WO2019103106A1 - Agent de régénération de protéine agrégée, et procédé de régénération de protéine agrégée l'utilisant - Google Patents

Agent de régénération de protéine agrégée, et procédé de régénération de protéine agrégée l'utilisant Download PDF

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WO2019103106A1
WO2019103106A1 PCT/JP2018/043222 JP2018043222W WO2019103106A1 WO 2019103106 A1 WO2019103106 A1 WO 2019103106A1 JP 2018043222 W JP2018043222 W JP 2018043222W WO 2019103106 A1 WO2019103106 A1 WO 2019103106A1
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protein
ion
regenerating
agent
aggregated
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PCT/JP2018/043222
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English (en)
Japanese (ja)
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恭子 藤田
大野　弘幸
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学校法人東京薬科大学
国立大学法人東京農工大学
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Priority to JP2019555369A priority Critical patent/JP6984828B2/ja
Publication of WO2019103106A1 publication Critical patent/WO2019103106A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution

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  • the present invention relates to an agent for regenerating aggregated proteins and a method for regenerating aggregated proteins using the same.
  • the proteins are easily denatured to form aggregates.
  • aggregated proteins insoluble aggregates; inclusion bodies
  • aggregated proteins are formed with a high probability even during large-scale expression in E. coli as a host.
  • As a method of regenerating such aggregated proteins into active proteins it is common to solubilize aggregated proteins with a high concentration of denaturing agent and then gradually remove the denaturing agent by dialysis or dilution. is there.
  • this method has the problems that it is time-consuming and complicated to operate, and a large amount of wastewater is generated.
  • solubilization is often attempted by denaturing using a fairly strong denaturant such as 6 M guanidine hydrochloride or 8 M urea.
  • a fairly strong denaturant such as 6 M guanidine hydrochloride or 8 M urea.
  • methods such as enhancing solubility by adding ⁇ -mercaptoethanol, a thiol reagent and a reducing agent are used.
  • the amount and type of the denaturing agent differ depending on the protein, and although dissolution occurs, there are many cases where subsequent regeneration becomes difficult.
  • the amount of modifying agent used is large due to experience, and there is no established method yet.
  • Unrolling begins by removing the denaturing agent from the solubilized aggregated protein. Dilution or dialysis is usually used to remove the denaturing agent. Rewinding is accomplished by directing the protein to fold more correctly in competition for proper folding or aggregation of the protein. It is said that the unwinding efficiency to the active form strongly depends on the intermediate state, but due to the interaction between the intermediates and the hydrophobic interaction of the denatured protein, reaggregation often occurs in the unwinding process. It is also performed to add a low molecular weight compound as a stabilizer in a buffer for efficient unwinding (see, for example, Patent Document 1).
  • the present invention is capable of solubilizing aggregated proteins without using a strong denaturant, and refolding to a native similar structure without using other additives.
  • the aim is to provide technology that can be consistently implemented.
  • an ion-binding salt ionic liquid having a predetermined chemical structure and containing a predetermined amount of water as hydration water is useful as a regenerating agent for aggregated proteins.
  • the present invention has been completed.
  • 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 represents a valence of the counter anion Represents a number
  • an agent for regenerating aggregated proteins which comprises as an active ingredient a hydrate of the ion binding salt represented by Here, it is characterized in that the molar ratio of the ion binding salt to the water of hydration in the hydrate is 1: 1 to 1:20.
  • a method of regenerating aggregated proteins comprising treating the aggregated proteins in the presence of the above-mentioned regenerating agent.
  • the present invention by designing the ionic structure of the ion binding salt (ionic liquid) and controlling the hydrophobicity, it is possible to solubilize aggregated proteins without using a strong denaturant, and others A technique can be provided that can be consistently implemented to refold into a native-like structure without the use of additives.
  • One embodiment of the present invention is represented by the following general formula (1) or the following general formula (2):
  • 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 represents a valence of the counter anion Represents a number, Containing the hydrate of the ion binding salt represented by At this time, the present invention relates to an agent for regenerating aggregated proteins, characterized in that the molar ratio of the ion binding salt to the hydration water in the hydrate is 1: 1 to 1:20.
  • the invention relates to a regenerating agent for aggregated protein.
  • aggregated protein refers to a state in which aggregates are formed by protein aggregation, and it does not matter by what cause unaggregated proteins are aggregated.
  • aggregated proteins include non-denatured (native) proteins aggregated by external stimuli such as heat and chemicals, and insoluble aggregates (so-called inclusion bodies) in which recombinant proteins produced using E. coli as a host are aggregated. These are all, of course, included in the concept of “aggregated protein” according to the present invention.
  • “regeneration” of aggregated protein is performed before aggregation of aggregated protein by a method including the steps of solubilizing (lysing) the aggregated protein and refolding the solubilized aggregated protein into a native structure. It means changing to a state having the same or similar structure and activity as
  • “rejuvenating agent” of aggregated protein is used to aid in both dissolution and refolding in “regeneration” of aggregated protein, and to improve the regeneration yield of aggregated protein.
  • Hydrate of ion binding salt The regenerating agent for aggregated protein according to the present embodiment is a hydrate (also referred to as “hydrated ionic liquid”) of the ion binding salt represented by the general formula (1) or the general formula (2) described above as an active ingredient It is
  • the ion binding salt which is a raw material of a 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 less, and is liquid at room temperature There are many that exhibit. When the hydrate is adjusted, almost all become liquid state.
  • X represents a nitrogen atom or a phosphorus atom. That is, the cation of the ion binding salt constituting the hydrated ionic liquid which is the active ingredient is an ammonium cation or a phosphonium cation.
  • the cation of the ion-binding salt represented by the general formula (1) has a structure in which four identical alkyl chains (-C n H 2n + 1 ) are covalently bonded to the central element (nitrogen atom or phosphorus atom). doing.
  • 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 the insufficient hydrophobicity of the cation, but the dissolved protein is refolded. It can not restore its activity.
  • n in the general formula (1) is an integer of 7 to 11, it is possible to exhibit excellent performance as a regenerating agent for aggregated protein.
  • X is a nitrogen atom
  • n is an integer of 8 to 10.
  • X is a nitrogen atom and n is 8 (see Examples described later).
  • p and q in the general formula (2) each independently represent an integer of 2 to 14.
  • p ⁇ q is further satisfied (that is, an alkyl chain differing only in one is longer than the other three alkyl chains) .
  • X is a phosphorus atom
  • p is an integer of 3 to 5
  • q is an integer of 9 to 13.
  • X is a phosphorus atom
  • p is 4 and q is 12 (see Examples described later).
  • a x- represents a counter anion (x represents the valence of the counter anion).
  • carboxylate ion examples include formate ion, acetate ion, propionate ion, butyrate ion, oxalate ion, citrate ion and tartrate ion.
  • the counter anion is preferably dihydrogen phosphate. All of these anions are common in that they all exhibit excellent cosmotropicity, and thus, when used as the anion of the ion-binding salt according to the present invention, they have excellent performance as a regenerating agent for aggregated proteins. It is considered to be effective.
  • the active ingredient of the regenerating agent for aggregated protein according to the present embodiment is a hydrate of the ion binding salt represented by the general formula (1) or the general formula (2) described above (hydrated ionic liquid) It is.
  • a hydrated ionic liquid is one in which the ion binding salt is hydrated in water, and includes hydrated water.
  • the hydrated ionic liquid which is the active ingredient of the aggregate protein regenerating agent according to the present embodiment, is characterized in that the amount of hydration water contained therein is controlled to a value within a predetermined range.
  • the molar ratio of the ion binding salt to water of hydration in the hydrate of the ion binding salt (hydrated ionic liquid) is 1: 1 to 1:20.
  • the above molar ratio is preferably 1: 2 to 1:17, more preferably 1: 3 to 1, since the presence of free water adversely affects the regeneration of aggregated protein. 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.
  • hydration ionic liquid as an active ingredient of the aggregation protein which concerns on this form, only 1 type may be used independently, the combination of the structure of the cation of a cation binding salt and an anion, or hydration water content. Two or more different amounts may be used in combination.
  • ion binding salt when a commercially available product exists, the commercially available product may be purchased and used. In addition, even when there is no commercial product, it can be manufactured by a conventionally known method (see Examples described later).
  • an ion binding salt as a hydrate is also known per se, and after adding a predetermined amount of water to the ion binding salt, it is subjected to processing such as mixing and stirring as necessary. , Hydration ions can be obtained.
  • Regeneration method of aggregated protein According to another aspect of the present invention, there is also provided a method of regenerating aggregated proteins. That is, another aspect of the present invention includes aggregating protein, which comprises dissolving and refolding the aggregated protein by treating the aggregated protein in the presence of the regenerating agent for aggregated protein according to one aspect of the present invention described above Is a reproduction method of
  • the protein to be regenerated includes peptides, polypeptides, regardless of the origin of natural or man-made (chemical synthesis method, fermentation method, genetic recombination method), etc. Proteins, and complexes of these (eg, (poly) peptide or complex of protein and compound, (poly) peptide or complex of protein and saccharide, complex of (poly) peptide or protein and metal, ( Poly) and the like, and complexes of proteins or proteins with coenzymes.
  • proteins or proteins with coenzymes regardless of the type of protein, for example, intracellular protein, extracellular protein, membrane protein, and nuclear protein are all included.
  • a disulfide bond may be involved in protein aggregation
  • the target protein is not necessarily a protein having a disulfide bond.
  • suitable proteins include proteins containing at least one disulfide bond.
  • the protein of interest is preferably recombinantly produced genetically using a prokaryotic expression such as E. coli or a eukaryotic expression such as yeast or a heterologous expression system such as a cell-free extraction system.
  • a prokaryotic expression such as E. coli or a eukaryotic expression such as yeast
  • a heterologous expression system such as a cell-free extraction system.
  • Such recombinants are often obtained as insoluble and inactive aggregates (so-called inclusion bodies), and the regeneration method according to the present invention can be suitably used.
  • the molecular weight of the target protein in the present embodiment is not particularly limited, but usually about 1,000 to 10,000,000 proteins can be mentioned. From the viewpoint of the regeneration effect, a protein having a molecular weight of 10,000 to 250,000 is preferred. In general, there is a correlation between the size of the molecular weight and the difficulty of regeneration, and a protein with a large molecular weight (approximately 10,000 or more) may be extremely difficult to regenerate. According to the regeneration method of the present embodiment, since a high regeneration effect can be obtained, the method is also effective for high molecular weight proteins having a molecular weight of 10,000 or more.
  • the regeneration method according to this embodiment can be particularly suitably used for a protein having a molecular weight of 1,000 or more.
  • the molecular weight of the protein can be measured by a general gel electrophoresis method or the like.
  • the method for regenerating aggregated proteins according to the present embodiment dissolves and refolds the aggregated proteins by treating the aggregated proteins in the presence of the regenerating agent for aggregated proteins according to one embodiment of the present invention. It is characterized by the points it contains.
  • dissolution and refolding in this regeneration method it is possible to achieve both the dissolution and the refolding of the aggregated protein consistently by using the regenerating agent according to the present invention as a solvent, which is extremely simple. It can be said that the method is an industrially very useful regeneration method in that the aggregated protein can be regenerated by the method.
  • conventionally known solvents and additives other than the regenerating agent according to the present invention may, of course, be used in at least one of dissolution and refolding of the aggregated protein.
  • dissolution and refolding of the aggregated protein is performed without using solvents or additives other than the regenerating agent according to the present invention.
  • the aggregated protein regenerating agent (hydrated ionic liquid) according to the present invention is contacted with the aggregated protein.
  • Dissolution and refolding of the aggregated protein proceed only by (it is preferable that the aggregated protein is dissolved in the hydrated ionic liquid as a solvent and stirred if necessary).
  • the mixture is preferably allowed to stand for a certain period of time.
  • the standing time may be, for example, 1 to 50 hours.
  • the temperature condition at the time of standing 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 of regenerating aggregated proteins according to the present embodiment can be reworded as a method of preparing normal proteins.
  • the method for regenerating aggregated proteins according to the present embodiment may be any method as long as the aggregated proteins are dissolved and refolded by treating the aggregated proteins in the presence of the regenerating agent according to the present invention, and the presence or absence of other steps There is no particular restriction.
  • the method for regenerating the aggregation protein according to this embodiment is ) (3), (1) to (3) or (1) to (4) may be a method comprising: (1) Cultivating step of protein-producing bacteria: culturing protein-producing bacteria such as E.
  • Lysing step taking out protein inclusions from protein-producing cells using a lysing agent or the like;
  • Dissolution step The above-mentioned protein inclusion body is dissolved in the aggregated protein regenerating agent (hydrated ionic liquid) according to the present invention as a solvent, and allowed to stand at room temperature for several hours if necessary.
  • the refolding step is performed subsequently to the lysing step to regenerate the protein having the original activity.
  • a desired normal protein (refolded protein) is isolated from the solution of the protein obtained above in the hydrated ionic liquid by using column chromatography or the like.
  • bacterial cells can be illustrated as a protein production microbe in the protein production microbe culture process of said (1).
  • bacterial cells include Streptococcus, Staphylococci, Escherichia, Streptomyces, and Bacillus cells, fungal cells: for example, yeast cells and Aspergillus.
  • Genus (Aspergillus) cells insect cells: eg Drosophila S2 (Drosophila S2), Spodoptera Sf9 (Spodoptera Sf9) cells
  • animal cells eg CHO, COS, Hela, C127, 3T3, BHK, 293 and Bows melanoma cells And plant cells.
  • an expression vector containing cDNA encoding a target protein is (i) separating messenger RNA (mRNA) from the target protein-producing cells and The stranded cDNA is then synthesized into double stranded DNA and the complementary DNA is incorporated into a phage or plasmid. (Ii) A host is transformed with the obtained recombinant phage or plasmid, and after culture, a phage containing the target DNA by hybridization with a DNA probe encoding a part of the target protein or immunoassay using an antibody Alternatively, isolate the plasmid.
  • mRNA messenger RNA
  • (Iii) It can be produced by cutting out the desired cloned DNA from the recombinant DNA and ligating the cloned DNA or a part thereof downstream of the promoter in the expression vector. Thereafter, the host is transformed with the expression vector and cultured by an appropriate method. Culturing is usually carried out at 15 to 43 ° C. for 3 to 24 hours, and if necessary, aeration and agitation can be added.
  • any of physical disruption by ultrasonic waves, treatment with a lysis enzyme such as lysozyme, treatment with a lysis agent such as a surfactant and the like can be used.
  • Treatment with a lytic agent is preferred from the viewpoint of productivity.
  • bacteriolytic agents such as quaternary ammonium type cationic surfactants whose counter ion is a carboxylate ion such as formic acid or acetic acid can be mentioned.
  • silica dextran, agarose, cellulose, acrylamide, vinyl polymer and the like can be mentioned.
  • Commercially available commercially available products include Sephadex series, Sephacryl series, Sepharose series (above, Pharmacia), Bio-Gel series (Bio-Rad) and the like.
  • a disulfide bond may be generated between molecules at the time of aggregation.
  • the refolded protein is used as a reducing agent. It is preferable to further conduct the operation of reducing the disulfide bond by contacting.
  • the reducing agent used at this time and conventionally known reducing agents used for cleaving disulfide bonds in peptides and proteins may be used in the same manner. Examples of such reducing agents include 1,4-dithiothreitol, ⁇ -mercaptoethanol, reduced glutathione, tris (2-carboxyethyl) phosphine hydrochloride and the like.
  • this disulfide bond is cleaved during aggregation or the regeneration according to the present invention It may be cleaved at the time of refolding (dissolution and refolding) by an agent or the like, and when refolding is completed, a plurality of thiol groups (—SH groups) may be exposed.
  • 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 following operation.
  • oxidizing agents used for the formation of disulfide bonds in peptides and proteins may be used in the same manner.
  • examples of such oxidizing agents include oxidized glutathione and cystine.
  • the following ion binding salts were prepared by exchanging the anions for the dihydrogen phosphate ion (dhp) for the ion binding salts (9) to (22) prepared above. 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 to OH ⁇ . Then, an equimolar amount of phosphoric acid was added, and after neutralization reaction, dehydration was performed with an evaporator to obtain an ion binding salt in which the anion was exchanged to dihydrogen phosphate (dhp).
  • an anion exchange column resin Amberlite IRN 78
  • ion binding salts (1) to (22) and (9 ') to (15') prepared above were made into hydrates by adding water. Specifically, water is added such that the number of water molecules is 3, 7 or 15 per 1 ion pair of the ion binding salt, to obtain an ion binding salt in the form of a hydrate.
  • ConA concanavalin A which is a sugar chain recognition protein
  • ConA was mixed with Milli-Q water to a concentration of 10 mg / mL, and incubated at 70 ° C. for 10 minutes to obtain aggregates of ConA.
  • the solubility of the sample after overnight stirring was confirmed by visual observation and fluorescence measurement.
  • the supernatant obtained by centrifuging the sample after agitation (10,000 g, 30 minutes) is obtained by excitation at an excitation wavelength of 280 nm, where tryptophan residue in the protein is to be measured.
  • the maximum fluorescence wavelength of the fluorescence spectrum and its fluorescence intensity were measured. The results are shown in Table 1 below.
  • FIG. 2A is a graph showing the results of evaluating the binding activity of a sample (positive control) in which native ConA was dissolved in the above 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.
  • FIGS. 2B to 2I are graphs showing the results of evaluation of the binding activity of the following samples: FIG. 2B: (11 ') P44412 dhp (1: 3) FIG. 2C: (11 ') P44412 dhp (1: 7) FIG. 2D: (11 ') P44412 dhp (1: 15) FIG. 2E: (14 ') N 8888 dhp (1: 3) FIG. 2F: (9 ') P4444 dhp (1: 3) FIG. 2G: (10 ') P4448 dhp (1: 3) FIG. 2H: (12 ') N 4444 dhp (1: 3) FIG. 2I: (15 ′) P6666 dhp (1: 3).
  • Cytochrome c Lysozyme Fructose Dehydrogenase Specifically, each protein was mixed with Milli Q water to a concentration of 10 mg / mL, and incubated at 70 ° C. for 10 minutes to obtain an aggregate of each protein.
  • the ion binding salt according to the present invention functions as a system capable of performing dissolution and refolding consistently regardless of the type of aggregated protein.
  • the activity was restored by the addition of a reducing agent ( ⁇ -mercaptoethanol) for fructose dehydrogenase because the disulfide bond generated between the molecules of the protein at the time of formation of the aggregate was cleaved by the addition of the reducing agent, and the refolding was normal. It is thought that this is because it became possible.
  • the ion binding salt according to the present invention can perform refolding and recovery of activity thereby more efficiently by using it in combination with a reducing agent as necessary.
  • the regenerating agent (ion-binding salt) of the aggregated protein according to the present invention achieves dissolution and refolding of the aggregated protein, and then adding the oxidizing agent without particularly requiring a separate operation. It can be understood that it can also be used as a necessary site for forming a disulfide bond.

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Abstract

Le problème décrit par la présente invention est de fournir une technologie avec laquelle une protéine agrégée peut être solubilisée sans utiliser d'agent de modification puissant, et la protéine agrégée peut être constamment réenroulée (repliée) en une structure similaire à la structure native, sans utiliser d'autres additifs. La solution selon l'invention porte sur un hydrate d'un sel de liaison d'ions représenté par la formule générale (1), à savoir {[X(CnH2n+1)4]+}XAX-, ou la formule générale (2), à savoir {[X(CpH2p+1)3(CqH2q+1)]+}XAX-, qui est utilisé en tant qu'agent de régénération d'une protéine agrégée (dans les formules : X représente un atome d'azote ou un atome de phosphore ; n représente un nombre entier de 7 à 11 ; p et q représentent chacun indépendamment un nombre entier de 2 à 14 ; |p - q| ≥ 6 est satisfaite ; Ax- représente un contre-anion choisi dans le groupe constitué d'ions de phosphate, d'ions d'hydrogénophosphate, d'ions de dihydrogénophosphate, d'ions de sulfate, d'ions d'hydrogénosulfate , et d'ions d'acide carboxylique ; et x représente la valence du contre-anion). Le rapport molaire du sel de liaison d'ions à l'eau d'hydratation dans l'hydrate est une valeur dans la plage de 1 : 1 à 1 : 20.
PCT/JP2018/043222 2017-11-22 2018-11-22 Agent de régénération de protéine agrégée, et procédé de régénération de protéine agrégée l'utilisant WO2019103106A1 (fr)

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Cited By (2)

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CN110625060A (zh) * 2019-10-15 2019-12-31 湖北工业大学 羟基乙叉二膦酸改性的磷酸盐粘结剂及其制备方法
JP7562094B2 (ja) 2020-11-20 2024-10-07 学校法人東京薬科大学 生体分子の回収方法

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110625060A (zh) * 2019-10-15 2019-12-31 湖北工业大学 羟基乙叉二膦酸改性的磷酸盐粘结剂及其制备方法
CN110625060B (zh) * 2019-10-15 2020-11-03 湖北工业大学 羟基乙叉二膦酸改性的磷酸盐粘结剂及其制备方法
JP7562094B2 (ja) 2020-11-20 2024-10-07 学校法人東京薬科大学 生体分子の回収方法

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