JP6984828B2 - Aggregated protein regenerating agent and method for regenerating aggregated protein using this - Google Patents

Description

The present invention relates to a regenerating agent for aggregated protein and a method for regenerating aggregated protein using the same.

Proteins are easily denatured to form aggregates. In addition, aggregate proteins (insoluble aggregates; inclusion bodies) are formed with high probability even during large-scale expression using Escherichia coli as a host. As a method of regenerating such an aggregated protein into an active protein, a method of solubilizing the aggregated protein with a high-concentration denaturing agent and then gradually removing the denaturing agent by dialysis or dilution is common. be. However, this method has problems that it is time-consuming and complicated to operate, and a large amount of wastewater is generated. There is also a problem that there is a high probability that aggregated proteins will be formed again during the operation.

Here, two processes, solubilization and refolding, are important for the regeneration of aggregated proteins. For lysis (solubilization) of protein aggregates, solubilization is often attempted by denaturing with a fairly strong denaturing agent such as 6M guanidine hydrochloride or 8M urea. Depending on the protein, a method such as increasing the solubility by adding β-mercaptoethanol, a thiol reagent, or a reducing agent is used. However, the amount and type of denaturant used varies depending on the protein, and although it dissolves, it is often difficult to regenerate it thereafter. In addition, the amount of denaturant used is largely based on experience, and there is no established method yet.

Furthermore, the process of removing the denaturant is important for unwinding after solubilization. Rewinding begins by removing the denaturant from the solubilized aggregate protein. Dilution or dialysis methods are usually used to remove the denaturant. Rewinding is achieved by guiding the protein in the more correctly folded direction in the competition for proper folding or aggregation. It is said that the efficiency of rewinding to the active form strongly depends on the intermediate state, but it is not uncommon for aggregation to occur again in the rewinding process due to the interaction between intermediates and the hydrophobic interaction of denatured proteins. For efficient rewinding, a low molecular weight compound is also added to the buffer as a stabilizer (see, for example, Patent Document 1). However, the current situation is that there is still no established method for the rewinding process. In addition, protein unfolding (refolding) in the living body proceeds in the chaperone. It is believed that this chaperone has a pocket with a hydrophobic region inside, where the protein folds to suppress inappropriate binding. This indicates that there is a limit to the method of slowly lowering the concentration of the denaturant.

By the way, a technique of utilizing an ionic bonding salt (ionic liquid) containing an imidazolium ion having a predetermined structure in a cation structure at the time of protein refolding has also been proposed (for example, Non-Patent Document 1 and Non-Patent Document 2). reference).

Japanese Unexamined Patent Publication No. 2016-108264

Protein Science (2005), 14: 2693-2701. Journal of Biotechnology 150 (2010) 64-72

As described above, various techniques for refolding solubilized proteins by adding various additives to the buffer have been conventionally proposed. However, none of the above-mentioned techniques can consistently perform solubilization and refolding of aggregated proteins, and there is still a problem that complicated operations are involved. Here, in the industrial production by mass expression of proteins using Escherichia coli as a host, the regeneration of aggregated proteins is almost inevitable in most cases. Therefore, if a technology that can perform the regeneration process by solubilizing and rewinding aggregated proteins by a simple operation is developed, its industrial usefulness will be immeasurable.

Therefore, the present invention can solubilize aggregated proteins without the use of strong denaturants and can rewind to a native-like structure without the use of other additives. The purpose is to provide technology that can be implemented consistently.

The present inventors have made diligent studies to solve the above problems. As a result, it was surprisingly found that an ion-binding salt (ionic liquid) having a predetermined chemical structure containing a predetermined amount of water as hydrated water is useful as a regenerating agent for aggregated proteins. The present invention has been completed.

That is, according to one embodiment of the present invention, the following general formula (1) or the following general formula (2):

During the ceremony
X represents a nitrogen atom or a phosphorus atom.
n represents an integer of 7 to 11 and represents
p and q each independently represent an integer of 2 to 14, and satisfy | p−q | ≧ 6.
A ^x− represents a counter anion selected from the group consisting of phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, sulfate ion, hydrogen sulfate ion and carboxylate ion, and x is the valence of the counter anion. Represents a number
Provided is an agent for regenerating an aggregated protein containing a hydrate of an ion-binding salt represented by the above as an active ingredient. Here, the molar ratio of the ion-binding salt to the hydrated water in the hydrate is 1: 1 to 1:20.

Further, according to another aspect of the present invention, there is also provided a method for regenerating the aggregated protein, which comprises treating the aggregated protein in the presence of the above-mentioned regenerating agent.

According to the present invention, by designing the ionic structure of an ionic binding salt (ionic liquid) and controlling the hydrophobicity, it is possible to solubilize aggregated proteins without using a strong denaturing agent, and other Techniques can be provided that can consistently rewind to a native-like structure without the use of additives.

To explain the change in the maximum fluorescence wavelength when the concentration of the denaturing agent is changed when the agglutinating protein is denatured (unfolded) using guanidine hydrochloride (guanidine chloride), which is a denaturing agent for the agglutinating protein. It is a graph of. It is a graph which shows the result of having evaluated the binding activity about the sample (positive control) in which the undenatured (native) sugar chain binding protein concanavalin A (ConA) was dissolved in the above buffer. It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "P44412 · dhp" which is an ion-binding salt (11'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 7) of "P44412 · dhp" which is an ion-binding salt (11'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 15) of "P44412 dhp" which is an ion-binding salt (11'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "N8888 · dhp" which is an ion-binding salt (14'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "P4444 · dhp" which is an ion-binding salt (9'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "P4448 dhp" which is an ion-binding salt (10'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "N4444 · dhp" which is an ion-binding salt (12'). It is a graph which shows the result of having evaluated the binding activity about the hydrate (salt: water molar ratio = 1: 3) of "P6666 · dhp" which is an ion-binding salt (15').

One embodiment of the present invention is the following general formula (1) or the following general formula (2):

During the ceremony
X represents a nitrogen atom or a phosphorus atom.
n represents an integer of 7 to 11 and represents
p and q each independently represent an integer of 2 to 14, and satisfy | p−q | ≧ 6.
A ^x− represents a counter anion selected from the group consisting of phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, sulfate ion, hydrogen sulfate ion and carboxylate ion, and x is the valence of the counter anion. Represents a number
Contains the hydrate of the ionic bond salt represented by, as an active ingredient,
At this time, the present invention relates to a regenerating agent for aggregated protein, characterized in that the molar ratio of the ion-binding salt to the hydrated water in the hydrate is 1: 1 to 1:20. Hereinafter, embodiments of the present invention will be described.

[Regenerating agent for aggregated protein]
The invention according to this embodiment relates to a regenerating agent for aggregated proteins. As used herein, the term "aggregated protein" refers to a state in which an aggregate is formed by agglutination of a protein, regardless of the cause of the non-aggregated protein to aggregate. Aggregated proteins include undenatured (native) proteins that aggregate under external stimuli such as heat and chemical substances, and insoluble aggregates (so-called encapsulaters) in which recombinant proteins produced using Escherichia coli as a host aggregate. Of course, all of these are included in the concept of "aggregating protein" according to the present invention.

As used herein, the term "regeneration" of an aggregated protein means that the aggregated protein is solubilized (dissolved) and the solubilized aggregated protein is refolded into a native structure before the aggregated protein is aggregated. Means to change to a state having the same or similar structure and activity as. As used herein, the "regenerating agent" for an aggregated protein is used to assist in both dissolution and refolding in the "regeneration" of the aggregated protein and to improve the regeneration yield of the aggregated protein.

<Hydrate of ionic bond salt>
The regenerating agent for aggregated protein according to this embodiment contains an ionic hydrate (also referred to as “hydrated ionic liquid”) of an ionic-binding salt represented by the above-mentioned general formula (1) or general formula (2) as an active ingredient. It is something to do.

(Ionic binding salt)
The ionic bond salt, which is the raw material of the hydrate, is a salt having a structure represented by the following general formula (1) or the following general formula (2), has a melting point of 100 ° C. or lower, and is in a liquid state at room temperature. There are many things that present. When the hydrate is adjusted, almost all of it becomes liquid.

In the general formulas (1) and (2), X represents a nitrogen atom or a phosphorus atom. That is, the cation of the ion-binding salt constituting the hydrated ionic liquid as an active ingredient is an ammonium cation or a phosphonium cation.

Cation of the ionic binding salt represented by the general formula (1) are four identical alkyl chain with respect to the central element (nitrogen atom or a phosphorus atom) (-C n H _2n + ₁₎ have a covalent bond structure is doing. Here, n represents an integer of 7 to 11. If n is too small in the general formula (1), the aggregated protein can be solubilized (dissolved) due to insufficient hydrophobicity of the cation, but the dissolved protein is refolded. The activity cannot be restored. On the other hand, if n is too large in the general formula (1), the hydrophobicity of the alkyl chain is too large, and as a result, the aggregated protein cannot be sufficiently solubilized (dissolved) even in the form of a hydrate. .. On the other hand, when n is an integer of 7 to 11 in the general formula (1), it is possible to exhibit excellent performance as a regenerating agent for aggregated proteins. In a preferred embodiment, in the ion-bonding salt represented by the general formula (1), X is a nitrogen atom and n represents an integer of 8 to 10. Further, in a more preferable embodiment, in the ion-binding salt represented by the general formula (1), X is a nitrogen atom and n is 8 (see Examples described later).

Further, the cation of the ionic bond salt represented by the general formula (2) has four alkyl chains bonded to the central element (nitrogen atom or phosphorus atom), but one of the four is bonded. The carbon number (q) of the alkyl chain (-C _q H _{2q + 1} ) is different from the carbon number (p) _{of the other three alkyl chains (-C p} H _{2p + 1).} Further, these differences in carbon number must be different by 6 or more (that is, | p−q | ≧ 6 is satisfied). In the case of the ion-binding salt represented by the general formula (2) having an asymmetric structure, having such a structure makes it exhibit properties similar to those of a surfactant, and is also a regenerating agent for aggregated proteins. It is considered that it will be possible to demonstrate excellent performance. Further, from the viewpoint of exhibiting a certain degree of hydrophobicity as in the general formula (1), it is preferable that p and q in the general formula (2) independently represent integers of 2 to 14 respectively. In another preferred embodiment, in the ion-bonding salt represented by the general formula (2), p <q is further satisfied (that is, the alkyl chain having only one difference is longer than the other three alkyl chains). .. In still another preferred embodiment, in the ion-bonding salt represented by the general formula (2), X is a phosphorus atom, p is an integer of 3 to 5, and q is an integer of 9 to 13. In a more preferred embodiment, in the ion-bonding salt represented by the general formula (2), X is a phosphorus atom, p is 4, and q is 12 (see Examples described later).

In the general formulas (1) and (2), A ^{x −} represents a counter anion (x represents the valence of the counter anion). In this embodiment, the counter anion is a phosphate ion which is a trivalent anion (x = 3), a hydrogen phosphate ion and a sulfate ion which are divalent anions (x = 2), and a monovalent anion. It is selected from the group consisting of dihydrogen phosphate ion and hydrogen sulfate ion of (x = 1). That is, x is an integer of 1 to 3. Examples of the carboxylic acid ion include formic acid ion, acetate ion, propionic acid ion, butyrate ion, oxalate ion, citrate ion, and tartaric acid ion. The counter anion is preferably dihydrogen phosphate ion. All of these anions have in common that they exhibit excellent cosmotropicity, and therefore, when used as an anion for an ion-binding salt according to the present invention, they have excellent performance as a regenerating agent for aggregated proteins. It is thought that it will be demonstrated.

The specific structure of the ion-binding salt according to the present invention is not particularly limited, and any ion-binding salt obtained by combining the above-mentioned structures can be used. Examples of the ion-binding salt include those represented by the following general formula (3) or the following general formula (4).

(Hydration water)
As described above, the active ingredient of the regenerating agent for the aggregated protein according to the present embodiment is a hydrate (hydrated ionic liquid) of an ionic-binding salt represented by the above-mentioned general formula (1) or general formula (2). Is. The hydrated ionic liquid is obtained by hydrating the above ion-binding salt with water, and contains hydrated water.

Here, the hydrated ionic liquid which is the active ingredient of the regenerating agent for the aggregated protein according to the present embodiment is characterized in that the amount of hydrated water contained therein is controlled to a value within a predetermined range.

Specifically, it is characterized in that the molar ratio of the ion-binding salt to the hydrated water in the hydrate of the ion-binding salt (hydrated ionic liquid) is 1: 1 to 1:20. With such a configuration, excellent performance as a regenerating agent for aggregated proteins can be exhibited. In addition, when the content of water with respect to the ionic bond salt becomes large, there is an increased possibility that water (free water) which is not involved in hydration is generated. Since the presence of free water adversely affects the regeneration of aggregated proteins, the molar ratio is preferably 1: 2 to 1:17, more preferably 1: 3 to 1, from the viewpoint of preventing the generation of free water. It is 1:15, more preferably 1: 3 to 1: 7, particularly preferably 1: 3 to 1: 5, and most preferably 1: 3 to 1: 4.

The hydrated ionic liquid as an active component of the aggregated protein according to this embodiment may be used alone, or may contain a combination of cation and anion structure of an ion-binding salt and hydrated water. Two or more kinds having different amounts may be used together.

<Method for producing ion-binding salt and its hydrate>
As for the ion-binding salt according to this embodiment, if a commercially available product exists, the commercially available product may be purchased and used. Further, even when a commercially available product does not exist, it can be manufactured by a conventionally known method (see Examples described later).

Furthermore, the method of using an ionic bond salt as a hydrate is also known by itself, and after adding a predetermined amount of water to the ionic bond salt, it is mixed, stirred, etc. as necessary. , Hydration ions can be obtained.

[Regeneration method of aggregated protein]
According to another embodiment of the invention, a method for regenerating aggregated proteins is also provided. That is, another form of the present invention comprises lysing and refolding the aggregated protein by treating the aggregated protein in the presence of the regenerating agent for the aggregated protein according to the above-mentioned aspect of the present invention. It is a reproduction method of.

In the method for regenerating aggregated protein according to this embodiment, the protein to be regenerated includes a peptide, a polypeptide, regardless of the origin or production method such as natural or artificial (chemical synthesis method, fermentation method, gene recombination method). Proteins and complexes thereof (eg, (poly) peptides or complexes of proteins with compounds, (poly) peptides or complexes of proteins with saccharides, (poly) peptides or complexes of proteins with metals, ( Poly) includes complexes of peptides or proteins with coenzymes). The type of protein is not limited, and for example, intracellular protein, extracellular protein, membrane protein, and nuclear protein are all included. It is known that a disulfide bond may be involved in protein aggregation, but the target protein does not necessarily have to be a protein having a disulfide bond. However, suitable proteins include proteins containing at least one disulfide bond.

The protein of interest is preferably a recombinant produced genetically engineered using a prokaryote such as Escherichia coli, a eukaryote such as yeast, or a heterologous expression system such as a cell-free extraction system. Since such recombinants are often obtained as insoluble and inactive aggregates (so-called inclusion bodies), the regeneration method according to the present invention can be preferably used.

The molecular weight of the target protein in this embodiment is not particularly limited, but usually, a protein of about 1,000 to 1,000,000 can be mentioned. From the viewpoint of regenerative effect, it is preferably a protein having a molecular weight of 10,000 to 250,000. Generally, there is a correlation between the size of the molecular weight and the difficulty of regeneration, and a protein having a large molecular weight (molecular weight of about 10,000 or more) may be extremely difficult to regenerate. According to the regeneration method according to this embodiment, since a high regeneration effect can be obtained, it is also effective for a high molecular weight protein having a molecular weight of 10,000 or more. Since proteins having a molecular weight of less than 1,000 can be easily rewound, the regeneration method according to this embodiment can be particularly preferably used for proteins having a molecular weight of 1,000 or more. The molecular weight of the protein can be measured by a general gel electrophoresis or the like.

As described above, the method for regenerating an aggregated protein according to this embodiment is to dissolve and refold the aggregated protein by treating the aggregated protein in the presence of a regenerating agent for the aggregated protein according to one embodiment of the present invention. It is characterized by including.

In the dissolution and refolding in this regeneration method, it is extremely simple because both the dissolution and the refolding of the aggregated protein can be consistently achieved by using the regenerating agent according to the present invention as a solvent. It can be said that it is an industrially very useful regeneration method in that the aggregated protein can be regenerated by the method. However, conventionally known solvents and additives other than the regenerating agent according to the present invention may of course be used for at least one of the dissolution and refolding of the aggregated protein. However, in a preferred embodiment, the aggregation protein is dissolved and refolded without using a solvent or additive other than the regenerating agent according to the present invention.

In any case, in order to dissolve and refold the aggregated protein in the presence of the aggregated protein regenerating agent according to the present invention, the aggregated protein regenerating agent (hydrated ion liquid) according to the present invention is brought into contact with the aggregated protein. Dissolution and refolding of the aggregated protein proceeds only by allowing the aggregated protein to be dissolved therein (preferably using a hydrated ion liquid as a solvent and stirring if necessary). From the viewpoint of sufficiently advancing dissolution and refolding (particularly refolding), it is preferable to allow the mixture to stand for a certain period of time. The standing time can be, for example, 1 to 50 hours. The temperature condition for standing still can be appropriately selected in the range of 0 to 100 ° C. according to the heat resistance of the target protein, but is usually in the range of 4 to 30 ° C.

The method for regenerating the aggregated protein according to this embodiment can be paraphrased as a method for preparing a normal protein.

The method for regenerating the aggregated protein according to this embodiment may be any method as long as it dissolves and refolds the aggregated protein by treating the aggregated protein in the presence of the regenerating agent according to the present invention, and the presence or absence of other steps may be used. There are no particular restrictions. When the target protein is a recombinant produced by genetic engineering using a heterologous expression system such as Escherichia coli, yeast, or cell-free extraction system, the method for regenerating the aggregated protein according to this embodiment is as follows (2). )-(3), (1)-(3) or (1)-(4) may be included:
(1) Culturing process of protein-producing bacteria: Culturing protein-producing bacteria such as Escherichia coli to produce recombinants;
(2) Lysis step: A protein inclusion body is taken out from the protein-producing cells using a lytic agent or the like;
(3) Dissolution step: The protein encapsulater is dissolved in the aggregated protein regenerating agent (hydrated ionic liquid) according to the present invention as a solvent, and if necessary, left at room temperature for several hours. According to the regeneration method according to the present invention, the refolding step is performed following the dissolution step, and the protein having the original activity is regenerated.

(4) Isolation step: The target normal protein (refolding protein) is isolated from the solution of the protein obtained above in the hydrated ionic liquid by using column chromatography or the like.

The following bacterial cells can be exemplified as the protein-producing bacteria in the above-mentioned (1) protein-producing bacteria culturing step. Bacterial cells include Streptococci, Staphylococci, Escherichia, Streptomyces and Bacillus cells, fungal cells such as yeast cells and Aspergillus. Aspergillus cells, insect cells: eg Drosophila S2, Spodoptera Sf9 cells, animal cells: eg CHO, COS, Hela, C127, 3T3, BHK, 293 and Bows melanoma cells. , As well as plant cells and the like.

Further, in the method for culturing the protein-producing bacterium in the above step (1), the expression vector containing the cDNA encoding the target protein (i) separates the messenger RNA (mRNA) from the target protein-producing cell and simply separates the messenger RNA (mRNA) from the mRNA. The stranded cDNA is then synthesized into double-stranded DNA and the complementary DNA is integrated into the phage or plasmid. (Ii) The host is transformed with the obtained recombinant phage or plasmid, and after culturing, the phage containing the target DNA is hybridized with a DNA probe encoding a part of the target protein or by an immunoassay method using an antibody. Alternatively, isolate the plasmid. (Iii) It can be produced by cutting out a cloned DNA of interest from the recombinant DNA and ligating the cloned DNA or a part thereof downstream of a promoter in an expression vector. Then, the host is transformed with an expression vector and cultured by an appropriate method. Culturing is usually carried out at 15 to 43 ° C. for 3 to 24 hours, and aeration and stirring can be added if necessary.

Further, as the lysis method adopted in the lysis step of (2) above, any of physical crushing by ultrasonic waves, treatment with a lysis enzyme such as lysozyme, and treatment with a lysis agent such as a surfactant can be used. From the viewpoint of productivity, treatment with a bacteriolytic agent is preferable. Further, from the viewpoint of not denaturing useful proteins, bacteriolytic agents such as quaternary ammonium-type cationic surfactants whose counterions are carboxylic acid ions such as formic acid and acetic acid can be mentioned.

Further, examples of the filler used in the column chromatography in the isolation step (4) above include silica, dextran, agarose, cellulose, acrylamide, vinyl polymer and the like. Commercially available commercially available products include Sephadex series, Sephacryl series, Sepharose series (Pharmacia), Bio-Gel series (Bio-Rad) and the like.

When a protein having a thiol group (-SH group) derived from a cysteine residue or the like aggregates, a disulfide bond may be formed between the molecules during the aggregation. When such an aggregated protein is dissolved and refolded using the refolding agent according to the present invention, when the refolded protein forms a disulfide bond between molecules, the refolded protein is used as a reducing agent. It is preferable to further carry out the operation of reducing the disulfide bond by contacting them. The reducing agent used at this time is not particularly limited, and conventionally known reducing agents used for cleaving disulfide bonds in peptides and proteins can be used in the same manner. Examples of such reducing agents include 1,4-dithiothreitol, β-mercaptoethanol, reduced glutathione, tris (2-carboxyethyl) phosphine hydrochloride and the like.

Also, if the native protein has a disulfide bond between a plurality of cysteine residues in the molecule, this disulfide bond is either cleaved during aggregation or regenerated according to the present invention. A plurality of thiol groups (-SH groups) may be exposed when the refolding is completed, for example, by being cleaved during regeneration (dissolution and refolding) by the agent. Thus, when the refolded protein has a plurality of thiol groups (-SH groups), the refolded protein is brought into contact with an oxidizing agent to form a disulfide bond between the plurality of thiol groups. It is preferable to further carry out the operation to be performed. The reducing agent used in this case is not particularly limited, and a conventionally known oxidizing agent used for forming a disulfide bond in a peptide or protein can be used in the same manner. Examples of such an oxidizing agent include oxidized glutathione and cystine.

Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to examples, but the technical scope of the present invention is not limited by the following examples.

[Dissolution of aggregated proteins using ion-binding salts]
(Preparation of ionic bond salt and its hydrate)
In order to confirm the solubility of the aggregated protein of the ion-binding salt, the following ion-binding salt was prepared by purchasing a commercially available product.
(1) 1-Butyl-3-methylpyridinium bromide (2) 1-butyl-3-methylimidazolium bromide (3) 1-butyl-3-methylpiperidinium bromide (4) Choline dihydrogen phosphate (5) 1-Butylpyridinium bromide (6) 1-decyl-3-methylimidazolium bromide (7) benzyltripropylammonium chloride (8) 1,3-didecil-2-methylimidazolium chloride (9) tetrabutylphosphonium bromide (P4444)・ Br)
(10) Tributyl-n-octylphosphonium bromide (P4448 · Br)
(11) Tributyldodecylphosphonium bromide (P44412 · Br)
(12) Tetrabutylammonium bromide (N4444 · Br)
(13) Tetrahexyl ammonium bromide (N6666 · Br)
(14) Tetra-n-octylammonium bromide (N8888 · Br)
(15) Tetrahexylphosphonium bromide (P6666 · Br)
(16) Dodecyltrimethylammonium chloride (17) Methyltri-n-octylammonium chloride (18) Trihexyl (tetradecyl) phosphonium chloride (19) Dimethyldioctylammonium bromide (20) Ethylhexadecyldimethylammonium bromide (21) Tributylhexadecylphosphonium bromide (22) Octyltrimethylammonium bromide.

Next, with respect to the ion-binding salts (9) to (22) prepared above, the following ion-binding salts were also prepared by exchanging the anion with dihydrogen phosphate ion (dhp). Specifically, first, the ion-binding salts prepared above were each dissolved in water and passed through an anion exchange column resin (Amberlite IRN 78) to exchange ^{anions with OH −.} Then, an equimolar amount of phosphoric acid was added, and after the neutralization reaction, dehydration was carried out with an evaporator to obtain an ionic bond salt in which the anion was exchanged for dihydrogen phosphate ion (dhp).
(9') Tetrabutylphosphonium dihydrogen phosphate (P4444 · dhp)
(10') Tributyl-n-octylphosphonium dihydrogen phosphate (P4448 · dhp)
(11') Tributyldodecylphosphonium dihydrogen phosphate (P44412 · dhp)
(12') Tetrabutylammonium dihydrogen phosphate (N4444 · dhp)
(13') Tetrahexylammonium dihydrogen phosphate (N6666 · dhp)
(14') Tetra-n-octylammonium dihydrogen phosphate (N8888 · dhp)
(15') Tetrahexylphosphonium dihydrogen phosphate (P6666 · dhp).

Further, a part of the ion-binding salts (1) to (22) and (9') to (15') prepared above was made into a hydrate form by adding water. Specifically, water is added so that the number of water molecules is 3, 7 or 15 molecules per one ion pair of the ion-binding salt to obtain an ion-binding salt in the form of a hydrate. rice field.

(Confirmation of solubility of aggregated protein)
As an aggregate protein, an aggregate of concanavalin A (ConA), which is a sugar chain recognition protein, was prepared. Specifically, ConA was mixed with milliQ water to a concentration of 10 mg / mL and incubated at 70 ° C. for 10 minutes to obtain ConA aggregates.

Then, about 0.5 mg of the aggregated protein (Agglomerate of ConA) prepared in this manner and 50 μL of the ion-binding salt (or its hydrate) prepared above were mixed and stirred overnight. ..

The solubility of the sample after stirring overnight was confirmed by visual inspection and fluorescence measurement. In the fluorescence measurement, the supernatant obtained by centrifuging the stirred sample (10,000 g, 30 minutes) was excited by exciting the tryptophan residue in the protein at an excitation wavelength of 280 nm. The maximum fluorescence wavelength of the fluorescence spectrum and its fluorescence intensity were measured. The results are shown in Table 1 below.

Here, the results shown in Table 1 will be considered. First, when undenatured (native) ConA is dissolved in buffer, a maximum fluorescence wavelength is observed at 336 nm. On the other hand, when the aggregated protein was denatured (unfolded) with guanidine hydrochloride (guanidine chloride), which is a typical denaturant, the maximum fluorescence wavelength was shifted to the long wavelength side, and a high concentration of guanidine hydrochloride was used. In some cases, it shifts to about 350 nm (Fig. 1). Considering this, it can be seen that some of the ion-binding salts according to the present invention were able to dissolve (solubilize) aggregated proteins with almost no shift of the maximum fluorescence wavelength toward the long wavelength side. .. This suggests that some of the hydrates of the ion-binding salts according to the present invention can function as a system capable of consistently lysing and refolding aggregated proteins. When an ion-binding salt containing an imidazolium structure or another aromatic ring-containing structure previously proposed as an additive for protein refolding in a cation is used, the aggregated protein is solubilized (dissolved). I couldn't.

[Confirmation of lysed protein activity]
In the above, it was confirmed whether or not the original activity (sugar chain binding ability) of ConA was maintained for some of the samples in which the aggregate protein (aggregate of ConA) was solubilized (dissolved). Specifically, the binding ability of the protein to mannose-added bovine serum albumin (BSA) immobilized on the sensor chip was evaluated using the biolayer interferometry (Blitz: Prime Tech Co., Ltd.). At the time of evaluation, the supernatant obtained by stirring the aggregated protein (ConA aggregate) in the hydrate of the ion-binding salt and then centrifugation by the same method as described above was used in PBS buffer. It was diluted 2,000 times and the binding activity was evaluated.

FIG. 2A is a graph showing the results of evaluation of binding activity of a sample (positive control) in which undenatured (native) ConA was dissolved in the buffer. The protein concentration in the positive control sample was adjusted to be equal to that evaluated using the hydrate of the ion-binding salt.

In addition, FIGS. 2B to 2I are graphs showing the results of evaluation of the binding activity of the following samples, respectively:
FIG. 2B: (11') P44412 · dhp (1: 3)
FIG. 2C: (11') P44412 · dhp (1: 7)
FIG. 2D: (11') P44412 · dhp (1:15)
FIG. 2E: (14') N8888 · dhp (1: 3)
FIG. 2F: (9') P4444 · dhp (1: 3)
FIG. 2G: (10') P4448 · dhp (1: 3)
FIG. 2H: (12') N4444 · dhp (1: 3)
FIG. 2I: (15') P6666 · dhp (1: 3).

From the results shown in FIGS. 2B to 2I, when treated with the ion-binding salt (11') (P44412 · dhp) and the ion-binding salt (14') (N8888 · dhp), the aggregated protein (ConA). It can be seen that not only the agglomerates of the protein can be solubilized (dissolved), but also the original activity of the protein can be restored. Thus, it was clearly demonstrated that these ion-binding salts function as a system capable of consistently dissolving and refolding aggregated proteins.

On the other hand, when treated with other ion-binding salts, even if the aggregated protein (ConA aggregate) can be solubilized (dissolved), the original activity of the protein is restored. I couldn't get it. This is to give hydrophobicity to the cation structure when it has a nitrogen atom or a phosphorus atom as the central element of the cation and has a symmetric structure in which all four alkyl chains bonded to the central element are the same. It is shown that the number of carbon atoms of the alkyl chain needs to be large to some extent. The hydrophobic environment formed by such a structure is considered to be important as a refolding environment. This is consistent with the fact that protein refolding in vivo proceeds in the hydrophobic region within the chaperone. In addition, when the chain length of the alkyl chain bonded to the central element of the cation (nitrogen atom or phosphorus atom) differs by only one, the difference in carbon number between the short chain length and the long chain length is large to some extent. It also shows that is necessary. It is presumed that an environment formed of asymmetrical molecules capable of forming a certain amount of space or domain structure, rather than a uniform environment formed of non-symmetrical molecules, is also suitable as a place for protein regeneration. Based on these results and discussions, the present inventors presume that the hydrate of the ion-binding salt according to the present invention will be useful as a regenerating agent for aggregated proteins.

(Examination when the type of aggregated protein is changed)
As the aggregated protein, instead of concanavalin A, aggregates of the following three kinds of proteins were prepared respectively.

・ Cytochrome c
-Lysozyme-fructose dehydrogenase Specifically, each protein was mixed with milliQ water to a concentration of 10 mg / mL and incubated at 70 ° C. for 10 minutes to obtain aggregates of each protein.

Then, about 0.5 mg of the aggregated protein (aggregate of each protein) prepared in this manner and 50 μL of the hydrate of the following ion-binding salt were mixed, and the mixture was stirred overnight. Then, the solubility of the sample after stirring overnight was confirmed by visual inspection and fluorescence measurement. In the fluorescence measurement, the supernatant obtained by centrifuging the stirred sample (10,000 g, 30 minutes) was excited by exciting the tryptophan residue in the protein at an excitation wavelength of 280 nm. The maximum fluorescence wavelength of the fluorescence spectrum and its fluorescence intensity were measured.

(Regenerating agent used for regeneration of cytochrome c aggregates)
・ N4444 ・ dhp hydrate (1: 3)
-P44412-dhp hydrate (1: 3)
・ P4448 ・ dhp hydrate (1: 3)
(Regenerating agent used to regenerate lysozyme aggregates)
・ P6666 ・ dhp hydrate (1: 3)
・ P4448 ・ dhp hydrate (1: 3)
(Regenerating agent used to regenerate fructose dehydrogenase aggregates)
-P44412-dhp hydrate (1: 3)
As a result, lysis (solubilization) of aggregated protein was confirmed in all the samples.

Subsequently, it was confirmed whether or not the activity of each of the above solubilized (solubilized) proteins was restored according to a conventional method. As a result, recovery of activity was confirmed in all the samples. For the dissolved (solubilized) fructose dehydrogenase, the recovery of activity was promoted by adding β-mercaptoethanol, which is a reducing agent, to the sample at a concentration of 10 mM.

As described above, by treating with the ion-binding salt according to the present invention, it is possible not only to realize solubilization (lysis) of various aggregated proteins but also to restore the original activity of each protein. Recognize. That is, it was clearly demonstrated that the ion-binding salt according to the present invention functions as a system capable of consistently performing lysis and refolding regardless of the type of aggregated protein. The activity of fructose dehydrogenase was restored by the addition of the reducing agent (β-mercaptoethanol) because the disulfide bonds generated between the protein molecules during the formation of aggregates were cleaved by the addition of the reducing agent, resulting in normal refolding. It is thought that this is due to the fact that As described above, the ion-binding salt according to the present invention can be used in combination with a reducing agent as needed to more efficiently perform refolding and restoration of activity thereof.

[Formation of disulfide bonds in peptide molecules in ionic salt]
A peptide (oxytocin) with a reduced disulfide bond was dissolved in a hydrate (1: 3) of P44412 / dhp, which is an ionic bond salt according to the present invention, at a concentration of 5 mM. An oxidizing agent functioning as a disulfide agent was added thereto, and the mixture was reacted at 25 ° C. for 1 hour. When the samples before and after the reaction were analyzed by HPLC, it was confirmed that a disulfide bond was formed in the peptide molecule. From this, the regenerating agent (ion-binding salt) for the aggregated protein according to the present invention can be obtained only by adding an oxidizing agent without requiring a special operation after achieving the lysis and refolding of the aggregated protein. It can be seen that it can also be used as a site for forming necessary disulfide bonds.

This application is based on Japanese Patent Application No. 2017-225090 filed on November 22, 2017, the disclosure of which is cited in its entirety by reference.

Claims (15)

The following general formula (1) or the following general formula (2):

During the ceremony
X represents a nitrogen atom or a phosphorus atom.
n represents an integer of 7 to 11 and represents
p and q each independently represent an integer of 2 to 14, and satisfy | p−q | ≧ 6.
A ^x- represents phosphate ion, a counter anion is selected from hydrogen phosphate ion and phosphate monobasic ion or Ranaru group, x is, represents the valence of the counter anion,
Contains the hydrate of the ionic bond salt represented by, as an active ingredient,
At this time, a regenerating agent for aggregated protein, characterized in that the molar ratio of the ion-binding salt to the hydrated water in the hydrate is 1: 1 to 1:20. The regenerating agent for an aggregated protein according to claim 1 , wherein the counter anion is a dihydrogen phosphate ion. The regenerating agent for an aggregated protein according to claim 1 or 2 , wherein the hydrate contains an ion-binding salt represented by the general formula (1) as an active ingredient. The regenerating agent for aggregated protein according to claim 3 , wherein X is a nitrogen atom and n represents an integer of 8 to 10. The regenerating agent for an aggregated protein according to claim 1 or 2 , wherein the hydrate contains an ion-binding salt represented by the general formula (2) as an active ingredient. The regenerating agent for aggregated protein according to claim 5 , which further satisfies p <q. The regenerating agent for aggregated protein according to claim 6 , wherein p is an integer of 4 to 8. The regenerating agent for aggregated protein according to claim 6 , wherein X is a phosphorus atom, p is an integer of 3 to 5, and q is an integer of 9 to 13. The ion-binding salt is the following general formula (3) or the following general formula (4):

The regenerating agent for aggregated protein according to claim 1. The regenerating agent for aggregated protein according to any one of claims 1 to 9 , wherein the molar ratio of the ion-binding salt to the hydrated water in the hydrate is 1: 3 to 1:15. A method for regenerating an aggregated protein, which comprises lysing and refolding the aggregated protein by treating the aggregated protein in the presence of the regenerating agent according to any one of claims 1 to 10. The method for regenerating an aggregated protein according to claim 11 , wherein the aggregated protein is treated in the presence of the regenerating agent as a solvent. The method for regenerating an aggregated protein according to claim 12 , wherein the aggregated protein is treated without using a solvent or an additive other than the regenerating agent. Any one of claims 11-13 , further comprising contacting the protein with a reducing agent to reduce the disulfide bond when the refolded protein forms a disulfide bond between the molecules. The method for regenerating an aggregated protein according to. A claim further comprising contacting the refolded protein with an oxidizing agent to form a disulfide bond between the plurality of thiol groups when the refolded protein has a plurality of thiol groups (-SH groups). The method for regenerating an aggregated protein according to any one of 11 to 14.

Images