JPH0363300A - Formation of macroproteliposome - Google Patents

Abstract

PURPOSE:To form the subject liposome controllable of orientation of membrane protein recombined in macroproteoliposome by modifying a specific position in membrane protein to carrier, subjecting salt solution with membrane lipid to freezing and thawing and dialyzing. CONSTITUTION:A specific position in membrane protein is modified to a carrier and a salt solution containing the said membrane protein and membrane lipid is prepared, then the said salt solution is subjected to freezing and thawing thus dialyzed to a solution of salts having an osmotic pressure lower than the said salt solution to afford the objective liposome. Besides, further the membrane protein is preferably dissociated from the carrier after dialysis. Furthermore, preferably, shape of the said carrier is spherical and the size is equivalent or larger than the diameter of the formed liposome.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for forming giant proteoribosomes into which membrane proteins are incorporated by controlling directionality.

[Conventional technology] Liposomes have traditionally been used as a simple model system for biological membranes.
In addition to being widely used in research on the physical properties and functions of biomembranes, applications are being considered for use on construction beams and in the medical field by encapsulating various functional materials and drugs and using them as microcapsules.

Liposomes in which proteins are incorporated into the lipid bilayer membrane are called proteolibosomes. Since proteolibosomes contain proteins, which are the main functional substances in living organisms, they can be endowed with a wide variety of properties in addition to liposomes. Therefore, if proteolibosomes with such excellent functions can be produced in large quantities through a simple process, they could be used in the industrial field as functional materials that take advantage of the ingenious functions of living organisms, and in medical applications. In this field, it can be expected to be applied to artificial organs, immunological preparations, diagnostic reagents, etc. Among them, giant proteolibosomes, which have a diameter of 5:1 or more, have the same size as cells, so they can easily simulate various functions of cells, and can be used as artificial cells.

In addition, due to its large internal volume, it has advantages such as high substance retention efficiency and high substance uptake efficiency, and can be used for the development of high-performance microcapsules, chemical sensors, etc. Furthermore, since the diameter exceeds 5 μm, it is not only possible to observe with an optical IA microscope, but also
Because they can be mechanically manipulated using micromanipulators, microinjectors, etc., they are expected to have qualitatively different uses compared to conventional liposomes that have a small diameter.

However, conventionally known methods for forming giant proteoribosomes include electric field fusion, static hydration, and reversed-phase evaporation, but all of these methods have various limitations on the formation conditions. , especially difficult to form under physiological conditions.

Regarding these methods, the present inventors previously proposed a patent application filed in 1983-2.
No. 11910 discloses a method that allows large numbers of giant proteoribosomes to be formed under physiological conditions using simple and mild steps. That is, in this method, an alkali metal salt solution containing membrane proteins and membrane lipids is frozen and thawed, and then dialyzed against a salt solution or buffer having a lower osmotic pressure than the alkali metal salt solution. By the above procedure, it is possible to form a large number of giant proteoribosomes with a diameter of 5 to 100 μι.

[Problem to be solved by the invention] On the other hand, membrane proteins are embedded in biological membranes with constant orientation, and due to this anisotropic membrane structure,
vector-like substances such as stimulus reception and transmission, and material transport;
The movement of information is realized.

Therefore, when applying proteolibosomes in medical or industrial applications, it is important that membrane proteins are incorporated in a certain orientation in membranes reconstituted by artificial methods to efficiently express their functions. It is advantageous to do so.

However, in all conventional methods for forming giant proteoribosomes, there is no means to control the directionality of membrane proteins in the formation process, so the directionality of membrane proteins in the formed giant proteoribosome membrane cannot be controlled. is usually random. Therefore, even in the case of giant proteolibosomes, there has been a desire for a formation method that can control the orientation of membrane proteins and incorporate membrane proteins into the membrane.

The present invention was made to improve such conventional techniques, and aims to provide a method for forming giant proteoribosomes in which membrane proteins are incorporated by controlling directionality.

[Means and effects for solving the problem] The inventors of the present invention have previously filed a patent application filed in 1983-21 as a means of controlling the directionality of membrane proteins in the membrane of giant proteolibosomes.
The inventors discovered that the modification of the membrane protein carrier disclosed in No. 5924 is still basically effective, leading to the present invention.

That is, in the present invention, after modifying a specific site of a membrane protein into a carrier and preparing a salt solution containing the membrane protein and membrane lipid,
This method of forming giant proteolibosomes is characterized in that the salt solution is frozen and thawed, and then dialyzed against a salt solution having a lower osmotic pressure than the salt solution.

In addition, one aspect of the present invention is to modify a specific site of a membrane protein into a carrier,
After preparing a salt solution containing the membrane protein and membrane lipid, the salt solution is frozen and thawed, and then dialyzed against a salt solution having a lower osmotic pressure than the salt solution, and then the membrane protein is removed from the carrier. This is a method for forming giant proteoribosomes, which is characterized by dissociation.

The present invention will be explained in detail below.

The method for forming giant proteoribosomes according to the present invention is based on the method for forming giant proteoribosomes disclosed in the above-mentioned Japanese Patent Application No. 63-211910, in which a specific site of a membrane protein is modified with a carrier, and the membrane protein is modified with a carrier. By preventing the formation of giant proteoribosomes on both sides, it is possible to incorporate membrane proteins from one side, thereby controlling directionality.

In the method for forming giant proteoribosomes of the present invention, a specific site of a membrane protein is first modified and immobilized on a carrier.

The membrane proteins used in the present invention include membrane proteins that exist in biological membranes, especially integral membrane proteins that penetrate through membranes, and also include natural and synthetic peptides that exist in membranes. Substances that can be used can also be applied. A specific example is an enzyme that catalyzes material metabolism (
Cytochrome oxidase, cytochrome P-450,! binding phospholipase, ATP synthesis, etc.), channels that transport substances through membranes (potassium channel, sodium channel, calcium channel, etc.), pumps (sodium-potassium pump, proton pump, etc.), and reception of information in the body. - Neurotransmitter receptors involved in transmission (acetylcholine receptors, glutamate receptors, etc.), hormone receptors (thyrolimb stimulating hormone (TH3) receptors, glucagon (GC) receptors, etc.), etc.

These membrane proteins are generally insoluble in water and may have membrane lipids attached to them, but they can be used as is or dissolved in a surfactant to the extent that the directionality can be limited by the modified carrier. Can be used.

The giant proteoribosomes formed in the present invention have a large diameter of 5 to 300 IL■, and when the diameters of the giant proteoribosomes formed are distributed within a certain range, it is necessary to limit the directionality of all proteoribosomes. , one is used in which the volume of the carrier is equal to or greater than the volume of the proteoribosomes formed. The shape of the carrier does not need to be particularly limited, but for example, it may be spherical and its size is equal to or larger than the diameter of the proteoribosome to be formed, or it may be planar or curved and The radius of curvature may be equal to or greater than the radius of the liposome of the proteolibosome being formed.

For example, a spherical carrier has a diameter of at least 100 gm, and it is preferable that the diameter is at least 3001L1 in consideration of protein modification efficiency, void space for proteoribosome formation, etc.; There are products with a length exceeding 3004 m that can be used.

As mentioned above, the shape of the carrier does not necessarily have to be spherical, and any other shape can be used.
Specific examples of carriers that have a planar or curved shape include those that have a plate-like structure or a structure in which plates are laminated, and that have an active group on the surface to which proteins can bind, or that can be provided with an active group. For example, the hydroxyl groups on the surface of cellulose type membrane filters and 1 mm square pieces of nitrocellulose membrane used in dialysis membranes can be activated as appropriate with cyanogen bromide, etc. It can be preferably used to modify specific sites of membrane proteins. In addition, the pore size is coarse (1001411
The porous glass piece described above is also a carrier with high modification efficiency for membrane proteins.

Next, in the method of modifying and dissociating a specific site of a membrane protein with a carrier, it is preferable to bind and dissociate the carrier and the protein under mild conditions that do not destroy proteoribosomes or denature the protein.

In this regard, for example, protein chromatography techniques can be applied. Protein chromatography aims to purify proteins by detecting electrostatic attraction (ion exchange chromatography), hydrophobic interactions (hydrophobic chromatography), and hydrogen bonds (hydrogen bonds) between the protein and an appropriately chemically modified carrier. Chromatography), specific affinity (affinity chromatography), etc. are used to bind and dissociate.

Therefore, the carrier used in the present invention may be made of any material that can cause the above-mentioned interaction and modify a specific site of a membrane protein.

As such a carrier, those mentioned above are applicable.

The membrane lipid in the present invention can be used without any restriction as long as it has the property of incorporating membrane proteins and has a bilayer membrane lamellar structure, such as various phospholipids, α-lipids, and neutral lipids. etc. As another example, synthetic bilayer-forming lipids that do not occur naturally can also be used.

Furthermore, various additives can be added as long as the bilayer membrane structure is not destroyed.

Next, specific examples of membrane lipids include phosphatidylcholine (lecithin), phosphatidylethanolamine, glycerophospholipids such as diphosphatidylglycerol, sphingophospholipids such as nisphingomyelin and ceramide syriatin; cerebroside, sulfatide, ceramide oligohexoside, etc. glycosphingolipids; and glyceroglycolipids such as glycosyldiacylglycerol containing a carbohydrate as a parent group. Further, examples of additives include membrane structure reinforcing factors such as cholesterol, and charge-imparting substances such as stearylamine and dicetyl phosphate.

Next, in the formation method of the present invention, a salt solution containing a membrane protein and a membrane lipid modified into specific carriers as described above is prepared.

The salt solution in the present invention mainly contains alkali metal ions at a high concentration, and also contains other metal ions.

It can contain inorganic/organic compounds, etc. However, sucrose and other anti-freezing agents are not preferred, and the preferred alkali metals are potassium,
Rubidium and sodium are also used. Lithium is not suitable.

Further, the concentration of the salt varies depending on the type of salt, but for example, when potassium chloride is used, a high concentration of 2M or more is desirable. Further, the pH of the solution can be freely selected within a range that does not deactivate membrane proteins and membrane lipids.

In the salt solution, the concentration of membrane protein is based on the weight of lipid: membrane protein:lipid=l:1-1:200, preferably 1
:10~1:120 is desirable, and the concentration of lipid is usually 1~30mg/wj? +preferably lO~2
0 g/ml is desirable.

In the next step, after freezing and thawing the above salt solution,
A giant proteoribosome can be formed by dialysis against a salt solution having a lower osmotic pressure than the salt solution.

In this process, when a salt solution containing membrane proteins and membrane lipids is frozen, the membrane proteins and membrane lipids form a micellar complex and dissolve in the solution concentrated by freezing, and then the frozen salt solution is dissolved. When the solution is melted at room temperature, the protein-lipid complex undergoes micelle-lamella transition and forms a bilayer membrane.
Furthermore, by dialyzing the salt solution against a salt solution with a lower osmotic pressure than the solution,
Depending on the conditions, proteoribosomes with a diameter of 300 #Ls are produced.

The number of times of freezing and thawing is desirably 3 or more times, and 6 times is sufficient. Furthermore, after freezing and thawing, portex (v
It is desirable to perform stirring using an oltex mixer.

In addition, the salt solution used for dialysis usually contains 0.1 to 100
(2) An alkali metal or alkaline earth metal salt solution of M, preferably 1 to 20, can be used.

The giant proteoribosome formed in the above process is in a state in which the membrane protein is modified with a carrier, but the present invention can also form a giant proteoribosome by dissociating the membrane protein from the carrier. Methods for dissociation include adding to the column a substance that acts antagonistically on the binding between the carrier and membrane proteins, and changing the physicochemical conditions of the solution (pL salt concentration, etc.). In the former case, for example, if a lectin, a protein that has the property of binding to sugar, is bound to a carrier, taking advantage of the fact that most membrane proteins have sugar chains at their ends, if excess sugar is added, Sugars competitively bind to lectins and release membrane proteins. In addition, when binding to Ifi bodies is performed site-specifically using a monoclonal antibody specific to a specific site of a membrane protein using an antigen-antibody reaction, adding an excess of habten can also cause It can be competitively dissociated from membrane protein carriers. However, the bond between antigen and antibody is very strong, and it is difficult to dissociate it even with a large excess of Habten.
In addition, the method using lectin is superior because it is often difficult to prepare a large amount of habten.

In addition, the method of dissociating by changing the physicochemical conditions of the solution often destroys the formed proteoribosomes depending on the conditions, so its applicability is quite limited.

[Example] Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Highly halophilic bacterium Halobacterium halobium (11alo
Violet membrane, a membrane fragment containing bacteriorhodopsin, was obtained from Bacterium halobiu+s by P.
US) method [Methods of Enzymology (M
methods of Enzymology), 31
．． 667-678, (1974)]. In addition, the purple membrane can be added to Canis Hung (in, S,
Huang et al. [Proceedings of the National Academy of Sciences]
ng of National Academy of
5science.

USA, ) 88.323 pages, (1,980)
] Method was used to delipidate and purify bacteriorhodopsin.

A monoclonal antibody that specifically binds to the carboxyl terminus of bacteriorhodopsin was developed by Kei Kimura (K.
@ura) et al. [Journal of Biological Chemistry (J, Biol, Chew.

), OK, pp. 2859-2867, (1980)]. This monoclonal antibody specifically binds to a fragment consisting of 39 residues including the carboxyl terminus among peptide fragments obtained by treating bacteriorhodopsin with cyanogen bromide.

Millipore's cellulose type membrane filter (pore size 0.22IL) was cut into squares and treated with cyanogen bromide to activate the hydroxyl groups on the surface, making it possible to bind proteins with amino groups. .

To this, add 1 mg of the previously prepared CNBr-6 monoclonal antibody per 100 mg of the above membrane filter.
The monoclonal antibody was bound to the filter by additional reaction. Excess antibody was removed by washing.

Next, a chloroform solution of 15 mg of soybean phospholipid was placed in a test tube, and the solvent was distilled off using a rotary evaporator and a vacuum pump to create a lipid is on the test wall. 3'MKCj+ solution (pHa, o) 1 mj) was added, suspended using a portex mixer, and treated with a probe-type ultrasonic oscillator to obtain a liposome dispersion having a diameter of 10 nm or less.

A piece of membrane filter bound with a monoclonal antibody was added to this liposome dispersion, frozen in liquid nitrogen or dry ice-acetone, thawed at room temperature, volL
The operation of processing with an ex mixer for 30 seconds was repeated three times.

Thereafter, the suspension was transferred to a dialysis tube and dialyzed against a 10mM potassium chloride aqueous solution for 2 days, producing a large amount of giant proteoribosomes with a diameter exceeding 1101 L.

In the giant proteoribosomes obtained by the above procedure, bacteriorhodopsin is incorporated with the carboxyl terminus facing outward. This was confirmed by the fact that a peptide containing a carboxyl terminus was produced as a degradation product when ribosomes were treated with enzymes.

Example 2 After obtaining giant proteolibosomes by the same method as in Example 1, the pH was adjusted to 11 using 1 normal ammonia water to combine bacteriorhodopsin and CNBr-6 monoclonal antibody incorporated into the liposomes. was dissociated to obtain giant proteolibosomes with a diameter exceeding 5-1.

The resulting giant proteoribosome incorporates bacteriorhodopsin with its carboxyl terminus facing outward.
This was confirmed by the fact that the pH of the external fluid became alkaline when the proteoribosome FJ suspension was irradiated with light, and that a peptide containing a carboxyl terminus was produced as a decomposition product when proteoribosomes were treated with enzymes.

Example 3 Bovine rhodopsin was isolated and purified by the following procedure.

All operations were performed under dark red light (Eastman Kodak Red Filter No. 1).

Discs, which are membranous structures containing rhodopsin, were extracted from bovine retina using the Ficoll flotation method (S■ith et al., Experimental Eye Research (Exp.
Eye Res, ), 20.211-217. (1
975)). The purified disk was mixed with 50 μg of octyl glucoside, 0.1i1 N
aC1゜1i+M MnCIz, 1mM CaCl2
．． After solubilizing with a solution of 10 sil Mops-NaOH (pH 7,0), Wheat gerta Lec
Rhodopsin was purified by affinity chromatography using tin-3 epharose 6 MB (Pharmacia, average particle size approximately 300 um) in a batch process (hot volume 1112 ml). That is, a solubilized disk solution (5 mg as protein) was added to the gel to allow rhodopsin to bind with lectin, and then the gel was soaked in a buffer solution (0.1M NaCl, 1mM MnCl).
2. 1mM CaC1, 10sil0ml-
Impurities were removed by thorough washing with NaOH (pH 7,0). Rhodopsin was used to form proteoribosomes while remaining bound to the carrier gel particles via lectin.

Next, a chloroform solution of soybean phospholipid 15B was placed in a test tube, and the solvent was distilled off using a rotary evaporator and a vacuum pump to form a thin lipid film on the test tube wall. Add to this 3t1 NaC1, 1mM MnC12, 1mM
CaCl2. 0.1M Mops-Na0)+
(pH 7,0) lso2 was added, suspended using a portex mixer, and treated with a probe-type ultrasonic oscillator to obtain a liposome dispersion having a diameter of 100 nm or less.

Next, the rhodopsin-bound gel is added to the liposome dispersion, stirred for 30 minutes, and then the dispersion is frozen with liquid nitrogen or dry ice-acetone, thawed at room temperature, and treated with a VO1tex mixer for 30 seconds. The operation was repeated three times. After this, the suspension was transferred to a dialysis tube and
0d Na1l, 10mM Mops-NaOH (
Dialysis against pH 7.0) for 2 days resulted in a diameter of 10
A large amount of giant proteoribosomes, which exceeds that of Iruri, was produced.

Next, in order to dissociate rhodopsin from the carrier and separate the giant proteolibosome from the carrier, 20 -N-acetyl-α-D-glucosamine, 10 volumes of NaCl, 10
After adding 2 mM of mM IIIops-NaOH (pH 7,0) to 1 mM of the dispersion and incubating for 1 hour, the supernatant was collected and a giant proteoribosome dispersion was collected. Furthermore, l0wM NaCl, 10m
N-acetyl-α-D-glucosamine was removed by dialysis against M Mops-NaOH (pH 7,0).

The yield was about 30% as protein.

In order to confirm that rhodopsin was incorporated into the generated giant proteoribosome membrane, the giant proteoribosome dispersion was rapidly frozen in a liquid helium quick freezing device (Eiko Engineering Co., Ltd.: RF-23 model). A replica of the membrane surface was created using a freeze-fracture replica making device (Eiko Engineering Co., Ltd.: Model FD-5A), and a transmission electron microscope (JEOL Co., Ltd.:
JEM 1000 model) was observed. Approximately 4n in diameter on WA surface
m particles were observed, confirming that rhodopsin was incorporated.

In addition, when β-N-acetylgelcosaminidase (Sigma) is applied to a giant proteoribosome dispersion and an external solution is analyzed, free sugars are detected, and the sugar residues are directed toward the outside of the proteoribosome and incorporated. It was confirmed that

[Effects of the Invention] As explained above, according to the present invention C1, in the method for forming giant proteoribosomes, the effect of being able to control the directionality of membrane proteins incorporated into giant proteoribosomes can be obtained.

Furthermore, according to the present invention, since the directionality of membrane proteins is controlled, the functions of membrane proteins can be expressed correctly and efficiently in giant proteolibosomes, which have the same size as cells. The industrial and technical value of the present invention is extremely high, as it opens the way to a new field of application of giant proteolibosomes, which have not been put into practical use due to the lack of an appropriate production method.

Claims (5)

[Claims] (1) After modifying a specific site of a membrane protein into a carrier and preparing a salt solution containing the membrane protein and membrane lipid, the salt solution is frozen and thawed, and then the osmotic pressure is lower than that of the salt solution. A method for forming giant proteolibosomes, which comprises dialysis against a saline solution. (2) After modifying a specific site of a membrane protein into a carrier and preparing a salt solution containing the membrane protein and membrane lipid, the salt solution is frozen and thawed, and then the osmotic pressure is lower than that of the salt solution. A method for forming giant proteolibosomes, which comprises dissociating membrane proteins from a carrier after dialysis against a saline solution. (3) The method for forming giant proteoribosomes according to claim 1 or 2, wherein the volume of the carrier is equal to or larger than the volume of the proteoribosomes to be formed. (4) The method for forming giant proteoribosomes according to claim 1, 2 or 3, wherein the carrier has a spherical shape and a size that is equal to or larger than the diameter of the proteoribosome to be formed. (5) Formation of giant proteoribosomes according to claim 1, 2 or 3, wherein the shape of the carrier is planar or curved, and the radius of curvature is equal to or greater than the radius of the proteoribosomes to be formed. Method.