Method for synthesizing proteins having disulfide bonds within or between molecules

Abstract

To provide a method for synthesizing a protein having an intramolecular or intermolecular disulfide bond.SOLUTION: Provided is a protein synthesis reaction method in which a reaction phase containing a synthetic reaction solution and a supply phase containing an energy source supply solution including a substrate and an energy source molecule are brought into direct or indirect contact with each other to supply the substrate and the energy source molecule in the supply phase to the reaction phase. The supply phase is compared to the reaction phase and brought under oxidative conditions, thereby synthesizing an activity-maintained protein having an intramolecular or intermolecular disulfide bond.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for synthesizing a protein having a disulfide bond within or between molecules.

Background Art

[0002] In the fields of disease research and drug discovery, etc., analysis of the interaction between biochemical substances and chemical substances is widely used as a very important approach for drug discovery research. In particular, protein-protein interaction is a general term for the interactions that occur between proteins in vivo. It is well known that the structural changes of proteins induced by this interaction are controlled by reactions and are involved in the control of the mechanisms underlying life such as signal transduction, transport, and metabolism. Such interactions have extremely diverse modes, and characteristics such as the softness of the action surface, the breadth, the length of the contact lifetime, and the presence or absence of structural changes vary widely depending on the protein species.

[0003] Proteins involved in the above protein-protein interactions include enzymes, antibodies, growth factors, membrane proteins, etc. Many of these proteins have disulfide bonds within or between molecules. Therefore, it is necessary to establish a method for synthesizing proteins that maintain the three-dimensional structure of these proteins and further have activity.

[0004] Patent Document 1 discloses "the multilayer method which is one of the methods for synthesizing proteins". Patent Document 2 discloses "a method for synthesizing a protein in which a disulfide bond within a protein molecule is retained". However, these documents do not disclose the optimal synthesis conditions of the multilayer method for synthesizing proteins having disulfide bonds within or between molecules.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

[0006] The objective is to provide a method for synthesizing proteins that have disulfide bonds within or between molecules. [Means for solving the problem]

[0007] The inventors of the present invention have completed the present invention by confirming that by comparing the supply phase with the reaction phase and subjecting it to oxidative conditions, it is possible to synthesize proteins that maintain activity and have intramolecular or intermolecular disulfide bonds. That is, the present invention is as follows.

[0008] 1. In a protein synthesis reaction method having intramolecular or intermolecular disulfide bonds, the reaction phase containing a synthesis reaction solution and the supply phase containing an energy source supply solution containing substrates and energy source molecules are brought into direct or indirect contact, and the substrates and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion across the contact interface between the two phases, A method for protein synthesis characterized in that the supply phase is under oxidative conditions compared to the reaction phase. 2. The protein synthesis method according to paragraph 1, characterized in that, under the aforementioned oxidation conditions, the redox potential of the supply phase is higher than that of the reaction phase. 3. The protein synthesis method according to item 1 or 2 above, wherein the oxidation-reduction potential of the reaction phase is -250 mV to -100 mV. 4. The protein synthesis method according to item 1 or 2 above, wherein the redox potential of the supply phase is -90 mV to 50 mV. 5. The protein synthesis method according to item 1 or 2 above, wherein the redox potential of the reaction phase is -250 mV to -100 mV, and the redox potential of the feed phase is -90 mV to 50 mV. 6. A protein synthesis method according to any one of items 1 to 5 above, characterized in that a reaction phase containing a synthesis reaction solution is brought into contact with a supply phase containing a substrate and an energy source supply solution containing an energy source molecule, and the substrate and energy source molecule from the supply phase are supplied to the reaction phase by free diffusion across the contact interface between the two phases. 7. A protein synthesis method according to any one of paragraphs 1 to 5 above, characterized in that a reaction phase containing a synthesis reaction solution, which is an intramembrane phase, and a supply phase containing an energy source supply solution containing a substrate and energy source molecules, which is an extramembrane phase, are brought into contact via a membrane, and the substrate and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion through the contact interface between the two phases. 8. A protein synthesis method according to any one of paragraphs 1 to 5 above, characterized in that, in a container having a membrane at its bottom, the supply phase is placed vertically above the reaction phase which is the in-membrane phase, and the container is immersed in an energy source supply solution which is the out-membrane phase, thereby supplying the substrate and energy source molecules of the supply phase to the reaction phase by free diffusion through the contact interface between the supply phase and the reaction phase and by free diffusion through the membrane between the reaction phase and the out-membrane phase. 9. Under the aforementioned oxidation conditions, the supply phase consists of oxidized glutathione, cystine, oxygen molecules, and NADP. + The method according to any one of items 1 to 8 of the preceding paragraph, characterized in that it is controlled by including and / or hydrogen peroxide. 10. The method according to any one of items 1 to 8 above, characterized in that the oxidation conditions are controlled by the supply phase not containing a reducing agent. 11. The method according to any one of items 1 to 10 above, characterized in that the reaction phase is under reducing conditions. 12. The method according to paragraph 11, characterized in that the reducing conditions are controlled by the reaction phase containing dithiothreitol, reduced glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, dithiobutylamine, cysteine, tris(2-carboxyethyl)phosphine, tributylphosphine, lipoic acid, NADPH, and / or NADH. 13. The method according to any one of items 1 to 12 above, characterized in that the reaction phase and / or the supply phase contain a PDI family protein including protein disulfide isomerase, BMC, aromatic thiol compounds, and / or an enzyme that reoxidizes PDI family proteins. 14. The method according to any one of items 1 to 13 above, characterized in that the chaperone is included in the reaction phase and / or the supply phase. 15. The method according to any one of paragraphs 1 to 14 above, characterized in that the protein is an enzyme, antibody, growth factor, or membrane protein. 16. The method according to any one of items 1 to 15 above, characterized in that the synthesis reaction solution is treated with a resin or column capable of chelating metal ions. [Effects of the Invention]

[0009] The synthesis method of the present invention can synthesize proteins that retain their activity, particularly enzymes that catalyze decarboxylation reactions, hydrolytic enzymes, and antibodies that retain antigen-binding ability. [Brief explanation of the drawing]

[0010] [Figure 1] The composition of the protein synthesis system and a schematic diagram of the layering method used in Example 1 are shown. [Figure 2] The results of the enzyme activity measurement in Example 1 are shown. The vertical axis of the graph shows the luciferase activity (relative luminescence intensity (%)) produced by Gaussia princeps. [Figure 3] The results of the enzyme activity measurement in Example 2 are shown. The vertical axis of the graph shows the human-derived acetylcholinesterase activity (Arbitrary Unit). [Figure 4] The results of the enzyme activity measurement in Example 3 are shown. The vertical axis of the graph shows tPA protease activity (Abs(405nm) / min / μg). [Figure 5]The results of confirming the ability of anti-AGIA-IgG Fab of Example 4 to bind to the AGIA sequence are shown. A shows the CBB staining, and B shows the results of Western blot. The vertical axis of the electrophoresis diagram indicates the molecular weight (kDa). [Figure 6] The measurement results of the enzyme activity of Example 5 are shown. A shows acetylcholinesterase, and B shows the results of the human tPA protease domain. The vertical axis of the graph indicates the relative value when the absorbance at a wavelength of 405 nm minus the negative control is set to 100% under the condition without nickel resin treatment. [Figure 7] The schematic diagram of the dialysis bilayer method is shown. [Figure 8] The comparison results of the addition conditions of the substance that catalyzes the disulfide bond exchange reaction and the catalyst that re-oxidizes the substance that catalyzes the disulfide bond exchange reaction are shown. The activities of tPApro synthesized under each condition are shown in a bar graph. From left to right, NC: negative control, - / -: no addition of PDI and Ero1α in both the supply phase and the reaction phase, + / -: addition of PDI and Ero1α only in the supply phase, - / +: addition of PDI and Ero1α only in the reaction phase, + / +: addition of PDI and Ero1α in both the supply phase and the reaction phase are shown. [Figure 9] The comparison results of the addition conditions of the oxidizing agent to the supply phase are shown. The activities of tPApro synthesized under each condition are shown in a bar graph. From left to right, NC: negative control, 0 - 五百: conditions with 0 μM, 20 μM, 100 μM, and 500 μM oxidized glutathione added are shown. [Figure 10] The conceptual diagram of the repeated batch method is shown. [Figure 11] The measurement results of the redox potential of the supply phase solution are shown. A shows the Nernst equation representing the relationship of factors related to the redox potential. B shows the time change of the measured redox potential under the solution conditions where DTT is added as a reducing agent or oxidized glutathione is added as an oxidizing agent at the concentrations indicated in the graph in the supply phase. C shows the average value in the time domain where the numerical values are stable under each solution condition, described as the measured value. [Figure 12]The measurement results of the redox potential of the reaction phase solution are shown. A shows the time change of the measured value of the redox potential under the solution conditions in which DTT is added at the concentration shown in the graph as a reducing agent in the reaction phase. The portion enclosed by the dotted line indicates the region from 1 to 5 minutes after the start of measurement. B shows the values obtained by averaging the observed values from 1 to 5 minutes after the start of measurement in each solution as the measured values. C shows the result of plotting the measured redox potential values of each solution against the natural logarithm value of the DTT concentration. The dotted line and the mathematical formula in the graph represent the regression line by the least squares method. [Figure 13] The measurement results of the redox potential of the solution obtained by diluting the reaction phase solution in Fig. 12 five-fold with a buffer solution (40 mM HEPES (pH 7.0) & 500 mM NaCl) are shown. A shows the time change of the measured value of the redox potential in each of the five-fold diluted solutions. B shows the average value in the time range where the values are stable in each solution as the measured value. C shows the result of plotting the measured redox potential values of each solution against the natural logarithm value of the DTT concentration. The dotted line and the mathematical formula in the graph represent the regression line by the least squares method. [Figure 14] The results of comparing the measured values of the redox potential of the reaction phase solution and the measured values of the redox potential of its five-fold diluted solution are shown. A shows the result of plotting the measured values of the redox potential of the reaction phase solution in Fig. 12 and the five-fold diluted solution in Fig. 13 against the natural logarithm value of the DTT concentration. The dotted line represents the regression line in Fig. 13. [Figure 15] The results of the redox potential in the supply phase and the reaction phase and the activity values of the target protein (tPApro or AChE) at that potential are shown.

Mode for Carrying Out the Invention

[0011] (Synthesis Method of the Present Invention) The present invention is a method for synthesizing proteins having intramolecular or intermolecular disulfide bonds, wherein a reaction phase containing a synthesis reaction solution and a supply phase containing an energy source supply solution containing a substrate and energy source molecules are brought into direct or indirect contact, and the substrate and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion across the contact interface between the two phases, and furthermore, the supply phase is characterized by being under oxidizing conditions compared to the reaction phase (hereinafter sometimes abbreviated as "the synthesis method of the present invention"). The synthesis method of the present invention includes at least a multilayer method, a dialysis method, a dialysis multilayer method, and a repeated batch method.

[0012] (Synthesis method utilizing the multilayer method of the present invention) In the conventional multilayer method {a cell-free protein synthesis method using a diffusion continuous batch method (see Patent Document 1)}, in which a synthesis reaction solution containing a biological extract (reaction phase) and a substrate and energy source supply solution (supply phase) are directly brought into contact, and substrate and energy source molecules from the supply phase are continuously supplied to the translation reaction system of the reaction phase by free diffusion across the contact interface between the two phases, while byproducts generated in the reaction phase are removed (by diffusion of byproducts into the supply phase), thereby extending the duration of the synthesis reaction and increasing the efficiency of the synthesis reaction, the reaction phase and the supply phase were in the same reduced state. However, in the synthesis method of the present invention, the supply phase is controlled under oxidative conditions compared to the reaction phase.

[0013] The interface between the two phases may be formed as a horizontal plane or as a vertical plane. To form the interface as a horizontal plane, for example, the reaction phase may be added to the reaction vessel first to form a lower layer, and then the feed phase may be gently overlaid on top of the reaction phase without disturbing the interface between the two phases (see Figure 1). The reaction vessel can be any shape and size that allows for a sufficient diffusion rate of the solute between the two phases; for example, a test tube or a multi-well microtiter plate can be used, but it is not limited to these. Furthermore, it is possible to form a vertical interface between the two phases by layering the synthesis reaction solution (reaction phase) and the energy source supply solution, and then centrifuging the reaction vessel containing these. A larger contact interface area between the two phases leads to a higher rate of mass exchange by diffusion and thus higher protein synthesis efficiency. Therefore, the optimal volume ratio of the supply phase to the reaction phase varies depending on the interface area of ​​the two phases. There are no particular restrictions on the volume ratio of the supply phase to the reaction phase, but for example, if the interface is circular and its diameter is 7 mm, a ratio of 1:4 to 1:8 is preferred, and 1:5 is more preferred.

[0014] (Synthesis method utilizing the dialysis method of the present invention) Conventional dialysis methods (see: Kikawa et al., 21st Annual Meeting of the Molecular Biology Society of Japan, WID6) are used to synthesize proteins using a dialysis membrane that allows mass transfer between the two phases, with the reaction solution as the intradialysis fluid (reaction phase), the energy source supply solution containing the substrate and energy source molecules as the extradialysis fluid (supply phase), and the reaction phase and supply phase being in equivalent reducing states. However, in the synthesis method of the present invention, the supply phase is controlled under oxidizing conditions compared to the reaction phase.

[0015] (Synthesis method using the dialysis multilayer method of the present invention) The conventional dialysis overlay method (see Takeda et al. ScientificReports 2015) is a diffusion-continuous batch cell-free protein synthesis method (see Figure 7) characterized by immersing a known dialysis cup (having a dialysis membrane at the bottom) in a supply phase, which is an extra-membrane phase containing an energy source supply solution with substrates and energy source molecules, and performing the conventional overlay method within the cup, thereby arranging the supply phase above and below the reaction phase, which allows for more efficient supply of substrates such as amino acids and release of reaction byproducts. In this method, the reaction phase and the supply phase were in equivalent reducing states. However, in the synthesis method of the present invention, the supply phase is controlled under oxidizing conditions compared to the reaction phase.

[0016] (Synthesis reaction solution) The "synthetic reaction solution constituting the reaction phase" in the synthesis method of the present invention includes a translation reaction system and a translation template for protein synthesis (e.g., mRNA) necessary for protein synthesis (particularly cell-free protein synthesis), and optionally includes liposomes and components used in conventionally known protein synthesis (particularly batch cell-free protein synthesis). Examples of components include amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for protein synthesis reactions. Specifically, when used in a reaction phase containing wheat germ extract, examples include creatine kinase, HEPES-KOH, ATP, GTP, creatine phosphate, spermidine, potassium acetate, magnesium acetate, 20 types of L-type amino acids, reducing agents, PDI family proteins including protein disulfide isomerase, BMC, aromatic thiol compounds, and / or enzymes and chaperones that reoxidize PDI family proteins. In addition, the protein synthesis reaction can be further stabilized by adding sugar alcohols such as inositol, xylitol, and / or ficol to the synthesis reaction solution to increase its viscosity and density, and by controlling the mixing rate between the two phases, the reaction phase and the feed phase.

[0017] (Energy source supply solution) In the synthesis method of the present invention, the "energy source supply solution containing substrates and energy source molecules constituting the supply phase" contains components that were consumed or deficient in the protein synthesis reaction in the reaction phase. Examples of components include substrates and energy source molecules such as cAMP, amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for protein synthesis reactions. Specifically, when used in a reaction phase containing wheat germ extract, examples include creatine kinase, HEPES-KOH, ATP, GTP, creatine phosphate, spermidine, potassium acetate, magnesium acetate, 20 types of L-type amino acids, reducing agents, PDI family proteins including protein disulfide isomerases, BMC, aromatic thiol compounds, and / or enzymes and chaperones that reoxidize PDI family proteins. In addition, the protein synthesis reaction can be further stabilized by adding sugar alcohols such as inositol, xylitol, and / or ficol to the energy source supply solution to increase the viscosity and density of the synthesis reaction solution, and by controlling the mixing rate between the two phases, the reaction phase and the supply phase.

[0018] (Synthesis method using the repeated batch method of the present invention) Conventional repeated batch methods are synthesis methods that make maximum use of the characteristics of the initial phase of a synthesis reaction, which has a high reaction rate, in general batch or diffusion continuous batch methods that use a template material as a raw material (see: WO2004 / 097014). More specifically, before or after the synthesis rate has decreased, or before or after the synthesis reaction has stopped, or during these processes, a dilution or concentration treatment is performed using an energy source supply solution (supply phase). Then, if the solution has been diluted, a concentration treatment is performed, and if the solution has been concentrated, a dilution treatment is performed again. These dilution and concentration treatments are repeated discontinuously, enabling the synthesis of large quantities of protein. In the synthesis method utilizing the repeated batch method of the present invention, an energy source supply solution (containing a reducing agent as needed), which is a supply phase in which the oxidation conditions are changed stepwise, is added to the reaction phase under reducing conditions. The reaction phase is characterized by gradually transitioning to oxidation conditions optimal for the synthesis and maintenance of proteins having intramolecular or intermolecular disulfide bonds, while maintaining the efficiency of protein synthesis.

[0019] The synthesis method using the repeated batch method of the present invention includes, for example, the following steps. 1) Proteins are synthesized in a reaction phase containing the synthesis reaction solution. 2) The reaction phase is diluted before or after the synthesis rate has decreased, before or after the synthesis reaction has stopped, or during these processes. 3) Following the dilution process, a concentration process is carried out. 4) The synthesis reaction is carried out using the concentrated reaction phase. or 1) Proteins are synthesized in a reaction phase containing the synthesis reaction solution. 2) The reaction phase is concentrated before or after the synthesis rate has decreased, before or after the synthesis reaction has stopped, or during these processes. 3) Following the concentration process, a dilution process is carried out. 4) The synthesis reaction is carried out using the diluted reaction phase. The concentration process may include a step to remove by-products from the reaction phase. In the dilution process, the energy source supply solution (containing a reducing agent as needed), which is the supply phase with gradually altered oxidation conditions, is added (replenished) to the reaction phase. You may repeat steps 1) to 4) above multiple times. More specifically, for example, the synthesis method using the repeated batch method of the present invention can employ the following conditions. Total response time: 24 hours Reaction phase: Contains 4 mM DTT, the reducing agent at the start of the reaction. Repeat batch conditions: Replace the energy source supply solution every 30 minutes. 0-3 hours: Add the energy source supply solution containing 3.5 mM DTT to the reaction phase. 3-6 hours: Add the energy source supply solution containing 3.0 mM DTT to the reaction phase. 6-9 hours: Add the energy source supply solution containing 2.5 mM DTT to the reaction phase. 9-12 hours: Add the energy source supply solution containing 2.0 mM DTT to the reaction phase. 12-15 hours: Add the energy source supply solution containing 1.5 mM DTT to the reaction phase. 15-18 hours: Add the energy source supply solution containing 1.0 mM DTT to the reaction phase. 18-21 hours: Add the energy source supply solution containing 0.5 mM DTT to the reaction phase. 21-24 hours: Add the energy source supply solution containing 0 mM DTT to the reaction phase.

[0020] (Translation response system) The "translation reaction system" in the synthesis method of the present invention can be any known cell-free or non-cell-free translation reaction system. For example, Escherichia coli, Bacillus subtilis, Sf9 insect cells, CHO cells, human cells, yeast, Brevibacillus, filamentous fungi (Aspergillus oryzae), tobacco BY-2 cells, or transient expression systems of plants such as Bethamiana tobacco, lettuce, tomato (fruit and leaves), rice, barley, Phalaenopsis orchid, and chili pepper can be used. More specifically, suitable examples of translation reaction systems using cell-free proteins include Escherichia coli, Escherichia coli reconstitutes, wheat, insects, yeast, tobacco, rabbit reticulocytes, and human cells. For the purpose of comprehensively obtaining a wide variety of proteins, the wheat cell-free system is particularly excellent, offering an extremely high probability of synthesizing proteins in a soluble state and being cost-effective.

[0021] The synthesis reaction solution, which includes the translation reaction system (particularly wheat germ extract) used in the synthesis method of the present invention, can improve the activity of the synthesized protein by treating it with a resin or column capable of chelating metal ions (e.g., Ni, Co, Cu, Fe, Ga, Al, Zr, Zn, particularly Ni) before protein synthesis, as shown in the results of Example 5.

[0022] (Translation template for protein synthesis) In the synthesis method of the present invention, the "translational template for protein synthesis" can be mRNA encoding the protein to be expressed. Furthermore, the mRNA can be used not only in a purified state, such as by ethanol precipitation, but also in an unpurified state.

[0023] (Synthesis reaction conditions) The synthesis reaction conditions for the synthesis method of the present invention can be those of the conventional multilayer method, dialysis method, and dialysis multilayer method. For example, the protein synthesis reaction is preferably carried out under static conditions, and the reaction temperature is 10°C to 35°C, which is the optimal temperature usually used in cell-free protein synthesis. Since the reaction phase, the feed phase, and the mixed phase formed when both phases are mixed during the synthesis process contain the target protein, the target protein can be easily obtained from these phases by known separation and purification methods such as dialysis, ion exchange chromatography, affinity chromatography, and gel filtration.

[0024] (Proteins that have disulfide bonds within or between molecules) The "protein having intramolecular or intermolecular disulfide bonds" in the synthesis method of the present invention is not particularly limited as long as it is a protein having intramolecular and / or intermolecular disulfide bonds. Examples include enzymes, antibodies, growth factors, and membrane proteins.

[0025] (Control of supply phase under oxidative conditions) The correctness of the oxidation-reduction potential can be determined by whether it conforms to Nernst's formula. For example, the measurement can be performed and determined using the following procedure. In the supply phase or reaction phase solution, a series of dilution solutions are prepared by systematically varying only the concentration of the reducing agent, while keeping the concentrations of other solution components constant. The oxidation-reduction potential measurements of these dilution series solutions confirm the following two points, based on the requirements of Nernst's equation: 1) For solutions in an equal dilution series (e.g., a 2x dilution series), the oxidation-reduction potential measurements show values ​​that are approximately equally spaced. 2) When plotted against the logarithm of the reducing agent concentration, the oxidation-reduction potential measurements show a downward-sloping linearity. However, if the reducing agent (or oxidizing agent) concentration is 0 mM, Nernst's equation breaks down, so a value that remains stable for a certain period of time is adopted as the redox potential value. The oxidation-reduction potential of the feed phase solution can be measured using known equipment (e.g., electrodes: ORP electrode 9300-10D (HORIBA), pH meter F-52 (HORIBA)) in accordance with the instructions in their manuals. For example, as shown in Figure 11, a series of feed phase solutions with varying reducing agent concentrations are prepared in approximately 1 ml each. After keeping them warm at 23±1°C, the potential measuring electrode is immersed in the solution for a certain period of time (e.g., 50-90 minutes). The average value over the stable time range is then read as the measured oxidation-reduction potential of the solution. The obtained measurement is then compared with a reference to confirm that it conforms to Nernst's formula. As is clear from the results of Examples 1 to 4 below, the synthesis method of the present invention allows for the synthesis of proteins that retain their activity by controlling (setting) the supply phase to be under oxidative conditions compared to the reaction phase. One example of the supply phase being under oxidative conditions compared to the reaction phase is that the redox potential of the supply phase is higher than that of the reaction phase. For example, when DTT is used alone as a reducing agent, the final concentration of the reaction phase is typically 0.0 mM to 0.5 mM (see Figure 15), and when GSSG is used alone as an oxidizing agent, the final concentration of the reaction phase is typically 0.02 mM to 0.5 mM (see Figure 9). Furthermore, for example, the redox potential of the supply phase (preferably the redox potential of the pre-synthesis stage (synthesis solution preparation stage)) is exemplified as being -90 mV to 50 mV, preferably -50 mV to 40 mV, more preferably -30 mV to 30 mV, and most preferably -10 mV to 20 mV (see Examples 8 and 9). In the synthesis method of the present invention, "controlling the supply phase under oxidative conditions" can be achieved by any method that allows the redox potential of the supply phase to be set and controlled to be higher than that of the reaction phase. For example, known oxidizing agents include oxidized glutathione, cystine, oxygen molecules, and NADP. + This can be achieved by including hydrogen peroxide or the like in the supply phase. Unlike conventional synthesis methods, the synthesis method of the present invention can also be achieved by not including a reducing agent in the supply phase. In addition, the synthesis method of the present invention can improve the activity of the synthesized protein by including an oxidizing agent in the supply phase, as described in the results of Example 7 below.

[0026] (Control of the reaction phase under reducing conditions) In the synthesis method of the present invention, "controlling the reaction phase under reducing conditions" can be achieved by any method that allows the oxidation-reduction potential of the reaction phase to be set and controlled to be lower than that of the supply phase. For example, this can be achieved by including known reducing agents such as dithiothreitol (DTT), reduced glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, dithiobutylamine, cysteine, tris(2-carboxyethyl)phosphine, tributylphosphine, lipoic acid, NADPH, NADH, etc., in the reaction phase. Measuring the redox potential of the reaction phase solution can be difficult because the potential response may differ from that of the supply phase due to the influence of cell extracts, making it challenging to observe a stable redox potential value. In such cases, the redox potential of the reaction phase solution can be measured using any of the redox potential measurement methods described in Example 8. For example, when DTT is used alone as a reducing agent, the final concentration of the reaction phase may be 0.2 mM to 40 mM, preferably 0.2 mM to 10 mM. Furthermore, the oxidation-reduction potential of the reaction phase (preferably the oxidation-reduction potential of the pre-synthesis stage (synthesis solution preparation stage)) is preferably in the range of -500 mV to -100 mV, more preferably -250 mV to -100 mV, more preferably -230 mV to -100 mV, even more preferably -210 mV to -100 mV, and most preferably -195 mV to -100 mV (see Examples 8 and 9).

[0027] (Substances that catalyze disulfide bond exchange reactions) In the synthesis method of the present invention, it is preferable to include a substance that catalyzes the disulfide bond exchange reaction, and further, a catalyst that reoxidizes the catalyst, in the reaction phase and / or feed phase. In addition, as described in the results of Example 6 below, it is particularly preferable that the synthesis method of the present invention includes a substance that catalyzes the disulfide bond exchange reaction, and further, a catalyst that reoxidizes the catalyst, in the reaction phase. This makes it possible to synthesize proteins in which intramolecular disulfide bonds are correctly formed (maintained) with higher efficiency. Examples of substances that catalyze disulfide bond exchange reactions include protein disulfide isomerase (PDI), an enzyme present in the endoplasmic reticulum of eukaryotic cells; GroEL and GroES, chaperone proteins derived from E. coli; and various proteins that catalyze refolding reactions, such as DnaK, DnaJ, and GrpE, as well as their small-molecule mimics (for example, BMC, a mimic of PDI (Chem Biol., 6, 871-879, 1999), and aromatic thiol compounds (4-mercaptobenzene acetate; J.Am.Chem.Soc.124, 3885-3892, 2002). Examples of catalysts for reoxidizing substances that catalyze disulfide bond exchange reactions include reoxidizing enzymes such as ER oxidase (hereinafter sometimes referred to as Ero1α or Ero1β), peroxiredoxin IV, glutathione peroxidase 7 and 8, and vitamin K epoxydriductase, all of which are present in the endoplasmic reticulum of eukaryotic cells.

[0028] (Proteins involved in the folding reaction of polypeptide chains) In the synthesis method of the present invention, it is preferable to include a protein involved in the polypeptide chain folding reaction in the reaction phase and / or supply phase, if necessary. This allows for the more efficient synthesis of proteins in which the intramolecular polypeptide chain folding is correctly formed (maintained). Examples of proteins involved in polypeptide chain folding include the HSP70 family chaperones (e.g., BIP(GRP78)), HSP90 family chaperones (e.g., GRP94), nucleotide exchange factors (e.g., BAP(SIL1), GRP170), DnaJ family chaperones (e.g., ERdj3, ERdj6), and peptidyl prolyl cis-trans isomerases (e.g., cyclophyllin B), all of which are present in eukaryotic cells and involved in the folding of newly synthesized polypeptide chains.

[0029] (Method for measuring the redox potential of the supply phase and reaction phase) 〇 Oxidation-reduction potential measurement method 1 The following are examples of methods for measuring the redox potentials of the supply phase and the reaction phase, but are not particularly limited. (1) Prepare the supply phase solution and the reaction phase solution. (2) Prepare a series of diluted solutions of the supply phase and a series of diluted solutions of the reaction phase, each with a different reducing agent concentration. (3) For each diluted solution, measure the oxidation-reduction potential for a certain period of time (e.g., 1 to 2 hours) using a known apparatus. Identify the time range (for example, 1 to 5 minutes from the start of measurement) that shows the redox potential value according to Nernst's formula (Figure 11A), and adopt the average value of that time range as the redox potential value (see Figures 11B, C, and 12). Method 2 for measuring oxidation-reduction potential In the measurement method 1 described above, if it is difficult to measure an accurate (stable) oxidation-reduction potential, the solution is diluted and the oxidation-reduction potential is measured. The regression line for the diluted series of solutions is used as a calibration curve, and the oxidation-reduction potential value at the reducing agent concentration of the reaction phase solution before dilution is determined by extrapolating it. For details, a series of reaction phase solutions with varying reducing agent concentrations are prepared by diluting them with an appropriate buffer solution (for example, a 5-fold dilution). It is preferable to confirm in advance that the buffer solution used for dilution has a composition that allows for stable measurement of the oxidation-reduction potential. Measurements are taken for each diluted solution over a set period of time (e.g., 60-90 minutes). The average value within the stable time range is read as the oxidation-reduction potential value, and it is compared with a reference to confirm that it conforms to Nernst's formula. By creating a regression line for these measured values ​​and using it as a calibration curve for extrapolation, the oxidation-reduction potential value at the reducing agent concentration of the reaction phase solution before dilution is determined.

[0030] The present invention will be described in more detail below with reference to experimental examples. However, these experimental examples should be considered merely as aids in gaining a concrete understanding of the present invention, and the scope of the present invention is not limited in any way by these experimental examples. [Examples]

[0031] (Synthesis of luciferase from Gaussia princeps by a multilayer synthesis method using PDI and Ero1α) The luciferase produced by the marine plankton Gaussia princeps (hereinafter referred to as "GLuc") consists of 168 amino acid residues (molecular weight 18170 kDa), excluding the signal sequence, and is an enzyme that catalyzes the reaction in which the luminescent substrate Coelenterazine reacts with oxygen molecules to produce photons. Since the formation of the three-dimensional structure of this enzyme requires the formation of five disulfide bonds, it was used in this example.

[0032] (1) Production of DNA encoding GLuc We commissioned GENEWIZ to produce it through gene synthesis.

[0033] (2)mRNA synthesis mRNA synthesis was performed on the template DNA obtained in (1) above using SP6 RNA polymerase (Promega) at a reaction temperature of 37°C for 6 hours. The reaction mixture (in aqueous solution) consisted of 80 mM HEPES-KOH (pH 7.6), 8 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, 2.5 mM NTPs each, 1.0 U / μl RNase inhibitor, 1.0 U / μl SP6 RNA polymerase, and 100 ng / μl plasmid.

[0034] (3) Protein synthesis (see Figure 1) Protein synthesis was carried out using the mRNA synthesis solution prepared in (2) above as a translation template and 10 μl of wheat germ extract. The reaction phase was composed of 10 μl of mRNA solution, 33 ng / μl creatine kinase, 30 mM HEPES-KOH (pH 7.6), 1.2 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate, 0.4 mM spermidine, 100 mM potassium acetate, 2.7 mM magnesium acetate, 0.3 mM L-type amino acids (20 types), and 4 mM DTT, added to the reaction phase. Furthermore, two different reaction phases were prepared by adding 10 μM PDI and 2 μM Ero1α or by adding them. The volume of the reaction phase was adjusted to 30 μl using the supply phase without DTT as described below. The supply phase was 200 μl per 30 μl of reaction phase. The supply phase contained 30 mM HEPES-KOH, 1.2 mM ATP, 1.0 mM cAMP, 16 mM creatine phosphate, 0.3 mM L-type amino acids (20 types), 2.7 mM magnesium acetate, 100 mM potassium acetate, and 0.4 mM spermidine. Furthermore, two different supply phases were prepared by adding or removing 4 mM DTT. As described above, the synthetic reaction system, consisting of a reaction phase and a feed phase, was prepared under four different conditions by combining the reaction phase (with or without PDI and Ero1α addition) and the feed phase (with or without 4 mM DTT addition) under two different conditions. The procedure for constructing the synthetic reaction system was as follows: first, 200 μl of the feed phase was added to the wells of a 96-well plate, and then 30 μl of the reaction phase was gently added to the bottom of the wells. The 96-well plate was placed in an incubator set to 16°C, and the synthesis reaction was carried out for 20 to 24 hours. After the synthesis reaction, the reaction phase and feed phase were uniformly mixed using a pipette, and then separated into soluble and insoluble fractions by centrifugation (21000 xg, 10 minutes).

[0035] (4)Activity measurement The activity of GLuc synthesized in (3) above was measured using the Pierce Gaussia Luciferase Glow Assay Kit (Thermo Fisher Scientific). The soluble fraction from (3) above was diluted 100-fold with the buffer provided in the kit. For the measurement, 30 μl of the diluted soluble fraction, 50 μl of the substrate solution (luminescent substrate Coelenterazine diluted 330-fold with the buffer provided in the kit), and 20 μl of HEPES buffer (50 mM HEHEP-NaOH (pH 7.0), 150 mM NaCl) were added to the wells of a 96-well plate and mixed. The luminescence intensity was immediately measured using a multi-label counter ARVO-MX (PerkinElmer). The measured luminescence intensity was converted to a value per 1 μg of luciferase contained in the soluble fraction, and then converted to a relative value (%). The results are shown in Figure 2. The bar graph in Figure 2 shows the activity measurement results of GLuc synthesized under four different synthesis reaction conditions. The two left columns show the results with a feed phase DTT concentration of 4 mM and with or without the addition of PDI and Ero1α, while the two right columns show the results with a feed phase DTT concentration of 0 mM and with or without the addition of PDI and Ero1α. As is clear from the comparison of these four conditions, when the feed phase DTT concentration was the conventional 4 mM (reducing conditions), GLuc retaining its activity could not be obtained even with the addition of PDI and Ero1α. On the other hand, under the condition where the feed phase DTT concentration was 0 mM (oxidizing conditions), GLuc retaining its activity was obtained. Furthermore, under the condition with the addition of PDI and Ero1α, the activity increased by about 30%. Based on the above, we were able to synthesize an active protein by subjecting the supply phase to oxidative conditions. Furthermore, by adding PDI and Ero1α to the reaction phase, we were able to enhance the activity by approximately 30%. [Examples]

[0036] (Synthesis of human-derived acetylcholinesterase by a multilayer synthesis method using PDI and Ero1α) Human acetylcholinesterase (hereinafter referred to as "AChE") consists of 583 amino acid residues (molecular weight 64576 kDa), excluding the signal sequence, and is an enzyme that hydrolyzes the neurotransmitter acetylcholine. Since its three-dimensional structure requires the formation of four disulfide bonds, it was used in this example.

[0037] (1) Production of DNA encoding AChE The DNA was amplified using a standard PCR method and then introduced into a circular plasmid to create the plasmid.

[0038] (2)mRNA synthesis The procedure was carried out in the same manner as in Example 1 above.

[0039] (3) Protein synthesis The procedure was carried out in the same manner as in Example 1 above.

[0040] (4)Activity measurement The activity of AChE synthesized in (3) above was measured using the Acetylcholinesterase Activity Assay Kit (Sigma-Aldrich). The soluble fraction from (3) above was diluted 100-fold with the buffer provided in the kit. For the measurement, 5 μl of the diluted soluble fraction and 95 μl of the substrate solution (10 μg / μl, dissolved in the buffer provided in the kit) were added to the wells of a 96-well plate and mixed. The absorbance at a wavelength of 405 nm was measured for 2 hours using a multi-label counter ARVO-MX (PerkinElmer). The difference in absorbance between 2 hours and 0 hours was divided by the difference in absorbance between the calibrator provided in the kit and ultrapure water (unit: "Arbitrary Unit") and used for activity comparison. The results are shown in Figure 3. The bar graph in Figure 3 shows the activity measurement results of AChE synthesized under four different synthesis reaction conditions. The two left columns show the results with and without PDI and Ero1α added when the supply phase DTT concentration was 4 mM, while the two right columns show the results with and without PDI and Ero1α added when the supply phase DTT concentration was 0 mM. As is clear from the comparison of these four conditions, when the supply phase DTT concentration was the conventional 4 mM (reducing conditions), AChE with retained activity could not be obtained even with the addition of PDI and Ero1α. On the other hand, under the condition where the supply phase DTT concentration was 0 mM (oxidizing conditions), AChE with retained activity was obtained. Furthermore, under the condition with the addition of PDI and Ero1α, the activity increased by about three times. Based on the above, we were able to synthesize a hydrolytic enzyme that retained its activity by subjecting the supply phase to oxidative conditions. Furthermore, by adding PDI and Ero1α to the reaction phase, we were able to enhance the activity by approximately three times. [Examples]

[0041] (Synthesis of protease domains of human-derived tissue-type plasminogen activators by a multilayer synthesis method using PDI and Ero1α) Tissue-type plasminogen activators are proteases that hydrolyze specific sequences of plasminogen to convert them into plasmin, and are used as drugs to dissolve blood clots that cause conditions such as cerebral infarction. The fragment containing the protease domain (amino acid residues 297-562, molecular weight 29774 kDa) (hereinafter referred to as "tPApro") was used in this example because it requires the formation of six disulfide bonds to form its three-dimensional structure.

[0042] (1) Production of DNA encoding tPApro The procedure was carried out in the same manner as in Example 2.

[0043] (2)mRNA synthesis The procedure was carried out in the same manner as in Example 1 above.

[0044] (3) Protein synthesis The procedure was carried out in the same manner as in Example 1 above.

[0045] (4)Activity measurement The reaction substrate used was Chromozym t-PA (Roche), which was dissolved in buffer (50 mM Tris-HCl (pH 8.0), 0.075% Tween 80) to a final concentration of 0.25 mM. 97 μl of this reaction substrate solution and 3 μl of the soluble fraction from (3) above were quickly mixed using a pipette, and the absorbance at 405 nm was immediately measured for 30 minutes using a spectrophotometer (Beckmann). The slope of the reaction curve obtained from the measurement (Abs(405nm) / min) was determined, and the value converted to per 1 μg of tPApro contained in the soluble fraction was used as the activity, and was used for activity comparison. These results are shown in Figure 4. The bar graph in Figure 4 shows the activity measurement results of tPApro synthesized under four different synthesis reaction conditions. The two bars on the left show the results with a feed phase DTT concentration of 4 mM and with or without the addition of PDI and Ero1α, while the two bars on the right show the results with a feed phase DTT concentration of 0 mM and with or without the addition of PDI and Ero1α. As is clear from the comparison of these four conditions, when the feed phase DTT concentration was the conventional 4 mM (reducing conditions), tPApro retaining its activity could not be obtained even with the addition of PDI and Ero1α. On the other hand, under the condition where the feed phase DTT concentration was 0 mM (oxidizing conditions), tPApro retaining its activity was obtained. Furthermore, under the condition in which PDI and Ero1α were added, the activity increased threefold. Based on the above, we were able to synthesize a hydrolytic enzyme that retained its activity by subjecting the supply phase to oxidative conditions. Furthermore, by adding PDI and Ero1α to the reaction phase, we were able to increase the activity threefold. [Examples]

[0046] (Synthesis of anti-AGIA-IgG antibody Fab fragments by a multilayer synthesis method using PDI and Ero1α) Anti-AGIA-IgG antibodies are monoclonal antibodies that specifically recognize the AGIA sequence (EEAAGIARP) as an antigen (Yano, T., et al., PLoS One, 11(6), e0156716 (2016)). The Fab fragment containing the antigen-binding site of this antibody (hereinafter referred to as "anti-AGIA-IgG Fab") is composed of an H chain and a L chain, each requiring three disulfide bonds to form its three-dimensional structure. Furthermore, the H chain and L chain are linked by one disulfide bond, and a total of seven disulfide bonds are required to form the Fab fragment.

[0047] (1) Preparation of DNA encoding the H chain and L chain of anti-AGIA-IgG Fab. The DNA encoding the H chain and L chain that make up the Fab fragment was prepared in the same manner as in Example 2.

[0048] (2)mRNA synthesis The mRNA synthesis of the H chain and L chain was carried out in the same manner as in Example 1 above.

[0049] (3) Protein synthesis 5 μl each of H-chain and L-chain mRNA solutions (10 μl total) were used. Furthermore, there are reports that adding peptidyl prolyl cis-trans isomerase is effective in the unwinding reaction of IgG antibody Fab fragments (Lilie, Hauke, et al., Protein Sci., 2, 1490-1496 (1993)). Therefore, we investigated whether the addition or absence of cyclophyllin B (hereinafter referred to as "CypB"), a type of cyclophyllin, to the reaction phase has an effect on the formation of the three-dimensional structure of the newly synthesized polypeptide chain of anti-AGIA-IgG Fab, along with the presence or absence of PDI and Ero1α addition. On the other hand, only 0 mM DTT conditions were used for the supply phase. Thus, four different conditions for the synthesis reaction system were prepared, and anti-AGIA-IgG Fab was synthesized. The other conditions were carried out in the same manner as in Example 1 above.

[0050] (4) Antigen binding experiment (pull-down assay) The binding experiment of the anti-AGIA-IgG Fab synthesized in (3) above to the AGIA sequence was performed using the pull-down assay method. The AGIA sequence used was one that had been pre-purified and fused with a GST tag at its N-terminus (hereinafter referred to as "GST-AGIA"). The GST tag alone used as the negative control (hereinafter referred to as "GST") was also pre-purified. The full-length anti-AGIA-IgG used as the positive control (hereinafter referred to as "anti-AGIA-IgG") was provided by Associate Professor Takeda (Ehime University). In the pull-down assay, GST-AGIA or GST was mixed with glutathione resin in Tris buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl and 0.1% Tween 80) (4°C, 30 minutes). Then, anti-AGIA-IgGFab synthesis reaction solution or anti-AGIA-IgG was added to each mixture and mixed for a further 1 hour. The recovered glutathione resin was washed three times with Tris buffer. Proteins bound to the resin were eluted with SDS sample buffer without a reducing agent and then subjected to SDS-PAGE. Detection of bound anti-AGIA-IgG Fab was performed by CBB staining or Western blotting. For detection by Western blotting, the antibody "Anti-His-tag mAb-HRP-DirecT (MBL)" (indicated as "αHis-Ab-HRP" in Figure 5) that recognizes the His tag was used against the His tag attached to the C-terminus of the H chain. These results are shown in Figure 5. Figure 5 shows the results of a pull-down assay confirming the AGIA sequence binding ability of anti-AGIA-IgG Fab synthesized under each condition. Anti-AGIA-IgG Fab synthesized under conditions with PDI and Ero1α added (GST-AGIA Lane 2 and 4 in Figure 5A) bound to the AGIA sequence similarly to anti-AGIA-IgG (GST-AGIA Lane 5 in Figure 5A). However, anti-AGIA-IgG Fab synthesized without the addition of PDI and Ero1α (GST-AGIA Lane 1 and 3 in Figure 5A) did not. This result was also confirmed by Western blotting (GST-AGIA Lane 2 and 4 in Figure 5B). On the other hand, the addition of CypB had no effect (GST-AGIA Lane 1 and 3 and Lane 2 and 4 in Figures 5A and 5B). Based on the above, we were able to synthesize antibodies that retained antigen-binding ability by adding PDI and Ero1α to the reaction phase and placing the supply phase under oxidative conditions. [Examples]

[0051] (Characteristics of wheat germ extract treated with nickel resin) Acetylcholinesterase was synthesized in the same manner as in Example 2, except that wheat germ extract treated with nickel resin (Ni Sepharose High Performance, GE Healthcare) was used, and its activity was further measured. Furthermore, human tPA protease domains were synthesized using the same method as in Example 3, except that wheat germ extract treated with nickel resin (Ni Sepharose High Performance, GE Healthcare) was used, and their activity was measured. As shown in Figure 6, by using wheat germ extract treated with a resin or column (particularly nickel resin or column) that can chelate metal ions, proteins that retain disulfide bonds can be obtained in a more active state. [Examples]

[0052] (Confirmation of the conditions for adding the substance that catalyzes the disulfide bond exchange reaction and the catalyst that reoxidizes the substance that catalyzes the disulfide bond exchange reaction) There are four possible conditions for adding the substance that catalyzes the disulfide bond exchange reaction (e.g., 10 μM PDI) and the catalyst that reoxidizes the substance that catalyzes the disulfide bond exchange reaction (e.g., 2 μM Ero1α), by adding them to the supply phase and / or the reaction phase. Therefore, tPApro was synthesized under these four different conditions, based on the conditions described in Example 3. More specifically, the procedure was the same as in the previous experiment, except for the addition conditions of PDI and Ero1α. After synthesis, each sample was separated into soluble and insoluble fractions by centrifugation. The soluble fraction was used for activity measurement. Activity measurement was performed by mixing the soluble fraction of each sample with the necessary reagents and immediately starting the measurement using ARVO-MX (PerkinElmer). Activity comparison was performed using the absorbance value at the time when the difference between each reaction curve was widest. As shown in Figure 8, it was confirmed that the activity of the substance catalyzing the disulfide bond exchange reaction and the catalyst for reoxidizing the catalyst is improved by including them in the supply phase and / or the reaction phase, but it was confirmed that it is more preferable to include them only in the reaction phase. [Examples]

[0053] (Confirmation of conditions for adding oxidizing agents to the supply phase) To actively maintain the supply phase under oxidative conditions, the concentration of the oxidizing agent (oxidized glutathione) added to the supply phase was varied to confirm its effect. The synthesis conditions were the same as in Example 3, except for the addition of oxidized glutathione to the supply phase. After synthesis, each sample was separated into soluble and insoluble fractions by centrifugation. The soluble fraction was used for activity measurement. Activity measurement was performed by mixing the soluble fraction of each sample with the necessary reagents and immediately starting the measurement using ARVO-MX (PerkinElmer). Activity comparison was performed using the absorbance value at the time when the difference between each reaction curve was widest. The results shown in Figure 9 confirm that including an oxidizing agent in the supply phase can improve the activity of the synthesized protein. [Examples]

[0054] (Measurement of redox potential of reaction phase and feed phase) The correct measurement of the redox potential can be determined by its adherence to Nernst's equation (Figure 11A). In this example, it was confirmed that the redox potentials of the reaction phase and the feed phase can be measured by following the procedure below.

[0055] 〇Method 1 for measuring oxidation-reduction potential (See Figures 11B, 11C, and 12) The procedure for this method is shown below. A series of diluted solutions were prepared by varying only the concentration of the reducing agent. Measurements were taken for each solution for a set period of time (e.g., 1-2 hours). We confirmed that the time range in which the redox potential value follows Nernst's formula (for example, 1 to 5 minutes from the start of measurement) can be identified, and that the average value of that time range can be adopted as the redox potential value.

[0056] ○ Redox potential measurement method 2 (shown in Figures 13 and 14) Method 1 is a measurement method for when it is difficult to accurately (stable) measure the oxidation-reduction potential. The basis for the effectiveness of this method is that, as shown in Figure 14A, the measured oxidation-reduction potentials of the reaction phase solution and its five-fold diluted solution lie on the same straight line. When the oxidation-reduction potential measurement of the reaction phase solution is affected by the type of cell extract used in the reaction phase, diluting the solution minimizes this effect, making oxidation-reduction potential measurement possible. Furthermore, as shown in Figure 14A, since the oxidation-reduction potential values ​​of the solutions before and after dilution lie on the same straight line, the regression line for the dilution series of solutions can be used as a calibration curve, and by extrapolating it, the oxidation-reduction potential value of the reaction phase solution at the reducing agent concentration before dilution can be determined. The results are shown in Figure 14B. The measured and extrapolated values ​​for the undiluted reaction phase solution show good agreement.

[0057] The procedure for this method is shown below. A series of reaction phase solutions, each varying only in reducing agent concentration, were prepared by diluting (e.g., 5-fold) them with a suitable buffer solution (e.g., 40 mM HEPES (pH 7.0) & 500 mM NaCl). The buffer solutions used for dilution were pre-examined to ensure they had a composition that allowed for stable measurement of the redox potential. Measurements were taken for each diluted solution over a set period of time (e.g., 60-90 minutes). The average value within the time range where the numerical value stabilized was read as the oxidation-reduction potential value, and it was confirmed that it conformed to Nernst's formula by comparing it with the above criteria. • By creating regression lines for these measured values ​​and using them as calibration curves for extrapolation, the redox potential values ​​at the reducing agent concentration of the reaction phase solution before dilution were determined.

[0058] The following shows the results of calculating the oxidation-reduction potentials of the supply phase and reaction phase used in Examples 1-4, 6, and 7 before synthesis, using any one of the above oxidation-reduction potential measurements. For calculating the redox potential of the reaction phase, the regression lines in Figure 12 were used for "1)" and Figure 13 for "2)". As is clear from the results in the table below, it was confirmed that there was no significant difference between the redox potential values ​​obtained from the two measurement methods.

[0059] [Table 1] [Examples]

[0060] (Measurement of redox potential and activity level of the target protein in the supply and reaction phases) The redox potentials in the supply phase and reaction phase, as well as the activity levels of the target protein at those potentials, were measured. Method for measuring tPApro activity: The DNA used was the same as in Example 3, and mRNA synthesis and protein synthesis were carried out in the same manner as in Example 3. The reaction substrate used was Chromozym t-PA (Roche), dissolved in buffer (50 mM Tris-HCl (pH 8.0), 0.075% Tween 80) to a final concentration of 0.25 mM. For activity measurement, 97 μl of this reaction substrate solution and 3 μl of the soluble fraction of the protein synthesis reaction mixture were added to the wells of a 96-well plate and quickly mixed with a pipette. Immediately afterward, the absorbance at a wavelength of 405 nm was measured using a multi-label counter ARVO-MX (PerkinElmer). The activity value was defined as the absorbance at 20 minutes after the start of measurement, minus the absorbance of the negative control (protein synthesis reaction mixture identical in all respects except that it did not express tPApro) (unit: OD (405 nm)). Method for measuring AChE activity: The DNA used was the same as in Example 2, and mRNA synthesis and protein synthesis were carried out in the same manner as in Example 2. Activity measurements were performed using the Acetylcholinesterase Activity Assay Kit (Sigma-Aldrich). 3 μl of the soluble fraction of the protein synthesis reaction mixture and 97 μl of the reaction substrate solution (10 μg / μl, dissolved in the kit's buffer) were added to the wells of a 96-well plate and quickly mixed with a pipette. Immediately afterward, the absorbance at 405 nm was measured using a multi-label counter ARVO-MX (PerkinElmer). The reaction substrate used in this activity measurement reacts with residual reducing agent, affecting the absorbance. Therefore, the measured absorbance was corrected so that the value at the start of the reaction was zero. Then, the activity value was calculated by subtracting the absorbance of the negative control (protein synthesis reaction mixture identical in all respects except for not expressing AChE) at 20 minutes after the start of measurement (unit: OD (405 nm)).

[0061] Figure 15 shows the results of measuring the activity level of the target protein. We confirmed that the target protein with activity can be synthesized when the redox potential of the supply phase is in the range of -90 mV to 50 mV and the redox potential of the reaction phase is in the range of -250 mV to -100 mV. [Industrial applicability]

[0062] The protein synthesis method of the present invention can provide proteins that retain their activity, particularly enzymes that catalyze decarboxylation reactions, hydrolytic enzymes, and antibodies that retain antigen-binding ability.

Claims

1. In a protein synthesis reaction method having intramolecular or intermolecular disulfide bonds, a reaction phase containing a synthesis reaction solution including a translation reaction system and a translation template is brought into direct or indirect contact with a supply phase containing an energy source supply solution including a substrate and energy source molecules, and the substrate and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion across the contact interface between the two phases, A method for protein synthesis characterized in that the redox potential of the reaction phase is -195 mV to -100 mV, and the redox potential of the supply phase is -90 mV to 20 mV.

2. The protein synthesis method according to claim 1, characterized in that a reaction phase containing a synthesis reaction solution and a supply phase containing an energy source supply solution containing a substrate and energy source molecules are brought into direct contact, and the substrate and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion through the contact interface between the two phases.

3. The protein synthesis method according to claim 1, characterized in that a reaction phase containing a synthesis reaction solution, which is an intramembrane phase, and a supply phase containing an energy source supply solution containing a substrate and energy source molecules, which is an extramembrane phase, are brought into contact via a membrane, and the substrate and energy source molecules of the supply phase are supplied to the reaction phase by free diffusion through the contact interface between the two phases.

4. The protein synthesis method according to claim 1, characterized in that, in a container having a membrane at its bottom, the supply phase is placed vertically above the reaction phase which is the membrane-internal phase, and the container is immersed in an energy source supply solution which is the membrane-external phase, thereby supplying the substrate and energy source molecules of the supply phase to the reaction phase by free diffusion through the contact interface between the supply phase and the reaction phase and by free diffusion through the membrane between the reaction phase and the membrane-external phase.

5. The supply phase consists of oxidized glutathione, cystine, oxygen molecules, and NADP. + The method according to any one of claims 1 to 4, characterized in that the oxidation-reduction potential is controlled by and / or by containing hydrogen peroxide.

6. The method according to any one of claims 1 to 5, characterized in that the oxidation-reduction potential is controlled by the supply phase not containing a reducing agent.

7. The method according to any one of claims 1 to 6, characterized in that the reaction phase is under reducing conditions.

8. The method according to claim 7, characterized in that the reduction conditions are controlled by the reaction phase containing dithiothreitol, reduced glutathione, 2-mercaptoethanol, 2-mercaptoethylamine, dithiobutylamine, cysteine, tris(2-carboxyethyl)phosphine, tributylphosphine, lipoic acid, NADPH, and / or NADH.

9. The method according to any one of claims 1 to 8, characterized in that the reaction phase and / or the feed phase include a PDI family protein containing protein disulfide isomerase, BMC, aromatic thiol compounds, and / or an enzyme that reoxidizes PDI family proteins.

10. The method according to any one of claims 1 to 9, characterized in that the chaperone is included in the reaction phase and / or the supply phase.

11. The method according to any one of claims 1 to 10, characterized in that the protein is an enzyme, antibody, growth factor, or membrane protein.

12. The method according to any one of claims 1 to 11, characterized in that the synthesis reaction solution is treated with a resin or column capable of chelating metal ions.

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