JP7253198B2 - Method for manufacturing lipid membrane device - Google Patents

Description

TECHNICAL FIELD The present invention relates to a lipid membrane device and a method for manufacturing a lipid membrane device.

Membrane proteins present in cell membranes are diverse and are targets for drug discovery. Since membrane proteins are difficult to handle, there is a problem that their functional analysis requires a huge amount of time and money. If we can create an ultra-compact biochip in which membrane proteins are arrayed on a semiconductor substrate, it will be possible to analyze the functions of many membrane proteins simultaneously at high speed, thereby shortening the time and cost required for new drug development. Many other effects are expected.

As a method of analyzing membrane proteins on a substrate, there is a method of arranging cells themselves on a substrate and performing functional measurement by a patch clamp method. However, there are usually multiple membrane proteins other than the target membrane protein on the cell membrane, and it is unclear whether the response obtained by stimulation with a certain drug is due to the target membrane protein. I had a problem. Therefore, construction of an in vitro measurement system using purified membrane proteins is demanded from the industrial and practical aspects of research.

As a representative method for arranging membrane proteins in vitro, a black membrane is formed by dissolving lipid molecules in an organic solvent such as n-decane and applying the lipid solution in an aqueous solution to small holes provided on a substrate. In addition, there is a method of fusing membrane proteins (see, for example, Non-Patent Document 1). However, it has been pointed out that the residual organic solvent and heterogeneity of the formed black membrane may affect the bioactivity of the membrane protein in this method.

In order to solve these problems, the present inventors artificially produced well structures on a substrate such as silicon, developed giant lipid membrane vesicles in the well openings, and artificially A technique for introducing membrane proteins onto the lipid bilayer membrane was proposed (Non-Patent Document 2). The artificial lipid bilayer membrane substrate thus prepared does not contain an organic solvent because it uses giant lipid membrane vesicles. The activity of the membrane protein reconstituted in this lipid membrane can be confirmed by an optical technique (fluorescence observation).

"Latest patch clamp experimental technique" edited by Yasunobu Okada (Yoshioka Shoten, 2011) Sumitomo K et al., Biosensors and Bioelectronics, vol.31, 445-450, 2012.

In order to introduce membrane proteins into artificial lipid bilayer membranes, there is a method of fusing membrane fractions containing membrane proteins or proteoliposomes (lipid vesicles in which membrane proteins are reconstituted) to artificial lipid bilayer membranes. However, this technique has the problem that the orientation of membrane proteins in the artificial lipid bilayer membrane cannot be controlled. Since membrane proteins are related to their function and orientation (orientation), orientation control is necessary to correctly analyze the function of membrane proteins.

In view of the above circumstances, an object of the present invention is to provide a technique for controlling the orientation of membrane proteins in an artificial lipid bilayer membrane.

One aspect of the present invention is a lipid membrane comprising a substrate having a well structure with an opening on one side, and a lipid bilayer membrane formed on the one side of the substrate and sealing the opening of the well structure. fusing the lipid bilayer membrane of the device with a lipid vesicle comprising a membrane protein within the lipid membrane, such that the membrane protein is introduced into the lipid bilayer membrane. A method for producing a lipid membrane device into which a membrane protein is introduced.

In one aspect of the present invention, a lipid vesicle containing a membrane protein in a lipid membrane is brought into contact with the one surface of a substrate having a well structure that is open on one surface, and as a result, the lipid vesicle is cleaved. to form a lipid bilayer membrane containing the membrane protein, to form a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane formed on the one surface to seal the opening of the well structure. A method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane, comprising the step of

Another aspect of the present invention is a method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane described above, wherein the lipid vesicle is a budding vesicle derived from a virus expressing the membrane protein. is.

Another aspect of the present invention is a method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane described above, wherein the lipid vesicle is a budding vesicle derived from a virus expressing the membrane protein. and giant lipid vesicles.

In one aspect of the present invention, a first lipid vesicle is brought into contact with the one surface of a substrate having a plurality of well structures opening on one surface, and as a result, the first lipid vesicle is cleaved. forming a first lipid bilayer membrane and sealing a part of the openings of the well structure formed on the one surface; fusing a second lipid vesicle containing a first membrane protein within the membrane, such that the first membrane protein is introduced into the first lipid bilayer membrane at the opening; The one side of the substrate is contacted with a third lipid vesicle containing a second membrane protein within a lipid membrane, such that the third lipid vesicle is cleaved to contain the second membrane protein. forming a second lipid bilayer membrane on the one surface to seal the opening of the well structure, and introducing a second membrane protein into the second lipid bilayer membrane; A method for producing a lipid membrane device in which the first membrane protein and the second membrane protein are introduced into a lipid bilayer membrane, comprising:

Further, one aspect of the present invention comprises a substrate having a well structure with an opening on one surface, and a lipid bilayer membrane sealing the opening of the well structure, wherein the lipid bilayer membrane contains a membrane protein. , wherein the extracellular domain of the membrane protein is arranged outside the sealed well structure and the intracellular domain of the membrane protein is arranged inside the sealed well structure. is.

Further, one aspect of the present invention comprises a substrate having a well structure with an opening on one surface, and a lipid bilayer membrane sealing the opening of the well structure, wherein the lipid bilayer membrane contains a membrane protein. , wherein the extracellular domain of the membrane protein is arranged inside the sealed well structure, and the intracellular domain of the membrane protein is arranged outside the sealed well structure. is.

Further, one aspect of the present invention provides a substrate having a first well structure and a second well structure that are open on one surface, a first lipid bilayer membrane that seals the first opening, a second lipid bilayer that seals the second opening, wherein the first lipid bilayer includes a first membrane protein; and the second lipid bilayer is a second lipid bilayer. a membrane protein, wherein the extracellular region of the first membrane protein is located outside the sealed well structure, and the intracellular region of the first membrane protein is located outside the sealed well structure. wherein the extracellular domain of the second membrane protein is located inside the sealed well structure and the intracellular domain of the second membrane protein is located inside the sealed well structure It is a lipid membrane device that is located outside the structure.

The present invention makes it possible to control the orientation of membrane proteins in an artificial lipid bilayer membrane.

When a membrane protein expressed in cells, viruses, etc. is simply solubilized and then reconstituted on a lipid membrane, the membrane protein on the lipid membrane can assume two orientations. In this case, it is difficult to control the orientation of the membrane protein in the lipid membrane, and the orientation of the membrane protein becomes random.

According to the present invention, a membrane protein is introduced into a lipid bilayer membrane using a virus-derived budding vesicle (a budding virus particle formed by an enveloped virus and/or its enucleated particle) expressing a desired membrane protein on its surface. can control the orientation (biased orientation) of the introduced membrane protein.

As used herein, “controlling the orientation” means controlling the orientation of membrane proteins in the lipid membrane so that they are biased in a single direction. It can be said that the higher the orientation of the membrane protein, that is, the stronger the orientation of the membrane protein and the more biased toward a single direction, the better the orientation can be controlled.

Membrane proteins expressed in viruses are present on the surface of budding vesicles in the same orientation as in cells in which the membrane proteins are naturally expressed.
On the surface of budding vesicles, besides the desired expressed membrane proteins, there is GP64, an envelope protein required for viral infection. GP64 is a protein involved in membrane fusion.
GP64 specifically fuses to negatively charged lipids such as PS (phosphatidylserine). Upon fusion with the lipid membrane, the orientation of the introduced protein maintains the orientation it had when present in the budding vesicle.

When a budding virus is fused to a lipid bilayer membrane containing negatively charged lipids formed on the well structure, the orientation of the membrane proteins is maintained, and the orientation outside the well structure is considered to be the outside of the cell.
On the other hand, when a budding virus is fused to a giant lipid vesicle, the orientation of membrane proteins is similarly maintained and introduced. When budding virus-fused giant lipid vesicles are spread on the well structure, the outer side of the giant lipid vesicle contacts the substrate side and the inner side appears on the surface, thus reversing the orientation of the membrane proteins.
In the case of fusing budding vesicles to lipid membranes bridging well structures (1) and in the case of developing giant lipid vesicles fusing budding vesicles (2), membrane proteins can be arranged in opposite directions. By selectively using these two introduction methods or by combining them, it becomes possible to control the orientation of membrane protein introduction into the lipid membrane that bridges the well structure.

(a) is a cross-sectional view of a budding vesicle 50a containing a membrane protein 60 within a lipid membrane 50. FIG. (b) is a front sectional view near the well structure 20 of the lipid membrane device 1. FIG. (c) is a front sectional view near the well structure 20 of the lipid membrane device 1A into which the membrane protein 60 is introduced. (a) is a schematic diagram showing how a budding vesicle 50a fuses with a giant lipid vesicle 70 to synthesize a giant lipid vesicle 70a containing a membrane protein 60 in a lipid membrane 70. FIG. (b) is a front cross-sectional view of the vicinity of the well structure 20 of the substrate 10. FIG. (c) is a schematic diagram of lipid membrane device 1B after opening 21 of well structure 20 of substrate 10 is sealed by giant lipid vesicle 70a having membrane protein 60 in the lipid membrane. It is a front cross-sectional view near the well structure 20 of the lipid membrane device 1C. FIG. 2 is a schematic diagram of a lipid membrane device in which electrodes are arranged; (a) is a fluorescence image obtained by photographing a device in which a well structure is sealed with a lipid membrane, excited by a 488 nm laser, and photographed from above. (b) is a fluorescence image excited by a 561 nm laser. (a) is a fluorescence image obtained by photographing a device in which a well structure is sealed with a lipid membrane containing membrane proteins, excited by a 488 nm laser, and photographed from above. (b) is a fluorescence image excited by a 561 nm laser. It is the result of having measured the fluorescence intensity of RFP in the dashed line of FIG.5(b) and FIG.6(b).

[Production method]
(First embodiment)
In one embodiment, the present invention provides a lipid membrane device comprising a substrate having a well structure with an opening on one side, and a lipid bilayer membrane formed on one side of the substrate and sealing the opening of the well structure. introducing a membrane protein into a lipid bilayer membrane, comprising fusing a lipid bilayer membrane of the lipid membrane with a lipid vesicle containing a membrane protein within the lipid membrane, resulting in introduction of the membrane protein into the lipid bilayer membrane. A method for manufacturing a lipid membrane device is provided.

A method for manufacturing a lipid membrane device 1A according to one embodiment of the present invention will be described with reference to the drawings. A lipid membrane device has a well structure on one surface (upper surface) of a substrate.
FIG. 1(a) is a cross-sectional view of a budding vesicle 50a containing a membrane protein 60 within a lipid membrane 50. FIG. FIG. 1(b) is a front sectional view of the vicinity of the well structure 20 of the lipid membrane device 1. FIG. FIG. 1(c) is a front sectional view near the well structure 20 of the lipid membrane device 1A into which the membrane protein 60 is introduced.
In addition, in FIG.1(b), (c), it demonstrates as an upper side and a lower side as a bottom.
One side refers to the direction in which the well structure 20 of the substrate 10 is open.

In this embodiment, the lipid vesicle containing the membrane protein may be a budding vesicle 50a whose secretion is induced by a virus, as described below.
As illustrated in FIG. 1( a ), a budding vesicle 50 a contains membrane proteins 60 within a lipid membrane 50 . The transmembrane region of membrane protein 60 is embedded within lipid membrane 50 .
In the budding vesicle 50a where the virus induces secretion, the orientation of the membrane proteins 60 is uniform. That is, the outer portion of the lipid membrane 50 of the membrane protein 60 possessed by the budding vesicle 50a is the extracellular region of the membrane protein 60 . The inner portion of the lipid membrane 50 of the membrane protein 60 of the budding vesicle 50 a is the intracellular region of the membrane protein 60 .

As exemplified in FIG. 1(b), the lipid membrane device 1 has a substrate 10 and a well structure 20 which is a recess in the upper surface of the substrate 10, and the opening 21 of the well structure 20 is the lipid bilayer 30. is sealed by
A thin film layer 11 may be laminated between the upper surface of the substrate 10 and the lipid bilayer membrane 30 . The end (overhang portion 11 a ) of the thin film layer 11 may extend into the opening 21 of the well structure 20 .

By carrying out a step of fusing budding vesicles (lipid vesicles) 50 a containing membrane proteins 60 in the lipid membrane 50 to the lipid bilayer membrane 30 at the opening 21 of the well structure 20 , the membrane protein 60 is transferred to the opening 21 . can be introduced into the lipid bilayer membrane 30 of

FIG. 1(c) is a schematic diagram showing a state after the membrane protein 60 of the budding vesicle (lipid vesicle) 50 fuses with the lipid bilayer membrane 30 sealing the opening 21. FIG.
When the budding vesicle 50a is fused to the lipid bilayer membrane 30, the extracellular domain of the membrane protein 60 is arranged outside the lipid bilayer membrane 30 in the opening 21 (outside the well structure 20), and the intracellular domain of the membrane protein 60 is The region is located inside the lipid bilayer membrane 30 of the opening 21 (inside the well structure 20).
In the present specification, the inside of the well structure 20 means an internal space below the lipid bilayer membrane 30 and closed by the well structure 20 and the lipid bilayer membrane 30 . Also, the outside of the well structure 20 means the space above the lipid bilayer membrane 30 .
That is, the membrane protein 60 of the lipid bilayer membrane 30 in the opening 21 maintains the orientation in the budding vesicle 50a and the orientation of the membrane protein in the cell.

In this embodiment, in order to fuse budding vesicles 50a containing membrane protein 60 within lipid membrane 50 to lipid bilayer membrane 30 of opening 21, the external dimensions of budding vesicles 50a are larger than the external dimensions of opening 21. is preferably set to be small.

According to the manufacturing method of this embodiment, the following lipid membrane device (lipid membrane device 1A) can be manufactured.
A substrate having a well structure open on one side and a lipid bilayer membrane sealing the opening of the well structure, the lipid bilayer membrane containing a membrane protein, the extracellular region of the membrane protein being sealed A lipid membrane device, wherein the intracellular region of the membrane protein is located inside the sealed well structure, and the membrane protein is located outside the well structure.

(Second embodiment)
In one embodiment, the present invention brings lipid vesicles containing a membrane protein into a lipid membrane into contact with one side of a substrate having a well structure that is open on one side, and as a result, the lipid vesicles are cleaved. forming a lipid bilayer membrane containing a membrane protein, forming a lipid membrane device on one side to seal the opening of the well structure, and forming a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane. Provided is a method for producing a lipid membrane device in which a membrane protein is introduced into the double membrane.

A method for manufacturing a lipid membrane device 1B according to one embodiment of the present invention will be described with reference to the drawings. FIG. 2(a) is a schematic diagram showing how a budding vesicle 50a fuses with a giant lipid vesicle 70 to synthesize a giant lipid vesicle 70a containing a membrane protein 60 in a lipid membrane 70. FIG. FIG. 2(b) is a front cross-sectional view of the vicinity of the well structure 20 of the substrate 10. FIG. FIG. 2(c) is a schematic diagram of the lipid membrane device 1B after the opening 21 of the well structure 20 of the substrate 10 is sealed by a giant lipid vesicle 70a having membrane proteins 60 within the lipid membrane.
In addition, in FIG.2(b), (c), it demonstrates as an upper side and a lower side as a bottom.
One side refers to the direction in which the well structure 20 of the substrate 10 is open.

In this embodiment, lipid vesicles containing membrane proteins may be giant lipid vesicles (lipid vesicles) 70a, as described below.
The giant lipid vesicle 70a may be obtained, for example, by fusing the budding vesicle 50a containing the membrane protein 60 described above with the giant lipid vesicle 70 described below. Since the orientation of the membrane proteins 60 is uniform in the budding vesicles 50a, the orientation of the membrane proteins 60 is maintained in the giant lipid vesicles 70a obtained by fusing the budding vesicles 50a with the giant lipid vesicles 70 as well. The outer portion of the lipid membrane 70 of the membrane protein 60 of the resulting giant lipid vesicle 70a is the extracellular region of the membrane protein 60 . The inner portion of the lipid membrane 70 of the membrane protein 60 of the resulting giant lipid vesicle 70 a is the intracellular region of the membrane protein 60 .

The giant lipid vesicle 70a introduced with the membrane protein 60 described above is brought into contact with the upper surface of the substrate 10 to cleave the giant lipid vesicle 70a.
As a result, the lipid bilayer 30 containing the membrane protein 60 seals the opening 21 of the well structure 20, as illustrated in FIG. 2( c ). In this way, a lipid membrane device 1B in which the membrane protein 60 is introduced into the lipid bilayer membrane 30 is formed.

In the lipid membrane device 1B described above, the extracellular region of the membrane protein 60 is arranged inside the lipid bilayer membrane 30 of the opening 21 (inside the well structure 20), and the intracellular region of the membrane protein 60 is arranged inside the opening 21. It is arranged outside the lipid bilayer membrane 30 (outside the well structure 20).

In this embodiment, the outer dimensions of the lipid vesicle 70a are such that the lipid vesicle 70a is opened to cleave and seal the lipid bilayer 30 at the opening 21, which contains the membrane protein 60 within the lipid membrane 70. It is necessary to set it larger than the outer dimensions of the portion 21 . If the outer dimensions of the giant lipid vesicle 70 a are smaller than the outer dimensions of the opening 21 , the giant lipid vesicle 70 a falls inside the well structure 20 and cannot cover the opening 21 .

By the manufacturing method of this embodiment, the following lipid membrane device (lipid membrane device 1B) can be manufactured.
A substrate having a well structure open on one side and a lipid bilayer membrane sealing the opening of the well structure, the lipid bilayer membrane containing a membrane protein, the extracellular region of the membrane protein being sealed A lipid membrane device, wherein the intracellular region of the membrane protein is located outside the sealed well structure.

(Third Embodiment)
In one embodiment, the present invention brings a first lipid vesicle into contact with one side of a substrate having a plurality of well structures open on one side, as a result of which the first lipid vesicle is cleaved. A step of forming a first lipid bilayer membrane and sealing an opening of a part of the well structure formed on one surface; fusing a second lipid vesicle containing a membrane protein such that the first membrane protein is introduced into the first lipid bilayer membrane at the opening; contacting a third lipid vesicle containing a second membrane protein, such that the third lipid vesicle is cleaved into a second lipid bilayer containing the second membrane protein, and forming to seal the opening of the well structure and introducing the second membrane protein into the second lipid bilayer membrane. Provided is a method for producing a lipid membrane device into which a membrane protein is introduced.

A method for manufacturing a lipid membrane device 1C according to one embodiment of the present invention will be described with reference to the drawings. FIG. 3 is a front cross-sectional view near the well structure 20 of the lipid membrane device 1C formed from the budding vesicle 50a and the giant lipid vesicle 70. FIG. In the lipid membrane device 1C, a first membrane protein 60 and a second membrane protein 61 are introduced into the lipid bilayer membrane 30 that seals the opening 21 .
In this embodiment, the membrane proteins 60 and 61 can be arranged in different directions for each well structure 20 on the same substrate 10 . In each well structure 20, the membrane proteins 60 and 61 are aligned.
Membrane protein 60 and membrane protein 61 may be the same protein or may be different proteins.

In this embodiment, the same substrate 10 is used, and the membrane protein 60 is introduced into the lipid bilayer membrane of the opening 21 of the well structure 20 by the manufacturing method described above in the first embodiment. A membrane protein 61 is introduced into the opening 21 of the well structure 20 by the manufacturing method described above. This will be explained in detail.

First, the one surface of the substrate having a plurality of well structures 20 is brought into contact with the first lipid vesicles 70 having no membrane protein 60 . The first lipid vesicle 70 may be a giant lipid vesicle as described below.
As a result, the first lipid vesicles 70 are cleaved to form the first lipid bilayer membrane 30, sealing the openings 21 of some of the well structures 20 on the substrate 10. . That is, at this stage, on the substrate 10, the well structure 20 in which the opening 21 is sealed by the lipid bilayer 30 and the well structure 20 in which the opening 21 is not sealed by the lipid bilayer are formed. , are present on the substrate 10 .

Subsequently, the first lipid bilayer membrane sealing the opening 21 is fused with a second lipid vesicle containing the first membrane protein 60 within the lipid membrane. The second lipid vesicle may be a budding vesicle 50a as described above.
As a result, the first membrane protein 60 is introduced into the first lipid bilayer membrane sealing the opening 21 . The orientation of the first membrane protein 60 is the same as the orientation of the membrane protein 60 in the opening 21 in the first embodiment.
At this stage, there is a well structure 20 on the substrate 10 in which the openings 21 are not sealed by the lipid bilayer membrane.

Subsequently, one side of the substrate 10 is brought into contact with a third lipid vesicle containing a second membrane protein 61 in the lipid membrane. The third lipid vesicle may be the giant lipid vesicle 70a described above.
As a result, the third lipid vesicle is cleaved into a second lipid bilayer membrane containing the second membrane protein 61 . The opening not sealed by the lipid bilayer membrane is sealed by the second lipid bilayer membrane, and the second membrane protein 61 is introduced into the second lipid bilayer membrane.
The extracellular region of the introduced membrane protein 61 is arranged inside the well structure 20 and the intracellular region of the membrane protein 61 is arranged outside the well structure 20 .

By the following method, the membrane proteins 60 can be arranged in different orientations on the same substrate 10, and the orientation of the membrane proteins introduced into the lipid bilayer membrane 30 of each well structure 20 can be identified. .
A case where a plurality of well structures 20 are formed in the substrate 10 will be described below.
First, the fluorescent material 23 is brought into contact with the upper surface of the substrate 10 to flow into the well structure 20 of the substrate 10 . Subsequently, the lipid membrane is cleaved near the opening 21 of the well structure 20 . Then, part of the opening 21 is sealed by the lipid bilayer membrane, so the fluorescent substance 23 is confined inside the well structure 20 .
Subsequently, the budding vesicle 50a containing the membrane protein 60 within the lipid membrane is fused to the lipid bilayer membrane 30 of the opening 21. FIG. Then, the fluorescent substance 23 is included in the well structure 20 and the membrane protein 60 is introduced into the lipid bilayer membrane 30 in the opening 21 of the well structure 20 .
At this time, the extracellular domain of the introduced membrane protein 60 is placed outside the well structure 20 and the intracellular domain of the membrane protein 60 is placed inside the well structure 20 .

Subsequently, the fluorescent substance 23 is removed from the upper surface of the substrate 10, the fluorescent substance 24 is brought into contact with the upper surface of the substrate 10, and the fluorescent substance is applied to the inside of the well structure 20 where the opening 21 is not sealed with the lipid bilayer membrane. 24 inflow. Subsequently, giant lipid vesicles 70 a containing membrane proteins 61 within the lipid membrane are brought into contact with the upper surface of substrate 10 . The opening 21 is then sealed by the lipid bilayer 30 containing the membrane protein 61 and the fluorescent substance 24 is confined inside the well structure 20 .
At this time, the extracellular domain of the introduced membrane protein 61 is placed inside the well structure 20 and the intracellular domain of the membrane protein 61 is placed outside the well structure 20 .

As described above, the orientation of the membrane protein 60 of the lipid bilayer membrane at the opening 21 can be distinguished by using the fluorescent substances 23 and 24 that emit different fluorescence.

According to the manufacturing method of this embodiment, the following lipid membrane device (lipid membrane device 1C) can be manufactured.
A substrate having a first well structure and a second well structure that are open on one surface, a first lipid bilayer membrane that seals the first opening, and a second lipid bilayer that seals the second opening. two lipid bilayer membranes, the first lipid bilayer membrane comprising a first membrane protein, the second lipid bilayer membrane comprising a second membrane protein, the extracellular portion of the first membrane protein The region is located outside the sealed well structure, the intracellular region of the first membrane protein is located inside the sealed well structure, and the extracellular region of the second membrane protein is A lipid membrane device disposed within the sealed well structure and wherein the intracellular region of the second membrane protein is disposed outside the sealed well structure.

(Fourth embodiment)
FIG. 4 is a schematic diagram of a lipid membrane device in which electrodes are arranged. As illustrated in FIG. 4, by arranging electrodes 40 on the substrate 10 on which the lipid bilayer membrane 30 is formed, currents inside and outside the well structure 20 can be measured.
Since the lipid bilayer membrane 30 has a high resistance, the current value changes when the opening 21 of the well structure 20 is sealed by the lipid bilayer membrane 30 .

As described above in the third embodiment, the orientation of membrane proteins 60, 61 in lipid bilayer membrane 30 at opening 21 can be controlled.
Here, when the budding vesicle 50a having the membrane protein 60 is fused after sealing the opening 21 with the giant lipid vesicle 70, the current value changes when the opening 21 is sealed with the giant lipid vesicle 70. .
In addition, when the opening 21 is sealed with the giant lipid 70a vesicle having the membrane protein 61, the current value changes at that time.
Furthermore, by associating the change in the current value inside and outside the well structure 20 when sealed with the lipid bilayer 30 with the positional information of the well structure 20, the orientation of the membrane protein in each well structure 20 can be identified. .

Further, by disposing the channel 50 inside the electrode 40 as shown in FIG. 4 or the well structure 20, the lipid bilayer membrane 30 having the membrane protein 60 can be manipulated.

The electrode 40 can generate a potential difference between the surface of the substrate 10 and the outside of the well structure 20 and between the inside of the well structure 20 and the outside of the well structure 20 . Channels 50 are capable of producing flow of solution into the well.

As shown in FIG. 4, the lipid bilayer substrate has a substrate 10 with a well structure 20 formed therein. An overhang (eaves) 11a is provided in the opening (aperture) 21 of the well structure 20 in order to increase the sealing efficiency of the well by the lipid bilayer membrane and to keep the crosslinked structure of the lipid membrane stable. It is desirable to be Inside the well structure 20, a fluorescent material 23 or an electrode 40 is arranged for functional measurement of membrane proteins. Either the fluorescent material 23 or the electrode 40 can be arranged, or both can be arranged.

The substrate 10 is preferably made of a material that does not interfere with fluorescence observation of the well structure 20 with a fluorescence microscope and that the surface of the substrate is negatively charged within the pH range (3 to 10) of buffer solutions normally used in physiological experiments.
Examples of materials for the substrate 10 include silicon, silicon oxide, silicon nitride, quartz, mica, and glass.

The number of well structures 20 that the substrate 10 has is not particularly limited. Instead of the well structure 20, a through hole without a bottom surface may be used.
The shape of the opening 21 of the well structure 20 is desirably circular or square from the viewpoint of stably forming the lipid bilayer membrane. The diameter of circular opening 21 or one side of square opening 21 is preferably in the range of 100 nm to 10 μm.
The size of the membrane protein 60 is generally on the order of 10-20 nm. The reason why the lower limit is 100 nm is to retain the membrane protein 60 in the lipid bilayer membrane 30 of the opening 21 . The reason why the upper limit is 10 μm is that the upper limit of the well size that can be sealed by the lipid bilayer membrane of the lipid vesicle is about 10 μm.

As a method for forming the well structure 20 on the substrate 10, microfabrication techniques such as photolithography, electron beam lithography, and dry etching can be applied.
A thin film layer 11 may be provided on the surface of the substrate 10 to form an overhang 11a in the opening 21 of the well structure 20 . The overhang portion 11a is formed so as to extend the upper surface of the substrate 10 in the direction of narrowing the opening of the opening portion 21 of the well structure 20 .
The material of the thin film layer 11 may be the same as or different from the material of the substrate 10 as long as the lipid bilayer membrane 30 adheres thereto. Since a selective etching method can be applied in the manufacturing process of the overhang portion 11a, it is desirable to use different materials. The thickness of the thin film layer 11 is desirably 50 nm to 500 nm.

When a membrane protein is expressed in a cell or the like, the obtained membrane protein is solubilized using a detergent, and then reconstituted by removing the detergent, it is difficult to control the orientation of the membrane protein. .
However, as described above, in virus-derived budding vesicles containing membrane proteins, the orientation of the membrane proteins can be aligned. In this case, the activity of the membrane protein is stably maintained because the surfactant does not contaminate the lipid membrane containing the membrane protein. In addition, since the orientation of membrane proteins is uniform, the functions of membrane proteins can be utilized with high efficiency.

In addition, when this budding vesicle is fused to a giant lipid vesicle, the orientation of membrane proteins can be aligned even in the fused vesicle (for example, Kamiya K et al., Confocal microscopic observation of fusion between baculovirus budded virus envelopes and single giant unilamellar vesicles., Biochim Biophys Acta. 2010 Sep;1798(9):1625-31.).
In budding vesicles and fused vesicles, the extracellular domains of membrane proteins are located outside their respective membranes. Thus, in budding vesicles and fused vesicles, the orientation of membrane proteins is identical to that in native cells.

Viruses that induce budding vesicles and secrete them (viruses that express the membrane protein 60) may be, for example, retroviruses and paramyxoviruses. Baculovirus is an insect pathogenic virus that has a circular double-stranded DNA gene. Specifically, in addition to two types of nucleopolyhedronviruses (NPVs) and granoloviruses (GVs), non-occluded viruses are known. Of these, NPV (nuclear polyhedrosis virus) is widely used in biotechnology because it produces a large amount of inclusion bodies called nuclear polyhedrosis in the nucleus of infected cells, reaching 40 to 50% of the total cellular protein. there is

Baculovirus is attracting attention as a protein expression system using eukaryotes because it can be expressed in large amounts while maintaining biological integrity. A convenient kit is commercially available, and it is becoming a routine technique. By using baculovirus, the budding vesicle can be obtained easily and inexpensively, and the membrane fusion protein described below is a well-known protein. Therefore, the fusion conditions and the like are known, and it is easy to use for the purpose.

Membrane protein 60 may be a membrane receptor having a site that penetrates the lipid membrane. The membrane receptor may be, for example, a 1-transmembrane receptor, a 4-transmembrane receptor, or a 7-transmembrane receptor. Membrane receptors accept a variety of ligands. Ligands are diverse and include, for example, amino acids, low-molecular-weight organic compounds, steroids, amino acids and their derivatives, peptides, and proteins.

Single transmembrane receptors include, for example, type I cytokine receptors and enzyme-coupled receptors having enzymatic activity on the cytoplasmic side. With this type of receptor, the degree of phosphorylation of the receptor changes upon binding of the ligand, resulting in the expression of enzymatic activity such as kinase activity and phosphatase activity. There are receptors with tyrosine kinase and serine-threonine kinase activity.

Examples of four-transmembrane receptors include those that form subunit structures and function as ion channels. When a ligand binds to an ionotropic receptor, the ion channel opens, causing the influx and outflow of ions, resulting in the expression of specific effects.

Seven-transmembrane receptors include, for example, those that exhibit actions by coupling with various G proteins. G protein-coupled receptors (GPCRs) include biogenic amines such as dopamine and serotonin, lipid derivatives such as prostaglandins, nucleic acids such as adenosine, amino acids such as GABA, and bioactive peptides (e.g., angiotensin, bradykinin, cholesterol, etc.). Cystokinin, etc.) form a receptor family. In addition, GPCRs are receptors for ex vivo signaling substances related to light, taste, and smell. GPCRs are important membrane proteins that play a central role in signal transduction. By analyzing the human genome sequence, it is expected that many orphan receptors belonging to GPCRs will be found. It is believed that the discovery of ligands corresponding to such GPCRs will enable effective drug development.
Specific examples of seven transmembrane receptors include muscarinic acetylcholine receptors, A1 adrenergic receptors, dopamine receptors, serotonin receptors, histamine receptors, group I metabotropic glutamate receptors (mGluR1/5 ) GABAB receptors, ATP receptors, leukotriene receptors, platelet activating factor (PAF) receptors, opioid receptors, orexin receptors, endothelin receptors, neuropeptide PACAP receptors, CRH receptors, chemokine receptors, non Neuromuscarinic receptors, adrenergic receptors, prostanoid receptors, prostaglandin E receptors, prostaglandin E2 receptors, nociceptin receptors, calcitonin receptors, bradykinin receptors, glucagon family peptide hormone receptors, other There are orphan seven transmembrane receptors.
Among the above-mentioned membrane receptors, seven-transmembrane receptors in particular bind to various ligands and are deeply involved in diseases and pharmaceuticals.

When arranging the electrode 40 in the well structure 20, there is a method of embedding the electrode 40 before forming the well structure 20 described above. Materials for the electrode 40 include silver/silver chloride, gold, and platinum. The substrate 10 in which the electrodes 40 are embedded must have excellent insulating properties. Examples of the material include silicon oxide, silicon nitride, alumina, tantalum oxide, resist film, and the like.

Types of lipid molecules in the lipid bilayer membrane 30 include neutral lipid molecules such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE), and negatively charged lipid molecules such as phosphatidylserine (PS) and phosphoglycerol (PG). A mixture with lipid molecules is used. In addition, cholesterol or the like may be mixed.
As a method for forming the lipid bilayer membrane 30, for example, a giant lipid vesicle composed of a three-component system of neutral lipid/negatively charged lipid/cholesterol having a diameter of 10 μm or more is developed on a substrate to form a lipid bilayer. A heavy film 30 may be obtained. By adopting this formation method, when a plurality of well structures 20 are provided on the substrate 10, the plurality of well structures 20 can be stably covered with the lipid bilayer membrane 30 without gaps at one time.

When the fluorescent substance is placed inside the well structure 20 , the giant lipid vesicles are developed in the same manner as described above while the solution containing the fluorescent substance is in contact with the inside of the well structure 20 . As a result, the opening 21 of the well structure 20 can be covered with the lipid bilayer membrane 30 while enclosing the fluorescent substance inside the well structure 20 . The fluorescent substance remaining outside the well structure 20 can be removed by replacing the solution outside the well structure 20 with a solution containing no fluorescent substance.

Typical techniques for producing giant lipid vesicles for sealing openings 21 include static hydration and electric field formation. The method for producing vesicles is not particularly limited, but it is preferable to employ the electric field formation method because it facilitates the production of giant lipid vesicles and the simplicity of the reaction time and reaction process. The electric field formation method is a technique in which a lipid molecule is formed into a thin film on an electrode such as indium tin oxide (ITO) and then an alternating electric field is applied to form giant lipid membrane vesicles in an aqueous solution.
In order to obtain lipid vesicles of uniform size, it is preferable to form a uniform thin film of lipid molecules with a thickness of several tens of nm to several μm on an ITO substrate, and an alternating electric field of several hundred mV to 2V. is preferable. This is because if the electric field strength is lower than the electric field range, the yield of vesicles is low, and if the electric field strength is higher than the electric field range, structural destruction of vesicles and electrolysis of water occur, and vesicles may not be produced.

EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Preparation of Substrate Having Well Structure)
A substrate was formed with a well structure and an overhang portion at the opening of the well structure.

First, a silicon substrate was prepared as the substrate 10 . A silicon oxide film layer (thin film layer) 11 having a thickness of 120 nm was formed on the upper surface of the substrate 10 by thermal oxidation. Subsequently, a well structure 20 having a circular opening (1 μm in diameter) was formed by photolithography and dry etching. The depth of the well structure was set to 1 μm.

Subsequently, the substrate 10 having the well structure 20 formed thereon was brought into contact with a potassium hydroxide solution (10% by weight). As a result, the substrate 10 under the silicon oxide film layer 11 was selectively etched to form overhangs 11 a at the four corners of the opening 21 of the well structure 20 .

[Experimental example 2]
(Preparation of giant lipid membrane vesicles and development on substrates)
On the substrate obtained in Experimental Example 1, the lipid membrane was cleaved and the opening of the well structure was sealed with a lipid bilayer membrane.

First, a mixed chloroform solution (concentration: 2.5 mM) of dioleoylphosphatidylcholine (DOPC) (80 mol%) and dioleoylphosphatidylserine (DOPS) (20 mol%) was prepared, and the resulting solution was 0.5 mol % of NBD (NBD-DOPE) was contained as a fluorescent labeling agent for a heavy film.

Subsequently, 200 μL of the chloroform solution was uniformly applied onto an ITO substrate (substrate in which ITO was thinned to a film thickness of 100 nm on glass, size 40×40 mm, 50 to 100Ω). This substrate was dried under reduced pressure at room temperature for 2 hours to completely remove the chloroform solvent, thereby forming a uniform lipid molecule thin film on the ITO substrate.

Subsequently, on the ITO substrate, a silicon rubber having a window (having a window of 20×20 mm in which a silicon rubber having an outer dimension of 30×30 mm and a thickness of 1 mm is hollowed out) is placed in close contact with the window. 500 μL of 30 mM sucrose aqueous solution was dropped into the part. Subsequently, an ITO substrate was placed on top of this so as not to contain air bubbles, and the solution in the silicone rubber window was sandwiched between the ITO substrates.

Subsequently, a clip electrode was attached to the ITO substrate, and an alternating electric field (sine wave, 1 V, 10 Hz) was applied at room temperature for 2 hours to form giant lipid membrane vesicles dispersed in the sucrose solution by the electric field formation method. rice field.

100 μL of a solution (20 mM acetate buffer, pH 4.5) was dropped onto the substrate 10 having the well structure 20 . Furthermore, 2 μL of the giant lipid membrane vesicle dispersion liquid was dropped into the solution and allowed to stand for 2 minutes to spread the giant lipid membrane vesicles on the substrate. A vesicle having a spherical structure collides with the substrate, and the spherical structure is destroyed to form a lipid bilayer membrane covering the well structure 20 .

Subsequently, a fluorescence observation image was obtained using lasers with wavelengths of 488 nm and 561 nm as excitation light. The results are shown in FIG. FIG. 5(a) is a fluorescence image of fluorescence derived from NBD-DOPE, which labels the lipid bilayer membrane, taken from above the lipid membrane device. FIG. 5(b) is a fluorescence image of the same position as FIG. 5(a) obtained by excitation with a 561 nm laser.

[Experimental example 3]
(Protein introduction by budding vesicles)
Baculovirus-derived budding vesicles (BV) were added to the substrate covered with the lipid bilayer membrane 30 prepared in Experimental Example 2. As BV, human adrenergic receptor β2 (ADRB2), which is a membrane protein, was expressed by fusing a fluorescent probe RFP to the C-terminus of the receptor.

After adding BV, the mixture was allowed to stand at room temperature, and fluorescence images were acquired with a confocal microscope. The results are shown in FIG. FIG. 6(a) is a fluorescence image obtained by photographing a device in which a well structure is sealed with a lipid membrane containing membrane proteins, excited by a 488 nm laser, and photographed from above. FIG. 6(b) is a fluorescence image excited by a 561 nm laser. Red bright spots were observed in FIG. 6(b).

FIG. 7 shows the results of measuring the fluorescence intensity of RFP along the dashed lines in FIGS. 5(b) and 6(b). In FIG. 7, "before fusion" indicates the fluorescence intensity along the dashed line in FIG. 5(b), and "after fusion" indicates the fluorescence intensity along the dashed line in FIG. 6(b). "Self-supporting membrane portion" indicates a portion where the opening of the well structure on the dashed line is sealed with a lipid bilayer membrane.

As a result, it was found that the fluorescence intensity in FIG. 7(b) was larger than that in FIG. 5(b) not only in the lipid bilayer membrane on the substrate but also in the self-supporting membrane portion.

This was derived from the fluorescent tag RFP labeling human adrenergic receptor β2 excited by a 561 nm laser, indicating that human adrenergic receptor β2 was introduced into the lipid bilayer membrane. RFP-derived fluorescence labeling ADRB2 was observed in the well structure 20 covered with the lipid bilayer membrane indicated by the arrow.

By this technique using BV, we succeeded in introducing the protein into the well structure 20 covered with the lipid bilayer membrane, which is a site where the protein function can be analyzed.

The present invention makes it possible to control the orientation of membrane proteins in an artificial lipid bilayer membrane.

Reference Signs List 1 Lipid membrane device 1A, 1B, 1C Lipid membrane device 10 Substrate 11 Thin film layer 11a Overhang 20 Well structure 21 Opening 23, 24 Fluorescent substance 30 Lipid bilayer membrane 40 Electrode (electrode layer) 50 Budding vesicle (lipid membrane) 50a Budding vesicle (including membrane protein 60) 60 (first) membrane protein 61 Second membrane Protein, 70... giant lipid vesicle, 70a... giant lipid vesicle (including membrane protein 60), 80... channel

Claims (8)

The lipid bilayer membrane of a lipid membrane device comprising a substrate having a well structure opening on one side, and a lipid bilayer membrane formed on the one side of the substrate and sealing the opening of the well structure. to the
fusing a lipid vesicle containing a membrane protein within a lipid membrane such that the membrane protein is introduced into the lipid bilayer membrane;
A thin film layer is laminated between the one surface of the substrate and the lipid bilayer membrane, and the thin film layer has an overhang extending to the opening of the well structure.
A method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane. On the one side of the substrate having a well structure opening on one side,
A lipid vesicle containing a membrane protein is brought into contact with a lipid membrane, as a result of which the lipid vesicle is cleaved to form a lipid bilayer membrane containing the membrane protein, and formed on the one surface to open the well structure. Sealing the part to form a lipid membrane device in which a membrane protein is introduced into the lipid bilayer membrane,
A method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane. 3. The method for producing a lipid membrane device in which the membrane protein is introduced into the lipid bilayer membrane according to claim 1, wherein the lipid vesicle is a virus-derived budding vesicle expressing the membrane protein. 3. The lipid into which the membrane protein is introduced into the lipid bilayer membrane according to claim 1 or 2, wherein the lipid vesicle is a fusion of a virus-derived budding vesicle expressing the membrane protein and a giant lipid vesicle. A method for manufacturing a membrane device. A first lipid vesicle is brought into contact with the one surface of a substrate having a plurality of well structures open on one surface, and as a result, the first lipid vesicle is cleaved to form a first lipid bilayer membrane. a step of sealing an opening of a portion of the well structure formed on the one surface;
fusing a second lipid vesicle comprising a first membrane protein within a lipid membrane to said first lipid bilayer membrane of said opening, resulting in said first lipid bilayer membrane of said opening; introducing said first membrane protein into
contacting the one surface of the substrate with a third lipid vesicle containing a second membrane protein in a lipid membrane, whereby the third lipid vesicle is cleaved to release the second membrane protein; forming a second lipid bilayer membrane comprising a second lipid bilayer membrane formed on the one surface to seal the opening of the well structure, and introducing a second membrane protein into the second lipid bilayer membrane; A method for producing a lipid membrane device in which the first membrane protein and the second membrane protein are introduced into the lipid bilayer membrane. A substrate having a well structure with an opening on one side, and a lipid bilayer membrane sealing the opening of the well structure,
The lipid bilayer membrane comprises a membrane protein,
the extracellular region of the membrane protein is located outside the sealed well structure;
the intracellular region of the membrane protein is located inside the sealed well structure;
A thin film layer is laminated between the one surface of the substrate and the lipid bilayer membrane, and the thin film layer has an overhang extending to the opening of the well structure.
Lipid membrane device. A substrate having a well structure with an opening on one side, and a lipid bilayer membrane sealing the opening of the well structure,
The lipid bilayer membrane comprises a membrane protein,
the extracellular region of the membrane protein is located inside the sealed well structure;
A lipid membrane device, wherein the intracellular region of the membrane protein is located outside the sealed well structure. A substrate having a first well structure and a second well structure that are open on one surface, a first lipid bilayer membrane that seals the opening of the first well structure , and the second well structure . and a second lipid bilayer membrane that seals the opening of
said first lipid bilayer membrane comprising a first membrane protein;
said second lipid bilayer membrane comprising a second membrane protein;
the extracellular region of the first membrane protein is located outside the sealed first well structure;
the intracellular region of the first membrane protein is disposed within the sealed first well structure;
the extracellular region of the second membrane protein is located inside the sealed second well structure;
A lipid membrane device, wherein the intracellular region of the second membrane protein is located outside the sealed second well structure.

Images