Method for manufacturing lipid bilayer in which membrane proteins are encapsulated

Abstract

To provide a method for manufacturing a lipid bilayer in which membrane proteins are encapsulated, which does not need sophisticated techniques such as fabrication of proteoliposome, can make the probability of encapsulating membrane proteins into a lipid bilayer higher, and can make orientation of encapsulated membrane proteins uniform.SOLUTION: A method for manufacturing a lipid bilayer in which membrane proteins are encapsulated has bringing an artificially fabricated lipid bilayer in contact with an exosome containing desired membrane proteins.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for producing a lipid bilayer membrane in which a membrane protein is embedded. [Background technology]

[0002] A technique for embedding membrane proteins is known, in which liposomes incorporating membrane proteins (proteoliposomes) are fused with lipid bilayer membranes.

[0003] Proteoliposomes are prepared as follows: Membrane proteins of interest are solubilized from cultured cells expressing the protein using a detergent. The solubilized membrane proteins are then mixed with phospholipids. When the detergent concentration is reduced by dialysis or other procedures, the membrane proteins are reconstituted into liposome membranes during the liposome formation process, forming proteoliposomes.

[0004] Patent Document 1 discloses a lipid bilayer substrate having a hole formed in a substrate, the opening of the hole being covered with a lipid bilayer membrane, the hole opening having an overhanging portion extending in a direction narrowing the opening, and a membrane protein disposed in the portion of the lipid bilayer membrane covering the opening. Here, the lipid bilayer membrane on which the membrane protein is disposed is prepared by a method in which proteoliposomes are added to the lipid bilayer membrane, causing the proteoliposomes and the lipid bilayer membrane to fuse and reconstitute the membrane protein into the lipid bilayer membrane. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Japanese Patent Application Laid-Open No. 2011-160718 Summary of the Invention [Problem to be solved by the invention]

[0006] However, when using proteoliposomes to embed membrane proteins in lipid bilayer membranes, the following problems arise: The preparation of proteoliposomes requires skilled techniques; Furthermore, the activity of proteoliposomes may be reduced by treatment with surfactants; Furthermore, proteoliposomes do not easily fuse with lipid bilayer membranes, and the success rate of embedding membrane proteins is generally considered to be less than 10%.

[0007] Therefore, an object of the present invention is to provide a method for producing a lipid bilayer membrane with an embedded membrane protein, which does not require skilled techniques such as those required for producing proteoliposomes, can increase the probability of embedding a membrane protein in a lipid bilayer membrane, and can achieve uniform orientation of the embedded membrane protein. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems, and as a result have found that the above problems can be solved by a method for producing a membrane protein-embedded lipid bilayer membrane, which comprises contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein, thereby completing the present invention. [Effects of the Invention]

[0009] According to the present invention, a method for producing a lipid bilayer membrane with an embedded membrane protein can be provided, which does not require skilled techniques such as those used for producing proteoliposomes, can increase the probability of embedding a membrane protein in a lipid bilayer membrane, and can make the embedded membrane protein uniformly oriented. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram of the preparation of an artificial lipid bilayer membrane by the monolayer lamination method. [Figure 2] 10 is a graph showing the measurement results of channel current in an example. DETAILED DESCRIPTION OF THE INVENTION

[0011] Hereinafter, an embodiment according to one aspect of the present invention will be described, but the present invention is not limited to only the following embodiment.

[0012] In this specification, the range "X to Y" means "X or more and Y or less." Unless otherwise specified, operations and measurements of physical properties are performed at room temperature (20 to 25°C) and a relative humidity of 40 to 50% RH.

[0013] One aspect of the present invention is a method for producing a lipid bilayer membrane having a membrane protein embedded therein, the method comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein.

[0014] Another aspect of the present invention is a method for embedding a membrane protein in a lipid bilayer membrane, comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein.

[0015] Another aspect of the present invention is a method for controlling the orientation of a membrane protein in a lipid bilayer membrane, comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein and embedding the membrane protein in the lipid bilayer membrane.

[0016] According to the present invention, it is possible to increase the probability of embedding a membrane protein in a lipid bilayer membrane and to make the orientation of the embedded membrane protein uniform, without requiring skilled techniques such as those used to prepare proteoliposomes.

[0017] The present inventors have found for the first time that a desired membrane protein (e.g., a GABA receptor) is expressed in the membrane of exosomes secreted from cells expressing the desired membrane protein. Contacting these exosomes with a lipid bilayer membrane results in spontaneous fusion between the exosomes and the lipid bilayer membrane, resulting in the embedding of the membrane protein within the lipid bilayer membrane. Proteoliposomes do not readily fuse with lipid bilayer membranes, and the success rate of membrane protein embedding is generally considered to be less than 10%. On the other hand, exosomes fuse more readily with lipid bilayer membranes than proteoliposomes, resulting in a success rate of membrane protein embedding in the present invention exceeding 80%. Furthermore, since exosomes containing the desired membrane protein are secreted from cells expressing the desired membrane protein, as described above, they are easily recovered and do not require the skilled techniques required for the preparation of proteoliposomes. Furthermore, the present inventors have found that the embedded membrane protein is uniformly oriented. This is believed to be made possible by exosome membrane fusion. It is recognized that membrane proteins in exosomes secreted from cells have a uniform orientation. In this invention, by artificially reproducing the in vivo phenomenon of membrane fusion, a method for embedding membrane proteins with controlled orientation can be constructed. Although the mechanism of membrane fusion has not been elucidated, it is thought to be as follows: When lipid bimolecules and exosomes come into contact, membrane fusion occurs and a fusion pore is formed. As this pore widens, the membrane fusion region expands. If membrane fusion proceeds via this mechanism, the orientation of the embedded membrane proteins depends on the orientation of the membrane proteins within the exosome membrane; that is, the embedded membrane proteins are thought to have a uniform orientation, similar to that of the membrane proteins within the exosome membrane.

[0018] The above mechanism is speculation, and the technical scope of the present invention is not limited to the above mechanism.

[0019] The constituent elements of the method for producing a lipid bilayer membrane in which a membrane protein is embedded according to the present invention will be described below.

[0020] The present invention involves contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein.

[0021] As used herein, the term "lipid bilayer membrane" refers to a membrane formed by amphipathic lipid molecules, each having a polar head group and a hydrophobic hydrocarbon chain, arranged with the polar head group on the outside and the hydrophobic hydrocarbon chain on the inside to form a bilayer structure.

[0022] In this specification, an "artificially produced lipid bilayer membrane" is also simply referred to as an "artificial lipid bilayer membrane."

[0023] In the present invention, the lipid molecules used in the artificial lipid bilayer membrane are not particularly limited as long as they can form a lipid bilayer membrane. Examples of lipid molecules include 1,2-dioleoyl-3-trimethylammonium propane, 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), diphytanylphosphatidylcholine (DPhPC), phosphatidylcholine (PC), egg yolk phosphatidylcholine, phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol phosphate (PIP), phosphatidic acid (PA), phosphatidylglycerol (PG), sphingolipids, cholesterol, and ergosterol. The lipid molecule is preferably selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), diphytanylphosphatidylcholine (DPhPC), phosphatidylcholine (PC), egg yolk phosphatidylcholine and cholesterol, more preferably 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), diphytanylphosphatidylcholine (DPhPC), phosphatidylcholine (PC), egg yolk phosphatidylcholine and cholesterol. The lipid molecules may be a combination of cholesterol and at least one selected from the group consisting of diphytanyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dihexadecanoyl-sn-glycero-3-phosphocholine (DPPC), diphytanylphosphatidylcholine (DPhPC), diphytanylphosphatidylcholine (DPhPC), phosphatidylcholine (PC), and egg yolk phosphatidylcholine, and more preferably a combination of diphytanylphosphatidylcholine (DPhPC), egg yolk phosphatidylcholine, and cholesterol. Only one type of lipid molecule may be used, or two or more types may be used.

[0024] The artificial lipid bilayer may be planar or vesicle-shaped. In one embodiment of the present invention, the artificially produced lipid bilayer is a planar lipid bilayer.

[0025] The method for producing an artificial lipid bilayer membrane is not particularly limited, and conventionally known methods can be used. When the artificial lipid bilayer membrane is a planar lipid bilayer membrane, the planar lipid bilayer membrane can be produced by a method such as a monolayer lamination method or a tip-dip method.

[0026] The monolayer lamination method is a method for creating a lipid bilayer membrane by spreading a lipid solution on the water surface of two aqueous phases separated by a film (e.g., Teflon film) with pores (e.g., 50-100 μm in diameter). The water surface is then gently raised, and the monolayers are laminated together within the pores.

[0027] A lipid solution can be prepared by mixing lipid molecules with an organic solvent (e.g., decane, hexadecane, hexane, chloroform, etc.). The type and concentration of lipid molecules can be adjusted appropriately depending on the lipid molecules used (or the combination thereof if two or more types are used). The concentration of lipid molecules in the lipid solution is, for example, 4 to 40 mg / mL. When two or more types of lipid molecules are used, the molar ratio between them can be adjusted appropriately. For example, when diphytanyl phosphatidylcholine (DPhPC), egg yolk phosphatidylcholine, and cholesterol are used as lipid molecules, the molar ratio between them (diphytanyl phosphatidylcholine (DPhPC):egg yolk phosphatidylcholine:cholesterol) is 1 to 4:5 to 8:1, preferably 2 to 3:6 to 7:1.

[0028] The tip-dip method involves forming a lipid membrane on the water surface while the tip of a pipette is immersed in the solution in a chamber, then lifting the pipette into the air and pressing it back down onto the water surface to create a lipid bilayer membrane at the tip of the pipette.

[0029] In one embodiment, the artificial lipid bilayer membrane according to the present invention is prepared by a monolayer lamination method.

[0030] Figure 1 shows an outline of the fabrication of an artificial lipid bilayer membrane by the monolayer lamination method. In Figure 1, the device has a chamber (preferably a Teflon chamber) consisting of two compartments, and the two compartments are separated by a film (preferably a Teflon film) with holes of 50 to 100 μm in diameter.

[0031] An example of the process for preparing an artificial lipid bilayer is shown below (see Figure 1): (1) Apply hexadecane around the pores of the film; (2) A solution (usually a buffer) is added to the compartments on both sides of the chamber; (3) placing a stirrer in compartment 1 and electrodes (e.g., Ag / AgCl electrodes) and syringes (not shown) in both compartments 1 and 2; (4) Using two syringes, adjust the water level of the solution in the chamber so that it is below the pores in the film; (5) The lipid solution is added to compartments 1 and 2. After a predetermined time (e.g., 10 minutes) has elapsed since the addition, the syringe is slowly depressed to raise the buffer water level in the chamber and create an artificial lipid bilayer membrane.

[0032] The formation of a planar lipid bilayer can be confirmed by checking whether there is a small change in the baseline when the voltage is changed to 100 mV and then to 0 mV.

[0033] The solution used in the above preparation step is not particularly limited, and examples thereof include an aqueous potassium chloride solution, a phosphate buffer solution, an acetate buffer solution, and a HEPES buffer solution.

[0034] In the present invention, exosomes contain a desired membrane protein, which is not particularly limited and includes, for example, ion channel receptors, voltage-dependent channels (sodium channels, potassium channels, calcium channels, chloride channels, etc.), transporters, G protein-coupled receptors, etc.

[0035] Examples of ionotropic receptors include GABA receptors, TRP (transient receptor potential) channels, ATP receptors, serotonin receptors, NMDA receptors, AMPA receptors, and kainate receptors.

[0036] The cells from which exosomes are derived are not particularly limited and can be appropriately selected depending on the desired protein. For example, when exosomes containing GABA receptors are used, the cells from which exosomes are derived can be a human embryonic kidney cell line (e.g., human embryonic kidney cell 293 (HEK293)) in which GABA receptors are overexpressed, or Chinese hamster ovary (CHO) cells.

[0037] The method for expressing a desired membrane protein in cells from which exosomes are derived is not particularly limited, and conventionally known methods can be used. For example, a method for expressing a desired membrane protein includes a method in which the desired protein is forcibly expressed by genetic recombination in cells. By forcibly expressing the desired protein, the desired membrane protein can be expressed in the exosome membrane.

[0038] If the desired protein is naturally expressed in exosomes, the above procedure does not need to be performed.

[0039] Whether the exosomes contain the desired membrane protein can be confirmed by immunoassay.

[0040] In the present invention, the method for contacting an artificial lipid bilayer membrane with exosomes containing a desired membrane protein is not particularly limited as long as the artificial lipid bilayer membrane and exosomes containing the desired membrane protein can be brought into contact with each other. When the artificial lipid bilayer membrane and exosomes containing the desired membrane protein come into contact with each other, the desired membrane protein is incorporated by membrane fusion.

[0041] For example, when using the device shown in Figure 1, after preparing an artificial lipid bilayer, exosomes containing a membrane protein are added to one compartment (e.g., compartment 1). This allows the artificial lipid bilayer to come into contact with exosomes containing the desired membrane protein. When adding exosomes containing a membrane protein, exosomes containing recovered membrane proteins may be added, or culture medium from cells from which the exosomes containing membrane proteins are derived may be added. After adding the exosomes containing membrane proteins, they may be stirred as needed. The exosomes added may contain only one type of membrane protein, two or more types of membrane proteins, or exosomes containing different membrane proteins, or a combination of these.

[0042] The method for preparing a culture medium for cells from which exosomes containing membrane proteins are derived is not particularly limited, and conventionally known methods can be appropriately adopted depending on the cells used.

[0043] When using exosomes containing recovered membrane proteins, the exosome recovery method is not particularly limited and includes ultracentrifugation, PEG precipitation, immunoprecipitation, affinity precipitation with magnetic beads using Tim4 (an exosome receptor expressed in macrophages), density gradient centrifugation, etc. Commercially available exosome isolation reagents (kits) may be used to isolate and recover exosomes.

[0044] The time for which the artificial lipid bilayer membrane is brought into contact with exosomes containing a desired membrane protein is not particularly limited and is, for example, 1 to 2 hours. By extending the contact time, the number of membrane proteins embedded in the artificial lipid bilayer membrane can be increased.

[0045] The temperature at which the artificial lipid bilayer membrane is brought into contact with exosomes containing a desired membrane protein may be room temperature (20 to 25°C) or 15 to 40°C.

[0046] The pH when contacting the artificial lipid bilayer with exosomes containing desired membrane proteins is 3.0 to 8.0, preferably 4.5 to 7.5, and more preferably 5.0 to 6.0. Lowering the pH allows for a greater number of embedded membrane proteins.

[0047] Whether a desired membrane protein has been embedded in an artificial lipid bilayer membrane can be confirmed by referring to a conventionally known method, depending on the type of the embedded membrane protein. For example, if the desired membrane protein is an ionotropic receptor, the structure of the ionotropic receptor changes upon binding to a signal molecule, allowing specific ions to pass through and resulting in a change in membrane potential. Therefore, whether the ionotropic receptor has been embedded in an artificial lipid bilayer membrane can be confirmed by measuring the change in membrane potential.

[0048] The production method of the present invention makes it possible to embed a desired membrane protein in a lipid bilayer. Furthermore, the lipid bilayer membrane with an embedded membrane protein obtained by the production method of the present invention can achieve uniform orientation of the embedded membrane protein. The artificial lipid bilayer membrane of the present invention is suitable for applications such as the development of drug discovery screening methods targeting membrane proteins; the creation of artificial cells and the establishment of cell-free assay methods for evaluating the biological effects of environmental pharmaceuticals using these cells; the development of taste sensors; the establishment of technology for modifying cellular functions by delivering membrane proteins into cells; the development of ultrasensitive measurement technology that mimics the amplification function of neurons; and the creation of intracellular and extracellular compartment devices.

[0049] <Embodiment> The following describes an embodiment of the present invention. [1] A method for producing a lipid bilayer membrane having a membrane protein embedded therein, the method comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein; [2] The method according to the above [1], wherein the artificially produced lipid bilayer membrane is a planar lipid bilayer membrane; [3] A method for embedding a membrane protein in a lipid bilayer membrane, comprising contacting an artificially prepared lipid bilayer membrane with exosomes containing a desired membrane protein; [4] A method for controlling the orientation of a membrane protein in a lipid bilayer, comprising contacting an artificially produced lipid bilayer with exosomes containing a desired membrane protein and embedding the membrane protein in the lipid bilayer. [Example]

[0050] The present invention will be described below using specific examples, but the present invention is not limited to these examples.

[0051] Preparation of planar lipid bilayers Planar lipid bilayer membranes were fabricated by the monolayer lamination method using the device (length 28.60 mm, width 37.10 mm, height 18.55 mm) shown in Figure 1. The device has a Teflon chamber consisting of two compartments, separated by a Teflon film with pores of 50 to 100 μm in diameter.

[0052] Planar lipid bilayers were prepared by the following method: (1) Hexadecane was applied around the pores of the Teflon film; (2) 70 μL of 150 mM potassium chloride aqueous solution (buffer) was added to the compartments on both sides of the chamber; (3) A stirrer was placed in compartment 1, and Ag / AgCl electrodes and syringes were placed in compartments 1 and 2; (4) Using two syringes, the buffer water level in the chamber was adjusted to be below the pores in the Teflon film; (5) 5 μL of a 7.0 mg / mL lipid solution (diphytanylphosphatidylcholine (DPhPC):egg yolk phosphatidylcholine:cholesterol = 2.25:6.75:1 (molar ratio)) in hexane / chloroform solution (hexane:chloroform = 1:1 (volume ratio)) was added to compartments 1 and 2. 10 minutes after addition, the syringe was slowly depressed to raise the water level of the buffer in the chamber, creating a planar lipid bilayer membrane.

[0053] The formation of a planar lipid bilayer was confirmed by checking whether there was a small change in the baseline when the voltage was changed to 100 mV and then to 0 mV.

[0054] Exosome purification A qEV original35 column (Meiwafosis Co., Ltd.) was attached to a qEV Auto Fraction Collector (AFC) (Meiwafosis Co., Ltd.). According to the AFC manual, 500 μL of culture medium from a human embryonic kidney cell line (HEK293) overexpressing gamma-aminobutyric acid (GABA) receptors was added, followed by the addition of 5 mL of 150 mM potassium chloride solution (buffer) to purify (recover) exosomes.

[0055] The expression of GABA receptors on the exosome membrane was confirmed by immunoassay. Specifically, exosomes were added to a flat substrate (microplate well) on which anti-CD63 antibodies were immobilized. Fluorescently labeled anti-GABAa receptors were then bound to the exosomes captured on the flat substrate. After washing away unbound fluorescently labeled antibodies, the fluorescence intensity was measured. Fluorescence observation confirmed the expression of GABA receptors on the exosome membrane.

[0056] Embedding of membrane proteins into planar lipid bilayers 25 μL of purified exosomes were added to compartment 1 of the device in which the planar lipid bilayer had been prepared. The pH in compartment 1 was 5.5. After addition, the mixture was stirred for 2 hours to obtain a planar lipid bilayer with embedded GABA receptors.

[0057] Confirmation of membrane protein embedding and evaluation of orientation GABA was added to compartment 1 or 2 of the device containing a planar lipid bilayer membrane embedded with GABA receptors at a concentration of 1.4 mM and stirred for 20 minutes. A voltage of 100 mV was then applied, and the channel current was measured for 15 minutes. The results are shown in Figure 2.

[0058] As shown in Figure 2, when GABA was added to compartment 1 containing exosomes, a square-wave channel current was observed (Figure 2A). This indicates that a planar lipid bilayer membrane embedded with GABA receptors was obtained.

[0059] On the other hand, when GABA was added to compartment 2, which did not contain exosomes, no square-wave channel current was observed (Figure 2B).

[0060] When GABA binds to its receptor outside the cell, the channel opens and Cl - flows from the outside of the cell into the inside of the cell. The current that flows at this time is the channel current. The fact that channel current was observed only when GABA was added to compartment 1 indicates that the orientation of the embedded membrane protein (GABA receptor) can be uniformly controlled. Therefore, it is clear that the artificial outside of the cell (compartment 1) and the inside of the artificial cell (compartment 2) can be clearly distinguished.

[0061] Using the same procedure as above, 104 planar lipid bilayer membranes embedded with GABA receptors were prepared, and the embedding of membrane proteins and the orientation were evaluated. 88 out of 104 experiments were similar to the results above, so that planar lipid bilayer membranes embedded with GABA receptors were obtained, and the orientation of GABA receptors was uniform. From this, it can be seen that the method for producing lipid bilayer membranes embedded with membrane proteins according to the present invention can increase the probability of embedding membrane proteins in lipid bilayer membranes, and can also make the orientation of membrane proteins uniform.

Claims

1. A method for producing a lipid bilayer membrane having a membrane protein embedded therein, comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein.

2. The method of claim 1 , wherein the artificially prepared lipid bilayer is a planar lipid bilayer.

3. A method for embedding a membrane protein in a lipid bilayer membrane, comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein.

4. A method for controlling the orientation of a membrane protein in a lipid bilayer membrane, comprising contacting an artificially produced lipid bilayer membrane with exosomes containing a desired membrane protein and embedding the membrane protein in the lipid bilayer membrane.

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