JPS6222796A - Separation and purification of biomembrane protein - Google Patents

Abstract

PURPOSE:To solubilize a biomembrane protein without denaturing the protein and separate and purify the above-mentioned protein in high purity without damaging the biomembrane, by carrying out gel electrophoresis in the presence of a specific sulfuric acid ester type anionic surfactant at a specified temperature or below. CONSTITUTION:A biomembrane protein, e.g. thylakoid membrane protein of spinach, is subjected to the gel electrophoresis in the presence of an alkyl(alkylphenyl) sulfuric acid ester salt type anionic surfactant, having at least one N atom as a counter ion in the molecule thereof and expressed by the formula ROSO3M (R is 6-22C alkyl or alkylphenyl; M is cation having one or N atoms), e.g. triethanolamine salt of dodecylsulfuric acid, preferably in 0.1-3wt% concentration expressed in terms of aqueous solution concentration, at <=15 deg.C, preferably 0-10 deg.C. Preferably, polyacrylamide gel is used as the gel for the electrophoresis and a buffer solution containing the same ion species as the above-mentioned counter ion is used as the buffer solution. The pH is preferably 4-9, particularly preferably 7.

Description

[Detailed Description of the Invention] Industrial Application Fields] The present invention is directed to the production of biomembrane proteins, which have been attracting attention and whose usefulness is being recognized in various fields such as pharmacy, medicine, and engineering, without impairing their biological functions. This invention relates to a method of separation and purification.

[Conventional technology]

Biological membranes are mainly composed of polar lipids and membrane proteins. Among these, membrane proteins maintain biological functions, and these are inserted into the bilayer membrane of polar lipids, especially phosphorus membranes, which make up the majority of the membrane proteins. Therefore,
Biological membrane proteins, such as E. coli outer membrane porin, cytochronoJb5, (Na+, K'')-ΔT Pa5e
', (Ca”) −A T Pa5e, (t(
When separating and purifying ATPase, etc., it is necessary to solubilize membrane proteins as the first step in separation and purification, as most of these are poorly water-soluble and are different from water-soluble globular proteins. be.

Solubilization of membrane proteins requires a medium with an environment similar to that of a lipid bilayer, and various organic solvents and aqueous surfactant solutions are used. Typical examples of organic solvents include acetone, butanol, ethanol, and pyridine, and examples of surfactants include anionic surfactants such as sodium dodecyl sulfate and 1 to 3-dimethyldodecylmonium chloride. There are cationic surfactants and nonionic surfactants typified by polyoxyethylene dodecyl ether.

However, since many organic solvents act as strong denaturants for proteins, it is often difficult to separate and purify membrane proteins without impairing biological functions. In addition, sodium dodecyl sulfate (hereinafter abbreviated as SDS), which is one of the anionic surfactants conventionally used in the biochemical field, acts as a strong protein denaturing agent. However, it is often difficult to separate and purify membrane proteins without impairing biological functions.

Therefore, attempts have been made to use nonionic surfactants with low protein denaturation properties as a solubilization medium.

However, many nonionic surfactants have a drawback that their critical micelle concentration is low, making it difficult to remove the surfactant bound to membrane proteins by dialysis after separation and purification.

On the other hand, it is possible to use lIO sulfate salts as anionic surfactants with low protein denaturation ability, but they belong to biosurfactants and are expensive, so they cannot be used in large quantities industrially. There is a fatal flaw. In addition, cationic surfactants are often used as bactericidal agents because they bond more strongly with the lipids that make up biological membranes than other surfactants, and their denaturing effects on proteins are not weak. There are few examples of successful separation and purification, and it cannot be said that it is a surfactant with a wide range of applications.・In contrast, separation and purification methods that focus on the physical and chemical properties of proteins themselves include thermal or PL+ treatment methods, fractional precipitation methods, adsorption/desorption methods, chromatography methods using ion exchangers, and isoelectric point fractionation. method, density gradient centrifugation method, electrophoresis method, chromatography method, molecular sieve method, two-phase partition method, and crystallization method, but all of these methods have advantages and disadvantages and are not satisfactory. There wasn't.

[Problems to be Solved by the Invention] The present invention provides a method for solubilizing biological membrane proteins without denaturing them and separating and purifying them with high purity without impairing biological functions.

[Means for solving problems]

The present invention shows that when gel electrophoresis is performed at low temperature in the presence of an alkyl (alkylphenyl) sulfate salt type anionic surfactant having a cation having at least one nitrogen atom in the molecule as a counterion, as a phenomenon.”
This is based on the knowledge that the above problems can be resolved.

That is, the present invention provides an anion represented by the general formula RO8'DJ (wherein R is an alkyl group having 6 to 22 carbon atoms or an alkylphenyl group, and M is a cation having at least one nitrogen atom). In the coexistence of a surfactant,
Provided is a method for separating and purifying biological membrane proteins, characterized in that gel electrophoresis is performed at 15° C. or lower.

The anionic surfactant used in the present invention is represented by the general formula 2, where R has 8 to 8 carbon atoms.
A linear alkyl group having 16 carbon atoms or a branched alkylphenyl group having 6 to 14 carbon atoms is preferred, and particularly preferably a C 1 straight-chain alkyl group.
It is a linear alkyl group having 0 to 14 carbon atoms or a branched alkylphenyl group having 8 to 12 carbon atoms. On the other hand, in the formula, M is preferably ammonia l or quaternary ammonium having at least one hydrocarbon group having 1 to 6 carbon atoms in the molecule (each having no surface-active ability). Specifically, M includes ammonium, monomethylammonium, dimethylammonium, trimethylammonium, tetramethylammonium, monoethylammonium, diethylammonium, triethylammonium, tetraethylammonium, monoethanolammonium, jetanolammonium, triethanolammonium, and tetraethanolammonium. , tris(hydroxymethyl)
Methylammonium, tris(hydroxyethyl)methylammonium, monoisopropanol ammonium,
Examples include diisopropanol ammonium, triisopropanol ammonium, tetraisopropanol ammonium, pyridinium, and morpholinium J.

Particularly preferred as the anionic surfactant of the present invention are:
These are tris(hydroxymethyl)methylammonium salt, triethanolamine salt, and triisopropanolamine salt of decyl sulfate, dodecyl sulfate, tetradecyl sulfate, and hexidecyl sulfate.

In the anionic surfactant used in the present invention, the number of carbon atoms in R in the formula must be 6 to 22, but this is because when the number of carbon atoms in R is less than 6, the surfactant ability is poor and the film This is because proteins cannot be solubilized efficiently, and on the other hand, proteins with a carbon number of 22
This is because if the amount exceeds this amount, the water solubility deteriorates, making it difficult to achieve the desired purpose.

In the present invention, in the presence of the above-mentioned anionic surfactant,
The following is characterized by using gel electrophoresis. Although known methods can be used for gel electrophoresis, the following method is preferred.

(a) Support for gel electrophoresis Starch gel, agarose gel, polyacrylamide gel, etc. can be used. Polyacrylamide gel is chemically stable, the gel concentration and degree of crosslinking can be freely controlled, that is, the pore size can be freely changed according to the purpose, and it is not electroosmotic and does not deform due to PLL or temperature changes. It is particularly preferable because it has many advantages such as being able to be molded into any mold with a small amount and having very good reproducibility.

(b) Apparatus and equipment A container for supporting gel such as polyacrylamide gel;
It is preferable to use a tank for filling both ends with a buffer solution, and the capacity, length, etc. of the tank can be arbitrarily set depending on the type and amount of the protein to be separated and purified. In addition, when performing gel electrophoresis at 0 to 15° C., it is desirable to use an air cooling or water cooling device.

(c) Power supply device It is particularly preferable to use direct current and a constant current/constant voltage generator because separation and purification performance and reproducibility are improved.

(d) Preparation of polyacrylamide gel for electrophoresis Contains 1 to 30% by weight of acrylamide, 005 to 10% of N,N'-methylenebisacrylamide to the acrylamide, and 0 to 1% by weight of the surfactant of the present invention. After preparing an aqueous solution, it is preferable to use the aqueous solution by gelling it by a polymerization reaction. The preferred gel concentration is such that the total amount of acrylamide and N,N'-methylenebisacrylamide is 3.
~15% by weight (hereinafter abbreviated as %) and N, N'
- Methylenebisacrylamide is in the range of 1 to 5% relative to acrylamide. Also, the surfactant concentration is 0.0
Particularly preferred is 5 to 0.2%.

Note that it is also possible to gel a polymerization catalyst, a polymerization accelerator, a p++ buffer, a preservative, etc. by mixing the aqueous solution described in 1 above. Riboflavin or the like can be used as a photopolymerization catalyst, or ammonium persulfate or the like can be used as a radical polymerization catalyst in any amount, preferably 0.04 to 0.12%. As a polymerization accelerator, for example, N,N,N',N'-tetramethylethylenediamine in an arbitrary amount, preferably 0.03 to 5%, particularly preferably 0.03 to 0.12%.
It can be used in Since dissolved oxygen may inhibit gelation, it is desirable to cover the top of the acrylamide aqueous solution with water to block air during degassing or gelation. As the p■ buffer, one containing the same ionic species as the counter ion M of the anionic surfactant used in the present invention can be used, and gel electrophoresis can be performed at the desired pH. Additionally, ρ11 buffers such as thorium phosphate may also be used, but their original effects tend to decrease. p) I buffer concentration 10-500mM, preferably 50-200mM
mM, particularly preferably 100mM.

(e) Temperature and ρ11 during separation and purification The temperature during separation and purification is preferably 0 to r5t, particularly preferably 0 to 10°C, and pll is preferably 4 to 9, particularly preferably 7. This is because if the temperature is lower than 0°C, the water in the gel may freeze, making migration impossible, and if the temperature exceeds 15°C, the membrane protein aggregate may denature and its function may be impaired, which is undesirable. Also, p+
If + is less than 4 or more than 9, acid/alkali denaturation may occur, which is also undesirable. The anionic surfactant of the present invention does not cause precipitation (10-) even when used at low temperatures, that is, 0° to 15°C. Therefore, when gel electrophoresis is performed at this temperature, denaturation of protein aggregates can be suppressed. It is particularly preferable to perform gel electrophoresis at 0°C.

) It is best not to fill both ends of the polyacrylamide gel for aqueous solution migration in the buffer tank with buffer solution. This buffer solution is composed of a buffer, a surfactant, and water contained in an aqueous solution for producing polyacrylamide gel. The buffering agent concentration is preferably the same as that in the aqueous solution for preparing polyacrylamide gel.

(g) Aqueous solution containing the membrane protein to be separated and purified. In addition to the membrane protein, the surfactant of the present invention is contained in an amount of 0.01 to
5%, preferably 0.1-3%, particularly preferably 0.5
Contains ~2%. Further, for the purpose of improving the separation and purification ability, a viscous liquid such as chrycerin can be added in an amount of 1 to 30%, but preferably 5 to 20%.

Further, this aqueous solution can contain a gel and a buffer in a buffer solution, and the concentration thereof is preferably 5 (10 m M or less) and a mild (Φ) buffer agent in the solution 8 degrees or less. Particularly preferably 1/2 to 1/2 of the buffer concentration in the buffer solution
The concentration is 1/20.

Furthermore, in this aqueous solution, a water-soluble anionic dye, pigment such as bromophenol blue was added to calculate the relative mobility, and a water-insoluble dye soluble in the micelles was added to indicate the position of the surfactant micelles. It is also possible to mix dyes such as yellow OB, which is an oily dye. In addition, cationic dyes such as malachite green form insoluble complexes with dodecyl sulfate ions, resulting in dodecyl sulfate micelles.
Since it is immediately solubilized and migrates with micelles, it is convenient for determining the mobility of micelles. These concentrations can be used within any range, but preferably 0.00 in the former case.
1 to 0.05%, and in the latter case 0.01 to 0.5%.

Electrophoresis is started by placing this aqueous solution on both ends of a polyacrylamide gel for electrophoresis.

Membrane proteins are separated and purified into disk shapes in the gel.

Membrane proteins separated into disks are stained with a dye and are recognized as colored particles. The dyes used here are, for example, Amidopura Tsuku, Coomassie Brilliant Blue, etc., and conventional methods (for example, Katsuya Hayashi, Ikuzo Uriya, Kensuke Toramura, Noriyoshi Nakamura, Katsuji Funatsu, editors, "Biochemical Experimental Methods - Protein Electrical properties''′□Society Publishing Center)
The degree of separation and purification of membrane proteins can be confirmed according to the following. Moreover, it is possible to dye fffl, δ as a green color without dyeing with dye.

Objects that can be separated and purified by the method of the present invention include all biological membrane proteins extracted and solubilized from animal organs, cultured cells, microbial cells, plant cells, etc.

(I) Separation and Purification The supernatant containing all of the solubilized membrane protein components can be directly separated and purified by the method of the present invention. In addition, the solubilized solution is subjected to conventional treatments such as nucleic acid removal, and the solution or the solution that has gone through the initial stages of purification is highly separated and purified depending on the purpose, such as fractional precipitation or density gradient centrifugation. It is also possible. In this case, in the rough separation and purification step prior to the separation and purification of the present invention, the membrane protein can be reversibly restored to its activity without impairing biological functions and even if denaturation occurs, the factors that bring about the denaturation are removed. It is desirable to adopt a method that allows for As described above, the method for separating and purifying biological membrane proteins according to the present invention is applicable to all biological membrane proteins and can be applied at any purification stage, so it is commercially advantageous. .

〔Effect of the invention〕

(1) Unlike bile salts, the anionic surfactant used in the present invention is commercially advantageous because it can be synthesized industrially at low cost and easily.

(2) The anionic surfactant used in the present invention can be used at low temperatures without precipitating, and when it acts on membrane proteins, it does not decompose into its constituent components (blade units), so it can be used in its raw state. It is advantageous because it can be separated and purified.

(3) Each membrane protein solubilized by the anionic surfactant has a specific amount of anionic surfactant bound to it, and each protein-surfactant complex has a different number of negative charges. ing. They each move at their own speed through the electrophoretic support toward the anode.

At this time, since the molecular sieving effect of the support is also added, the separation and purification ability is much improved compared to the gel filtration method, which can only expect a simple molecular sieving effect.

(4) The second drawback of separating and purifying membrane proteins solubilized with surfactants by gel filtration is that a large amount of solvent is required. The separation and purification method according to the present invention is advantageous in that this is not necessary.

(5) Since nonionic surfactants have a low critical micelle concentration, it is often difficult to remove nonionic surfactant particles bound to membrane proteins by dialysis. In the separation and purification method of the present invention, the critical micelle concentration of the surfactant used is higher than that of nonionic surfactants, so the surfactant can be easily removed by dialysis or even electrodialysis.

Next, the present invention will be explained with reference to Examples, but the present invention is not limited thereto.

〔Example〕

The method according to the present invention was applied to spinach thylakoid membrane protein, and will be described below with reference to Examples and Comparative Examples. Incidentally, gel electrophoresis was performed as follows.

0 Gel Electrophoresis Gel electrophoresis was performed according to the 5DS-polyacrylamide gel electrophoresis method described in the paper. [For example, 1. ■, V. Maizel, Jr.

″Methods in Virology
Il, Academic Press.

(197]), P, 179: Toshio Takagi, Jun Miyake, ゛′
New Experimental Chemistry Course Volume 20 Biological Chemistry ■ (Chemical Society of Japan)
Pamaruzen (1978), P., 109) However, no SS bond cleavage agent is added, S and DS are used as the surfactants in the present invention, and the buffer contains counterions of the surfactants in the present invention. I changed it to something. The gel composition and buffer composition are shown in Table 11 and Table 2.

The internal temperature of the gel during electrophoresis was controlled at approximately 0° C. by cooling the gel with ice water.

Table-1 Gel composition Acrylamide 5%N
, N'-Methylenebisacrylamide (relative to acrylamide) 2.7% ammonium persulfate 0.07% N,N,N',N,'-
Tetramethylenediamine 0.12% buffer (pH 7) 100mM surfactant 0.1% water
Balance Ding 7 Table-2 Buffer Tissue buffer (pH?) 100mM surfactant 0.1% water
Balance Example 1 A thylakoid membrane suspension (1.5 mg/mj2 concentration as chlorophyll) fractionated from spinach was directly subjected to 6% polyacrylamide gel electrophoresis.

Solubilization and electrophoresis of thylakoid pancreas was performed at 0°C.

As a result, 5 wires (CF2, CF2, C
A sharp green chlorophyll band (abbreviated as F2, CF2, CF2) and a broad free chlorophyll band were obtained. Five chlorophyll bands were cut out, and each band was treated with lithium dodecyl sulfate (hereinafter referred to as 1), which is a stronger protein denaturant than SDS.
Polyacrylamide gel electrophoresis was further performed in the presence of DS (abbreviated as DS). 1. , DS exhibits an affinity (binding) for polyacrylamide, so in order to perform normal electrophoresis of proteins, 11136 degrees in the gel composition and buffer composition were set to 1% and 0.4%, respectively. The tubercles are 10 to 13 in CPI and Ce2, and 1 in Ce3.
Five to six major protein polypeptide bands were obtained for Ce4 and five major protein polypeptide bands for Ce5. I cut it out again 5
When the chlorophyll hand in the book was subjected to rearyl electrophoresis again, the chlorophylls of each strand other than Ce4 showed the same mobility as a single band as in the -th electrophoresis, and almost no dissociation of chlorophyll was observed. I couldn't. That is, the chlorophyll band obtained by the method of the present invention is a band of a chlorophyll-protein molecule assembly, and furthermore, as is clear from the results of repeated electrophoresis, it has been found that the protein molecule assembly does not denature at 0°C. Ta. Previous research has shown that the structural intermediates of photosynthesis in green plants include the photosystem ■, the photosystem ■, and the photochemically inactive light-harvesting chlorophyll-protein complex (
It is roughly divided into three types: L l-I Cp).

(Kiyuki Sato, “Proteins, Nucleic Acids, Enzymes, Separate Volume N0121 Mechanism of Photosynthesis” Kyoritsu Shuppan (] 979), p. 4O-5
1) The construction of the chlorophyll-protein molecular assembly of the photosystem has been well studied.
, Chem, 252.4564-4569 (19
77): , J.E., Mullet, J.J., Bur.
ke and C,,1,8rntzen.

Plant Physiol, 15,814-
822. 823-827 (1980)), the CPI obtained in the present invention was confirmed to be a chlorophyll-protein molecule assembly of photosystem I from the measurement of the protein polypeptide composition, chlorophyll a/bl:H, and absorption spectrum. Ta.

Regarding L HCP, there is one type of polypeptide chain that constitutes it (R, RlJ, Hoarau and 1.
C1 Leclerc.

Photochem, Photobiol, 26
．． 151-158 (1977); 5atoh, P.
lant Ce1l Physiol.

20.499-512 (1979)) and 3-5 types (J, , 1. Burke, K, B
, 5teinback and C,, 1, Arntz
Plant Physiol, 63.
237-2 4 3 (1979); B, Ande
rsson, J.M., anderson andl.
, 1. Ryrie, I! ur, J., Biochem.
, 1 2 3, 465-472 (19B2>). However, according to the results of the present invention, there are two types of polypeptide chains corresponding to L HCP, and based on the number of polypeptide chains, chlorophyll a/b ratio, and absorption spectrum measurements, it is thought that Ce3 corresponds to the former and Ce5 corresponds to the latter.

Although there is currently no unified description of photosystem ①, there is a strong possibility that Ce4 is a chlorophyll-protein molecule assembly of photosystem H based on the polypeptide composition.

Comparative Example 1 When polyacrylamide gel electrophoresis was performed by replacing the surfactant in Example 1 with LDS, all the chlorophylls migrated as one broad band. This result indicates that all chlorophyll was dissociated from the protein molecule assembly. Furthermore, the electrophoresis pattern of the protein band detected by staining was the same as the electrophoresis pattern for the sample that had been heat denatured in advance. This shows that the chlorophyll-protein molecular assembly is completely denatured by LDS even at 0°C.

When using LDS, it is impossible to separate and purify the chlorophyll-protein molecule aggregate state (raw state).

Comparative Example 2 The same operation as in Example 1 was performed at 25°C. As a result, three types of chlorophyll-protein molecular aggregates were separated. The chlorophyll-protein molecular assembly CPI obtained in Example 1 was completely denatured and dissociated, and the corresponding chlorophyll band and protein band disappeared. Most of the denatured and dissociated CPI migrated at Ce2 and Ce5 positions.

In other words, the band obtained by this operation can be said to be a collection of chlorophyll-protein molecular aggregates that are far removed from their original appearance. Therefore, separation and purification with high purity is impossible.

Claims (1)

[Claims] In the coexistence of an anionic surfactant represented by the general formula ROSO_3M (wherein R is an alkyl group or alkylphenyl group having 6 to 22 carbon atoms, and M is a cation having at least one nitrogen atom), A method for separating and purifying biological membrane proteins, characterized by performing gel electrophoresis at 15°C or lower.