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WO2018198543A1 - Mélange réactionnel pour la synthèse de protéines acellulaires, procédé de synthèse de protéines acellulaires utilisant ce melange et kit pour la synthèse de protéines acellulaires - Google Patents

Mélange réactionnel pour la synthèse de protéines acellulaires, procédé de synthèse de protéines acellulaires utilisant ce melange et kit pour la synthèse de protéines acellulaires Download PDF

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WO2018198543A1
WO2018198543A1 PCT/JP2018/008797 JP2018008797W WO2018198543A1 WO 2018198543 A1 WO2018198543 A1 WO 2018198543A1 JP 2018008797 W JP2018008797 W JP 2018008797W WO 2018198543 A1 WO2018198543 A1 WO 2018198543A1
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cell
acid
reaction mixture
protein synthesis
free protein
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Japanese (ja)
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俊将 本間
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Ｓｐｉｂｅｒ株式会社
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a reaction mixture for cell-free protein synthesis, a cell-free protein synthesis method using the same, and a cell-free protein synthesis kit.
  • Recombinant protein production methods include in vivo methods including culturing transformed cells and in vitro using cell-derived extracts (cell extracts) called so-called cell-free protein synthesis methods. The method is known.
  • in vitro cell-free protein synthesis methods (1) can direct resources for protein synthesis to the exclusive production of the protein of interest ( 2) Since it does not relate to cell growth or survival, the synthesis environment can be flexibly changed, such as conditions such as tRNA level changes reflecting the codon usage of genes, redox potential, pH, or ionic strength, 3) A protein product that has been purified and properly folded can be easily recovered as it is, (4) A non-naturally-isotopically labeled amino acid can be incorporated, and (5) In vivo unstable, insoluble or cellular The ability to synthesize proteins that are toxic, (6) proteins that are difficult to make in vivo because they require unique cofactors Point may be synthesized, and (7) is believed to have the advantages of cloning, such that it is possible to avoid the process of transformation, such as the cell for expression in vivo. For this reason,
  • cell extracts used in cell-free protein synthesis methods cell extracts prepared from plants, animals, cultured cells, microorganisms and the like are known, and kits are currently commercially available from several companies. ing.
  • the cell-free protein synthesis method has a low production efficiency because the reaction duration for synthesizing the protein is short.
  • a continuous flow system using a dialysis membrane has been developed that supplies the substrate consumed by the translation reaction and removes by-products that inhibit the reaction by dialysis.
  • it is necessary to continuously add a reagent in order to increase the amount of protein synthesis by this continuous flow system, it is necessary to continuously add a reagent, and there is a problem that the cost of the reagent increases.
  • the reagents used in the cell-free protein synthesis method include ATP and GTP necessary for protein synthesis reaction, and UTP and CTP necessary for the synthesis of mRNA when DNA is used as a template nucleic acid. Since it is a rather expensive reagent, the cell-free protein synthesis method is very expensive.
  • a cell-free protein synthesis method using a nucleotide cheaper than the above-mentioned nucleoside triphosphates such as AMP, GMP, UMP and CMP (Patent Document 1), or a nucleoside such as adenosine, guanosine
  • Patent Document 2 a cell-free protein synthesis method using uridine and cytidine
  • these reagents are also considerably more expensive than reagents used for in vivo methods.
  • Adenosine, guanosine and the like are not suitable for industrial use because of their low solubility in water.
  • Non-Patent Document 3 a certain result has been obtained by reviewing the energy regeneration system in the in vitro reaction (Patent Document 3, Non-Patent Document 2), and granulocyte macrophage colony stimulation Succeeded in synthesizing 700 mg / L of factor in 10 hours (Non-patent Document 3).
  • An object of the present invention is to provide a novel approach for inexpensive protein synthesis in a cell-free protein synthesis method.
  • inosinic acid is an alternative to nucleotides and nucleosides corresponding to ATP, GTP, UTP and CTP. It has been found that proteins can be synthesized at low cost by a cell-free protein synthesis method by using the method, and the present invention has been completed.
  • a reaction mixture for cell-free protein synthesis comprising a cell extract and inosinic acid.
  • the cell extract is a cell extract derived from at least one cell selected from the group consisting of plants, animals and microorganisms.
  • the microorganism is at least one selected from the group consisting of E. coli and yeast.
  • reaction mixture according to any one of [1] to [5], further comprising a template nucleic acid, a substrate for target protein synthesis, and an energy source.
  • the template nucleic acid comprises DNA encoding at least one promoter and a target protein.
  • the energy sources are ATP, GTP, glucose, ribose, pyruvate, phosphoenolpyruvate, carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and glucose-6 -At least one selected from the group consisting of phosphate, citric acid, cis-aconitic acid, isocitric acid, ⁇ -ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid and glutamic acid, The reaction mixture according to [6].
  • a cell-free protein synthesis kit comprising a cell extract and inosinic acid, a template nucleic acid, a substrate for protein synthesis, and / or an energy source.
  • the reaction mixture for cell-free protein synthesis of the present invention contains a cell extract and inosinic acid that can be industrially mass-produced, so that it can be produced at the same productivity as AMP, GMP, UMP, and CMP at low cost. Proteins can be synthesized. Inosinic acid also has the advantage of being easy to handle due to its high solubility compared to adenosine and guanosine. Furthermore, according to the cell-free protein synthesis method of the present invention, the cost of protein synthesis in the method can be reduced by using the reaction mixture for cell-free protein synthesis of the present invention, and the protein can be efficiently and industrially produced. Production can be done.
  • Test Example 1 a growth curve (black triangle) of BL21 strain cultured in a modified 2 ⁇ YTPG medium using glycerol as a carbon source and a growth curve of BL21 strain cultured in a normal 2 ⁇ YTPG medium using glucose as a carbon source It is a graph which shows the comparison of (black circle). In Test Example 2, it is a graph showing the results of measuring the fluorescence intensity of GFP synthesized by the cell-free protein synthesis method over time.
  • Example 1 it is a graph which shows the result of having compared the fluorescence intensity of GFP synthesized by the cell-free protein synthesis method.
  • the black bars show the results for the reaction mixture containing AMP, GMP, UMP and CMP, and the shaded bars show the results for the reaction mixture containing inosinic acid.
  • reaction mixture for cell-free protein synthesis contains a cell extract and inosinic acid.
  • Cell extract As long as the cell extract according to the present embodiment can generate a protein encoded by the template nucleic acid, those skilled in the art appropriately use those derived from any cell such as plants, animals, cultured cells, microorganisms, and the like. Can be selected and used.
  • plant-derived cell extracts include gramineous plants such as wheat, barley, rice, and corn, and cell extracts derived from seed germs such as spinach.
  • cell extracts derived from seed germs such as spinach.
  • commercially available cell extracts derived from plants include wheat germ-derived wheat extract (manufactured by Promega), PROTEIOS (manufactured by Cell Free Science).
  • animal-derived cell extracts include cell extracts derived from blood cells and gonad-derived cells of mammals such as rabbits, humans, rats, mice, and monkeys.
  • animal-derived cell extracts include rabbit reticulocyte lysate systems (manufactured by Promega).
  • cell extracts derived from cultured cells include lymphoma (lymphoma) -derived cells, tumor cells, stem cells, and insect culture cells such as High Five (manufactured by Invitrogen) and Sf21 (manufactured by Invitrogen). And cell extracts derived from silkworm tissue and the like.
  • lymphoma lymphoma
  • insect culture cells such as High Five (manufactured by Invitrogen) and Sf21 (manufactured by Invitrogen).
  • cell extracts derived from silkworm tissue and the like cell extracts derived from silkworm tissue and the like.
  • insect cell-derived cell extracts commercially available insect cell-derived cell extracts such as Transdirect insect cell (manufactured by Shimadzu Corporation) can also be used.
  • a cell extract derived from microorganisms for example, a cell extract derived from yeast, Escherichia coli or the like can be used.
  • a cell extract of yeast for example, Yeast, 1986, Vol. 2, pp. 35-52, Protein Nucleic Acid Enzyme, 1991, Vol. 36, pp. 2548-2554, Japanese Patent Application Laid-Open No. 2004-208640, and the like.
  • any of spore yeast, basidiomycetous yeast, and incomplete yeast can be used, but spore yeast is preferred.
  • Fission yeast is preferable as the spore yeast. Examples of the fission yeast include Schizosaccharomyces cerevisiae, Schizosaccharomyces pombe, and the like.
  • preparation of a cell extract derived from yeast can be performed, for example, as follows.
  • the yeast cells are rapidly frozen using an inert gas such as liquid nitrogen, acetone-dry ice or the like, and the cells are crushed with the cells frozen.
  • an inert gas such as liquid nitrogen, acetone-dry ice or the like
  • a method of crushing the frozen yeast cells As a method of crushing the frozen yeast cells, a method of crushing the yeast cells using a pestle in a mortar, a method of using a multi-bead shocker (manufactured by Yasui Kikai Co., Ltd.) using a metal cone as a crushing medium, etc. Can be mentioned.
  • a solution for extracting yeast cells can be prepared by adding an extraction solution to the crushed yeast cells.
  • the extraction liquid a liquid containing a protease inhibitor in the liquid for extraction is preferable, and a liquid containing at least a potassium salt, a magnesium salt, dithiothreitol and a buffer is more preferable.
  • potassium salt examples include potassium acetate, potassium carbonate, potassium hydrogen carbonate, potassium chloride, dipotassium hydrogen phosphate, dipotassium hydrogen citrate, potassium sulfate, potassium dihydrogen phosphate, potassium iodide, potassium phthalate and the like. Potassium acetate is preferred.
  • the potassium salt content is preferably 1 mM to 500 mM, more preferably 10 mM to 300 mM.
  • magnesium salt examples include magnesium acetate, magnesium sulfate, magnesium chloride, magnesium citrate, magnesium hydrogen phosphate, magnesium iodide, magnesium lactate, magnesium nitrate, and magnesium oxalate, with magnesium acetate being preferred.
  • the content of the magnesium salt is preferably 0.01 mM to 10 mM, and more preferably 0.1 mM to 5 mM.
  • the content of dithiothreitol (hereinafter sometimes referred to as “DTT”) is preferably 0.01 mM to 10 mM, and more preferably 0.1 mM to 5 mM.
  • the buffer examples include HEPES-KOH, Tris-HCl, acetic acid-sodium acetate, citric acid-sodium citrate, phosphoric acid, boric acid, MES, and PIPES.
  • a buffer that maintains the pH of the extraction solution at 4 to 10, more preferably a pH of 6 to 8.
  • the content of the buffer is preferably 5 mM to 200 mM, more preferably 10 mM to 100 mM.
  • the cell extract of E. coli can be obtained, for example, according to a known method described in Non-Patent Document 1 or Non-Patent Document 3, for example, 1) a step of culturing E. coli, 2) recovering E. coli, and It can be obtained through a step of obtaining an extract.
  • the gene associated with the endogenous protein may be removed from the E. coli used in advance by a known genetic engineering technique. Good.
  • a medium for culturing Escherichia coli it is a liquid medium containing a carbon source, a nitrogen source and inorganic salts that can be assimilated by Escherichia coli. It may be used.
  • the carbon source for example, glucose, sucrose, maltose, glycerol can be used.
  • glucose and glycerol are preferable, and glycerol is more preferable.
  • nitrogen source examples include ammonium salts of inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic acids or organic acids such as ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • the medium for culturing Escherichia coli include, for example, a modified 2 ⁇ YTPG medium (Table 1) in which the glucose in the 2 ⁇ YTPG medium and the 2 ⁇ YTPG medium are replaced with glycerol.
  • a known antifoaming agent examples include ADEKA (registered trademark) LG-295S.
  • the addition amount of the antifoaming agent may be, for example, 0.5 to 2 ml / L, and preferably 1 ml / L.
  • the pH of the medium may be 6 to 8, for example, and is preferably pH 7.
  • the pH of the medium can be adjusted using NaOH or the like.
  • T7 RNA polymerase or the like when E. coli capable of expressing T7 RNA polymerase or the like is used, a cell extract containing T7 RNA polymerase or the like in advance can be obtained.
  • an inducing agent such as IPTG
  • an inducing agent such as IPTG may be added to the medium.
  • the culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature can be, for example, 15 to 40 ° C.
  • the pH of the culture medium during the culture is preferably maintained at 3.0 to 9.0.
  • the pH during culture can be adjusted using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • Cultivation may be performed, for example, until the initial to late logarithmic growth is reached, and is preferably performed until the middle phase of logarithmic growth is reached.
  • the culture time is within these ranges, a cell extract with better protein synthesis efficiency can be obtained.
  • the middle phase of logarithmic growth is usually reached by culturing at a culture temperature of 36 ° C. for 4 to 6 hours.
  • fed-batch culture in which a medium (feed solution) is fed into the culture solution continuously or intermittently according to the passage of the culture time may be performed.
  • the fed-batch culture may be performed in E. coli according to a known method.
  • the carbon source mentioned in the above culture medium can be used as the carbon source in the feed solution.
  • glycerol when used in the culture medium, it is preferable to use glycerol as the carbon source of the feed solution.
  • a feed solution prepared by adjusting the concentration of the carbon source according to the growth of the cells may be prepared. However, a feed solution containing 30 to 70% of the carbon source is cultured in an appropriate amount according to the growth of the cells. It may be added inside.
  • Step 2 Step of recovering cultured E. coli and obtaining cell extract
  • the step of recovering E. coli cultured in step 1) and obtaining the cell extract from the recovered E. coli is used in the art for the purpose of cell-free protein synthesis. Can be carried out by known methods. For example, it can be performed according to the protocols described in Non-Patent Document 1 and Non-Patent Document 2. Specific methods for obtaining the cell extract include, for example, the following method of recovering cultured E. coli cells and destroying the recovered E. coli cells.
  • the cells are collected from the culture solution and kept at a low temperature until a cell extract is obtained. Specifically, it can be carried out at a low temperature of, for example, 0 to 15 ° C., preferably 2 to 10 ° C.
  • Examples of the method for recovering E. coli cells from the E. coli culture solution include a centrifugation method and a filtration method.
  • E. coli cells can be recovered by centrifuging at 4 ° C. and 7,000 to 14,000 ⁇ g for 20 to 50 minutes.
  • the washing solution a solution containing an inorganic salt and a compound having a buffering action, for example, an S30 buffer solution or the like is used.
  • the S30 buffer can be prepared as follows. For example, 6.06 g of 2-amino-2-hydroxymethyl-1,3-propanediol, 15.0 g of magnesium acetate tetrahydrate and 29.4 g of potassium acetate were added to about 450 ml of pure water, preferably milli-Q or the like. Dissolves in ultrapure water.
  • the obtained S30 buffer solution can be stored at 4 ° C., diluted 10-fold with ultrapure water immediately before use, and added with 1M DTT aqueous solution to a final concentration of 2 mM.
  • the step of washing E. coli cells using a solution containing the above-mentioned inorganic salt and a compound exhibiting a buffering action is performed by, for example, suspending the recovered cells in the solution and centrifuging.
  • the recovering step can be performed by repeating, for example, 2 to 3 times.
  • the suspension can be efficiently homogenized by using a homogenizer or the like.
  • the cells can be collected from the homogenized suspension by, for example, centrifuging for 10 to 50 minutes at 4 ° C. and 9,000 to 14,000 ⁇ g.
  • the washed cells can be stored at -80 ° C. Freezing at ⁇ 80 ° C. is preferably performed by rapid cooling using liquid nitrogen or the like.
  • Examples of means for destroying E. coli cells include means using a lytic enzyme such as an ultrasonic crusher, a French press, a high-pressure homogenizer, a dynomill, a mortar, glass beads, and lysozyme.
  • a lytic enzyme such as an ultrasonic crusher, a French press, a high-pressure homogenizer, a dynomill, a mortar, glass beads, and lysozyme.
  • a lytic enzyme such as an ultrasonic crusher, a French press, a high-pressure homogenizer, a dynomill, a mortar, glass beads, and lysozyme.
  • a lytic enzyme such as an ultrasonic crusher, a French press, a high-pressure homogenizer, a dynomill, a mortar, glass beads, and lysozyme.
  • it may be performed at a pressure of 600 to 1,700 bar and a flow rate of about 1 ml / min. From a
  • the state of destruction can be confirmed by analyzing the liquid before and after destruction with a particle size distribution meter. It is preferable to add DTT to the bacterial cell disruption solution so that the final concentration is 1 mM.
  • a cell suspension obtained by resuspending the collected cells in a solution (for example, S30 buffer) containing the above-described inorganic salt and a compound having a buffering action is used. It is preferable.
  • the bacterial cell suspension include a bacterial cell suspension obtained by adding a solution such as 1 to 2 mL of S30 buffer per 1 g of recovered bacterial cells.
  • a solution such as 1 to 2 mL of S30 buffer per 1 g of recovered bacterial cells.
  • a cell extract from which cell debris and genomic DNA have been removed It is preferable to use a cell extract from which cell debris and genomic DNA have been removed.
  • the removing method include a centrifugal separation method and a filtration method. For example, the process of centrifuging for 20 to 50 minutes at 4 ° C under conditions of 10,000 to 30,000 xg and obtaining the supernatant is repeated 2 to 3 times, for example, to remove cell debris and genomic DNA. can do.
  • a cell extract excluding endogenous nucleic acids Insoluble components generated by the treatment with the activated mix added can be removed by a centrifugal separation method or the like under the same conditions as described above. By this operation, endogenous RNA can be removed. Endogenous nucleic acid can be decomposed by further adding a nuclease or the like. In that case, it is preferable to add a nuclease inhibitor after the nucleic acid degradation. Endogenous amino acids, nucleic acids, nucleosides and the like can also be removed by dialysis.
  • the cell extract can be stored at ⁇ 80 ° C. Freezing at ⁇ 80 ° C. is preferably performed by rapid cooling using liquid nitrogen or the like.
  • the content of the cell extract in the reaction mixture according to this embodiment may be 5 to 70% (volume / volume) or 10 to 50% (volume / volume) based on the total amount of the reaction mixture. It may be 20-40% (capacity / capacity) or 20-30% (capacity / capacity).
  • the wet cell (g) / reaction mixture (mL) ratio used to obtain the cell extract may be 0.01-1 g / mL, and may be 0.05-0.3 g / mL. .
  • the reaction mixture for cell-free protein synthesis according to the present invention contains inosinic acid as an energy source and a substrate supply source required for mRNA synthesis.
  • Examples of the energy source include ATP, GTP, glucose, ribose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phospho Examples include glycerate, glucose-6-phosphate, citric acid, cis-aconitic acid, isocitric acid, ⁇ -ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid, and glutamic acid. In the case of an energy source that does not contain a phosphate group, it is preferable to add inorganic phosphoric acid to the reaction mixture.
  • inosinic acid has an industrial production process established as an umami component, and is very cheap compared to nucleotides used for normal cell-free protein synthesis. Furthermore, it is more than 1000 times more soluble in water than adenosine and guanosine, which can be nucleotide precursors. Therefore, the reaction mixture for cell-free protein synthesis according to the present invention can be easily used not only as a reaction mixture in a batch method but also as an external liquid component in a permeation method.
  • inosinic acid as a salt to the reaction mixture according to the present embodiment.
  • the salt of inosinic acid include a sodium salt, such as disodium inosinate.
  • the content of inosinic acid in the cell-free protein synthesis reaction mixture according to this embodiment may be 0.1 to 10 mM, 0.5 to 8 mM, or 1 to 8 mM based on the total amount of the reaction mixture. It may be 5 mM and may be 2-4 mM.
  • the inosinic acid when used as a source of ATP, GTP, UTP and CTP necessary for mRNA synthesis, the inosinic acid is added to the reaction mixture containing the cell extract and the energy source before the template nucleic acid is added. Is preferably added.
  • the reaction mixture for cell-free protein synthesis can contain at least one of the following components (i) to (viii) as necessary.
  • Template nucleic acid The template nucleic acid only needs to contain DNA or RNA encoding the target protein to be expressed and DNA or RNA containing an appropriate expression control region, and can be in either linear or circular form. May be. Examples of the expression control region include a promoter sequence, terminator sequence, enhancer sequence, poly A addition signal, and ribosome binding sequence.
  • the template nucleic acid preferably contains at least one promoter and DNA encoding the target protein.
  • the amount of template nucleic acid to be added is preferably 0.1 to 50 ⁇ g / mL, more preferably 1 to 20 ⁇ g / mL, based on the total volume of the reaction mixture.
  • the template nucleic acid may be designed so that a fusion protein incorporating a tag sequence can be synthesized so that the synthesized protein can be easily detected or purified.
  • a tag sequence for example, an affinity tag such as a histidine tag (His tag) utilizing specific affinity (binding, affinity) with other molecules, glutathione that specifically binds to glutathione-S- Examples include tag sequences such as transferase (GST) and maltose binding protein (MBP) that specifically binds to maltose.
  • GST transferase
  • MBP maltose binding protein
  • an “epitope tag” using an antigen-antibody reaction may be used. Examples of the epitope tag include HA (peptide sequence of influenza virus hemagglutinin) tag, myc tag, and FLAG tag. Further, a tag sequence that can be separated with a specific protease can also be used.
  • RNA polymerase When the template nucleic acid is DNA, RNA polymerase can be included. As the RNA polymerase, an RNA polymerase that recognizes one or more transcription factors targeting the template nucleic acid can be used.
  • the reaction mixture of the present invention preferably contains T7 RNA polymerase from the viewpoint of improving the production efficiency of the target protein. T7 RNA polymerase may be added when preparing the reaction mixture. As described above, T7 RNA polymerase is added to the cell extract using a strain such as BL21 (DE3) capable of expressing T7 RNA polymerase. It may be included.
  • the reaction mixture according to this embodiment may contain amino acids necessary for synthesizing the target protein.
  • the amino acids may include all 20 types of amino acids constituting the protein, and may be appropriately selected from 20 types in consideration of the target protein, the reagent to be used, and the like. Unnatural amino acids may also be used. When an unnatural amino acid is used, it is necessary to add factors such as tRNA and aminoacylated tRNA synthetase that have been modified to introduce the unnatural amino acid into a protein.
  • a substrate for RNA synthesis can be included in order to further increase the amount of protein synthesis.
  • the substrate for RNA synthesis include ribonucleotides such as ribonucleotide triphosphate (rNTP) and ribonucleotide monophosphate (rNMP), and ribonucleosides such as adenosine.
  • the reaction mixture according to the present embodiment can contain polyamines.
  • polyamines that can be included in the reaction mixture include spermine, spermidine, and putrescine.
  • (V) Salt The reaction mixture according to this embodiment can contain a salt.
  • Salts that can be included in the reaction mixture include, for example, potassium, magnesium, ammonium, and manganese salts of acetic acid, glutamic acid, or sulfuric acid.
  • the reaction mixture according to this embodiment can include an oxidation / reduction regulator.
  • the oxidation / reduction regulator include DTT, ascorbic acid, glutathione, and / or oxides thereof.
  • ribosome transfer RNA
  • optional translation factors eg, translation initiation factor, elongation factor termination factor, ribosome recycling factor, etc.
  • cofactors thereof aminoacyl tRNA synthetase (ARS), methionyl tRNA formyl transfer Enzyme (MTF), polymer compound (eg, polyethylene glycol, dextran, diethylaminoethyl dextran, quaternary aminoethyl and aminoethyldextran), nuclease, nuclease inhibitor, protein stabilizer, chaperone, solubilizer, non-denaturing interface
  • An active substance for example, Triton X100 or the like may be added to the reaction mixture as necessary.
  • the cell-free protein synthesis method according to the present invention can be carried out using the reaction mixture according to the present invention in both dialysis methods and batch methods.
  • protein synthesis is performed in a closed system consisting of an internal solution containing the above reaction mixture and an external solution containing a substrate and an energy source for synthesis of the target protein separated by a dialysis membrane such as an ultrafiltration membrane.
  • a dialysis membrane such as an ultrafiltration membrane.
  • a substrate for synthesizing a target protein, an energy source, and the like are supplied from an external solution to the reaction mixture via a dialysis membrane, and extra by-products in the reaction mixture can be diffused into the external solution. Therefore, the reaction can be continued for a longer time.
  • the batch method is a synthesis method in which the reaction mixture containing all the components necessary for protein synthesis is mixed with the reaction solution and uniformly contained in the reaction solution, and the reaction is performed.
  • the reaction time is shorter than that of the dialysis method. The result is immediately available.
  • Protein synthesis may be performed at 20 to 40 ° C., for example, and is preferably performed at 30 ° C.
  • the reaction time for protein synthesis by the batch method is preferably 2 hours or more after mixing the cell extract and inosinic acid, regardless of whether or not the template nucleic acid is added.
  • the reaction time is preferably 3 to 40 hours, more preferably 5 to 25 hours after mixing the cell extract and inosinic acid.
  • the kit for cell-free protein synthesis according to the present invention contains a cell extract obtained from E. coli and inosinic acid. Inosinic acid may be pre-mixed with the cell extract (cell extract obtained from E. coli) and included in the kit as a cell extract containing inosinic acid, and independently of the cell extract. It may be included in this kit.
  • the kit may contain a cell extract and inosinic acid obtained from E. coli, a template nucleic acid, a substrate for target protein synthesis, and / or an energy source.
  • the template nucleic acid, the substrate for synthesizing the target protein, and the energy source may be mixed in advance with the cell extract, and may be included in the kit independently of the cell extract.
  • This kit may further contain the above-mentioned RNA polymerase, polyamines, salt, oxidation / reduction regulator, and other additives. These additives may be mixed in advance with the cell extract, and may be included in the kit independently of the cell extract.
  • E. coli culture (effect of carbon source)
  • Escherichia coli BL21 strain (Novagen Co., Ltd.) was cultured in a flask with 2 ⁇ YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until OD 600 was 4.2.
  • the culture solution was modified 2 ⁇ YTPG medium with the composition of Table 1 using glycerol as a carbon source (Adeka (registered trademark) LG-295S was added as an antifoaming agent at 1 ml / L and adjusted to pH 7 with NaOH). 500 mL OD 600 was added to a 0.05 is entered a 1L jar fermenter.
  • the same strain was cultured in the same manner using a normal 2 ⁇ YTPG medium using glucose (18 g / L) as a carbon source.
  • the culture temperature was maintained at 36 ° C., and the culture was performed at a constant pH of 7.0.
  • the dissolved oxygen concentration was maintained at 2.4 mg / L or more.
  • the growth curve is shown in FIG. Modified 2 ⁇ YTPG medium and specific growth rate when cultured in a conventional 2 ⁇ YTPG medium was respectively 1.08H -1 and 0.74h -1. This result is presumed to suggest that ribosomes in cells cultured with glycerol and enzymes of the energy regeneration system have higher activity.
  • a culture solution having a preferable cell concentration can be obtained in a shorter time than culturing using glucose as a carbon source, so that a cell extract can be obtained in a shorter time. It was shown that there is.
  • the culture solution 2L of the BL21 strain was centrifuged under conditions of 7,000 ⁇ g, 4 ° C., and 20 minutes to recover 31 g of wet cells.
  • the cells were washed with an S30 buffer solution (pH 8.2) containing 10 mM Tris-acetic acid, 14 mM magnesium acetate, 60 mM potassium acetate and 2 mM DTT, and then rapidly cooled to ⁇ 80 ° C. using liquid nitrogen.
  • the microbial cell crushed material was centrifuged twice under the conditions of 20,400 ⁇ g, 4 ° C. and 30 minutes to remove the microbial cell residue.
  • 20 types comprising 300 mM Tris-acetic acid, 13.2 mM magnesium acetate, 13.2 mM ATP, 4.4 mM DTT, 6.7 U / mL pyruvate kinase, 84 mM phosphoenolpyruvate and protein in 200 ⁇ L of the centrifugation supernatant 60 ⁇ L of an activation mix containing all of the amino acids (0.04 mM each) was added and incubated at 30 ° C. for 150 minutes.
  • the mixture was centrifuged at 20,400 ⁇ g, 4 ° C. for 30 minutes, and the insoluble fraction was removed to obtain 230 ⁇ L of cell extract.
  • a DNA fragment (2) containing the replication origin and the ampicillin resistance gene sequence was prepared by the PCR method.
  • DNA fragment (1) and DNA fragment (2) were fused by a known In-Fusion reaction and transformed into E. coli HST08. After culturing the transformed E. coli, the plasmid was extracted using QIAGEN Plasmid Kit (manufactured by Qiagen).
  • a DNA fragment (3) containing the T7 promoter, RBS, T7 terminator, origin of replication and ampicillin resistance gene sequence was prepared by PCR.
  • a DNA fragment (4) containing a Holly-GFP gene was prepared by PCR using T5-HollyGFP (Cosmo Bio Inc.).
  • DNA fragment (3) and DNA fragment (4) were fused by a known In-Fusion reaction and transformed into E. coli HST08. After culturing the transformed E. coli, pUC-GFP was extracted using QIAGEN Plasmid Kit (manufactured by Qiagen) and used as a template nucleic acid for cell-free protein synthesis reaction.
  • PUC-GFP for template nucleic acid has high purity (A 260 / A 280 is 1.8 or more, A 260 / A 230 is 2.0 or more) using a spectrophotometer, and further, a DNA sequencer is used. The nucleotide sequence was read and it was confirmed that no unnecessary mutation was introduced.
  • Cell-free protein synthesis reaction 10 ⁇ L of the master mix containing the compound of Table 2, 6 ⁇ L of the prepared cell extract, 1 ⁇ L of 40 mM DTT aqueous solution, 2 ⁇ L of 0.2 g / L pUC-GFP (template nucleic acid) and 1 ⁇ L of 1000 U / ⁇ L T7 RNA polymerase were added to the reaction in a total volume of 20 ⁇ L.
  • a mixture (liquid) was prepared.
  • a negative control (NC) prepared by adding 2 ⁇ L of RO water instead of pUC-GFP was prepared.
  • the reaction mixture was transferred to a 384-well microplate, and the amount of GFP synthesis was compared by measuring the fluorescence intensity over time while incubating at 30 ° C. using a microplate reader TECANinfinite F200 (manufactured by Tecan Japan Co., Ltd.).
  • FIG. 2 shows the measurement result of the fluorescence intensity.
  • the fluorescence intensity of GFP in the case of using a cell extract derived from E. coli cultured in a medium containing glycerol as a carbon source is a cell derived from E. coli cultured in a medium containing glucose as a carbon source.
  • the value was about 2 times higher than the fluorescence intensity of GFP when the extract was used (FIG. 2).
  • the amount of GFP synthesized by the cell-free protein synthesis method using cell extracts derived from E. coli cultured in a medium containing glycerol as a carbon source is the same as the cell extract derived from E. coli cultured in a medium containing glucose as a carbon source. Compared with the cell-free protein synthesis method using, it was shown to be about twice as much.
  • Example 1 Cell-free protein synthesis method
  • the BW25113 strain lacking the rna gene was prepared by the method of Datsenko and Wanner, and the deletion was introduced into the T7 Express strain (New England Biolabs) by P1 transduction to prepare the T7 Express strain lacking the rna gene.
  • the rna gene is a gene involved in the synthesis of ribonuclease I and is involved in the stability of mRNA.
  • the T7 Express strain lacking the rna gene was cultured in a flask in 2 ⁇ YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until the OD 600 was 3.5.
  • the culture broth contained 2.1 L of modified 2 ⁇ YTPG medium (added 1 ml / L of ADEKA (registered trademark) LG-295S as an antifoam agent and adjusted to pH 7 using NaOH) with the composition shown in Table 1
  • the 3 L jar fermenter was added to an OD 600 of 0.05 and main culture was performed.
  • the main culture was performed while maintaining the culture temperature at 36 ° C., controlling the pH of the medium to be constant at 7.0, and maintaining the dissolved oxygen concentration of the medium at 2.4 mg / L or more.
  • OD 600 reached 2
  • 1 mM IPTG was added to the medium to induce T7 RNA polymerase. Thereafter, the culture was continued until the mid-log OD 600 reached 12.
  • the microbial cell crushed material was centrifuged twice under the conditions of 20,400 ⁇ g, 4 ° C. and 30 minutes to remove the microbial cell residue.
  • the obtained supernatant was used as a cell extract.
  • FIG. 3 shows the measurement result of the fluorescence intensity.

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Abstract

La présente invention concerne un mélange réactionnel pour la synthèse de protéines acellulaire, ledit mélange réactionnel comprenant un extrait cellulaire et de l'acide inosinique. La présente invention concerne également un procédé de synthèse de protéine acellulaire dans lequel ledit mélange réactionnel est utilisé. La présente invention concerne en outre un kit pour la synthèse de protéines acellulaire, ledit kit comprenant un extrait cellulaire, un acide inosinique, un acide nucléique matrice, et un substrat et/ou une source d'énergie pour la synthèse de la protéine cible.
PCT/JP2018/008797 2017-04-28 2018-03-07 Mélange réactionnel pour la synthèse de protéines acellulaires, procédé de synthèse de protéines acellulaires utilisant ce melange et kit pour la synthèse de protéines acellulaires WO2018198543A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020135747A1 (fr) * 2018-12-28 2020-07-02 康码（上海）生物科技有限公司 Système de synthèse de protéine acélullaire in vitro optimisé et application correspondante
WO2021241546A1 (fr) * 2020-05-28 2021-12-02 Ｓｐｉｂｅｒ株式会社 Procédé de production de protéine cible
CN115851558A (zh) * 2023-02-27 2023-03-28 百葵锐(天津)生物科技有限公司 一种新的原核无细胞蛋白质合成系统及其应用
CN117143895A (zh) * 2023-08-30 2023-12-01 态创生物科技(广州)有限公司 一种抗菌肽的无细胞合成体系、试剂盒及其应用

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020135747A1 (fr) * 2018-12-28 2020-07-02 康码（上海）生物科技有限公司 Système de synthèse de protéine acélullaire in vitro optimisé et application correspondante
WO2021241546A1 (fr) * 2020-05-28 2021-12-02 Ｓｐｉｂｅｒ株式会社 Procédé de production de protéine cible
CN115851558A (zh) * 2023-02-27 2023-03-28 百葵锐(天津)生物科技有限公司 一种新的原核无细胞蛋白质合成系统及其应用
CN117143895A (zh) * 2023-08-30 2023-12-01 态创生物科技(广州)有限公司 一种抗菌肽的无细胞合成体系、试剂盒及其应用

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