WO2018159647A1 - Reaction mixture for cell-free protein synthesis, cell-free protein synthesis method in which same is used, and kit for cell-free protein synthesis - Google Patents

Reaction mixture for cell-free protein synthesis, cell-free protein synthesis method in which same is used, and kit for cell-free protein synthesis Download PDF

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WO2018159647A1
WO2018159647A1 PCT/JP2018/007397 JP2018007397W WO2018159647A1 WO 2018159647 A1 WO2018159647 A1 WO 2018159647A1 JP 2018007397 W JP2018007397 W JP 2018007397W WO 2018159647 A1 WO2018159647 A1 WO 2018159647A1
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cell
protein synthesis
reaction mixture
acid
coli
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French (fr)
Japanese (ja)
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俊将 本間
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Ｓｐｉｂｅｒ株式会社
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    • C12P21/00—Preparation of peptides or proteins

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  • 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.
  • the in vitro cell-free protein synthesis method (1) can direct resources for protein synthesis to the exclusive production of the target protein, (2 ) Since it is not involved in 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) ) The ability to easily recover the purified and properly folded protein product as is, (4) the incorporation of non-naturally isotopically labeled amino acids, and (5) in vivo instability, insolubility or cytotoxicity.
  • Non-Patent Document 1 Proteins that are difficult to make in vivo because they require unique cofactors And (7) it is considered to have advantages such as that it can avoid processes such as cloning for expression in vivo and transformation of cells, etc. It is becoming an established method to some extent (Non-Patent Document 1).
  • 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.
  • Patent Document 1 As another approach to increase the reaction duration, a certain result has been obtained by reviewing the energy regeneration system in the in vitro reaction (Patent Document 1, 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 increasing the amount of protein synthesis in a cell-free protein synthesis method.
  • the present inventors have obtained cell extractions obtained from E. coli cultured in a medium using glycerol as a carbon source. It has been found that the amount of protein synthesis can be increased by using a reaction mixture containing a product, and the present invention has been completed.
  • a reaction mixture for cell-free protein synthesis comprising a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source.
  • the reaction mixture according to [1] further comprising a template nucleic acid, a substrate for synthesizing the target protein, and an energy source.
  • the template nucleic acid comprises DNA encoding at least one promoter and a target protein.
  • the energy source is ATP, GTP, glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and Selected from the group consisting of 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, The reaction mixture according to [2] or [3].
  • a cell-free protein synthesis kit comprising a cell extract obtained from E.
  • the amount of protein synthesis in a cell-free protein synthesis method can be increased by using a reaction mixture containing a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source. , Thereby improving protein production efficiency.
  • glycerol is inexpensive, the amount of protein synthesis in the cell-free protein synthesis method can be increased at low cost.
  • a medium containing glycerol as a carbon source does not cause a Maillard reaction when autoclaving, and thus has an advantage that proteins are hardly colored.
  • Comparison of growth curve (black triangle) of BL21 strain cultured in modified 2 ⁇ YTPG medium with glycerol as carbon source and growth curve (black circle) of BL21 strain cultured in normal 2 ⁇ YTPG medium with glucose as carbon source It is a graph which shows. 6 is a graph showing the results of measuring the fluorescence intensity of GFP synthesized by the cell-free protein synthesis method of Example 2 over time.
  • the black triangles show the results using the BL21 strain cultured in the modified 2 ⁇ YTPG medium, the black circles show the results using the BL21 strain cultured in the usual 2 ⁇ YTPG medium, and the white triangles and white circles are used instead of pUC-GFP.
  • the result of the negative control to which water was added is shown (white triangle: modified 2 ⁇ YTPG medium, white circle: normal 2 ⁇ YTPG medium).
  • the reaction mixture for cell-free protein synthesis of the present invention is characterized in that it contains a cell extract derived from E. coli cultured in a medium containing glycerol.
  • the cell-free protein synthesis method of the present invention includes a step of culturing E. coli in a medium using glycerol as a carbon source, a step of recovering the cultured E. coli and obtaining a cell extract from the recovered E. coli, and a reaction mixture containing the cell extract.
  • synthesize a protein using Cell-free protein synthesis using the reaction mixture of the present invention is performed in, for example, Methods in molecular biology, 267, 169-182, Mol. Syst. Biol, except that cell extracts from E. coli cultured in a medium containing glycerol are used. ., 2008; 4: 220 or Biotechnol. Bioeng., 2011; 108: 1570.
  • Any Escherichia coli microorganism can be used as the E. coli cultured in a medium using glycerol as a carbon source, for example, Escherichia coli.
  • a medium for culturing Escherichia coli is a liquid medium containing glycerol as a carbon source and containing a nitrogen source and inorganic salts that can be assimilated by Escherichia coli. Any of the media may be used.
  • the content of glycerol as a carbon source may be, for example, 2.5 to 100 g / L based on the total volume of the medium.
  • the lower limit of the glycerol content based on the total volume of the medium is 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10 , 12, 15 or 20 g / L.
  • the upper limit of the glycerol content based on the total volume of the medium may be 30, 40, 50, 60, 70, 80 or 90 g / L.
  • the glycerol content may be at least 2.5 g / L based on the total volume of the medium, and may be 5.0-40 g / L or 10-30 g / L.
  • the 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.
  • peptone and yeast extract can be used as the nitrogen source.
  • the content of the nitrogen source may be, for example, 5 to 60 g / L, 10 to 40 g / L, or 20 to 30 g / L based on the total volume of the medium.
  • inorganic salts examples include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.
  • monopotassium phosphate, dipotassium phosphate and sodium chloride can be used as an inorganic salt.
  • the content of the inorganic salt may be, for example, 1 to 50 g / L, 5 to 40 / L, or 10 to 20 g / L based on the total volume of the medium.
  • the modified 2 ⁇ YTPG medium shown in Table 1 in which glucose in the 2 ⁇ YTPG medium is replaced with glycerol can be exemplified.
  • an antifoaming agent such as Adeka (registered trademark) LG-295S
  • the addition amount of the antifoaming agent can be 0.5 to 2 ml / L, and preferably 1 ml / L.
  • the pH of the medium during the preparation of the medium is preferably 6 to 8, and 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.
  • the culture time can be the culture time from the beginning to the late phase of logarithmic growth, preferably the time until reaching the middle phase of logarithmic growth.
  • 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 as the culture time elapses may be performed.
  • the fed-batch culture may be performed according to a known method in E.
  • a feed solution with a glycerol concentration adjusted according to the growth of the cells may be prepared, but a feed solution containing 30-70% glycerol is added to the culture solution according to the growth of the cells. You may do it.
  • the cell extract means a solid, liquid or mixture thereof obtained by disrupting E. coli cultured in the step 1).
  • the cell extract contains factors necessary for transcription of RNA using DNA as a template and factors necessary for protein translation, which are contained in E. coli cells. Examples of these factors include ribosome, aminoacylated tRNA synthetase, translation initiation factor and translation termination factor.
  • a transcription and translation reaction system can be reconstructed in vitro to synthesize proteins.
  • the step of recovering E. coli and obtaining a cell extract from the recovered E. coli can be performed by methods known in the art for the purpose of cell-free protein synthesis. For example, it can be carried out in accordance with the protocol of James R. Swartz et al. Described in Methods molecular ology biology, 267, 169-182, Mol. Syst.
  • As a specific method for obtaining the cell extract for example, the following method for recovering E. coli cells and destroying the recovered E. coli cells can be given.
  • the cells are recovered from the culture solution and kept at a low temperature until the cell extract is obtained.
  • it can be carried out at a low temperature of 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 centrifugal separation method and a filtration method.
  • E. coli cells can be recovered by centrifuging for 20 to 50 minutes at 4 ° C. and 7,000 to 14,000 g.
  • 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. About 450 ml of pure water, preferably ultra-pure, such as mili-Q, containing 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 Dissolve in water.
  • the S30 buffer 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 an inorganic salt and a compound having a buffering action is performed by, for example, suspending the recovered cells in the solution and collecting them by centrifugation.
  • the process 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 recovered 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 Escherichia 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.
  • a cell suspension obtained by resuspending the collected cells in a solution containing an inorganic salt and a compound having a buffering action (for example, S30 buffer) is used.
  • a compound having a buffering action for example, S30 buffer
  • the bacterial cell suspension include a bacterial cell suspension to which a solution such as 1 to 2 mL of S30 buffer is added 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.
  • cell debris, genomic DNA, etc. are removed by repeating the step of centrifuging for 20-50 minutes at 4 ° C., 10,000-30,000 g, and obtaining the supernatant, for example, 2-3 times. Can do.
  • Cell extracts include 1/3 to 1/4 volume activation mix (300 mM Tris-acetate, pH 8.2, 13.2 mM magnesium acetate, 13.2 mM ATP, 4.4 mM DTT, 6.7 U / mL pyruvate kinase, 84 mM phosphoenolpyruvate and 20 kinds of amino acids (each 0.04 mM)) are added and incubated at 15 to 42 ° C., preferably 20 to 37 ° C. for 10 to 300 minutes, preferably 30 to 180 minutes. Is preferred. Protein synthesis can be performed efficiently by using such a cell extract.
  • a cell extract from which endogenous amino acids, nucleic acids, nucleosides and the like have been removed Insoluble components generated by the treatment with the activated mix added can be removed by a centrifugation method 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.
  • reaction mixture for cell-free protein synthesis was obtained in step 2.
  • the content of the cell extract in the reaction mixture may be 5 to 70% (volume / volume), 10 to 50%, or 20 to 40% based on the whole reaction mixture. 20-30%.
  • 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 of the present invention can contain the following components (i) to (viii) as necessary, in addition to the cell extract.
  • 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 regulatory region include a promoter sequence, terminator sequence, enhancer sequence, poly A addition signal, and ribosome binding sequence.
  • the template nucleic acid preferably includes DNA encoding at least one promoter and a 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-S-transferase that specifically binds to glutathione (GST), and tag sequences such as maltose binding protein (MBP) that specifically binds to maltose.
  • GST histidine tag
  • MBP maltose binding protein
  • an “epitope tag” using an antigen-antibody reaction may be used.
  • HA peptide sequence of hemagglutinin of influenza virus
  • myc tag peptide sequence of myc tag
  • FLAG tag peptide sequence of hemagglutinin of influenza virus
  • 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.
  • (Iii) Energy source If the cell extract lacks the energy required for protein synthesis, it is preferable to include an additional energy source in the reaction mixture.
  • the energy source can also be added or supplemented during the protein synthesis reaction.
  • Examples of energy sources include ATP, GTP, glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, Examples include 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.
  • the reaction mixture 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.
  • the reaction mixture can contain a substrate for RNA synthesis as a substrate for target protein synthesis.
  • a substrate for RNA synthesis include ribonucleotides such as ribonucleotide triphosphate (rNTP) and ribonucleotide monophosphate (rNMP).
  • the reaction mixture can contain polyamines.
  • polyamines that can be included in the reaction mixture include spermine, spermidine, and putrescine.
  • the reaction mixture 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 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 may be added as necessary.
  • the reaction mixture can be used to synthesize cell-free proteins.
  • a dialysis method using the reaction mixture of the present invention
  • 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. Done.
  • 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.
  • 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.
  • the protein synthesis system can be produced at a lower cost than the PANOx type using NTP or PEP. It can be performed. Protein synthesis may be performed at 20 to 40 ° C, preferably 30 ° C.
  • the reaction in the batch method is preferably performed for 2 to 8 hours.
  • the cell-free protein synthesis kit of the present invention includes a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source, a template nucleic acid, It includes a substrate and / or an energy source for target protein synthesis.
  • 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.
  • the 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.
  • Example 1 (Culture using glycerol) BL21 strain (Novagen) was cultured in a flask with 2 ⁇ YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until OD600 was 4.2.
  • 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. Specific growth rate when cultured in a modified 2xYTPG medium and normal 2xYTPG medium was respectively 1.08H -1 and 0.74h -1. This result is presumed that ribosomes in cells cultured in glycerol and enzymes of the energy regeneration system have higher activity.
  • a cell extract can be obtained in a shorter time because a culture solution having a preferable cell concentration can be obtained in a shorter time than in a culture using glucose by culturing with glycerol. .
  • Example 2 (Preparation of cell extract) By the same method as in Example 1, a cell extract was prepared by the following method using BL21 strain cultured in modified 2xYTPG medium and normal 2xYTPG medium until the log mid-phase OD600 was 12.
  • 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 S30 buffer (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 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 (1) containing T7 promoter, ribosome binding site (RBS) and T7 terminator sequences was prepared by PCR using pET3a (Novagen).
  • pUC19 a DNA fragment (2) containing the replication origin and the ampicillin resistance gene sequence was prepared by PCR.
  • DNA fragment (1) and DNA fragment (2) were fused by In-Fusion reaction and transformed into E. coli HST08.
  • the plasmid was extracted using QIAGEN Plasmid Kit.
  • 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). DNA fragment (3) and DNA fragment (4) were fused by In-Fusion reaction and transformed into E. coli HST08. After culturing the E. coli, pUC-GFP was extracted using QIAGEN Plasmid Kit and used as a template nucleic acid for cell-free protein synthesis reaction. The pUC-GFP for template nucleic acid has high purity (A260 / A280 is 1.8 or more, A260 / A230 is 2.0 or more) using a spectrophotometer, and the nucleotide sequence is determined using a DNA sequencer. Reading and confirming that there were no unnecessary mutations.
  • Cell-free protein synthesis reaction 10 ⁇ L of master mix containing the compounds of Table 2, 6 ⁇ L of cell extract, 1 ⁇ L of 40 mM DTT aqueous solution, 2 ⁇ L of 0.2 g / L pUC-GFP and 1 ⁇ L of 1000 U / ⁇ L T7 RNA polymerase were added to prepare a total reaction mixture (liquid) of 20 ⁇ L. .
  • 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 TECANinfine F200.
  • FIG. 2 shows the measurement result of the fluorescence intensity.
  • the fluorescence intensity of GFP by a cell-free protein synthesis method using a medium containing glycerol as a carbon source for culturing E. coli cells was determined using a medium containing glucose as a carbon source for culturing E. coli cells. The value was approximately twice as high as the fluorescence intensity of GFP obtained by the cell-free protein synthesis method.
  • the amount of GFP synthesized by the cell-free protein synthesis method using a medium containing glycerol as a carbon source for culturing E. coli cells is the same as the method for cell-free protein synthesis using a medium containing glucose as a carbon source for culturing E. coli cells. It was shown to be about twice as high as that of.

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Abstract

Disclosed is a reaction mixture for cell-free protein synthesis, wherein the reaction mixture includes a cell extract obtained from Escherichia coli cultured in a medium having glycerol as a carbon source.

Description

無細胞タンパク質合成用反応混合物、これを用いた無細胞タンパク質合成方法及び無細胞タンパク質合成用キットCell-free protein synthesis reaction mixture, cell-free protein synthesis method using the same, and cell-free protein synthesis kit
　本発明は、無細胞タンパク質合成用反応混合物、これを用いた無細胞タンパク質合成方法及び無細胞タンパク質合成用キットに関する。 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.
　組換えタンパク質の生産方法としては、形質転換された細胞を培養することによるインビボによる方法と、いわゆる無細胞タンパク質合成方法と呼ばれる、細胞由来の抽出物を用いるインビトロでの方法が知られている。 As a method for producing a recombinant protein, an in vivo method by culturing transformed cells and an in vitro method using a cell-derived extract called a so-called cell-free protein synthesis method are known.
　工業規模でのタンパク質の生産方法としては、現段階ではインビボによる方法が優れていると考えられている。しかしながら、インビボによる方法と比較して、インビトロでの無細胞タンパク質合成方法は、（１）タンパク質の合成のための資源を、目的とするタンパク質の独占的な産生に向けることができる点、（２）細胞の増殖又は生存に関わることがないため、遺伝子のコドン使用を反映させたｔＲＮＡレベルの変更、酸化還元電位、ｐＨ、あるいはイオン強度といった条件等、合成環境を柔軟に変更できる点、（３）精製されて適切に折りたたまれたタンパク質産物をそのまま簡単に回収できる点、（４）非天然で同位体標識されたアミノ酸を組み入れことができる点、（５）インビボで不安定、不溶性又は細胞毒性であるタンパク質を合成することができる点、（６）独特の補因子を必要とするためにインビボでは作ることが難しいタンパク質を合成することができる点、及び（７）インビボにおける発現のためのクローニング、細胞の形質転換等のプロセスを回避することができる点等の利点を有すると考えられるため、この分野の基盤技術になりつつあり、ある程度、確立された方法となっている（非特許文献１）。 As a protein production method on an industrial scale, an in vivo method is considered to be superior at this stage. However, compared with the in vivo method, the in vitro cell-free protein synthesis method (1) can direct resources for protein synthesis to the exclusive production of the target protein, (2 ) Since it is not involved in 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) ) The ability to easily recover the purified and properly folded protein product as is, (4) the incorporation of non-naturally isotopically labeled amino acids, and (5) in vivo instability, insolubility or cytotoxicity. (6) Proteins that are difficult to make in vivo because they require unique cofactors And (7) it is considered to have advantages such as that it can avoid processes such as cloning for expression in vivo and transformation of cells, etc. It is becoming an established method to some extent (Non-Patent Document 1).
　しかしながら、インビボによる方法と比較して、無細胞タンパク質合成方法は、タンパク質を合成するための反応持続時間が短いため、生産効率が低い。この反応持続時間を増加させるため、翻訳反応により消費される基質を供給するとともに、反応を阻害する副産物を透析により除去する透析膜を使用した連続的流系が開発された。しかしながら、この連続的流系によりタンパク質合成量を増加させるためには連続的に試薬を追加することが必要となり、試薬等のコストが嵩む等の問題があった。 However, compared to the in vivo method, the cell-free protein synthesis method has a low production efficiency because the reaction duration for synthesizing the protein is short. In order to increase the reaction duration, 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. However, 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.
　また、反応持続時間を増加させる別のアプローチとして、インビトロでの反応におけるエネルギー再生系を見直すことにより、一定の成果が得られており（特許文献１、非特許文献２）、顆粒球マクロファージコロニー刺激因子を１０時間で７００ｍｇ／Ｌ合成することに成功している（非特許文献３）。 As another approach to increase the reaction duration, a certain result has been obtained by reviewing the energy regeneration system in the in vitro reaction (Patent Document 1, Non-Patent Document 2), and granulocyte macrophage colony stimulation Succeeded in synthesizing 700 mg / L of factor in 10 hours (Non-patent Document 3).
　しかしながら、工業規模での生産においては、生産量及びコストにおいて更なる改善が求められている。 However, in production on an industrial scale, further improvements in production volume and cost are required.
特許第５２５９０４６号Japanese Patent No. 5259046
　本発明は、無細胞タンパク質合成方法においてタンパク質合成量を増加させるための新規なアプローチを提供することを目的とする。 An object of the present invention is to provide a novel approach for increasing the amount of protein synthesis in a cell-free protein synthesis method.
　本発明者らは、種々の培養条件で培養した大腸菌より得られる細胞抽出物を用いてタンパク質合成の方法を鋭意検討した結果、炭素源としてグリセロールを用いた培地で培養した大腸菌より得られる細胞抽出物を含む反応混合物を用いることにより、タンパク質合成量を増加させることができることを見出し、本発明を完成するに至った。 As a result of intensive studies on protein synthesis methods using cell extracts obtained from E. coli cultured under various culture conditions, the present inventors have obtained cell extractions obtained from E. coli cultured in a medium using glycerol as a carbon source. It has been found that the amount of protein synthesis can be increased by using a reaction mixture containing a product, and the present invention has been completed.
　すなわち、本発明は、例えば、以下の各発明に関する。
［１］　無細胞タンパク質合成用反応混合物であって、グリセロールを炭素源とした培地で培養した大腸菌から得られた細胞抽出物を含む、反応混合物。
［２］　鋳型核酸、目的タンパク質合成のための基質、及びエネルギー源をさらに含む、上記［１］に記載の反応混合物。
［３］　鋳型核酸が、少なくとも１つのプロモーター及び目的タンパク質をコードするＤＮＡを含む、上記［２］に記載の反応混合物。
［４］　エネルギー源が、ＡＴＰ、ＧＴＰ、グルコース、ピルベート、ホスホエノールピルベート（ＰＥＰ）、カルバモイルホスフェート、アセチルホスフェート、クレアチンホスフェート、ホスホピルベート、グリセルアルデヒド－３－ホスフェート、３－ホスホグリセレート及びグルコース－６－ホスフェート、クエン酸、cis-アコニット酸、イソクエン酸、α－ケトグルタル酸、スクシニルＣoＡ、コハク酸、フマル酸、リンゴ酸、オキサロ酢酸、グリオキシル酸及びグルタミン酸からなる群より選ばれる、上記［２］又は［３］に記載の反応混合物。
［５］　大腸菌が、Ｔ７　ＲＮＡポリメラーゼを発現する大腸菌である、上記［１］～［４］のいずれかに記載の反応混合物。
［６］　上記［１］～［５］のいずれかに記載の反応混合物を用いた無細胞タンパク質合成方法。
［７］　グリセロールを炭素源とした培地で大腸菌を培養する工程、
　培養した大腸菌を回収し、回収した大腸菌から細胞抽出物を得る工程、及び
　細胞抽出物を含む反応混合物を用いてタンパク質を合成する工程
を含む、無細胞タンパク質合成方法。
［８］　グリセロールを炭素源とした培地で培養した大腸菌から得られた細胞抽出物、　並びに
鋳型核酸、目的タンパク質合成のための基質、及び／又はエネルギー源
を含む、無細胞タンパク質合成用キット。
［９］　上記［６］記載の無細胞タンパク質合成方法により得られるタンパク質。 That is, the present invention relates to the following inventions, for example.
[1] A reaction mixture for cell-free protein synthesis, comprising a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source.
[2] The reaction mixture according to [1], further comprising a template nucleic acid, a substrate for synthesizing the target protein, and an energy source.
[3] The reaction mixture according to the above [2], wherein the template nucleic acid comprises DNA encoding at least one promoter and a target protein.
[4] The energy source is ATP, GTP, glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and Selected from the group consisting of glucose-6-phosphate, citric acid, cis-aconitic acid, isocitric acid, α-ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid and glutamic acid, The reaction mixture according to [2] or [3].
[5] The reaction mixture according to any one of [1] to [4] above, wherein the E. coli is an E. coli expressing T7 RNA polymerase.
[6] A cell-free protein synthesis method using the reaction mixture according to any one of [1] to [5].
[7] A step of culturing E. coli in a medium using glycerol as a carbon source,
A cell-free protein synthesis method comprising the steps of recovering cultured E. coli, obtaining a cell extract from the recovered E. coli, and synthesizing a protein using a reaction mixture containing the cell extract.
[8] A cell-free protein synthesis kit comprising a cell extract obtained from E. coli cultured in a medium containing glycerol as a carbon source, and a template nucleic acid, a substrate for synthesizing a target protein, and / or an energy source.
[9] A protein obtained by the cell-free protein synthesis method according to [6] above.
　本発明の方法によれば、炭素源としてグリセロールを用いた培地で培養した大腸菌から得られる細胞抽出物を含む反応混合物を用いることにより、無細胞タンパク質合成方法におけるタンパク質合成量を増加させることができ、それによりタンパク質の生産効率を向上することができる。 According to the method of the present invention, the amount of protein synthesis in a cell-free protein synthesis method can be increased by using a reaction mixture containing a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source. , Thereby improving protein production efficiency.
　また、グリセロールは安価であるため、低コストで無細胞タンパク質合成方法におけるタンパク質合成量を増加させることができる。 Also, since glycerol is inexpensive, the amount of protein synthesis in the cell-free protein synthesis method can be increased at low cost.
　また、グリセロールを用いた培養はグルコースを用いた培養よりも大腸菌細胞の増殖速度が速いため、グルコースを用いた場合と比較してより短時間で細胞抽出物を得ることができる。 In addition, since culture using glycerol has a faster growth rate of E. coli cells than culture using glucose, a cell extract can be obtained in a shorter time than when glucose is used.
　さらに、炭素源としてグリセロールを含む培地は、オートクレーブする際にメイラード反応が起こらないため、タンパク質が着色されにくいという利点も有する。 Furthermore, a medium containing glycerol as a carbon source does not cause a Maillard reaction when autoclaving, and thus has an advantage that proteins are hardly colored.
グリセロールを炭素源とした改変２×ＹＴＰＧ培地により培養したＢＬ２１株の増殖曲線（黒三角）と、グルコースを炭素源とした通常の２×ＹＴＰＧ培地により培養したＢＬ２１株の増殖曲線（黒丸）の比較を示すグラフである。Comparison of growth curve (black triangle) of BL21 strain cultured in modified 2 × YTPG medium with glycerol as carbon source and growth curve (black circle) of BL21 strain cultured in normal 2 × YTPG medium with glucose as carbon source It is a graph which shows. 実施例２の無細胞タンパク質合成方法により合成したＧＦＰの蛍光強度を経時的に測定した結果を示すグラフである。黒三角は、改変２×ＹＴＰＧ培地により培養したＢＬ２１株を用いた結果、黒丸は通常の２×ＹＴＰＧ培地により培養したＢＬ２１株を用いた結果を示し、白三角及び白丸はｐＵＣ－ＧＦＰの代わりに水を加えたネガティブコントロールの結果を示す（白三角：改変２×ＹＴＰＧ培地、白丸：通常の２×ＹＴＰＧ培地）。6 is a graph showing the results of measuring the fluorescence intensity of GFP synthesized by the cell-free protein synthesis method of Example 2 over time. The black triangles show the results using the BL21 strain cultured in the modified 2 × YTPG medium, the black circles show the results using the BL21 strain cultured in the usual 2 × YTPG medium, and the white triangles and white circles are used instead of pUC-GFP. The result of the negative control to which water was added is shown (white triangle: modified 2 × YTPG medium, white circle: normal 2 × YTPG medium).
無細胞タンパク質合成用反応混合物を用いたタンパク質合成
　本発明の無細胞タンパク質合成用反応混合物は、グリセロールを含む培地で培養された大腸菌由来の細胞抽出物を含むことを特徴とする。本発明の無細胞タンパク質合成方法は、グリセロールを炭素源とした培地で大腸菌を培養する工程、培養した大腸菌を回収し、回収した大腸菌から細胞抽出物を得る工程、及び細胞抽出物を含む反応混合物を用いてタンパク質を合成する工程を含む。本発明の反応混合物を用いた無細胞タンパク質合成は、グリセロールを含む培地で培養された大腸菌からの細胞抽出物を用いる以外は、例えばMethods in molecular biology, 267, 169-182、Mol. Syst. Biol., 2008; 4: 220あるいはBiotechnol. Bioeng., 2011; 108: 1570に記載の公知の方法に準じて行うことができる。 Protein synthesis using reaction mixture for cell-free protein synthesis The reaction mixture for cell-free protein synthesis of the present invention is characterized in that it contains a cell extract derived from E. coli cultured in a medium containing glycerol. The cell-free protein synthesis method of the present invention includes a step of culturing E. coli in a medium using glycerol as a carbon source, a step of recovering the cultured E. coli and obtaining a cell extract from the recovered E. coli, and a reaction mixture containing the cell extract. And synthesize a protein using Cell-free protein synthesis using the reaction mixture of the present invention is performed in, for example, Methods in molecular biology, 267, 169-182, Mol. Syst. Biol, except that cell extracts from E. coli cultured in a medium containing glycerol are used. ., 2008; 4: 220 or Biotechnol. Bioeng., 2011; 108: 1570.
１）グリセロールを炭素源とした培地で大腸菌を培養する工程
　グリセロールを炭素源とした培地で培養する大腸菌としては、エシェリヒア・コリに属する微生物であればいずれも用いることができ、例えば、エシェリヒア・コリ　Ａ１９、ＢＬ２１（ノバジェン社）、ＢＬ２１（ＤＥ３）（ライフテクノロジーズ社）、ＢＬＲ（ＤＥ３）（メルクミリポア社）、ＤＨ１、ＧＩ６９８、ＨＢ１０１、ＪＭ１０９、Ｋ５（ＡＴＣＣ２３５０６）、Ｋ－１２、ＫＹ３２７６、ＭＣ１０００、ＭＧ１６５５（ＡＴＣＣ４７０７６）、ＮＭＲ２、Ｎｏ．４９、Ｒｏｓｅｔｔａ（ＤＥ３）（ノバジェン社）、ＴＢ１、Ｔｕｎｅｒ（ノバジェン社）、Ｔｕｎｅｒ（ＤＥ３）（ノバジェン社）、Ｗ１４８５、Ｗ３１１０（ＡＴＣＣ２７３２５）、ＸＬ１－Ｂｌｕｅ、ＸＬ２－Ｂｌｕｅ等の株を挙げることができる。 1) Step of culturing Escherichia coli in a medium using glycerol as a carbon source Any Escherichia coli microorganism can be used as the E. coli cultured in a medium using glycerol as a carbon source, for example, Escherichia coli. A19, BL21 (Novagen), BL21 (DE3) (Life Technologies), BLR (DE3) (Merck Millipore), DH1, GI698, HB101, JM109, K5 (ATCC23506), K-12, KY3276, MC1000, MG1655 (ATCC 47076), NMR2, No. 49, Rosetta (DE3) (Novagen), TB1, Tuner (Novagen), Tuner (DE3) (Novagen), W1485, W3110 (ATCC27325), XL1-Blue, XL2-Blue, etc. .
　大腸菌を培養するための培地としては、炭素源としてグリセロールを用い、大腸菌が資化し得る窒素源及び無機塩類等を含有する液体培地であり、培養を効率的に行える培地であれば天然培地、合成培地のいずれを用いてもよい。 As a medium for culturing Escherichia coli, it is a liquid medium containing glycerol as a carbon source and containing a nitrogen source and inorganic salts that can be assimilated by Escherichia coli. Any of the media may be used.
　炭素源としてのグリセロールの含有量は、例えば、培地全体の容量を基準として２．５～１００ｇ／Ｌであってよい。培地全体の容量を基準としたグリセロールの含有量の下限値は、２．５、３．０、４．０、５．０、６．０、７．０、８．０、９．０、１０、１２、１５又は２０ｇ／Ｌであってよい。培地全体の容量を基準としたグリセロールの含有量の上限値は、３０、４０、５０、６０、７０、８０又は９０ｇ／Ｌであってよい。グリセロールの含有量は培地全体の容量を基準として少なくとも２．５ｇ／Ｌであってよく、５．０～４０ｇ／Ｌ又は１０～３０ｇ／Ｌであってもよい。 The content of glycerol as a carbon source may be, for example, 2.5 to 100 g / L based on the total volume of the medium. The lower limit of the glycerol content based on the total volume of the medium is 2.5, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10 , 12, 15 or 20 g / L. The upper limit of the glycerol content based on the total volume of the medium may be 30, 40, 50, 60, 70, 80 or 90 g / L. The glycerol content may be at least 2.5 g / L based on the total volume of the medium, and may be 5.0-40 g / L or 10-30 g / L.
　窒素源としては、例えば、アンモニア、塩化アンモニウム、硫酸アンモニウム、酢酸アンモニウム及びリン酸アンモニウム等の無機酸又は有機酸のアンモニウム塩、その他の含窒素化合物、並びにペプトン、肉エキス、酵母エキス、コーンスチープリカー、カゼイン加水分解物、大豆粕及び大豆粕加水分解物、各種発酵菌体及びその消化物を用いることができる。例えば、窒素源として、ペプトン及び酵母エキスを用いることができる。窒素源の含有量は、例えば、培地全体の容量を基準として５～６０ｇ／Ｌであってよく、１０～４０ｇ／Ｌであってよく、２０～３０ｇ／Ｌであってよい。 Examples of the nitrogen source 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. For example, peptone and yeast extract can be used as the nitrogen source. The content of the nitrogen source may be, for example, 5 to 60 g / L, 10 to 40 g / L, or 20 to 30 g / L based on the total volume of the medium.
　無機塩としては、例えば、リン酸第一カリウム、リン酸第二カリウム、リン酸マグネシウム、硫酸マグネシウム、塩化ナトリウム、硫酸第一鉄、硫酸マンガン、硫酸銅及び炭酸カルシウムを用いることができる。例えば、無機塩として、リン酸第一カリウム、リン酸第二カリウム及び塩化ナトリウムを用いることができる。無機塩の含有量は、例えば、培地全体の容量を基準として１～５０ｇ／Ｌであってよく、５～４０／Ｌであってよく、１０～２０ｇ／Ｌであってよい。 Examples of inorganic salts that can be used include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate. For example, as an inorganic salt, monopotassium phosphate, dipotassium phosphate and sodium chloride can be used. The content of the inorganic salt may be, for example, 1 to 50 g / L, 5 to 40 / L, or 10 to 20 g / L based on the total volume of the medium.
　具体的な培地としては、例えば、２×ＹＴＰＧ培地のグルコースをグリセロールに替えた、表１に示した改変２×ＹＴＰＧ培地をあげることができる。培養時の発泡を防ぐために、当該培地に消泡剤、例えばアデカ（登録商標）ＬＧ－２９５Ｓ等を添加することが好ましい。消泡剤の添加量としては０．５～２ｍｌ／Ｌをあげることができ、１ｍｌ／Ｌが好ましい。培地調製時の培地のｐＨは、６～８が良く、ｐＨ７が好ましい。培地のｐＨの調整はＮａＯＨ等を用いて行うことができる。
Figure JPOXMLDOC01-appb-T000001
 As a specific medium, for example, the modified 2 × YTPG medium shown in Table 1 in which glucose in the 2 × YTPG medium is replaced with glycerol can be exemplified. In order to prevent foaming during culture, it is preferable to add an antifoaming agent such as Adeka (registered trademark) LG-295S to the medium. The addition amount of the antifoaming agent can be 0.5 to 2 ml / L, and preferably 1 ml / L. The pH of the medium during the preparation of the medium is preferably 6 to 8, and preferably pH 7. The pH of the medium can be adjusted using NaOH or the like.
Figure JPOXMLDOC01-appb-T000001
　また、Ｔ７　ＲＮＡポリメラーゼ等の発現が可能な大腸菌を用いた場合には、Ｔ７　ＲＮＡポリメラーゼ等をあらかじめ含んだ細胞抽出物が得られる。Ｔ７　ＲＮＡポリメラーゼ等の発現がＩＰＴＧのような誘導剤により惹起される場合には培地にＩＰＴＧ等の誘導剤を加えれば良い。 In addition, 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. When expression of T7 RNA polymerase or the like is induced by an inducing agent such as IPTG, an inducing agent such as IPTG may be added to the medium.
　培養は、振盪培養又は深部通気攪拌培養等の好気的条件下で行うことが好ましい。培養温度は、例えば、１５～４０℃で行うことができる。培養中の培養培地のｐＨは３．０～９．０に保持することが好ましい。培養中のｐＨの調整は、無機酸、有機酸、アルカリ溶液、尿素、炭酸カルシウム及びアンモニア等を用いて行うことができる。 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.
　培養時間は、タンパク質合成効率の良い細胞抽出物を得るために、対数増殖初期～後期、好ましくは対数増殖中期に達する時点までの培養時間をあげることができる。改変２×ＹＴＰＧ培地を用いる場合には、培養温度３６℃で、４～６時間培養することで通常対数増殖中期に達する。
　高濃度の菌体培養液を得るために、培養時間の経過に応じて連続的または断続的に培地（フィード液）を培養液中へ流加する流加培養を行っても良い。流加培養は大腸菌において公知の方法に準じて行えば良いが、この流加培養においても、フィード液中の炭素源としてグリセロールを用いることが重要である。グリセロール濃度を菌体の増殖に応じて調製したフィード液を準備しても良いが、３０～７０％グリセロールを含むフィード液を、菌体の増殖に合わせて、適切な量を培養液中へ添加しても良い。 In order to obtain a cell extract with high protein synthesis efficiency, the culture time can be the culture time from the beginning to the late phase of logarithmic growth, preferably the time until reaching the middle phase of logarithmic growth. When a modified 2 × YTPG medium is used, the middle phase of logarithmic growth is usually reached by culturing at a culture temperature of 36 ° C. for 4 to 6 hours.
In order to obtain a high-concentration bacterial cell culture solution, fed-batch culture in which a medium (feed solution) is fed into the culture solution continuously or intermittently as the culture time elapses may be performed. The fed-batch culture may be performed according to a known method in E. coli, but it is important to use glycerol as a carbon source in the feed solution also in this fed-batch culture. A feed solution with a glycerol concentration adjusted according to the growth of the cells may be prepared, but a feed solution containing 30-70% glycerol is added to the culture solution according to the growth of the cells. You may do it.
２）大腸菌を回収し、細胞抽出物を得る工程
　細胞抽出物は、１）の工程で培養した大腸菌を破壊して得られた、固体、液体又はその混合物を意味する。細胞抽出物には、大腸菌細胞内に含まれていた、ＤＮＡを鋳型としたＲＮＡの転写に必要な因子、並びにタンパク質の翻訳に必要な因子が含まれる。これらの因子としては、例えば、リボソーム、アミノアシル化ｔＲＮＡ合成酵素、翻訳開始因子及び翻訳終結因子が挙げられる。この細胞抽出物を含む反応混合物を用いて、インビトロで転写及び翻訳の反応系を再構成してタンパク質を合成させることができる。 2) Step of recovering E. coli and obtaining cell extract The cell extract means a solid, liquid or mixture thereof obtained by disrupting E. coli cultured in the step 1). The cell extract contains factors necessary for transcription of RNA using DNA as a template and factors necessary for protein translation, which are contained in E. coli cells. Examples of these factors include ribosome, aminoacylated tRNA synthetase, translation initiation factor and translation termination factor. Using the reaction mixture containing the cell extract, a transcription and translation reaction system can be reconstructed in vitro to synthesize proteins.
　大腸菌を回収し、回収した大腸菌から細胞抽出物を得る工程は、無細胞タンパク質合成の目的のために当該分野において既知の方法により行うことができる。例えば、Methods in molecular biology, 267, 169-182、Mol. Syst. Biol 2008 4 220に記載のJames R. Swartzらのプロトコルに準じて行うことができる。細胞抽出物を得るための具体的な方法としては、例えば以下のような、大腸菌の菌体を回収し、回収した大腸菌の菌体を破壊する方法等をあげることができる。 The step of recovering E. coli and obtaining a cell extract from the recovered E. coli can be performed by methods known in the art for the purpose of cell-free protein synthesis. For example, it can be carried out in accordance with the protocol of James R. Swartz et al. Described in Methods molecular ology biology, 267, 169-182, Mol. Syst. As a specific method for obtaining the cell extract, for example, the following method for recovering E. coli cells and destroying the recovered E. coli cells can be given.
　大腸菌を回収し、回収した大腸菌から細胞抽出物を得る工程において、培養液から菌体を回収し、細胞抽出物を得るまでは低温に保って行うことが好ましい。例えば、０～１５℃、好ましくは２～１０℃の低温に保って行うことができる。 In the step of recovering E. coli and obtaining the cell extract from the recovered E. coli, it is preferable that the cells are recovered from the culture solution and kept at a low temperature until the cell extract is obtained. For example, it can be carried out at a low temperature of 0 to 15 ° C., preferably 2 to 10 ° C.
　大腸菌培養液から大腸菌の菌体を回収する方法としては、遠心分離法、濾過法をあげることができる。例えば、４℃、７，０００～１４，０００ｇの条件で２０～５０分間、遠心分離することにより大腸菌の菌体を回収することができる。 Examples of the method for recovering E. coli cells from the E. coli culture solution include a centrifugal separation method and a filtration method. For example, E. coli cells can be recovered by centrifuging for 20 to 50 minutes at 4 ° C. and 7,000 to 14,000 g.
　回収後に、培地成分等を除去するため、回収した大腸菌の菌体を洗浄する工程を含むことが好ましい。洗浄溶液としては、無機塩及び緩衝作用を示す化合物を含んだ溶液、例えば、Ｓ３０緩衝液等が用いられる。Ｓ３０緩衝液は以下のように調製することができる。２－アミノ－２－ヒドロキシメチル－１，３－プロパンジオール６．０６ｇ、酢酸マグネシウム四水和物１５．０ｇ及び酢酸カリウム２９．４ｇを約４５０ｍｌの純水、好ましくはｍｉｌｉ－Ｑ等の超純水に溶解する。溶解後、酢酸でｐＨ８．２に調整し、５００ｍｌにメスアップし、０．２２μｍのフィルターで滅菌する。Ｓ３０緩衝液は４℃で保存することができ、使用直前に超純水で１０倍に希釈し、最終濃度が２ｍＭになるよう１Ｍ　ＤＴＴ水溶液を加えたものを使用する。 After the collection, it is preferable to include a step of washing the collected E. coli cells in order to remove medium components and the like. As 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. About 450 ml of pure water, preferably ultra-pure, such as mili-Q, containing 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 Dissolve in water. After dissolution, adjust to pH 8.2 with acetic acid, make up to 500 ml, and sterilize with a 0.22 μm filter. The S30 buffer 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.
　無機塩及び緩衝作用を示す化合物を含んだ溶液（例えばＳ３０緩衝液）を用いて大腸菌の菌体を洗浄する工程は、例えば、回収した菌体を、当該溶液に懸濁し、遠心分離で回収する工程を例えば２～３回繰り返すことにより行うことができる。懸濁はホモジナイザー等を用いることにより効率的に均質化できる。均質化した懸濁液からの菌体の回収は、例えば４℃、９，０００～１４，０００ｇの条件で１０～５０分間遠心分離することにより行うことができる。当該洗浄菌体は－８０℃で保存することができる。－８０℃での凍結は、液体窒素等を用いた急冷による方法が好ましい。 The step of washing E. coli cells using a solution containing an inorganic salt and a compound having a buffering action (for example, S30 buffer) is performed by, for example, suspending the recovered cells in the solution and collecting them by centrifugation. The process 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 recovered 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.
　大腸菌の菌体を破壊する手段としては、例えば、超音波破砕機、フレンチプレス、高圧ホモゲナイザー、ダイノミル、乳鉢、ガラスビーズ及びリゾチーム等の溶菌酵素を用いる手段をあげることができる。例えば、高圧ホモゲナイザーを用いて菌体を破砕する場合には、６００～１，７００ｂａｒの圧力で、約１ｍｌ／分の流速の条件で行えば良い。実用的な観点では、６００～１０００ｂａｒの圧力で数回破砕を繰り返すことが好ましい。
　破壊前後の液を粒度分布計で分析することにより破壊の状況を確認することができる。菌体破壊液にはＤＴＴを最終濃度１ｍＭになるように添加することが好ましい。 Examples of means for destroying Escherichia 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. For example, when disrupting cells using a high-pressure homogenizer, it may be performed at a pressure of 600 to 1,700 bar and a flow rate of about 1 ml / min. From a practical viewpoint, it is preferable to repeat crushing several times at a pressure of 600 to 1000 bar.
The state of destruction can be confirmed by analyzing the liquid before and after the 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.
　大腸菌の菌体を破壊する方法においては、無機塩及び緩衝作用を示す化合物を含んだ溶液（例えばＳ３０緩衝液）に、回収した菌体を再懸濁した、菌体懸濁液を用いることが好ましい。菌体懸濁液としては、例えば、回収菌体１ｇあたり１～２ｍＬのＳ３０緩衝液等の溶液を添加した菌体懸濁液をあげることができる。－８０℃で保存した回収菌体を用いる場合には氷上で融解させて懸濁液を作製することが好ましい。 In the method for destroying Escherichia coli cells, a cell suspension obtained by resuspending the collected cells in a solution containing an inorganic salt and a compound having a buffering action (for example, S30 buffer) is used. preferable. Examples of the bacterial cell suspension include a bacterial cell suspension to which a solution such as 1 to 2 mL of S30 buffer is added per 1 g of recovered bacterial cells. When using the collected cells stored at −80 ° C., it is preferable to prepare a suspension by thawing on ice.
　細胞抽出物としては、細胞破片及びゲノムＤＮＡ等を除去したものを用いることが好ましい。除去する方法としては、遠心分離法、濾過法を挙げることができる。例えば、４℃、１０，０００～３０，０００ｇの条件で２０～５０分間、遠心分離し、上清を取得する工程を、例えば２～３回繰り返すことにより細胞破片及びゲノムＤＮＡ等を除去することができる。 It is preferable to use a cell extract from which cell debris and genomic DNA have been removed. Examples of the removing method include a centrifugal separation method and a filtration method. For example, cell debris, genomic DNA, etc. are removed by repeating the step of centrifuging for 20-50 minutes at 4 ° C., 10,000-30,000 g, and obtaining the supernatant, for example, 2-3 times. Can do.
　細胞抽出物としては、１／３～１／４容量の活性化ミックス（３００ｍＭＴｒｉｓ－酢酸、ｐＨ８．２、１３．２ｍＭ酢酸マグネシウム、１３．２ｍＭＡＴＰ、４．４ｍＭＤＴＴ、６．７Ｕ／ｍＬピルビン酸キナーゼ、８４ｍＭホスホエノールピルビン酸及び２０種類のアミノ酸（各０．０４ｍＭ））を加え、１５～４２℃、好ましくは２０～３７℃で、１０～３００分、好ましくは３０～１８０分間インキュベートしたものを用いることが好ましい。このような処理をした細胞抽出物を用いることにより効率よくタンパク質合成を行うことができる。 Cell extracts include 1/3 to 1/4 volume activation mix (300 mM Tris-acetate, pH 8.2, 13.2 mM magnesium acetate, 13.2 mM ATP, 4.4 mM DTT, 6.7 U / mL pyruvate kinase, 84 mM phosphoenolpyruvate and 20 kinds of amino acids (each 0.04 mM)) are added and incubated at 15 to 42 ° C., preferably 20 to 37 ° C. for 10 to 300 minutes, preferably 30 to 180 minutes. Is preferred. Protein synthesis can be performed efficiently by using such a cell extract.
　細胞抽出物としては、内在性のアミノ酸、核酸及びヌクレオシド等を除いたものを用いることが好ましい。活性化ミックスを加えた処理によって発生した不溶性成分は上記と同条件の遠心分離法等で除去できる。当該操作によって内在性のＲＮＡを除去できる。内在性の核酸は、ヌクレアーゼ等を更に添加することにより分解できる。その場合、核酸の分解後にヌクレアーゼ阻害剤を加えて処理することが好ましい。また、透析により内在性のアミノ酸、核酸及びヌクレオシド等を除くこともできる。 It is preferable to use a cell extract from which endogenous amino acids, nucleic acids, nucleosides and the like have been removed. Insoluble components generated by the treatment with the activated mix added can be removed by a centrifugation method 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.
　細胞抽出物は－８０℃で保存することができる。－８０℃での凍結は、液体窒素等を用いた急冷による方法が好ましい。 Cell extract can be stored at -80 ° C. Freezing at −80 ° C. is preferably performed by rapid cooling using liquid nitrogen or the like.
３）細胞抽出物を含む反応混合物を用いてタンパク質を合成する工程
　本発明の細胞抽出物を含む反応混合物（「無細胞タンパク質合成用反応混合物」ともいう）は、２）の工程で得られた細胞抽出物を含む。反応混合物中の細胞抽出物の含有量は、反応混合物全体を基準として、５～７０％（容量／容量）であってよく、１０～５０％であってよく、２０～４０％であってよく、２０～３０％であってよい。細胞抽出物を得るために用いた湿菌体（ｇ）／反応混合物（ｍＬ）の割合は、０．０１～１ｇ／ｍＬであってよく、０．０５～０．３ｇ／ｍＬであってよい。 3) Step of synthesizing protein using reaction mixture containing cell extract The reaction mixture containing the cell extract of the present invention (also referred to as “reaction mixture for cell-free protein synthesis”) was obtained in step 2). Contains cell extract. The content of the cell extract in the reaction mixture may be 5 to 70% (volume / volume), 10 to 50%, or 20 to 40% based on the whole reaction mixture. 20-30%. 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. .
　本発明の無細胞タンパク質合成用反応混合物は、細胞抽出物に加え、必要に応じて以下の（ｉ）～（ｖｉｉｉ）の成分を含むことができる。 The reaction mixture for cell-free protein synthesis of the present invention can contain the following components (i) to (viii) as necessary, in addition to the cell extract.
（ｉ）鋳型核酸
　鋳型核酸は、発現させたい目的タンパク質をコードするＤＮＡ又はＲＮＡと、適当な発現調節領域を含むＤＮＡ又はＲＮＡを含んでいればよく、直鎖状及び環状の何れの形態であってもよい。発現調節領域としては、プロモーター配列、ターミネーター配列、エンハンサー配列、ポリＡ付加シグナル及びリボソーム結合配列などをあげることができる。鋳型核酸は、少なくとも１つのプロモーター及び目的タンパク質をコードするＤＮＡを含むことが好ましい。添加する鋳型核酸の量は、反応混合物全体の容量を基準として、０．１～５０μｇ／ｍＬであることが好ましく、１～２０μｇ／ｍＬであることがより好ましい。 (I) 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 regulatory region include a promoter sequence, terminator sequence, enhancer sequence, poly A addition signal, and ribosome binding sequence. The template nucleic acid preferably includes DNA encoding at least one promoter and a target protein. The amount of template nucleic acid to be added is preferably 0.1 to 50 μg / mL, more preferably 1 to 20 μg / mL, based on the total volume of the reaction mixture.
　合成されたタンパク質を容易に検出あるいは精製できるよう、タグ配列を組み込んだ融合タンパク質が合成できるように鋳型核酸を設計しても良い。タグ配列として、例えば、他の分子との特異的親和性（結合性、アフィニティ）を利用した、例えばヒスチジンタグ（Ｈｉｓタグ）のようなアフィニティタグ、グルタチオンに特異的に結合するグルタチオン－Ｓ－トランスフェラーゼ（ＧＳＴ）、マルトースに特異的に結合するマルトース結合タンパク質（ＭＢＰ）等のタグ配列をあげることができる。また、抗原抗体反応を利用した「エピトープタグ」を利用しても良い。エピトープタグとして、ＨＡ（インフルエンザウイルスのヘマグルチニンのペプチド配列）タグ、ｍｙｃタグ、ＦＬＡＧタグ等を挙げることができる。さらにタグ配列を特定のプロテアーゼで切り離せるようにしたものも利用できる。 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. As a tag sequence, for example, an affinity tag such as a histidine tag (His tag) utilizing specific affinity (binding, affinity) with other molecules, glutathione-S-transferase that specifically binds to glutathione (GST), and tag sequences such as maltose binding protein (MBP) that specifically binds to maltose. Further, an “epitope tag” using an antigen-antibody reaction may be used. As the epitope tag, HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like can be mentioned. Further, a tag sequence that can be separated with a specific protease can also be used.
（ｉｉ）ＲＮＡポリメラーゼ
　上記鋳型核酸がＤＮＡである場合には、ＲＮＡポリメラーゼを含むことができる。ＲＮＡポリメラーゼとしては、上記鋳型核酸を対象とする１つ又は複数の転写因子を認識するＲＮＡポリメラーゼを用いることができる。本発明の反応混合物は、目的タンパク質の生産効率を向上させる観点から、Ｔ７　ＲＮＡポリメラーゼを含むことが好ましい。Ｔ７　ＲＮＡポリメラーゼは、反応混合物を調製する際に添加してもよく、上述したように、Ｔ７　ＲＮＡポリメラーゼ等の発現が可能なＢＬ２１（ＤＥ３）等の株を用いて細胞抽出物にＴ７　ＲＮＡポリメラーゼが含まれるようにしてもよい。 (Ii) 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.
（ｉｉｉ）エネルギー源
　細胞抽出物にタンパク質合成に必要なエネルギーが不足している場合には、反応混合物に追加的なエネルギー源を含めることが好ましい。また、エネルギー源は、タンパク質合成反応の間に添加又は補充することもできる。エネルギー源としては、例えば、ＡＴＰ、ＧＴＰ、グルコース、ピルベート、ホスホエノールピルベート（ＰＥＰ）、カルバモイルホスフェート、アセチルホスフェート、クレアチンホスフェート、ホスホピルベート、グリセルアルデヒド－３－ホスフェート、３－ホスホグリセレート、グルコース－６－ホスフェート、クエン酸、cis-アコニット酸、イソクエン酸、α－ケトグルタル酸、スクシニルＣoＡ、コハク酸、フマル酸、リンゴ酸、オキサロ酢酸、グリオキシル酸及びグルタミン酸等が挙げられる。 (Iii) Energy source If the cell extract lacks the energy required for protein synthesis, it is preferable to include an additional energy source in the reaction mixture. The energy source can also be added or supplemented during the protein synthesis reaction. Examples of energy sources include ATP, GTP, glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, Examples include glucose-6-phosphate, citric acid, cis-aconitic acid, isocitric acid, α-ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid, and glutamic acid.
（ｉｖ）目的タンパク質合成のための基質
　目的タンパク質合成のための基質として、反応混合物は、目的タンパク質を合成するために必要なアミノ酸を含むことができる。アミノ酸としては、タンパク質を構成する２０種類の全てのアミノ酸を含んでもよく、目的タンパク質や用いる試薬等を考慮して２０種類から適宜選択して用いてもよい。 (Iv) Substrate for target protein synthesis As a substrate for target protein synthesis, the reaction mixture 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.
　上記鋳型核酸がＤＮＡである場合には、反応混合物は目的タンパク質合成のための基質として、ＲＮＡ合成のための基質を含むことができる。ＲＮＡ合成のための基質としては、例えば、リボヌクレオチド三リン酸（ｒＮＴＰ）、リボヌクレオチド一リン酸（ｒＮＭＰ）等のリボヌクレオチドが挙げられる。 When the template nucleic acid is DNA, the reaction mixture can contain a substrate for RNA synthesis as a substrate for target protein synthesis. Examples of the substrate for RNA synthesis include ribonucleotides such as ribonucleotide triphosphate (rNTP) and ribonucleotide monophosphate (rNMP).
（ｖ）ポリアミン類
　反応混合物は、ポリアミン類を含むことができる。反応混合物に含め得るポリアミン類としては、例えば、スペルミン、スペルミジン及びプトレシン等が挙げられる。 (V) Polyamines The reaction mixture can contain polyamines. Examples of polyamines that can be included in the reaction mixture include spermine, spermidine, and putrescine.
（ｖｉ）塩
　反応混合物は、塩を含むことができる。反応混合物に含め得る塩としては、例えば、酢酸、グルタミン酸、又は硫酸のカリウム塩、マグネシウム塩、アンモニウム塩、及びマンガン塩が挙げられる。 (Vi) Salt The reaction mixture 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.
（ｖｉｉ）酸化／還元調整剤
　反応混合物は、酸化／還元調整剤を含むことができる。酸化／還元調整剤としては、例えば、ＤＴＴ、アスコルビン酸、グルタチオン、及び／又はそれらの酸化物が挙げられる。 (Vii) Oxidation / Reduction Modifier The reaction mixture can include an oxidation / reduction regulator. Examples of the oxidation / reduction regulator include DTT, ascorbic acid, glutathione, and / or oxides thereof.
（ｖｉｉｉ）その他の添加物
　また、必要に応じて反応混合物に、下記の成分を補充あるいは添加しても良い。
　即ち、リボソーム、転移ＲＮＡ（ｔＲＮＡ）、任意選択の翻訳因子（例えば、翻訳開始因子、伸長因子終結因子、リボソームリサイクリング因子等）及びその補因子、アミノアシルｔＲＮＡ合成酵素（ＡＲＳ）、メチオニルｔＲＮＡホルミル転移酵素（ＭＴＦ）、高分子化合物（例えば、ポリエチレングリコール、デキストラン、ジエチルアミノエチルデキストラン、四級アミノエチル及びアミノエチルデキストラン等）、ヌクレアーゼ、ヌクレアーゼ阻害剤、タンパク質安定剤、シャペロン、可溶化剤、非変性界面活性物質（例えば、トリトンＸ１００）等を必要に応じて添加しても良い。 (Viii) Other additives In addition, the following components may be supplemented or added to the reaction mixture as necessary.
That is, ribosome, transfer RNA (tRNA), optional translation factors (eg, translation initiation factor, elongation factor termination factor, ribosome recycling factor, etc.) and 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) may be added as necessary.
　反応混合物の成分に関しては、例えば米国特許第７，３３８，７８９号及び同第７，３５１，５６３号、及び米国特許出願公開第２０１０／０１８４１３５号及びＵＳ２０１０／００９３０２４の記載を参考とすることができる。 Regarding the components of the reaction mixture, for example, the descriptions in US Pat. Nos. 7,338,789 and 7,351,563, and US Patent Application Publication Nos. 2010/0184135 and 2010/0093024 can be referred to. .
　上記反応混合物を用いて無細胞タンパク質の合成を行うことができる。
　本発明の反応混合物を用いた無細胞タンパク質合成方法は、透析法及びバッチ法いずれも利用することができる。透析法の場合は、上記反応混合物を含む内液と限外濾過膜のような透析膜により隔離した目的タンパク質合成のための基質及びエネルギー源等を含む外液からなる閉鎖系でタンパク質の合成が行われる。透析法では、透析膜を介して、目的タンパク質合成のための基質及びエネルギー源等が外液から反応混合物に供給されるとともに、反応混合物中の余計な副産物を外液中に拡散させることができるため、より長時間、反応を持続させることができる。バッチ法は、タンパク質合成に必要な成分すべてを含む上記反応混合物を反応溶液と混合し、反応溶液中に均一に含ませ、反応を行う合成法であり、透析法と比較すると反応時間は短いが、結果がすぐに得られる。例えば、Mol. Syst. Biol 2008 4 220（非特許文献３）に記載のＣｙｔｏｍｉｍ型無細胞タンパク質合成系を利用することにより、ＮＴＰやＰＥＰを使用するＰＡＮＯｘ型と比較して低コストでタンパク質合成系を行うことができる。タンパク質の合成は、２０～４０℃で行えば良く、３０℃が好ましい。バッチ法における反応は２～８時間行うことが好ましい。 The reaction mixture can be used to synthesize cell-free proteins.
As the cell-free protein synthesis method using the reaction mixture of the present invention, both a dialysis method and a batch method can be used. In the case of dialysis, 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. Done. In the dialysis method, 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. For example, by using the Cytom type cell-free protein synthesis system described in Mol. Syst. Biol 2008 4 220 (Non-patent Document 3), the protein synthesis system can be produced at a lower cost than the PANOx type using NTP or PEP. It can be performed. Protein synthesis may be performed at 20 to 40 ° C, preferably 30 ° C. The reaction in the batch method is preferably performed for 2 to 8 hours.
無細胞タンパク質合成用キット
　本発明の無細胞タンパク質合成用キット（以下、「本キット」とも表す）は、グリセロールを炭素源とした培地で培養した大腸菌から得られた細胞抽出物、並びに鋳型核酸、目的タンパク質合成のための基質、及び／又はエネルギー源を含む。鋳型核酸、目的タンパク質合成のための基質、及びエネルギー源は、上記細胞抽出物と予め混合されていてもよく、上記細胞抽出物とは独立して本キットに含まれていてもよい。また、本キットは、上述したＲＮＡポリメラーゼ、ポリアミン類、塩、酸化／還元調整剤、その他の添加物をさらに含んでいてもよい。これらの添加物は、上記細胞抽出物と予め混合されていてもよく、上記細胞抽出物とは独立して本キットに含まれていてもよい。 Cell-free protein synthesis kit The cell-free protein synthesis kit of the present invention (hereinafter also referred to as “this kit”) includes a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source, a template nucleic acid, It includes a substrate and / or an energy source for target protein synthesis. 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. The 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.
［実施例１］
（グリセロールを用いた培養）
　ＢＬ２１株（ノバジェン社）を２ｘＹＴ培地（１．６％Ｂａｃｔｏ　Ｔｒｉｐｔｏｎ、１％　Ｙｅａｓｔ　Ｅｘｔｒａｃｔ、０．５％ＮａＣｌ）でＯＤ６００が４．２になるまでフラスコ培養した。 [Example 1]
(Culture using glycerol)
BL21 strain (Novagen) was cultured in a flask with 2 × YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until OD600 was 4.2.
　当該培養液を、グリセロールを炭素源とした、表１の組成の改変２ｘＹＴＰＧ培地（消泡剤としてアデカ（登録商標）ＬＧ－２９５Ｓを１ｍｌ／Ｌ添加し、ＮａＯＨを用いてｐＨ７に調整）５００ｍＬが入った１ＬジャーファメンターにＯＤ６００が０．０５になるように添加した。 500 mL of this culture broth containing glycerol as a carbon source and a modified 2xYTPG medium (added 1 ml / L of Adeka (registered trademark) LG-295S as an antifoaming agent and adjusted to pH 7 using NaOH) with a composition of Table 1 It added so that OD600 might be set to 0.05 to the 1L jar fermenter which entered.
　比較として、グルコース（１８ｇ／Ｌ）を炭素源とした通常の２ｘＹＴＰＧ培地を用いて、同じ株を同様に培養した。培養温度は３６度に保ち、ｐＨ７．０で一定に制御して培養した。溶存酸素濃度は２．４ｍｇ／Ｌ以上に維持した。増殖曲線を図１に示す。改変２ｘＹＴＰＧ培地と通常の２ｘＹＴＰＧ培地で培養した際の比増殖速度は、それぞれ１．０８ｈ－１と０．７４ｈ－１であった。この結果は、グリセロールで培養した細胞内のリボソームやエネルギー再生系の酵素群がより高い活性を有すると推察される。 For comparison, 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. Specific growth rate when cultured in a modified 2xYTPG medium and normal 2xYTPG medium was respectively 1.08H -1 and 0.74h -1. This result is presumed that ribosomes in cells cultured in glycerol and enzymes of the energy regeneration system have higher activity.
　グリセロールを用いた培養により、グルコースを用いた培養よりもより短時間で好ましい菌体濃度の培養液が得られることから、より短時間で細胞抽出物を得ることが可能であることが示された。 It was shown that a cell extract can be obtained in a shorter time because a culture solution having a preferable cell concentration can be obtained in a shorter time than in a culture using glucose by culturing with glycerol. .
［実施例２］
（細胞抽出物の調製）
　上記実施例１と同様の方法により、改変２ｘＹＴＰＧ培地及び通常の２ｘＹＴＰＧ培地のそれぞれにおいて、対数中期ＯＤ６００が１２になるまで培養したＢＬ２１株を用いて、下記の方法で細胞抽出物を調製した。 [Example 2]
(Preparation of cell extract)
By the same method as in Example 1, a cell extract was prepared by the following method using BL21 strain cultured in modified 2xYTPG medium and normal 2xYTPG medium until the log mid-phase OD600 was 12.
　当該ＢＬ２１株の培養液２Ｌを７，０００×ｇ、４℃、２０分間の条件で遠心分離し、湿菌体３１ｇを回収した。１０ｍＭＴｒｉｓ－酢酸、１４ｍＭ酢酸マグネシウム、６０ｍＭ酢酸カリウム及び２ｍＭＤＴＴを含むＳ３０緩衝液（ｐＨ８．２）で菌体を洗浄した後、液体窒素を用いて－８０℃に急冷した。 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 S30 buffer (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.
　菌体１ｇあたりＳ３０緩衝液２ｍＬを加えて解凍、懸濁し、ＧＥＡ.Ｎｉｒｏ　ｓｏａｖｉ社製の高圧ホモジナイザー（ＰａｎｄａＰＬＵＳ２０００）を使用して１３００ｂａｒ以上の圧で菌体を破砕した。
　２０，４００×ｇ、４℃、３０分間の条件で２回遠心分離を行い、菌体残渣を取り除いた。 2 mL of S30 buffer was added per 1 g of microbial cells, thawed and suspended, and the microbial cells were crushed at a pressure of 1300 bar or higher using a high-pressure homogenizer (PandaPLUS 2000) manufactured by GEA.Niro soavi.
Centrifugation was performed twice under the conditions of 20,400 × g, 4 ° C. and 30 minutes to remove the cell residue.
　当該遠心分離上清２００μＬに、３００ｍＭＴｒｉｓ－酢酸、１３．２ｍＭ酢酸マグネシウム、１３．２ｍＭＡＴＰ、４．４ｍＭＤＴＴ、６．７Ｕ／ｍＬピルビン酸キナーゼ、８４ｍＭホスホエノールピルビン酸及び２０種類のアミノ酸（各０．０４ｍＭ）を含む活性化ミックス６０μＬを加え、３０℃で１５０分間インキュベートした。 To 200 μL of the centrifuged supernatant, 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 20 kinds of amino acids (each 0.04 mM ) Was added and incubated at 30 ° C. for 150 minutes.
　インキュベート後、２０，４００×ｇ、４℃、３０分間の条件で遠心分離を行い、不溶性画分を取り除くことで細胞抽出物２３０μＬを得た。 After the incubation, the mixture was centrifuged at 20,400 × g, 4 ° C. for 30 minutes, and the insoluble fraction was removed to obtain 230 μL of cell extract.
（ｐＵＣ－ＧＦＰの作製）
　ｐＥＴ３ａ（ノバジェン社）を利用し、Ｔ７プロモーター、リボソーム結合サイト（ＲＢＳ）及びＴ７ターミネーターの配列を含むＤＮＡ断片（１）をＰＣＲ法によって調製した。
　ｐＵＣ１９（タカラバイオ株式会社）を利用し、複製起点及びアンピシリン耐性遺伝子の配列を含むＤＮＡ断片（２）をＰＣＲ法によって調製した。
　ＤＮＡ断片（１）及びＤＮＡ断片（２）をＩｎ－Ｆｕｓｉｏｎ反応により融合し、大腸菌ＨＳＴ０８に形質転換した。前記大腸菌を培養後、ＱＩＡＧＥＮ　Ｐｌａｓｍｉｄ　Ｋｉｔを使用し、プラスミドを抽出した。
　前記プラスミドを利用し、Ｔ７プロモーター、ＲＢＳ、Ｔ７ターミネーター、複製起点及びアンピシリン耐性遺伝子の配列を含むＤＮＡ断片（３）をＰＣＲ法によって調製した。
　Ｔ５－ＨｏｌｌｙＧＦＰ（コスモバイオ社）を利用し、Ｈｏｌｌｙ－ＧＦＰ遺伝子を含むＤＮＡ断片（４）をＰＣＲ法によって調整した。
　ＤＮＡ断片（３）及びＤＮＡ断片（４）をＩｎ－Ｆｕｓｉｏｎ反応により融合し、大腸菌ＨＳＴ０８に形質転換した。前記大腸菌を培養後、ＱＩＡＧＥＮ　Ｐｌａｓｍｉｄ　Ｋｉｔを使用し、ｐＵＣ－ＧＦＰを抽出し、無細胞タンパク質合成反応の鋳型核酸として利用した。
　鋳型核酸用のｐＵＣ－ＧＦＰは、分光光度計を用いて高純度（Ａ２６０／Ａ２８０が１．８以上、Ａ２６０／Ａ２３０が２．０以上）であること、さらに、ＤＮＡシーケンサーを用いて塩基配列を読み取り、不必要な変異が入っていないことを確認した。 (Preparation of pUC-GFP)
A DNA fragment (1) containing T7 promoter, ribosome binding site (RBS) and T7 terminator sequences was prepared by PCR using pET3a (Novagen).
Using pUC19 (Takara Bio Inc.), a DNA fragment (2) containing the replication origin and the ampicillin resistance gene sequence was prepared by PCR.
DNA fragment (1) and DNA fragment (2) were fused by In-Fusion reaction and transformed into E. coli HST08. After culturing the E. coli, the plasmid was extracted using QIAGEN Plasmid Kit.
Using the plasmid, 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).
DNA fragment (3) and DNA fragment (4) were fused by In-Fusion reaction and transformed into E. coli HST08. After culturing the E. coli, pUC-GFP was extracted using QIAGEN Plasmid Kit and used as a template nucleic acid for cell-free protein synthesis reaction.
The pUC-GFP for template nucleic acid has high purity (A260 / A280 is 1.8 or more, A260 / A230 is 2.0 or more) using a spectrophotometer, and the nucleotide sequence is determined using a DNA sequencer. Reading and confirming that there were no unnecessary mutations.
（無細胞タンパク質合成反応）
　表２の化合物を含むマスターミックス１０μＬ、細胞抽出物６μＬ、４０ｍＭ　ＤＴＴ水溶液１μＬ、０．２ｇ／Ｌ　ｐＵＣ－ＧＦＰ２μＬ及び１０００Ｕ／μＬ　Ｔ７　ＲＮＡポリメラーゼ１μＬを加え、全量２０μＬの反応混合物（液体）を用意した。ネガティブコントロール（ＮＣ）としてｐＵＣ－ＧＦＰの代わりにＲＯ水２μＬを加えたものを用意した。３８４ウェルマイクロプレートに反応混合物を移し、マイクロプレートリーダーＴＥＣＡＮｉｎｆｉｎｉｔｅＦ２００を使用して３０℃でインキュベートしながら、経時的に蛍光強度を測定することで、ＧＦＰ合成量を比較した。図２に蛍光強度の測定結果を示す。
Figure JPOXMLDOC01-appb-T000002
 (Cell-free protein synthesis reaction)
10 μL of master mix containing the compounds of Table 2, 6 μL of cell extract, 1 μL of 40 mM DTT aqueous solution, 2 μL of 0.2 g / L pUC-GFP and 1 μL of 1000 U / μL T7 RNA polymerase were added to prepare a total reaction mixture (liquid) of 20 μL. . A negative control (NC) prepared by adding 2 μL 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 TECANinfine F200. FIG. 2 shows the measurement result of the fluorescence intensity.
Figure JPOXMLDOC01-appb-T000002
　反応を開始して５時間後、炭素源にグリセロールを含む培地を大腸菌細胞の培養に用いた無細胞タンパク質合成方法によるＧＦＰの蛍光強度は、炭素源にグルコースを含む培地を大腸菌細胞の培養に用いた無細胞タンパク質合成方法によるＧＦＰの蛍光強度よりも約２倍高い値を示した。これにより、炭素源にグリセロールを含む培地を大腸菌細胞の培養に用いた無細胞タンパク質合成方法によるＧＦＰの合成量は、炭素源にグルコースを含む培地を大腸菌細胞の培養に用いた無細胞タンパク質合成方法と比較して、約２倍多いことが示された。 Five hours after the start of the reaction, the fluorescence intensity of GFP by a cell-free protein synthesis method using a medium containing glycerol as a carbon source for culturing E. coli cells was determined using a medium containing glucose as a carbon source for culturing E. coli cells. The value was approximately twice as high as the fluorescence intensity of GFP obtained by the cell-free protein synthesis method. Thus, the amount of GFP synthesized by the cell-free protein synthesis method using a medium containing glycerol as a carbon source for culturing E. coli cells is the same as the method for cell-free protein synthesis using a medium containing glucose as a carbon source for culturing E. coli cells. It was shown to be about twice as high as that of.

Claims (9)

  1. 　無細胞タンパク質合成用反応混合物であって、グリセロールを炭素源とした培地で培養した大腸菌から得られた細胞抽出物を含む、反応混合物。 A reaction mixture for cell-free protein synthesis, comprising a cell extract obtained from E. coli cultured in a medium using glycerol as a carbon source.
  2. 　鋳型核酸、目的タンパク質合成のための基質、及びエネルギー源をさらに含む、請求項１に記載の反応混合物。 The reaction mixture according to claim 1, further comprising a template nucleic acid, a substrate for synthesizing the target protein, and an energy source.
  3. 　鋳型核酸が、少なくとも１つのプロモーター及び目的タンパク質をコードするＤＮＡを含む、請求項２に記載の反応混合物。 The reaction mixture according to claim 2, wherein the template nucleic acid comprises DNA encoding at least one promoter and a target protein.
  4. 　エネルギー源が、ＡＴＰ、ＧＴＰ、グルコース、ピルベート、ホスホエノールピルベート（ＰＥＰ）、カルバモイルホスフェート、アセチルホスフェート、クレアチンホスフェート、ホスホピルベート、グリセルアルデヒド－３－ホスフェート、３－ホスホグリセレート及びグルコース－６－ホスフェート、クエン酸、cis-アコニット酸、イソクエン酸、α－ケトグルタル酸、スクシニルＣoＡ、コハク酸、フマル酸、リンゴ酸、オキサロ酢酸、グリオキシル酸及びグルタミン酸からなる群より選ばれる、請求項２又は３に記載の反応混合物。 Energy sources are ATP, GTP, glucose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and glucose-6 -Selected from the group consisting of phosphate, citric acid, cis-aconitic acid, isocitric acid, α-ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid and glutamic acid The reaction mixture described in 1.
  5. 　大腸菌が、Ｔ７　ＲＮＡポリメラーゼを発現する大腸菌である、請求項１～４のいずれか一項に記載の反応混合物。 The reaction mixture according to any one of claims 1 to 4, wherein the E. coli is E. coli expressing T7 RNA polymerase.
  6. 　請求項１～５のいずれか一項に記載の反応混合物を用いた無細胞タンパク質合成方法。 A cell-free protein synthesis method using the reaction mixture according to any one of claims 1 to 5.
  7. 　グリセロールを炭素源とした培地で大腸菌を培養する工程、
    　培養した大腸菌を回収し、回収した大腸菌から細胞抽出物を得る工程、及び
    　細胞抽出物を含む反応混合物を用いてタンパク質を合成する工程
    を含む、無細胞タンパク質合成方法。     Culturing E. coli in a medium using glycerol as a carbon source,
    A cell-free protein synthesis method comprising the steps of recovering cultured E. coli, obtaining a cell extract from the recovered E. coli, and synthesizing a protein using a reaction mixture containing the cell extract.
  8. 　グリセロールを炭素源とした培地で培養した大腸菌から得られた細胞抽出物、並びに
    　鋳型核酸、目的タンパク質合成のための基質、及び／又はエネルギー源
    を含む、無細胞タンパク質合成用キット。     A cell-free protein synthesis kit comprising a cell extract obtained from E. coli cultured in a medium containing glycerol as a carbon source, and a template nucleic acid, a substrate for synthesizing a target protein, and / or an energy source.
  9. 請求項６記載の無細胞タンパク質合成方法により得られるタンパク質。 A protein obtained by the cell-free protein synthesis method according to claim 6.
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