Protein manufacturing method

Abstract

To provide a method for producing protein that can improve the solubility of protein in protein synthesis using an acellular protein synthetic pathway.SOLUTION: In a reaction liquid of an acellular protein synthetic pathway comprising one or both of cold shock protein and a nucleic acid comprising a code region encoding cold shock protein, a protein having a glutathione-s-transferase tag is synthesized. In a method for synthesizing a protein having a glutathione-s-transferase tag in a reaction liquid of an acellular protein synthetic pathway, a base sequence of a region encoding a sequence of 11 or 12 amino acids continuous from an N terminus of the glutathione-s-transferase tag is optimized in a templated nucleic acid for use in the synthesis.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a protein. [Background technology]

[0002] Known methods for producing proteins include a cell-free protein synthesis system in which factors necessary for protein translation are extracted as a cell extract and the intracellular reaction system is reconstituted in a test tube (Patent Documents 1 and 2).Known cell extracts include those derived from Escherichia coli, insect cells, wheat germ, tobacco cells, and animal cells, and kits are commercially available.

[0003] Cell-free protein synthesis systems have the following advantages: rapid protein synthesis, the ability to synthesize labeled or cytotoxic proteins, the ability to freely change the composition of the reaction mixture, the ability to use PCR products as templates, and the lack of genetic recombination experiments. However, cell-free protein synthesis systems can sometimes produce proteins with insufficient solubility. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Special Publication No. 7-110236 [Patent Document 2] Japanese Patent Application Publication No. 4-200390 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a method for producing a protein that can improve the solubility of a protein in protein synthesis using a cell-free protein synthesis system. [Means for solving the problem]

[0006] The present invention has the following aspects. [1] A method for producing a protein, in which a protein having a glutathione-S-transferase tag is synthesized in a reaction solution of a cell-free protein synthesis system containing either or both of a cold shock protein and a nucleic acid containing a coding region encoding the cold shock protein. [2] The method for producing a protein according to [1], wherein the sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag is the amino acid sequence represented by SEQ ID NO: 1 or 2. [3] The method for producing a protein according to [1] or [2], wherein the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 3 to 11. [4] The method for producing a protein according to any one of [1] to [3], wherein the base sequence of the region encoding the glutathione-S-transferase tag in the template nucleic acid used for synthesis is optimized by software including an algorithm for avoiding RNA secondary structures and an algorithm for optimizing codons and GC contents in accordance with the expression organism species. [5] The method for producing a protein according to any one of [1] to [4], wherein the cold shock protein is CspA derived from Escherichia coli, and the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 7 to 9. [6] The method for producing a protein according to any one of [1] to [5], wherein the reaction temperature is 4 to 30°C. [7] A method for synthesizing a protein having a glutathione-S-transferase tag in a reaction solution of a cell-free protein synthesis system, wherein the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag in a template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 4, 6 to 11. [8] The method for producing a protein according to any one of [1] to [6], wherein the reaction solution is a solution containing a cell extract. [9] The method for producing a protein according to [7], wherein the cell extract is a cell extract derived from Escherichia coli. [Effects of the Invention]

[0007] According to the present invention, a method for producing a protein that can improve the solubility of a protein in protein synthesis using a cell-free protein synthesis system can be provided. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows the electrophoresis results of GST synthesis in Example 1. [Figure 2] 2(a) and 2(b) show the amounts of GST fusion proteins synthesized (fluorescence values) in Example 2, where FIG. 2(a) shows the results at a reaction temperature of 30°C, FIG. 2(b) shows the results at a reaction temperature of 23°C, and FIG. 2(c) shows the results at a reaction temperature of 16°C. [Figure 3] 1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 3. [Figure 4] 4 shows the amount of GST fusion protein synthesized (fluorescence value) in Example 4, where FIG. 4(a) shows the results at a reaction temperature of 23°C, and FIG. 4(b) shows the results at a reaction temperature of 16°C. [Figure 5] 5 shows the amounts of GST fusion protein synthesized (fluorescence values) in Example 5, where FIG. 5(a) shows the results for Sq12, Sq18, Sq23, and Sq24, and FIG. 5(b) shows the results for Sq15, Sq18, and Sq25. [Figure 6] 1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 6. [Figure 7] 1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 7. [Figure 8] 1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 8. [Figure 9] 1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 9. [Figure 10]1 shows the electrophoresis results of the synthesis of GST fusion proteins in Example 10. [Figure 11] Electrophoresis results of the synthesis of GST fusion proteins in Example 11, including His tag, Sq12, Sq13, and Sq15. [Figure 12] Electrophoresis results of the synthesis of GST fusion proteins in Example 11, including His tag, Sq18, Sq19, and Sq20. [Figure 13] Electrophoresis results of the synthesis of GST fusion proteins in Example 12, including His-tag, Sq12, Sq13, and Sq15. [Figure 14] Electrophoresis results of the synthesis of GST fusion proteins in Example 12, including His tag, Sq18, Sq19, and Sq20. [Figure 15] Electrophoresis results of the synthesis of GST fusion proteins in Example 13, including His tag, Sq12, Sq13, and Sq15. [Figure 16] Electrophoresis results of the synthesis of GST fusion proteins in Example 13, including His tag, Sq18, Sq19, and Sq20. DETAILED DESCRIPTION OF THE INVENTION

[0009] In the specification and claims, the following terms have the following meanings: A "cell-free protein synthesis system" is a system in which protein factors necessary for protein translation are extracted as a cell extract and the intracellular reaction system is reconstituted in a test tube to synthesize a target protein. Cell-free protein synthesis systems include both cell-free transcription / translation systems, including cell-free transcription systems that synthesize RNA using DNA as a template and cell-free translation systems that read information from mRNA and synthesize proteins on ribosomes, and cell-free translation systems. A "cold shock protein" is a protein or protein domain having an amino acid sequence that matches the CSD family (PF00313) of the Pfam database (version 32.0) with a score of 100 or greater. A numerical range indicated by "to" means a numerical range that includes the numbers before and after "to" as the lower and upper limits.

[0010] [First embodiment] The protein production method according to the first embodiment is a method for synthesizing a glutathione-S-transferase (GST)-tagged protein in a cell-free protein synthesis system (CF system) containing either or both of a cold shock protein (Csp) and a nucleic acid containing a coding region encoding Csp. That is, this method involves protein synthesis in a CF system reaction solution in the presence of Csp to obtain a GST fusion protein in which a GST tag has been added to a target protein. The first embodiment is particularly effective in improving the solubility of the resulting protein in protein synthesis at low temperatures.

[0011] The CF system can be a cell extract extracted from cells containing the components necessary for the CF system, or a solution used in reconstituted cell-free protein synthesis, which is a mixture of individually purified factors necessary for protein synthesis, such as ribosomes, aminoacyl-tRNA synthetases, tRNAs, and translation termination factors. The reaction solution is preferably a solution containing a cell extract.

[0012] Examples of cell extracts include cell extracts extracted from plant cells, animal cells, fungal cells, and bacterial cells that contain components necessary for the translation system involved in protein synthesis, such as ribosomes, aminoacyl-tRNA synthetases, and tRNAs, or components necessary for the transcription and translation systems. Specific examples include cell extracts from Escherichia coli, wheat germ, tobacco cells, rabbit reticulocytes, mouse L-cells, Ehrlich ascites tumor cells, HeLa cells, CHO cells, and budding yeast.

[0013] As the cell extract, a cell extract derived from Escherichia coli is preferred in terms of stability and scalability of extract preparation. Examples of cell extracts derived from Escherichia coli include S30 extract (hereinafter also referred to as "E. coli S30 extract") and S12 extract from E. coli (BL21, etc.) cells. E. coli S30 extract contains all of the enzymes and factors of E. coli required for transcription and translation.

[0014] The method for preparing the cell extract is not particularly limited, and known methods can be used. For example, an Escherichia coli S30 extract can be prepared by the method described in Zubay G., Ann Rev Genet, (1973) 7, 267-287, Seki E., Matsuda N., Yokoyama S., and Kigawa T. (2008), Anal. Biochem., etc. Specifically, cell extracts are prepared by culturing E. coli and recovering the cells by centrifugation or other methods. The recovered cells are washed, resuspended in a buffer solution, and disrupted using a French press, glass beads, or a Waring blender. Insoluble material from the disrupted E. coli is removed by centrifugation, and the cells are mixed with a preincubation mixture and incubated. This procedure degrades endogenous nucleic acids (DNA, RNA), but calcium salts, micrococcal nuclease, or other agents may be added to further degrade endogenous nucleic acids. Subsequently, endogenous amino acids, nucleic acids, nucleosides, etc. are removed by dialysis, and the extract is dispensed into appropriate aliquots and stored in liquid nitrogen or at -80°C. Commercially available cell extracts may also be used.

[0015] The content of the cell extract in the reaction solution, expressed as a final concentration in the reaction solution (equivalent to the absorbance A260 of the cell extract at 260 nm), is preferably 40 to 200, more preferably 50 to 150. When the content of the cell extract is within the above range, the target protein can be easily synthesized with high efficiency.

[0016] The cell extract contains all the enzymes and factors necessary for transcription and translation, but when used in a protein synthesis reaction solution, further supplementary components may be added. Examples of supplementary components include L-amino acids that serve as substrates, energy sources, salts, various ions, buffer solutions, energy regeneration systems, nuclease inhibitors, reducing agents, and antibacterial agents.

[0017] Examples of L-amino acids include the 20 naturally occurring amino acids and their derivatives. When an isotope-labeled protein is produced as the target protein, a labeled amino acid labeled with a stable isotope or a radioactive isotope is used. Examples of energy sources include adenosine triphosphate (ATP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), uridine triphosphate (UTP) (hereinafter, these four are collectively referred to as NTP), creatine phosphate (CP), phosphoenolpyruvate (PEP), and glucose. Alternatively, adenosine monophosphate (AMP), cytidine monophosphate (CMP), guanosine monophosphate (GMP), and uridine monophosphate (UMP) (hereinafter, these four are collectively referred to as NMP) may be added to the reaction solution instead of NTP, and NTP may be synthesized in situ using metabolic enzymes (Calhoun KA and Swartz JR, Biotechnol. Prog., 2005, 21, 1146-1153). Examples of salts include ammonium acetate, potassium acetate, potassium glutamate, magnesium acetate, magnesium chloride, potassium acetate, and calcium chloride. Examples of buffer solutions include Tris-acetate and HEPES-KOH.

[0018] The energy regeneration system is preferably an ATP regeneration system. Examples of ATP regeneration systems include a combination of 10 to 100 mM CP and 0.02 to 5 μg / μL creatine kinase (CK), or a combination of 1 to 20 mM PEP and 0.01 to 1 μg / μL pyruvate kinase (PK). Both PK and CK are enzymes that regenerate ATP from ADP and require PEP and CP as substrates, respectively.

[0019] Nuclease inhibitors include, for example, RNase inhibitors. An example of a reducing agent is dithiothreitol (DTT). Antibacterial agents include, for example, sodium azide and ampicillin.

[0020] To enhance protein synthesis activity, polyethylene glycol (PEG), folic acid, cAMP, tRNA, and the like may be added. Furthermore, when DNA is used as a template for the target protein, NTP or its precursor (e.g., NMP, nucleoside), which is a substrate for RNA synthesis, and RNA polymerase (e.g., T7, T3, and SP6 RNA polymerase) may be included. Chaperone proteins (e.g., DnaJ, DnaK, GroE, GroEL, GroES, and HSP70) that help proteins form three-dimensional structures, and disulfide bond isomerases (e.g., DsbC) may also be added. The number of supplementary ingredients may be one or more.

[0021] When an E. coli S30 extract is used, the reaction solution preferably contains the E. coli S30 extract, L-amino acids, a buffer solution, salts, NTPs, and an energy source, for example, an E. coli S30 extract, HEPES-KOH, DTT, NTPs (ATP, CTP, GTP, UTP), CP, CK, and at least one amino acid (one of the 20 naturally occurring amino acids or a derivative thereof).

[0022] It is preferable to store the supplementary components separately from the cell extract and mix them immediately before use. Alternatively, the supplementary components can be mixed with the cell extract in advance and then freeze-thawed to remove the RNase complex (WO 2000 / 183805).

[0023] In this embodiment, the CF-based reaction solution contains either or both of Csp and a Csp template nucleic acid. In the present invention, it is preferable to use Csp. Csps are thought to be involved in adaptation to low-temperature environments and have been found in many organisms, from bacteria to higher animals and plants. Examples of Csps include CspA, CspB, CspC, CspE, CspG, and CspI derived from Escherichia coli, SliCspC derived from Shewanella livingstonensis, and BsuCspB derived from Bacillus subtilis. CspA derived from Escherichia coli is a Csp that is most significantly expressed in low-temperature environments. The reaction solution may contain one type of Csp, or two or more types of Csp.

[0024] When Csp is used, the content of Csp in the reaction solution is preferably 0.5 to 1.5 μg / μL, more preferably 0.8 to 1.2 μg / μL when the reaction temperature is 16 to 23° C., and more preferably 1.2 to 1.5 μg / μL when the reaction temperature is lower than 16° C. If the Csp content is within the above range, the target protein can be easily synthesized with high efficiency.

[0025] A Csp template nucleic acid is a nucleic acid that serves as a template for synthesizing Csp in a CF system. When a Csp template nucleic acid is contained in a reaction solution of a CF system, Csp is synthesized during the reaction, resulting in a reaction solution containing Csp. Because Csp is rapidly synthesized even in a low-temperature environment, even when a Csp template nucleic acid is used, the target protein is synthesized in the presence of Csp. The Csp template nucleic acid contained in the reaction solution may be one type or two or more types.

[0026] When a Csp template nucleic acid is used, the content of the Csp template nucleic acid in the reaction solution is preferably 0.1 to 1.0 ng / μL, more preferably 0.2 to 0.8 ng / μL, and even more preferably 0.2 to 0.4 ng / μL. When the content of the Csp template nucleic acid is within the above range, the target protein can be easily synthesized with high efficiency.

[0027] In this embodiment, either template DNA or template RNA may be used as the template nucleic acid. That is, a GST fusion protein may be synthesized by adding template DNA to a cell-free transcription / translation system, or a GST fusion protein may be synthesized by adding template RNA to a cell-free translation system. When template DNA is used, the form of the template DNA for the GST fusion protein is not particularly limited, and it may be either a circular double-stranded DNA such as a plasmid DNA produced by recombinant DNA technology, or a linear DNA prepared by PCR. In the present invention, GST fusion proteins can be synthesized stably and efficiently even when linear template DNA is used.

[0028] In this embodiment, the GST tag can be GST (molecular weight: 26 kDa) expressed from a gene derived from Schistosoma japonicum. In addition to wild-type GST, mutant GST containing a mutation that can increase the expression level of the GST tag can also be used. In the GST fusion protein to be synthesized, the GST tag is preferably added to the N-terminus of the target protein.

[0029] Examples of GST tags to be added to a protein of interest include a GST tag in which a region of 11 consecutive amino acids from the N-terminus has the same amino acid sequence as the wild-type GST tag, as shown in SEQ ID NO: 1, and a GST tag in which a region of 12 consecutive amino acids from the N-terminus has the amino acid sequence shown in SEQ ID NO: 2, in which a lysine has been inserted at the second position from the N-terminus. Inserting a lysine at the second position from the N-terminus of the GST tag, as in the amino acid sequence shown in SEQ ID NO: 2, facilitates the expression of a protein with improved solubility. Hereinafter, in the GST tag, the region from the N-terminus to the 11th amino acid (lysine) represented by SEQ ID NO: 1 and the region from the N-terminus to the 12th amino acid (lysine) represented by SEQ ID NO: 2 will be collectively referred to as "region A."

[0030] Furthermore, in order to facilitate the expression of a protein with improved solubility, it is preferable that a silent mutation be introduced into the template nucleic acid around the initiation codon of the GST tag, more specifically, into the region encoding region A of the GST tag of the template nucleic acid.

[0031] Examples of the nucleotide sequence of the region encoding region A of the GST tag of the template nucleic acid include the nucleotide sequences shown in SEQ ID NOs: 3 to 11. Among these, the nucleotide sequences shown in SEQ ID NOs: 4 to 11 are preferred, and the nucleotide sequences shown in SEQ ID NOs: 4, 6 to 11 are more preferred, since proteins with improved solubility can be easily expressed. When the base sequence of the region encoding region A of the GST tag of the template nucleic acid is a sequence represented by SEQ ID NO: 4, 6, 8, 10, or 11, a GST tag is expressed in which the region of 12 consecutive amino acids from the N-terminus has the amino acid sequence represented by SEQ ID NO: 2.

[0032] The base sequence of the region encoding the GST tag in the template nucleic acid, including the region from region A to the C-terminus of the GST tag (hereinafter also referred to as "region B"), is preferably optimized using software including the following algorithms (a) and (b). This makes it easier to express a protein with improved solubility. The software may further include at least one of the following algorithms (c) to (e). (a) Avoiding RNA secondary structures. (b) Optimize the codon and GC content to suit the expression species. (c) Remove potential splice sites and RNA-destabilizing sequences. (d) Adding sequences that stabilize mRNA. (e) Removing introns.

[0033] The software that can be used is software that can optimize gene sequences to suit the species of organism, and specific examples include GeneOptimizer (registered trademark) (Thermo Fisher Scientific) and GENEius (EUROFINS).

[0034] In this embodiment, only one type of template nucleic acid may be used, or two or more types of template nucleic acids with different base sequences in the region encoding the GST tag may be used in combination. When CspA is used as the Csp, the base sequence of the region encoding region A of the GST tag in the template nucleic acid used for synthesis is preferably the base sequence shown in any one of SEQ ID NOs: 7 to 9, since this facilitates the expression of a protein with improved solubility.

[0035] The content of template nucleic acid in the reaction solution can be appropriately set depending on the protein synthesis activity of the cell extract, the type of target protein, etc., and can be, for example, about 0.5 to 10 ng / μL.

[0036] In this embodiment, the protein can be synthesized by dialysis, batch synthesis, or bilayer synthesis (Sawasaki, T., Hasegawa, Y., Tsuchimochi, M., Kamura, N., Ogasawara, T., Kuroita, T., and Endo, Y. (2002) FEBS Lett. 514, 102-105), with dialysis being preferred. Dialysis is a synthesis method in which an internal reaction solution and an external solution containing reaction substrates are separated by a dialysis membrane (ultrafiltration membrane) and the synthesis is carried out in a closed system that can be shaken or stirred. In dialysis, the substrates required for synthesis are supplied from the external solution to the reaction solution via the dialysis membrane, and excess by-products in the reaction solution are diffused into the external solution, allowing the reaction to continue for a longer period of time. This makes it possible to obtain a higher amount of protein synthesis.

[0037] The reaction temperature is preferably 4 to 30°C, more preferably 16 to 23°C. The reaction time is preferably 2 to 6 hours in the case of a batch method, and more preferably 4 to 6 hours in the case of a reaction at 16° C. or below. In the case of a dialysis method, the reaction time is preferably 1 to 40 hours, and more preferably 5 to 40 hours.

[0038] The molecular weight cutoff of the dialysis membrane separating the internal dialysis solution from the external dialysis solution is preferably 3,500 to 100,000, more preferably 10,000 to 50,000. The shaking or stirring speed can be, for example, 100 to 300 rpm.

[0039] After synthesis, the GST fusion protein is preferably purified. Methods for purifying the GST fusion protein include known protein purification methods, such as ammonium sulfate or acetone precipitation, acid extraction, anion or cation exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, gel filtration chromatography, hydroxyapatite chromatography, isoelectric focusing chromatography, and chromatofocusing. These purification methods may be performed alone or in combination. Affinity purification using an adsorbent that specifically recognizes the GST tag may also be used. The GST fusion protein can be identified and quantified by activity measurement, immunological measurement, spectroscopic measurement, amino acid analysis, or the like, while comparing with a standard sample as necessary.

[0040] [Second embodiment] The protein production method according to the second embodiment is a method for synthesizing a GST-tagged protein in a CF-based reaction solution, in which the base sequence of a region encoding 11 or 12 consecutive amino acids from the N-terminus of the GST tag in a template nucleic acid used for synthesis is set to any one of SEQ ID NOs: 4, and 6 to 11. That is, the protein production method according to the second embodiment is a method in which the base sequence encoding region A of the GST tag in a template nucleic acid in a CF-based protein synthesis is set to any one of SEQ ID NOs: 4, and 6 to 11. This improves the solubility of proteins synthesized in the CF system.

[0041] The description of the CF-based reaction solution in the first embodiment also applies to the CF-based reaction solution in the second embodiment, except that it contains Csp. The template nucleic acid used in the second embodiment, including region B, is preferably optimized using the above-mentioned software, such as GeneOptimizer (registered trademark) (Thermo Fisher Scientific) or GENEius (EUROFINS), similar to the template nucleic acid used in the first embodiment.

[0042] In this embodiment, only one type of template nucleic acid may be used, or two or more types of template nucleic acids with different base sequences in the region encoding the GST tag may be used in combination. The content of template nucleic acid in the reaction solution can be appropriately set depending on the protein synthesis activity of the cell extract, the type of target protein, etc., and can be, for example, about 0.5 to 10 ng / μL.

[0043] Examples of methods for synthesizing proteins in this embodiment include the same methods as in the first embodiment, and dialysis is preferred. The reaction temperature is preferably 4 to 30°C, more preferably 23 to 30°C. The reaction time is preferably 2 to 6 hours in the case of a batch method, and more preferably 4 to 6 hours in the case of a reaction at 16° C. or below. In the case of a dialysis method, the reaction time is preferably 1 to 40 hours, and more preferably 5 to 40 hours.

[0044] In this embodiment as well, it is preferable to purify the GST fusion protein after its synthesis. Examples of methods for purifying the GST fusion protein include the same methods as those exemplified in the first embodiment.

[0045] The uses of proteins produced by the protein production method of the present invention are not particularly limited. For example, they can be used for three-dimensional structural analysis by X-ray crystallography or NMR measurement, enzymatic substance production, etc. The present invention enables highly efficient production of proteins with improved solubility. Therefore, they are suitable for, for example, three-dimensional structural analysis and enzymatic substance production, which require large amounts of protein. [Example]

[0046] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following descriptions.

[0047] [Example 1] A linear DNA containing a T7 promoter, a ribosome binding sequence, any one of the nucleotide sequences represented by SEQ ID NO: 12, 14, 16, 18, or 20, and a T7 terminator was prepared by PCR as a template DNA for synthesizing GST having the amino acid sequence represented by SEQ ID NO: 21. Furthermore, a linear DNA containing a T7 promoter, a ribosome binding sequence, any one of the nucleotide sequences represented by SEQ ID NO: 13, 15, 17, or 19, and a T7 terminator was prepared by PCR as a template DNA for synthesizing GST having the amino acid sequence represented by SEQ ID NO: 22 (GST with a lysine inserted at the second position from the N-terminus). Using these linear DNAs containing the nucleotide sequences represented by SEQ ID NO: 12 to 20 as templates, GSTs (Sq12 to Sq20) were synthesized in a CF reaction mixture. The GST expressed using a linear DNA containing the nucleotide sequence represented by SEQ ID NO: 12 as a template is designated Sq12, and the same is true for GSTs expressed using other linear DNAs as templates. The E. coli S30 extract was a stable isotope-labeled cell-free enzyme solution (Taiyo Nippon Sanso) prepared from the E. coli BL21 codon plus strain. LMCP (D-Glu) was used as a protein synthesis reagent, excluding enzymes and amino acids. Protein synthesis reactions using each template DNA were carried out for 18 hours by dialyzing a reaction solution having the composition shown in Table 1 below against an external dialysis solution having the composition shown in Table 2. The reaction temperature was 30°C, 23°C, or 16°C. The reaction scale was 30 μL of the internal solution (reaction solution) and 500 μL of the external dialysis solution.

[0048] "LMCP (D-Glu)" in Tables 1 and 2 refers to a mixture containing HepesKOH (pH 7.5) as a buffer solution, potassium D-glutamate and ammonium acetate as salts, ATP as an energy source, GTP, CTP, and UTP as transcription substrates, and cyclic AMP, folinic acid, DTT, and polyethylene glycol as other reagents.

[0049] [Table 1]

[0050] [Table 2]

[0051] The total fraction (T) and soluble fraction (S) were subjected to SDS (sodium dodecyl sulfate)-polyacrylamide gel electrophoresis (15% Tricine-SDS-PAGE) and then stained with CBB. The results are shown in Figure 1.

[0052] As shown in Figure 1, a comparison of Sq12 and Sq13, and Sq14 and Sq15, showed that the insertion of lysine at the second position from the N-terminus increased the amount of GST expression. Furthermore, Sq18, which does not have a lysine at the second position from the N-terminus, also showed high GST expression levels, even at 16°C. Among Sq12 to Sq20, the amount of GST expression was particularly high in Sq15 to Sq20.

[0053] [Example 2] Plasmid DNA was prepared as a template for a GST fusion protein containing a GST tag at the N-terminus of GFP (green fluorescent protein). This plasmid DNA consisted of a T7 promoter, a ribosome binding sequence, any of the sequences shown in SEQ ID NOS: 12 to 20, a GFP protein variant (GFPS1) gene, and a T7 terminator. GFP (GST fusion protein) having a GST tag (Sq12 to Sq20) was synthesized in the same manner as in Example 1, except that the plasmid DNA was used at 1.0 ng / μL. The amount of GST fusion protein synthesized was determined from the GFP fluorescence intensity. After diluting the reaction mixture with buffer (20 mM Tris-HCl, pH 7.5, 300 mM NaCl), the GFPS1 fluorescence intensity was measured using a multimode microplate reader, SpectraMax i3 (Molecular Devices), at excitation 485 nm and emission 535 nm. The results are shown in Figure 2.

[0054] As shown in Figure 2, a comparison of Sq12 and Sq13, and a comparison of Sq14 and Sq15, showed that the insertion of lysine at the second position from the N-terminus of GST increased the amount of GST fusion protein synthesized. Furthermore, even in Sq18, which does not have lysine at the second position from the N-terminus, a high amount of GST fusion protein was synthesized, even at 16°C. Among Sq12 to Sq20, the amount of GST fusion protein with the GST tag Sq15 to Sq20 synthesized was particularly high. These trends were similar to those observed in Example 1, where only GST was expressed.

[0055] [Example 3] GST (Sq12 to Sq20) was synthesized using linear DNA containing the nucleotide sequences represented by SEQ ID NOs: 12 to 20 as templates in the same manner as in Example 1, except that purified CspA derived from Escherichia coli was added to the reaction solution to a concentration of 1.0 μg / μL. Purified CspA was synthesized in a CF system. The reaction temperature was 23°C or 16°C. The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 3.

[0056] As shown in Figure 3, GST was expressed sufficiently in the CF system in the presence of Csp. The expression level of GST Sq16-18 was particularly high.

[0057] [Example 4] GFP (GST fusion protein) having a GST tag (Sq12 to Sq20) was synthesized in the same manner as in Example 3, except that 1.0 ng / μL of plasmid DNA of a GST fusion protein having a GST tag at the N-terminus of GFP (green fluorescent protein) was used. The reaction temperature was 23°C or 16°C. The amount of GST fusion protein synthesis (GFP fluorescence value) measured in the same manner as in Example 2 is shown in FIG.

[0058] As shown in Figures 4(a) and 4(b), the GST fusion proteins were expressed sufficiently in the CF system in the presence of Csp. In particular, the GST fusion proteins with GST tags of Sq16 to 18 were expressed at high levels.

[0059] [Example 5] Plasmid DNA was prepared as a template for a GST fusion protein containing a GST tag at the N-terminus of GFP. This plasmid DNA consisted of a T7 promoter, a ribosome binding sequence, a sequence shown in SEQ ID NO: 12, 15, 18, 23, 24, or 25, a modified GFP protein (GFPS1) gene, and a T7 terminator. GFP (GST fusion protein) having a GST tag (Sq12, Sq15, Sq18, Sq23, Sq24, Sq25) was synthesized in the same manner as in Example 4, except that the reaction temperature was 30°C, 23°C, or 16°C. The amount of GST fusion protein synthesis (GFP fluorescence value) measured in the same manner as in Example 2 is shown in FIG.

[0060] The template DNA for the GST tag of Sq23 (SEQ ID NO: 23) has a silent mutation introduced into the nucleotide sequence encoding region A of the GST tag, but the nucleotide sequence encoding region B is the same as that of the wild-type. The template DNA for the GST tag of Sq24 (SEQ ID NO: 24) has the same nucleotide sequence encoding region A of the GST tag as that of the wild-type, but the nucleotide sequence encoding region B has been optimized. The template DNA for the GST tag of Sq18 (SEQ ID NO: 18) has the same nucleotide sequence encoding region A of the GST tag as that of Sq23, and the same nucleotide sequence encoding region B as that of Sq24. The template DNA for the GST tag of Sq25 (SEQ ID NO: 25) is a GST tag in which a lysine is inserted at the second position from the N-terminus of Sq23. The template DNAs for the GST tags of Sq15, Sq18, and Sq25 (SEQ ID NOs: 15, 18, and 25) differ in the nucleotide sequence encoding region A of the GST tag, but share the same nucleotide sequence encoding region B.

[0061] As shown in Figure 5(a), the amount of GST fusion protein synthesized with the Sq23 GST tag was greater than that with the Sq24 GST tag, and was closer to that of the GST fusion protein with the Sq18 GST tag. This indicates that optimizing the base sequence encoding region A of the GST tag in the template nucleic acid is more effective in increasing the amount of soluble GST fusion protein synthesized than optimizing the base sequence encoding region B of the GST tag. As shown in Figure 5(b), in the CF system in the presence of Csp, the amount of GST fusion protein synthesized was greater with the Sq25 GST tag than with the Sq15 GST tag, and was closer to that of the GST fusion protein with the Sq18 GST tag. This indicates that the base sequence encoding region A of the GST tag in the template nucleic acid contains an optimal sequence for increasing the amount of GST fusion protein synthesized in the CF system in the presence of Csp.

[0062] [Example 6] A linear DNA containing a T7 promoter, a ribosome binding sequence, a His tag sequence (N11), the MAP2K6 gene, and a T7 terminator was prepared by PCR as a template DNA for a GST fusion protein of kinase (MAP2K6) with a His tag (N11) at its N-terminus. A kinase with a His tag was synthesized in the same manner as in Example 1, except that the template DNA was used at 1.5 ng / μL. As template DNA for a GST fusion protein having a GST tag at the N-terminus of kinase (MAP2K6), a linear DNA containing a T7 promoter, a ribosome binding sequence, either the nucleotide sequence shown in SEQ ID NO: 12 or 18, the MAP2K6 gene, and a T7 terminator was prepared by PCR. A kinase having a GST tag (GST fusion protein) was synthesized in the same manner as in Example 1, except that the template DNA was used at 1.5 ng / μL and a reaction solution containing no polyethylene glycol was used. Furthermore, a His-tagged kinase and GST fusion protein was synthesized in the same manner, except that purified CspA derived from Escherichia coli was added to the reaction solution to a concentration of 1.0 μg / μL. The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 6.

[0063] [Example 7] A His-tag fusion protein and a GST fusion protein were synthesized in the same manner as in Example 6, except that a linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), the AURKB gene, and a T7 terminator, and a linear DNA containing a T7 promoter, a ribosome binding sequence, either the nucleotide sequence shown in SEQ ID NO: 12 or 18, the AURKB gene, and a T7 terminator, were used as template DNA for a GST fusion protein having a His-tag sequence (N11) or a GST tag at the N-terminus of kinase (AURKB). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 7.

[0064] [Example 8] A His-tag fusion protein and a GST fusion protein were synthesized in the same manner as in Example 6, except that a linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), the SYK gene, and a T7 terminator, and a linear DNA containing a T7 promoter, a ribosome binding sequence, either the nucleotide sequence shown in SEQ ID NO: 12 or 18, the SYK gene, and a T7 terminator, were used as template DNA for a GST fusion protein having a His-tag sequence (N11) or a GST tag at the N-terminus of a kinase (SYK). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 8.

[0065] [Example 9] A His-tag fusion protein and a GST fusion protein were synthesized in the same manner as in Example 6, except that a linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), a TRIB2 gene, and a T7 terminator, and a linear DNA containing a T7 promoter, a ribosome binding sequence, either the nucleotide sequence shown in SEQ ID NO: 12 or 18, a TRIB2 gene, and a T7 terminator were used as template DNA for a GST fusion protein having a His-tag sequence (N11) or a GST tag at the N-terminus of a protein kinase inhibitor (TRIB2). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 9.

[0066] [Example 10] A His-tag fusion protein and a GST fusion protein were synthesized in the same manner as in Example 6, except that a linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), an MCM5 gene, and a T7 terminator, and a linear DNA containing a T7 promoter, a ribosome binding sequence, either the nucleotide sequence shown in SEQ ID NO: 12 or 18, an MCM5 gene, and a T7 terminator, were used as template DNA for a GST fusion protein having a His-tag sequence (N11) or a GST tag at the N-terminus of helicase (MCM5). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figure 10.

[0067] As shown in Figures 6 to 10, the GST-tagged GST fusion protein was synthesized in greater amounts and was more soluble than the His-tagged protein. Furthermore, the addition of Csp to the reaction mixture increased the amount of soluble GST fusion protein synthesized.

[0068] [Example 11] His-tag fusion proteins and GST fusion proteins were synthesized in the same manner as in Example 6, except that linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), a TRIB2 gene, and a T7 terminator, and linear DNA containing a T7 promoter, a ribosome binding sequence, any of the nucleotide sequences shown in SEQ ID NOs: 12, 13, 15, 18, 19, and 20, a TRIB2 gene, and a T7 terminator were used as template DNA for GST fusion proteins having a His-tag sequence (N11) or a GST tag at the N-terminus of a protein kinase inhibitor (TRIB2). The reaction temperature was 30°C or 16°C. The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figures 11 and 12.

[0069] [Example 12] A His-tag fusion protein and a GST fusion protein were synthesized in the same manner as in Example 11, except that a linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), the AURKC gene, and a T7 terminator, and a linear DNA containing a T7 promoter, a ribosome binding sequence, any of the nucleotide sequences represented by SEQ ID NOs: 12, 13, 15, 18, 19, and 20, the AURKC gene, and a T7 terminator were used as template DNAs for GST fusion proteins having a His-tag sequence (N11) or a GST tag at the N-terminus of the kinase (AURKC). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figures 13 and 14.

[0070] [Example 13] His-tag fusion proteins and GST fusion proteins were synthesized in the same manner as in Example 11, except that linear DNA containing a T7 promoter, a ribosome binding sequence, a His-tag sequence (N11), an MCM5 gene, and a T7 terminator, and linear DNA containing a T7 promoter, a ribosome binding sequence, any of the nucleotide sequences shown in SEQ ID NOs: 12, 13, 15, 18, 19, and 20, an MCM5 gene, and a T7 terminator were used as template DNAs for GST fusion proteins having a His-tag sequence (N11) or a GST tag at the N-terminus of helicase (MCM5). The total fraction (T) and soluble fraction (S) from the reaction with and without CspA (Csp+ and Csp-) using various template DNAs were subjected to 15% Tricine-SDS-PAGE and stained with CBB. The results are shown in Figures 15 and 16.

[0071] As shown in Figures 11 to 16, the solubility of GST fusion proteins, which tend to precipitate at 30°C, was improved by performing the synthesis reaction at 16°C, and they were more soluble than proteins with His tags. Furthermore, the addition of Csp to the reaction mixture increased the amount of soluble GST fusion protein synthesized. In particular, the expression levels of GST fusion proteins with GST tags Sq13, Sq15, Sq18, and Sq19 were high.

Claims

1. A method for producing a protein, comprising synthesizing a protein having a glutathione-S-transferase tag at its N-terminus in a reaction solution of a cell-free protein synthesis system containing either or both of a cold shock protein and a nucleic acid containing a coding region encoding the cold shock protein, the method comprising: the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 4, 6 to 11; the reaction solution is a solution containing a cell extract, A method for producing a protein, wherein the cell extract is a cell extract derived from Escherichia coli.

2. The method for producing a protein according to claim 1, wherein the sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag is the amino acid sequence represented by SEQ ID NO: 1 or 2.

3. The method for producing a protein according to claim 1 or 2, wherein the nucleotide sequence of a region encoding the amino acid sequence of the glutathione S-transferase tag in the template nucleic acid used for synthesis is a nucleotide sequence represented by any one of SEQ ID NOs: 13, 15 to 20.

4. 3. The method for producing a protein according to claim 1 or 2, wherein the cold shock protein is CspA derived from Escherichia coli, and the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 7 to 9.

5. The method for producing a protein according to claim 4, wherein the base sequence of a region encoding the amino acid sequence of the glutathione S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 16 to 18.

6. The method for producing a protein according to any one of claims 1 to 5, wherein the reaction temperature is 4 to 30°C.

7. A method for synthesizing a protein having a glutathione-S-transferase tag at its N-terminus in a reaction solution of a cell-free protein synthesis system, comprising: the base sequence of a region encoding a sequence of 11 or 12 consecutive amino acids from the N-terminus of the glutathione-S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 4, 6 to 11; the reaction solution is a solution containing a cell extract, A method for producing a protein, wherein the cell extract is a cell extract derived from Escherichia coli.

8. The method for producing a protein according to claim 7, wherein the base sequence of a region encoding the amino acid sequence of the glutathione S-transferase tag in the template nucleic acid used for synthesis is a base sequence represented by any one of SEQ ID NOs: 13, 15 to 20.

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