JP2727469B2 - Method for forming proteoliposomes - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for forming a proteoliposome in which a membrane protein is incorporated into a membrane of a membrane vesicle such as a liposome, and more particularly to a method for controlling the direction of the membrane protein. The present invention relates to a method for forming a proteoliposome into which is incorporated.

[Prior art] Liposomes are lipids that are one of the constituent molecules of biological membranes.
A biofunctional molecular assembly that is formed by dispersing and re-associating in an aqueous solution environment and has a structure similar to a cell membrane. It produces useful substances as a diagnostic or therapeutic agent in the medical field, and also in the industrial field. The application to is considered.

On the other hand, a proteoliposome in which a protein is incorporated into the liposome has a function-added molecule.
Liposomes can have more advanced functions.

By the way, various known methods have been devised for the production of proteoliposomes, which are properly used depending on the purpose of use, the type of protein, and the like. The main ones include dialysis, sonication, freeze-thawing, and the like.

Dialysis is a method in which proteins are solubilized together with lipids using a surfactant and then dialyzed to remove the surfactant.
Small proteoliposomes less than 100 nm in diameter are produced. In the sonication method, a protein is sonicated together with a lipid liposome, and a small proteoliposome having a diameter of 100 nm or less is also generated. Furthermore, the freeze-thaw method is a method in which a mixture of a protein and a liposome is repeatedly frozen and thawed to incorporate the protein into a lipid membrane, and a relatively large proteoliposome having a diameter of several hundred nm is generated.

[Problems to be Solved by the Invention] On the other hand, in a biological membrane, a membrane protein is embedded with a fixed orientation, and this anisotropic membrane configuration allows the stimulus reception, transmission, and substance transport. Vector material / information transfer is realized. Therefore, even when applying proteoliposomes to medical or industrial applications, the membrane protein is incorporated with a certain orientation in the membrane reconstructed by an artificial technique, and the function is efficiently expressed. It is advantageous in doing so.

However, conventional methods for producing proteoliposomes employ a method in which a lipid membrane is dissolved or partially destroyed by chemical or physical action, and a membrane protein is inserted to reconstitute the membrane. Did not include any means for controlling the direction of the membrane protein.

In fact, the membrane proteins in the proteoliposome membrane prepared by the conventional manufacturing method only have a random orientation.

In addition, the diameter of the proteoliposome formed by each production method was defined, and the size could not be arbitrarily selected.

On the other hand, membrane proteins in the formation of proteoliposomes,
In particular, the insertion of integral membrane proteins into lipid bilayers has not been studied in detail. However, few cases have been reported. That is, Zaki
m) et al., “Biokimika e Biophysica Actor (B
iochimica et Biophysica Acta), 906 , pp. 33-68,
(1987), cytochrome, UDP glucuronosyltransferase, bacteriorhodopsin, etc., to a membrane protein solubilized by a surfactant or to a lipid bilayer of a membrane protein excluding both surfactant and membrane lipid. Examines the spontaneous insertion of
They report that insertion of membrane proteins into lipid bilayers occurs at lower activation energies than fusion between liposomes. However, the directions of the inserted membrane proteins are not uniform, and no control of the direction is performed.

Accordingly, an object of the present invention is to provide a novel method for producing a proteoliposome having a process of controlling the membrane protein to be able to distinguish and orient inside or outside the vesicle, and the incorporated membrane protein is effectively used. To make it available.

However, there are various methods for producing one liposome constituting the proteoliposome, depending on the constituent lipids, solution conditions, and the like. Liposomes having an arbitrary size in the range of several tens nm to several hundreds of μm in diameter are available. Obtainable. Therefore, the present inventors have paid attention to the fact that if a membrane protein can be inserted into a previously prepared liposome with uniform orientation, a proteoliposome having an arbitrary diameter and having a controlled protein orientation can be obtained. did.

The present inventors have tried variously to bind a membrane protein, and when binding to the membrane protein using an appropriate carrier, the site where the membrane protein is inserted into the lipid bilayer is limited, and thereby the orientation is reduced. The inventors have discovered that the properties can be controlled, and have completed the present invention.

[Means and Actions for Solving the Problems] That is, the present invention relates to a step of binding a membrane protein to a carrier that does not penetrate into a lipid bilayer membrane; a step of preparing a liposome; and a step of preparing a membrane protein bound to the carrier. Inserting the proteoliposome into the liposome.

 Hereinafter, the present invention will be described in detail.

The method for forming a proteoliposome of the present invention comprises the steps of: binding a specific site of a hydrophilic side chain of a membrane protein with a carrier having a property of not invading a lipid bilayer membrane; And the membrane protein bound to the carrier is incorporated into the liposome, so that the insertion site of the membrane protein into the lipid bilayer is limited, and a certain direction is maintained. And will be incorporated into the membrane.

In the present invention, since the process of incorporating the membrane protein into the liposome and the process of forming the liposome are independent, the liposome may be any as long as it has the property of incorporating the membrane protein. , The diameter of the liposome, the lipid composition and the like can be arbitrarily set.

Therefore, by selecting a site to be bound by a carrier in a membrane protein and a method for forming a liposome, in a proteoliposome having an arbitrary diameter, the direction of the membrane protein is:
It can be arbitrarily controlled. As a result, the membrane protein incorporated into the proteoliposome is right side-out
Or it is suitable for in side-out.

As the membrane protein in the present invention, a membrane protein present in a biological membrane having a hydrophilic side chain, among which, among others, an integral membrane protein existing through a membrane is used, and natural and synthetic peptides and the like are used. Substances that can be present in the membrane can also be applied. Specific examples include enzymes that catalyze substance metabolism (cytochrome oxidase, cytochrome P-450, membrane-bound phospholipase, ATP synthase, etc.), channels that transport substances through membranes (potassium channel, sodium channel, Calcium channels, etc.), pumps (sodium-potassium pumps, proton pumps, etc.), photoreceptor chromoproteins (rhodopsin, bacteriorhodopsin), neurotransmitter receptors (acetylcholine receptor, glitamate receptor) involved in the reception and transmission of information in the body Body receptors), hormone receptors (thyroid stimulating hormone (THS) receptor, glycagon (GC) receptor, etc.).

These membrane proteins are generally insoluble in water and may have attached membrane lipids.However, as long as the orientation can be limited by the carrier to be bound, the membrane proteins can be used as such or dissolved in a surfactant. Can be used.

In general, when the ratio of the membrane protein to the lipid is increased, the amount to be incorporated should increase, but the amount remaining without being actually incorporated increases. Thus, the protein:
Lipids (weight ratio) is at most 1: 1 and preferably 1:10
A range of ~ 1: 100 is desirable.

The concentration of the lipid during the formation of the liposome depends on the formation method, but is preferably in the range of 1 to 20 mM (0.75 to 15 mg / ml).

As the liposome in the present invention, any liposome having a property of incorporating a membrane protein and having a bilayer lamellar structure can be used without limitation. For example, various phospholipids, glycolipids, neutral lipids, etc. Is mentioned. As another example, non-naturally occurring synthetic bilayer-forming liposomes can also be used. Further, various additives can be added as long as the bilayer structure is not destroyed.

The size and lipid composition of the liposome can be arbitrarily set without any particular limitation.

As a method for forming such a liposome, a method for forming a liposome having an arbitrary diameter using various lipids and adducts can be applied. For example, sonication,
Surfactant dialysis, freeze-thaw, reverse-phase evaporation, and the like. (For example, Seikagaku Experimental Lecture, Vol. 3, “Membrane Lipids and Plasma Lipoproteins (Part 2),” edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin, 1987) The carrier that binds membrane proteins does not penetrate into lipid bilayer membranes. Is used. For this purpose, in the present invention, a carrier in which the volume of the carrier is equal to or larger than the volume of the liposome prepared in advance is used. Further, the shape of the carrier is not particularly limited, for example, a spherical shape, the size of which is equal to or larger than the diameter of the liposome prepared in advance,
Alternatively, a liposome having a flat or curved surface and a radius of curvature equal to or larger than the radius of a liposome prepared in advance may be used.

For example, when the diameter of the liposome is usually 10 nm to 1 μm, and as large as 10 μm to several hundred μm, the size of the carrier is 1 μm to 10 μm or 100 μm to 1 mm depending on the diameter of the liposome. What should be used.
As described above, the shape of the carrier is not necessarily spherical, and any other shape can be used.
Further, a continuous curved surface, a curved surface inside a hollow tube, and the like also correspond to the carrier defined by the present invention.

Next, a method for binding and dissociating a specific site of the hydrophilic side chain of the membrane protein with the carrier involves binding and dissociation of the carrier and the protein under mild conditions without destroying the proteoliposome or denaturing the protein. It is preferred to do so.

In this regard, for example, a protein chromatography technique can be applied. In protein chromatography, electrostatic attraction (ion-exchange chromatography), hydrophobic interaction (hydrophobic chromatography), and hydrogen bonding (hydrophobic chromatography) between an appropriately chemically bonded carrier and protein are used for the purpose of protein purification. Hydrogen bond chromatography), specific hydrophilicity (affinity chromatography)
The binding and dissociation are carried out using such methods.

Therefore, the carrier used in the present invention includes, for example, a material capable of causing the above-described interaction and binding to a specific site of the hydrophilic side chain of the membrane protein; for example, an amino group, a hydroxyl group, an epoxy group, a sulfin sulfide group on the surface. Any material having an active functional group such as the above can be used. For example, various gels such as agarose and cellulose used for chromatography, organic materials such as plastics, and inorganic materials such as glass, ceramics, and metal oxide surfaces having oxygen atoms on the surface are also used with cyanogen bromide. Since it can be activated, it can be used as a carrier. Above all, gels for chromatography are usually about 10 μm in diameter, and are extremely easy to use because a protein chromatography column can be used as it is. In some cases, reconstitution of proteoliposomes is possible simultaneously with purification of membrane proteins.

The method for dissociating the membrane protein incorporated in the liposome from the carrier is to add a substance that acts to prevent binding between the carrier and the membrane protein to the column, and to change the physicochemical conditions of the solution (pH, salt concentration Etc.) methods. In the former, for example, utilizing the fact that most of the membrane protein has a sugar chain at the end, using a protein lectin having the property of binding to sugar bound to a carrier, if excess sugar is added,
Sugar binds lectin repulsively and releases membrane proteins.
In addition, when binding to a carrier is performed site-specifically using a monoclonal antibody specific to a specific site of a membrane protein using an antigen-antibody reaction, the excess The membrane protein can be dissociated from the carrier in an anti-antagonistic manner. However, the binding between antigen and antibody is very strong, and it is difficult to dissociate even with a large excess of papten, and it is often difficult to prepare a large amount of hapten. Is excellent.

In addition, the method of dissociating by changing the physicochemical conditions of the solution often destroys the formed proteoliposome depending on the conditions, and its application is considerably limited.

Next, the steps of forming the proteoliposome of the present invention are schematically shown in the drawings as shown in FIGS. 1, 2 and 3.

1 (a) to 1 (d) are explanatory views schematically illustrating the basic concept of the method for forming a proteoliposome of the present invention. In the figure, the operations are performed in the order of (a) → (b) → (c) → (d). First, the specific site of the hydrophilic side chain of the membrane protein 2 is bound and fixed by the carrier 1. Next, the dispersion of the liposome 3 is mixed with the membrane protein 2 held by the carrier 1, the membrane protein 2 is incorporated into the liposome, and then the proteoliposome 4 is formed by dissociating the membrane protein 2 from the carrier 1.

The above forming step is performed as shown in FIGS. 2 (a) and 2 (b).
The reaction can be carried out by filling the column container 5 with the carrier 1, or by using a curved membrane sheet 6 as the carrier as shown in FIG.

[Example] Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 Bovine rhodopsin was isolated and purified by the following procedure. All operations were performed under dark red (Eastman Kodak Red Filter No. 1).

From the bovine retina, the disc, which is a membrane structure containing rhodopsin, was analyzed by the Ficoll flotation method [Smith et al., Experimental Eye Research (Exp.
s.), 20 , 211-217, (1975)]. Purify the disc with 50 mM octyl glucoside, 0.1
M NaCl, 1 mM MnCl ₂ , 1 mM CaCl ₂ , 10 mM Mops-NaOH (pH 7.
After solubilization with the solution of 0), Con A-Sephar-ose 4B;
Agarose gel (Pharmacia, average particle size of gel is about 100μ)
Rhodopsin was purified by affinity chromatography using the column (m) (5 mm × 10 cm). That is, 5 mg of solubilized disk protein was added to the column, and rhodopsin was allowed to bind to concanavalin A, and then the column was buffered (0.1 M NaCl, 1 mM MnCl ₂ , 1 mM CaCl ₂ ,
The impurities were removed by sufficiently washing with 10 mM Mops-NaOH (pH 7.0). Rhodopsin was used for forming proteoliposomes while being bound to the gel particles of the carrier via concanavalin A.

Liposomes were prepared by the freeze-thaw method as follows. 15mg of aglectin (S
igma: A thin film of soybean phosphatidylcholine, type IV S) was prepared. To this, 1 ml of 0.1 M NaCl, 10 mM Mops-Na
OH (pH 7.0) was added, and the mixture was treated with a vortex mixer for 1 minute (power maximum) to disperse the lipid thin film. The lipid dispersion was transferred to a 12 × 75 mm capped polypropylene tube (Becton Dickinson: Falkon 2063) and saturated with nitrogen gas. Water-soluble ultrasonic oscillator (Branson:
Sonifier B-15 type (200W) with cup horn)
And sonicated liposome dispersion (average particle diameter 100n) (power 2, duty cycle 60%, water cooling)
m).

This liposome dispersion was frozen in a dry ice-acetone bath, thawed at room temperature, and then treated with an aqueous sonicator for 30 seconds (power 1) three times.
Desired liposome dispersion (average particle size about 1 μm, volume 0.5 μm
³ ) Got it.

The liposome dispersion was added to the column and incubated at room temperature for 1 hour to form proteoliposomes incorporating rhodopsin into the liposome membrane.

Next, in order to dissociate rhodopsin from the carrier and elute the proteoliposome from the column, methyl-α-D-mannoside and methyl-α-, which bind to concanavalin A with the sugar chain of rhodopsin in a antagonistic manner, are used. Buffer containing D-glucoside (50 mM methyl-α-D-mannoside, 50 mM methyl-α-D-glycoside, 0.1 mM NaCl, 10 mM
Mops-NaOH (pH 7.0) was passed through the column. The effluent was collected and dialyzed against a buffer (0.1 M NaCl, 10 mM Mops-NaOH (pH 7.0)) to remove free sugar molecules.

In order to confirm that rhodopsin is incorporated in the produced proteoliposome membrane, a proteoliposome dispersion was added to a liquid helium quick-freezing device (Eiko
After freezing quickly with Engineering Company: RF-23 type, freeze-fracture replica making device (Eiko Engineering Company: F
D-5A), a replica of the membrane surface was created, and observed with a transmission electron microscope (JEOL: JEM 100U).
Particles with a diameter of about 4 nm were observed on the membrane surface, confirming that rhodopsin was incorporated.

Further, when β-N-acetylglucosaminidase (Sigma) was allowed to act on the proteoliposome dispersion to analyze the external solution, free sugars were detected, and the sugar residues were directed toward the outside of the proteoliposomes and incorporated. It was confirmed that.

Example 2 Halobacterium halobium (Halobact)
erium halobium), purple membrane, a membrane fragment containing bacteriorhodopsin, was purified by P. Oesterhe
lt) and W. Stoeckeniu
s) method [Methods of Enzymology (Methos
ds of Enzymology), 31 , pp. 667-678, (1974)]. In addition, purple membrane was used for KS hang (K.
S. Huang) et al. [Proceeding of National Academy of Science (Proceeding of Nati)
onal Academy of Science.USA,) 88 , 323, (1980)]
Bacteriorhodopsin was purified by delipidation using the method described above.

A monoclonal antibody that specifically binds to the carboxyl terminus of bacteriorhodopsin was developed by K.Kimu
ra) et al. [Journal of Biological Chemistry (J. Biol. Chem.), 257 , 2859-2867, (19)
80)]. This monoclonal antibody
Among the peptide fragments obtained by treating bacteriorhodopsin with cyanogen bromide, it specifically binds to a fragment consisting of 39 residues including a carboxyl terminus.

This CNBr-6 monoclonal antibody was covalently immobilized on Pharmacia's agarose gel, CNBr-activated Sepharose 4B (gel particle diameter about 100 μm). Using 2 mg of the antibody per 1 ml of the swollen gel, the mixture was reacted overnight at 4 ° C. under the conditions of a coupling buffer, 0.5 M NaCl and 0.2 M NaCO ₃ (pH 8.5). After blocking unreacted active groups and washing away the excess antibody, the antibody-bound gel is applied to a column (5 mm
× 10 cm) and the column was washed with 0.1 M NaCl.

0.1 M NaCl containing 0.2 mg of purified bacteriorhodopsin
The solution was applied to the column to bind the carboxyl terminus of bacteriorhodopsin to the CNBr-6 monoclonal antibody immobilized on the gel.

A liposome dispersion was prepared in the same manner as in Example 1.

0.2 mg of this liposome suspension was bacteriorhodopsin.
Is applied to the fixed column and incubated for 1 hour.
Bacteriorhodopsin was incorporated into the liposomes.

The column was flushed with 0.1 M NaCl adjusted to pH 11 with ammonia, and bacteriorhodopsin incorporated in the liposome was added.
It was dissociated from the CNBr-6 monoclonal antibody, and the proteoliposome was eluted. The eluted proteoliposome suspension was immediately dialyzed against 0.1 M NaCl. Approximately 40% of the bacteriorhodopsin was incorporated into the proteoliposomes and recovered.

The proteoliposomes obtained by the above procedure incorporate bacteriorhodopsin with the carboxyl terminus on the outside, which means that the pH of the external solution becomes alkaline when the proteoliposome suspension is irradiated with light, and that the proteoliposomes Was confirmed by the generation of a peptide containing a carboxyl terminus as a degradation product upon enzymatic treatment.

Example 3 Bacteriorhodopsin and CNBr-6 monoclonal antibodies were prepared by the method described in Example 2. A liposome dispersion was prepared by the method described in Example 1.

Millipore's cellulose type membrane filter (pore size 0.22μm) is cut into about 1mm squares and used as a planar carrier. Cyanogen bromide treatment is used to activate hydroxyl groups on the surface and bind proteins with amino groups. I made it possible.
To this, the previously prepared CNBr-6 monoclonal antibody was added in an amount of 1 mg per 100 mg of the above-mentioned membrane filter and reacted to bind the monoclonal antibody to the filter. Excess antibody was washed away.

0.1 M NaCl containing 0.2 mg of purified bacteriorhodopsin
To 1 ml of the solution, a membrane filter with a monoclonal antibody bound (dry weight 10 mg,
0.1 mg), and the carboxyl terminal of bacteriorhodopsin was bound to a CNBr-6 monoclonal antibody immobilized on a membrane filter. Thereafter, the membrane filter was washed with 0.1 M NaCl to remove free proteins, added to the previously prepared liposome dispersion, and incubated for 1 hour to incorporate bacteriorhodopsin into the liposomes.

Next, 1N aqueous ammonia is added to the dispersion to adjust the pH.
After dissociating the bacteriorhodopsin and the antibody as 11,
The pH was returned to neutral by adding 1N hydrochloric acid. Thereafter, the membrane filter was removed by centrifugation.

 The bacteriorhodopsin recovery was about 20%.

The proteoliposomes obtained by the above procedure incorporate bacteriorhodopsin with the carboxyl terminus on the outside, which means that the pH of the external solution becomes alkaline when the proteoliposome suspension is irradiated with light, and that the proteoliposomes Was confirmed by the generation of a peptide containing a carboxyl terminus as a degradation product upon enzymatic treatment.

Example 4 A method for introducing bacteriorhodopsin into a lipid bilayer membrane in a uniform manner using an affinity chromatography column having a bacteriorhodopsin antibody immobilized as a ligand on a cyanogen bromide-activated agarose carrier.

Bacteriorhodopsin is available from P. Oe
sterhelt) and W. Stoe
ckenius) [Methods of Enzymology, 31 , pp. 667-678, (1974).
), And extracted as a purple membrane from Halobacterium halobium, and furthermore, KSHuang et al. [Proceeding of National Academy of Sciences]
Science. USA), 88 , 323, (1980)].

A monoclonal antibody that specifically binds to the carboxyl terminus of bacteriorhodopsin was developed by K.Kimu
ra) et al. [Journal of Biological Chemistry (J. Biol. Chem.), 257 , 2859-2867, (19)
80)]. This monoclonal antibody
Among the peptide fragments obtained by treating bacteriorhodopsin with cyanogen bromide, it specifically binds to a fragment consisting of 39 residues including a carboxyl terminus.

This CNBr-6 monoclonal antibody was covalently immobilized on Pharmacia's agarose gel, CNBr-activated Sepharose 4B (gel particle diameter about 100 μm). The antibody was reacted at 4 ° C. overnight under the conditions of a coupling buffer, 0.5 M NaCl, and 0.2 M NaCO ₃ (pH 8.5), using 2 ml of the antibody per 1 ml of the swollen gel. After blocking unreacted active groups and washing away excess antibody, the gel was washed with 0.1 M NaCl.

0.1 M NaCl containing 0.2 mg of purified bacteriorhodopsin
The carboxyl terminus of bacteriorhodopsin was immobilized on the gel by adding the antibody-bound gel to the solution.
Bound to CNBr-6 monoclonal antibody. Gel again
Unreacted bacteriorhodopsin was removed by washing with 0.1 M NaCl.

15M soybean phospholipid (azo lectin) thin film 0.1MN
Hydration was performed in 1 ml of aCl using a vortex mixer, and the mixture was treated for about 30 minutes with a prog-type ultrasonic oscillator. Next, after mixing with a gel bound with 0.2 mg of bacteriorhodopsin, frozen with dry ice-acetone, thawed at room temperature,
Proteoliposomes incorporating bacteriorhodopsin were prepared by repeating the operation of gentle sonication three times.

To this, 0.1N ammonia water was added to adjust the pH to 11, and
Bacteriorhodopsin and CNBr incorporated into liposomes
After dissociation from the -6 monoclonal antibody and separation of the proteoliposome from the carrier gel, the pH was returned to neutral again using 0.1 N hydrochloric acid. Finally, only the proteoliposomes were collected by centrifugation.

The proteoliposomes obtained by the above procedure incorporate bacteriorhodopsin with the carboxyl terminus outside, which indicates that the pH of the external solution becomes alkaline when the proteoliposome suspension is irradiated with light, and that the proteoliposomes Was confirmed by the generation of a peptide containing a carboxyl terminus as a degradation product upon enzymatic treatment.

[Effects of the Invention] As described above, according to the present invention, when a proteoliposome is formed by incorporating a membrane protein into a liposome, it is possible to restrict the directionality of the protein to and from the vesicle membrane. I do. Therefore, when a membrane protein such as mass transfer ATPase, which has a function of transporting a substance through a membrane in a certain direction, is incorporated into a liposome membrane by the method of the present invention, the direction of mass transfer from the inside of the vesicle to the outside. Alternatively, the control can be performed in the opposite manner, and the membrane protein that does not meet the purpose can be reduced, so that it is much more efficient.

In addition, since it becomes possible to embed a protein such as an enzyme or an antibody which specifically binds to a substrate or an antigen-antibody or the like into a liposome membrane, by embedding the liposome so that the binding site is oriented outside of the liposome, it becomes unsuitable. The number of proteins having orientation can be reduced, and the required amount of protein can be reduced.

[Brief description of the drawings]

FIGS. 1 (a) to 1 (d) are explanatory views schematically showing the basic concept of the method for forming a proteoliposome of the present invention, and FIGS. 2 (a) and 2 (b) show a method of carrying out the present invention by column chromatography. FIG. 3 and FIG. 3 are explanatory diagrams showing a method for carrying out the present invention using a carrier having a continuous curved surface. DESCRIPTION OF SYMBOLS 1 ... Carrier 2 ... Membrane protein 3 ... Liposome 4 ... Proteoliposome 5 ... Column container 6 ... Membrane sheet

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Masanori Sakuranaga 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Hiroyoshi Kishi 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (72) Inventor Nobuko Yamamoto 3-30-2 Shimomaruko, Ota-ku, Tokyo Canon Inc. (72) Inventor Kinya Kato 3-30-2 Shimomaruko 3-chome, Ota-ku, Tokyo Canon Inc. (72) Inventor Harumi Iwashita 3-30-2 Shimomaruko, Ota-ku, Tokyo Inside Canon Inc. (56) References JP-A-63-111428 (JP, A) Biochemistry, vol. 2374-2385 (1980) Biochem. , Vol. 101, No. 2, pp. 433-440 (1987) Biol. Chem. , Vol. 252, pp. 7384-7390 (1977) Biochem. Biophys. ACTA, 812 (3), pp. 752-766

Claims (6)

(57) [Claims] 1. A method comprising: binding a membrane protein to a carrier that does not penetrate into a lipid bilayer membrane; preparing a liposome; and inserting the membrane protein bound to the carrier into the liposome. A method for forming a proteoliposome. 2. The method according to claim 1, wherein the volume of the carrier is equal to or greater than the volume of the liposome. 3. The method for forming a proteoliposome according to claim 1, wherein the carrier has a spherical shape, and the size is equal to or larger than the diameter of the liposome. 4. The method for forming a proteoliposome according to claim 1, wherein the shape of the carrier is planar or curved, and the radius of curvature is equal to or larger than the radius of the liposome. 5. The method for forming a proteoliposome according to claim 1, wherein the carrier is bound to a site of a hydrophilic side chain of the membrane protein. 6. The method for forming a proteoliposome according to claim 1, further comprising the step of dissociating the membrane protein inserted into the liposome from the carrier.