WO2018159647A1

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

Info

Publication number: WO2018159647A1
Application number: PCT/JP2018/007397
Authority: WO
Inventors: 俊将本間
Original assignee: Ｓｐｉｂｅｒ株式会社
Priority date: 2017-02-28
Filing date: 2018-02-28
Publication date: 2018-09-07
Also published as: JP2020092604A

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

Cell-free protein synthesis reaction mixture, cell-free protein synthesis method using the same, and cell-free protein synthesis kit

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 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.

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).

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.

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).

However, in production on an industrial scale, further improvements in production volume and cost are required.

Japanese Patent No. 5259046

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.

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.

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.

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.

Also, since glycerol is inexpensive, the amount of protein synthesis in the cell-free protein synthesis method can be increased at low cost.

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.

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.

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).

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) 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. .

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.

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.

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.

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.

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.

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.

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.

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) 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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) 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. .

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) 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.

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 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, α-ketoglutaric acid, succinyl CoA, succinic acid, fumaric acid, malic acid, oxaloacetic acid, glyoxylic acid, and glutamic acid.

(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.

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

(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) 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) 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.

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. .

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.

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.

[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.

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.

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.

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. .

[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.

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.

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.

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.

(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.

(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.

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

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 claim 1, further comprising a template nucleic acid, a substrate for synthesizing the target protein, and an energy source.
The reaction mixture according to claim 2, wherein the template nucleic acid comprises DNA encoding at least one promoter and a target protein.
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.
The reaction mixture according to any one of claims 1 to 4, wherein the E. coli is E. coli expressing T7 RNA polymerase.
A cell-free protein synthesis method using the reaction mixture according to any one of claims 1 to 5.
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.
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.
A protein obtained by the cell-free protein synthesis method according to claim 6.