Method for producing membrane protein-nanodisc complex

Abstract

[Problem] The problem to be addressed is to provide a method for producing a membrane protein-nanodisc complex which is less prone to fragmentation caused by decomposition of a membrane scaffold protein and which maintains the activity of a membrane protein. [Solution] The above problem can be solved by a method for producing a membrane protein-nanodisc complex involving a step for co-expressing a membrane protein and a membrane scaffold protein in a wheat cell-free protein synthesis system containing a lipid and a wheat germ extract.

Description

Method for producing membrane protein-nanodisc complexes

[0001] The present disclosure relates to methods for producing membrane protein-Nanodisc complexes. This application claims priority to Japanese Application No. 2023-150619, which is incorporated herein by reference.

[0002] Membrane proteins are a collective term for proteins that penetrate or attach to the membranes of all cells and organelles, from microorganisms to animals and plants, and function within the membrane. Membrane proteins transport vital substances, such as ions and nutrients, and function as highly sensitive sensors of extracellular signals, known as sensory receptors for hormones, light, heat, and sound. Membrane proteins are central molecules in biological signaling pathways that regulate the transmission of information and energy through cell membranes, and are said to account for approximately 60% of therapeutic drug targets for various diseases. However, membrane proteins are difficult to isolate and prepare because they aggregate and become insoluble when separated from their native lipid bilayer, making evaluation of membrane protein function and evaluation of drugs targeting membrane proteins extremely challenging. While detergents can be used to solubilize membrane proteins, this often disrupts the original structure of the membrane protein and results in loss of functionality.

[0003] Nanodisc technology, developed by Professor Stephen Sligar of the University of Illinois, enables the preparation of membrane proteins within water-soluble, near-native lipid bilayers, making it applicable to a variety of technical fields, including structural biology, electron microscopy, NMR, EPR, and spectroscopic detection. Patent Document 1 (JP 2007-525490) discloses a "method for producing membrane protein-nanodisc complexes, including a process for self-organizing a phospholipid bilayer with amphipathic helical segments of apolipoprotein A-1 (a membrane scaffold protein) and embedding a membrane protein therein." However, the method described in Patent Document 1 has the drawback of poor membrane protein embedding within the nanodiscs, limiting the types of membrane proteins that can be used.

[0004] A method for co-expressing a membrane protein and a membrane protein scaffold protein in the presence of lipids is known (Non-Patent Document 1).

[0005] Special Table 2007-525490

[0006] Front. Pharmacol., 03 July 2019 Sec.Translational Pharmacology Volume 10 - 2019 | https: / / doi.org / 10.3389 / fphar.2019.00744

[0007] Regarding the method disclosed in Non-Patent Document 1, examples have been described in which cell-free systems such as insect cell extracts, mammalian extracts, and Escherichia coli cell extracts were used. However, the issues of insect cell extracts and mammalian extracts, such as very low synthesis yields and high costs, have been cited. E. coli extracts are unsuitable for synthesizing eukaryotic membrane proteins; they typically require a refolding step after membrane protein synthesis, making them unsuitable for coexpression. Furthermore, E. coli cell extracts retain the activity to degrade membrane scaffold proteins in addition to mRNA degradation activity. Degradation of membrane scaffold proteins results in membrane scaffold protein fragments of various molecular weights adhering to the hydrophobic regions of the lipid bilayer, resulting in a loss of uniformity of the membrane protein-nanodisc complex. As a result, the physicochemical stability of the membrane protein-nanodisc complex is significantly impaired. Therefore, the objective of this study was to provide a method for producing membrane protein-nanodisc complexes that is less susceptible to fragmentation due to degradation of the membrane scaffold protein and maintains the activity of the membrane protein.

[0008] The present inventors have confirmed that the above-mentioned problems can be solved by a "method for producing a membrane protein-nanodisc complex, which comprises a step of co-expressing a membrane protein and a membrane scaffold protein in a wheat cell-free protein synthesis system containing lipids and a wheat germ extract," and have completed the production method of the present disclosure.

[0009] 1. A method for producing a membrane protein-nanodisc complex, comprising a step of co-expressing a membrane protein and a membrane scaffold protein in a wheat cell-free protein synthesis system containing a lipid and a wheat germ extract. 2. The method according to the preceding item 1, wherein the wheat germ extract has been treated with an inorganic compound. 3. The method according to the preceding item 2, wherein the inorganic compound is at least one selected from alpha-alumina, titanium oxide, silicon nitride, titanium nitride, aluminum hydroxide, gamma-alumina, silicon dioxide, aluminum silicate, zinc oxide, cesium oxide, and zirconium oxide. 4. The method according to the preceding item 1 or 2, wherein the co-expression method is a dialysis overlay method, a dialysis method, or a overlay method. 5. The method according to the preceding item 1 or 2, wherein the lipid is a phospholipid, a glycerophospholipid, or a sphingolipid. 6. The method according to the preceding item 5, wherein the lipid is a soybean phospholipid. 7. The method according to the preceding item 2, wherein the inorganic compound is aluminum hydroxide, the co-expression method is a dialysis overlay method, and the lipid is a soybean phospholipid. 8. 8. The method according to item 7, wherein the wheat cell-free protein synthesis system comprises a PDI family protein including protein disulfide isomerase and a catalyst for reoxidizing a substance that catalyzes a disulfide bond exchange reaction. 9. The method according to item 8, wherein the membrane scaffold protein is a modified sequence of apolipoprotein (apo) AI. 10. The method according to item 9 above, wherein the amino acid sequence of the membrane scaffold protein is any one of the following 1) to 13): 1) MSP1: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 2) MSP2: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 3) MSP1E1: FX-H1-H2-H3-H4-H4-H5-H6-H7-H8-H9-H10 4) MSP1E2: FX-H1-H2-H3-H4-H5-H4-H5-H6-H7-H8-H9-H10 5) MSP1E3: FX-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 6) MSP1D1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H107) MSP1E3D1: H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 8) MSP1D2: H2-H3-H4-H5-H6-H7-H8-H9-H10 9) MSP2N1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-11)-H2-H3-H4-H5-H6- H7-H8-H9-H10 10) MSP2N2: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-H4-H5-H6-H7-H8-H9-H10 11) MSP2N3: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-17)-H2-H3-H4-H5-H6-H7-H8-H9-H10 12) A sequence having an amino acid sequence that is 90% identical to any one of 1) to 11) above and that has the ability to form nanodiscs, 13) A sequence having 1 to 20 amino acid substitutions, deletions, insertions and / or additions in the sequences 1) to 11) above and that has the ability to form nanodiscs, wherein the amino acid sequences of the above sequences are as follows: H1: LKLLDNWDSVTSTFSKLREQLG (SEQ ID NO: 1) H1△(1-11): STFSKLREQLG (SEQ ID NO: 2) H1△(1-17): REQLG (SEQ ID NO: 3) H2: PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO: 4) H3: KDLEEVKAKVQ (SEQ ID NO: 5) H4: PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 6) H5: PLRAELQEGARQKLHELQEKLS (SEQ ID NO: 7) H6: PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 8) H7: PYSDELRQRLAARLEALKENGG (SEQ ID NO: 9) H8: ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 10) H9: PALEDLRQGLL (SEQ ID NO: 11) H10: PVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO: 12) FX: MGHHHHHHIEGR (SEQ ID NO: 13). 11. The method according to item 7 above, wherein the membrane protein is a membrane protein having an intramolecular or intermolecular disulfide bond. 12. A kit for producing a membrane protein-Nanodisc complex, comprising:1) A translation template or transcription template for a membrane scaffold protein; 2) A lipid; and 3) A wheat cell-free protein synthesis system containing an inorganic compound-treated wheat germ extract.

[0010] The method for producing a membrane protein-Nanodisc complex of the present disclosure and the membrane protein-Nanodisc complex obtained by said production method have one or more of the following effects: (1) A high synthesis success rate. (2) A high yield. (3) The three-dimensional structure of the membrane protein in the membrane protein-Nanodisc complex is maintained. (4) The activity of the membrane protein in the membrane protein-Nanodisc complex is maintained. (5) The membrane protein-Nanodisc complex is highly homogeneous. (6) The membrane protein-Nanodisc complex maintains its structure even after repeated freezing and thawing.

[0011] Image of protein-nanodisc complex Confirmation of membrane scaffold protein fragments in each synthesis system Results of GFP protein synthesis amount and fluorescence activity using inorganic compound-treated wheat germ extract Confirmation of membrane scaffold protein fragments using conventional wheat germ extract and inorganic compound-treated wheat germ extract 1 Confirmation of membrane scaffold protein fragments using conventional wheat germ extract and inorganic compound-treated wheat germ extract 2 Sequence of membrane scaffold protein Composition of 1xSUB-AMIX-SGC Composition of reaction phase Results of SDS-PAGE, scattering intensity distribution, and particle number distribution of soluble fraction DLS measurement results AlphaScreen overview AlphaScreen results Pull-down assay results Results of scattering intensity distribution and particle number distribution after one-step and two-step purification Overview of dialysis overlay method

[0012] (Production Method of the Present Disclosure) The present disclosure relates to a method for producing a membrane protein-Nanodisc complex, comprising the step of co-expressing a membrane protein and a membrane scaffold protein in a wheat cell-free protein synthesis system containing lipids and wheat germ extract (hereinafter sometimes abbreviated as the "production method of the present disclosure"). Specifically, the present disclosure relates to a method for producing a membrane protein-Nanodisc complex, comprising the step of co-expressing a membrane protein and a membrane scaffold protein by directly or indirectly contacting a reaction phase containing a synthesis reaction solution with a feed phase containing an energy source supply solution containing substrates and energy source molecules, thereby supplying the substrates and energy source molecules from the feed phase to the reaction phase. The production method of the present disclosure has a high yield and a high synthesis success rate, and the membrane protein-Nanodisc complex obtained by this method is highly uniform and maintains the three-dimensional structure and activity of the membrane protein.

[0013] (Membrane Protein-Nanodisc Complex) The membrane protein-nanodisc complex has a structure exemplified in Figure 1. Specifically, the complex has a structure in which the membrane protein is embedded in a disk-shaped nanoparticle (nanodisc) in which two membrane scaffold proteins surround a lipid bilayer in a belt-like manner.

[0014] (Co-expression) In the present disclosure, "co-expression" refers to the synthesis (particularly simultaneous synthesis) of multiple proteins, namely, a membrane protein and a membrane scaffold protein, in the same synthesis system. In the present disclosure, both a translation template (mRNA) or transcription template (cDNA) for the membrane scaffold protein and a translation template (mRNA) or transcription template (cDNA) for the membrane protein are added to the synthesis reaction solution. When membrane proteins form heterogeneous complexes, it is also possible to add translation templates for multiple membrane proteins. In co-expression, it is desirable to adjust the amount of mRNA for both proteins added so that the membrane scaffold protein is synthesized in excess relative to the membrane protein ultimately synthesized. This allows for maximum solubilization of membrane proteins that are prone to insolubilization. As shown in the following examples, it is preferable to add approximately twice the amount of membrane scaffold protein mRNA to the membrane protein mRNA adjusted to a concentration of 1 mg / ml.

[0015] (Membrane Protein) The term "membrane protein" in the present disclosure is not particularly limited, but includes proteins attached to the biological membranes of all cells and intracellular organelles, from microorganisms to animals and plants. Membrane proteins are classified into integral membrane proteins and peripheral membrane proteins. Membrane proteins are further classified into several types based on the direction of insertion into the membrane, the presence or absence of a signal peptide, and the location of the transmembrane domain within the protein. Functions of membrane proteins include transporting substances necessary for life support, such as ions and nutrients, and highly sensitive sensors of extracellular signals, known as sensory receptors for hormones, light, heat, sound, etc. Examples include GHSR, Claudin 4, rhodopsin, two-component regulatory systems, transporters, GPCRs, ion channels, and enzyme-coupled proteins. Specific examples include TetA, GR (Gloeobacter-derived rhodopsin), channelrhodopsin ChR, various histidine kinases (PhoQ, RcsC, EnvZ, ArcB), transport proteins TetA, LacY, COX, aquaporins, and various GPCRs (α1B-adrenergic receptor α1BAR, adenosine A2A receptor A2AR, serotonin 2A receptor (5H2TR), dopamine D2 receptor (D2R), etc.). In addition, the number of transmembrane domains of the membrane protein is not particularly limited, and examples include 7, 4-5, and 1 transmembrane domains.

[0016] (Membrane Scaffold Protein) The "membrane scaffold protein (MSP)" of the present disclosure is not particularly limited, but may be a protein (peptide) in which a portion of the apolipoprotein (apo) AI sequence has been modified or deleted. An example of an MSP that surrounds the nanodisc in a belt-like fashion is a mutant of apoA-I in which the relatively low lipid affinity of the N-terminal 43 residues has been removed. This mutant protein is composed of 10 amphipathic helices separated primarily by prolines. The amphipathic helices have a primary sequence in which hydrophilic amino acids are located every 2-3 residues. Alpha-helix formation places these hydrophilic residues on one side, forming a hydrophilic surface. A hydrophobic surface composed of nonpolar amino acids is formed on the opposite side. In nanodiscs, this hydrophobic surface faces the lipid side (inward) and interacts with the acyl chains of the lipids, preventing their exposure to water. For example, the following sequence shown in Figure 6 may be used.1) MSP1: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 2) MSP2: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 3) MSP1E1: FX-H1-H2-H3-H4-H4-H5-H6-H7-H8-H9-H10 4) MSP1E2: FX-H1-H2-H3-H4-H5-H4-H5-H6-H7-H8-H9-H10 5) MSP1E3: FX-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 6) MSP1D1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10 7) MSP1E3D1: H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 8) MSP1D2: H2-H3-H4-H5-H6-H7-H8-H9-H10 9) MSP2N1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-11)- H2-H3-H4-H5-H6- H7-H8-H9-H10 10) MSP2N2: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H2-H3-H4-H5-H6-H7-H8-H9-H10 11) MSP2N3: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-17)-H2-H3-H4-H5-H6-H7-H8-H9-H10 12) A sequence having an amino acid sequence that is 90% (95%, 96%, 97%, 98% or 99%) identical to any one of 1) to 11) above and that has the ability to form nanodiscs. 13) A sequence in any one of the above sequences 1) to 11), in which 1 to 20 (1 to 15, 1 to 10, 1 to 8, 1 to 5, or 1 to 3) amino acids have been substituted, deleted, inserted, and / or added, and which has the ability to form nanodiscs. The amino acid sequences of the above sequences are as follows:H1: LKLLDNWDSVTSTFSKLREQLG (SEQ ID NO: 1) H1△(1-11): STFSKLREQLG (SEQ ID NO: 2) H1△(1-17): REQLG (SEQ ID NO: 3) H2: PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO: 4) H3: KDLEEVKAKVQ (SEQ ID NO: 5) H4: PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 6) H5: PLRAELQEGARQKLHELQEKLS (SEQ ID NO: 7) H6: PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 8) H7: PYSDELRQRLAARLEALKENGG (SEQ ID NO: 9) H8: ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 10) H9: PALEDLRQGLL (SEQ ID NO: 11) H10: PVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO: 12) FX: MGHHHHHHIEGR (SEQ ID NO: 13). Regarding the above 11) to 13), the protein having a mutation may be a naturally occurring protein, or may be one obtained by introducing a mutation into a naturally occurring gene. Methods for introducing mutations are well known, and include, for example, site-directed mutagenesis, homologous recombination, primer extension, and polymerase chain reaction (hereinafter abbreviated as PCR), which can be used alone or in combination. For example, mutations can be introduced according to methods described in textbooks (Sambrook et al., eds., "Molecular Cloning, A Laboratory Manual, 2nd Edition," 1989, Cold Spring Harbor Laboratory; Muramatsu Masami, ed., "Laboratory Manual of Genetic Engineering," 1988, Maruzen Co., Ltd.), or by modifying these methods. Ulmer's technique (Ulmer, KM, Science, 1983, Vol. 219, pp. 666-671) can also be used. In the case of peptides, from the viewpoint of not changing the basic properties (physical properties, functions, physiological activity, immunological activity, etc.) of the peptide when introducing mutations, for example, mutual substitutions between homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) can easily be envisioned.

[0017] (Lipids) The "lipids" of the present disclosure are not particularly limited. Examples of phospholipids that can be used in the Nanodisc assembly method include phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, dipalmitoyl-phosphatidylcholine, dimyristoylphosphatidylcholine, 1-palmitoyl-2-oleyl-phosphatidylcholine, dihexanoylphosphatidylcholine, dipalmitoylphosphatidylethanolamine, dipalmitoylphosphatidylinositol; dimyristoylphosphatidylethanolamine; dimyristoylphosphatidylinositol; dihexanoylphosphatidylethanolamine; dihexanoylphosphatidylinositol; 1-palmitoyl-2-oleyl-phosphatidylethanolamine; and 1-palmitoyl-2-oleyl-phosphatidylinositol. These can be used singly or in mixtures. Phospholipids can contain a glycerol backbone or sphingolipids. Generally, phospholipids have saturated fatty acids with 6 to 20 carbon atoms and commonly used head groups, such as those listed below, including, but not limited to, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The head groups can be positively or negatively charged, or even bipolar. Phospholipids can be natural (those occurring in nature), synthetic (those not occurring in nature), or a mixture of natural and synthetic phospholipids. Plant-derived lipids, in particular, are soft and tend to easily incorporate membrane proteins. Examples include those derived from olives, macadamia nuts, soybeans, corn, evening primrose, flaxseed, and perilla. Among these, soybean-derived asolectin and soybean phospholipids are the most suitable.

[0018] In the production method of the present disclosure, the membrane protein and / or membrane scaffold protein of the membrane protein-Nanodisc complex may be fused with a known tag (particularly a tag for protein production), such as SBP, PA, DYKDDDDK (FLAG: SEQ ID NO: 14), 6xHis, HA, V5, c-Myc, MBP, etc.

[0019] (Applications) The membrane protein-nanodisc complexes produced by the production method of the present disclosure can be used to evaluate the structure, function, or pharmacological efficacy of membrane proteins.

[0020] (Translation Reaction System) The "wheat cell-free protein synthesis system" in the production method of the present disclosure can be a publicly known wheat cell-free protein synthesis system. The wheat germ cell-free system sold by Self-Science Co., Ltd. in Japan and overseas is ISO 9001 compliant, produces high protein yields, has excellent inhibitory properties against degradation of the produced protein, and exhibits extremely stable quality between lots, making it ideal. Information on protein synthesis methods is provided on the company's website, and although methods such as the batch method, bilayer method, dialysis bilayer method, and repeated batch method can be used, the bilayer method and dialysis bilayer method are preferred in terms of forming disulfide bonds in the membrane protein to be synthesized (described below) and providing a high degree of freedom in the synthesis system, and the dialysis bilayer method is optimal in terms of the ease of increasing yield.

[0021] The results of the following examples show that the synthesis reaction solution containing the wheat cell-free protein synthesis system (wheat germ extract) used in the production method of the present disclosure can improve the activity of membrane proteins by treating it with an inorganic compound before protein synthesis. Examples of inorganic compounds include alpha-alumina, titanium oxide, silicon nitride, titanium nitride, aluminum hydroxide, gamma-alumina, silicon dioxide, aluminum silicate, zinc oxide, cesium oxide, and zirconium oxide.

[0022] (Translation template for protein synthesis) In the production method of the present disclosure, mRNA encoding the protein to be expressed can be used as the "translation template for protein synthesis." Furthermore, mRNA can be used not only after purification by ethanol precipitation or the like, but also in an unpurified state.

[0023] (Synthesis Method Using the Bilayer Method of the Present Disclosure) The bilayer method is a diffusion-based continuous batch cell-free protein synthesis method characterized by directly contacting a synthesis reaction solution (reaction phase) containing a biological extract with a substrate and energy source supply solution (supply phase), continuously supplying substrate and energy source molecules from the supply phase to a translation reaction system in the reaction phase by free diffusion through the contact interface between the two phases, and eliminating by-products generated in the reaction phase (by-products diffusing into the supply phase), thereby extending the duration of the synthesis reaction and increasing the efficiency of the synthesis reaction. The interface between the two phases may be formed as a horizontal or vertical surface. To form a horizontal interface, for example, the reaction phase may be first added to a reaction vessel to form a lower layer, and then the supply phase may be gently layered on top of the reaction phase without disturbing the interface between the two phases. The shape and size of the reaction vessel may be any suitable size that allows for sufficient diffusion of solutes between the two phases, such as, but not limited to, a test tube or a multi-well microtiter plate. It is also possible to form a vertical interface between the synthesis reaction solution (reaction phase) and the energy source supply solution by layering them and then centrifuging the reaction vessel containing them. The larger the contact interface area between the two phases, the higher the rate of material exchange by diffusion and the higher the efficiency of protein synthesis. 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 has a diameter of 7 mm, a ratio of 1:4 to 1:8 is preferred, and 1:5 is even more preferred.

[0024] (Synthesis Method Using Dialysis Method of the Present Disclosure) Dialysis method (see Kikawa et al., 21st Meeting of the Molecular Biology Society of Japan, WID6) is a method for protein synthesis using a dialysis inner solution (reaction phase) containing a synthesis reaction solution, a dialysis outer solution (supply phase) containing an energy source supply solution containing substrates and energy source molecules, and a dialysis membrane capable of transferring substances between the two phases.

[0025] (Synthesis Method Using the Dialysis Bilayer Method of the Present Disclosure) The dialysis bilayer method (see Takeda et al., Scientific Reports 2015) is a diffusion continuous batch cell-free protein synthesis method (see FIG. 15) characterized in that a known dialysis cup (having a dialysis membrane on the bottom) is immersed in a feed phase, which is an extramembrane phase containing an energy source feed solution containing substrates and energy source molecules, and a conventional bilayer method is performed in the cup, thereby arranging the feed phases above and below the reaction phase, thereby more efficiently supplying substrates such as amino acids and releasing reaction by-products.

[0026] (Synthesis Reaction Solution) In the production method of the present disclosure, the "synthesis reaction solution constituting the reaction phase" contains a translation reaction system and a translation template (e.g., mRNA) necessary for protein synthesis, and, if necessary, liposomes and components used in conventionally known protein synthesis (particularly, batch-type cell-free protein synthesis). Examples of components include amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for the protein synthesis reaction. Specifically, when used in a reaction phase containing a wheat germ extract, examples of components include creatine kinase, HEPES-KOH, ATP, GTP, creatine phosphate, spermidine, potassium acetate, magnesium acetate, 20 types of L-amino acids, a reducing agent, PDI family proteins including protein disulfide isomerase, BMC, an aromatic thiol compound, and / or an enzyme that reoxidizes PDI family proteins, and a chaperone group. In addition, the protein synthesis reaction can be further stabilized by adding sugar alcohols such as inositol, xylitol, and / or ficoll to the synthesis reaction solution to increase the viscosity and density of the synthesis reaction solution and controlling the mixing rate between the reaction phase and the supply phase.

[0027] (Energy Source Supply Solution) In the production method of the present disclosure, the "energy source supply solution comprising substrates and energy source molecules constituting the supply phase" includes components consumed or in short supply during the protein synthesis reaction in the reaction phase. Examples of components, such as substrates and energy source molecules, include amino acids, ATP, GTP, creatine phosphate, and other ions and buffers necessary for the protein synthesis reaction. Specifically, when used in a reaction phase containing a wheat germ extract, examples of components include creatine kinase, HEPES-KOH, ATP, GTP, creatine phosphate, spermidine, potassium acetate, magnesium acetate, 20 types of L-amino acids, a reducing agent, PDI family proteins including protein disulfide isomerase, BMC, aromatic thiol compounds, and / or enzymes that reoxidize PDI family proteins, and chaperones. In addition, the protein synthesis reaction can be further stabilized by adding sugar alcohols such as inositol, xylitol, and / or ficoll to the energy source supply solution to increase the viscosity and density of the synthesis reaction solution and controlling the mixing rate between the reaction phase and the supply phase.

[0028] (Synthesis Method Using the Repeated Batch Method of the Present Disclosure) The repeated batch method is a synthesis method characterized by maximizing the high reaction rate characteristics of the initial phase of a synthesis reaction using a template material in a typical batch or diffusion continuous batch method (see WO 2004 / 097014). Specifically, a dilution or concentration treatment is performed using an energy source supply solution (feed phase) around the time when the synthesis rate is approximately slowing down or the synthesis reaction is approximately halting, or during these processes. If the solution is diluted, a concentration treatment is then performed, and if the solution is concentrated, a dilution treatment is performed. These dilution and concentration treatments are then discontinuously repeated, enabling the synthesis of large amounts of protein. In the synthesis method using the repeated batch method of the present disclosure, an energy source supply solution (containing a reducing agent, if necessary) is added to a reaction phase under reducing conditions, which is a feed phase in which oxidation conditions are gradually changed. The reaction phase is characterized by a gradual transition to oxidation conditions optimal for the synthesis and maintenance of proteins with intramolecular or intermolecular disulfide bonds while maintaining protein synthesis efficiency.

[0029] The synthesis method using the repeated batch method of the present disclosure includes, for example, the following steps: 1) synthesizing a protein in a reaction phase containing a synthesis reaction solution; 2) diluting the reaction phase approximately around the time when the synthesis rate drops, approximately around the time when the synthesis reaction stops, or during any of these steps; 3) performing a concentration treatment following the dilution treatment; 4) performing a synthesis reaction in the concentrated reaction phase; or 1) synthesizing a protein in a reaction phase containing a synthesis reaction solution; 2) concentrating the reaction phase approximately around the time when the synthesis rate drops, approximately around the time when the synthesis reaction stops, or during any of these steps; 3) performing a dilution treatment following the concentration treatment; 4) performing a synthesis reaction in the diluted reaction phase; The concentration treatment may include a step of removing by-products from the reaction phase; and the dilution treatment involves adding (replenishing) an energy source supply solution (containing a reducing agent, if necessary), which is a supply phase in which the oxidation conditions are gradually changed, to the reaction phase. The above steps 1) to 4) may be repeated multiple times.

[0030] (Synthesis Reaction Conditions) The synthesis reaction conditions for the production method of the present disclosure can be those of the bilayer method, dialysis method, and dialysis bilayer method. For example, the protein synthesis reaction is preferably carried out under static conditions, at a reaction temperature of 10°C to 35°C, which is the optimum temperature typically used in cell-free protein synthesis. The target protein is contained in the reaction phase, the supply phase, and a mixed phase in which both phases are mixed over the course of synthesis, and therefore 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.

[0031] (Membrane Protein Having Intramolecular or Intermolecular Disulfide Bonds) In the production method of the present disclosure, the "membrane protein having intramolecular or intermolecular disulfide bonds" is not particularly limited as long as it is a membrane protein having intramolecular and / or intermolecular disulfide bonds. Examples thereof include the membrane proteins disclosed herein.

[0032] (Oxidizing Conditions in the Supply Phase) In the production method of the present disclosure, membrane proteins that retain their activity can be synthesized by preferably controlling (setting) the "supply phase under oxidizing conditions compared to the reaction phase." An example of a supply phase being under oxidizing conditions compared to the reaction phase is when the redox potential of the supply phase is higher than that of the reaction phase. The redox potential of the reaction phase and supply phase can be measured using known devices (e.g., ORP electrode 9300-10D (manufactured by HORIBA) or pH meter F-52 (manufactured by HORIBA)) according to the instructions in their instruction manuals. For example, approximately 3 ml of solution is prepared and kept at 26°C. After that, a potential measurement electrode is immersed in the solution, and the measurement value is allowed to stabilize (approximately 15 to 30 minutes), and the stabilized value can be used as the redox potential value.

[0033] (Controlling the Supply Phase to an Oxidizing Condition) In the production method of the present disclosure, "controlling the supply phase to an oxidizing condition" can be performed by any method as long as the oxidation-reduction potential of the supply phase can be set and controlled to be higher than that of the reaction phase. For example, known oxidizing agents such as oxidized glutathione, cystine, oxygen molecules, and NADP can be used. + This can be achieved by including hydrogen peroxide, etc. in the feed phase.

[0034] (Controlling the Reaction Phase to Reduced Conditions) In the production method of the present disclosure, "controlling the reaction phase to reduced conditions" can be achieved by any method that allows the redox potential of the reaction phase to be set or controlled 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, and NADH in the reaction phase. For example, when DTT is used alone as a reducing agent, the final concentration in the reaction phase is 0.4 mM to 40 mM, preferably 1.0 mM to 10 mM. Furthermore, the redox potential of the reaction phase is preferably in the range of -500 mV to -100 mV.

[0035] (Substance that catalyzes a disulfide bond exchange reaction) In the production method of the present disclosure, it is preferable that a substance that catalyzes a disulfide bond exchange reaction, and further a catalyst that reoxidizes the catalyst, be included in the reaction phase and / or supply phase. In addition, it is particularly preferable that the production method of the present disclosure includes a substance that catalyzes a disulfide bond exchange reaction, and further a catalyst that reoxidizes the catalyst, in the reaction phase. This makes it possible to more efficiently synthesize membrane proteins in which intramolecular disulfide bonds are correctly formed (retained). 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, which are chaperone proteins derived from Escherichia coli; various proteins that catalyze refolding reactions, such as DnaK, DnaJ, and GrpE; and low-molecular-weight mimics of these proteins (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 that reoxidize substances that catalyze disulfide bond exchange reactions include reoxidizing enzymes present in the endoplasmic reticulum of eukaryotic cells, such as ER oxidase (hereinafter referred to as Ero1α or Ero1β), peroxiredoxin IV, glutathione peroxidases 7 and 8, and vitamin K epoxyreductase.

[0036] (Proteins Involved in Polypeptide Chain Folding Reaction) In the production method of the present disclosure, it is preferable to include a protein involved in the polypeptide chain folding reaction in the reaction phase and / or supply phase, as necessary. This allows for more efficient synthesis of membrane proteins in which the intramolecular polypeptide chain folding is correctly formed (maintained). Examples of proteins involved in the polypeptide chain folding reaction include 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., cyclophilin B), which are proteins present in eukaryotic cells and involved in the folding reaction of nascent polypeptide chains.

[0037] The present disclosure will be described in more detail below using examples. However, the following examples should be considered as an aid to gain a concrete understanding of the present disclosure, and the scope of the present disclosure is not limited by the following examples in any way.

[0038] (Improvement of Wheat Germ Extract) The present inventors have confirmed that the wheat cell-free protein synthesis system has a much higher success rate for synthesizing soluble membrane proteins through nanodisc formation than other synthesis systems (E. coli synthesis systems). For example, it is recognized by those skilled in the art that the synthesis success rate for E. coli synthesis systems is approximately 10%. On the other hand, the present inventors have confirmed that the synthesis success rate for over a dozen membrane proteins, including those in this example, is approximately 100%. Therefore, in this example, we investigated why the wheat cell-free protein synthesis system has a higher synthesis success rate than the E. coli synthesis system.

[0039] (Confirmation of membrane scaffold protein synthesis in various systems) Figure 2 shows the results of synthesizing a membrane scaffold protein (MSP1E3D1) using the commercially available E. coli synthesis system 1, E. coli synthesis system 2, and a commercially available wheat cell-free protein synthesis system (WEPRO1240: Cell-Free Sciences). As is clear from the electrophoresis pattern shown in Figure 2, the E. coli synthesis system exhibited numerous ladder-like degradation products (fragments) on the low molecular weight side. The membrane scaffold protein is crucial for maintaining the shape and uniformity of the nanodiscs in the membrane protein-nanodisc complex. It is believed that residual proteases and nucleases contribute to the degradation of the membrane scaffold protein. These fragments bind to the hydrophobic portions of lipids, resulting in heterogeneous and unstable membrane protein-nanodisc complexes, which can cause significant problems in binding assays of sample compounds and in observations using cryo-EM. On the other hand, synthesis using a wheat cell-free protein synthesis system significantly reduced and improved the fragmentation of membrane scaffold proteins, but faint fragmentation bands were still observed. During the synthesis reaction, membrane scaffold protein and membrane protein mRNAs are degraded by nucleases present in the extract or bind to nucleic acid-binding proteins, resulting in degradation and fragmentation, and loss of their function as translation templates.

[0040] The present inventors have investigated various treatment methods to suppress the generation of the fragmented products, and have found that this can be avoided by contacting the product with a specific inorganic compound.

[0041] (Production of inorganic compound-treated wheat germ extract) The method for producing wheat germ extract is described in detail in Springer Protocols, Cell-Free Protein Production, Methods and Protocol, pp. 23-30s. The extract produced by this method was passed through a 10,000 cut-off ultrafiltration filter to remove low-molecular-weight components of 10,000 or less, and then contacted with the inorganic compounds shown in Figure 3, followed by decantation using a centrifugal separator to produce an inorganic compound-treated wheat germ extract.

[0042] (Performance Verification of Wheat Germ Extract Treated with Inorganic Compounds 1) Figure 3 shows the results of GFP protein expression in a wheat cell-free protein synthesis system containing wheat germ extract treated with various inorganic compounds. Figure 3 shows the GFP fluorescence intensity and band intensity. Alpha-alumina, aluminum hydroxide, silicon nitride, titanium nitride, silicon dioxide, cesium oxide, zirconium oxide, and gamma-alumina all exhibited similar fluorescence values. The difference in electronegativity between these inorganic compounds was confirmed to be 1.5 or greater. Zirconium oxide and cesium oxide were identified as compounds that exhibited a decrease in fluorescence intensity relative to the amount of GFP synthesized (i.e., inactivation). Silicon nitride and titanium nitride were identified as compounds that exhibited a high fluorescence intensity relative to the amount of GFP synthesized (GFP fluorescence was present, but the amount synthesized was not very high). Aluminum silicate and zirconium oxide were identified as compounds with high processing activity even in small amounts. Aluminum hydroxide, alpha-alumina, gamma-alumina, and cesium oxide were identified as compounds with moderate processing activity. Considering the above, alpha-alumina and aluminum hydroxide were confirmed to offer a good balance between performance and process efficiency. Furthermore, taking the above into consideration, it was confirmed that aluminum hydroxide, α-alumina, silicon dioxide, silicon nitride, cesium oxide, titanium nitride, and zirconium oxide are preferable, and therefore, in the following examples, a wheat germ extract treated with aluminum hydroxide was used.

[0043] (Performance Verification 2 of Wheat Germ Extract Treated with Inorganic Compounds) We confirmed whether wheat germ extract treated with inorganic compounds (particularly aluminum hydroxide) is superior to wheat germ extract untreated with inorganic compounds. As is clear from the electrophoresis diagram in Figure 4, the aluminum hydroxide-treated wheat germ extract showed significantly lower levels of ladder-like degradation products (fragmented products) on the low molecular weight side compared to wheat germ extract untreated with inorganic compounds. The pixel position in Figure 5 confirmed that the fragmented product region of the aluminum hydroxide-treated wheat germ extract was 8.0%, while that of the wheat germ extract untreated with inorganic compounds was 14.2%.

[0044] (Performance verification of inorganic compound-treated wheat germ extract 3) Changes in the types of total proteins were confirmed before and after aluminum hydroxide treatment. Specifically, 20 ul of wheat germ extract (3 replicates) was subjected to TCA precipitation, trypsin digestion, Ziptip column purification, and proteome analysis. Approximately 900 proteins were detected. Approximately 40% (32 / 76) of the proteins adsorbed to aluminum hydroxide and undetected by the WEPRO7240H were confirmed to be nucleic acid (DNA, RNA) binding proteins.

[0045] (Calculation of the yield of membrane protein-Nanodisc complexes in various synthesis systems) Based on the above results, the degradation rates of the membrane scaffold proteins were as follows: Commercially available E. coli synthesis system 1: approximately 30% Commercially available E. coli synthesis system 2: approximately 60% Inorganic compound-untreated wheat germ extract: approximately 14% Inorganic compound-treated wheat germ extract: approximately 8% As shown in Figure 1 , in the membrane protein-Nanodisc complex, considering the structure in which the two membrane scaffold protein bands surround the lipid, the yield is expected to be as follows, assuming the inorganic compound-treated wheat germ extract is 100: Commercially available E. coli synthesis system 1: approximately 7% Commercially available E. coli synthesis system 2: approximately 14% Inorganic compound-untreated wheat germ extract: approximately 33%. Therefore, the yield of membrane protein-Nanodisc complexes in the wheat cell-free protein synthesis system containing inorganic compound-treated wheat germ extract is approximately seven times higher than that in the commercially available E. coli synthesis system. In addition, as mentioned above, the success rate of synthesizing membrane protein-nanodisc complexes using the wheat cell-free protein synthesis system containing wheat germ extract is approximately 10 times (100 / 10) higher than that of the commercially available E. coli synthesis system.

[0046] (Production of membrane protein-Nanodisc complex) In this example, a membrane protein-Nanodisc complex was produced using the dialysis overlay method. <Membrane proteins> GHSR: Growth Hormone Secretagogue Receptor (belonging to the seven-transmembrane G-protein coupled receptor 1 family, consisting of 366 amino acids) Claudin 4: A four-transmembrane protein consisting of 209 amino acids in total, which forms tight junctions) <Tags> SBP tag: MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 15) <Membrane scaffold protein> MSP1E3D1 with Hisx6 fused to the N-terminus: H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 (see Figure 6)

[0047] (1) Preparation of lipids (Asolectin-Liposome solution) 1) 200 μl of commercially available 1xSUB-AMIX-SGC (see Figure 7, sold by Cell Free Science Co., Ltd.) was added to 10 mg of commercially available soybean-derived asolectin liposome, lyophilized (lyophilized product, sold by Cell Free Science Co., Ltd.) and thoroughly suspended. 2) The suspension was transferred to a 0.6 ml tube and subjected to ultrasonic treatment (20% output, 1 to 3 minutes; within 5 minutes). It was confirmed that the solution was not turbid. 3) Debris was precipitated by centrifugation (15,000 rpm, 10 minutes). 4) The supernatant was used below.

[0048] (2) Preparation of dialysis solution For the following dialysis overlay synthesis, DTT was added to the solution (HO: 4.86 ml, 40x SUB-AMIX-SGC: 135 µl each) to prepare a dialysis solution (5.40 ml) containing 4 mM DTT. The internal dialysis solution was 0.8 ml, and the external dialysis solution was 4.6 ml. The internal dialysis solution and external dialysis solution had the same composition.

[0049] (3) Preparation of Dialysis Bilayer Synthesized Membrane Protein-Nanodisc Complexes As shown in Figure 8, 30 μl of the reaction phase, 200 μl of the feed phase (dialysis internal solution), and 1.5 ml of the external dialysis solution were used. The wheat germ extract was treated with aluminum hydroxide, which had produced good results in Example 1 above. The membrane scaffold protein was set at approximately twice the amount (molar concentration) of the membrane protein. Synthesis: Synthesis was performed at 16°C for 4 days. Recovery: After synthesis, the upper and lower layers (reaction phase) were mixed with a pipette and recovered. Purification: The soluble and insoluble fractions were separated by centrifugation. The soluble fraction was used for purification.

[0050] (Purification) The detailed conditions for the above purification are as follows. Washing buffer: A pH 7-8 buffer, such as PBS, Tris, or HEPES, was prepared. The salt concentration, such as NaCl, was kept below 500 mM. Elution buffer: Approximately 5 mM d-desthiobiotin was added to the washing buffer. Because desthiobiotin has low solubility, a stock solution was prepared by suspending it in MilliQ water or an appropriate buffer to a concentration equivalent to approximately 200-500 mM once fully dissolved. This was then frozen and stored as a stock solution. Streptavidin Sepharose HP (SA resin): After washing twice with an appropriate amount of MilliQ water, the resin was suspended in washing buffer to form a 50% slurry. <Binding, washing, and elution> An appropriate amount of SA resin (50% slurry) was added to the sample. The mixture was gently mixed using a rotator at 4°C for approximately 1 hour. The resin (SA resin) was recovered using an open column or spin column. The resin was washed multiple times with an appropriate amount of washing buffer. An appropriate amount of elution buffer was added and mixed with the resin. After leaving it to stand for 5 minutes, the eluate was collected. This process was repeated 3-5 times. If necessary, the eluate was concentrated and buffer exchanged using a concentrator.

[0051] Soluble fractions of the GHSR-Nanodisc complex and Claudin4-Nanodisc complex were obtained using the SBP tag. Bands for both proteins were detected by SDS-PAGE of the soluble fraction. This confirmed the formation of complexes (see Figure 9). The particle size distribution was confirmed using a nanoparticle size measurement device using dynamic light scattering (DSL). A single band representing the complex was confirmed (see Figure 9). These results confirmed the synthesis of highly uniform membrane protein-Nanodisc complexes.

[0052] (Confirmation of the optimal amount of lipid added) In the production of Claudin4-nanodisc complexes in Example 2, the amount of lipid added was changed, and the particle size of the membrane protein-nanodisc complexes was measured by DLS measurement. The DLS measurement results are shown in Figure 10. It was confirmed that the amount of lipid added does not affect the particle size of the membrane protein-nanodisc complexes.

[0053] (Verification of the Function of Claudin4-Nanodisc Complex 1) The Claudin4-Nanodisc complex produced in Example 2 was examined to determine whether it maintained the functional activity of Claudin4. The interaction between Claudin4 and its ligand, CPE (Clostridium perfringens enterotoxin), was examined using AlphaScreen. The Clostridium perfringens-derived CPE used was synthesized using a wheat cell-free synthesis method, and it was further confirmed that it was successfully synthesized.

[0054] Samples: Claudin4-SBP (purified product, desthiobiotin was removed by buffer exchange) GST-CPE (purified product, GSH was removed by buffer exchange) Both proteins were adjusted to 10 ng / μl, and 1 μl of each was used in the assay system. Assay Buffer: 100 mM Tris-HCl (pH 8.0), 100 mM NaCl, 0.01% Tween-20, 1 mg / ml BSA

[0055] Assay 1) A Claudin4-SBP protein mix (6 μl of Claudin4-SBP, 1 μl of Assay Buffer) was prepared, and 7 μl was added to each well of a 384-well plate. 2) A GST-CPE protein mix (1 μl of GST-CPE, 7 μl of Assay Buffer) was prepared, and 8 μl was added to each well of 1). 3) Each well was mixed by pipetting or vortexing, and centrifuged. 4) The plate was incubated at 26°C for 1 hour. 5) A bead mix (9.8 μl of Assay Buffer, 0.1 μl of Streptavidin Donor beads, and 0.1 μl of anti-GST Acceptor Beads) was prepared using the AlphaScreen GST detection kit (Revivity), and 10 μl was added to each well. 6) The 384-well plate was protected from light and incubated at 26°C for 1 hour. 7) Measurements were performed. As shown in Figure 11, when CPE binds to Claudin4 in the Claudin4-nanodisc complex, the streptavidin donor beads and acceptor beads come into close proximity, resulting in an increase in fluorescence intensity.

[0056] The assay results are shown in Figure 12. As shown in Figure 12, when equal amounts (25 ng, 12 ng, and 6.25 ng) of GST-CPE and CLDN4-SBP were added, a strong signal was observed, confirming that CLDN4 and CPE interacted with each other.

[0057] (Confirmation of the Function of Claudin4-Nanodisc Complex 2) We confirmed whether the Claudin4-Nanodisc complex produced in Example 2 maintained the functional activity of Claudin4. A pull-down assay was performed using Claudin4 and its ligand, CPE.

[0058] Samples used: Claudin4 alone, MSP (membrane scaffold protein: MSP1E3D1) alone, or unpurified synthetic supernatant of Claudin4 and MSP co-expression, GST-CPE (purified product), GST-only (purified product). Experimental procedure: 1) Each unpurified synthetic supernatant was mixed with GST-CPE (purified product) or GST-only (purified product) and mixed by end-over-end mixing at 4°C for 30 minutes. 2) Glutathione resin was added and mixed by end-over-end mixing at 4°C for another hour. 3) The resin was recovered by centrifugation and the supernatant was removed. 4) To wash the recovered resin, it was suspended in washing buffer and then centrifuged to remove the supernatant. This procedure was repeated three times. 5) The resin was eluted with a buffer containing 10 mM glutathione, and the results were confirmed by SDS-PAGE.

[0059] The samples co-expressing Claudin4 and MSP (Fig. 13: synthesis conditions 3 and 4) bound to GST-CPE. On the other hand, samples expressing MSP alone (Fig. 13: synthesis condition 1) and Claudin4 alone (Fig. 13: synthesis condition 2) did not bind to GST-CPE. This is thought to be because the co-expression of Claudin4 with MSP resulted in the formation of solubilized nanodiscs, which were present in the synthesis supernatant (see Fig. 13). On the other hand, under synthesis condition 1, which did not contain Claudin4, or synthesis condition 2, in which Claudin4 was insolubilized as proteoliposomes, Claudin4 was absent from the synthesis supernatant and therefore did not bind to GST-CPE (see Fig. 13).

[0060] (Verification of the function of the Claudin4-Nanodisc complex 3) Two-step affinity purification (SBP and His tag) of the nanodisc-formed Claudin4 and CPE complex was performed. Specifically, a one-step streptavidin purification and a two-step nickel column purification were performed. Samples used: SBP-tagged Claudin4, His-fused membrane scaffold protein (MSP1E3D1).

[0061] The scattering intensity distribution and particle number distribution results after one-step purification and two-step purification are shown in Figure 14. As is clear from the results in Figure 14, the particle size and polydispersity (monodispersity) of the Claudin4-nanodisc complex were maintained even after CPE binding, confirming its homogeneity.

[0062] Taking into account the results of Examples 2, 4, and 5 above, it was confirmed that the membrane protein-nanodisc complex obtained by the production method of the present disclosure maintained the three-dimensional structure and activity of the membrane protein.

[0063] (Confirmation of Stability of Protein-Nanodisc Complex) In this example, repeated freezing and thawing was performed to confirm the stability of the Claudin4-Nanodisc complex produced in Example 2. It was confirmed that the structure of the Claudin4-Nanodisc complex was maintained even after repeated freezing and thawing. Specifically, the Claudin4-Nanodisc complex was subjected to four or more freeze-thaw cycles. On the other hand, it was confirmed that the structure of the complex synthesized in the E. coli synthesis system was not maintained after repeated freeze-thawing. The protein-Nanodisc complex obtained by the production method of the present disclosure was able to maintain its structure even after two (three, four, five, six, seven, eight, nine, or ten) or more freeze-thaw cycles.

[0064] The production method of the present disclosure can provide a membrane protein-nanodisc complex that maintains the activity of the membrane protein.

Claims

1. A method for producing a membrane protein-nanodisc complex, comprising the step of co-expressing a membrane protein and a membrane scaffold protein in a wheat cell-free protein synthesis system comprising lipids and a wheat germ extract.

2. The method of claim 1, wherein the wheat germ extract has been treated with an inorganic compound.

3. The method according to claim 2, wherein the inorganic compound is at least one selected from alpha alumina, titanium oxide, silicon nitride, titanium nitride, aluminum hydroxide, gamma alumina, silicon dioxide, aluminum silicate, zinc oxide, cesium oxide and zirconium oxide.

4. The method according to claim 1 or 2, wherein the co-expression method is a dialysis overlay method, a dialysis method, or a overlay method.

5. The method of claim 1 or 2, wherein the lipid is a phospholipid, a glycerophospholipid, or a sphingolipid.

6. The method of claim 5, wherein the lipid is a soybean phospholipid.

7. The method of claim 2, wherein the inorganic compound is aluminum hydroxide, the co-expression method is a dialysis overlay method, and the lipid is soybean phospholipid.

8. The method according to claim 7, wherein the wheat cell-free protein synthesis system comprises a PDI family protein including protein disulfide isomerase and a catalyst for reoxidizing a substance that catalyzes a disulfide bond exchange reaction.

9. The method of claim 8, wherein the membrane scaffold protein is a modified sequence of apolipoprotein (apo) AI.

10. The method according to claim 9, wherein the amino acid sequence of the membrane scaffold protein is any one of the following 1) to 13): 1) MSP1: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 2) MSP2: FX-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 3) MSP1E1: FX-H1-H2-H3-H4-H4-H5-H6-H7-H8-H9-H10 4) MSP1E2: FX-H1-H2-H3-H4-H5-H4-H5-H6-H7-H8-H9-H10 5) MSP1E3: FX-H1-H2-H3-H4-H5-H6-H4-H5-H6-H7-H8-H9-H10 6) MSP1D1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10 7) MSP1E3D1: H1-H2-H3-H4-H5-H6-H7-H8-H9-H10 8) MSP1D2: H2-H3-H4-H5-H6-H7-H8-H9-H10 9) MSP2N1: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-11)- H2-H3-H4-H5-H6- H7-H8-H9-H10 10) MSP2N2: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT- H2-H3-H4-H5-H6-H7-H8-H9- H10 11) MSP2N3: H1Δ(1-11)-H2-H3-H4-H5-H6-H7-H8-H9-H10-GT-H1Δ(1-17)- H2-H3-H4-H5-H6-H7-H8-H9-H10 12) A sequence having an amino acid sequence that is 90% identical to any one of 1) to 11) above and has the ability to form nanodiscs, 13) A sequence comprising any one of the above sequences 1) to 11) in which 1 to 20 amino acids have been substituted, deleted, inserted and / or added, and which has the ability to form nanodiscs, wherein the amino acid sequence of the sequence is as follows: H1: LKLLDNWDSVTSTFSKLREQLG (SEQ ID NO: 1) H1△(1-11): STFSKLREQLG (SEQ ID NO: 2) H1△(1-17): REQLG (SEQ ID NO: 3)H2: PVTQEFWDNLEKETEGLRQEMS (SEQ ID NO: 4) H3: KDLEEVKAKVQ (SEQ ID NO: 5) H4: PYLDDFQKKWQEEMELYRQKVE (SEQ ID NO: 6) H5: PLRAELQEGARQKLHELQEKLS (SEQ ID NO: 7) H6: PLGEEMRDRARAHVDALRTHLA (SEQ ID NO: 8) H7: PYSDELRQRLAARLEALKENGG (SEQ ID NO: 9) H8: ARLAEYHAKATEHLSTLSEKAK (SEQ ID NO: 10) H9: PALEDLRQGLL (SEQ ID NO: 11) H10: PVLESFKVSFLSALEEYTKKLNTQ (SEQ ID NO: 12) FX: MGHHHHHHIEGR (SEQ ID NO: 13).

11. The method according to claim 7, wherein the membrane protein is a membrane protein having intramolecular or intermolecular disulfide bonds.

12. A kit for producing a membrane protein-nanodisc complex, comprising: 1) A translation or transcription template for a membrane scaffold protein; 2) A lipid; 3) A wheat cell-free protein synthesis system containing an inorganic compound-treated wheat germ extract.

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