTT), 100 ⁇ ⁇ 25 mM 4NTPs, 12.5 1 RNAsin (1200 Units) ⁇ 100 1 pEU (including GFP gene clones, 100 ⁇ g), 12.5 ⁇ 1 SP6 polymerase (2400 units) was added with 575 ⁇ 1 of water to make a total volume of 1 ml.
  • the transcription solution containing unpurified mRNA was not purified after the transcription reaction.
  • the transcription solution containing the purified mRNA was subjected to ethanol precipitation after the transcription reaction, and the supernatant was removed and dried.
  • reaction solution concentration 60A260nm, for example, the amount of the transferred solution is the 1Z2 amount at 120A260nm in the proportional calculation.
  • the liquid concentration is the same). All reactions were allowed to stand at 26 degrees for 5 hours o
  • the amount of protein synthesis was determined by measuring the incorporation of radioactivity into the acid-insoluble fraction of 14 C-labeled leucine as follows: 5 microliters of the reaction mixture was spotted on a 3MM Whatman filter paper and 10% After soaking in ice-cold TCA (triclo-mouth acetic acid) for 1 hour, it was boiled in 5% TCA solution for 10 minutes. Take out this filter, remove TCA and water with ethanol ether (50:50 volume), dry, and measure the radioactivity incorporated into the hot TCA insoluble fraction with a liquid scintillation counter (toluene scintillator). did.
  • Fig. 1: B Even when unpurified mRNA (as-is transcription solution) is used (Fig. 1: B), as with protein synthesis using purified mRNA (Fig. 1: A), V and misplaced embryos are extracted. There was no significant difference in the reaction system between the two mRNAs (purified or unpurified) in each synthesis efficiency using the solution.
  • the symbols a-e in Fig. 1 are: a: extract concentration at the start of 40OD, b: extract concentration at the start of 60OD, c: extract concentration at the start of 80OD, d: start Extraction concentration at time is 100 OD, e: Extraction concentration at start is 120 OD. Each concentration after synthesis (after about 5 hours) was 1Z12 at the start.
  • ⁇ indicates that the protein synthesis reaction was performed after mixing the mixture with the extract concentration of 120 OD with complete stirring and mixing at the start of the reaction (the concentration of the extract in the reaction solution is 10 OD).
  • the amount of protein synthesis is 1Z3 or less of the synthesis amount of the multilayer method (e).
  • Transcription molds are prepared by the two methods described in the transcription reaction step of Example 1 (methods of making DNA clones amplified and synthesized by PCR methods into transcription molds or introducing them into plasmids to form transcription molds). did. In both cases, mRNA in the transcription solution after the transcription reaction was not purified.
  • the reaction phase and the supply phase were mixed. Subsequently, using an Amicon Ultra concentrated membrane (manufactured by Millipore) with a molecular weight of 10000 cut-off, centrifugal concentration was carried out to 0.5 ml which was the starting liquid volume of the reaction phase. Next, the synthesis was resumed by overlaying the feed solution to be the feed phase on the concentrated reaction phase. This was repeated three times (the synthesis reaction was four times). The second and subsequent feeds do not contain creatine kinase.
  • Amicon Ultra concentrated membrane manufactured by Millipore
  • the GFP synthesis lasts up to 4 times because the increase in the staining intensity of the CBB-stained GFP band (arrow in Fig. 2) on polyacrylamide native gel electrophoresis. It could be confirmed. Before the start of the synthesis reaction, there is no GFP band (in Fig. 2: 0 lane).
  • the protein synthesis method using transcripts from PCR is the same as the protein synthesis method using a transcription solution obtained by using a plasmid as a transcription cage from the comparison of GFP staining intensity on PCR and plasmid electrophoresis gel in FIG. It was found that the GFP synthesis efficiency was equivalent or better.
  • the cell-free protein synthesis method according to the present invention includes complicated methods such as an ultrafiltration membrane method using a semipermeable membrane, a dialysis membrane method, and a column chromatography method in which a translation cage is fixed on a resin.
  • each method can be used in a cell-free system that uses a tissue cell extract. It was shown that protein synthesis can be performed with high efficiency.
  • the cell-free protein synthesis method according to the present invention has been conventionally performed! / There are no drawbacks such as low membrane material strength, reduced membrane function due to clogging, and complicated operation, as seen in continuous cell-free protein synthesis using a membrane.
  • the technology according to the present invention will be a basic element technology for automating the production of gene products (proteins) that will serve as the basis for functional analysis and structural analysis of a huge number of genes that will be brought forward with the completion of future genome projects. .
  • it can be said that it is indispensable as an elemental technology for automating cell-free protein synthesis systems, such as the development of fully automated cell-free protein synthesis robots for multiple specimens.
  • Fig. 1 shows the purified and unpurified mRNA (transcription solution as it is) of the transcription reaction solution.
  • the amount of synthetic product was measured over time by a multi-layer system using cell-free protein solutions containing various concentrations of wheat germ extract.
  • Fig. 2 compares the efficiency of protein synthesis between a DNA template amplified and synthesized by PCR and a transcription template using a plasmid.

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Abstract

L’invention concerne un procédé de synthèse de protéines sans cellules, selon lequel une étape essentielle du procédé classique de synthèse des protéines sans cellules peut être évitée dans les moyens de synthèse des dites protéines. L’invention concerne également un appareil approprié pour réaliser la synthèse des dites protéines. On a étudié l’importance, dans le cadre de ce qui précède, des modèles de transcription et des moyens de préparation des substrats d’une solution de réaction de transcription. Ces études ont montré que même si l’on ne purifie pas le ARNm d’une solution de réaction de transcription après que ladite réaction a eu lieu, une solution de transcription contenant du ARNm en tant que produit de la réaction de transcription peut être introduite telle quelle dans le système de synthèse des protéines. Cette découverte a mené à la présente invention.
PCT/JP2005/020715 2004-11-12 2005-11-11 Procede de synthese de proteines sans cellules Ceased WO2006051901A1 (fr)

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WO2015141649A1 (fr) * 2014-03-20 2015-09-24 ユニバーサル・バイオ・リサーチ株式会社 Dispositif d'automatisation de l'amplification d'acide nucléique et dispositif d'automatisation de l'analyse d'une amplification d'acide nucléique

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WO2013080811A1 (fr) 2011-11-28 2013-06-06 国立大学法人名古屋大学 Biomarqueur pour une infundibulo-neuro hypophysite lymphocytaire et applications d'utilisation associées

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Publication number Priority date Publication date Assignee Title
WO2002018586A1 (fr) * 2000-08-30 2002-03-07 Wakenyaku Co Ltd Conception et construction d'un modele de transcription de synthese d'une proteine acellulaire, et methode de dilution de type discontinue utilisee dans la synthese d'une proteine de germe de ble acellulaire
WO2004009639A1 (fr) * 2002-07-18 2004-01-29 Cellfree Sciences Co., Ltd. Anticorps a chaine unique et utilisation de ce dernier
WO2004070047A1 (fr) * 2003-02-10 2004-08-19 Cellfree Sciences Co., Ltd. Procede automatique de synthese d'une proteine et appareil associe

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018586A1 (fr) * 2000-08-30 2002-03-07 Wakenyaku Co Ltd Conception et construction d'un modele de transcription de synthese d'une proteine acellulaire, et methode de dilution de type discontinue utilisee dans la synthese d'une proteine de germe de ble acellulaire
WO2004009639A1 (fr) * 2002-07-18 2004-01-29 Cellfree Sciences Co., Ltd. Anticorps a chaine unique et utilisation de ce dernier
WO2004070047A1 (fr) * 2003-02-10 2004-08-19 Cellfree Sciences Co., Ltd. Procede automatique de synthese d'une proteine et appareil associe

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015141649A1 (fr) * 2014-03-20 2015-09-24 ユニバーサル・バイオ・リサーチ株式会社 Dispositif d'automatisation de l'amplification d'acide nucléique et dispositif d'automatisation de l'analyse d'une amplification d'acide nucléique
JPWO2015141649A1 (ja) * 2014-03-20 2017-04-13 ユニバーサル・バイオ・リサーチ株式会社 核酸増幅自動化装置、及び核酸増幅分析自動化装置

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