WO2012133695A1 - タンパク質結晶化条件探索剤及びタンパク質結晶化条件探索方法 - Google Patents
タンパク質結晶化条件探索剤及びタンパク質結晶化条件探索方法 Download PDFInfo
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- WO2012133695A1 WO2012133695A1 PCT/JP2012/058451 JP2012058451W WO2012133695A1 WO 2012133695 A1 WO2012133695 A1 WO 2012133695A1 JP 2012058451 W JP2012058451 W JP 2012058451W WO 2012133695 A1 WO2012133695 A1 WO 2012133695A1
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- protein
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- crystallization condition
- condition search
- saponite
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N23/00—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
- G01N23/20—Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00 by using diffraction of the radiation by the materials, e.g. for investigating crystal structure; by using scattering of the radiation by the materials, e.g. for investigating non-crystalline materials; by using reflection of the radiation by the materials
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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
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
- C07K1/306—Extraction; Separation; Purification by precipitation by crystallization
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- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
- C30B29/54—Organic compounds
- C30B29/58—Macromolecular compounds
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- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions
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- C—CHEMISTRY; METALLURGY
- C30—CRYSTAL GROWTH
- C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
- C30B29/00—Single crystals or homogeneous polycrystalline material with defined structure characterised by the material or by their shape
- C30B29/54—Organic compounds
Definitions
- the present invention relates to a protein crystallization condition search agent and a protein crystallization condition search method using the same. More specifically, the present invention relates to a protein crystallization condition search agent having improved protein crystal formation promoting characteristics and operability and a protein crystallization condition search method using the same.
- Crystallization passes through a process of forming crystal nuclei in a supersaturated solution and a process of growing crystal nuclei into crystals.
- the obstacle in structural analysis is the crystallization process.
- the search for protein crystallization conditions consists of a primary screening and a secondary screening.
- the primary screening is a search process of physical conditions and chemical conditions for the purpose of determining the crystallization start conditions of the protein by extensive search of the crystallization conditions of the target protein. For example, in this step, the protein concentration effective for crystallization, the type and concentration of the precipitant, the type and pH of the buffer solution, etc. are determined, and conditions under which crystallization can be started are found.
- the secondary screening a precise search process of physical conditions and chemical conditions for the purpose of determining the final crystallization conditions of the target protein by modifying the crystallization start conditions selected through the primary screening. It is.
- the modification of the crystallization start condition in the second screening may be a partial change of the physical condition and the chemical condition constituting the crystallization start condition, or a new physical condition and chemistry not included in the crystallization start condition. This is done by selectively adding conditions. For example, the conditions such as the concentration of the precipitating agent and the pH of the buffer solution are engraved, or the additives and surfactants not used in the primary screening are used. Crystallization conditions, specifically, conditions for obtaining as large and high quality single crystals as possible are found.
- Both screenings require trial and error conditions of combinations of myriad physical and chemical conditions depending on the protein of interest. For example, multidimensional conditions such as the type of precipitant, the pH of the solution, the concentration of the protein solution, and the temperature are examined. Crystallization conditions are determined, and the obtained crystal is subjected to structural analysis by X-ray irradiation. Prior to X-ray irradiation, the crystals may be immersed in an antifreeze containing a cryoprotectant. Moreover, when it is desired to omit the dipping step, protein crystallization is attempted using a precipitant solution containing a cryoprotectant in the primary or secondary screening. In any case, each screening is repeated until the desired crystals are obtained.
- multidimensional conditions such as the type of precipitant, the pH of the solution, the concentration of the protein solution, and the temperature are examined. Crystallization conditions are determined, and the obtained crystal is subjected to structural analysis by X-ray irradiation. Prior to X-ray irradiation, the crystals
- the conventional primary screening sometimes fails to determine not only crystals but also crystalline precipitates and cannot determine the crystallization start conditions. Since protein crystallization conditions change the presence or absence of crystal nucleation due to slight changes in the setting conditions such as solvent and temperature, the conventional secondary screening is a modification of the crystallization start conditions selected by the primary screening. As a result, crystals may not be obtained.
- the conventional secondary screening Since it is difficult to control the formation of protein crystal nuclei by changing the setting conditions such as the solvent and temperature, the conventional secondary screening has a tendency not to produce crystals of a desired size and number.
- the conventional secondary screening produces crystals under conditions called a metastable region that has a lower supersaturation condition than the crystallization start condition and does not cause crystal nucleation but has the potential for crystal growth. I did not. It is well known that when protein crystals form in the metastable region, the crystals tend to be of sufficiently high quality. Since cryoprotectants suppress the formation of protein crystal nuclei, the use of reagents containing cryoprotectants in the primary and secondary screens tended not to crystallize proteins.
- the conventional screening system has a tendency to fail to obtain a target crystal or waste a valuable sample protein in spite of a heavy examination of conditions. Accordingly, there is a demand for the development of a method for searching for protein crystallization conditions that is simple, economical, and highly reliable.
- Patent Document 1 discloses a screening chip for searching protein crystallization conditions using a layered silicate, wherein the silicate is coated in a film form on a support.
- the layered silicate of Patent Document 1 has a problem in that it does not sufficiently promote the crystal nucleation of a wide range of proteins in screening based on the search for various protein crystallization conditions. Further, since it is not a uniform thin film, it may be easily detached.
- the invention of Patent Document 1 requires the user of the screening chip to select the optimum type and application amount of the layered silicate. Such a condition study consumes a large amount of protein solution.
- silicate is present at the non-contact portion between the screening chip and the protein solution, extensive penetration of the protein solution tends to cause a decrease in supersaturation and protein denaturation.
- Patent Documents 2 and 3 disclose protein crystal formation control agents and control methods using layered silicates.
- the layered silicates of Patent Documents 2 and 3 also do not sufficiently promote the formation of protein crystal nuclei in screening, similar to that of Patent Document 1.
- the layered silicates of Patent Documents 2 and 3 have a weak surface negative charge and low water swellability, they tend to aggregate and settle upon contact with a screening reagent containing a high concentration of precipitant.
- Non-Patent Document 1 discloses a lysozyme crystal formation control agent containing a fluorine-containing layered silicate.
- Non-Patent Document 1 discloses the use of layered silicates in lysozyme crystallization, but does not describe the use of layered silicates in screening based on the search for various protein crystallization conditions. . Further, the layered silicate of Non-Patent Document 1 cannot be said to sufficiently promote the formation of protein crystal nuclei in screening.
- Non-Patent Document 2 discloses a protein crystal formation control agent containing fluorine mica. This fluoric mica is synthesized by a solid reaction method in the absence of moisture using talc as a starting material. However, since the fluorine mica of Non-Patent Document 2 does not have a hydroxyl group, the rate of crystal nucleation cannot be adjusted, and there are cases where crystal nucleation is not promoted. Absent. Moreover, since it is non-swellable with respect to water, there exists a tendency to aggregate and settle by contact with the reagent containing a high concentration precipitant. Therefore, the use of the fluorinated mica dispersion requires stirring for uniform dispersion.
- Non-Patent Document 3 discloses a protein crystal formation control agent comprising a mineral containing a layered silicate.
- the layered silicate constituting the mineral of Non-Patent Document 3 cannot be said to sufficiently promote the formation of protein crystal nuclei.
- Non-Patent Document 4 discloses a protein crystal formation control agent composed of mica containing an amino group.
- the mica of Non-Patent Document 4 forms a sheet for use in sealing wells in the hanging drop method. This sheet-like product cannot be said to sufficiently promote the formation of protein crystal nuclei.
- the present inventors include a water-swellable layered silicate having a fluorine atom and a hydroxyl group, and the fluorine atom is covalently bonded to the silicate by isomorphous substitution with the hydroxyl group.
- the present inventors have found that the strong surface negative charge and the excellent water swellability of the water-swellable layered silicate having a fluorine atom contribute to the improvement of the operability of the protein crystallization condition search agent. It was. Based on these findings, the present inventors have made further studies and have come to achieve the present invention.
- the present invention provides a protein crystallization condition search agent comprising a water-swellable layered silicate having a fluorine atom and a hydroxyl group, wherein the fluorine atom is covalently bonded to the silicate by isomorphous substitution. .
- this invention provides the protein crystallization condition search method including the process of mixing the protein crystallization condition search agent and the solution in which the protein is dissolved.
- the present invention also provides use of the protein crystallization condition search agent for mixing the protein crystallization condition search agent and a solution in which the protein is dissolved to recover the crystallized protein.
- the search for protein crystallization conditions refers to the setting of physical conditions and chemical conditions to search for the crystallization conditions of the target protein, whether or not crystals are formed, whether or not crystalline precipitates are formed, and non-crystallized. Based on the presence or absence of crystalline precipitate formation, success rate of crystallization, formation time, shape, size, crystal nucleus density (number of crystals per unit area) or X-ray diffraction data of the formed protein, Determining physical and chemical conditions suitable for crystallization.
- the protein crystallization condition search agent of the present invention it is possible to search for crystallization conditions efficiently and practically by examining conditions with relatively little load. Moreover, according to the protein crystallization condition searching method of the present invention, conditions suitable for protein crystallization can be set efficiently and practically in a process with relatively little load.
- FIG. 1 is a diagram showing the results of X-ray diffraction of saponite of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- 1 shows the X-ray diffraction pattern of the saponite of Preparation Example 1
- (b) shows the X-ray diffraction pattern of the saponite of Preparation Example 2
- (c) shows the X-ray diffraction pattern of the saponite of Preparation Example 3.
- FIG. 2 shows the nitrogen adsorption / desorption isotherms of the saponites of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- 2 shows the nitrogen adsorption / desorption isotherms of the saponites of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- 2 shows the nitrogen adsorption / desorption isotherms of the saponites of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- (a) shows the isother
- screening refers to the setting of physical and chemical conditions for searching for crystallization conditions of an object, whether or not crystals are formed, whether or not crystalline precipitates are formed, and non-crystalline. Determine the physical and chemical conditions suitable for crystallization of the target object based on the presence / absence of precipitate formation, crystallization success rate, formation time, shape, size, crystal nucleus density or X-ray diffraction data of the formed crystal. Refers to a process.
- metalstable region refers to a set of supersaturated conditions that have the potential for crystal growth but not crystal nucleation of the object.
- the term “layered silicate” is a compound having a two-dimensional layer structure, and the layer structure is composed of at least a silicon atom and an electronegative ligand. Compound.
- the layered silicate may have a single layer of a tetrahedral layer and / or an octahedral layer, or a mixed layer thereof.
- the tetrahedral sheet has a structure in which silicon ions (Si 4+ ) or aluminum ions (Al 3+ ) are surrounded by four oxygen ions (O 2 ⁇ ), and the three vertices are shared with the adjacent tetrahedron, and the structure is a hexagonal network. It is connected in a sheet form.
- the octahedron sheet spreads two-dimensionally with the octahedron surrounded by magnesium ions (Mg 2+ ) or aluminum ions surrounded by six oxygen ions (O 2 ⁇ ) or hydroxide ions (OH ⁇ ). It is a thing. When the tetrahedral sheet and the octahedral sheet are combined, oxygen ions at the apex of the tetrahedral sheet are shared. In addition, hydroxide ions may be isomorphously substituted by fluorine ions (F ⁇ ).
- isomorphic substitution refers to the replacement of one atom of a compound having a crystal structure with another atom of the same or similar ionic radius without changing the crystal structure of the compound. Refers to reaction.
- the protein crystallization condition search agent of the present invention includes a water-swellable layered silicate having a fluorine atom and a hydroxyl group at a specific ratio.
- the fluorine atom is covalently bonded to the layered silicate by isomorphous substitution.
- Layered silicates when in contact with proteins, produce aggregates of the proteins that function as crystal nuclei.
- the layer structure, fluorine atom and hydroxyl group of the layered silicate can promote the formation of protein crystal nuclei.
- the fluorine atom and water swellability of the layered silicate impart uniform dispersibility and film formability to the crystallization condition search agent.
- the crystallization condition search agent of the present invention is suitable for searching for crystallization conditions for crystallization of protein in an amount from nanogram units to microgram units.
- the crystallization condition search agent may be composed only of a layered silicate or a mixture of a layered silicate and other components. Examples of other components include pure water, a precipitating agent, a buffering agent, an additive, an organic solvent, and a mixed component thereof.
- the crystallization condition search agent may include a single or a plurality of layered silicates.
- the layered silicate used for the crystallization condition search agent of the present invention is not particularly limited, but preferably montmorillonite, talc, phlogopite, mica, kaolin, chlorite, pyrophyllite, vermiculite, enderite, chrysotile, beidellite, Examples include halloysite, nontronite, sericite, dickite, antigolite, hectorite (fluorine hectorite), soconite, kanemite, macatite, octosilicate, magadiite, keniaite, stevensite and saponite. More preferred are mica, montmorillonite, stevensite, hectorite and saponite, and particularly preferred is saponite.
- the layered silicate natural products or synthetic products may be mentioned.
- the natural product may contain impurities, but from the viewpoint of strict crystallization control of nucleation, the impurities are preferably removed.
- the synthesized product has high purity, and the isomorphous substitution efficiency can be freely controlled during synthesis. Therefore, strict control in nucleation is possible.
- the layered silicate may have any average particle size.
- the average particle diameter is preferably 2 to 50 ⁇ m from the viewpoint of uniform dispersibility.
- the average particle size may be determined by laser particle size distribution measurement using water as a dispersion medium.
- the layered silicate may have any cation exchange capacity.
- the cation exchange capacity is preferably at least 10 meq / 100 g, more preferably at least 60 meq / 100 g.
- a suitable range of cation exchange capacity imparts a negative charge suitable for promoting nucleation to the layered silicate.
- the specific surface area of the layered silicate is preferably 1 to 1000 m 2 / g, more preferably 10 to 500 m 2 / g, and still more preferably 100 to 200 m 2 / g.
- the specific surface area imparts a surface charge density suitable for promoting nucleation to the layered silicate.
- the layered silicate Since the layered silicate has water swellability, it is difficult to spontaneously settle in a reagent containing a high concentration of precipitant. Accordingly, it is possible to provide a dispersion type crystallization condition search agent having improved uniform dispersion, long-term stability in a uniform dispersion state, and redispersibility. In addition, since the water-swellable layered silicate has excellent film forming properties, it is possible to provide a uniform thin-film crystallization condition search agent.
- the layered silicate is preferably a smectite group.
- the smectite family has a 2: 1 type layer structure.
- the use of the smectite group makes it possible to provide a crystallization condition search agent having improved uniformity and operability.
- the layered silicate is more preferably saponite among the smectite group.
- Saponite has a permanent negative charge because it has a layered structure in which silicon atoms in the tetrahedral layer are replaced with aluminum atoms.
- Saponite imparts nucleation promoting properties attributed to mesopores to the search agent for crystallization conditions.
- the use of saponite enables the crystallization condition search agent to promote crystal nucleation of more diverse proteins in a single product, and to give improved uniformity and operability.
- the bonding form of the hydroxyl group does not matter.
- the layered silicate may have a hydroxyl group bonded by a covalent bond.
- the layered silicate may bind a compound containing a hydroxyl group directly or indirectly.
- the hydroxyl group is covalently bonded within the crystal structure of the layered silicate.
- a layered silicate having a fluorine atom and a hydroxyl group adsorbs a protein in a protein solution, and produces an aggregate of the protein that functions as a crystal nucleus.
- the molar ratio of the hydroxyl group to the fluorine atom can be controlled by the amount of added fluorine raw material during synthesis.
- the fluorine atoms are covalently bonded within the crystal structure of the layered silicate by isomorphous substitution.
- a layered silicate in which fluorine atoms are isomorphously substituted has a relatively large cation exchange capacity, and thus increases the surface charge density of the layered silicate. Therefore, the layered silicate into which fluorine atoms are introduced by isomorphous substitution effectively adsorbs proteins in a protein solution and effectively produces protein aggregates that function as crystal nuclei.
- the crystallization condition search agent is less likely to aggregate even when contacted with a reagent containing a high concentration of precipitant, and has improved uniformity.
- the layered silicate having a fluorine atom and a hydroxyl group can be synthesized by an ordinary method.
- the synthesis method include a hydrothermal synthesis method, a melt synthesis method, a high-pressure synthesis method, a solid reaction method, a flame melting method, and an alteration method.
- hydrothermal synthesis using a fluorine source is preferable.
- Hydrothermal synthesis using a fluorine source can easily introduce fluorine atoms and hydroxyl groups into the product by isomorphous substitution.
- a layered silicate having a fluorine atom and a hydroxyl group may be synthesized by a high pressure synthesis method using a fluorine source, a solid reaction method, a flame melting method, and an alteration method. Furthermore, a layered silicate having a fluorine atom and a hydroxyl group may be synthesized by introducing a hydroxyl group into the fluorine-containing layered silicate.
- the molar ratio of fluorine atoms and hydroxyl groups in the layered silicate affects the protein crystallization success rate, crystallization speed, crystal nucleus density, and crystal size.
- the fluorine content and the molar ratio between fluorine atoms and hydroxyl groups are determined by using a layered silicate that has been freed of free fluorine and soluble ions by filtering or centrifuging several times after dispersing the synthesized sample in distilled water. Then, it may be quantified and calculated with a fluorescent X-ray apparatus (RIX1000, manufactured by Rigaku).
- the molar ratio of fluorine atoms to hydroxyl groups in the layered silicate is preferably 3% to 15%. More preferably, it is 4% to 12%, and most preferably 5 to 10%.
- the molar ratio of the hydroxyl group is preferably 85% to 97%, more preferably 88% to 96%, and most preferably 90% to 95%.
- the success rate of crystallization in the primary screening using the crystallization condition search agent can be improved.
- the success rate of crystallization, the rate of crystal formation, the size of the crystal, and the crystal nucleus density can be controlled.
- the layered silicate does not effectively promote crystal nucleation.
- the desired crystal may not be obtained.
- the crystal size may be small, or high-quality crystals for X-ray diffraction may not be obtained.
- the crystallization condition search agent may contain a layered inorganic compound other than the layered silicate.
- layered inorganic compounds include layered metal hydroxides and layered double hydroxides.
- the crystallization condition search agent may contain any protein crystal formation control agent.
- Suitable crystal formation control agents are disclosed in Japanese Patent Application Laid-Open No. 2007-55931, International Publication No. 2004/041847, International Publication No. 02/088835 and Japanese Patent Application Laid-Open No. 8-294601. These suitable crystal formation control agents are incorporated herein by reference.
- the crystallization condition search agent may contain any precipitating agent for decreasing the solubility of the protein.
- the precipitating agent include salts, water-soluble polymer compounds, organic solvents, and mixed components thereof.
- the salt include ammonium sulfate, magnesium sulfate, sodium sulfate, lithium chloride, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, sodium nitrate, sodium fluoride, sodium tartrate, sodium formate, ammonium phosphate, lithium sulfate, and chloride.
- Examples include cobalt, zinc sulfate, and sodium malonate.
- water-soluble polymer compound examples include polyethylene glycol having a molecular weight of 400 to 20000, polyethylene glycol monoalkyl ether, and polyethyleneimine.
- organic solvent examples include ethanol, methanol, acetone, 2-methyl-2,4-pentanediol, isopropanol, butanol, dioxane, dimethyl sulfoxide, and dimethylformamide.
- the crystallization condition search agent may contain a surfactant.
- the surfactant include sodium dodecyl sulfate, lithium dodecyl sulfate, dodecyldimethylamine-N-oxide, N-decyl- ⁇ -D-maltoside, N-dodecyl- ⁇ -D-maltopyranoside, N-octyl- ⁇ - Examples include D-glucopyranoside, N-octyl- ⁇ -D-thioglucopyranoside, N-nonyl- ⁇ -D-thiomaltoside, polyoxyethylene-octylphenyl ether, decanoyl-N-methylglucamide and sucrose monolaurate.
- the crystallization condition search agent may include a cryoprotectant.
- cryoprotectant include glycerol, ethylene glycol, sucrose, and trehalose.
- the crystallization condition search agent may contain any additive.
- Additives include, for example, ligands, inhibitors, coenzymes, substrates, amino acids, gels, antibodies and mixtures thereof. Suitable amino acids are disclosed in WO 2008/102469. A suitable gel is disclosed in Japanese Patent Application Laid-Open No. 2007-45668. These suitable amino acids and gels are incorporated herein by reference.
- the crystallization condition search agent may contain an ionic liquid as long as it does not adversely affect protein crystallization.
- Suitable ionic liquids are Russell A. Judge et al., “The Effect of Ionic Liquids on Protein Crystallization and X-ray Diffraction Resolution”, CRYSTAL GROWTH & DESIGN, 2009, Vol. 9, No. 8, p.3463- 3469. This suitable ionic liquid is incorporated herein by reference.
- the crystallization condition search agent may contain an arbitrary reagent.
- a suitable reagent is disclosed in JP-A-2006-83126. This suitable reagent is incorporated herein by reference.
- the dosage form of the crystallization condition search agent may be any.
- a solid agent, a dispersion liquid agent, and a clay-like semi-solid agent are mentioned.
- the crystallization condition search agent is a dispersion agent obtained by adding a powdery layered silicate to a dispersion medium, or a thin-film solid agent obtained by applying a layered silicate on a support.
- a dispersion of a water-swellable layered silicate having a fluorine atom hardly aggregates and settles in a reagent containing a high concentration precipitant. Since the thin-film solid agent does not require the preparation of a dispersion, the operability in searching for crystallization conditions can be improved.
- the support for supporting the thin film-like solid agent may be any, but is preferably a cover glass, a slide glass, a plate, a bridge or a rod, and particularly preferably a bridge.
- the thin-film solid agent applied to the bridge enables a rapid search for crystallization conditions by a sitting drop vapor diffusion method.
- an ordinary dispersion medium can be used as the crystallization condition search agent.
- the dispersion medium include pure water, buffer solution, precipitant solution, surfactant solution, alcohol, organic solvent, chelating agent solution, reducing agent solution, cryoprotectant solution, ionic liquid, mineral oil, and mixed solvents thereof. Is mentioned.
- the dispersion medium is an aqueous solution of a nonionic water-soluble polymer compound.
- nonionic water-soluble polymer compounds include polyethylene glycol and polyethylene glycol monoalkyl ether.
- the use of this dispersion medium effectively prevents aggregation and sedimentation of the layered silicate particles, and makes it possible to separate a uniform dispersion without stirring. Therefore, the use of the dispersion medium not only enables efficient crystallization condition search by hand, but also allows efficient crystallization conditions by combining with a known crystallization automation apparatus not equipped with a stirrer. Allows searching.
- Suitable automation devices are disclosed in JP 2010-151821, JP 2006-281139, JP 2003-14596, JP 2004-194647, and JP 2003-502652. . These suitable automated devices are hereby incorporated by reference.
- the crystallization condition search agent is a solid agent.
- solid agents include powders, granules, thick films, thin films, tablets, and lyophilizers.
- the solid agent in the form of a thick film or a thin film may be a self-supporting film or a film supported on a support. Thick film or thin film crystallization condition search agents do not require any agitation and thus have improved operability.
- the crystallization condition search agent in the form of a thick film or a thin film has improved uniformity because it does not foam even when a reagent containing a surfactant is used. Accordingly, a thick film or thin film crystallization condition search agent is suitable for large-scale high-throughput screening.
- the thick film or thin film solid agent may be formed by any film forming method. Suitable film forming methods are disclosed in JP-A-2008-119659, JP-A-2008-18299, JP-A-2006-150160, JP-A-2005-163058, and JP-A-2001-220244. . These suitable deposition methods are incorporated herein by reference.
- the crystallization condition search agent is a thin film-like solid agent coated on a support.
- the thin-film solid agent has not unevenness on the application surface of the layered silicate, so that it is not easily detached from the support, and crystal nucleus formation can be strictly controlled.
- the thin-film solid agent has further improved uniformity and operability.
- the thin film is preferably formed only on the portion of the support that contacts the protein. This configuration not only reduces the amount of layered silicate used, but also prevents excessive wasting of protein and rapid loss of moisture due to extensive penetration of the protein solution.
- the support is a cover glass, a slide glass, a plate, a bridge or a rod.
- Suitable supports are disclosed in JP 2002-179500 A, JP 2002-233702 A, JP 2006-500960 A, US Pat. No. 5,419,278, and US Pat. No. 5,096,676. These suitable supports are incorporated herein by reference. This configuration not only enables efficient manual screening, but also enables efficient screening in combination with a known crystallization automation device that does not include a stirring device.
- the type of protein targeted by the crystallization condition search agent is not particularly limited.
- proteins include acidic protein, basic protein, neutral protein, glycoprotein, lipoprotein, membrane protein, enzyme, antibody, receptor, transcription factor, oligopeptide, polypeptide, protein-protein complex, protein and Examples include complexes with nucleic acids, complexes between proteins and low-molecular compounds, complexes between proteins and coenzymes, complexes between proteins and sugars, complexes between proteins and metals, and complexes between enzymes and substrates.
- Preferred are acidic proteins, basic proteins, and neutral proteins. These proteins may have any molecular weight and isoelectric point.
- the protein may be soluble or insoluble in pure water or a buffer solution.
- the insoluble protein may be dissolved using any solubilizing agent.
- Suitable solubilizers are disclosed in JP-A-2007-314526, JP-T-2001-519836, JP-A-5-125092 and JP-A-5-32689. These suitable solubilizers are incorporated herein by reference.
- the protein may be a natural protein or an artificial protein.
- the protein may be an artificial one synthesized by various methods.
- Examples of the artificial protein include an expressed protein synthesized by a gene recombination technique, a protein synthesized by a chemical synthesis method, and a protein synthesized by a fermentation method.
- the expressed protein may be synthesized using any expression system.
- Examples of expression systems include E. coli expression systems, yeast expression systems, insect cell expression systems, mammalian cell expression systems, and cell-free expression systems.
- Suitable expression systems include JP2010-63386, JP2010-11851, JP2008-193953, JP2008-538503, JP2007-501007, JP2006-. No. 502691, No. 2006-508684, No. 2005-517385, No. 2004-506428 and No. 2003-511667. These suitable expression systems are incorporated herein by reference.
- the expressed protein may be appropriately fused with a tag protein.
- Suitable tag proteins are disclosed in JP 2010-75090 A, JP 2004-532025 A, JP 2003-289882 A, and JP 2003-38195 A. These suitable tag proteins are incorporated herein by reference.
- the solvent for dissolving the protein is not particularly limited.
- the protein dissolving solvent include pure water, buffer solution, precipitant solution, surfactant solution, alcohol solution, organic solvent, chelating agent solution, reducing agent solution, cryoprotectant solution, ionic liquid, and a mixed solution thereof.
- the dissolution solvent is a buffer.
- the buffer component include sodium acetate, sodium phosphate, sodium citrate, sodium cacodylate, sodium carbonate, imidazole, trishydroxymethylaminomethane, Good buffer, and mixed components thereof.
- the buffer can be at any pH, for example, in the range of pH 3.5-10.0.
- the crystallization condition search method of the present invention includes a step of mixing the above-described crystallization condition search agent containing a water-swellable layered silicate having a fluorine atom and a hydroxyl group with a solution in which a protein is dissolved.
- the concentration of the protein solution is not particularly limited as long as the protein is dissolved.
- the initial protein concentration is preferably high, preferably 2 mg / mL or more, more preferably 5 mg / mL to 200 mg / mL, and particularly a concentration in the vicinity of saturation solubility. preferable.
- the amount of the crystallization condition search agent added to the protein solution is not particularly limited, but the final concentration of the layered silicate in the protein solution is preferably at least 0.01% (w / v), 0.01% More preferably, the content is set to ⁇ 1.0% (w / v).
- the final concentration of the layered silicate is preferably 0.01% to 0.5% (w / v), preferably 0.05% to 0.3% (w / v). v) is more preferable, and 0.1% to 0.2% (w / v) is even more preferable.
- the method for crystallization of the protein is not particularly limited.
- the crystallization method include a batch method, a vapor diffusion method, a dialysis method, a free interface diffusion method, a concentration method, a light irradiation method, a vibration method, and a stirring method.
- a vapor diffusion method a hanging drop method, a sitting drop method, and a sandwich drop method are preferably used.
- the vapor diffusion method enables easy and rapid screening using a trace amount of protein.
- Suitable crystallization methods are disclosed in JP 2010-13420 A, JP 2010-1220 A, JP 2003-306497 A, JP 4029987 A and JP 6-1116098 A. These suitable crystallization methods are incorporated herein by reference.
- the protein may be crystallized using an arbitrary container.
- Suitable containers include JP2009-172530A, JP2008-309617A, JP2008-126135A, JP2007-1788A, JP2006-124238A, and JP2006-308607A. Gazette, JP-T 2005-502571, JP-T 2005-515151, JP-T 2006-525864, JP-A 2004-307335, JP-A 2003-248004, JP-T 2002-528701 No. 2002-536255, JP-A-11-130600, US Pat. No. 5,096,676, US Pat. No. 5,130,105, US Pat. No. 5,221,410 It is disclosed in the specification and International Publication No. 01/088821. These suitable containers are incorporated herein by reference.
- the protein may be crystallized under any temperature condition.
- the temperature is preferably 4 to 37 ° C, and more preferably 4 to 20 ° C.
- the formed crystal may be observed using a microscope.
- the microscope include a stereoscopic microscope, an optical microscope, a polarizing microscope, and an electron microscope.
- the crystal may be observed with the naked eye or may be observed using a crystal detection device.
- Suitable crystal detection devices are disclosed in JP 2007-45663 A, JP 2005-9949 A, JP 2006-242690 A, JP 2004-323336 A, and JP 2007-248280 A. . These suitable crystal detection devices are incorporated herein by reference.
- the formed crystal may be analyzed by an X-ray diffractometer. X-ray diffraction can be performed regardless of whether or not the crystallization condition search agent is attached to the crystal.
- the crystallization condition search agent of the present invention does not adversely affect the X-ray diffraction experiment, and a good X-ray diffraction image can be obtained even using a crystal to which the crystallization condition search agent is attached.
- the formed crystal may be used as a seed crystal for searching for new crystallization conditions.
- the crystallization condition search agent of the present invention may be used as a protein crystal formation control agent or a protein crystal formation accelerator.
- the crystallization condition search agent of the present invention may be applied to industrial protein purification using protein crystallization, or may be applied to crystallization of other biopolymers other than proteins. These uses include the case where the crystallization condition search agent of the present invention acts as a nucleating agent or a crystal nucleating agent.
- the reaction gel was put into a 3 L autoclave container, and hydrothermal synthesis was performed at 250 ° C. for 4 hours.
- the product after hydrothermal synthesis (1 L) was suction filtered with a filter paper to remove moisture.
- the obtained cake was redispersed by adding 2 L of water and stirring, and this was refiltered. Unreacted substances were washed away by repeating the redispersion and refiltration operations three times.
- the obtained cake was dried to obtain a synthetic saponite having a fluorine atom and a hydroxyl group (hereinafter sometimes referred to as saponite of Preparation Example 1).
- Preparation Example 2 A synthetic saponite having a fluorine atom and a hydroxyl group (hereinafter sometimes referred to as saponite of Preparation Example 2) was obtained in the same manner as Preparation Example 1 except that the blending amount of sodium fluoride was changed in Preparation Example 1. .
- Preparation Example 3 Synthetic saponite having a hydroxyl group (hereinafter referred to as saponite of Preparation Example 3) in the same manner as Preparation Example 1, except that sodium hydroxide (manufactured by Kanto Chemical Co., Inc.) was blended in place of sodium fluoride in Preparation Example 1. Got).
- sodium hydroxide manufactured by Kanto Chemical Co., Inc.
- FIG. 1 shows an analysis result by an X-ray diffractometer (manufactured by Rigaku, MiniFlex).
- FIG. 1 shows the X-ray diffraction pattern of the saponite of Preparation Example 1
- (b) shows the X-ray diffraction pattern of the saponite of Preparation Example 2
- (c) shows the X-ray diffraction pattern of the saponite of Preparation Example 3.
- Table 1 shows the molar ratio and molar ratio of fluorine atoms and hydroxyl groups of the saponites of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- the fluorine content is the content of fluorine atoms obtained by a fluorescent X-ray measurement apparatus (RIX1000, manufactured by Rigaku). From Table 1, it turns out that the saponite of the preparation example 1 and the preparation example 2 contains 0.86 mass% and 0.53 mass% of fluorine atoms, respectively.
- the molar ratio in the chemical formula of a fluorine atom and a hydroxyl group was obtained by calculating the saponite chemical formula of the adjustment example by the method described in “Clay Handbook 3rd Edition” from the result of fluorescent X-ray analysis.
- Table 1 shows the molar ratio when the total molar ratio of fluorine atoms and hydroxyl groups is 100%.
- FIG. 2 shows a nitrogen adsorption / desorption isotherm under liquid nitrogen temperature conditions.
- (a) shows the isotherm of the saponite of Preparation Example 1
- (b) shows the isotherm of the saponite of Preparation Example 2
- (c) shows the isotherm of the saponite of Preparation Example 3.
- the isotherm of each sample has a shape corresponding to type IV in the IUPAC classification, indicating that it is a typical layered compound.
- the H2 hysteresis loop seen in the relative pressure range of 0.40 to 1.00 indicates the presence of mesopores with a diameter of 2-50 nm. These mesopores are thought to originate from the saponite card house structure. This card house structure is formed by irregular stacking of end surfaces and side surfaces of flat nanoparticles.
- Table 2 shows the analysis results of the cation exchange capacity, specific surface area and surface charge density of the saponites of Preparation Example 1, Preparation Example 2 and Preparation Example 3.
- the cation exchange capacity was measured based on the Schollenberger method using ammonium acetate.
- the nitrogen adsorption isotherm was obtained using a nitrogen adsorption amount measuring apparatus (manufactured by Quantachrome Instruments, AUTOSORB-1). The moisture of each sample was removed by degassing at 200 ° C. for 3 hours when measuring the volume of nitrogen adsorbed (nitrogen adsorption amount).
- the specific surface area is calculated using Brunar-Emmett-Teller (BET) using the data of the volume of nitrogen adsorbed in the range of relative pressure in the nitrogen adsorption isotherm of 0.050 to 0.30 in FIG. Determined by law.
- BET Brunar-Emmett-Teller
- the surface charge density was defined as the cation exchange capacity relative to the specific surface area.
- Example 1 Evaluation test example 1-1 of crystallization promotion using lysozyme 1-1
- the saponite of Preparation Example 1 in the form of a thin film was applied to the primary screening of hen egg white lysozyme. Crystallization was performed by a sitting drop vapor diffusion method using a lysozyme solution having a predetermined concentration.
- a commercially available screening kit Crystal Screen I (manufactured by Hampton Research), was used. This kit contains 50 kinds of precipitant solutions shown in Table 3.
- a protein solution chicken egg white lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in 0.020 mol / L sodium phosphate buffer (pH 7.0) containing 0.150 mol / L sodium chloride, and 8 mg / mL.
- a lysozyme solution was prepared.
- the saponite of Preparation Example 1 was added to pure water to prepare a 0.2% (w / v) saponite dispersion. 5 ⁇ L of the dispersion was dropped onto a microbridge (Hampton Research), degassed at room temperature, and dried to form a saponite thin film on the microbridge.
- Test Example 1-2 The test was performed in the same manner as in Test Example 1-1 except that the saponite of Preparation Example 2 was used in place of the saponite of Preparation Example 1 in Test Example 1-1.
- Comparative Test Example 1-1 The test was performed in the same manner as in Test Example 1-1 except that the saponite of Preparation Example 3 was used instead of the saponite of Preparation Example 1 in Test Example 1-1.
- Example 1-1 Comparative Test Example 1-2 Except that in Example 1-1, pure water was dropped on the microbridge instead of the 0.2% (w / v) saponite dispersion, and a microbridge not coated with saponite was used. Tested in the same way.
- Table 4 shows the results of Test Example 1-1, Test Example 1-2, Comparative Test Example 1-1, and Comparative Test Example 1-2.
- the numbers shown in Table 4 are the numbers of the crystallized solutions among the 50 kinds of precipitant solutions used.
- the success rate of crystallization indicates the ratio of the crystallized solution among the 50 kinds of precipitant solutions used.
- Table 4 shows that Test Example 1-1 forms crystals with three precipitant solutions (No. 17, No. 20, and No. 40) that are not crystallized in Comparative Test Example 1-2.
- Test Example 1-2 forms crystals with the four precipitant solutions (No. 15, No. 17, No. 21, and No. 29) that are not crystallized in Comparative Test Example 1-2. From this, by using saponite having a fluorine atom and a hydroxyl group for the primary screening, it is possible to find a crystallization initiation condition that is not found in the absence of saponite.
- Test Example 1-1 forms crystals with two precipitant solutions (No. 20 and No. 40) that are not crystallized in Comparative Test Example 1-1.
- Test Example 1-2 forms crystals with three precipitant solutions (No. 15, No. 21, and No. 29) that are not crystallized in Comparative Test Example 1-1.
- a saponite having a fluorine atom and a hydroxyl group for primary screening, it is possible to find a crystallization initiation condition that is not found in the presence of a saponite that does not contain a fluorine atom.
- Test Example 1-1 and Test Example 1-2 do not form crystals with the two precipitant solutions (number 30 and number 34) crystallized in Comparative Test Example 1-2. This is considered to be because lysozyme was adsorbed with the addition of saponite, and the supersaturation degree in the drop was significantly reduced.
- Example 2 Test Example 2-1 for Influence of Saponite on Crystal Growth of Lysozyme
- the dispersion of saponite of Preparation Example 1 was applied for the purpose of obtaining crystals for X-ray diffraction measurement of chicken egg white lysozyme.
- the dispersion contains a cryoprotectant. Crystallization was performed by a hanging drop vapor diffusion method using a lysozyme solution and a dispersion having a predetermined concentration.
- a precipitant solution containing cryoprotectant As a precipitant solution containing cryoprotectant, a 0.2 mol / L sodium acetate buffer solution (pH 4.7) containing 1.2 mol / L sodium chloride and 30% (w / v) glycerol was prepared.
- a protein solution chicken egg white lysozyme (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 0.020 mol / L sodium acetate buffer (pH 4.7) to prepare a 55 mg / mL lysozyme solution.
- a dispersion was prepared by dispersing the saponite of Preparation Example 1 at a concentration of 0.2% (w / v).
- X-ray diffraction data of lysozyme crystals were collected at the beam line BL6A station of the Institute for Materials Structure Science, High Energy Accelerator Research Organization, and the Synchrotron Radiation Science Research Facility (Photon Factory). From the drop containing saponite, a crystal with adhering saponite was scooped using a mount loop for crystal freezing (Cryoloop, manufactured by Hampton Research). The crystal was cooled in a nitrogen stream at ⁇ 173 ° C. and irradiated with synchrotron radiation X-ray having a wavelength of 0.97800 mm. The X-ray diffraction image acquired by the CD camera was analyzed using DPS / MOSFLM which is program package software.
- Test Example 2-2 The test was performed in the same manner as in Test Example 5-1, except that the saponite of Preparation Example 2 was used instead of the saponite of Preparation Example 1.
- Comparative Test Example 2-1 The test was performed in the same manner as in Test Example 2-1, except that the saponite of Preparation Example 3 was used instead of the saponite of Preparation Example 1.
- Comparative Test Example 2-2 Instead of using a dispersion in which the saponite of Preparation Example 1 was dispersed at a concentration of 0.2% (w / v) in Test Example 2-1, a precipitant solution not added with saponite was used. The test was performed in the same manner as in Test Example 2-1.
- Table 5 shows the results of Test Example 2-1, Test Example 2-2, Comparative Test Example 2-1, and Comparative Test Example 2-2.
- both the test example and the comparative test example have the same space group and unit cell constant of the formed crystal. This indicates that saponite does not affect the space group and unit cell constants of lysozyme crystals. It can be seen that the mosaicity range of Test Example 2-2 is smaller than that of Comparative Test Example 2-2. Furthermore, it can be seen that the integrity of Test Example 2-2 is almost the same as that of Comparative Test Example 2-2. These results indicate that the saponite of Test Example 2-2 does not adversely affect the crystal growth of lysozyme and forms high quality crystals. From the above, it can be seen that protein crystal X-ray diffraction data can be obtained even when the crystallization condition search agent is added in the second screening step in the search for crystallization conditions.
- Example 3 Influence test example 3-1 of crystallization condition search agent in protein crystallization from various protein solutions (dispersions)
- the saponite dispersion of Preparation Example 1 was applied as a search example for the crystallization conditions of each of the 11 types of proteins. Crystallization was performed by the hanging drop vapor diffusion method using each protein solution and dispersion at a predetermined concentration.
- Bovine liver catalase, Tritillachuum albumin proteinase K and bovine pancreatic trypsin were purchased from Wako Pure Chemical Industries, Ltd.
- Nata bean-derived concanavalin A, aprotinin, Thamatococcus daniellii-derived thaumatin and human serum albumin were purchased from Sigma-Aldrich Japan.
- Streptomyces rubiginosus-derived glucose isomerase and Trichoderma longibrachiatum-derived xylanase were purchased from Hampton Research.
- Egg white-derived avidin was purchased from Nacalai Tesque. The above protein was crystallized without purification.
- Thermus caldophilus GK24-derived L-lactate dehydrogenase is available from the prior art literature (Koide S. et al., “Crystallization of Allosteric L-Lactate Dehydrogenase from Thermus caldophilus and Preliminary Crystallographic Data 1991, The Jp. It was expressed and purified by E. coli expression system according to 6-7.).
- Tables 6A, 6B and 6C The crystallization conditions from each protein solution are shown in Tables 6A, 6B and 6C.
- Table 6A shows the composition of each protein solution used for crystallization of each protein.
- Table 6B shows the composition of each precipitant solution used for crystallization of each protein.
- Table 6C shows the composition of each reservoir solution used for crystallization of each protein.
- each protein solution described in Table 6A, each precipitant solution described in Table 6B, and each reservoir solution described in Table 6C were prepared.
- a dispersion liquid in which the saponite of Preparation Example 1 was dispersed at a concentration of 0.2% (w / v) was prepared.
- the formed crystals were evaluated by the crystal formation time.
- the crystal formation time was defined as the elapsed time (days) until the first crystal appeared under a polarizing microscope (40 times magnification), that is, the crystal of about 10 ⁇ m to 20 ⁇ m appeared.
- Test Example 3-2 The test was performed in the same manner as in Test Example 3-1, except that the saponite of Preparation Example 2 was used instead of the saponite of Preparation Example 1.
- Comparative Test Example 3-1 The test was performed in the same manner as in Test Example 3-1, except that the saponite of Preparation Example 3 was used in place of the saponite of Preparation Example 1 in Test Example 3-1.
- Comparative Test Example 3-2 Instead of using a dispersion in which the saponite of Preparation Example 1 was dispersed at a concentration of 0.2% (w / v) in Test Example 3-1, a precipitant solution not added with saponite was used. The test was performed in the same manner as in Test Example 3-1.
- Table 7 shows the crystal formation times from the protein solutions of Test Example 3-1, Test Example 3-2, Comparative Test Example 3-1, and Comparative Test Example 3-2.
- the numerical values described in Table 9 indicate the number of days (unit: days) when the first crystal was observed.
- the N.I. C. Indicates that crystals do not form after standing for 2 months.
- Comparative Test Example 3-2 does not form crystals, whereas Comparative Test Example 3-1 forms crystals. It can be seen that Comparative Test Example 3-1 accelerates the crystal formation time compared to Comparative Test Example 3-2 in the crystallization of catalase and thaumatin.
- Test Example 3-2 accelerates the crystal formation time compared to Test Example 3-1. This indicates that the molar ratio between the fluorine atom and the hydroxyl group of the saponite of Preparation Example 2 is suitable for promoting the crystallization of thaumatin.
- this crystallization condition search agent greatly improves the possibility of crystallization even under solution conditions that could not be crystallized under existing crystallization conditions including a metastable region. .
- the crystallization condition search agent accelerates the crystallization speed, but it does not succeed in crystallization only by promoting the speed, but effectively gives the protein a place to form crystal nuclei and allows crystallization. The effect is given.
- the method for searching for crystallization conditions of the present invention it can be easily predicted that the crystallization conditions can be determined only by studying the conditions with relatively little load.
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Abstract
Description
また、本発明は、上記のタンパク質結晶化条件探索剤とタンパク質が溶解している溶液とを混合する工程を含む、タンパク質結晶化条件探索方法を提供する。
また、本発明は、前記タンパク質結晶化条件探索剤とタンパク質が溶解している溶液とを混合し、結晶化タンパク質を回収するタンパク質結晶化条件探索剤の使用を提供する。
本発明においてタンパク質の結晶化条件の探索とは、対象となるタンパク質の結晶化条件を探索するために物理条件及び化学条件を設定し、結晶形成の有無、結晶性沈殿物形成の有無、非結晶性沈殿物形成の有無、結晶化成功率、形成した結晶の形成時間、形状、大きさ、結晶核密度(単位面積あたりの結晶の個数)又はX線回折データを判断基準として、対象となるタンパク質の結晶化に適する物理条件及び化学条件を決定することをいう。
また、本発明のタンパク質結晶化条件探索方法によれば、比較的負荷の少ない工程でタンパク質の結晶化に適した条件を効率的且つ実用的に設定できる。
サポナイトは、結晶化条件探索剤にメソ細孔に起因する核形成促進特性を付与する。サポナイトの使用は、結晶化条件探索剤に、単一品でより多様なタンパク質の結晶核形成を促進することを可能とし、且つ改善された均一性及び操作性を付与することを可能とする。
また、フッ素原子を同形置換させることで層状ケイ酸塩の永久負電荷が同形置換していないものに比べ増加し、層状ケイ酸塩の粒子間に強い静電的反発力を作用させる。従って、結晶化条件探索剤は、高濃度の沈殿剤を含む試薬と接触しても凝集しにくく、改善された均一性を有する。
塩としては、例えば、硫酸アンモニウム、硫酸マグネシウム、硫酸ナトリウム、塩化リチウム、塩化ナトリウム、塩化カリウム、塩化マグネシウム、塩化カルシウム、硝酸ナトリウム、フッ化ナトリウム、酒石酸ナトリウム、ギ酸ナトリウム、リン酸アンモニウム、硫酸リチウム、塩化コバルト、硫酸亜鉛及びマロン酸ナトリウムが挙げられる。
水溶性高分子化合物としては、例えば、分子量400~20000のポリエチレングリコール、ポリエチレングリコールモノアルキルエーテル及びポリエチレンイミンが挙げられる。
有機溶媒としては、例えば、エタノール、メタノール、アセトン、2-メチル-2,4-ペンタンジオール、イソプロパノール、ブタノール、ジオキサン、ジメチルスルホキシド及びジメチルホルムアミドが挙げられる。
従って、前記分散媒の使用は、手動による効率的な結晶化条件探索の実施を可能とするだけでなく、攪拌装置を具備しない公知の結晶化自動化装置との組み合わせによる効率的な結晶化条件の探索を可能とする。好適な自動化装置は、特開2010-151821号公報、特開2006-281139号公報、特開2003-14596号公報、特開2004-194647号公報及び特表2003-502652号公報に開示されている。これらの好適な自動化装置は、参照によって本明細書に組み込まれる。
また、本発明の結晶化条件探索剤は、タンパク質結晶形成制御剤又はタンパク質結晶形成促進剤として使用してもよい。さらに、本発明の結晶化条件探索剤は、タンパク質結晶化を用いた工業的なタンパク質精製に適用してもよいし、タンパク質以外の他の生体高分子の結晶化に適用してもよい。これらの用途には、本発明の結晶化条件探索剤を造核剤や結晶核形成剤として作用させる場合も包含される。
Na型サポナイト理論化学式Na0.33(Mg3)(Al0.33Si3.67)O10(OH)2に基づき、水ガラス(北越化学工業株式会社製)、硫酸マグネシウム(関東化学株式会社製)及び硫酸アルミニウム(関東化学株式会社製)を、モル比率がSi:Mg:Al=3.67:3:0.33となるように反応させ、水和ゲル1.5Lを調製した。この水和ゲルにフッ化ナトリウム(関東化学株式会社製)を溶解させた水溶液0.5Lを加え、2Lの反応ゲルを調製した。反応ゲルを3Lオートクレーブ容器に入れ、250℃にて4時間水熱合成を行った。水熱合成後の生成物(1L)を濾紙で吸引濾過して、水分を除去した。得られたケーキに水2Lを加えて攪拌することにより再分散し、これを再濾過した。再分散及び再濾過操作を3回繰り返すことで未反応物を洗浄除去した。得られたケーキを乾燥することによって、フッ素原子と水酸基とを有する合成サポナイト(以下、調製例1のサポナイトと呼ぶことがある)を得た。
調製例1においてフッ化ナトリウムの配合量を変えたこと以外は調製例1と同じ方法で、フッ素原子と水酸基とを有する合成サポナイト(以下、調製例2のサポナイトと呼ぶことがある)を得た。
調製例1においてフッ化ナトリウムの代わりに水酸化ナトリウム(関東化学株式会社製)を配合したこと以外は調製例1と同じ方法で、水酸基を有する合成サポナイト(以下、調製例3のサポナイトと呼ぶことがある)を得た。
試験例1-1
鶏卵白リゾチームの第1次スクリーニングに薄膜状の調製例1のサポナイトを適用した。所定の濃度のリゾチーム溶液を用い、シッティングドロップ蒸気拡散法により結晶化を行った。
タンパク質溶液として、鶏卵白リゾチーム(和光純薬工業(株)製)を、0.150mol/L塩化ナトリウムを含む0.020mol/Lリン酸ナトリウム緩衝液(pH7.0)に溶解し、8mg/mLリゾチーム溶液を調製した。
試験例1-1において調製例1のサポナイトの代わりに調製例2のサポナイトを使用したこと以外は、試験例1-1と同じ方法で試験した。
試験例1-1において調製例1のサポナイトの代わりに調製例3のサポナイトを使用したこと以外は、試験例1-1と同じ方法で試験した。
試験例1-1においてマイクロブリッジ上に0.2%(w/v)サポナイト分散液の代わりに純水を滴下し、サポナイトを塗布しないマイクロブリッジを使用したこと以外は、試験例1-1と同じ方法で試験した。
この現象は、フッ素原子と水酸基とを有するサポナイトが、結晶核として作用するリゾチームの凝集体を効率的に形成する場として機能したことに起因すると考えられる。
試験例2-1
鶏卵白リゾチームのX線回折測定用の結晶を得ることを目的に調製例1のサポナイトの分散液を適用した。分散液は、クライオプロテクタントを含む。所定の濃度のリゾチーム溶液及び分散液を用い、ハンギングドロップ蒸気拡散法により結晶化を行った。
タンパク質溶液として、鶏卵白リゾチーム(和光純薬工業株)製)を0.020mol/L酢酸ナトリウム緩衝液(pH4.7)に溶解し、55mg/mLリゾチーム溶液を調製した。
クライオプロテクタントを含む沈殿剤溶液を用い、調製例1のサポナイトを0.2%(w/v)の濃度で分散させた分散液を調製した。
試験例2-1において調製例1のサポナイトの代わりに調製例2のサポナイトを使用したこと以外は、試験例5-1と同じ方法で試験した。
調製例1のサポナイトの代わりに調製例3のサポナイトを使用したこと以外は、試験例2-1と同じ方法で試験した。
試験例2-1において調製例1のサポナイトを0.2%(w/v)の濃度で分散させた分散液を使用する代わりに、サポナイトを添加していない沈殿剤溶液を使用したこと以外は、試験例2-1と同じ方法で試験した。
試験例3-1
11種類のタンパク質におけるそれぞれのタンパク質の結晶化条件の探索例として調製例1のサポナイトの分散液を適用した。所定濃度のそれぞれのタンパク質溶液及び分散液を用い、ハンギングドロップ蒸気拡散法により結晶化を行った。
沈殿剤溶液を用い、調製例1のサポナイトを0.2%(w/v)の濃度で分散させた分散液を調製した。
試験例3-1において調製例1のサポナイトの代わりに調製例2のサポナイトを使用したこと以外は、試験例3-1と同じ方法で試験した。
試験例3-1において調製例1のサポナイトの代わりに調製例3のサポナイトを使用したこと以外は、試験例3-1と同じ方法で試験した。
試験例3-1において調製例1のサポナイトを0.2%(w/v)の濃度で分散させた分散液を使用する代わりに、サポナイトを添加していない沈殿剤溶液を使用したこと以外は、試験例3-1と同じ方法で試験した。
この現象は、フッ素原子と水酸基とを有するサポナイトが、結晶核として作用するタンパク質の凝集体を効率的に形成する場として機能したことに起因すると考えられる。
この現象は、水酸基を有するサポナイトが、結晶核として作用するタンパク質の凝集体を形成する場として機能したことに起因すると考えられる。
Claims (13)
- フッ素原子と水酸基とを有する水膨潤性層状ケイ酸塩を含み、前記フッ素原子は水酸基との同形置換により前記ケイ酸塩に共有結合していることを特徴とする、タンパク質結晶化条件探索剤。
- 前記フッ素原子と水酸基とのモル比率がそれぞれ3%~15%、85%~97%である、請求項1に記載のタンパク質結晶化条件探索剤。
- 前記フッ素原子と水酸基とのモル比率がそれぞれ4%~12%、88%~96%である、請求項2に記載のタンパク質結晶化条件探索剤。
- 前記ケイ酸塩がスメクタイトである、請求項1~3のいずれか一項に記載のタンパク質結晶化条件探索剤。
- 前記ケイ酸塩がサポナイトである、請求項4に記載のタンパク質結晶化条件探索剤。
- 分散液剤である、請求項1~5のいずれか一項に記載のタンパク質結晶化条件探索剤。
- 固形剤である、請求項1~5のいずれか一項に記載のタンパク質結晶化条件探索剤。
- 前記固形剤が支持体上に塗布した薄膜である、請求項7に記載のタンパク質結晶化条件探索剤。
- 前記支持体がカバーガラス、スライドガラス、プレート、ブリッジ又はロッドである請求項8に記載のタンパク質結晶化条件探索剤。
- ナノグラム単位からマイクログラム単位までの量のタンパク質の結晶化条件を探索するための結晶化条件探索剤である、請求項1~9のいずれか一項に記載のタンパク質結晶化条件探索剤。
- 遺伝子組換え技術により合成した発現タンパク質の結晶化条件を探索するための結晶化条件探索剤である、請求項1~10のいずれか一項に記載のタンパク質結晶化条件探索剤。
- 請求項1~11のいずれか一項に記載のタンパク質結晶化条件探索剤とタンパク質が溶解している溶液とを混合する工程を含む、タンパク質結晶化条件探索方法。
- 請求項1~11のいずれか一項に記載のタンパク質結晶化条件探索剤とタンパク質が溶解している溶液とを混合し、結晶化タンパク質を回収する請求項1~11のいずれか一項に記載のタンパク質結晶化条件探索剤の使用。
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EP2692911B1 (en) | 2017-02-22 |
US20130344523A1 (en) | 2013-12-26 |
JP5972866B2 (ja) | 2016-08-17 |
US10175183B2 (en) | 2019-01-08 |
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EP2692911A1 (en) | 2014-02-05 |
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