JPH0366699A - Separation and purification of protein - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for solubilizing a desired protein without denaturing it and separating and purifying it with high purity without impairing biological functions.

Conventional Techniques Highly purified proteins are not only important for studying protein function and structure, but are also widely used in the fields of pharmaceutical science and engineering. In particular, biological membrane proteins are essential for maintaining biological functions, and they mainly constitute biological membranes by being inserted into a bilayer membrane of polar lipids, especially phospholipids, which make up the majority of them.

Biological membrane proteins, such as red blood cell membrane glycophorin, Escherichia coli outer membrane porin, cytochrome b s (Na”, K”)A
TPase, (Ca”)ATPase, (H”)A
Many such as TPases are poorly water-soluble themselves, and unlike water-soluble globular biomembrane proteins, biomembrane proteins must be solubilized as the first step of separation and purification. media such as various organic solvents such as acetone, butanol, ethanol, and pyridine, anionic surfactants such as sodium dodecyl sulfate, cationic surfactants such as trimethyldodecyl ammonium chloride, and polyoxyethylene dodecyl ether. Aqueous solutions of surfactants such as nonionic surfactants are used.

However, many organic solvents act as strong denaturing agents for biological membrane proteins, so it is often difficult to separate and purify biological membrane proteins without impairing biological functions.

Furthermore, nonionic surfactants conventionally used in the biochemical field have a low critical micelle concentration, making it difficult to remove the surfactant bound to biological membrane proteins by dialysis after separation and production.

In addition, there are known anionic surfactants that use alkyl sulfates (Japanese Patent Application Laid-open No. 6136296, Japanese Patent Application Laid-open No. 62-22796), but these are susceptible to hydrolysis; The conditions are inevitably restricted, and although sulfonates such as alkylbenzene sulfonates and σ-olefin sulfonates are known (Japanese Patent Application Laid-open No. 76500/1983), these Although it is not easily affected by hydrolysis, it has drawbacks such as causing biomembrane protein denaturation depending on sensitive biomembrane proteins. Although it is possible to use bile salts as anionic surfactants with low protein denaturation properties, they belong to biosurfactants and have the disadvantage that they cannot be industrially mass-produced.

In addition, cationic surfactants are often used as bactericidal agents because they have a stronger bond with the lipids that make up biological membranes than other surfactants and do not have a weak denaturing effect on biological membrane proteins. There are few examples of its use in the separation and purification of membrane proteins, and the scope of application is inevitably limited.

On the other hand, separation and purification methods that focus on the physicochemical properties of biological membrane proteins themselves include heat or pH treatment methods, fractional precipitation methods, adsorption/desorption methods, chromatography methods using ion exchangers, isoelectric focusing methods, density gradient centrifugation, electrophoresis,
Affinity chromatography method, molecular sieve method,
There are two-phase distribution methods, crystallization methods, etc., but each of these methods has advantages and disadvantages and cannot be said to be completely satisfactory.

Problems to be Solved by the Invention The present invention overcomes the drawbacks of such conventional protein separation and purification methods, solubilizes proteins such as necessary biological membrane proteins and water-soluble proteins without denaturing them, and improves biological functions. The purpose of this work is to provide a method that can separate and purify to high purity with good reproducibility without damaging.

Means for Solving the Problems The present inventors have conducted various studies in order to develop a method for separating and purifying proteins having the above-mentioned preferable characteristics. or by using a mixture of this and at least one or two surfactants selected from cationic surfactants and amphoteric surfactants of different ionic species to achieve the purpose. Based on this finding, the present invention was completed.

That is, the present invention provides - formula % formula % (1) (wherein R is an alkyl group having 6 to 22 carbon atoms or a (1)
(In the formula, R is a hydrocarbon group having 1 to 4 carbon atoms, Q and m are each 0 to 20.12+m is 1 to 40.n is 2 to 4, and M is an alkali metal, alkaline earth metal, ammonium or quaternary ammonium), a surfactant selected from a sulfonic acid surfactant represented by , a cationic surfactant, and an amphoteric surfactant. The present invention provides a method for separating and purifying proteins, which is characterized by subjecting proteins to gel electrophoresis in the presence of a mixed system with at least one type of agent.

The present invention will be explained in detail below.

The protein to be separated and purified by the method of the present invention is not particularly limited as long as it can be extracted and solubilized from animal organs, cultured cells, microbial cells, plant cells, etc.
Examples include TPage and lactoglobulin.

Formula (1) representing the anionic surfactant used in the present invention
R has 10 to 14 carbon atoms, Q and m have 0 to 1O1Q+
m is preferably 3 to 15, n is preferably 2 to 3, M is preferably an alkali metal, an alkaline earth metal, ammonium, etc.
Particularly preferred are sodium and ammonium.

Examples of the cationic surfactant used in the present invention include long-chain alkyl tri-short-chain alkyl quaternary ammonium salts, long-chain alkyl di-short-chain alkyl benzyl quaternary Examples include quaternary ammonium salts such as ammonium salts, long-chain alkyl amine salts, and long-chain amine oxides. Quaternary ammonium salts and amine oxides in which the long chain alkyl group has to-18 carbon atoms are preferred.

As counter ions for these quaternary ammonium groups, halogen ions and monoalkyl sulfate groups are preferable, and chloride ions and bromide ions are particularly preferable.

Specific examples of these surfactants include dodecyltrimethylammonium salt (chloride salt, monomethyl sulfate), dodecyldimethylamine oxide, hexadecyljetanolamine oxide, and the like.

Examples of the amphoteric surfactant used in the present invention include long-chain alkyldialkylbetaines, 2-long-chain alkyl imidazoline derivatives, and long-chain alkyldialkylsulfobetaines, among which the long-chain alkyl group has 10 to 10 carbon atoms. 14 long-chain alkyl dialkyl betaines are preferred, and specific examples include dodecyl dimethyl betaine and tetradecyl dimethyl sulfobetaine.

In the present invention, the specific anionic surfactant of the formula (I) may be used alone, but preferably selected from the anionic surfactant, a cationic surfactant, and an amphoteric surfactant. It is desirable to use at least one type of surfactant in combination to form a mixed system.

By using such a surfactant of the present invention, for the first time, proteins generated by using an anionic surfactant such as sodium dodecyl sulfate (hereinafter abbreviated as SDS) alone, a cationic surfactant, or an amphoteric surfactant can be removed. This eliminates disadvantages such as degeneration. Although the details of its mechanism of action are unknown, it is presumed that the cicada nature of the specific anionic surfactant or the ionic complex of the mixed surfactant are involved in some way.

In the present invention, when a mixed system of the anionic surfactant and cationic surfactant is used, the mixing ratio thereof is preferably in the range of 9515 to 50150 in molar ratio, particularly in the range of 90/lo to 70/30. selected.

In particular, when dodecyltrimethylammonium salt is used in combination with polyoxyethylene (average number of added moles of 8) sodium lauryl ether propiosulfonate, the mixing ratio thereof is extremely preferably 90/10 in terms of molar ratio.

In the present invention, when a mixed system of the anionic surfactant and amphoteric surfactant is used, the mixing ratio thereof is as follows:
Preferably the molar ratio is 9515 to 50150, especially 90/
Selected in the range of 10 to 60/4Q. In particular, when long-chain alkyl dimethylamine oxide is mixed with polyoxyethylene (average number of added moles of 8) sodium lauryl ether propiosulfonate, the mixing ratio is extremely preferably 70/30 in terms of molar ratio. .

In the present invention, proteins are separated and purified by subjecting them to gel electrophoresis treatment in the presence of the above surfactant.

Liquid chromatography treatment can also be applied instead of this gel electrophoresis treatment.

This gel electrophoresis method is not particularly limited as long as it is a method commonly used for protein separation and purification. As a support for gel electrophoresis, for example, polyacrylamide gel, cellulose acetate, Sephadex, vinyl chloride-vinyl acetate copolymer, polyvinyl chloride, starch granules, starch gel, agarose gel, etc. are used, among which polyacrylamide gel The gel is chemically stable, and the gel concentration and degree of crosslinking can be freely controlled, so the pore size etc. can be changed freely.It is not electroosmotic and changes little due to pH and temperature changes, so it can be formed into any shape. It is particularly preferred because it has many advantages such as being moldable and having very good reproducibility.

To prepare this polyacrylamide gel, preferably 1 to 30% by weight of acrylamide and 0.05 to 30% of N,N'' methylene bisacrylamide to the acrylamide.
After preparing an aqueous solution containing 10% by weight and 0.01 to 1% by weight of the surfactant of the present invention, a method is used in which the aqueous solution is gelled by a polymerization reaction. In this preferred gel, acrylamide and N,N'-
Methylenebisacrylamide has a total amount of acrylamide of 3 to 15% by weight (hereinafter abbreviated as %), and N,
N'-methylenebisacrylamide is selected in proportions ranging from 1 to 5% relative to acrylamide. Moreover, the surfactant concentration is particularly preferably 0.05 to 0.2%. Note that it is also possible to gel the aqueous solution by mixing a polymerization catalyst, a polymerization promoter, a pH buffer, a preservative, etc. with the aqueous solution.

As the polymerization catalyst, for example, riboflavin is used when polymerization is initiated by light such as a fluorescent lamp, and ammonium persulfate is used in an arbitrary amount when polymerization is initiated at room temperature, preferably 0.04%. It is used in the range of ~0.12%. Examples of the polymerization accelerator include N, N,
N',N'-tetramethylethylenediamine is used in any amount, preferably in the range of 0.1-0.5%. Since dissolved oxygen may inhibit gelation, it is desirable to further cover the upper layer of the aqueous solution with water during degassing or gelation to prevent contact between air and the aqueous solution. pH
As the buffer, for example, sodium dihydrogen phosphate-disodium hydrogen phosphate system, sodium carbonate-sodium hydrogen carbonate system, etc. are used, and the concentration thereof is 10 to 500 mM.
.

Preferably it is adjusted to 50-200mM.

Electrophoresis treatment is usually carried out at 1 to 40°C, preferably 4 to 20°C.
It is carried out at a temperature of 4 to 9, preferably 6 to 8 pt+. If this temperature is below 0°C, the water in the gel may freeze, and if it exceeds 40°C, the protein may be thermally denatured and its function may be impaired, which is not preferable. Furthermore, if the pH is less than 4 or more than 9, acid/alkali denaturation may occur, which is also undesirable.

Further, it is preferable to fill both ends of the support such as the polyacrylamide gel with a buffer solution. This buffer solution is composed of a buffer, a surfactant, and water that are normally included in an aqueous solution for producing polyacrylamide gel. The concentrations of buffering agents and surfactants are preferably similar to those in the aqueous solutions for producing polyacrylamide gels.

The protein to be treated is then usually used in the form of an aqueous solution. This aqueous solution contains the surfactant of the present invention in a range of usually 0.1 to 10%, preferably 0.2 to 3%, and more preferably 0.5 to 2%. In addition, this aqueous solution can also contain a gel and a buffer in the buffer solution, and the concentration thereof is usually 500 mM or less, and preferably less than the buffer concentration in the buffer solution. The ratio is preferably 1/2 to 1/20 of the buffer concentration.

Functions and Effects of the Invention According to the method of the present invention, a specific surfactant with extremely low protein denaturation ability is used, so it can be applied to proteins that are easily denatured, and it can be applied to proteins that are easily denatured. In addition to not requiring a large amount of solvent as in the case of legal methods, the surfactant of the present invention has a higher critical micelle concentration than nonionic surfactants, so it is easy to remove the surfactant by dialysis or electrodialysis. It can be easily removed, and since the anion part has a sulfonate structure, the pH in the aqueous solution state is low.
Even if it deviates from the neutral range, it has remarkable effects such as being less susceptible to hydrolysis compared to surfactants having a sulfate structure.

In particular, in the present invention, when a mixed surfactant is used, the protein denaturation ability of the surfactant is extremely low, equivalent to that of a nonionic surfactant, and each solubilized protein has a unique property. amount of surfactant is bound,
Since the protein-surfactant complexes each have a different number of negative charges, they each move toward the anode in the electrophoretic support at a unique speed, and at this time, the molecular sieving effect of the support is This has the advantage that the separation and purification ability is much improved compared to the conventional gel filtration method for proteins solubilized with nonionic surfactants.

EXAMPLES Next, the present invention will be explained in more detail by examples.
The present invention is not limited to these.

Below, canine kidney (Na", K") ATPase or β-
Lactoglobulin was separated and purified.

The ATPase activity after separation and purification and the degree of separation and purification were determined as follows.

Determination of ATPage activity 4mM ATP, 100mM NaCl2.25mMKC
l213.9raMMgCQs and 0.2+++MED
An appropriate amount of soybean-derived phospholipid was added to a 30mM imidazole/30mM glycylglycine buffer (pH 7, 2, 20°C) containing TA, and treated at 37°C for 2.5 to 4 minutes.
The reaction was then terminated by adding a highly concentrated aqueous SDS solution. After that, H
The method of egyvary et al. [Anal, Biochem
, .

94, p., 397-401 (1979)].

The degree of separation and purification was determined by taking out the gel after gel electrophoresis and performing conventional 5DS-polyacrylamide gel electrophoresis using a slab type two-dimensional electrophoresis apparatus. In other words, (Na'', K'') ATPase is composed of a and β subunits with different molecular weights, and it is known that several of these subunits aggregate to form oligomers [
Y., Hayashi et al., Biochim.

Biophys, Acta, 748. p, 153
~167 (1983)], pure (Na”, Nitsu AT
When Pase is subjected to 5DS-polyacrylamide gel electrophoresis, two bands corresponding to σ and β are obtained,
You can't get any other band. Therefore, the degree of separation and purification was determined by measuring the presence or absence of the third band.

The following abbreviations were used.

POE: Polyoxyethylene P: Average number of added moles pop: Polyoxypropylene Example 1 Jorgensen's method [Bioc
Him.

Biophys, Acta, 356. p36-5
2 (1974)] (Na", K") ATPa
se, the first dimension in the presence of POE (P8) sodium lauryl ether propiosulfonate/dodecyltrimethylammonium chloride (molar ratio 90/10) and the second dimension in the presence of SOS, which is conventionally performed. Polyacrylamide gel electrophoresis was performed. - gel compositions subjected to dimensional and two-dimensional polyacrylamide gel electrophoresis;
The buffer compositions are shown in Tables 1 and 2 below, respectively.

Table 1 Acrylamide N,N'-methylenebisacrylamide ammonium persulfate N,N,N',N'-tetramethylenediamine phosphate buffer (pH 7) Surfactant water Table 2 Phosphate buffer (pH 7) Surface activity Solution water 5% 2.7% 0.07% 0.15% 00mM 0.1% Balance 00mM 0.1% Balance gel electrophoresis was performed as follows.

The ATPase was adjusted to an aqueous solution satisfying the following conditions and used as a sample solution.

The conditions are that the ATPase concentration is 0.1 me or more, the buffer concentration is 0.1 M or less, and the surfactant weight concentration is several times that of the protein.

After layering this sample solution on the top of the gel, 7 mA
A current was applied for 90 minutes. [For example, J, V, Maiz
el.

Jr., “Methods in Virology
, Academic Press.

(1971), P, 109, edited by the Chemical Society of Japan, Toshio Takagi, Jun Miyake, 'New Experimental Chemistry Course Volume 20 Biological Science I'
, Maruzen (1978), p. 109, etc. However,
When using the mixed surfactant according to the invention, S-
It is necessary to not add an S-bond cleavage agent and to read SDS as the surfactant in the present invention. ] As a result, two clear bands were obtained. At the same time, gel electrophoresis was performed, and the proteins contained in the regions corresponding to the two distinct bands (not subjected to staining treatment) were run out of the gel, and the presence or absence of ATPase activity was determined, and the result was positive. In addition, when the gel that had been subjected to the same operation was taken out and subjected to conventional SDS-polyacrylamide gel electrophoresis using a slab-type two-dimensional electrophoresis device, two bands were detected, σ and β, respectively. Two spots corresponding to 1 were obtained, and no other spots were observed. In this case, a difference in band mobility was observed between -dimensional and two-dimensional gel electrophoresis. Also,
When the presence or absence of ATPase activity was determined for this product, the activity was negative. This indicates that there is a difference in the effect of the mixed surfactant according to the present invention and SDS on the hydrodynamic properties of the a1β subunit. That is, the two bands obtained by polyacrylamide gel electrophoresis according to the present invention are σ and β, and it can be seen that biological functions can be restored by reconstitution.

Example 2 POE (P 7
) The same operation as in Example was performed except that sodium lauryl ether propiosulfonate/dodecyldimethylamine oxide (molar ratio 70/30) was used.

However, in polyacrylamide gel electrophoresis according to the present invention, the migration speed of (Na", K") ATPase varies depending on the surfactant used, so a preliminary test was conducted in advance to determine the migration position of the target membrane protein. . As a result, (Na“,K”)ATPas
The e activity was positive and the purity was also very good.

Example 3 POE(P3)P was used instead of the surfactant in Example 1.
As a result of performing the same operation as in Examples 1 and 2 except for using OP(P 3 ) sodium lauryl ether propiosulfonate/dodecyldimethylamine oxide (molar ratio 65/35), (Na", K" ) The ATPase activity was positive and the purity was also extremely good.

Example 4 PoE (P 3
) As a result of carrying out the same operations as in Examples 1 and 2 except for using sodium lauryl ether propiosulfonate/dodecyldimethylbetaine (molar ratio 65/35),
The ATPase activity was positive and the purity was extremely good.

Example 5 Purified β-lactoglobulin and P instead of (Na'', K'') ATPase and surfactant in Example 1
OE (P7) Sodium lauryl ether propiosulfonate/dodecyldimethylamine oxide (molar ratio 70
As a result of carrying out the same operation as in Example 1 except that each of the following proteins was used: /30), only one protein band was observed after electrophoresis. The molecular weight of β-lactoglobulin under these electrophoresis conditions was determined by light scattering method [Toshio Takagi, “Biochemistry J.
57. p, 202 (1985)], it was found to be about 37.200, confirming that it migrated well without dissociating into subunits.

Example 6 POE (P 3
) pop(P 3 ) lauryl ether Sodium propiosulfonate/dodecyldimethylamine oxide (molar ratio 65/35) was carried out in the same manner as in Example 5, and the results were the same as in Example 5. It was confirmed that the electrophoresis was successful without dissociating into subunits.

Example 7 POE (P 8
') Sodium lauryl ether propiosulfonate/
Dodecyltrimethylammonium chloride (molar ratio 90
As a result of carrying out the same operation as in Example 5 except that 1/10) was used, it was confirmed that the electrophoresis was performed well without being dissociated into subunits, similar to the results of Example 5.

Example 8 The same operation as in Example 5 was performed except that POE (P8) sodium lauryl ether globiosulfonate/dodecyl dimethyl betaine (molar ratio 65/35) was used in place of the surfactant in Example 5. As a result, similar to the results of Example 5, it was confirmed that the electrophoresis was performed well without dissociating into subunits.

Comparative Example 1 When the same operation as in Example was performed using sodium dodecyl sulfate (SO5), two bands were obtained. After these were excised, they were reconstituted and the presence or absence of ATPase activity was determined, and the activity was negative. In addition, as a result of confirming the purity by slab two-dimensional gel electrophoresis, the one with high mobility among the two clear bands becomes a single spot with high mobility even in two-dimensional electrophoresis, A clear band with relatively low mobility became a relatively small single spot in two-dimensional electrophoresis, and there was almost no difference in mobility from that obtained in two-dimensional gel electrophoresis. That is, using sDs,
It can be said that (Na", K") changes the hydrodynamic volume of the α and β subunits of ATPase, and as a result changes to the point where recovery of biological functions becomes impossible.

Comparative Example 2 A POE (P3) sodium lauryl ether sulfate aqueous solution (10% by weight) was boiled for 10 to 20 hours, and this was mixed with dodecyltrimethylammonium chloride in a molar ratio of 90/10.
As a result of operating in the same manner as in Example 1 using a gel prepared to have the following properties, (Na'', K'') ATPase did not migrate into the gel. This is because the anionic activator was hydrolyzed.

Comparative Example 3 As a result of performing the same operation as in Example 5 using C1, ~, α-sodium finsulfonate, β-lactoglobulin was dissociated into subunits and denatured.

Comparative example 4 CI! Linear sodium sodium alkylbenzenesulfonate/dodecyldimethylamine oxide ratio 90/10)
As a result of performing the same operation as in Comparative Example 3 using .beta.-lactoglobulin, it was dissociated into subunits and denatured.

Claims (1)

[Claims] 1 General formula RO-(XO)_l-(YO)_m-(CH_2)_n
-SO_3M (1) (wherein, R is an alkyl group or alkylphenyl group having 6 to 22 carbon atoms, and X and Y are 1 to 22 carbon atoms
4 hydrocarbon group, l and m are each 0 to 20, l+m
is 1 to 40, n is 2 to 4, and M is an alkali metal, alkaline earth metal, ammonium, or quaternary ammonium); A protein characterized by subjecting the protein to gel electrophoresis treatment in the presence of a mixed system of a surfactant and at least one surfactant selected from cationic surfactants and amphoteric surfactants. separation and purification method.