WO2009091053A1 - Procédé de production de cristaux, procédé de production de cristaux congelés, cristaux, méthode d'analyse structurelle des cristaux, méthode de contrôle de la cristallisation et appareil de contrôle de la cristallisation - Google Patents

Procédé de production de cristaux, procédé de production de cristaux congelés, cristaux, méthode d'analyse structurelle des cristaux, méthode de contrôle de la cristallisation et appareil de contrôle de la cristallisation Download PDF

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WO2009091053A1
WO2009091053A1 PCT/JP2009/050592 JP2009050592W WO2009091053A1 WO 2009091053 A1 WO2009091053 A1 WO 2009091053A1 JP 2009050592 W JP2009050592 W JP 2009050592W WO 2009091053 A1 WO2009091053 A1 WO 2009091053A1
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Prior art keywords
crystal
gel
crystallization
biological material
solution
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PCT/JP2009/050592
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English (en)
Japanese (ja)
Inventor
Shigeru Sugiyama
Hiroaki Adachi
Hiroyoshi Matsumura
Kazufumi Takano
Satoshi Murakami
Tsuyoshi Inoue
Yusuke Mori
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Sosho, Inc.
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Priority to JP2009550072A priority Critical patent/JP5351771B2/ja
Publication of WO2009091053A1 publication Critical patent/WO2009091053A1/fr

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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
    • 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
    • C30B5/00—Single-crystal growth from gels

Definitions

  • the present invention relates to a crystal production method, a frozen crystal production method, a crystal, a crystal structure analysis method, a crystallization screening method, and a crystallization screening apparatus.
  • a method for producing a crystal that precipitates (crystallizes) a crystal from a solution of protein, nucleic acid or the like requires a very advanced technique and is difficult. Furthermore, crystals of proteins, nucleic acids, and the like are very brittle and are likely to be distorted inside the crystals, so that there is a problem that they are easily destroyed unless handled carefully.
  • an object of the present invention is to provide a method for producing a crystal, in which a crystal can be easily produced and the produced crystal is easy to handle.
  • the inventors of the present invention have made extensive studies to solve the above problems. As a result, it has been found that in the method for producing crystals of biological materials such as proteins and nucleic acids, crystals are precipitated from the gel instead of the biological material solution. As described above, in the method for producing crystals of biological substances such as proteins and nucleic acids, conventionally, crystals are generally precipitated from a solution. The inventors of the present invention have made the first attempt to deposit crystals directly from the gel in these biological substances.
  • the crystal production method of the present invention is: A method for producing a crystal of biological material, The method includes a crystallization step of crystallizing the biological material in a gel.
  • the present invention provides: A method for producing a frozen crystal comprising a freezing step of freezing a crystal of biological material,
  • the method for producing a crystal according to the present invention further includes a coated crystal production process for producing the coated crystal coated with the gel, In the freezing step, a frozen crystal production method is provided, wherein the coated crystal is frozen.
  • the present invention provides: A method for producing a crystal of a biological material, comprising a crystal growth step of further growing a biological material crystal that is a seed crystal in a solution of the biological material, In the crystal growth step, there is provided a crystal manufacturing method, wherein the seed crystal is a coated crystal coated with the gel manufactured by the crystal manufacturing method of the present invention.
  • this crystal production method may be referred to as “growth crystal production method of the present invention” or simply “growth crystal production method”.
  • the present invention provides a crystal produced by the crystal production method of the present invention, the frozen crystal production method of the present invention, or the growth crystal production method of the present invention.
  • the present invention provides: A method for structural analysis of a crystal of a biological material, There is provided a structural analysis method comprising a structural analysis step of structural analysis of the coated crystal manufactured by the crystal manufacturing method of the present invention or the frozen crystal manufactured by the frozen crystal manufacturing method of the present invention.
  • the present invention provides: A crystallization screening method for screening crystallization conditions of a biological material, A crystal production process for producing crystals of the biological material; Including a screening step of screening crystallization conditions in the crystal manufacturing step, In the crystal production step, a crystallization screening method is provided, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
  • the present invention provides: A crystallization screening apparatus for screening crystallization conditions of a biological material, Crystal manufacturing means for manufacturing crystals of the biological material; Screening means for screening the crystallization conditions of the biological material,
  • the crystallization screening apparatus is characterized in that the crystal production means includes a crystallization means for precipitating crystals of the biological material in a gel.
  • the crystal manufacturing method of the present invention it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution.
  • the generated crystal is fixed by a gel. More specifically, for example, since the generated crystal is fixed to the gel, it is possible to prevent damage due to collision with the wall of the container, polycrystallization due to collision between the generated crystals, and the like. It is thought that it is easy to grow into a few single crystals.
  • these considerations are merely examples of possible mechanisms and do not limit the present invention in any way.
  • the crystal is not easily damaged by freezing. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
  • the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics.
  • the manufacturing method of the crystal of the present invention is not particularly limited, and may be a crystal manufactured by any other manufacturing method as long as it has similar characteristics.
  • the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
  • FIG. 1A is a photograph of a crystal obtained according to an embodiment of the present invention.
  • FIG. 1B is a photograph of the crystal of FIG. 1A taken at a different angle.
  • FIG. 2A is a diagram showing an X-ray diffraction image of the crystal of FIG.
  • FIG. 2B is an enlarged view of a part of FIG. 2A.
  • FIG. 3A is a diagram showing an X-ray diffraction image of a crystal obtained by another example of the present invention.
  • FIG. 3B is an enlarged view of a part of FIG. 3A.
  • FIG. 4 is a diagram showing an X-ray diffraction image when the crystal of FIG. 3 is measured under different conditions.
  • FIG. 1A is a photograph of a crystal obtained according to an embodiment of the present invention.
  • FIG. 1B is a photograph of the crystal of FIG. 1A taken at a different angle.
  • FIG. 2A is a diagram showing an X-ray diffraction image of
  • FIG. 5A is a diagram showing an X-ray diffraction image when the crystal of FIG. 4 is measured under still another condition.
  • FIG. 5B is an enlarged view of a part of FIG. 5A.
  • FIG. 6A is a photograph showing a state immediately before the crystals of FIGS. 3 to 5 are immersed in the cryoprotectant.
  • FIG. 6B is a photograph showing a state after 5 seconds from immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6C is a photograph showing a state one minute after immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6D is a photograph showing a state after 5 minutes from immersing the crystal of FIG. 6A in the cryoprotectant.
  • FIG. 6A is a diagram showing an X-ray diffraction image when the crystal of FIG. 4 is measured under still another condition.
  • FIG. 5B is an enlarged view of a part of FIG. 5A.
  • FIG. 7A is a photograph of a crystal obtained by yet another example of the present invention.
  • FIG. 7B is a diagram showing an X-ray diffraction image of the crystal of FIG. 7A.
  • FIG. 7C is an enlarged view of a part of FIG. 7B.
  • FIG. 8A is a photograph of a crystal obtained according to yet another example of the present invention.
  • FIG. 8B is a diagram showing an X-ray diffraction image of the crystal of FIG. 8A.
  • FIG. 8C is an enlarged view of a part of FIG. 8B.
  • FIG. 9A is a diagram showing a state immediately before the crystal of the comparative example is immersed in the cryoprotectant.
  • FIG. 9B is a diagram showing a state immediately after the crystal of FIG.
  • FIG. 10A is a diagram showing a state immediately before dipping a crystal of another comparative example in an antifreezing agent.
  • FIG. 10B is a diagram showing a state immediately after the crystal of FIG. 10A is immersed in an anti-freezing agent.
  • FIG. 11 is a diagram showing photographs and data of crystals of still another example and comparative example of the present invention.
  • FIG. 12 is a diagram showing an X-ray diffraction image of a crystal according to still another example of the present invention.
  • FIG. 13 is a schematic diagram illustrating an example of a result of crystal structure analysis of a biomolecule.
  • FIG. 14 is a schematic diagram showing still another example of the result of analyzing the crystal structure of a biomolecule.
  • FIG. 15 is a graph showing the crystallization screening result in one example of the present invention.
  • FIG. 16 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 17 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 18 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 19 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 20 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 21 is a graph showing a crystallization screening result in still another example of the present invention.
  • FIG. 22 is a photograph and a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 23 is a photograph and a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 24 is a graph showing crystallization screening results in still another example of the present invention.
  • FIG. 25 is a photograph showing a crystallization screening result in still another example of the present invention.
  • FIG. 26 is a photograph showing an E. coli-derived protein crystal in still another example of the present invention.
  • FIG. 27 is a photograph showing a DNA crystal in still another example of the present invention.
  • FIG. 28 is a graph showing a crystallization screening result and a photograph of a crystal in still another example of the present invention.
  • FIG. 29 is a photograph showing an example in which the coated gel is removed by lowering the temperature.
  • FIG. 30 is a photograph showing the drying tolerance evaluation of the crystals of the example and the crystals of the comparative example.
  • FIG. 31A is a photograph showing the drying tolerance evaluation of crystals of still another example and comparative example.
  • FIG. 31B is a photograph showing the drying tolerance evaluation of the crystals of the same example and the comparative example after the elapse of the evaluation time of FIG. 31A.
  • FIG. 32 is a photograph showing crystals before and after gelation of the surrounding solution in crystals produced in yet another example.
  • FIG. 33 is a diagram showing an X-ray diffraction image of the crystal of the example.
  • FIG. 34 is a diagram showing an X-ray diffraction image of a crystal of still another example.
  • FIG. 35 is a photograph showing processing of a gel-coated crystal with a femtosecond laser.
  • FIG. 36 is a photograph showing removal of a gel-coated crystal that has been subjected to femtosecond laser processing.
  • FIG. 37 shows a photograph of the crystal of FIG. 36 and X-ray crystal structure analysis data.
  • FIG. 38 is a photograph showing one example of a method for producing a grown crystal.
  • FIG. 39 is a photograph showing another example of the method for producing a grown crystal.
  • FIG. 40 is a diagram schematically illustrating an example of a crystallization screening method using a concentration gradient by centrifugation.
  • FIG. 41 is a photograph showing a crystallization screening result by the method of FIG. FIG.
  • FIG. 42 is a schematic view illustrating the crystal mounting operation in the crystal structure analysis.
  • FIG. 43 is a schematic view illustrating an apparatus used for a crystal mounting operation in crystal structure analysis.
  • FIG. 44 is a schematic diagram showing an example of the crystallization screening apparatus of the present invention.
  • FIG. 45 is a schematic diagram showing still another example of the crystallization screening apparatus (crystallization kit) of the present invention.
  • FIG. 46 is a schematic diagram showing an example of crystallization screening according to the present invention.
  • FIG. 47 is a graph showing an example of a gel strength measurement result of an agarose gel.
  • FIG. 48 is a graph obtained by extracting a part of the data of the graph of FIG.
  • the crystal production method of the present invention is a method for producing a crystal of a biological material, which includes a crystallization step of crystallizing the biological material, wherein the biological material is crystallized in a gel in the crystallization step. It is characterized by making it.
  • the “biological substance” may be a biological substance, but may be a synthetic substance having the same structure as it, or a derivative or an artificial substance having a structure similar to the biological substance.
  • the biological substance when it is a biopolymer compound, it may be a polymer compound derived from a living body, a synthetic polymer compound having the same structure as it, or a derivative or artificial material having a structure similar to that of a bio-derived polymer compound. It may be a polymer compound.
  • the biological material is a protein, it may be a biologically derived (naturally-derived) protein, a synthetic protein, a naturally occurring natural protein, or a non-naturally occurring artificial protein.
  • the biological substance when it is a peptide, it may be a biologically derived (naturally-derived) peptide, a synthetic peptide, a naturally occurring peptide having a naturally occurring structure, or an artificial peptide having a non-naturally occurring structure.
  • the biological material when it is a nucleic acid, it may be a biologically derived (naturally-derived) nucleic acid, a synthetic nucleic acid, a naturally-occurring natural nucleic acid, or a non-naturally-occurring artificial nucleic acid.
  • the biological substance when it is a sugar chain, it may be a sugar chain derived from a living body (naturally derived), a synthetic sugar chain, a natural sugar chain having a naturally occurring structure, or an artificial sugar chain having a non-naturally occurring structure.
  • the biological material is not particularly limited, but for example, a biopolymer compound, protein, natural protein, artificial protein, peptide, natural peptide, artificial peptide, nucleic acid, natural nucleic acid, artificial nucleic acid, sugar chain, natural sugar chain, or artificial A sugar chain is preferred.
  • the natural nucleic acid is not particularly limited, and examples thereof include DNA and RNA.
  • the artificial nucleic acid is not particularly limited, and examples thereof include LNA and PNA.
  • the molecular weight of the biological material is not particularly limited.
  • the molecular weight of the “biopolymer compound” is, for example, 5000 or more, but is not limited thereto, and may be less than 5000.
  • the molecular weight is, for example, 1000 or more, but is not limited thereto, and may be less than 1000.
  • the crystal production method of the present invention further includes a solution preparation step of preparing a solution of the biological material prior to the crystallization step, and a gelation step of preparing the gel by gelling the solution.
  • the biological material solution further contains a gelling agent.
  • the gelling agent is not particularly limited, but is preferably at least one selected from the group consisting of polysaccharides, thickening polysaccharides, proteins, and gels at elevated temperature, agarose, agar, More preferably, it is at least one selected from the group consisting of carrageenan, gelatin, collagen, polyacrylamide, and gelled polyacrylamide gel at elevated temperature.
  • the gelation temperature is not particularly limited, but is, for example, 0 to 90 ° C., preferably 0 to 60 ° C., more preferably 0 to 35 ° C. from the viewpoint of ease of crystal production.
  • the gelling agent may be, for example, a gel that gels at a low temperature and forms a sol at a high temperature, or a gel that forms a sol at a low temperature and gels at a high temperature. Gels that sol at a low temperature and gel at a high temperature are referred to as “gels at elevated temperature”. Further, for example, a gel that returns to a sol again when the temperature of the gel obtained by cooling is increased again or when the temperature of the gel obtained by increasing temperature is cooled again is preferable. Such a gelling agent is called “thermo-reversible gel”.
  • the gelling agent may be, for example, a hydrogel or an organogel, but is preferably a hydrogel.
  • the hydrogel for example, it is more preferable to use a gelled hydrogel at elevated temperature.
  • the gelled hydrogel at elevated temperature has the property of solling at a low temperature and gelling at a high temperature as described above, contrary to a general gel that gels at a low temperature and sols at a high temperature.
  • the coated crystal coated with the gelled hydrogel at elevated temperature has advantages such as being particularly strong in drying and capable of easily removing the gel by cooling.
  • limit especially as a gelatinization type hydrogel at the time of temperature rising For example, a meviol gel is mentioned.
  • Mebiol gel is a trade name of a gelled hydrogel produced by Mebiol Co., Ltd. and has the following chemical structure, for example.
  • Meviol gel is a polyacrylamide gel having properties as a gelling hydrogel at elevated temperature and properties as a thermoreversible hydrogel.
  • crystal production method of the present invention can be performed, for example, as follows.
  • Solution preparation step First, the biological material is dissolved in a solvent to obtain a solution.
  • the said solvent is not restrict
  • Specific examples of the solvent include water, ethanol, methanol, acetonitrile, acetone, anisole, isopropanol, ethyl acetate, butyl acetate, chloroform, cyclohexane, diethylamine, dimethylacetamide, dimethylformamide, toluene, butanol, and butyl methyl ether.
  • the concentration of the biological substance is not particularly limited, but is, for example, 0.2 to 300 mg / mL, preferably 0.5 to 100 mg / mL, more preferably 1 to 50 mg / mL.
  • a gelling agent is added to the biological material solution to cause gelation, thereby preparing a gel containing the biological material.
  • the gelling agent may be added directly to the biological material solution, but it is preferable to prepare a gelling agent solution separately and then mix it with the biological material solution because it is easy to mix uniformly.
  • the solvent of the gelling agent solution is not particularly limited, but is the same as the biological material solution, for example.
  • the concentration of the gelling agent in the gelling agent solution is not particularly limited, but from the viewpoint of gel strength and the like described later, for example, 0.6 to 50% by mass, preferably The amount is 0.8 to 40% by mass, more preferably 1.0 to 30% by mass, still more preferably 1.0 to 25% by mass, and particularly preferably 1.0 to 20% by mass.
  • the mechanism of the correlation between the gelling agent concentration and the ease of crystallization of the biological substance is unknown, but for example, the gelling agent concentration can be obtained by utilizing the crystallization screening method of the present invention described later. Can be set as appropriate.
  • the method of gelling after adding a gelling agent to the biological material solution is not particularly limited.
  • the gelling agent solution may be prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the biological material solution, and then allowed to stand at a temperature equal to or lower than the gelation temperature. More specifically, for example, after the gelling agent solution and the biological material solution are mixed, they may be sealed in a capillary and gelled in the capillary. As a result, the gel is sealed in the capillary.
  • the gelling agent solution may be prepared at a low temperature, mixed with the biological material solution, and then gelled by increasing the temperature.
  • the gel strength after the gelation is, for example, 100 Pa or more, preferably 200 Pa or more, more preferably 300 Pa or more, from the viewpoint of easy protection of the biological material crystals deposited in the crystallization step described below from physical impact. Preferably it is 500 Pa or more, Most preferably, it is 1000 Pa or more.
  • the gel strength of the gelling agent is preferably as high as possible, and the upper limit is not particularly limited, but is, for example, 200,000 Pa or less.
  • the gel strength after gelation can be appropriately set by adjusting the gelling agent concentration.
  • the gel strength at the same gelling agent concentration varies depending on the type of the gelling agent. For example, as shown in the graph of FIG.
  • agarose III agarose SP (agarose sea plaque), and agarose 9A (all trade names of Takara Bio Inc.) are usually agarose after gelation at the same concentration.
  • the gel strength increases in the order of III> agarose SP> agarose 9A.
  • the graph of FIG. 48 shows the measurement results of only agarose 9A in FIG. 47 and 48, the horizontal axis represents the concentration (mass%) of each agarose, and the vertical axis represents the gel strength (g / cm 2 ). Note that 1 g / cm 2 corresponds to 98.0665 Pa. Further, when the gelling agent concentration is less than a certain critical concentration, the gel strength cannot be measured (0 on the graph) because the solution containing the gelling agent does not clearly gel.
  • the concentration of the gelling agent is not less than the critical concentration.
  • the critical concentration is about 0.6% by mass as shown in FIG. 47 and FIG.
  • the gel strength is a numerical value measured at a frequency of 1 Hz and a measurement temperature of 20 ° C. using a rheostress RS1 Rheometer (trade name of Rheometer manufactured by Eihiro Seiki Co., Ltd.) in a dynamic viscoelasticity measurement mode.
  • the measured values shown in FIGS. 47 and 48 were also measured by this method.
  • the gel strength is lower than the gel dissolution temperature and can be divided by 5 (example: : 15 ° C, 10 ° C, 5 ° C, 0 ° C, -5 ° C).
  • the gel strength is higher than the gel dissolution temperature and is divisible by 5 degrees Celsius.
  • the measured value at the lowest measurable temperature among the temperatures (example: 25 ° C, 30 ° C, 35 ° C) is used.
  • this measurement method is an example of the measurement method of the gel strength, and the present invention is not limited by the steps and conditions in this measurement method. Further, even if other rheometers are used, the same gel strength measurement value can be obtained except for errors if measurement is performed in the same measurement mode, measurement temperature and frequency.
  • Crystallization step Further, crystals of the biological material are precipitated from the gel.
  • This method is not particularly limited.
  • the gel may be left still and wait for crystals of the biological material to precipitate.
  • the gel may be allowed to stand in contact with or in close proximity to the precipitant solution. More specifically, for example, after the gel is sealed in a capillary, it may be immersed in the precipitant solution.
  • the precipitating agent is not particularly limited, and for example, the same precipitating agent as used in a known crystal production method may be used.
  • the precipitating agent examples include sodium chloride, calcium chloride, sodium acetate, ammonium acetate, ammonium phosphate, ammonium sulfate, potassium sodium tartrate, sodium citrate, PEG (polyethylene glycol), magnesium chloride, sodium cacodylate, HEPES (2 -[4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid), MPD (2-methyl-2,4-pentanediol), Tris-HCl (trishydroxymethylaminomethane hydrochloride) Is at least one.
  • the solvent for the precipitating agent solution is not particularly limited, and may be the same as the biological material solution, for example.
  • the concentration of the precipitating agent is not particularly limited, but is, for example, 0.0001 to 10M, preferably 0.0005 to 8M, and more preferably 0.0005 to 6M.
  • the said precipitating agent may contain the pH adjuster etc. suitably as needed.
  • the precipitating agent may also be referred to as “precipitating agent”.
  • the method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is described.
  • the crystallization method using the precipitant is not limited thereto.
  • the biological material solution is mixed with a precipitating agent together with a gelling agent, gelled to form a gel containing the precipitating agent, and left as it is after gelation to precipitate crystals.
  • the method may be used. Such a crystallization method is called “batch method”.
  • the solvent of the biological material solution, the concentration of the biological material, the concentration of the gelling agent, the concentration of the precipitating agent and the like are not particularly limited, but are the same as described above, for example.
  • Crystallization of biological materials such as proteins can be controlled by, for example, the mixing ratio of the biological material and the precipitating agent.
  • the mixing ratio in more detail, for example, it is possible to obtain a higher quality and larger single crystal.
  • a concentration gradient can be formed by diffusing a precipitating agent as described above in a gel having a network molecular structure (for example, agarose gel).
  • the diffusion rate of the precipitating agent is sufficiently slower than the crystal growth rate.
  • the production method of the present invention can also be applied to a crystallization screening method as a “combinatorial crystallization technique” capable of searching a mixture ratio of various combinations all at once.
  • a specific screening method using the concentration gradient formation and an apparatus used therefor are as described below, for example.
  • the crystal manufacturing method of the present invention can be carried out as described above, the present invention is not limited to this.
  • the crystal production method of the present invention may be performed, for example, by the following method (i) or (ii), or by other methods.
  • the following (i) is referred to as “counter diffusion method”, and the following (ii) is referred to as “liquid-solid method”.
  • the gelling agent solution is prepared at a temperature higher than the gelation temperature (for example, 20 to 45 ° C.), mixed with the solution of the biological material (protein etc.), and enclosed in a capillary. To gel. Thereafter, it is brought into contact with a precipitant solution as necessary.
  • a gel for example, solid agarose
  • a solution of the biological material protein, etc.
  • the precipitating agent may be used in the form of a solution or as a solid.
  • the biological material is crystallized in a gel.
  • This “crystallize in gel” may crystallize the biological material in the gel after the gelation step, for example, as described above.
  • the term “crystallize in gel” means, for example, that the biological material solution containing a gelling agent is not gelled and is in a sol state, the biological material in the gelling agent solution in the sol state. And crystallizing.
  • the mechanism is not clear, even if the solution is in a sol state, the inclusion of the gelling agent may lead to the effect of the present invention in that crystals are likely to precipitate and damage to the crystals is less likely to occur. .
  • the method for crystallizing the biological material in the gelling agent solution in the sol state is particularly useful when, for example, the gelling agent is a thermoreversible hydrogel.
  • the gelling agent is a thermoreversible hydrogel.
  • the biological material is crystallized, and then the solution is gelled, and the biological material crystal is further grown in the gel. It is particularly preferred that By stirring the sol-state solution, for example, the biological material crystals are more likely to grow and large crystals can be easily obtained.
  • the stirring speed is not particularly limited, but is, for example, 10 to 1000 rpm, preferably 30 to 200 rpm, more preferably 50 to 100 rpm.
  • the stirring time is not particularly limited, but is, for example, 5 minutes to 60 days, preferably 30 minutes to 30 days, and more preferably 1 hour to 20 days.
  • the temperature of the sol solution during the stirring is not particularly limited, but is, for example, 0 to 40 ° C., preferably 2 to 30 ° C., and particularly preferably 4 to 25 ° C.
  • the crystal produced by the crystal production method of the present invention is preferably, for example, a coated crystal coated with the gel.
  • the production conditions of the coated crystal are not particularly limited.
  • the crystal of the biological material is precipitated from the gel, so that the coated crystal covered with the gel is necessarily formed. Also good.
  • crystals of biological materials such as proteins are brittle and are easily altered by drying or the like. For this reason, biological material crystals are damaged by physical impact or drying unless they are operated carefully and quickly, for example, when they are used as samples for crystal structure analysis (mounting) and when they are used as seed crystals (seeding). In many cases, it will cause alteration.
  • the coated crystal in the present invention is improved in resistance to drying and physical impact because the crystal is coated with a gel, so that the crystal is hardly deteriorated or damaged. For this reason, for example, the mounting operation and the seeding operation are extremely easy to perform. Furthermore, the preservability of crystals is improved.
  • the gel covering the crystal is preferably removed in advance if there is a problem such as causing measurement noise in the crystal structure analysis described later.
  • the removal method is not particularly limited.
  • the gel is a thermoreversible hydrogel, as described above, the gel is made into a sol by cooling and can be easily removed.
  • the cooling temperature for making the sol is not particularly limited, but in the case of meviol gel, for example, it is 15 ° C. or lower.
  • the coated crystal can be processed by an appropriate method, and only the crystal not containing the gel can be taken out.
  • the processing method is not particularly limited, for example, there is processing using laser light.
  • the laser light is not particularly limited, but it is particularly preferable to use a femtosecond laser.
  • the femtosecond laser can be processed only in the vicinity of the condensing point, and thus has an advantage that it can be easily processed while the crystal manufacturing (growing) container is sealed, for example.
  • a crystal can be cut into a size and shape suitable for applications such as structural analysis.
  • the crystal having the appropriate size and shape can be manufactured by cutting the crystal into an appropriate size and shape by such a processing step.
  • the processing step it is possible to appropriately remove only the gel from the coated crystal and manufacture a processed crystal that leaves only the crystal portion. That is, the processed crystal may be a coated crystal coated with a gel or may not be coated with a gel.
  • a high-quality and large-sized crystal can be obtained with a higher probability than in the past, and according to the screening method (concentration gradient method) using the above-described concentration gradient formation, A wide range of search conditions (combinatorial search) is possible. With respect to these, for example, it is possible to obtain results superior to the vapor diffusion method that has been conventionally most commonly used for crystallization of proteins and the like.
  • the following advantages can be obtained by precipitating crystals of the biological material from the gel.
  • the crystal obtained in the solution freely moves in the solution when the crystal is mounted, it may be damaged before the X-ray measurement by indirect or direct contact with the loop or the like. For this reason, in order to perform measurement with high accuracy, a skilled skill in handling the loop is required.
  • the crystal production method of the present invention crystals of the biological substance (proteins, etc.) are precipitated from the gel.
  • the said biological material crystal will be in the state fixed with the gel. Therefore, the mounting operation is easy and the mounting operation is highly reproducible due to the fact that the biological material crystal is fixed by the gel and the movement is inhibited, and the biological material crystal is protected by the gel and is not easily damaged. According to this, for example, by automating the crystal mounting process, it is possible to achieve full automation of X-ray structural analysis of protein crystals and the like, which could not be achieved conventionally.
  • the obtained crystals are covered with the gel, so that the crystals can be frozen and a simple mounting operation can be performed. High accuracy data can be obtained because it can be frozen without degrading quality.
  • the frozen crystal manufacturing method of the present invention is a frozen crystal manufacturing method including a freezing step of freezing the crystal of biological material, as described above, and is coated with the gel by the crystal manufacturing method of the present invention.
  • the method further comprises a coated crystal manufacturing step for manufacturing the coated crystal, wherein the coated crystal is frozen in the freezing step.
  • the method for producing frozen crystals of the present invention is not particularly limited, but it is preferable that the method further includes an immersion step of immersing the coated crystals in an antifreezing agent prior to the freezing step.
  • a frozen state for example, 100K or less.
  • the damage to the crystal due to radiation for example, synchrotron radiation for obtaining high resolution data
  • the freezing instead of the encapsulation in the capillary suppresses damage to the crystal due to radiation.
  • the coated crystal and the frozen crystal are protected with a gel.
  • the gel is considered to suppress damage caused by X-rays or the like.
  • crystallization damage by freezing is further suppressed by further including the immersion process which immerses the said covering crystal
  • the cryoprotectant is not particularly limited, but is preferably an organic compound, such as glycerol (glycerin), MPD (2-Methyl-2,4-PentaneDiol), DMSO (dimethyl sulfoxide), It is at least one selected from the group consisting of PEG (polyethylene glycol) and lithium acetate. These may be used as they are or as an aqueous solution.
  • the amount (concentration) of the cryoprotectant added to the crystallization mother liquor is finely adjusted, and optimal conditions are screened. It was necessary.
  • the concentration of the cryoprotectant is not particularly limited. As described above, according to the present invention, the crystal damage caused by the cryoprotectant can be greatly suppressed. For example, 100% DMSO, PEG, or a high concentration aqueous solution thereof (for example, 50 to 60% by mass) can be used. Crystals of the biological material can also be directly immersed.
  • the type of anti-freezing agent itself may be, for example, the same as that conventionally used.
  • the cryoprotectant molecule may bind to the active site of the biological material and remain in the crystal.
  • This figure is a schematic diagram showing an example of a structural analysis result when glycerol is used as an anti-freezing agent and measurement is performed at a resolution of 0.94 mm in X-ray crystal structure analysis of glucose isomerase (GI).
  • GI glucose isomerase
  • 2GLK in the figure is a PDB ID (protein data bank registration number), which means glucose isomerase (GI).
  • the coated crystal of the present invention when used, for example, as shown in the schematic diagram of FIG. 14, the freezing structure is not bonded to the active site and free structure information is obtained, which is advantageous for drug design and the like. .
  • the crystal damage due to the cryoprotectant is significant, and therefore the types of antifreeze agents that are less likely to cause crystal damage have been limited. For this reason, it has been difficult to select an anti-freezing agent that hardly remains in the crystal from among the anti-freezing agents that hardly cause crystal damage.
  • the damage of the crystal caused by the cryoprotectant is suppressed, so that the range of the cryoprotectant that can be selected is wide, and the cryoprotectant that does not easily remain in the crystal is appropriately selected. You can also do it.
  • glucose isomerase although not necessarily clear, it is considered that lithium acetate is less likely to remain in the crystal than glycerol.
  • this explanation is merely an example, and does not limit the present invention at all. For example, it does not mean that glycerol is inappropriate as an antifreezing agent used in the present invention.
  • the growth crystal manufacturing method of the present invention includes a crystal growth step of further growing a biological material crystal as a seed crystal in a solution of the biological material, and the seed crystal is coated with the gel. It is.
  • the growth crystal production method of the present invention is not particularly limited.
  • various conditions such as the concentration and temperature of the biological material solution in the crystal growth step may be appropriately set with reference to a method of growing a crystal using a seed crystal in the prior art.
  • the preferable concentration of the biological material solution varies depending on the type of the biological material, but is preferably as high as possible from the viewpoint of ease of crystallization of growth.
  • the temperature is not particularly limited, and may be left at room temperature (about 5 to 35 ° C.), or may be cooled to a low temperature of 5 ° C. or lower or 0 ° C. or lower with a refrigerator or the like.
  • the time for the crystal growth step is not particularly limited, and may be allowed to stand as appropriate until a desired crystal is precipitated.
  • a crystal manufacturing method for further growing a seed crystal in a solution is widely performed.
  • a biological material such as a protein
  • the crystal since the crystal is brittle, a physical impact is caused when the seed crystal is moved into the biological material solution. There was a risk of damage.
  • the seed crystal after the seed crystal is moved into the biological material solution, the seed crystal may be dissolved due to a change in the concentration or osmotic pressure of the solution around the seed crystal, and crystal growth may not occur. It was.
  • the growth crystal manufacturing method of the present invention since the seed crystal is a coated crystal coated with the gel, the risk of damage due to physical impact is drastically reduced.
  • the seed crystal is a coated crystal coated with the gel, dissolution of the seed crystal is also suppressed, and the crystal is likely to grow.
  • a large crystal suitable for various applications such as neutron beam crystal structure analysis can be obtained with high reliability by a simple operation.
  • the structural analysis method of the present invention is a method for structural analysis of a crystal of a biological material, which is manufactured by a coated crystal manufactured by the crystal manufacturing method of the present invention or a frozen crystal manufacturing method of the present invention. And a structural analysis step for structural analysis of the frozen crystals.
  • a method for analyzing the structure of the crystal is not particularly limited, but it is preferable to use X-ray crystal structure analysis or neutron beam crystal structure analysis. According to the crystal manufacturing method of the present invention, as described above, damage to the crystal, crystallization, and the like can be prevented, and thus a large crystal suitable for neutron beam crystal structure analysis can be obtained.
  • the structure analysis method of the present invention is not particularly limited, and may be performed in the same manner as the conventional crystal structure analysis method, X-ray crystal structure analysis method, or neutron beam crystal structure analysis method.
  • Crystals of biological substances (proteins etc.) subjected to structural analysis in the structural analysis method of the present invention are coated crystals manufactured by the crystal manufacturing method of the present invention or frozen crystals manufactured by the frozen crystal manufacturing method of the present invention. High quality and large crystals.
  • highly accurate data for example, X-ray diffraction data
  • the frozen crystal production method of the present invention there are advantages as described in the section of the crystal production method and the frozen crystal production method of the present invention.
  • the structure analysis method of the present invention is not particularly limited as described above, and any method may be used.
  • the structure analysis method can be performed as follows.
  • the first scheme is shown separately in the upper part of FIG. 42 and the second scheme is shown separately in the lower part.
  • the first scheme shows a first example of the structural analysis method of the present invention
  • the second scheme shows a second example of the structural analysis method of the present invention.
  • the wording in the figure is an example for convenience of explanation, and does not limit the present invention.
  • a coated crystal 102 placed on a plate 101 is prepared.
  • the coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention.
  • the coated crystal is immersed in an anti-freezing agent and further scooped with a loop-shaped mounting device as described above.
  • the coated crystal thus picked up is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is subjected to structural analysis by X-ray crystal structure analysis or neutron beam crystal structure analysis.
  • the first example of the structural analysis method of the present invention can be implemented.
  • the coated crystal can be skimmed and frozen using the loop-shaped mounting device manually.
  • the loop-shaped mounting device may be referred to as “cryo loop”, for example.
  • a coated crystal 102 and a tube 103 are prepared.
  • the coated crystal 102 is coated with a gel and is a coated crystal manufactured by the crystal manufacturing method of the present invention.
  • the tube 103 may be provided with a holding portion for holding the tube 103 itself with another instrument or device at the base thereof.
  • the tip of the tube 103 is pierced into the coated crystal 102, and the portion containing the crystal is taken into the tip of the tube 103 together with the coated gel.
  • only the crystals and gel taken in at the tip of the tube 103 are separated from other parts of the coated crystal 102 by, for example, laser processing.
  • the tip of the tube 103 in which the crystals and gel are taken is immersed in an anti-freezing agent prepared in a separate container.
  • the tube 103 is placed with the tip facing upward (mount), and the coated crystal taken therein is frozen by a low-temperature gas spraying device or the like (freezing step), and the frozen crystal is analyzed by X-ray crystal structure analysis.
  • structural analysis is performed by neutron beam crystal structure analysis or the like.
  • This second example can be performed, for example, as an “automount” method in which the above steps are automated as necessary.
  • the coated crystal 102 may be prepared inside another tube.
  • the tip of the tube 103 may be pierced into the other tube, and the portion containing the crystal may be taken into the tip of the tube 103 together with the coated gel.
  • the subsequent steps can be performed in the same manner as in the second scheme of FIG.
  • the removal method is not particularly limited.
  • a method of cooling a thermoreversible hydrogel there are a method of processing with a laser beam such as a femtosecond laser, and the like.
  • the crystallization screening method of the present invention is a crystallization screening method for screening a crystallization condition of a biological material, the crystal manufacturing step for manufacturing the biological material crystal, and the crystal manufacturing step. Including the screening step of screening for crystallization conditions in the method, wherein the crystal is produced by the crystal production method of the present invention or the frozen crystal production method of the present invention.
  • the crystallization screening method of the present invention since the crystal can be easily manufactured by the crystal manufacturing method of the present invention, it is not necessary to set the crystal manufacturing conditions so strictly, the screening is also easy. It can be carried out. Specifically, for example, optimal conditions such as the concentration of the biological material and the concentration ratio (mixing ratio) of the biological material and the precipitating agent can be easily screened. Moreover, since the frozen crystal can be easily manufactured by the method for manufacturing a frozen crystal of the present invention, screening of the frozen crystal manufacturing condition can be easily performed. More specifically, for example, as described above, the crystal damage caused by the cryoprotectant can be greatly suppressed, so that screening of conditions such as the cryoprotectant concentration can be simplified.
  • the crystallization screening apparatus of the present invention is a crystallization screening apparatus for screening a crystallization condition of a biological material, the crystal manufacturing means for manufacturing the biological material crystal, and the biological material crystal. Screening means for screening crystallization conditions, wherein the crystal production means includes crystallization means for crystallizing the biological material in a gel.
  • the crystallization screening apparatus of the present invention is not particularly limited.
  • the configuration can be simplified as compared with the conventional crystallization screening apparatus.
  • it is possible to simplify the screening of conditions such as the concentration of the biological material, the concentration ratio of the biological material and the precipitating agent (mixing ratio), the concentration of the cryoprotectant, etc. Simplification is also possible.
  • the crystallization screening method of the present invention may be performed using any apparatus or instrument, but is preferably performed by the crystallization screening apparatus of the present invention.
  • FIG. 44 shows three examples of the crystallization screening apparatus of the present invention and the crystallization screening method of the present invention using the same.
  • Three examples of the crystallization screening method are referred to as a hanging drop method, a sitting drop method, and a concentration gradient method, respectively.
  • the upper left of FIG. 44 is an example of an apparatus used for the hanging drop method
  • the lower left is an example of an apparatus used for the sitting drop method
  • the upper right is an example of an apparatus used for the concentration gradient method.
  • this apparatus includes a lid 201 and a plate 203 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means.
  • the lid 201 has a plate shape, and a projection 202 is provided on one surface thereof, and the other surface has a flat shape.
  • the upper surface of the protrusion 202 has, for example, a flat shape so that a gel can be placed thereon.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 203.
  • a projection 202 formed on the lid 201 can be fitted into the well.
  • the number of wells formed on the protrusion 202 provided on the lid 201 and the plate 203 is arbitrary, and one or more wells may be provided, but a sufficient number is provided for efficient screening. Is preferred.
  • the protrusion 202 be removable from the lid 201 main body, for example, because an operation such as collecting a crystal can be easily performed.
  • the material for forming each component is not particularly limited.
  • a material that is generally used for physics and chemistry equipment, such as glass and plastic, and that does not interfere with crystal production and screening may be used.
  • the size of each component is not particularly limited, but for example, it is preferable that the entire device is of a size convenient for handling on an experimental table.
  • the crystallization screening method (hanging drop method) of the present invention using this apparatus can be performed as follows, for example. That is, first, the lid 201 is prepared with the protrusion 202 facing upward. Next, a gel containing a biological material is placed on the upper surface of the protruding portion 202, or is placed on the upper surface of the protruding portion 202 as a solution containing a biological material and solidified (gelled) there.
  • the method for producing this gel is as described above, for example. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used.
  • a precipitating agent (precipitating agent) solution is placed in the well of the plate 203.
  • the method for producing the precipitating agent solution is also as described above, for example.
  • the lid 201 is turned upside down so that the projection 202 faces downward, and the projection 202 is fitted into the plate well. Accordingly, the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel.
  • the crystal manufacturing process in the crystallization screening method of the present invention can be performed. In this crystal manufacturing process, various conditions such as the biological substance concentration and the precipitating agent concentration are changed, and after the crystal manufacturing process, the crystal formation state under each condition is observed. Thus, the screening step of screening for crystallization conditions is performed.
  • the screening can be performed in a single crystal manufacturing process by variously changing the biological material concentration and the precipitating agent concentration in each protrusion 202 and well. It is also possible to carry out the process. As described above, the crystallization screening method (hanging drop method) of the present invention using this apparatus can be carried out.
  • this apparatus has a plate 207 as a main component.
  • the plate 207 constitutes the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serves as the screening means.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 207.
  • the number of the wells is arbitrary and may be one or more, but it is preferable to provide a sufficient number for efficient screening. Note that the forming material and the size of this device are the same as those in the upper left of FIG.
  • the crystallization screening method (sitting drop method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed in the well of the plate 207 or placed in the well in the state of a biological material solution and solidified (gelled) there. The method for producing this gel is as described above, for example. Although it is described as “a mixture of protein and gel” in the drawing, it is not limited to protein, and any biological material may be used. Next, a precipitant (precipitant) solution is injected into the well. The method for producing the precipitating agent solution is also as described above, for example.
  • the gel containing the biological material is immersed in the precipitant (precipitating agent) solution, and the biological material is crystallized in the gel.
  • a seal or the like is preferably provided on the upper surface of the plate 207 in order to prevent foreign substances from entering the well, volatilization of the precipitant solution, and the like.
  • the screening step can be performed in a single crystal manufacturing step by variously changing the biological material concentration and the precipitating agent concentration in each well. is there.
  • the crystallization screening method (sitting drop method) of the present invention using this apparatus can be carried out.
  • this apparatus has a lid 209 and a plate 206 as main components. These constitute the crystallization means and the crystal production means in the crystallization screening apparatus of the present invention, and also serve as the screening means.
  • the lid 209 has a plate shape, and a part of one surface thereof protrudes to form a tube 205.
  • the inside of the tube 205 is hollow, and the end on the side in contact with the lid 209 main body is open.
  • the other end of the tube 205 (hereinafter referred to as “protruding end”) is closed, and, for example, a semipermeable membrane (dialysis membrane) or the like is stretched, so that solid matter does not permeate, and only particles having a certain size or less. It can be transmitted.
  • a well (hole) that does not penetrate to the opposite side is formed on the upper surface of the plate 206.
  • a tube 205 formed on the lid 209 can be fitted into the well.
  • the number of wells formed on the tube 205 and the plate 206 formed on the lid 209 is arbitrary, and may be one or more, but it is preferable to provide a sufficient number for efficient screening.
  • the forming material and the size of this device are the same as those in the upper left of FIG. 44 except for the semipermeable membrane (dialysis membrane) at the end of the tube 205.
  • the crystallization screening method (concentration gradient method) of the present invention using this apparatus can be performed, for example, as follows. That is, first, a gel containing a biological material is placed inside the tube 205 formed on the lid 209. The method for producing this gel is as described above, for example. The gel may be injected into the tube 205 after being in a gel state, but it is preferable that the gel is injected into the tube 205 in a solution (sol) state and gelled in the tube 205. In the figure, “a mixture of protein and gel” is described. However, the present invention is not limited to protein, and any biological substance may be used. On the other hand, a precipitant (precipitant) solution is placed in the well of the plate 206.
  • the method for producing the precipitating agent solution is also as described above, for example.
  • the tube 205 is fitted into the well of the plate 206.
  • the precipitating agent penetrates into the gel through the tip of the tube 205.
  • the gel 205 does not permeate due to a semipermeable membrane (dialysis membrane) or the like stretched around the protruding end of the tube 205.
  • a seal or the like is preferably installed on the upper surface of the lid 209 in order to prevent foreign matter from entering the well, drying of the gel, and the like. Thereby, the crystal manufacturing process in the crystallization screening method of the present invention can be performed.
  • the precipitating agent penetrates into the gel through the tip of the tube 205, a gradient is formed in the concentration of the precipitating agent (precipitating agent) in the gel. That is, the concentration of the precipitating agent increases on the side closer to the tip of the tube 205, and the concentration of the precipitating agent decreases as the distance from the tip increases.
  • the concentration gradient By forming this concentration gradient, the ratio between the concentration of the biological material and the concentration of the precipitating agent varies in various parts inside the tube 205. For this reason, after the completion of the crystal production process, the screening process for screening the crystallization conditions can be performed by observing the crystal formation state in the tube 205.
  • the screening step can be performed in more detail by changing the biological material concentration and the precipitant concentration in each tube 205 and well. It is.
  • the crystallization screening method (concentration gradient method) of the present invention using this apparatus can be carried out.
  • the crystallization screening method by the concentration gradient method can also be performed using a centrifuge, for example, as in Examples described later.
  • Various conditions such as the types and concentrations of the biological material and the gelling agent are not limited to the examples described later, and can be arbitrarily set. Specifically, it is the same as that of other embodiment, for example.
  • the centrifugation speed and the like are not particularly limited and can be set as appropriate.
  • the crystallization screening method by the concentration gradient method can be performed not only by forming a precipitant concentration gradient but also by forming a biological material concentration gradient, for example.
  • a gel containing no biological material is prepared in advance, and the biological material solution is gradually permeated from one side of the gel to form a gradient of the biological material concentration in the gel. You may do it.
  • the gel may or may not contain a precipitating agent in advance, for example.
  • the crystallization screening apparatus and the crystallization screening method of the present invention are not limited to these, and any apparatus and method may be used.
  • various conditions of the crystallization screening method are not limited to the above description, and can be changed as appropriate.
  • FIG. 44 as a crystallization method, a method of bringing the gel containing the biological material solution into contact with or in proximity to the precipitant solution is shown.
  • the crystallization method using a precipitating agent is, for example, mixing a precipitating agent together with a gelling agent in the biological material solution and gelling it to obtain a gel containing the precipitating agent.
  • the crystal may be precipitated by allowing it to stand as it is.
  • the crystallization screening apparatus for example, as shown in FIG. 45, an apparatus in which the tube 205 of the “concentration gradient method” apparatus (apparatus shown in the upper right of FIG. 44) is replaced with a shallow well 305 may be used. 45, the lid 304 and the plate 306 are the same as the “concentration gradient method” apparatus (the apparatus shown in the upper right of FIG. 44) except that the depth of the well is shallow to match the well 305.
  • the crystallization screening method using this apparatus is the same as the apparatus of the “concentration gradient method”, except that there is no concentration gradient formation in the gel, or is the same as the other apparatus shown in FIG.
  • 45 has an advantage that the overall operation is simpler than the apparatus of the “concentration gradient method” because the wells formed in the lid 304 and the plate 306 are shallow. 45, since the gel and the precipitating agent solution are present in different wells, the gel and the precipitation are compared with the devices of the “hanging drop method” and the “sitting drop method”. There is an advantage that the separation operation from the agent solution is simple.
  • the crystallization screening method of the present invention is performed automatically, for example.
  • the crystal manufacturing process may be automated by a method of automatically dispensing the solution (sol) of the biological material into each well or the inside of a tube by a dispensing device.
  • a dispensing device with a temperature controller
  • the biological material is not limited to proteins, and any material may be used. Any dispensing device may be used.
  • the screening process may be automated by automatically observing and photographing the crystal formation state with a microscope as illustrated.
  • the crystallization screening apparatus of the present invention includes a crystal manufacturing means for manufacturing a crystal of biological material, and therefore can be used as a crystal manufacturing apparatus for manufacturing a crystal of biological material.
  • Example 1 to 7 and Comparative Examples 1 to 7 below protein crystals of the following (1) to (3) were used as specimens (analyzed substances).
  • the following (1) to (2) and (4) to (7) protein crystals or (8) nucleic acid (DNA) crystals were used as specimens (analytes).
  • (1) Lysozyme (2) Glucose isomerase (3) Saumatine (4) Elastase (5) Synechococcus-derived phosphoribrokinase (PRK) (Phosphoribokinase (PRK) / Synechococcus) (6) Serine acetyltransferase (SAT) (7) E. coli foreign body excretion transporter (AcrB) (8) DNA
  • the trade name Ultrax-18 (Cu counter cathode) manufactured by Rigaku Corporation was used as the X-ray source.
  • the detector used was R-AXIS IV ++ manufactured by Rigaku Corporation.
  • the measurement voltage was 50 kV
  • the measurement current was 100 mA
  • the beam diameter was 0.3 mm.
  • Example 1 Agarose-III (manufactured by Dojindo Laboratories Co., Ltd., gelation temperature is about 37-39 ° C.) 2% by mass and 98% by mass of water dissolved by heating (A) 0.1 mL and chicken egg white lysozyme solution (B ) 0.1 mL of both were mixed at a constant temperature of 35 ° C. to obtain a crystallization solution (C). Before the crystallization solution (C) became a solidified gel, it was filled into a capillary (manufactured by Hirgenberg).
  • the capillary was made of glass (80 mm, inner diameter 0.7 mm), and filled with a crystallization solution (C) before solidifying into an internal tubular space.
  • the crystallization solution (C) solidified after several tens of seconds at room temperature, and the capillary inner space was filled with the gel in which the crystallization solution (C) was solidified.
  • the filled capillary was then inserted into a test tube container into which approximately 2 ml of the precipitant solution was injected.
  • This test tube container was made of glass having a diameter of about 16 mm and a length of about 133 mm. At this time, a contact interface between the solidified (gelled) crystallization solution (C) and the precipitating agent solution was formed at the insertion end of the capillary.
  • the open end of the test tube was sealed with a stopper.
  • the composition of the protein solution and the precipitating agent solution is shown below.
  • Precipitating agent solution 1.0M sodium chloride 0.1M acetate buffer pH 4.5
  • Crystallization temperature 20 ° C
  • lysozyme crystals were confirmed in the solidified gel within a few days.
  • the capillary was taken out from the test tube, and the lysozyme crystal crystallized in the solidified gel in the capillary was taken out by the following method. That is, first, a glass plate was prepared, and about 30 ⁇ L each of a precipitating agent solution and an anti-freezing agent solution (100% dimethyl sulfoxide solution in this example) were dispensed on different positions. .
  • the capillary was cut or crushed within a range of 5 to 10 mm before and after the crystal to be taken out using tweezers or a capillary cutter, and then the solidified gel was cut with a cutter or the like. Furthermore, the crystals in the taken-out gel were moved into the precipitating agent solution prepared in advance. Using a micro tool (manufactured by Hampton Research) or the like, the gel around the lysozyme crystal was carefully peeled off to a thickness of about 0.1 mm under a microscope.
  • FIG. 1A and FIG. 1B show photographs of the lysozyme crystals of Example 1 protected with a gel, taken at different angles.
  • Example 1 a large and high quality lysozyme single crystal was obtained.
  • 2A and 2B show X-ray diffraction images of the lysozyme crystal of Example 1.
  • FIG. FIG. 2A is an overall view
  • FIG. 2B is an enlarged view of a part (the frame line portion at the lower right of FIG. 2A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • Example 2 A glucose isomerase solution was used in place of the chicken egg white lysozyme solution, and crystals were produced in the same manner as in Example 1 except for the composition of the protein solution and the precipitating agent solution and the type of the cryoprotectant. About 1 week after setup at a crystallization temperature of 4 ° C., glucose isomerase crystals were confirmed in the solidified gel. The composition of the protein solution and the precipitating agent solution is shown below.
  • Protein solution 25 mg / mL glucose isomerase 0.1 M Hepes buffer pH 7.5 1 mM magnesium chloride
  • Precipitating agent solution 2.5M ammonium sulfate 0.1M Hepes buffer pH7.5 Crystallization temperature: 4 °C
  • the antifreezing agent solution was subjected to X-ray crystal structure analysis using a 2.5M lithium acetate solution.
  • the glucose isomerase crystal could be easily captured without damage, and it was resistant to liquid nitrogen treatment without being damaged by the antifreezing agent solution, and a good X-ray diffraction image was obtained.
  • FIG. 3A, 3B, 4, 5A and 5B show X-ray diffraction images of the glucose isomerase crystal of Example 2.
  • FIG. 3A is an overall view
  • FIG. 3B is an enlarged view of a part (the frame line portion at the upper right of FIG. 3A).
  • FIG. 4 is an X-ray diffraction image taken under different conditions.
  • FIG. 5A is an overall view of an X-ray diffraction image measured by further changing the conditions
  • FIG. 5B is an enlarged view of a part (the frame line part at the lower right of FIG. 5A). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • 6A to 6D show the lysozyme crystal of Example 2.
  • FIG. 1 is an overall view
  • FIG. 3B is an enlarged view of a part (the frame line portion at the upper right of FIG. 3A).
  • FIG. 4 is an X-ray diffraction image taken under
  • FIG. 6A is a diagram showing a state immediately before dipping in the antifreezing agent solution, and a single crystal is obtained.
  • FIG. 6B shows a state after 5 seconds from the immersion in the cryoprotectant solution
  • FIG. 6C shows a state after 1 minute from the immersion
  • FIG. 6D shows a state after 5 minutes after the immersion.
  • the crystals on the right side of FIGS. A to D collapsed gradually after immersion because they had some defects before immersion in the cryoprotectant solution, but the crystals on the left side were good single crystals and were completely disintegrated even after being immersed for 5 minutes. I did not.
  • Example 5 Crystallization was carried out in the same manner as in Example 1 except that a thaumatin solution was used in place of the chicken egg white lysozyme solution and the composition of the protein solution and the precipitating agent solution and the type of the antifreezing agent were used. After setting up at a crystallization temperature of 20 ° C., glucose isomerase crystals were confirmed in the gel solidified in about one week. Below, the composition of a protein solution and a precipitant solution is shown.
  • Protein solution 20 mg / mL thaumatin 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5
  • Precipitating agent solution 2.0 M potassium sodium tartrate 0.1 M N- (2-acetamido) iminodiacetic acid buffer pH 6.5
  • the cryoprotectant solution was subjected to X-ray crystal structure analysis using 100% glycerol.
  • thaumatin crystals could be easily captured without damage, and withstand damage to liquid nitrogen without damage caused by the antifreezing agent solution, and a good X-ray diffraction image was obtained.
  • FIG. 7A shows a photograph of the thaumatin crystal of Example 5 protected with a gel. As illustrated, in Example 5, a large and high quality thaumatin single crystal was obtained. 7B and 7C show X-ray diffraction images of the thaumatin crystals of Example 5. FIG. FIG. 7B is an overall view, and FIG. 7C is an enlarged view of a part (the frame line portion at the bottom of FIG. 7B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • FIG. 8A shows a photograph of the thaumatin crystal of Example 7 protected with a gel. As illustrated, in Example 7, a large and high quality thaumatin single crystal was obtained. 8B and 8C show X-ray diffraction images of the thaumatin crystal of Example 7. FIG. FIG. 8B is an overall view, and FIG. 8C is an enlarged view of a part (a frame portion at the bottom of FIG. 8B). As shown in the figure, it can be seen that a good X-ray diffraction image was obtained.
  • agarose-III manufactured by Dojindo Laboratories, Inc., gelation temperature is about 37 to 39 ° C.
  • agarose IX-A manufactured by Sigma, gelation temperature is about 8 to 17 ° C.
  • crystals and frozen crystals were produced in the same manner as in Examples 1 to 7 except that the temperature conditions were changed, and similarly good results were obtained.
  • Example 1 Crystallization was carried out in exactly the same manner as in Example 1, except that a lysozyme solution without addition of agarose was used. After setup, lysozyme crystals were confirmed within a few days. The lysozyme crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the antifreeze solution (100% dimethyl sulfoxide solution). Therefore, X-ray crystal structure analysis could not be performed.
  • 9A and 9B show photographs of the lysozyme crystal of Comparative Example 1.
  • FIG. FIG. 9A is a photograph showing a state immediately before dipping in the cryoprotectant solution, and a single crystal is obtained.
  • FIG. 9B is a photograph showing a state immediately after being immersed in the anti-freezing agent solution, and the crystals are shattered.
  • Example 2 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (2.5M lithium acetate solution). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 3 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 4 Crystallization was carried out in exactly the same manner as in Example 1 except that a glucose isomerase solution without adding agarose was used. Glucose isomerase crystals were confirmed about one week after setup. The glucose isomerase crystal taken out from the capillary was broken into pieces and melted at the moment of immersion in the antifreeze solution (50% glycerol solution). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 5 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in the cryoprotectant solution (100% glycerol). Therefore, X-ray crystal structure analysis could not be performed.
  • Example 6 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and dissolved at the moment of immersion in an anti-freezing agent solution (100% polyethylene glycol (average molecular weight 400)). Therefore, X-ray crystal structure analysis could not be performed. 10A and 10B show photographs of the lysozyme crystal of Comparative Example 6. FIG. FIG. 10A is a photograph showing a state immediately before dipping in an anti-freezing agent solution, and a single crystal is obtained. FIG. 10B is a photograph showing a state immediately after being immersed in the cryoprotectant solution, and the crystals are shattered.
  • Example 7 Crystallization was carried out in exactly the same manner as in Example 1 except that a thaumatin solution to which no agarose was added was used. After the setup, thaumatin crystals were confirmed in about one week. The thaumatin crystals taken out from the capillaries were broken into pieces and melted at the moment of immersion in an anti-freezing agent solution (60% polyethylene glycol (average molecular weight 4000) solution). Therefore, X-ray crystal structure analysis could not be performed.
  • an anti-freezing agent solution 60% polyethylene glycol (average molecular weight 4000) solution. Therefore, X-ray crystal structure analysis could not be performed.
  • Example 8 A coated crystal coated with a gel was produced, and an X-ray crystal structure analysis was performed using a 2.5 M aqueous lithium acetate solution as an antifreezing agent. Using a 2.5M aqueous lithium acetate solution as an antifreezing agent, setting the immersion time of the coated crystal in the antifreezing agent to 15 minutes, the biological material constituting the crystal to be analyzed, and the gel (Agarose III) The same procedure as in Example 1 was performed except for the concentration.
  • Example 8 is one in which elastase, which is a kind of protein, is used as the biological material, and the gel (agarose III) concentration is 2.0 mass%.
  • Example 9 uses lysozyme which is a kind of protein as the biological material, and the concentration of gel (agarose III) is 1.6% by mass.
  • Example 10 uses glucose isomerase, which is a kind of protein, as the biological material and the gel (agarose III) concentration is 1.6 mass%.
  • Example 8 is one in which elastase which is a kind of protein is used as the biological material, and the immersion time in the cryoprotectant is 2 seconds.
  • Example 9 uses lysozyme which is a kind of protein as the biological material, and the immersion time in the cryoprotectant is 15 seconds.
  • FIG. 11 shows photographs and data of the crystals of Examples 8 to 10 and Comparative Examples 8 to 9. It is a photograph of comparative example 8, example 8, comparative example 9, example 9, and example 10, respectively from left to right.
  • the numerical value in the lower part of the figure shows the amount of agarose added (mass%), the immersion time in the cryoprotectant (Cryo) solution, and the mosaic property in the X-ray crystal structure analysis.
  • the coated crystals of Examples 8 to 10 were not damaged even when immersed for a long time of 15 minutes in an anti-freezing agent (2.5 M lithium acetate aqueous solution).
  • the mosaic property of these crystals is a sufficiently small value of about 0.3, indicating that the crystals are of high quality.
  • the crystal of Comparative Example 8 had a large mosaic property of 1.17, and the crystal of Comparative Example 10 was damaged when immersed in an anti-freezing agent for 15 seconds, and X-ray crystal structure analysis could not be performed.
  • Example 11 Coated crystal production and X-ray crystal structure analysis were performed under the same conditions as in Example 10 except that the immersion time in the antifreezing agent (2.5 M lithium acetate aqueous solution) was changed to 10 minutes. As a result, the mosaic value is 0.08 ° including the beam dispersion, which is an extremely small value, indicating that the crystal has a very high quality over the prior art.
  • FIG. 12 shows an X-ray diffraction image of the crystal of this example. The resolution at this time was 0.93 mm, and the crystal to be analyzed was a large crystal having a diameter of 0.5 mm or more.
  • Example 12 Production of crystals under conditions of changing gel concentration and screening of crystallization conditions
  • the agarose-III concentration in the agarose-III solution (A) was variously changed from 0 to 2.0% by mass in increments of 0.2% by mass, and the lysozyme concentration in the protein solution (B) was changed from 30 mg / mL.
  • a coated crystal coated with agarose-III gel was produced in the same manner as in Example 1 except that it was carried out by the batch method using Imp @ ct plate (trade name) manufactured by Hampton Research Co., Ltd.
  • the crystallization conditions (crystal production conditions) under the changing conditions were screened.
  • description will be made using the reference numerals in the lower left of FIG. 44 (sitting drop method). Since this figure is a schematic diagram, the number of wells, the dimensional ratio of each part, etc. It is different from plate. That is, first, the agarose-III concentration in the agarose-III solution (A) was prepared at various agarose-III concentrations changed from 0 to 2.0% by mass in steps of 0.2% by mass as described above. This was mixed with the protein solution (B) to prepare crystallization solutions (C) having different agarose-III concentrations.
  • the crystallization solution (C) having different agarose-III concentrations was mixed with the precipitating agent solution, and 6 ⁇ L was injected into each well of the crystallization plate 207.
  • a total of eight crystallized samples with the same agarose-III concentration were prepared and injected into separate wells.
  • This crystallization solution (C) formed droplets in the well and immediately gelled.
  • a seal was affixed to the upper surface of the plate (crystallization plate) 207 to prevent the gel in each well from being exposed to the atmosphere, and left to stand at 20 ° C. for 3 days. Thereafter, the number of crystals formed in each sample was observed with a microscope. The number of produced crystals of eight samples at each concentration was averaged to obtain the average number of crystals (pieces).
  • the horizontal axis represents the agarose-III concentration
  • the vertical axis represents the average number of crystals (pieces).
  • Example 13 Crystal production and crystallization condition screening under gel concentration changing conditions
  • the type of agarose was agarose-III (Agarose III), agarose 9A, or agarose Sea Plaque (agarose SP), and agarose SeaKem (all are trade names of Takara Bio Inc.), and the agarose concentration was 0, 0. .2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5
  • the gel was changed in the same manner as in Example 12 except that it was variously changed to 4.0, 4.5, 5.0, 5.5, or 6.0% by mass (for SeaKem up to 2.0% by mass or less).
  • the crystallization conditions (crystal production conditions) under the concentration change conditions were screened.
  • the graph of FIG. 16 shows the results when the agarose concentration is 0 to 2.0% by mass.
  • the graph on the upper left of the figure is a graph showing the results of agarose 9A.
  • the graph in the upper right of the figure is a graph showing the results of agarose SP.
  • the graph on the lower left of the figure is a graph showing the results of agarose-III.
  • the graph on the lower right of the figure is a graph showing the results of agarose SeaKem.
  • the horizontal axis represents the agarose concentration
  • the vertical axis represents the average number of crystals (pieces). Further, the graph of FIG.
  • the graph 17 shows the results when the agarose concentration is 0 and 2.0 to 6.0 mass%.
  • the graph in the upper right of the figure is a graph showing the results of agarose-III.
  • the graph on the lower left of the figure is a graph showing the results of agarose 9A.
  • the lower right graph is a graph showing the results of agarose SP.
  • the horizontal axis represents the agarose concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (concentration other than 0), it was shown that good crystal production conditions were obtained. It was done.
  • Example 14 Crystal production under crystallization condition change and crystallization condition screening
  • the type of agarose is NuSieve 3: 1 (trade name of Takara Bio Inc.), and the agarose concentration is 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 1.4, 1.6, 1.8, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 or 6.0 mass%
  • the crystallization conditions (crystal production conditions) under gel concentration change conditions were screened in the same manner as in Example 12 except that various changes were made. The result is shown in the graph of FIG. In the figure, the horizontal axis represents NuSieve 3: 1 concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 15 Crystal production and crystallization condition screening under changing conditions of gel concentration and precipitant concentration
  • the crystallization conditions (crystal production conditions) under the conditions of changing the gel concentration and the precipitating agent were screened in the same manner as in Example 12 except that the immersion time was 6 days or 7 days.
  • the gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 12.
  • the result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 16 Crystal production and crystallization condition screening under conditions of changing gel concentration and precipitating agent concentration
  • high melting point agarose-III was used instead of the normal agarose-III used in each of the above Examples, the crystallization conditions (crystal production under the conditions of changing gel concentration and precipitating agent) Condition).
  • the gel concentration was changed from 0 to 2.0% by mass in increments of 0.2% by mass for each sodium chloride concentration as in Example 15.
  • the result is shown in the graph of FIG. In the figure, the horizontal axis represents the agarose-III concentration, and the vertical axis represents the average number of crystals (pieces).
  • Example 17 Crystal production under crystallization condition change and crystallization condition screening
  • agarose-III concentration 0 or 2.0
  • elastase was used instead of lysozyme as the protein
  • concentration of elastase in the protein solution (B) was changed.
  • Example 12 except that it was 12.5 mg / mL, that the type of agarose was agarose seaplaque (SP), and that the number of samples used to calculate the average number of crystals was 12 instead of 8.
  • crystallization conditions crystal production conditions under gel concentration changing conditions were screened. The result is shown in the graph of FIG.
  • the horizontal axis represents the agarose SP concentration
  • the vertical axis represents the average number of crystals (pieces).
  • Example 18 Crystal production under crystallization condition change and crystallization condition screening
  • Glucose isomerase was used instead of lysozyme as the protein
  • the concentration of glucose isomerase was 10 mg / mL in the protein solution (B)
  • the type of agarose was ultra-low molecular weight agarose 9A
  • the precipitating agent was chlorinated.
  • a 0.1 M calcium chloride (CaCl 2 ) aqueous solution containing 10% by mass of 2-methyl-2,4-pentanediol (MPD) was used.
  • Example 19 Crystal production under crystallization condition change and crystallization condition screening
  • the type of agarose being agarose SP and the precipitating agent being a 0.1 M calcium acetate (CaAc) aqueous solution containing 8.0% by mass of 2-methyl-2,4-pentanediol (MPD).
  • crystallization conditions under gel concentration change conditions were screened.
  • the results are shown in the photograph and graph of FIG.
  • the numerical value at the bottom of each photograph indicates the agarose SP concentration (% by mass).
  • the horizontal axis represents the agarose SP concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated.
  • Example 20 Crystal production and crystallization condition screening under changing conditions of protein concentration and gel concentration
  • the horizontal axis represents the agarose SP (Seaplaque) concentration
  • the vertical axis represents the average number of crystals (pieces).
  • the mechanism of the correlation between the agarose SP concentration (gel concentration) and the average number of crystals is unknown, but in the presence of agarose (the concentration is other than 0), good crystal production conditions were obtained in all cases. Indicated. Moreover, it was shown that good crystal production conditions were obtained regardless of whether the thaumatin concentration was 50 mg / mL or 25 mg / mL.
  • Example 21 Crystal production under crystallization condition change and crystallization condition screening
  • Crystal production and crystallization condition screening were performed in the same manner as in Example 12 except for the following (1) to (3).
  • (1) Crystals were produced by the hanging drop method using VDX plate (trade name of Hampton Research).
  • a mixed solution with 2 HgCl was used as a crystallization solution (C).
  • this crystallization solution (C) gelatinized immediately, it used for the crystal manufacturing process immediately after preparation.
  • the crystal manufacturing process by the hanging drop method was performed as follows. That is, first, 500 ⁇ L / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 ⁇ L of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other.
  • Example 22 Crystal production of E. coli-derived protein (crystallization)
  • Escherichia coli-derived serine acetyltransferase (SAT) 7.5 mg / mL was used instead of the above protein, and a precipitating agent (precipitating agent) solution was added to 4.5% by mass of PEG 8000 (Promega's polyethylene glycol) ) And 0.1M sodium cacodylate in pH 6.5 aqueous solution, except that the crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 21.
  • SAT Escherichia coli-derived serine acetyltransferase
  • Example 23 Crystal production of E. coli-derived protein (crystallization)] Same as Example 22 except that the precipitating agent (precipitating agent) solution was a pH 6.5 aqueous solution containing 7.5% by mass of PEG8000 (trade name of polyethylene glycol from Promega) and 0.1 M sodium cacodylate. Thus, crystallization conditions (crystal production conditions) under gel concentration change conditions were screened.
  • Example 24 Crystal production of E. coli-derived protein (crystallization)
  • E. coli foreign body excretion transporter (AcrB) 28 mg / mL was used instead of SAT, and a precipitating agent (precipitating agent) solution was 10% by mass of PEG2000 (trade name of polyethylene glycol of Promega), Crystallization conditions (crystal production conditions) under gel concentration changing conditions were screened in the same manner as in Example 12 except that the aqueous solution was pH 5.6 containing 80 mM sodium dihydrogen phosphate and 20 mM sodium citrate.
  • Example 25 Crystal production of E. coli-derived protein (crystallization)
  • the crystallization conditions under the gel concentration changing conditions were the same. Screened.
  • Example 26 Crystal production of nucleic acid (DNA) and screening of crystallization conditions
  • a crystal of nucleic acid (DNA) was produced as follows.
  • DNA was annealed. That is, first, a 1 mM aqueous solution of DNA1 (5′-AAGAAAAAAA-3 ′: SEQ ID NO: 1), which is a single-stranded DNA, and DNA2 (5′-TTTTTTTCTT-3 ′: SEQ ID NO: 2), which is a complementary strand of DNA1, are used. An equal amount of 1 mM aqueous solution was mixed, and left at 60 ° C. for 10 minutes and then at 20 ° C. for 15 minutes. Here, 1 mM aqueous solution of BNA (Bridged Nucleic Acid, Gene Design Co., Ltd.) was added in the same amount as the DNA1 aqueous solution, and allowed to stand at 20 ° C. overnight. In this way, DNA annealing was performed to obtain an annealed DNA aqueous solution. This annealed DNA aqueous solution was used for crystal production.
  • DNA1 5′-AAGAAAAAAA-3 ′: SEQ ID NO: 1
  • the crystallization solution (C) (mixed aqueous solution of gel and DNA) was prepared as follows. That is, first, agarose 9A was dissolved in water at 85 ° C., and an aqueous gelling agent solution (4 times the final concentration) kept at 37 ° C. was prepared. This gelling agent aqueous solution, a buffer containing MPD, sodium cacodylate and MgCl 2 at a concentration four times the final concentration, and the annealed DNA aqueous solution are mixed in this order at a volume ratio of 1: 1: 2. A crystallization solution (C) was obtained. Since this crystallization solution (C) was gelated immediately, it was subjected to the following crystal production process immediately after preparation.
  • the crystal manufacturing process was performed as follows. That is, first, 500 ⁇ L / well of the precipitant solution was prepared in advance in each well (plate well) of the plate 203. On the other hand, 2 ⁇ L of the crystallization solution (C) prepared as described above was dispensed on the protrusion 202 (cover glass) to be gelled. For each concentration of the crystallization solution (C), eight identical samples were placed on top of the eight protrusions 202. Thereafter, the lid 201 was turned upside down so that the projections 202 faced down, and the projections 202 were fitted into the plate well containing the precipitating agent solution to bring the gel and the precipitating agent solution close to each other. After setting in this way, the mixture was allowed to stand at 20 ° C. for 1 to 5 days, and DNA crystals were precipitated in the gel to produce crystals.
  • FIG. 27 shows a process of producing a DNA crystal (crystal growth) in this example.
  • the left column of FIG. 27 is an example in which agarose was not added (corresponding to a comparative example).
  • the middle column is an example with an agarose 9A final concentration of 1.0 mass%.
  • the right column is an example of a final concentration of 1.6% by mass of agarose 9A.
  • the upper stage is a photograph immediately after standing at 20 ° C. for 1 day
  • the lower stage is a photograph immediately after standing at 20 ° C. for 5 days.
  • the DNA crystal grows without adding agarose, but according to the example of the present invention to which agarose was added, it was shown that the crystal was more likely to grow.
  • the drawing when 5 days passed after standing at 20 ° C., the number of fine crystals decreased and the transparency of the gel increased, and the crystals grew larger than when 1 day after standing.
  • Example 27 Crystal production and crystallization screening using mebiol gel
  • agarose-III solution A
  • meviol gel solution in which the meviol gel concentration was variously changed to 0, 1, 3, and 5% by mass was used, and the concentration of lysozyme in the protein solution (B) was changed. Lysozyme crystals were produced in the same manner as in Example 12 except that the concentration was changed to 40 mg / mL, and crystallization screening was performed.
  • FIG. 28 shows the result. In the graph on the left side of the figure, the horizontal axis represents the amount of meviol gel added (mass%), and the vertical axis represents the average number of crystals (number of crystal precipitates).
  • the photograph on the right side shows the precipitated crystals when 1% by mass of mebiol gel is added and when no mebiol gel is added.
  • mebiol gel when no mebiol gel was added, almost no lysozyme crystals were precipitated, and the precipitated crystals were cracked and were not good quality crystals.
  • mebiol gel when mebiol gel was added, many high-quality crystals without cracks were obtained.
  • the amount of meviol gel added was increased to 5% by mass, the number of crystal precipitates increased rapidly. Furthermore, even when the mebiol gel was increased to 7% by mass, the same good results were obtained.
  • the above crystals are coated crystals coated with mebiol gel.
  • mebiol gel By cooling to 15 ° C. or lower, mebiol gel is liquefied (solified) and the gel coating can be removed very easily. It was. This is shown in the photograph of FIG.
  • the photograph on the left shows crystals coated with mebiol gel before cooling.
  • the photo on the right shows the crystal after cooling, with the gel coating removed.
  • Example 13 coated crystals produced using 2.0 mass% agarose 9A, 2.0 mass% agarose SP, or 2.0 mass% agarose-III were used for evaluation.
  • Example 27 a coated crystal produced using 1% by mass of meviol gel was used for evaluation. The evaluation is performed by removing the seal from the upper surface of the well (VDX plate (trade name) manufactured by Hampton Research Co., Ltd.) for the hanging drop method containing the crystal, exposing the crystal to normal temperature atmosphere, and observing the change with time under a microscope. went.
  • the leftmost “None” line shows the change with time of a crystal (comparative example) produced without adding gel.
  • the second “2.0% 9A” line from the left shows the change over time of crystals produced using 2.0 mass% agarose 9A.
  • the third “2.0% SP” line from the left shows the change over time of crystals produced using 2.0 mass% agarose SP.
  • the rightmost “2.0% III” line shows the time course of the crystals produced using 2.0 mass% agarose-III.
  • the number represented by “min” on the left side of the photograph represents the exposure time (minutes) of the crystal to the atmosphere.
  • the crystal produced without the addition of the gel started to dry at an exposure time of 10 minutes, a remarkable crack occurred at an exposure time of 16 minutes, and was already unusable, and the exposure time was 29 minutes. Then it was completely collapsed.
  • each coated crystal of the example withstood the drying for a long time of exposure time of 20 to 29 minutes.
  • FIGS. 31A and 31B show the results of evaluation of the crystal of Example 27 against dryness.
  • the “no gel addition” line in the upper part of FIG. 31A shows the change with time of the crystal (comparative example) produced without adding the gel.
  • the line of “addition of 1% mebiol” in the lower part of FIG. 31A shows the change over time of crystals produced using 1.0 mass% meviol gel.
  • the upper part of FIG. 31B shows the change with time of the crystal (comparative example) produced without adding the gel, following the upper part of FIG. 31A.
  • the lower part of FIG. 31B shows the change over time of crystals produced using 1.0 mass% meviol gel following the lower part of FIG. 31A.
  • the number represented by “min” represents the exposure time (minutes) of the crystal to the atmosphere.
  • the crystal produced without the addition of gel started to dry on the surface after exposure time of 4 minutes, began to crack after exposure time of 22 minutes, Cracks occurred and almost completely collapsed after 36 minutes of exposure.
  • the coated crystal of the example coated with mebiol gel does not cause any deterioration such as cracking and drying even at an exposure time of 36 minutes, and even a small amount of crystals have fine cracks even at an exposure time of 41 minutes. Only entered.
  • the coated crystal produced by the production method of the present invention was extremely resistant to drying and suitable for storage because it was coated with a gel.
  • Example 28 Crystal production using mebiol gel
  • a mixed solution of lysozyme concentration 35 mg / mL, sodium chloride concentration 3% by mass, sodium acetate concentration 0.2M, mebiol gel concentration 2% by mass is kept at 12 ° C. and allowed to stand at 12 ° C. for about 1 week.
  • the crystal production method of the present invention was carried out. Thereafter, the temperature of the mixed solution was raised to 20 ° C. and converted (moved) into a gel state. It was observed whether there was a change such as damage to the crystal before and after this state change (movement). The observation result is shown in the photograph of FIG. The left is a photograph of the sol state (before movement), and the right is a photograph of the gel state (after movement). As shown, no change was observed in the crystal state before and after the movement. Further, similar results were obtained even when the meviol gel concentration was changed to 3% by mass.
  • FIG. 33 shows the results of X-ray crystal structure analysis of the crystals of Examples 27 and 28 in the table and photograph of FIG. X-ray crystal structure analysis was carried out in the same manner as in the previous examples.
  • the cryoprotectant was 2.5M lithium acetate and the immersion time was 15 minutes.
  • Number 1 in the table indicates a crystal (comparative example) produced without adding mebiol gel.
  • Numbers 2 and 3 show the crystals of Example 28 (2% and 3% by weight meviol gel).
  • Numbers 4 and 5 indicate the crystals of Example 27 (2% and 5% by weight meviol gel).
  • the photograph shows an X-ray diffraction image, and the numbers 1, 2, 3, and 5 attached to the photograph respectively correspond to the numbers in the table.
  • FIG. 34 shows the result. As shown on the left side of FIG. 34, a good crystal was obtained, and as shown on the right side, a good X-ray diffraction image and a low mosaic property (0.3178) were obtained.
  • the processed part (processed crystal) cut out by processing could be taken out using tweezers, cryoloop, etc., and used for X-ray crystal structure analysis.
  • the left side of FIG. 36 is a photograph showing a state in which the processed part (processed crystal) is being taken out
  • the right part of FIG. 36 is a photograph of the processed part (processed crystal) after being taken out.
  • FIG. 37 shows the X-ray crystal structure analysis result of the processed part.
  • the left side of FIG. 37 is a photograph showing the crystal and gel of the processed part
  • the right side is the resolution and mosaic property of each part shown on the left side of the figure. As shown in the figure, it was confirmed that the crystal included in the processed part has a good structure.
  • Such laser beam processing can be performed on crystals not covered with gel. According to this laser beam processing, non-contact processing can be performed in a sealed environment. Furthermore, since the molecular bond is cut by light energy, processing can be performed without thermal diffusion to the peripheral portion. In particular, the femtosecond laser absorbs light energy only in the vicinity of, for example, a few ⁇ m near the condensing point, so that precise three-dimensional processing or the like is possible. However, when a crystal that is present in the liquid and is not coated with a gel is processed with a laser beam, the crystal is not fixed with the gel and moves, so that accurate processing may be difficult. Moreover, since the crystal is not protected by the gel, it may be damaged.
  • the crystal used for laser beam processing is a coated crystal coated with a gel (gel-coated crystal)
  • these problems can be solved.
  • the gel-coated crystal is processed using laser light, the crystal can be processed and taken out more easily than the processing by physical means, and the gel coating has an advantage that the processing is not hindered.
  • the subject of both gel coat crystal and laser beam processing can be supplemented.
  • Example 29 Production of growth crystal using coated crystal as seed crystal
  • a part of the lysozyme crystal (agarose-III concentration: 1.0% by mass) coated with the agarose gel produced in the crystallization screening of Example 12 was cut out with the above-mentioned femtosecond laser as a seed crystal.
  • This seed crystal was put in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 60 days to grow the crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG.
  • the left figure is the gel-coated crystal of Example 12, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 60 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 1 mm. Such a large crystal can be used for various applications, and is suitable for, for example, neutron beam crystal structure analysis.
  • Example 30 Production of growth crystal using coated crystal as seed crystal
  • a part of the lysozyme crystal coated with meviol gel (meviol gel concentration: 5.0% by mass) produced in the crystallization screening of Example 27 was cut out with the femtosecond laser as a seed crystal.
  • This seed crystal was placed in an aqueous solution containing 3% by mass of sodium chloride and 20 mg / mL of lysozyme, and allowed to stand at room temperature for 4 days to grow a crystal, and the growth crystal production method of the present invention was carried out. This is shown in the photograph of FIG.
  • the left figure is the gel-coated crystal of Example 27, the middle figure is a seed crystal (processed crystal) that is partly cut by femtosecond laser processing, and the right figure is after growth for 4 days at room temperature. It is a grown crystal. As shown in the figure, growth in a solution yielded a large crystal that was much larger than the seed crystal and had a diameter of about 0.5 mm. Such a large crystal can be used for various applications as described in Example 29, and is suitable for, for example, neutron beam crystal structure analysis.
  • Example 31 Crystallization screening using concentration gradient by centrifugation
  • Crystallization screening was performed by forming a gradient in the concentration of the precipitating agent (precipitating agent) between the upper and lower parts inside the centrifuge tube using a centrifuge.
  • precipitating agent precipitating agent
  • a centrifuge tube 405 (manufactured by BD, trade name: BD Falcon Cell Culture Inserts) was prepared.
  • This centrifuge tube has a PET semipermeable membrane (dialysis membrane) attached to the bottom, and the semipermeable membrane allows only particles of a certain size or less to pass through without passing through solids.
  • a 2.0 mass% agarose aqueous solution (agarose type is Seaplaque) was placed in the centrifuge tube 405 and gelled.
  • aqueous solution containing 50 mg / mL of lysozyme, 4.0% by mass of sodium chloride, 0.1M sodium acetate, pH 4.5 was poured onto the upper surface of the gel. This was centrifuged at 20,000 rpm for 15 hours at 20 ° C. using a centrifuge (trade name Plate Spin, manufactured by Kubota Corporation), and the lysozyme solution was uniformly diffused in the gel.
  • a precipitating agent (4.0 mass% sodium chloride aqueous solution) was placed in a cylindrical container 406 that was thicker than the centrifuge tube 405 and closed at the bottom. Further, a centrifuge tube 405 containing the gel was put therein. When left at 20 ° C. for 2 days, the precipitating agent gradually permeates into the centrifuge tube 405 through the bottom PET film, and the concentration of the precipitating agent is high at the bottom and the concentration of the precipitating agent is low at the top. A lysozyme crystal was precipitated in the gel. In this way, crystals were precipitated in the gel to produce lysozyme crystals.
  • a precipitating agent 4.0 mass% sodium chloride aqueous solution
  • FIG. 41 shows the photograph. 41, the upper part of FIG. 41 shows the upper part inside the centrifuge tube 405 (cup), and the lower part of FIG. 41 shows the lower part inside the centrifuge tube 405 (cup) (PET membrane (membrane) side).
  • the number of precipitated crystals was large at the lower part of the cup (membrane side) where the precipitant concentration was high.
  • the number of precipitated crystals was small. That is, using centrifuging, a concentration gradient of the precipitating agent was formed between the upper part and the lower part inside the centrifuge container, and the crystallization conditions could be screened.
  • this Example is a screening of the crystallization conditions using the concentration gradient of a precipitating agent, the screening of the crystallization conditions by forming a protein concentration gradient can also be performed simply. The same applies to biological substances other than proteins.
  • the crystal manufacturing method of the present invention it is possible to easily manufacture a crystal of the biological substance, rather than precipitating the crystal from a solution. Moreover, according to the frozen crystal manufacturing method of the present invention, the crystal is not easily damaged by freezing. Furthermore, according to the growth crystal manufacturing method of the present invention, since the crystal is grown using the coated crystal coated with the gel as a seed crystal, a larger crystal can be obtained. In addition, the crystal of the present invention is manufactured by the crystal manufacturing method of the present invention, the frozen crystal manufacturing method of the present invention, or the grown crystal manufacturing method of the present invention, so that it is suitable for crystal structure analysis, for example. It has special characteristics. Furthermore, according to the structure analysis method of the present invention, since the structure analysis of the coated crystal coated with the gel or the frozen crystal frozen from the gel is performed, there is an advantage that the crystal is easy to handle.
  • the crystallization screening method of the present invention can easily produce crystals by the crystal production method of the present invention, it is not necessary to set the crystal production conditions so strictly, and thus screening can be performed easily. . Furthermore, the crystallization screening apparatus of the present invention can simplify the configuration of the apparatus for the same reason.
  • the crystal production method and frozen crystal production method of the present invention are useful for crystal structure analysis, particularly X-ray crystal structure analysis, and can also be applied to crystallization screening methods. Furthermore, the method for producing crystals and the method for producing frozen crystals of the present invention and the uses of the crystals and frozen crystals produced thereby are not limited to the above-mentioned uses, and can be used for all uses.

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  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Materials Engineering (AREA)
  • Metallurgy (AREA)
  • Organic Chemistry (AREA)
  • Dispersion Chemistry (AREA)
  • Crystals, And After-Treatments Of Crystals (AREA)
  • Analysing Materials By The Use Of Radiation (AREA)
  • Peptides Or Proteins (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

Cette invention concerne un procédé de production de cristaux caractérisé en ce qu'un cristal peut être produit facilement et en ce qu'un cristal produit au moyen dudit procédé est facile à manipuler. Spécifiquement, l'invention concerne un procédé de production d'un cristal à partir d'une substance biologique, ledit procédé comprenant une étape de cristallisation consistant à cristalliser la substance biologique, ladite substance biologique étant cristallisée en forme de gel lors de l'étape de cristallisation.
PCT/JP2009/050592 2008-01-17 2009-01-17 Procédé de production de cristaux, procédé de production de cristaux congelés, cristaux, méthode d'analyse structurelle des cristaux, méthode de contrôle de la cristallisation et appareil de contrôle de la cristallisation WO2009091053A1 (fr)

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WO2012099180A1 (fr) 2011-01-18 2012-07-26 国立大学法人大阪大学 Procédé de conversion de substance cible, procédé de fabrication de cristaux, procédé de fabrication de composition et dispositif de conversion de substance cible
WO2012131847A1 (fr) * 2011-03-25 2012-10-04 株式会社クニムネ Appareil de fabrication d'un cristal de polymère organique
JP2012254969A (ja) * 2011-05-18 2012-12-27 Institute Of Physical & Chemical Research タンパク質結晶製造方法
CN104391018A (zh) * 2014-10-22 2015-03-04 西北大学 三维dna纳米结构、电化学生物传感器及其制备方法和应用
US9182216B2 (en) 2010-09-22 2015-11-10 Osaka University Method for observing protein crystal
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JP2017071590A (ja) * 2015-10-09 2017-04-13 東レ株式会社 含繊維結晶、含繊維結晶の製造方法、含繊維結晶の製造装置および薬剤ソーキング装置
CN110607551A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种制备食品添加剂单晶或无定型物的方法
CN110607555A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种制备紫杉醇单晶或无定型物的方法
CN110607552A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种利用水溶液制备单晶或无定型物的方法
CN110735177A (zh) * 2018-10-30 2020-01-31 中国科学院化学研究所 一种利用溶液冻结制备单晶或无定型物的方法

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JP2011116591A (ja) * 2009-12-03 2011-06-16 Kunimune:Kk 有機高分子結晶製造装置
US9182216B2 (en) 2010-09-22 2015-11-10 Osaka University Method for observing protein crystal
WO2012099180A1 (fr) 2011-01-18 2012-07-26 国立大学法人大阪大学 Procédé de conversion de substance cible, procédé de fabrication de cristaux, procédé de fabrication de composition et dispositif de conversion de substance cible
US9751068B2 (en) 2011-01-18 2017-09-05 Osaka University Target substance transfer method, crystal production method, composition production method, and target substance transfer device
WO2012131847A1 (fr) * 2011-03-25 2012-10-04 株式会社クニムネ Appareil de fabrication d'un cristal de polymère organique
JPWO2012131847A1 (ja) * 2011-03-25 2014-07-24 株式会社クニムネ 有機高分子結晶製造装置
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JP2012254969A (ja) * 2011-05-18 2012-12-27 Institute Of Physical & Chemical Research タンパク質結晶製造方法
CN104391018A (zh) * 2014-10-22 2015-03-04 西北大学 三维dna纳米结构、电化学生物传感器及其制备方法和应用
CN104391018B (zh) * 2014-10-22 2017-01-18 西北大学 三维dna纳米结构、电化学生物传感器及其制备方法和应用
WO2016093231A1 (fr) * 2014-12-12 2016-06-16 東洋インキＳｃホールディングス株式会社 Article pour culture cellulaire, et polymère biocompatible
WO2017061314A1 (fr) * 2015-10-09 2017-04-13 東レ株式会社 Cristal contenant des fibres, son procédé de préparation, appareil de préparation d'un cristal contenant des fibres, et appareil de trempage utilisé en médecine
JP2017071590A (ja) * 2015-10-09 2017-04-13 東レ株式会社 含繊維結晶、含繊維結晶の製造方法、含繊維結晶の製造装置および薬剤ソーキング装置
CN108137647A (zh) * 2015-10-09 2018-06-08 东丽株式会社 含纤维晶体、含纤维晶体的制造方法、含纤维晶体的制造装置和化学试剂浸泡装置
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CN108137647B (zh) * 2015-10-09 2022-03-15 东丽株式会社 含纤维晶体、含纤维晶体的制造方法、含纤维晶体的制造装置和化学试剂浸泡装置
CN110607551A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种制备食品添加剂单晶或无定型物的方法
CN110607555A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种制备紫杉醇单晶或无定型物的方法
CN110607552A (zh) * 2018-10-30 2019-12-24 中国科学院化学研究所 一种利用水溶液制备单晶或无定型物的方法
CN110735177A (zh) * 2018-10-30 2020-01-31 中国科学院化学研究所 一种利用溶液冻结制备单晶或无定型物的方法
CN110607552B (zh) * 2018-10-30 2024-02-20 中国科学院化学研究所 一种利用水溶液制备单晶或无定型物的方法
CN110607555B (zh) * 2018-10-30 2024-02-20 中国科学院化学研究所 一种制备紫杉醇单晶或无定型物的方法
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