JP2020127364A - Reaction mixture for cell-free protein synthesis, and cell-free protein synthesis method and cell-free protein synthesis kit using the same - Google Patents

Abstract

To provide a novel approach for increasing an amount of protein synthesis in a cell-free protein synthesis method.SOLUTION: A reaction mixture for cell-free protein synthesis contains a cell extract obtained from Escherichia coli, and glycerol.SELECTED DRAWING: None

Description

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.

As a method for producing a recombinant protein, an in vivo method including culturing a transformed cell and an in vitro method using a cell-derived extract (cell extract), which is a so-called cell-free protein synthesis method, can be used. Is known.

As a method for producing a protein on an industrial scale, an in vivo method is considered to be excellent at this stage. However, compared to the in vivo method, the in vitro method of cell-free protein synthesis (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, it is possible to flexibly change the synthetic environment such as changes in tRNA level reflecting the codon usage of genes, conditions such as redox potential, pH, and ionic strength. 3) Purified and properly folded protein products can be easily recovered as they are, (4) Non-naturally-isotopically labeled amino acids can be incorporated, (5) In vivo unstable, insoluble or cell The ability to synthesize toxic proteins, (6) the ability to synthesize proteins that are difficult to make in vivo due to the need for unique cofactors, and (7) for in vivo expression. It is considered to have advantages such as being able to avoid processes such as cloning and cell transformation. Therefore, the in vitro method of cell-free protein synthesis is becoming a basic technology in this field, and has been established to some extent (Non-Patent Document 1).

However, compared with the in vivo method, the cell-free protein synthesis method has a low reaction duration for synthesizing the protein, and thus the production efficiency is low. In order to increase the duration of this reaction, a continuous flow system using a dialysis membrane has been developed, which 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 reagents, and there is a problem that the cost of reagents and the like increases.

As another approach to increase the reaction duration, certain results have been obtained by reviewing the energy regeneration system in the reaction in vitro (Patent Document 1, Non-Patent Document 2), and granulocyte-macrophage colony stimulating factor was used. It has succeeded in synthesizing 700 mg/L in 10 hours (nonpatent literature 3).

Further, in a cell-free protein synthesis system using a cell extract derived from Escherichia coli, it is described that addition of high concentration salt and glycerol to template DNA is not preferable (Non-Patent Document 4).

In industrial scale production, an optimal composition of the reaction mixture for cell-free protein synthesis is sought in order to further improve the production amount and cost.

Japanese Patent No. 5259046

Methods in molecular biology, vol. 267, Recombinant Gene Expression: Reviews and Protocols, Second Edition, Humana Press Inc. , Totowa, NJ, pp. 169-182 Mol. Syst. Biol. , 2008, vol. 4, No. 220, pp. 1-10 Biotechnol. Bioeng. , 2011, vol. 108, No. 7, pp. 1570-1578 The S30 T7 High-Yield Protein Expression System, PROMEGA NOTES, September 2008, NUMBER 100, pp. 9-10

The present invention aims to provide a novel approach for increasing the amount of protein synthesis in a cell-free protein synthesis method.

As a result of diligent studies on the reaction conditions for protein synthesis in the cell-free protein synthesis method, the present inventors have found that the addition of glycerol to the reaction mixture for cell-free protein synthesis can increase the amount of synthesis of the target protein. The inventors have found that they can do so and have completed the present invention.

That is, the present invention relates to the following inventions, for example.
[1]
A reaction mixture for cell-free protein synthesis, which comprises a cell extract obtained from Escherichia coli and glycerol.
[2]
The reaction mixture according to [1], wherein the E. coli is E. coli expressing T7 RNA polymerase.
[3]
The reaction mixture according to [1] or [2], wherein the E. coli is E. coli cultured in a medium containing glycerol as a carbon source.
[4]
The reaction mixture according to any one of [1] to [3], further including a template nucleic acid, a substrate for synthesizing a target protein, and an energy source.
[5]
The reaction mixture according to [4], wherein the template nucleic acid contains at least one promoter and a DNA encoding a target protein.
[6]
The energy source is ATP, GTP, glucose, ribose, pyruvate, phosphoenolpyruvate, carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate and 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, at least one selected from the group consisting of: The reaction mixture according to [4].
[7]
A cell-free protein synthesis method using the reaction mixture according to any one of [1] to [6].
[8]
A cell-free protein synthesis kit comprising a cell extract obtained from Escherichia coli, glycerol, a template nucleic acid, a substrate for protein synthesis of interest, and/or an energy source.
[9]
A protein obtained by the cell-free protein synthesis method according to [7].

Since the reaction mixture for cell-free protein synthesis of the present invention contains the cell extract obtained from Escherichia coli and glycerol, the synthesis amount of the target protein can be increased. Further, according to the cell-free protein synthesis method of the present invention, by using a reaction mixture for cell-free protein synthesis in which glycerol is added to a cell extract obtained from Escherichia coli, the amount of protein synthesis in the method can be increased. It is possible to improve the protein production efficiency.

In Test Example 1, the growth curve of the BL21 strain cultured in the modified 2×YTPG medium containing glycerol as a carbon source (black triangle) and the growth curve of the BL21 strain cultured in the normal 2×YTPG medium containing glucose as the carbon source. It is a graph which shows the comparison of (black circle). 7 is a graph showing the results of time-dependent measurement of fluorescence intensity of GFP synthesized by the cell-free protein synthesis method in Test Example 2. Open triangles show the results using the BL21 strain cultured in the modified 2×YTPG medium, open circles show the results using the BL21 strain cultured in the usual 2×YTPG medium, and the filled triangles and filled circles represent the pUC-GFP instead. The results of a negative control (NC) to which water is added are shown (black triangle: modified 2×YTPG medium, black circle: normal 2×YTPG medium). 3 is a graph showing the results of time-dependent measurement of fluorescence intensity of GFP synthesized by the cell-free protein synthesis method in Example 1. The black circles show the results using the reaction mixture for cell-free protein synthesis without glycerol (0 mM), the black triangles show the results using the reaction mixture for cell-free protein synthesis with 100 mM glycerol added, and the black squares show the results. The result using the reaction mixture for cell-free protein synthesis which added glycerol 200mM is shown.

Hereinafter, modes for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.

[Reaction mixture for cell-free protein synthesis]
The reaction mixture for cell-free protein synthesis of the present invention comprises a cell extract obtained from Escherichia coli and glycerol.

(Cell extract)
The cell extract according to the present embodiment means a solid, a liquid, or a mixture thereof obtained by destroying Escherichia coli. The cell extract contains factors necessary for transcription of RNA using DNA as a template and factors necessary for protein translation, which were 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 this cell extract, the reaction system of transcription and translation can be reconstituted in vitro to synthesize the protein.

The cell extract according to the present embodiment can be obtained, for example, through the steps of 1) culturing E. coli and 2) collecting the cultured E. coli and obtaining a cell extract.

1) Step of culturing Escherichia coli Any Escherichia coli microorganism can be used as Escherichia coli used for producing a cell extract, and examples thereof include Escherichia coli A19, BL21 (Novagen), BL21 (DE3). ) (Life Technologies), BLR(DE3) (Merck Millipore), DH1, GI698, HB101, JM109, K5 (ATCC23506), K-12, KY3276, MC1000, MG1655 (ATCC47076), NMR2, No. 49, Rosetta (DE3) (Novagen), TB1, Tuner (Novagen), Tuner (DE3) (Novagen), W1485, W3110 (ATCC27325), XL1-Blue, XL2-Blue and the like can be mentioned. ..

When the cell extract contains an endogenous protein that has a detrimental effect on protein synthesis, a gene associated with the endogenous protein is previously removed from the E. coli strain used by a known genetic engineering technique. Good.

The medium for culturing Escherichia coli is a liquid medium containing a carbon source, a nitrogen source and inorganic salts that can be assimilated by Escherichia coli, and any natural medium or synthetic medium can be used as long as the medium can be cultured efficiently. You may use.

As the carbon source, for example, glucose, sucrose, maltose, glycerol can be used. As the carbon source, glucose and glycerol are preferable, and glycerol is more preferable.

As the nitrogen source, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium salts of inorganic acids or organic acids such as ammonium acetate and ammonium phosphate, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, corn steep liquor, Casein hydrolyzate, soybean meal, soybean meal hydrolyzate, various fermented cells and digested products thereof can be used.

As the inorganic salt, for example, potassium primary phosphate, potassium secondary phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate and calcium carbonate can be used.

Specific examples of the medium for culturing Escherichia coli include, for example, a modified 2×YTPG medium (Table 1) in which 2×YTPG medium and 2×YTPG medium glucose are replaced with glycerol. In order to prevent foaming during culture, it is preferable to add a known antifoaming agent to the medium. Examples of the defoaming agent include ADEKA (registered trademark) LG-295S and the like. The amount of defoaming agent added may be, for example, 0.5 to 2 ml/L, and is preferably 1 ml/L. The pH of the medium may be, for example, 6 to 8, preferably pH 7. The pH of the medium can be adjusted using NaOH or the like.

When Escherichia 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 the expression of T7 RNA polymerase or the like is induced by an inducer such as IPTG, an inducer 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 culturing is preferably maintained at 3.0 to 9.0. The pH adjustment during the culture can be performed using an inorganic acid, an organic acid, an alkaline solution, urea, calcium carbonate, ammonia or the like.

The culturing may be carried out, for example, until the early logarithmic phase to the late logarithmic growth, and preferably until the midlogarithmic growth phase is reached. When the culturing time is within these ranges, a cell extract having more excellent protein synthesis efficiency can be obtained. When a modified 2×YTPG medium is used as the culture medium, the medium logarithmic growth phase is usually reached by culturing at a culture temperature of 36° C. for 4 to 6 hours.

In order to obtain a high-concentration cell culture solution, fed-batch culture may be carried out in which the medium (feed solution) is fed into the culture solution continuously or intermittently according to the lapse of culture time. Fed-batch culture may be carried out according to a known method in E. coli. Further, in this fed-batch culture, the carbon source mentioned in the above culture medium can be used as the carbon source in the feed solution. For example, when glycerol is used in the culture medium, it is preferable to use glycerol as the carbon source of the feed solution. A feed solution may be prepared in which the concentration of the carbon source is adjusted according to the growth of the bacterial cells, but the feed solution containing 30 to 70% of the carbon source is a culture solution in an appropriate amount according to the growth of the bacterial cells. You may add in.

2) Step of recovering cultured Escherichia coli and obtaining cell extract The step of recovering Escherichia coli cultured in the step 1) and obtaining a cell extract from the recovered Escherichia coli is performed in the art for the purpose of cell-free protein synthesis. Can be carried out by a known method. For example, it can be performed according to the protocols described in Non-Patent Document 1 and Non-Patent Document 2. As a specific method for obtaining a cell extract, for example, the following method may be mentioned in which E. coli cells are collected and the collected E. coli cells are destroyed.

In the step 2), it is preferable that the cells are collected from the culture solution and kept at a low temperature until the cell extract is obtained. Specifically, it can be carried out, for example, at a low temperature of 0 to 15°C, preferably 2 to 10°C.

Examples of the method for recovering Escherichia coli cells from the E. coli culture solution include a centrifugation method and a filtration method. For example, the bacterial cells of Escherichia coli can be recovered by centrifugation for 20 to 50 minutes under the condition of 4° C. and 7,000 to 14,000×g.

After the collection, it is preferable to include a step of washing the collected Escherichia coli cells in order to remove the medium components and the like. As the washing solution, a solution containing an inorganic salt and a compound having a buffer action, for example, 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 are added to about 450 ml of pure water, preferably mili-Q. Dissolve in ultrapure water. After dissolution, the pH is adjusted to 8.2 with acetic acid, the volume is adjusted to 500 ml, and sterilized with a 0.22 μm filter. The obtained S30 buffer solution can be stored at 4° C., is diluted 10-fold with ultrapure water immediately before use, and 1 M DTT aqueous solution is added so that the final concentration becomes 2 mM.

The step of washing the bacterial cells of Escherichia coli using a solution containing the above-mentioned inorganic salt and a compound having a buffer action (for example, S30 buffer solution) includes, for example, suspending the recovered bacterial cells in the solution and centrifuging the suspension. It can be performed by repeating the collecting step, for example, 2-3 times. The suspension can be efficiently homogenized by using a homogenizer or the like. The cells can be recovered from the homogenized suspension by centrifugation at, for example, 4° C. and 9,000 to 14,000×g for 10 to 50 minutes.

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 the bacterial cells of Escherichia coli include means using ultrasonic crusher, French press, high-pressure homogenizer, dynomill, mortar, glass beads, and lysing enzyme such as lysozyme. For example, when the cells are disrupted by using a high-pressure homogenizer, the pressure may be 600 to 1,700 bar and the flow rate may be about 1 ml/min. From a practical viewpoint, it is preferable to repeat the 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 destruction with a particle size distribution analyzer. It is preferable to add DTT to the cell disruption solution so that the final concentration will be 1 mM.

In the method for destroying the bacterial cells of Escherichia coli, the recovered bacterial cells are resuspended in a solution (for example, S30 buffer solution) containing the above-mentioned inorganic salt and a compound having a buffer action, and a bacterial cell suspension is used. It is preferable. Examples of the bacterial cell suspension include a bacterial cell suspension obtained by adding 1 to 2 mL of a solution such as S30 buffer solution per 1 g of the recovered bacterial cells. When using the cells stored at −80° C., it is preferable to melt them on ice to prepare a suspension.

As the cell extract, it is preferable to use a cell extract from which cell debris, genomic DNA and the like have been removed. Examples of the method for removing may 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 to 50 minutes under conditions of 4° C. and 10,000 to 30,000×g for 20 to 50 minutes, for example 2 to 3 times can do.

As a cell extract, 1/3 to 1/4 volume of activation mix (300 mM Tris-acetic acid, 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 all 20 kinds of amino acids (0.04 mM each) that compose protein were added, and the temperature was 15 to 42° C., preferably 20 to 37° C., and 10 to 300 minutes, Preferably, the one incubated for 30 to 180 minutes can be used. Protein synthesis can be efficiently performed by using the cell extract treated as described above.

Furthermore, as the cell extract, it is preferable to use a cell extract from which endogenous nucleic acids have been removed. The insoluble component generated by the treatment with the activated mix added can be removed by a centrifugation method under the same conditions as above. By this operation, endogenous RNA can be removed. Endogenous nucleic acid can be decomposed by further adding nuclease or the like. When the addition treatment is performed, it is preferable to add a nuclease inhibitor after the treatment. In addition, endogenous amino acids, nucleic acids, nucleosides and the like can 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.

(Content of cell extract)
The content of the cell extract in the reaction mixture for cell-free protein synthesis according to the present embodiment may be 5 to 70% (volume/volume) based on the total amount of the reaction mixture, and 10 to 50% (volume/volume). Volume), 20-40% (volume/volume), and 20-30% (volume/volume). The wet cell (g)/reaction mixture (mL) ratio used to obtain the cell extract may be 0.01 to 1 g/mL, or 0.05 to 0.3 g/mL. ..

(Glycerol)
It is important to use the reaction mixture for cell-free protein synthesis of the present invention to which glycerol is further added.

(Content of glycerol)
The content of glycerol in the reaction mixture for cell-free protein synthesis according to the present embodiment is not particularly limited as long as the optimum addition concentration is appropriately determined according to the composition of the reaction mixture, and for example, the total amount of the reaction mixture 10 to 500 mM, 20 to 300 mM, 40 to 200 mM, 80 to 100 mM.

(Other components of reaction mixture)
The reaction mixture for cell-free protein synthesis according to the present embodiment may include at least one of the following components (i) to (viii), if necessary.

(I) Template Nucleic Acid The template nucleic acid only needs to contain DNA or RNA that encodes the desired protein to be expressed and DNA or RNA that contains an appropriate expression control region, and may be either linear or circular. May be. Examples of the expression control region include a promoter sequence, a terminator sequence, an enhancer sequence, a poly A addition signal and a ribosome binding sequence. Further, the template nucleic acid preferably contains at least one promoter and a DNA encoding the target protein. The amount of the template nucleic acid to be added is preferably 0.1 to 50 μg/mL, more preferably 1 to 20 μg/mL, based on the volume of the entire 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. As the tag sequence, for example, an affinity tag such as a histidine tag (His tag) that utilizes specific affinity (binding property) with other molecules, or glutathione-S- that specifically binds to glutathione is used. Examples include tag sequences such as transferase (GST) and maltose binding protein (MBP) that specifically bind to maltose. Moreover, you may utilize the "epitope tag" which utilized the antigen antibody reaction. Examples of the epitope tag include HA (peptide sequence of hemagglutinin of influenza virus) tag, myc tag, FLAG tag and the like. Furthermore, a tag sequence that can be cleaved with a specific protease can also be used.

(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 targeted for the template nucleic acid can be used. From the viewpoint of improving the production efficiency of the target protein, the reaction mixture of the present invention preferably contains T7 RNA polymerase. T7 RNA polymerase may be added when preparing a reaction mixture, and as described above, T7 RNA polymerase is added to cell extracts 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 the energy source include ATP, GTP, glucose, ribose, pyruvate, phosphoenolpyruvate (PEP), carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate. Examples thereof include rate, 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. For energy sources that do not contain phosphate groups, it is preferred to add inorganic phosphoric acid to the reaction mixture.

(Iv) Substrate for synthesis of target protein As a substrate for synthesis of target protein, the reaction mixture can contain amino acids necessary for synthesizing the target protein. As amino acids, all 20 types of amino acids that make up proteins (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, It means tryptophan, tyrosine and valine. The same shall apply hereinafter.), and may be appropriately selected from the 20 types in consideration of the target protein and the reagent to be used. Also, non-natural amino acids may be used. When an unnatural amino acid is used, factors such as tRNA and aminoacylated tRNA synthetase that have been modified to introduce the unnatural amino acid into a protein may be added.

When the template nucleic acid is DNA, the reaction mixture may include a substrate for RNA synthesis as a substrate for protein synthesis of interest. Examples of the substrate for RNA synthesis include ribonucleotides such as ribonucleotide triphosphate (rNTP) and ribonucleotide monophosphate (rNMP), and ribonucleosides such as adenosine.

(V) Polyamines The reaction mixture can include polyamines. Examples of polyamines that can be included in the reaction mixture include spermine, spermidine, putrescine and the like.

(Vi) Salt The reaction mixture can include a salt. Salts that may be included in the reaction mixture include, for example, potassium, magnesium, ammonium, and manganese salts of acetic acid, glutamic acid, or sulfuric acid.

(Vii) Oxidation/Reduction Modifier The reaction mixture may include an oxidation/reduction modifier. Examples of the oxidation/reduction modifier include DTT, ascorbic acid, glutathione, and/or their oxides.

(Viii) Other Additives If necessary, the following components may be supplemented or added to the reaction mixture. That is, ribosome, transfer RNA (tRNA), optional translation factor (eg, translation initiation factor, elongation factor termination factor, ribosomal recycling factor, etc.) and its cofactor, aminoacyl-tRNA synthetase (ARS), methionyl-tRNA formyl transfer. Enzyme (MTF), polymer compound (eg, polyethylene glycol, dextran, diethylaminoethyl dextran, quaternary aminoethyl and aminoethyl dextran, etc.), nuclease, nuclease inhibitor, protein stabilizer, chaperone, solubilizer, non-denaturing interface An active substance (eg Triton X100) or the like may be added to the reaction mixture as needed.

Regarding the components of the reaction mixture, see, for example, US Pat. Nos. 7,338,789 and 7,351,563, and US Patent Application Publication No. 2010/0184135 and US Patent Application Publication No. 2010/0093302. Can be used as a reference.

[Cell-free protein synthesis method]
The cell-free protein synthesis method according to this embodiment can be performed using the reaction mixture of the present invention. Cell-free protein synthesis using the reaction mixture of the present invention is performed according to a known method described in, for example, Non-Patent Document 1, Non-Patent Document 2 or Non-Patent Document 3, except that the composition of the reaction mixture is different. You can

As a 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 the dialysis method, protein synthesis is performed in a closed system consisting of an inner solution containing the above reaction mixture and an outer solution containing a substrate for energy synthesis and a substrate for target protein synthesis separated by a dialysis membrane such as an ultrafiltration membrane. Done. In the dialysis method, a substrate and an energy source for synthesizing a target protein are supplied from the external liquid to the reaction mixture through a dialysis membrane, and unnecessary byproducts in the reaction mixture can be diffused into the external liquid. Therefore, the reaction can be continued for a longer time. The batch method is a synthetic method in which the reaction mixture containing all of the components necessary for protein synthesis is mixed with a reaction solution and uniformly contained in the reaction solution to carry out the reaction, but the reaction time is shorter than that of the dialysis method. , The results are available immediately. Cell extract, glycerol, and an energy source other than glycerol can be mixed in advance, and glycerol can also be used as a source of other energy sources before the template nucleic acid is added. In this case, the mixed solution can be used as a reaction mixture of the batch method and/or an external solution of the dialysis method. For example, by using the Cytomim type cell-free protein synthesis system described in Non-Patent Document 3, the protein synthesis system can be performed at a lower cost than the PANOx type that uses NTP or PEP.

The protein may be synthesized at, for example, 20 to 40°C, preferably 30°C.

The reaction time of protein synthesis by the batch method reaches almost the maximum production amount in 2 to 8 hours when glycerol is not added. When the reaction mixture for cell-free protein synthesis to which glycerol is added is used, the protein synthesis is continued for 10 hours or more after mixing the cell extract and glycerol. It is preferably -40 hours, more preferably 10-25 hours.

[Kit for cell-free protein synthesis]
The cell-free protein synthesis kit according to the present embodiment (hereinafter, also referred to as “the present kit”) contains a cell extract obtained from Escherichia coli and glycerol. Glycerol may be premixed with the cell extract and included in the kit as a cell extract containing glycerol (cell extract obtained from Escherichia coli), or included in the kit independently of the cell extract. It may be. The kit may contain a template nucleic acid, a substrate for protein synthesis of interest, and/or an energy source in addition to a cell extract obtained from Escherichia coli and glycerol. The template nucleic acid, the substrate for synthesizing the target protein, and the energy source may be premixed with the cell extract, or may be contained in the kit independently of the cell extract. Further, the kit may further contain the above-mentioned RNA polymerase, polyamines, salt, oxidation/reduction adjusting agent, and other additives. These additives may be premixed with the cell extract, or may be contained in the kit independently of the cell extract.

Hereinafter, the present invention will be described more specifically based on Examples. However, the present invention is not limited to the following examples.

[Test Example 1: E. coli culture]
(Culture of Escherichia coli using glycerol as a carbon source)
Escherichia coli BL21 strain (Novagen) was subjected to flask culture in 2×YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until the OD ₆₀₀ reached 4.2.

A modified 2×YTPG medium having the composition shown in Table 1 using glycerol as a carbon source (Adeka (registered trademark) LG-295S (1 ml/L as an antifoaming agent was added, and the pH was adjusted to 7 using NaOH)) It was added to a 1 L jar fermentor containing 500 mL so that the OD ₆₀₀ was 0.05.

For comparison, the same strain was similarly cultured using a normal 2×YTPG medium containing glucose (18 g/L) as a carbon source. The culturing temperature was kept at 36° C., and the culturing was carried out 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. It 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. It is speculated that this result suggests that ribosomes in the cells cultured in glycerol and enzymes in the energy regeneration system have higher activity.

Culturing using glycerol as a carbon source can obtain a cell extract with a preferable cell concentration in a shorter time than culture using glucose as a carbon source, so that a cell extract can be obtained in a shorter time. Was shown.

[Test Example 2: Cell-free protein synthesis (effect of cell extract)]
(Preparation of cell extract)
By the same method as in Test Example 1 above, a cell extract was obtained by the following method using the BL21 strain cultivated until each of the modified 2×YTPG medium and the normal 2×YTPG medium until the mid-logarithmic OD ₆₀₀ was 12. Was prepared.

2 L of the culture broth of the BL21 strain was centrifuged under the conditions of 7,000 xg and 4°C for 20 minutes to recover 31 g of wet bacterial 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.

2 mL of S30 buffer was added per 1 g of the microbial cells, thawed and suspended, and GEA. The cells were disrupted at a pressure of 1300 bar or higher using a high pressure homogenizer (Panda PLUS2000) manufactured by Niro soavi.

Next, the disrupted cell bodies were centrifuged twice under the conditions of 20,400×g, 4° C. and 30 minutes to remove cell residue.

In 200 μL of the centrifugation 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 protein are constituted 20. 60 μL of activation mix containing all amino acids of each type (0.04 mM each) was added and incubated at 30° C. for 150 minutes.

After the incubation, centrifugation was performed under the conditions of 20,400×g, 4° C., and 30 minutes, and the insoluble fraction was removed to obtain 230 μL of cell extract.

(Preparation of pUC-GFP)
Using pET3a (Novagen), a DNA fragment (1) containing T7 promoter, ribosome binding site (RBS) and T7 terminator sequences was prepared by PCR.

Using pUC19 (Takara Bio Inc.), a DNA fragment (2) containing the origin of replication and the sequence of the ampicillin resistance gene was prepared by the PCR method.

The DNA fragment (1) and the DNA fragment (2) were fused by a known In-Fusion reaction and transformed into Escherichia coli HST08. After culturing the transformed Escherichia coli, a plasmid was extracted using QIAGEN Plasmid Kit (manufactured by Qiagen Co., Ltd.).

Using the extracted plasmid, a DNA fragment (3) containing the sequences of T7 promoter, RBS, T7 terminator, origin of replication and ampicillin resistance gene was prepared by the PCR method.

Using T5-HollyGFP (manufactured by Cosmo Bio Inc.), the DNA fragment (4) containing the Holly-GFP gene was prepared by the PCR method.

The DNA fragment (3) and the DNA fragment (4) were fused by a known In-Fusion reaction and transformed into Escherichia coli HST08. After culturing the transformed Escherichia coli, pUC-GFP was extracted using QIAGEN Plasmid Kit (manufactured by Qiagen Co., Ltd.) and used as a template nucleic acid for cell-free protein synthesis reaction.

PUC-GFP for template nucleic acid has a high purity (A260/A280 is 1.8 or more, A260/A230 is 2.0 or more) by using a spectrophotometer, and a base sequence is determined by using a DNA sequencer. It was read and confirmed that there were no unnecessary mutations.

(Cell-free protein synthesis reaction)
10 μL of master mix containing the compounds of Table 2, 6 μL of prepared cell extract, 1 μL of 40 mM DTT aqueous solution, 2 μL of 0.2 g/L pUC-GFP (template nucleic acid) and 1 μL of 1000 U/μL T7 RNA polymerase were added, and total reaction volume was 20 μL. A mixture (liquid) was prepared. A negative control (NC) was prepared by adding 2 μL of RO water instead of pUC-GFP. The reaction mixture was transferred to a 384-well microplate, and fluorescence intensity was measured over time while incubating at 30° C. using a microplate reader TECANinfinite F200 (manufactured by Tecan Japan Co., Ltd.) to compare the amount of GFP synthesis. FIG. 2 shows the measurement result of the fluorescence intensity.

5 hours after the reaction was started, the fluorescence intensity of GFP when the cell extract derived from Escherichia coli cultured in a medium containing glycerol as a carbon source was used. The value was about 2-fold higher than the fluorescence intensity of GFP when the extract was used (Fig. 2). Thus, the amount of GFP synthesized by the cell-free protein synthesis method using the cell extract derived from Escherichia coli cultured in the medium containing glycerol as the carbon source was the cell extract derived from Escherichia coli derived from the medium containing glucose as the carbon source. It was shown to be about 2-fold higher than that of the cell-free protein synthesis method using.

[Example 1: Cell-free protein synthesis (effect of glycerol)]
(Culture using glycerol as a carbon source)
A BW25113 strain deficient in the rna gene was prepared by the method of Datsenko and Wanner, and a deficiency was introduced into the T7 Express strain (New England Biolabs) by P1 transduction to prepare a T7 Express strain deficient in 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 subjected to flask culture in 2×YT medium (1.6% Bacto Tripton, 1% Yeast Extract, 0.5% NaCl) until the OD ₆₀₀ reached 3.5.

Next, 2.1 L of the culture solution was added to the modified 2×YTPG medium having the composition shown in Table 1 (adding 1 ml/L of ADEKA (registered trademark) LG-295S as an antifoaming agent and adjusting the pH to 7 using NaOH). It was added to another 3 L jar famentor so that the OD ₆₀₀ was 0.05, and main culture was performed.

The main culture was carried out by keeping the culture temperature at 36° C., controlling the medium at a constant pH of 7.0, and maintaining the dissolved oxygen concentration in the medium at 2.4 mg/L or more. When the OD ₆₀₀ reached 2, 1 mM IPTG was added to the medium to induce T7 RNA polymerase. Then, the culture was continued until the mid-logarithmic OD ₆₀₀ reached 12.

(Preparation of cell extract)
2 L of the culture broth of the T7 Express strain cultured as described above was centrifuged under the conditions of 7,000×g, 4° C. and 20 minutes to recover 31 g of wet bacterial 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.

2 mL of S30 buffer was added per 1 g of the microbial cells, thawed and suspended, and GEA. The cells were disrupted at a pressure of 1300 bar or higher using a high pressure homogenizer (Panda PLUS2000) manufactured by Niro soavi.

Next, the disrupted cell bodies were centrifuged twice under the conditions of 20,400×g, 4° C. and 30 minutes to remove cell residue. The obtained supernatant was used as a cell extract.

(Template nucleic acid: pUC-GFP)
As the template nucleic acid, pUC-GFP prepared in Test Example 2 was used.

(Cell-free protein synthesis reaction)
The master mix shown in Table 2 and three types of master mix in which glycerol was added to the master mix at 100 mM (0.1%) or 200 mM (0.2%) were prepared. 6 μL of the prepared cell extract, 1 μL of 40 mM DTT aqueous solution, 2 μL of 0.2 g/L pUC-GFP and 1 μL of ultrapure water were added to 10 μL of the master mix to prepare a total reaction mixture (liquid) of 20 μL.

When preparing the above reaction mixture, it was prepared on ice in order to prevent the progress of the protein synthesis reaction before observation. 20 μL of the reaction mixture was placed in a 384-well microplate, and the amount of GFP synthesis was compared by measuring fluorescence intensity over time while incubating at 30° C. using a microplate reader TECANinfinite F200. FIG. 3 shows the measurement results of fluorescence intensity.

As shown in FIG. 3, the productivity of the glycerol-added system was lower than that of the system without the additive for up to 10 hours, but thereafter the GFP synthesis rate was improved. In the system in which 100 mM glycerol was added, it was possible to finally obtain about twice the production amount of GFP, and it was possible to confirm the effect of adding glycerol.

On the other hand, no improvement in productivity was observed in the system in which 200 mM glycerol was added, but if the initial glycerol concentration was lowered or glycerol was added by the method of divided or continuous administration, the synthesis rate at the initial stage of the reaction stagnated. It can be avoided, and it is expected that the productivity will be sufficiently improved even in the system containing 200 mM glycerol.

Claims (9)

A reaction mixture for cell-free protein synthesis, which comprises a cell extract obtained from Escherichia coli and glycerol. The reaction mixture according to claim 1, wherein the E. coli is E. coli expressing T7 RNA polymerase. The reaction mixture according to claim 1 or 2, wherein the Escherichia coli is Escherichia coli cultured in a medium containing glycerol as a carbon source. The reaction mixture according to any one of claims 1 to 3, further comprising a template nucleic acid, a substrate for protein synthesis of interest, and an energy source. The reaction mixture according to claim 4, wherein the template nucleic acid comprises at least one promoter and DNA encoding a protein of interest. The energy source is ATP, GTP, glucose, ribose, pyruvate, phosphoenolpyruvate, carbamoyl phosphate, acetyl phosphate, creatine phosphate, phosphopyruvate, glyceraldehyde-3-phosphate, 3-phosphoglycerate, 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, at least one selected from the group consisting of: The reaction mixture according to claim 4. A cell-free protein synthesis method using the reaction mixture according to claim 1. A cell-free protein synthesis kit comprising a cell extract obtained from Escherichia coli, glycerol, a template nucleic acid, a substrate for synthesizing a target protein, and/or an energy source. A protein obtained by the cell-free protein synthesis method according to claim 7.

Images