WO2011115223A1 - Method for searching for condition for crystallization of biopolymer, and device for the method - Google Patents

Method for searching for condition for crystallization of biopolymer, and device for the method Download PDF

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Publication number
WO2011115223A1
WO2011115223A1 PCT/JP2011/056429 JP2011056429W WO2011115223A1 WO 2011115223 A1 WO2011115223 A1 WO 2011115223A1 JP 2011056429 W JP2011056429 W JP 2011056429W WO 2011115223 A1 WO2011115223 A1 WO 2011115223A1
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solution
biopolymer
concentration
reagent
crystallization
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PCT/JP2011/056429
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French (fr)
Japanese (ja)
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秀行 宮武
直 堂前
真吾 恩田
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独立行政法人理化学研究所
日機装株式会社
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Priority to JP2012505751A priority Critical patent/JPWO2011115223A1/en
Publication of WO2011115223A1 publication Critical patent/WO2011115223A1/en

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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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D9/00—Crystallisation
    • B01D9/0077—Screening for crystallisation conditions or for crystal forms
    • C—CHEMISTRY; METALLURGY
    • C30—CRYSTAL GROWTH
    • C30B—SINGLE-CRYSTAL GROWTH; UNIDIRECTIONAL SOLIDIFICATION OF EUTECTIC MATERIAL OR UNIDIRECTIONAL DEMIXING OF EUTECTOID MATERIAL; REFINING BY ZONE-MELTING OF MATERIAL; PRODUCTION OF A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; SINGLE CRYSTALS OR HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; AFTER-TREATMENT OF SINGLE CRYSTALS OR A HOMOGENEOUS POLYCRYSTALLINE MATERIAL WITH DEFINED STRUCTURE; APPARATUS THEREFOR
    • C30B7/00—Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions

Definitions

  • the present invention relates to a biopolymer crystallization condition exploration method and an apparatus used therefor.
  • biopolymers including proteins is analyzed by crystallizing the biopolymer and subjecting it to an X-ray diffraction test.
  • biopolymers there are a wide variety of biopolymers, and the process for crystallization is not constant, and some biopolymers may not be crystallized.
  • Currently employed biopolymer crystallization methods are performed by trying a large number of crystallization conditions in a random permutation combination and rely heavily on irrational techniques that rely on chance.
  • crystal structure analysis is also a useful means used in drug design and development based on protein three-dimensional structure, and many protein structure analyzes are awaited.
  • the development of high-performance X-ray diffractometers for the analysis of complex crystal structures is progressing, but the crystallization method of vital biopolymers depends on trial and error and chance, and the protein crystal structure It becomes the rate-limiting factor of the whole analysis.
  • Patent Document 1 a method for producing a biopolymer crystal under the crystallization conditions found by the exploration method is also provided.
  • the method for exploring the crystallization conditions of a biopolymer from a biopolymer-containing solution described in Patent Document 1 changes the physicochemical properties of the solution to a solution separated from the biopolymer-containing solution by a semipermeable membrane. Continuously or intermittently adding a substance to be changed to continuously or intermittently change the physicochemical properties of the biopolymer-containing solution, during which the biopolymer dispersion state in the biopolymer-containing solution By continuously or intermittently monitoring the conditions for the biopolymer to exist in a monodisperse state, the biopolymer crystallization conditions Is a method of exploring.
  • Patent Document 2 Similar to the invention described in Patent Document 1, Patent Document 2 also stores a biopolymer-containing solution and a crystallization agent-containing solution in two chambers partitioned by a semipermeable membrane, respectively, through the semipermeable membrane.
  • a method for producing a biopolymer crystal is described in which crystal growth conditions are determined by detecting crystal growth due to penetration of a crystallizing agent into a biopolymer-containing solution, and crystal growth is performed under these conditions.
  • Patent Documents 1 and 2 The entire description of Patent Documents 1 and 2 is hereby specifically incorporated by reference.
  • a crystallization cell that is adjacent to the solution mixing chamber via a semipermeable membrane is used, and the protein in the crystallization cell is concentrated by the semipermeable membrane.
  • concentration of a substance for example, a precipitant
  • the concentration of protein, precipitant, etc. in the solution in the crystallization cell can be unevenly distributed in the vicinity of the semipermeable membrane and at a position away from the semipermeable membrane, resulting in hindering protein crystallization. Or, it may hinder the formation and growth of stable crystals, which is a big problem.
  • precipitation on the semipermeable membrane is likely to occur as the protein is concentrated, and there is a problem that the accuracy of the condition search is lowered due to the influence of this precipitation.
  • an object of the present invention is to change the physicochemical properties of the biopolymer-containing solution continuously or intermittently under the condition that the biopolymer such as protein exists in a stable dispersed state, so that the biopolymer is crystallized.
  • An object of the present invention is to provide a method and an apparatus for exploring the conditions for conversion to physicochemical properties while maintaining the uniformity of the concentration and temperature of the biopolymer-containing solution as much as possible.
  • a new system that internally circulates a biopolymer-containing solution such as protein is employed, and a low-molecular component such as a precipitant is contained in the solution using a semipermeable membrane unit installed in the circulation path.
  • a semipermeable membrane unit installed in the circulation path.
  • the present invention for solving the above problems is as follows. [1] Pour the solution into the circulation channel, At least one concentration of the biopolymer in the solution and at least one substance (hereinafter referred to as a reagent) that changes the physicochemical properties of the solution is continuously or intermittently kept constant in the circulation channel. And continuously or intermittently monitoring crystal formation in solution to explore the conditions under which the biopolymer crystals are formed, The capacity in the circulation channel is maintained constant by discharging a part of the solution in the channel to the outside of the channel through the semipermeable membrane as the solution is injected into the circulation channel. , Method for exploring biopolymer crystal formation conditions.
  • the circulation channel has an addition site for the biopolymer-containing solution, a discharge site through the semipermeable membrane, an addition site for the reagent-containing solution, a dispersion state monitoring site, and a crystal formation monitoring site in this order.
  • the method according to any one of [1] to [2], wherein the dispersion state of the biopolymer contained in the solution is monitored continuously or intermittently in parallel with the monitoring of crystal formation.
  • the dispersion state is monitored by measuring a scattered light intensity distribution by a dynamic light scattering method.
  • the monitor for observing the dispersion state of a biopolymer is a scattered light intensity distribution measurement cell by a dynamic light scattering method coupled to a scattered light intensity distribution measurement device by a dynamic light scattering method [12] Equipment.
  • the generation of the biopolymer is suppressed while suppressing the occurrence of precipitation on the semipermeable membrane.
  • Concentration is possible. For example, it is possible to change the concentration of the precipitant etc. almost uniformly throughout the circulating solution while keeping the concentration of the biopolymer constant, and as a result, more strictly biopolymer that could not be provided by the prior art.
  • the biopolymer can be placed under conditions where the concentration of the precipitant is controlled.
  • the scattered light intensity distribution can be measured by the dynamic light scattering method for the circulating solution, the dispersion state of the biopolymer in the circulating solution can be appropriately measured.
  • the dispersion state of the biopolymer in the circulating solution can be appropriately measured.
  • FIG. 1 is a conceptual diagram of a solution circulation type protein automatic crystallization apparatus using a scattered light intensity measurement distribution apparatus by a dynamic light scattering method.
  • FIG. 2a shows the change in the electrical conductivity in the path when the liquid B is injected into the path and the liquid A is injected.
  • FIG. 2b shows the change in conductivity in the path when lysozyme solution is injected into the path.
  • FIG. 3a shows the results of a crystallization experiment with lysozyme.
  • FIG. 3b shows the change in the degree of dispersion of lysozyme molecules during crystallization.
  • FIG. 4a shows the change in conductivity with respect to the number of measurements in a glucose isomerase crystallization experiment.
  • FIG. 4 b shows the change in ultraviolet absorption value with respect to the number of measurements in glucose isomerase crystallization experiments.
  • FIG. 4 c shows the scattered light intensity distribution at the time of concentration (866 times) until the ultraviolet absorbance in the crystallization experiment of glucose isomerase reaches 0.38 (19 mg / ml).
  • FIG. 4d shows the scattered light intensity distribution when 2.3 ml of the B solution was rapidly injected (867 times) in the crystallization experiment of glucose isomerase.
  • FIG. 4e shows the scattered light intensity distribution after 1080 measurements (a glucose isomerase crystal having a size of about 0.1 mm was obtained) in a glucose isomerase crystallization experiment. A photograph of a glucose isomerase crystal having a size of about 0.1 mm obtained by 1080 times of measurement in the crystallization experiment of glucose isomerase is shown.
  • the biopolymer crystal formation condition exploration method of the present invention At least one concentration of the biopolymer in the solution and at least one substance (reagent) that changes the physicochemical properties of the solution is continuously or intermittently maintained while the volume in the circulation channel is kept constant. Changing and monitoring the crystal formation in solution continuously or intermittently to explore the conditions under which the biopolymer crystals are formed, The volume of liquid in the circulation channel is kept constant by discharging a part of the solution in the channel through the semipermeable membrane as the solution is injected into the circulation channel. Is done.
  • the biopolymer for searching for crystal generation conditions is not particularly limited as long as it is a biopolymer that requires crystallization.
  • biopolymers include water-soluble proteins, membrane proteins, nucleic acid-protein complexes, and the like.
  • the biopolymer capable of exploring the crystal formation conditions by the method of the present invention is not particularly limited as long as it has a molecular weight that does not penetrate the semipermeable membrane used.
  • the semipermeable membrane to be used is not particularly limited, but any semipermeable membrane that can be used can be used, considering the molecular weight of the biopolymer for which the crystallization conditions are probed by the method of the present invention. Can be appropriately selected.
  • a regenerated cellulose membrane (5 kDa cut off, 10 kDa cut off, 30 kDa cut off, 50 kDa cut off, 100 kDa cut off) manufactured by VIVA SCIENCE may be used, but is not limited to these. Absent.
  • a semipermeable membrane having a cutoff molecular weight of 5000 it is possible to concentrate or maintain the concentration if the polymer has a weight average molecular weight of 10,000 or more.
  • the circulation flow path in the crystal generation condition exploration method of the present invention includes a biopolymer-containing solution addition site, a discharge site through a semipermeable membrane, a reagent-containing solution addition site, a dispersion state monitoring site, and a crystal formation monitoring site. In this order. It is preferable that the biopolymer-containing solution addition site is provided next to the crystal formation monitor site so that the biopolymer concentration in the circulation channel is uniform in the circulation system.
  • the crystal generation condition exploration apparatus of the present invention will be further described as an example.
  • the biopolymer crystallization condition search apparatus of the present invention is an apparatus that can be used in the crystallization condition search method of the present invention, and has the following configuration.
  • a constant-volume channel communicating between (1) to (5) and a pump for circulating the solution in the channel are provided.
  • This device monitors the dispersion state of the biopolymer according to the above (4) and (5) continuously or intermittently adjusts the concentration of the biopolymer and the concentration of the reagent in the solution flowing in the crystallization cell. It is used to search for the conditions under which biopolymers crystallize.
  • the scattered light intensity distribution by the dynamic light scattering method is used for the scattered light intensity measured by the dynamic light scattering method (hereinafter referred to as the scattering intensity measurement cell).
  • the scattering intensity measurement cell Guide to the measuring device and monitor the dispersion state of the protein particles in the scattered light intensity measurement cell from the scattered light intensity distribution.
  • the scattered light intensity distribution measurement system using the dynamic light scattering method uses an optical fiber probe that inserts an optical fiber directly into the scattered light intensity measurement cell, directly irradiates the particle group with laser light, and can directly receive the scattered light. (For example, Nikkiso Co., Ltd. NanotracUPA) is preferable.
  • the monitoring of the dispersion state of the biopolymer can be performed by, for example, a scattered light intensity distribution measurement by a dynamic light scattering method, as a method for analyzing the association state of the biopolymer particles in the solution. Can measure the dispersion state of the biopolymer more accurately by measuring the scattered light intensity distribution by the dynamic light scattering method using the frequency analysis method.
  • the dispersion state monitoring by the scattered light intensity distribution measurement by the dynamic light scattering method is performed, for example, by temporarily stopping the circulation of the solution and temporarily stopping the movement of the solution in the scattered light intensity measurement cell. .
  • the circulation of the solution is not stopped, and the dynamic light scattering intensity distribution measurement can be performed by controlling the circulation speed within a range that does not hinder the scattered light intensity measurement by the dynamic light scattering method.
  • a bypass is provided in parallel with the scattered light intensity measurement cell, and the solution is circulated through this bypass to temporarily stop the movement of the solution in the scattered light intensity measurement cell.
  • the flow rate is lowered so that the dispersion state can be monitored. Therefore, when monitoring the dispersion state of the biopolymer by the scattered light intensity distribution measurement by the dynamic light scattering method, the dispersion state of the biopolymer is changed while performing the operation related to the solution circulation.
  • the pump is a peristaltic pump from the viewpoint that it is not easily affected by solid matter such as precipitates and can stably feed liquid.
  • FIG. 1 shows a conceptual diagram of a crystallization condition exploration device of the present invention using a scattered light intensity distribution device by a dynamic light scattering method.
  • the case where protein is used as the biopolymer will be described below as an example.
  • the biopolymer-containing solution storage and input part of (1) can be a syringe pump 10 having a syringe filled with the biopolymer-containing solution.
  • the continuous dialysis membrane 20 using the semipermeable membrane of (2) can be discharged from the flow path 60 as a filtrate through the semipermeable membrane.
  • the reagent-containing solution storage and input unit 30 of (3) has one or a plurality of reagent-containing solution storage units, from one or more of these storage units, into the flow path 60 from the syringe pump,
  • the reagent-containing solution is injected through an electric or manual switching valve (electromagnetic valve 61 in the figure).
  • the biopolymer dispersion state monitor 40 of (4) can be, for example, a scattered light intensity measurement cell 42 coupled to a scattered light intensity distribution measuring device 41 by a dynamic light scattering method.
  • the crystallization cell 50 of (5) is installed in the flow path 60.
  • the cell internal volume is, for example, several tens of microliters to several hundreds of microliters.
  • the flow path 60 communicating between the above (1) to (5) has a constant volume, for example, a tube made of a material whose inner surface in contact with the circulating solution is inert to the solution and its contents. Can be. Although all of the flow path 60 can be formed of a tube made of the same material, a tube made of a different material can be appropriately used depending on the location.
  • the channel 60 can be, for example, a flexible silicone tube, PCV, polypropylene, or the like.
  • the pump 70 is a pump for circulating the solution in the flow path, and is preferably a peristaltic pump for the reason described above.
  • a peristaltic pump is used as the pump 70
  • the flow path 60 in the vicinity of the peristaltic pump is suitably a flexible resin tube.
  • the syringe pump which has a syringe filled with the biopolymer-containing solution is located upstream of the peristaltic pump from the viewpoint of preventing backflow to the syringe.
  • the protein solution is injected from the syringe pump 10 into the path.
  • the protein concentration in the pathway is concentrated by the continuous dialysis membrane 20 and circulated in the pathway by the peristaltic pump 70.
  • Reagents such as salts and buffers are injected from the reagent loading unit 30 while being controlled in the path.
  • the dispersion state of the protein solution can be monitored with almost no time delay by the scattered light intensity measurement cell 42 coupled to the scattered light intensity distribution measuring device 40 by the dynamic light scattering method. Crystallization is observed in the observation part 51 in the crystallization cell 50.
  • a series of operations is controlled by the PC 90, for example.
  • the concentration of the reagent in the path can be monitored by using, for example, a conductivity meter 80.
  • the installation position of the conductivity meter 80 is not particularly limited, but can also be installed at a position other than immediately downstream of the syringe pump 10 shown in FIG.
  • the solution flows through the system at a constant speed, and the solution is filtered through the continuous dialysis membrane 20.
  • the protein concentration gradually increases.
  • the reagent concentration is gradually increased while the protein concentration is kept constant.
  • a solution containing no reagent can be injected into the system from the reagent charging unit 30. In this case, the reagent concentration gradually decreases while the protein concentration is maintained constant. Filtration of the solution in the continuous dialysis membrane 20 is performed while the solution flows at a constant speed.
  • the solution circulation flow rate is sufficiently ensured compared to the reagent injection rate and protein solution injection rate, and the amount of various solution injections is extremely small compared to the amount of solution in the circulation path.
  • Changes in the reagent concentration and protein concentration in the solution near the dialysis membrane 20 are gentle, and the solution circulates constantly and maintains a stirring state except during measurement, so the concentration in the system is almost uniform. The state can be maintained, and as a result, the dispersion state of the protein is extremely unlikely to change locally.
  • the uniform solution composition means that the solution composition in the entire circulation path becomes the same within a minute order time.
  • the circulation rate of the solution is not particularly limited as long as the system can maintain a substantially uniform concentration state, and further depends on the capacity of the system, the cross-sectional area of the flow path, the type and capacity of the pump used, etc. Can be appropriately determined in consideration of a change in conditions to be given to the biopolymer-containing solution for crystallization.
  • the circulation rate of the solution can be, for example, in the range of 0.0001 to 2 ml / min.
  • the relationship between the type of tube used in the peristaltic lick pump, the discharge amount, and the head rotation speed (rpm) is disclosed as technical information by the manufacturer of the peristaltic lick pump. For example, see http://www.technosaurus.co.jp/product/mp-flow_1.htm. The entire description is hereby specifically incorporated by reference. According to the published technical information, when PVC 0.5 mm is used as a tube, the precipitating agent is rapidly injected (about 1 ml / min) when the rotation speed of the pump is set to a maximum of 50 rpm. Even so, the solution in the path can circulate at a circulation rate of about 1 ml / min.
  • the composition in the path can reach a uniform equilibrium after a few minutes.
  • the solution circulates at about 0.1 ml / min by operating the head speed at about 1 to 5 rpm. Therefore, it is possible to change the solution composition while maintaining sufficient uniformity in the path.
  • the substance (reagent) that changes the physicochemical properties of the solution can be, for example, at least one substance selected from the group consisting of a pH buffer, a precipitating agent, salts, additives, and water.
  • the pH buffering agent, the precipitating agent, the salts, and the additives can be appropriately selected from substances that have been used so far for crystallization of biopolymers.
  • pH buffer examples include sodium citrate, sodium acetate, sodium phosphate / potassium, MES, sodium cacodylate, HEPES, Tris-HCl and the like.
  • precipitating agent examples include ammonium sulfate, sodium chloride, lithium chloride, polyethylene glycol, 2-methyl-2,4-pentanediol and the like. However, these are not limited.
  • Examples of the salts include calcium chloride, magnesium chloride, zinc chloride, cobalt chloride, lithium sulfate, ammonium acetate, magnesium acetate, zinc acetate, cesium chloride and the like.
  • Examples of the additive include ethylene glycol, glycerol, urea, ethanol, propanol, isopropanol, EDTA, DDT, hexanediol, octyl glucoside and the like. However, these are not limited.
  • the temperature of the solution in the circulation path can be controlled to be constant by circulating a temperature-controlled liquid through a water jacket (not shown) in the crystallization cell, or a biopolymer.
  • the temperature can also be changed in parallel with the change in the concentration and the addition of the solution.
  • the search operation under the same conditions can be performed by changing the temperature.
  • the temperature of the circulation path can be adjusted by, for example, installing a part or all of the circulation path in the constant temperature bath (chamber) 100.
  • the solution temperature at which the method of the present invention is performed is not particularly limited, and may be in a range where freezing does not occur when lowering, and may be within a range where denaturation or the like does not occur in biopolymers when increased. .
  • it can be in the range of 0 to 40 ° C.
  • a biopolymer crystal having quality and size suitable for structural analysis can be produced using the crystallization conditions.
  • the biopolymer crystal can be produced by a conventional method. For example, a vapor diffusion method, a dialysis method, a batch method, and the like are known.
  • the obtained biopolymer crystal can be used for crystal structure analysis.
  • Crystal structure analysis can be performed, for example, according to the MAD method.
  • X-ray diffraction data of a crystal is measured using synchrotron radiation.
  • the synchrotron radiation that can be used for this purpose can be generated, for example, by the large synchrotron radiation facility SPring-8 / RIKEN beamline IBL44B2 (BL45XU).
  • Example 1 Using the characteristics of the apparatus using the continuous flow type semipermeable membrane described in FIG. 1, it was confirmed by experiments that the concentration of the precipitating agent and the like in the path can be arbitrarily changed continuously. The results are shown in FIG.
  • Solution A is a buffer solution for dissolving lysozyme, one of the biopolymers.
  • the composition is 0.1 M sodium acetate, pH 4.3, and solution B is a lysozyme crystallization agent. This is a solution in which sodium is dissolved to a concentration of 2.0 M. Liquid A and liquid B were respectively injected into the channels, and the electrical conductivity of the filtrate flowing out of the semipermeable membrane was measured.
  • B solution (0.1 M sodium acetate buffer, pH 4.3, 2.0 M NaCl) is injected with 5.2 ⁇ ml (at an injection rate of 5 ⁇ ml / h), and then 10 ⁇ ml (50 ⁇ ml / h Infused at the infusion rate.
  • the conductivity was saturated at about 14 S / m.
  • 20 ml of A buffer was injected (at an injection rate of 10 ml / h).
  • the conductivity changes smoothly, indicating that the system can freely change the composition while maintaining the uniformity of the composition in the pathway. It was.
  • C (V) A ⁇ exp [-V / Vo] + C ' A, C ': Constant Vo: Path capacity
  • Example 2 The apparatus used in Example 1 was actually assembled, and lysotium crystallization experiments were performed using lysotium as a protein sample. The results are shown in FIGS. 3a and 3b.
  • the horizontal axis represents the number of times of measurement (time), and the vertical axis represents the radius obtained by measuring the scattered light intensity distribution by the dynamic light scattering method of lysozyme molecules.
  • lysozyme was concentrated to a concentration of 34 mg / ml.
  • the concentration of the precipitant NaCl
  • the concentration of the precipitant NaCl
  • the concentration of the precipitant was increased from 0 to 1 M in 5 minutes
  • an increase in the radius obtained by measuring the scattered light intensity distribution by the dynamic light scattering method of protein molecules was observed.
  • the precipitant concentration was further increased gradually, the formation of lysozyme crystals was confirmed as shown in the photograph. This continuous photograph is a measurement result of 50 to 65 times, and each measurement was performed at 10 minute intervals.
  • the magnified photograph in which the crystal can be observed is the 55th measurement photograph. It can be seen that crystals of about 0.2 to 0.4 mm are formed (see scale).
  • MV is a volume average diameter
  • MN is a number average diameter
  • MA is an area average diameter.
  • Fig. 3b shows the change in the degree of dispersion of lysotium molecules during this crystallization process.
  • a signal detected by a scattered light intensity distribution measuring apparatus using a dynamic light scattering method is converted into a power spectrum (frequency vs signal intensity) by a frequency analysis method.
  • the vertical axis represents the signal intensity (the intensity of the scattered light from the particle group multiplied by the incident light intensity), and the horizontal axis represents the number of sections specific to the scattering intensity measuring apparatus corresponding to the frequency.
  • the curve depicted in FIG. 3b is obtained by logarithmically converting the power spectrum and dividing it into frequency segments as appropriate (hereinafter this graph is referred to as Raw-data).
  • This curve in Raw-data has a specific peak corresponding to the particle size, and the peak position (horizontal axis: channel corresponding to frequency) is a typical solute (single molecule or molecular combination) present in the liquid. It shows the characteristics of the size. The larger the particle size, the smaller the number of channels at the peak position. Conversely, the higher the number of channels, the smaller the diameter.
  • the number of measurements shown in the series in Fig. 3b indicates the number of times when the scattered light intensity distribution by the dynamic light scattering method is periodically measured, with the time when lysotium is first introduced into the system as the 0th time.
  • the measurement result (dispersion state of lysozyme) at each measurement number (time) is plotted in one graph.
  • the dispersion state of lysozyme without adding a precipitant is the measurement result of the 14th to 16th times in the figure, and the measurement result at this time is a smooth curve with a scattered light intensity distribution peaking around 68ch. Is drawn.
  • the dispersion state of lysozyme at the time when the precipitant was first introduced is the measurement result of the 17th time in the figure, and a single curve is drawn as in the case of measuring only lysozyme, but the peak position is slightly shifted to the low ch side. The signal intensity at the peak ch increased.
  • lysozyme crystals were finally produced while maintaining the shape of the scattered light intensity distribution (17th to 50th times). Since the 50th measurement, lysozyme was consumed for crystal formation and the concentration of lysozyme in the solution was lowered, so that the scattered light intensity decreased (60th).
  • Example 3 Similarly to Example 2, the apparatus used in Example 1 was actually assembled, and glucose isomerase crystallization experiments were performed using glucose isomerase as a protein sample.
  • the experimental conditions are as follows. ⁇ Glucose isomerase: manufactured by Hampton Research Molecular weight: 173,000 ⁇ Protein solution: 30 mg / ml glucose isomerase, 50 mM HEPES, pH 7.0, 1 mM MgCl 2 ⁇ Solution A: 50 mM HEPES, pH 7.0, 1 mM MgCl 2 ⁇ B solution (precipitant solution): 3M NH 4 (SO 4 ) 2 , 50 mM HEPES, pH 7.0, 1 mM MgCl 2 ⁇ Crystallization cell temperature: 20 °C
  • FIG. 4a shows the change in conductivity with respect to the number of measurements
  • FIG. 4b shows the change in ultraviolet absorption value with respect to the number of measurements.
  • the protein solution was injected at a rate of 0.05 ml / hour and the precipitant solution B at a rate of 0.1 ml / hour.
  • the ultraviolet absorbance was 0.515 and the conductivity was 16.2 S / m, and a glucose isomerase crystal with a size of about 0.1 mm was obtained as shown in the figure (photo is shown in FIG. 4f) .
  • the scattered light intensity distribution at that time also maintained stable dispersion as shown in FIG. 4e.
  • the present invention is useful in the field related to crystallization production of biopolymers.

Abstract

Disclosed are: a method for searching for a condition for the crystallization of a biopolymer such as a protein by continuously or intermittently varying a physicochemical property of a solution containing the biopolymer under such conductions where the biopolymer can be present in the solution stably in a monodispersed state, in which the variation in the physicochemical property can be achieved while retaining the uniformity of the biopolymer-containing solution as possible; and a device. Specifically disclosed are: a method for searching for a condition for the production of crystals of a biopolymer, which comprises allowing a solution to flow in a circulation flow path, continuously or intermittently varying the concentration of at least one component selected from a biopolymer contained in the solution and at least one substance (referred to as "reagent", hereinbelow) capable of alternating a physicochemical property of the solution while maintaining the volume of the solution in the circulation flow path at a constant level, and continuously or intermittently monitoring the production of the crystals in the solution to find a condition under which the crystals of the biopolymer can be produced, wherein the volume of the solution in the circulation flow path can be maintained at a constant level by injecting the solution into the circulation flow path while discharging a part of the solution in the flow path to the outside of the flow path through a semipermeable film; and a device which can be used for the searching method.

Description

生体高分子の結晶化条件探査方法及びそれに用いる装置Biopolymer crystallization condition exploration method and apparatus used therefor 関連出願の相互参照Cross-reference of related applications
　本出願は、２０１０年３月１８日出願の日本特願２０１０－６２８６２号の優先権を主張し、その全記載は、ここに特に開示として援用される。 This application claims the priority of Japanese Patent Application No. 2010-62862 filed on Mar. 18, 2010, the entire description of which is specifically incorporated herein by reference.
　本発明は、生体高分子の結晶化条件探査方法及びそれに用いる装置に関する。 The present invention relates to a biopolymer crystallization condition exploration method and an apparatus used therefor.
　タンパク質を始めとした生体高分子の立体構造は、生体高分子を結晶化し、Ｘ線回折試験に付すことにより解析される。しかし、生体高分子は多種多様であり、その結晶化へのプロセスは一定ではなく、生体高分子によっては結晶化しない場合もある。現在採用されている生体高分子の結晶化方法は、非常に多数の結晶化条件をランダムな順列組合せで試みることにより行われ、多分に偶然性に依存する非合理的手法に依存している。 The three-dimensional structure of biopolymers including proteins is analyzed by crystallizing the biopolymer and subjecting it to an X-ray diffraction test. However, there are a wide variety of biopolymers, and the process for crystallization is not constant, and some biopolymers may not be crystallized. Currently employed biopolymer crystallization methods are performed by trying a large number of crystallization conditions in a random permutation combination and rely heavily on irrational techniques that rely on chance.
　ポストゲノム時代を迎えた現在、結晶構造解析はタンパク質の立体構造を基にした薬剤設計、開発等でも用いられる有用な手段でもあり、多くのタンパク質の構造解析が待たれている。複雑な結晶構造の解析のために、高性能のＸ線回折装置の開発も進んでいるものの、肝心な生体高分子の結晶化方法が、試行錯誤と偶然性に依存しており、タンパク質の結晶構造解析全体の律速要因となっている。 In the post-genomic era, crystal structure analysis is also a useful means used in drug design and development based on protein three-dimensional structure, and many protein structure analyzes are awaited. The development of high-performance X-ray diffractometers for the analysis of complex crystal structures is progressing, but the crystallization method of vital biopolymers depends on trial and error and chance, and the protein crystal structure It becomes the rate-limiting factor of the whole analysis.
　そこで本発明者らは、先に、比較的少量の生体高分子であっても、より確実に、かつより簡便に生体高分子の結晶化条件を探査できる方法及び装置を提供することに成功し、さらに、前記探査方法により見いだされた結晶化条件において生体高分子の結晶を製造する方法も提供した(特許文献１)。 Therefore, the present inventors have previously succeeded in providing a method and apparatus that can probe the crystallization conditions of a biopolymer more reliably and more easily even with a relatively small amount of biopolymer. Furthermore, a method for producing a biopolymer crystal under the crystallization conditions found by the exploration method is also provided (Patent Document 1).
　特許文献1に記載の生体高分子含有溶液からの生体高分子の結晶化条件を探査する方法は、生体高分子含有溶液と半透膜で隔てられた溶液に、溶液の物理化学的性質を変化させる物質を連続的または断続的に添加して、前記生体高分子含有溶液の物理化学的性質を連続的または断続的に変化させ、その間の、生体高分子含有溶液中の生体高分子の分散状態を、連続的または断続的にモニタリングすることで、生体高分子が安定に単分散状態で存在する条件を検索し、生体高分子が安定に単分散状態する条件で、生体高分子の結晶化条件を探査する方法である。 The method for exploring the crystallization conditions of a biopolymer from a biopolymer-containing solution described in Patent Document 1 changes the physicochemical properties of the solution to a solution separated from the biopolymer-containing solution by a semipermeable membrane. Continuously or intermittently adding a substance to be changed to continuously or intermittently change the physicochemical properties of the biopolymer-containing solution, during which the biopolymer dispersion state in the biopolymer-containing solution By continuously or intermittently monitoring the conditions for the biopolymer to exist in a monodisperse state, the biopolymer crystallization conditions Is a method of exploring.
　特許文献１に記載の発明と同様に、特許文献2にも、半透膜で仕切られた2つの室に生体高分子含有溶液と結晶化剤含有溶液をそれぞれ格納し、半透膜を介して、結晶化剤の生体高分子含有溶液への浸透による結晶成長を検出することで結晶成長の条件を求め、この条件により結晶育成を行う生体高分子結晶の製造方法が記載されている。 Similar to the invention described in Patent Document 1, Patent Document 2 also stores a biopolymer-containing solution and a crystallization agent-containing solution in two chambers partitioned by a semipermeable membrane, respectively, through the semipermeable membrane. A method for producing a biopolymer crystal is described in which crystal growth conditions are determined by detecting crystal growth due to penetration of a crystallizing agent into a biopolymer-containing solution, and crystal growth is performed under these conditions.
特開2004-45169号公報Japanese Patent Laid-Open No. 2004-45169 特開2004-26528号公報JP 2004-26528 A
特許文献１および２の全記載は、ここに特に開示として援用される。 The entire description of Patent Documents 1 and 2 is hereby specifically incorporated by reference.
　特許文献１及び２に記載の方法では、具体的には、半透膜を介して溶液混合室と隣接している結晶化セルを用い、前記半透膜により、結晶化セル内のタンパク質を濃縮し、あるいは結晶化セル内の溶液の物理化学的性質を変化させる物質(例えば、沈殿剤)の濃度を変化させている。そのため、結晶化セル内の溶液中のタンパク質や沈殿剤等の濃度に、半透膜近傍と半透膜から離れた位置で、不均一な濃度分布ができ、結果として、タンパク質の結晶化を妨げ、あるいは安定的な結晶の形成や成長を妨げることがあり、大きな問題となっていた。また、タンパク質の濃縮に伴い、半透膜上での沈殿も生じやすいことも懸念され、この沈殿の影響によって条件探査の精度が下がるという問題点があった。 In the methods described in Patent Documents 1 and 2, specifically, a crystallization cell that is adjacent to the solution mixing chamber via a semipermeable membrane is used, and the protein in the crystallization cell is concentrated by the semipermeable membrane. Alternatively, the concentration of a substance (for example, a precipitant) that changes the physicochemical properties of the solution in the crystallization cell is changed. Therefore, the concentration of protein, precipitant, etc. in the solution in the crystallization cell can be unevenly distributed in the vicinity of the semipermeable membrane and at a position away from the semipermeable membrane, resulting in hindering protein crystallization. Or, it may hinder the formation and growth of stable crystals, which is a big problem. In addition, there is a concern that precipitation on the semipermeable membrane is likely to occur as the protein is concentrated, and there is a problem that the accuracy of the condition search is lowered due to the influence of this precipitation.
　そこで、本発明の目的は、タンパク質等の生体高分子が安定な分散状態で存在する条件で生体高分子含有溶液の物理化学的性質を連続的または断続的に変化させて、生体高分子が結晶化する条件を探査する方法であって、前記生体高分子含有溶液の濃度、温度などの均一性を極力維持しながら物理化学的性質の変化を実施できる方法および装置を提供することにある。 Therefore, an object of the present invention is to change the physicochemical properties of the biopolymer-containing solution continuously or intermittently under the condition that the biopolymer such as protein exists in a stable dispersed state, so that the biopolymer is crystallized. An object of the present invention is to provide a method and an apparatus for exploring the conditions for conversion to physicochemical properties while maintaining the uniformity of the concentration and temperature of the biopolymer-containing solution as much as possible.
　本発明では、新たにタンパク質等の生体高分子含有溶液を内部循環させる方式を採用し、かつ、循環経路に設置された半透膜ユニットを用いて溶液中の沈殿剤等の低分子成分を含有する溶液を系外に排出することで、溶液中の生体高分子及び沈殿剤等の濃度の均一性を維持しながら、半透膜上での沈殿発生を抑制しつつ、生体高分子の濃縮を可能にした。かつ生体高分子の濃度を一定に保ちつつ沈殿剤等の濃度を自在に変化させることができることを見出し、または溶液中のタンパク質粒子の分散状態をモニタリングする為の工程も更に含めることにより、前記先行技術が有する課題を解決できることを見出して本発明を完成させた。 In the present invention, a new system that internally circulates a biopolymer-containing solution such as protein is employed, and a low-molecular component such as a precipitant is contained in the solution using a semipermeable membrane unit installed in the circulation path. By discharging the solution to the outside of the system, while maintaining the uniformity of the concentration of the biopolymer and the precipitant in the solution, the biopolymer is concentrated while suppressing the occurrence of precipitation on the semipermeable membrane. Made possible. In addition, it is found that the concentration of the precipitant can be freely changed while keeping the concentration of the biopolymer constant, or further including a step for monitoring the dispersion state of the protein particles in the solution. The present invention has been completed by finding that the problems of the technology can be solved.
　前記課題を解決するための本発明は以下のとおりである。
［1］
循環流路内に溶液を流し、
該溶液中の生体高分子および溶液の物理化学的性質を変化させる少なくとも1種の物質(以下、試薬と呼ぶ)の少なくとも一つの濃度を連続的または断続的に、循環流路内の容量を一定に維持しつつ変化させ、かつ溶液中の結晶生成を連続的または断続的にモニタリングして、前記生体高分子の結晶が生成する条件を探査することを含み、
前記循環流路内の容量は、循環流路内への溶液注入に伴って、流路内の溶液の一部を、半透膜を介して流路外に排出することで一定に維持される、生体高分子の結晶生成条件探査方法。
［2］
循環流路は、生体高分子含有溶液の添加部位、半透膜を介しての排出部位、試薬含有溶液の添加部位、分散状態モニター部位及び結晶生成モニター部位をこの順に有し、前記結晶生成モニター部位の次に前記生体高分子含有溶液の添加部位を有する［1］に記載の方法。
［3］
結晶生成のモニタリングと並行して溶液に含有される生体高分子の分散状態が連続的または断続的にモニタリングされる、［1］～[2]のいずれかに記載の方法。
［4］
分散状態のモニタリングは、動的光散乱法による散乱光強度分布測定により行う、［3］に記載の方法。
［5］
溶液は流路中をペリスタルティックポンプで循環する、［1］～[4]のいずれかに記載の方法。
［6］
ペリスタルティックポンプによる送液は、半透膜を介しての排出部位と試薬含有溶液の添加部位の間で行われる、［5］に記載の方法。
［7］
溶液中の生体高分子の濃度変化は、流路内の溶液に生体高分子含有溶液を注入することで行い、溶液中の試薬の濃度変化は、流路内の溶液に試薬含有溶液または試薬を含有しない溶液を注入することで行う、［1］～[6]のいずれかに記載の方法。
［8］
溶液中の生体高分子の濃度変化及び試薬の濃度変化は、独立に行われる［1］～[7]のいずれかに記載の方法。
［9］
溶液中の生体高分子の濃度を一定に維持しつつ、溶液中の試薬の濃度を変化させて結晶生成のモニタリングを行う［1］～[8]のいずれかに記載の方法。
［10］
循環溶液中の試薬濃度の変化は、濃度の上昇または減少である、［1］～[9]のいずれかに記載の方法。
［11］
生体高分子が、水溶性タンパク質、膜タンパク質、及び核酸－タンパク質複合体から成る群から選ばれる少なくとも1種である、［1］～[10]のいずれかに記載の方法。
［12］
［1］～[11]のいずれかに記載の生体高分子の結晶化条件探査方法に用いられる装置であって、
(1)生体高分子含有溶液貯蔵及び投入部、
(2)半透膜を用いた連続透析膜、
(3)物理化学的性質を変化させる物質(以下、試薬と呼ぶ)含有溶液貯蔵及び投入部、
(4)生体高分子の分散状態観察用のモニター、及び
(5)結晶化セル、
上記(1)～(5)の間を連絡する容量一定の流路、並びに
流路内に溶液を流通させるためのポンプを有し、
前記(4)による生体高分子の分散状態のモニタリング及び(5)結晶化セル内を流れる溶液中の前記生体高分子濃度及び前記試薬の少なくとも一つの濃度を連続的または断続的に変化させて、生体高分子が結晶化する条件を探査するために用いられる前記装置。
［13］
(4)生体高分子の分散状態観察用のモニターは、動的光散乱法による散乱光強度分布測定装置に結合された動的光散乱法による散乱光強度分布測定セルである［12］に記載の装置。
［14］
ポンプは、ペリスタルティックポンプである［12］または[13]に記載の装置。 The present invention for solving the above problems is as follows.
[1]
Pour the solution into the circulation channel,
At least one concentration of the biopolymer in the solution and at least one substance (hereinafter referred to as a reagent) that changes the physicochemical properties of the solution is continuously or intermittently kept constant in the circulation channel. And continuously or intermittently monitoring crystal formation in solution to explore the conditions under which the biopolymer crystals are formed,
The capacity in the circulation channel is maintained constant by discharging a part of the solution in the channel to the outside of the channel through the semipermeable membrane as the solution is injected into the circulation channel. , Method for exploring biopolymer crystal formation conditions.
[2]
The circulation channel has an addition site for the biopolymer-containing solution, a discharge site through the semipermeable membrane, an addition site for the reagent-containing solution, a dispersion state monitoring site, and a crystal formation monitoring site in this order. The method according to [1], wherein the biopolymer-containing solution addition site is located next to the site.
[3]
The method according to any one of [1] to [2], wherein the dispersion state of the biopolymer contained in the solution is monitored continuously or intermittently in parallel with the monitoring of crystal formation.
[Four]
The method according to [3], wherein the dispersion state is monitored by measuring a scattered light intensity distribution by a dynamic light scattering method.
[Five]
The method according to any one of [1] to [4], wherein the solution is circulated in the flow path by a peristaltic pump.
[6]
The method according to [5], wherein the liquid transfer by the peristaltic pump is performed between the discharge site through the semipermeable membrane and the addition site of the reagent-containing solution.
[7]
The concentration of the biopolymer in the solution is changed by injecting the solution containing the biopolymer into the solution in the flow channel, and the concentration change of the reagent in the solution is changed by adding the reagent-containing solution or the reagent The method according to any one of [1] to [6], which is carried out by injecting a solution that does not contain.
[8]
The method according to any one of [1] to [7], wherein the concentration change of the biopolymer and the concentration change of the reagent in the solution are performed independently.
[9]
The method according to any one of [1] to [8], wherein the crystal formation is monitored by changing the concentration of the reagent in the solution while maintaining the concentration of the biopolymer in the solution constant.
[Ten]
The method according to any one of [1] to [9], wherein the change in the reagent concentration in the circulating solution is an increase or decrease in the concentration.
[11]
The method according to any one of [1] to [10], wherein the biopolymer is at least one selected from the group consisting of a water-soluble protein, a membrane protein, and a nucleic acid-protein complex.
[12]
[1] An apparatus used for the biopolymer crystallization condition exploration method according to any one of [1] to [11],
(1) Biopolymer-containing solution storage and input section,
(2) continuous dialysis membrane using semipermeable membrane,
(3) Solution storage and input unit containing a substance (hereinafter referred to as a reagent) that changes physicochemical properties,
(4) a monitor for observing the dispersion state of a biopolymer, and
(5) crystallization cell,
A constant-volume channel communicating between the above (1) to (5), and a pump for circulating the solution in the channel,
Monitoring the dispersion state of the biopolymer according to (4) and (5) continuously or intermittently changing the concentration of the biopolymer and the concentration of the reagent in the solution flowing through the crystallization cell, The apparatus used for exploring conditions for crystallization of biopolymers.
[13]
(4) The monitor for observing the dispersion state of a biopolymer is a scattered light intensity distribution measurement cell by a dynamic light scattering method coupled to a scattered light intensity distribution measurement device by a dynamic light scattering method [12] Equipment.
[14]
The device according to [12] or [13], wherein the pump is a peristaltic pump.
　本発明によれば、上記構成を採用することで、溶液中の生体高分子及び沈殿剤等の濃度の均一性を維持しながら、半透膜上での沈殿発生を抑制しつつ生体高分子の濃縮が可能になる。例えば、生体高分子の濃度を一定に保ちつつ沈殿剤等の濃度を、循環する溶液全体でほぼ均一に変化させることもでき、その結果、先行技術では提供できなかった、より厳密に生体高分子および沈殿剤濃度が制御された条件に生体高分子を置くことが可能になる。さらに、本発明では、循環する溶液について動的光散乱法による散乱光強度分布測定を行うことができるため、循環溶液中の生体高分子の分散状態を適宜測定できる。これらの構成の結果、先行技術では探索することが難しかった非常に狭い組成領域でのみ結晶化し得る生体高分子であっても、結晶化条件を探索することが可能になる。 According to the present invention, by adopting the above configuration, while maintaining the uniformity of the concentration of the biopolymer and the precipitant in the solution, the generation of the biopolymer is suppressed while suppressing the occurrence of precipitation on the semipermeable membrane. Concentration is possible. For example, it is possible to change the concentration of the precipitant etc. almost uniformly throughout the circulating solution while keeping the concentration of the biopolymer constant, and as a result, more strictly biopolymer that could not be provided by the prior art. In addition, the biopolymer can be placed under conditions where the concentration of the precipitant is controlled. Furthermore, in the present invention, since the scattered light intensity distribution can be measured by the dynamic light scattering method for the circulating solution, the dispersion state of the biopolymer in the circulating solution can be appropriately measured. As a result of these configurations, it is possible to search for crystallization conditions even for biopolymers that can be crystallized only in a very narrow composition region, which was difficult to search with the prior art.
図１は、動的光散乱法による散乱光強度測定分布装置を利用した、溶液循環型タンパク質自動結晶化装置の概念図である。FIG. 1 is a conceptual diagram of a solution circulation type protein automatic crystallization apparatus using a scattered light intensity measurement distribution apparatus by a dynamic light scattering method. 図２ａは、経路内にＢ液を注入した際及びＡ液を注入した際の経路内の電導率の変化を示す。FIG. 2a shows the change in the electrical conductivity in the path when the liquid B is injected into the path and the liquid A is injected. 図２ｂは、経路内にリゾチウム溶液を注入した際の経路内の電導率の変化を示す。FIG. 2b shows the change in conductivity in the path when lysozyme solution is injected into the path. 図３ａは、リゾチウムによる結晶化実験の結果を示す。FIG. 3a shows the results of a crystallization experiment with lysozyme. 図３ｂは、結晶化中のリゾチウム分子の分散度の変化を示す。FIG. 3b shows the change in the degree of dispersion of lysozyme molecules during crystallization. 図４ａは、グルコースイソメラーゼの結晶化実験における測定回数に対する導電率変化を示す。FIG. 4a shows the change in conductivity with respect to the number of measurements in a glucose isomerase crystallization experiment. 図４ｂは、グルコースイソメラーゼの結晶化実験における測定回数に対する紫外吸収値変化を示す。FIG. 4 b shows the change in ultraviolet absorption value with respect to the number of measurements in glucose isomerase crystallization experiments. 図４ｃは、グルコースイソメラーゼの結晶化実験における紫外吸光度が0.38 (19mg/ml) になるまで濃縮した（866回）時点での散乱光強度分布を示す。FIG. 4 c shows the scattered light intensity distribution at the time of concentration (866 times) until the ultraviolet absorbance in the crystallization experiment of glucose isomerase reaches 0.38 (19 mg / ml). 図４ｄは、グルコースイソメラーゼの結晶化実験において2.3mlのB溶液を急速注入した（867回）際の散乱光強度分布を示す。FIG. 4d shows the scattered light intensity distribution when 2.3 ml of the B solution was rapidly injected (867 times) in the crystallization experiment of glucose isomerase. 図４ｅは、グルコースイソメラーゼの結晶化実験において測定回数1080回（0.1mm程度の大きさのグルコースイソメラーゼ結晶が得られた）における散乱光強度分布を示す。FIG. 4e shows the scattered light intensity distribution after 1080 measurements (a glucose isomerase crystal having a size of about 0.1 mm was obtained) in a glucose isomerase crystallization experiment. グルコースイソメラーゼの結晶化実験において測定回数1080回で得られた0.1mm程度の大きさのグルコースイソメラーゼ結晶の写真を示す。A photograph of a glucose isomerase crystal having a size of about 0.1 mm obtained by 1080 times of measurement in the crystallization experiment of glucose isomerase is shown.
[生体高分子の結晶生成条件探査方法]
本発明の生体高分子の結晶生成条件探査方法
　循環流路内に溶液を流し、
該溶液中の生体高分子および溶液の物理化学的性質を変化させる少なくとも1種の物質(試薬)の少なくとも一つの濃度を連続的または断続的に、循環流路内の容量を一定に維持しつつ変化させ、かつ溶液中の結晶生成を連続的または断続的にモニタリングして、前記生体高分子の結晶が生成する条件を探査することを含み、
前記循環流路内の容液量は、循環流路内への溶液注入に伴って、流路内の溶液の一部を、半透膜を介して流路外に排出することで一定に維持される。 [Probing method for crystal formation conditions of biopolymers]
The biopolymer crystal formation condition exploration method of the present invention.
At least one concentration of the biopolymer in the solution and at least one substance (reagent) that changes the physicochemical properties of the solution is continuously or intermittently maintained while the volume in the circulation channel is kept constant. Changing and monitoring the crystal formation in solution continuously or intermittently to explore the conditions under which the biopolymer crystals are formed,
The volume of liquid in the circulation channel is kept constant by discharging a part of the solution in the channel through the semipermeable membrane as the solution is injected into the circulation channel. Is done.
　本発明の結晶生成条件探査方法において、結晶生成条件を探査する生体高分子は、結晶化を要する生体高分子であれば特に制限はない。生体高分子としては、例えば、水溶性タンパク質、膜タンパク質、核酸－タンパク質複合体などを挙げることができる。 In the crystal generation condition search method of the present invention, the biopolymer for searching for crystal generation conditions is not particularly limited as long as it is a biopolymer that requires crystallization. Examples of biopolymers include water-soluble proteins, membrane proteins, nucleic acid-protein complexes, and the like.
　本発明の方法で結晶生成条件を探査できる生体高分子は、使用する半透膜を透過しない分子量を有するものであれば、特に制限はない。一方、使用する半透膜にも特に制限はないが、入手可能な半透膜であればいずれも使用でき、本発明の方法で結晶化条件を探査する生体高分子の分子量等を考慮して、適宜選択することができる。例えば、半透膜としては、ＶＩＶＡ ＳＣＩＥＮＣＥ社製の再生セルロース膜（５ｋＤａ ｃｕｔ ｏｆｆ、１０ｋＤａ ｃｕｔ ｏｆｆ、３０ｋＤａ ｃｕｔ ｏｆｆ、５０ｋＤａ ｃｕｔ ｏｆｆ、１００ｋＤａ ｃｕｔ ｏｆｆ）等を使用できるが、これらに限定されるものではない。例えば、カットオフ分子量が5000の半透膜を用いる場合、重量平均分子量が10,000以上の高分子であれば、濃縮または濃度の維持が可能である。 The biopolymer capable of exploring the crystal formation conditions by the method of the present invention is not particularly limited as long as it has a molecular weight that does not penetrate the semipermeable membrane used. On the other hand, the semipermeable membrane to be used is not particularly limited, but any semipermeable membrane that can be used can be used, considering the molecular weight of the biopolymer for which the crystallization conditions are probed by the method of the present invention. Can be appropriately selected. For example, as the semipermeable membrane, a regenerated cellulose membrane (5 kDa cut off, 10 kDa cut off, 30 kDa cut off, 50 kDa cut off, 100 kDa cut off) manufactured by VIVA SCIENCE may be used, but is not limited to these. Absent. For example, when a semipermeable membrane having a cutoff molecular weight of 5000 is used, it is possible to concentrate or maintain the concentration if the polymer has a weight average molecular weight of 10,000 or more.
　前記本発明の結晶生成条件探査方法における循環流路は、生体高分子含有溶液の添加部位、半透膜を介しての排出部位、試薬含有溶液の添加部位、分散状態モニター部位及び結晶生成モニター部位をこの順に有する。循環流路中の生体高分子濃度が循環系内で均一になるように、前記結晶生成モニター部位の次に前記生体高分子含有溶液の添加部位を有することが好ましい。以下、本発明の結晶生成条件探査装置を例にさらに説明する。 The circulation flow path in the crystal generation condition exploration method of the present invention includes a biopolymer-containing solution addition site, a discharge site through a semipermeable membrane, a reagent-containing solution addition site, a dispersion state monitoring site, and a crystal formation monitoring site. In this order. It is preferable that the biopolymer-containing solution addition site is provided next to the crystal formation monitor site so that the biopolymer concentration in the circulation channel is uniform in the circulation system. Hereinafter, the crystal generation condition exploration apparatus of the present invention will be further described as an example.
[生体高分子の結晶化条件探査装置]
　本発明の生体高分子の結晶化条件探査装置は、本発明の結晶化条件探査方法に用いることができる装置であって、以下の構成を有する。
(1)生体高分子含有溶液貯蔵及び投入部、
(2)半透膜を用いた連続透析膜、
(3)物理化学的性質を変化させる物質(試薬と呼ぶ)含有溶液貯蔵及び投入部、
(4)生体高分子の分散状態観察用のモニター、及び
(5)結晶化セル、
　前記(1)～(5)の間を連絡する容量一定の流路、並びに
流路内に溶液を流通させるためのポンプを有する。
　この装置は、前記(4)による生体高分子の分散状態のモニタリング及び(5)結晶化セル内を流れる溶液中の前記生体高分子濃度及び前記試薬の少なくとも一つの濃度を連続的または断続的に変化させて、生体高分子が結晶化する条件を探査するために用いられる。 [Biopolymer crystallization condition exploration equipment]
The biopolymer crystallization condition search apparatus of the present invention is an apparatus that can be used in the crystallization condition search method of the present invention, and has the following configuration.
(1) Biopolymer-containing solution storage and input section,
(2) continuous dialysis membrane using semipermeable membrane,
(3) A solution storage and input unit containing a substance (referred to as a reagent) that changes physicochemical properties,
(4) a monitor for observing the dispersion state of the biopolymer, and
(5) crystallization cell,
A constant-volume channel communicating between (1) to (5) and a pump for circulating the solution in the channel are provided.
This device monitors the dispersion state of the biopolymer according to the above (4) and (5) continuously or intermittently adjusts the concentration of the biopolymer and the concentration of the reagent in the solution flowing in the crystallization cell. It is used to search for the conditions under which biopolymers crystallize.
　さらに、本発明の装置においては、(4)動的光散乱法による散乱光強度測定セル（以下、散乱強度測定セルと記す）で測定された散乱光を動的光散乱法による散乱光強度分布測定装置に導き、散乱光の強度分布から散乱光強度測定セルの中のタンパク質粒子の分散状態をモニタリングする。動的光散乱法による散乱光強度分布測定装置としては、光ファイバーを直接散乱光強度測定セルに挿入し、粒子群に直接レーザ光を照射し、さらに散乱光を直接受光できる光ファイバープローブを利用する方式（例えば、日機装（株）社製　NanotracUPA ）が好ましい。 Further, in the apparatus of the present invention, (4) the scattered light intensity distribution by the dynamic light scattering method is used for the scattered light intensity measured by the dynamic light scattering method (hereinafter referred to as the scattering intensity measurement cell). Guide to the measuring device and monitor the dispersion state of the protein particles in the scattered light intensity measurement cell from the scattered light intensity distribution. The scattered light intensity distribution measurement system using the dynamic light scattering method uses an optical fiber probe that inserts an optical fiber directly into the scattered light intensity measurement cell, directly irradiates the particle group with laser light, and can directly receive the scattered light. (For example, Nikkiso Co., Ltd. NanotracUPA) is preferable.
　本発明では、生体高分子の分散状態をモニタリングしながら生体高分子が結晶化する条件を探査することで、生体高分子の分散状態と生体高分子の結晶化を関連づけて把握することができるとの利点がある。さらに、生体高分子の分散状態のモニタリングについては、溶液中での生体高分子の粒子の会合状態を解析する方法として、例えば動的光散乱法による散乱光強度分布測定で行うことができ、好ましくは周波数解析法を使用した動的光散乱法による散乱光強度分布測定で行うことで、生体高分子の分散状態をより正確に評価できる。尚、動的光散乱法による散乱光強度分布測定による分散状態のモニタリングは、例えば、溶液の循環を一時的に停止して散乱光強度測定セル内の溶液の動きを一時的に停止して行う。あるいは、溶液の循環は停止せずに、動的光散乱法による散乱光強度測定に支障のない範囲に循環速度を制御して、動的光散乱強度分布測定を行うこともできる。さらにまた、溶液の循環は維持するが、散乱光強度測定セルと並行してバイパスを設けて、このバイパスを介して溶液は流通させ、散乱光強度測定セル内の溶液の動きを一時的に停止、又は分散状態をモニタリングができる程度に流速を下げて行う。従って、生体高分子の分散状態のモニタリングを動的光散乱法による散乱光強度分布測定で行う場合には、このような溶液循環に関する操作を行いながら、生体高分子の分散状態を変化させる。 In the present invention, it is possible to grasp the biopolymer dispersion state and the crystallization of the biopolymer in association with each other by exploring the conditions for the crystallization of the biopolymer while monitoring the dispersion state of the biopolymer. There are advantages. Furthermore, the monitoring of the dispersion state of the biopolymer can be performed by, for example, a scattered light intensity distribution measurement by a dynamic light scattering method, as a method for analyzing the association state of the biopolymer particles in the solution. Can measure the dispersion state of the biopolymer more accurately by measuring the scattered light intensity distribution by the dynamic light scattering method using the frequency analysis method. The dispersion state monitoring by the scattered light intensity distribution measurement by the dynamic light scattering method is performed, for example, by temporarily stopping the circulation of the solution and temporarily stopping the movement of the solution in the scattered light intensity measurement cell. . Alternatively, the circulation of the solution is not stopped, and the dynamic light scattering intensity distribution measurement can be performed by controlling the circulation speed within a range that does not hinder the scattered light intensity measurement by the dynamic light scattering method. Furthermore, while the circulation of the solution is maintained, a bypass is provided in parallel with the scattered light intensity measurement cell, and the solution is circulated through this bypass to temporarily stop the movement of the solution in the scattered light intensity measurement cell. Alternatively, the flow rate is lowered so that the dispersion state can be monitored. Therefore, when monitoring the dispersion state of the biopolymer by the scattered light intensity distribution measurement by the dynamic light scattering method, the dispersion state of the biopolymer is changed while performing the operation related to the solution circulation.
　さらに、ポンプは、ペリスタルティックポンプであることが、沈殿等の固形物によって影響を受けにくく、安定して送液を行えるというという観点から好ましい。 Furthermore, it is preferable that the pump is a peristaltic pump from the viewpoint that it is not easily affected by solid matter such as precipitates and can stably feed liquid.
　図１に動的光散乱法による散乱光強度分布装置を利用した、本発明の結晶化条件探査装置の概念図を示す。生体高分子としてタンパク質を用いた場合を例に、以下説明する。
(1)の生体高分子含有溶液貯蔵及び投入部は、生体高分子含有溶液を満たしたシリンジを有するシリンジポンプ10であることができる。(2)の半透膜を用いた連続透析膜20は、半透膜を介して溶液をろ液として流路60内から排出できる。(3)の試薬含有溶液貯蔵及び投入部30は、1つまたは複数の試薬含有溶液の貯蔵部を有し、この貯蔵部の1つまたは複数から、流路60内には、シリンジポンプから、電動または手動切り替えバルブ(図中は電磁バルブ61)を経由することによって試薬含有溶液が注入される。(4)の生体高分子の分散状態のモニター40は、例えば、動的光散乱法による散乱光強度分布測定装置41に結合された散乱光強度測定セル42であることができる。(5)の結晶化セル50は、流路60内に設置する。セル内容量は、例えば、数十マイクロリットル～数百マイクロリットルである。また、セル内を観察するために、セル上部と下部は透明な観察窓を持つか、セル全体をアクリル樹脂で製作することにより、内部観察を可能とすることができる。また、セル内の結晶化セル外周辺にはウオータージャケット(図示せず)を設置することもでき、このジャケットに温度制御された流体を循環させることにより、セル全体の温度を制御することができる。上記(1)～(5)の間を連絡する流路60は、容量一定であり、例えば、少なくとも循環する溶液と接する内面が溶液及びその含有物に対して不活性である材料製のチューブであることができる。流路60の全てを同一の材質のチューブで形成することもできるが、場所に応じて異なる材質のチューブを適宜用いることができる。流路60は、例えば、フレキシブルなシリコーンチューブ、PCV、ポリプロピレンなどであることができる。 FIG. 1 shows a conceptual diagram of a crystallization condition exploration device of the present invention using a scattered light intensity distribution device by a dynamic light scattering method. The case where protein is used as the biopolymer will be described below as an example.
The biopolymer-containing solution storage and input part of (1) can be a syringe pump 10 having a syringe filled with the biopolymer-containing solution. The continuous dialysis membrane 20 using the semipermeable membrane of (2) can be discharged from the flow path 60 as a filtrate through the semipermeable membrane. The reagent-containing solution storage and input unit 30 of (3) has one or a plurality of reagent-containing solution storage units, from one or more of these storage units, into the flow path 60 from the syringe pump, The reagent-containing solution is injected through an electric or manual switching valve (electromagnetic valve 61 in the figure). The biopolymer dispersion state monitor 40 of (4) can be, for example, a scattered light intensity measurement cell 42 coupled to a scattered light intensity distribution measuring device 41 by a dynamic light scattering method. The crystallization cell 50 of (5) is installed in the flow path 60. The cell internal volume is, for example, several tens of microliters to several hundreds of microliters. Moreover, in order to observe the inside of a cell, internal observation can be enabled by having a transparent observation window in the upper part and the lower part of the cell or manufacturing the whole cell with an acrylic resin. In addition, a water jacket (not shown) can be installed around the crystallization cell in the cell, and the temperature of the entire cell can be controlled by circulating a temperature-controlled fluid through the jacket. . The flow path 60 communicating between the above (1) to (5) has a constant volume, for example, a tube made of a material whose inner surface in contact with the circulating solution is inert to the solution and its contents. Can be. Although all of the flow path 60 can be formed of a tube made of the same material, a tube made of a different material can be appropriately used depending on the location. The channel 60 can be, for example, a flexible silicone tube, PCV, polypropylene, or the like.
　ポンプ70は、流路内に溶液を流通させるためのポンプであり、ペリスタルティックポンプであることが、上記で説明した理由から好ましい。ポンプ70としてペリスタルティックポンプを用いる場合には、ペリスタルティックポンプ付近の流路60はフレキシブルな樹脂製のチューブであることが適当である。尚、生体高分子含有溶液を満たしたシリンジを有するシリンジポンプは、シリンジへの逆流を防止するという観点からペリスタルティックポンプの上流に位置することが好ましい。 The pump 70 is a pump for circulating the solution in the flow path, and is preferably a peristaltic pump for the reason described above. When a peristaltic pump is used as the pump 70, the flow path 60 in the vicinity of the peristaltic pump is suitably a flexible resin tube. In addition, it is preferable that the syringe pump which has a syringe filled with the biopolymer-containing solution is located upstream of the peristaltic pump from the viewpoint of preventing backflow to the syringe.
　上記装置において、タンパク質溶液は、シリンジポンプ10内から経路内に注入される。経路内のタンパク質濃度は、連続透析膜20により濃縮され、ペリスタルティックポンプ70によって経路内を循環させられる。試薬投入部30から塩、バッファーなどの試薬が経路内に制御されつつ注入される。タンパク質溶液の分散状態は、動的光散乱法による散乱光強度分布測定装置40に結合された散乱光強度測定セル42により、ほとんど時間遅れなくモニタリングすることができる。結晶化セル50内の観察部51おいて結晶化が観察される。一連の動作は、例えば、PC90により制御される。尚、経路内での試薬の濃度は、例えば、導電率計80を用いることでモニタリングすることができる。導電率計80の設置位置は、特に制限はないが、図1に示すシリンジポンプ10の直ぐ下流以外の位置に設置することもできる。 In the above apparatus, the protein solution is injected from the syringe pump 10 into the path. The protein concentration in the pathway is concentrated by the continuous dialysis membrane 20 and circulated in the pathway by the peristaltic pump 70. Reagents such as salts and buffers are injected from the reagent loading unit 30 while being controlled in the path. The dispersion state of the protein solution can be monitored with almost no time delay by the scattered light intensity measurement cell 42 coupled to the scattered light intensity distribution measuring device 40 by the dynamic light scattering method. Crystallization is observed in the observation part 51 in the crystallization cell 50. A series of operations is controlled by the PC 90, for example. The concentration of the reagent in the path can be monitored by using, for example, a conductivity meter 80. The installation position of the conductivity meter 80 is not particularly limited, but can also be installed at a position other than immediately downstream of the syringe pump 10 shown in FIG.
　本発明の方法及び装置においては、系内を溶液が一定の速度で流通しており、かつ連続透析膜20においては溶液がろ過される。タンパク質溶液が系内に注入されるときには、タンパク質濃度は徐々に上昇し、タンパク質溶液は注入されず、試薬溶液が注入される場合には、タンパク質濃度は一定に維持されつつ試薬濃度が徐々に上昇する。また、試薬を含まない溶液を試薬投入部30から系内に注入することもでき、この場合には、タンパク質濃度は一定に維持されつつ試薬濃度は徐々に低下する。連続透析膜20における溶液のろ過は、溶液が一定の速度で流通しながら行われる。また、溶液の循環流速が試薬注入速度やタンパク質溶液の注入速度に比べて十分に確保されており、かつ各種溶液の注入量が循環経路内の溶液量に比べて極微量な条件下では、連続透析膜20付近における溶液中の試薬濃度やタンパク質濃度の変化は穏やかであり、かつ溶液が、測定時以外は、常時循環し、攪拌状態を維持していることから、系内がほぼ均一な濃度状態を維持でき、その結果、タンパク質の分散状態が局所的に変化する恐れが極めて低く、タンパク質等の生体高分子が結晶化する条件を探査するに適している。本発明において、溶液組成が均一であるとは、分オーダーの時間内に、循環経路内部全体の溶液組成が同一になることを意味する。 In the method and apparatus of the present invention, the solution flows through the system at a constant speed, and the solution is filtered through the continuous dialysis membrane 20. When the protein solution is injected into the system, the protein concentration gradually increases. When the protein solution is not injected and the reagent solution is injected, the reagent concentration is gradually increased while the protein concentration is kept constant. To do. In addition, a solution containing no reagent can be injected into the system from the reagent charging unit 30. In this case, the reagent concentration gradually decreases while the protein concentration is maintained constant. Filtration of the solution in the continuous dialysis membrane 20 is performed while the solution flows at a constant speed. In addition, the solution circulation flow rate is sufficiently ensured compared to the reagent injection rate and protein solution injection rate, and the amount of various solution injections is extremely small compared to the amount of solution in the circulation path. Changes in the reagent concentration and protein concentration in the solution near the dialysis membrane 20 are gentle, and the solution circulates constantly and maintains a stirring state except during measurement, so the concentration in the system is almost uniform. The state can be maintained, and as a result, the dispersion state of the protein is extremely unlikely to change locally. In the present invention, the uniform solution composition means that the solution composition in the entire circulation path becomes the same within a minute order time.
　溶液の循環速度は、系内がほぼ均一な濃度状態を維持できる速度であれば特に制限はなく、系の容量や流路の断面積、使用されるポンプの種類や能力等に応じて、さらには、生体高分子含有溶液に結晶化のため与えたい条件変化等を考慮して適宜決定できる。溶液の循環速度は、例えば、0.0001～2 ml/minの範囲であることができる。 The circulation rate of the solution is not particularly limited as long as the system can maintain a substantially uniform concentration state, and further depends on the capacity of the system, the cross-sectional area of the flow path, the type and capacity of the pump used, etc. Can be appropriately determined in consideration of a change in conditions to be given to the biopolymer-containing solution for crystallization. The circulation rate of the solution can be, for example, in the range of 0.0001 to 2 ml / min.
　例えば、ペリスタルティックルティックポンプにおける使用チューブの種類と吐出量とヘッド回転速度（rpm）との関係は、ペリスタルティックルティックポンプのメーカーが技術情報として公開している。
　例えば、http://www.technosaurus.co.jp/product/mp-flow_1.htm参照。その全記載は、ここに特に開示として援用される。
　このような公開されている技術情報によれば、チューブとしてPVC0.5 mmを使用する場合、ポンプの回転速度を最大の50 rpmに設定すると、沈殿剤を急速に注入（1 ml/min程度）しても約1 ml/minの循環速度で経路内の溶液が循環できる。このような循環速度であれば、数分後には経路内の組成は均一な平衡状態に達することができる。一方、タンパク質や沈殿剤をゆっくり注入する（例えば、0.0002～0.07 ml/min）過程では、ヘッド速度を1～5 rpm程度で運転することで0.1 ml/min程度で溶液は循環する。そのため、十分経路内の均一性を保ちつつ溶液組成を変化させることが可能になる。 For example, the relationship between the type of tube used in the peristaltic lick pump, the discharge amount, and the head rotation speed (rpm) is disclosed as technical information by the manufacturer of the peristaltic lick pump.
For example, see http://www.technosaurus.co.jp/product/mp-flow_1.htm. The entire description is hereby specifically incorporated by reference.
According to the published technical information, when PVC 0.5 mm is used as a tube, the precipitating agent is rapidly injected (about 1 ml / min) when the rotation speed of the pump is set to a maximum of 50 rpm. Even so, the solution in the path can circulate at a circulation rate of about 1 ml / min. With such a circulation rate, the composition in the path can reach a uniform equilibrium after a few minutes. On the other hand, in the process of slowly injecting protein or precipitating agent (for example, 0.0002 to 0.07 ml / min), the solution circulates at about 0.1 ml / min by operating the head speed at about 1 to 5 rpm. Therefore, it is possible to change the solution composition while maintaining sufficient uniformity in the path.
　溶液の物理化学的性質を変化させる物質(試薬)は、例えば、ｐＨ緩衝剤、沈殿化剤、塩類、添加剤、及び水から成る群から選ばれる少なくとも１ 種の物質であることができる。ｐＨ緩衝剤、沈殿化剤、塩類、添加剤は、生体高分子の結晶化にこれまで使用されている物質から適宜選択することができる。 The substance (reagent) that changes the physicochemical properties of the solution can be, for example, at least one substance selected from the group consisting of a pH buffer, a precipitating agent, salts, additives, and water. The pH buffering agent, the precipitating agent, the salts, and the additives can be appropriately selected from substances that have been used so far for crystallization of biopolymers.
　ｐＨ緩衝剤としては、例えば、クエン酸ナトリウム、酢酸ナトリウム、リン酸ナトリウム／ カリウム、ＭＥＳ、カコジル酸ナトリウム、ＨＥＰＥＳ、Ｔｒｉｓ－ＨＣｌ等を挙げることができる。但し、これらの限定されるものではない。
　沈殿化剤としては、例えば、硫酸アンモニウム、塩化ナトリウム、塩化リチウム、ポリエチレングリコール、２－メチル－２，４－ペンタンジオール等を挙げることができる。但し、これらの限定されるものではない。 Examples of the pH buffer include sodium citrate, sodium acetate, sodium phosphate / potassium, MES, sodium cacodylate, HEPES, Tris-HCl and the like. However, these are not limited.
Examples of the precipitating agent include ammonium sulfate, sodium chloride, lithium chloride, polyethylene glycol, 2-methyl-2,4-pentanediol and the like. However, these are not limited.
　塩類としては、例えば、塩化カルシウム、塩化マグネシウム、塩化亜鉛、塩化コバルト、硫酸リチウム、酢酸アンモニウム、酢酸マグネシウム、酢酸亜鉛、塩化セシウム等を挙げることができる。但し、これらの限定されるものではない。添加剤としては、例えば、エチレングリコール、グリセロール、尿素、エタノール、プロパノール、イソプロパノール、ＥＤＴＡ、ＤＤＴ、ヘキサンジオール、オクチルグルコシド等を挙げることができる。但し、これらの限定されるものではない。 Examples of the salts include calcium chloride, magnesium chloride, zinc chloride, cobalt chloride, lithium sulfate, ammonium acetate, magnesium acetate, zinc acetate, cesium chloride and the like. However, these are not limited. Examples of the additive include ethylene glycol, glycerol, urea, ethanol, propanol, isopropanol, EDTA, DDT, hexanediol, octyl glucoside and the like. However, these are not limited.
　循環経路内の溶液の温度は、結晶化セル内のウオータージャケット(図示せず)に温調された液体を循環させることにより、一定になるように制御しておくこともできるし、生体高分子の濃度変化や溶液の添加と並行して温度を変化させることもできる。生体高分子の濃度変化時や溶液添加時の温度を一定にする場合、同一条件の検索操作を、温度を変えて行うこともできる。循環経路の温度調整は、例えば、循環経路の一部または全部を恒温槽(室)100に設置することで行うこともできる。本発明の方法を実施する溶液温度は、特に制限はなく、低くする場合、凍結が生じない範囲であればよく、また、高くする場合、生体高分子に変性等が生じない範囲であれば良い。例えば、０～４０℃の範囲とすることができる。 The temperature of the solution in the circulation path can be controlled to be constant by circulating a temperature-controlled liquid through a water jacket (not shown) in the crystallization cell, or a biopolymer. The temperature can also be changed in parallel with the change in the concentration and the addition of the solution. When the temperature at the time of changing the concentration of the biopolymer or at the time of adding the solution is made constant, the search operation under the same conditions can be performed by changing the temperature. The temperature of the circulation path can be adjusted by, for example, installing a part or all of the circulation path in the constant temperature bath (chamber) 100. The solution temperature at which the method of the present invention is performed is not particularly limited, and may be in a range where freezing does not occur when lowering, and may be within a range where denaturation or the like does not occur in biopolymers when increased. . For example, it can be in the range of 0 to 40 ° C.
　以上の方法により、良質な結晶が得られる条件が探査できたら、その結晶化条件を用いて、構造解析に適した品質とサイズを有する生体高分子結晶を製造することができる。この生体高分子結晶の製造は、常法により行うことができる。例えば、蒸気拡散法、透析法、バッチ法などが知られている。 If the conditions for obtaining good quality crystals can be explored by the above method, a biopolymer crystal having quality and size suitable for structural analysis can be produced using the crystallization conditions. The biopolymer crystal can be produced by a conventional method. For example, a vapor diffusion method, a dialysis method, a batch method, and the like are known.
　得られた生体高分子結晶は、結晶構造解析に使用することができる。結晶構造解析は、例えば、ＭＡＤ法に従って行うことができ、例えば、放射光を使用して、結晶のＸ 線回折データを測定する。この目的に使用できる放射光は、例えば、大型放射光施設ＳＰｒｉｎｇ－８／理研（ＲＩＫＥＮ）ビームラインＩＢＬ４４Ｂ２（ＢＬ４５ＸＵ）により発生させることかできる。 The obtained biopolymer crystal can be used for crystal structure analysis. Crystal structure analysis can be performed, for example, according to the MAD method. For example, X-ray diffraction data of a crystal is measured using synchrotron radiation. The synchrotron radiation that can be used for this purpose can be generated, for example, by the large synchrotron radiation facility SPring-8 / RIKEN beamline IBL44B2 (BL45XU).
　以下、本発明を実施例によりさらに詳細に説明する。 Hereinafter, the present invention will be described in more detail with reference to examples.
実施例１
　図１に記載した連続フロー型半透膜を利用する装置の特徴を用いて、経路内の沈殿剤濃度などを連続的に任意に変化させられることを実験により確認した。結果を図2aに示す。A液は生体高分子の一つであるリゾチウムを溶解させるための緩衝液であり、組成は0.1 M 酢酸ナトリウム, pH4.3であり、B液はリゾチウムの結晶化剤であり、A液に塩化ナトリウムを2.0 Mの濃度に溶解した溶液である。A液、B液をそれぞれ経路内に注入し、半透膜外に流出した濾液の電導度を測定した。 Example 1
Using the characteristics of the apparatus using the continuous flow type semipermeable membrane described in FIG. 1, it was confirmed by experiments that the concentration of the precipitating agent and the like in the path can be arbitrarily changed continuously. The results are shown in FIG. Solution A is a buffer solution for dissolving lysozyme, one of the biopolymers. The composition is 0.1 M sodium acetate, pH 4.3, and solution B is a lysozyme crystallization agent. This is a solution in which sodium is dissolved to a concentration of 2.0 M. Liquid A and liquid B were respectively injected into the channels, and the electrical conductivity of the filtrate flowing out of the semipermeable membrane was measured.
　沈殿剤注入ステップでは、B液（0.1 M 酢酸ナトリウム緩衝液、pH4.3、2.0 M NaCl）を5.2 ml(5 ml/hの注入速度で)注入し、その後、10 ml(50 ml/hの注入速度で)注入した。導電率は、約14 S/mで飽和した。その後、A緩衝液を20 ml (10 ml/hの注入速度で)注入した。図2aに示すように、導電率がスムーズに変化していることから、本システムにより、経路内の組成の均一性を保ちながら、組成を自由に変化されることが可能であることが示された。 In the precipitating agent injection step, B solution (0.1 M sodium acetate buffer, pH 4.3, 2.0 M NaCl) is injected with 5.2 μml (at an injection rate of 5 μml / h), and then 10 μml (50 μml / h Infused at the infusion rate. The conductivity was saturated at about 14 S / m. Thereafter, 20 ml of A buffer was injected (at an injection rate of 10 ml / h). As shown in Figure 2a, the conductivity changes smoothly, indicating that the system can freely change the composition while maintaining the uniformity of the composition in the pathway. It was.
　次に経路内にタンパク質溶液を注入した際の経路内のタンパク質濃度変化を測定した。結果を図2bに示す。10回目から48回目（測定は5分間隔）までの間に、リゾチウム溶液（20 mg/ml, 0.1 M 酢酸ナトリウム緩衝液, pH4.3）を5.36 ml(2 ml/hの注入速度で)経路内に注入した。タンパク質濃度に比例する動的光散乱法による散乱光強度分布の測定強度のピーク値がスムーズに上昇変化した。ピーク値がスムーズに上昇変化したことから本装置により経路内の均一性を保ちつつ、タンパク質濃度を変化させることが可能であることが示された。 Next, the change in protein concentration in the path when the protein solution was injected into the path was measured. The result is shown in FIG. Between 10th and 48th (measurement is every 5 minutes), 5.36 ml (at an injection rate of 2 ml / h) of lysotium solution (20 mg / ml, 0.1 M sodium acetate buffer, pH4.3) Injected into. The peak value of the measured intensity of the scattered light intensity distribution by the dynamic light scattering method proportional to the protein concentration smoothly increased and changed. Since the peak value smoothly increased and changed, it was shown that it is possible to change the protein concentration while maintaining uniformity in the path by this apparatus.
　経路内に沈殿剤をV量注入したときの経路内沈澱剤濃度C(V)（S/m)は、下記式で概算できる。
　　　C(V) = A・exp[-V/Vo] + C’
　　　A, C’: 定数
　　　Vo: 経路容量 The intra-route precipitant concentration C (V) (S / m) when a V amount of precipitant is injected into the route can be estimated by the following equation.
C (V) = A ・ exp [-V / Vo] + C '
A, C ': Constant Vo: Path capacity
　上記実験では、沈殿剤投入前またはタンパク質溶液注入前の経路には、A液（0.1 M 酢酸ナトリウム緩衝液, pH 4.3）が充填されており、C’は0.6 mS/m程度である。沈殿剤及びタンパク質溶液はあらかじめA液に溶解してあるため、全実験過程に渡って経路内のA液組成は維持される。
Vo = 2.5 mlであり、Aは経路内容量Voによって決まる。 In the above experiment, the route before the precipitating agent is charged or before the protein solution is injected is filled with solution A (0.1 M sodium acetate buffer, pH 4.3), and C ′ is about 0.6 mS / m. Since the precipitant and the protein solution are preliminarily dissolved in the liquid A, the composition of the liquid A in the route is maintained throughout the entire experimental process.
Vo = 2.5 ml, and A is determined by the path volume Vo.
　図2aに示す結果から、経路内の沈澱剤濃度C(V)を、経路内に沈澱剤を注入することにより上昇させることが可能であり、また、経路内にAバッファーを注入させることにより下降させることが可能であることを確認することができた。これにより、経路内の沈澱剤濃度C(V)を、経路外部から沈澱剤あるいはAバッファーを注入することにより、任意に変化させることが可能であることを確認した。この際の経路内の沈澱剤濃度C(V)は、経路内に注入した沈澱剤量(V)との関数として、上記式に従って数式により把握することが可能である。こうした特徴は、従来の結晶化装置にはない、本発明の装置に特有の特徴である。 From the results shown in FIG. 2a, it is possible to increase the concentration C (V) of the precipitant in the route by injecting the precipitant into the route, and to decrease by injecting the A buffer into the route. I was able to confirm that it was possible. As a result, it was confirmed that the concentration C (V) of the precipitant in the route can be arbitrarily changed by injecting the precipitant or A buffer from the outside of the route. At this time, the concentration C (V) of the precipitant in the route can be grasped by a mathematical formula according to the above equation as a function of the amount (V) of the precipitant injected into the route. These characteristics are unique to the apparatus of the present invention, which is not found in the conventional crystallization apparatus.
実施例２
　実施例１で使用した装置を実際に組み立てて、タンパク質試料としてリゾチウムを用いて、リゾチウムの結晶化実験を行った。結果を図3a及び図3bに示す。 Example 2
The apparatus used in Example 1 was actually assembled, and lysotium crystallization experiments were performed using lysotium as a protein sample. The results are shown in FIGS. 3a and 3b.
　クロマトグラムは、横軸が測定回数(時間)、縦軸がリゾチウム分子の動的光散乱法による散乱光強度分布測定より求めた半径である。タンパク質濃縮過程では、リゾチウムを34mg/mlの濃度まで濃縮した。次に、沈殿剤（NaCl）濃度を5分間で0→1Mまで上昇させると、タンパク質分子の動的光散乱法による散乱光強度分布測定より求めた半径の増加が観察された。更に徐々に沈殿剤濃度を増加させると、写真のようにリゾチウム結晶の生成が確認された。この連続写真は、50～65回の測定結果であり、各測定は10分間隔で行った。結晶が観察できる拡大写真は、測定55回目の写真である。0.2～0.4mm程度の結晶が生成していることが分かる(目盛り参照)。図中のMVは体積平均径、MNは個数平均径、MAは面積平均径である。 In the chromatogram, the horizontal axis represents the number of times of measurement (time), and the vertical axis represents the radius obtained by measuring the scattered light intensity distribution by the dynamic light scattering method of lysozyme molecules. In the protein concentration process, lysozyme was concentrated to a concentration of 34 mg / ml. Next, when the concentration of the precipitant (NaCl) was increased from 0 to 1 M in 5 minutes, an increase in the radius obtained by measuring the scattered light intensity distribution by the dynamic light scattering method of protein molecules was observed. When the precipitant concentration was further increased gradually, the formation of lysozyme crystals was confirmed as shown in the photograph. This continuous photograph is a measurement result of 50 to 65 times, and each measurement was performed at 10 minute intervals. The magnified photograph in which the crystal can be observed is the 55th measurement photograph. It can be seen that crystals of about 0.2 to 0.4 mm are formed (see scale). In the figure, MV is a volume average diameter, MN is a number average diameter, and MA is an area average diameter.
　この結晶化過程におけるリゾチウム分子の分散度の変化を図3bに示す。動的光散乱法による散乱光強度分布測定装置で検出された信号は、周波数解析法によってパワースペクトル（周波数vs信号強度）に変換される。図3b縦軸は信号強度(粒子群からの散乱光強度に入射光強度をかけたもの)、横軸は周波数に相当する散乱強度測定装置固有の区分数を表している。図3bに描かれている曲線は、このパワースペクトルを対数変換し、適宜周波数区分に切り分けたものである（以下、このグラフをRaw-dataと呼ぶ）。このRaw-data中の曲線は粒子径に応じた固有のピークを持ち、ピーク位置（横軸；周波数に対応したチャンネル）は液中に存在する代表的な溶質(単体分子あるいは分子の結合体）サイズの特徴を示している。粒子サイズが大きいほど、ピーク位置のチャンネル数が小さくなる。逆に、高いチャンネル数ほど径が小さい粒子に対応している。 Fig. 3b shows the change in the degree of dispersion of lysotium molecules during this crystallization process. A signal detected by a scattered light intensity distribution measuring apparatus using a dynamic light scattering method is converted into a power spectrum (frequency vs signal intensity) by a frequency analysis method. In FIG. 3b, the vertical axis represents the signal intensity (the intensity of the scattered light from the particle group multiplied by the incident light intensity), and the horizontal axis represents the number of sections specific to the scattering intensity measuring apparatus corresponding to the frequency. The curve depicted in FIG. 3b is obtained by logarithmically converting the power spectrum and dividing it into frequency segments as appropriate (hereinafter this graph is referred to as Raw-data). This curve in Raw-data has a specific peak corresponding to the particle size, and the peak position (horizontal axis: channel corresponding to frequency) is a typical solute (single molecule or molecular combination) present in the liquid. It shows the characteristics of the size. The larger the particle size, the smaller the number of channels at the peak position. Conversely, the higher the number of channels, the smaller the diameter.
　図3b中の系列に示した測定回数は、リゾチウムを系内に初めて投入した時点を0回目とし、続けて定期的に動的光散乱法による散乱光強度分布を測定したときの回数を示しており、図3bでは各々の測定回数(時間)での測定結果（リゾチウムの分散状態）を一つのグラフにプロットしている。沈殿剤を加えていない状態でのリゾチウムの分散状態は、図中の14～16回目の測定結果であり、このときの測定結果は68ch付近をピークに散乱光強度分布が一山のなめらかなカーブを描いている。沈殿剤を初めて投入した時点のリゾチウムの分散状態は図中の17回目の測定結果であり、リゾチウムのみを測定した場合と同様一山のカーブを描くが、そのピーク位置が低ch側へややシフトし、ピークchにおける信号強度が増加した。本結晶化試験では、17回目の測定以降、上記散乱光強度分布の形状を維持しつつ、最終的にリゾチウムの結晶が生成された(17～50回目)。50回目の測定以降、結晶生成にリゾチウムが消費され溶液中のリゾチウム濃度が低下したため、散乱光強度が下がった(60回目)。 The number of measurements shown in the series in Fig. 3b indicates the number of times when the scattered light intensity distribution by the dynamic light scattering method is periodically measured, with the time when lysotium is first introduced into the system as the 0th time. In FIG. 3b, the measurement result (dispersion state of lysozyme) at each measurement number (time) is plotted in one graph. The dispersion state of lysozyme without adding a precipitant is the measurement result of the 14th to 16th times in the figure, and the measurement result at this time is a smooth curve with a scattered light intensity distribution peaking around 68ch. Is drawn. The dispersion state of lysozyme at the time when the precipitant was first introduced is the measurement result of the 17th time in the figure, and a single curve is drawn as in the case of measuring only lysozyme, but the peak position is slightly shifted to the low ch side. The signal intensity at the peak ch increased. In this crystallization test, after the 17th measurement, lysozyme crystals were finally produced while maintaining the shape of the scattered light intensity distribution (17th to 50th times). Since the 50th measurement, lysozyme was consumed for crystal formation and the concentration of lysozyme in the solution was lowered, so that the scattered light intensity decreased (60th).
　他方、リゾチウムの沈殿を生じた際の分散状態を図3に示す。この場合、高チャンネル側に図3bと同様にリゾチウムのピークが存在しているが、0～40ch領域にスパイク状のピークが出力された。これは過剰の沈殿剤投入によりリゾチウムの凝集物ができたためである。またこの時のセル状態を観察すると、図3aの写真のように沈殿が生じていることが確認された。 On the other hand, the dispersion state when precipitation of lysozyme occurs is shown in FIG. In this case, a lysozyme peak was present on the high channel side as in FIG. 3b, but a spike-like peak was output in the 0-40 ch region. This is because lysozyme aggregates were formed by adding an excessive amount of precipitant. Further, when the cell state at this time was observed, it was confirmed that precipitation occurred as shown in the photograph of FIG. 3a.
　したがって、リゾチウムが結晶化する場合は、測定全般にわたって0～40chの領域にスパイク等の強度変化は見られず、結晶化過程全般にわたってリゾチウム分子の溶液状態は一定の分散状態を維持していたことが確認された。 Therefore, when lysozyme crystallizes, there is no change in intensity such as spikes in the 0-40 ch region throughout the measurement, and the solution state of lysotium molecules maintains a constant dispersion state throughout the crystallization process. Was confirmed.
実施例３
　実施例２と同様に、実施例１で使用した装置を実際に組み立てて、タンパク質試料としてグルコースイソメラーゼを用いて、グルコースイソメラーゼの結晶化実験を行った。実験条件は以下の通りである。
・　グルコースイソメラーゼ：Hampton Research社製　分子量:173,000
・　タンパク質溶液：30mg/ml グルコースイソメラーゼ、50mM HEPES, pH7.0, 1mM MgCl2
・　A溶液：50mM HEPES, pH7.0, 1mM MgCl2
・　B溶液（沈澱剤溶液）：3M NH4(SO4)2, 50mM HEPES, pH7.0, 1mM MgCl2
・　結晶化セル温度：20℃ Example 3
Similarly to Example 2, the apparatus used in Example 1 was actually assembled, and glucose isomerase crystallization experiments were performed using glucose isomerase as a protein sample. The experimental conditions are as follows.
・ Glucose isomerase: manufactured by Hampton Research Molecular weight: 173,000
・ Protein solution: 30 mg / ml glucose isomerase, 50 mM HEPES, pH 7.0, 1 mM MgCl 2
・ Solution A: 50 mM HEPES, pH 7.0, 1 mM MgCl 2
・ B solution (precipitant solution): 3M NH 4 (SO 4 ) 2 , 50 mM HEPES, pH 7.0, 1 mM MgCl 2
・ Crystallization cell temperature: 20 ℃
　結果を図4に示す。4aは測定回数に対する導電率変化、図4bは測定回数に対する紫外吸収値変化を示す。
(1)タンパク質溶液を結晶化装置に注入し、紫外吸光度（λ280 0.2cmセル使用）が0.38 (19mg/ml) になるまで濃縮した（866回）。その際の散乱光強度分布は、図4cに示すように低チャンネル側の信号がほとんどない安定な分散状態を示し、ピーク位置は59チャンネルであった。
(2)2.3mlのB溶液を急速注入したところ（867回）、導電率は13.0 S/mになり、動的光散乱プロファイルは図4dに示すように、安定な分散状態を維持しつつ、ピークチャンネルが56チャンネルにシフトした。
(3)その後、タンパク質溶液を0.05ml/hourおよび沈澱剤溶液Bを0.1ml/hourの速度でそれぞれ注入した。
(4)測定回数1080回において、紫外吸光度は0.515,導電率は16.2 S/mになり、図のように0.1mm程度の大きさのグルコースイソメラーゼ結晶が得られた（図4fに写真を示す）。その際の散乱光強度分布も、図4eに示すように安定な分散を維持していた。 The results are shown in FIG. 4a shows the change in conductivity with respect to the number of measurements, and FIG. 4b shows the change in ultraviolet absorption value with respect to the number of measurements.
(1) The protein solution was poured into a crystallizer and concentrated (866 times) until the ultraviolet absorbance (λ280 0.2 cm cell used) became 0.38 (19 mg / ml). The scattered light intensity distribution at that time showed a stable dispersion state with almost no signal on the low channel side as shown in FIG. 4c, and the peak position was 59 channels.
(2) When 2.3 ml of B solution was rapidly injected (867 times), the conductivity became 13.0 S / m, and the dynamic light scattering profile maintained a stable dispersion state as shown in FIG. Peak channel shifted to 56 channels.
(3) Thereafter, the protein solution was injected at a rate of 0.05 ml / hour and the precipitant solution B at a rate of 0.1 ml / hour.
(4) When the number of measurements was 1080, the ultraviolet absorbance was 0.515 and the conductivity was 16.2 S / m, and a glucose isomerase crystal with a size of about 0.1 mm was obtained as shown in the figure (photo is shown in FIG. 4f) . The scattered light intensity distribution at that time also maintained stable dispersion as shown in FIG. 4e.
　本発明は、生体高分子の結晶化製造に関する分野に有用である。 The present invention is useful in the field related to crystallization production of biopolymers.
10 生体高分子含有溶液を満たしたシリンジを有するシリンジポンプ
20 半透膜を用いた連続透析膜
30 試薬含有溶液貯蔵及び投入部
40 生体高分子の分散状態のモニター
41 動的光散乱法による散乱光強度測定装置
42 動的光散乱強度測定セル
50 結晶化セル
51 結晶観察部
60 流路
61 電磁バルブ
70 ペリスタルティックポンプ
80 導電率計
90 制御PC
100 恒温槽 10 Syringe pump with syringe filled with biopolymer-containing solution
20 Continuous dialysis membrane using semipermeable membrane
30 Reagent-containing solution storage and input section
40 Monitoring the dispersion state of biopolymers
41 Scattered light intensity measurement device by dynamic light scattering method
42 Dynamic light scattering intensity measurement cell
50 Crystallization cell
51 Crystal observation part
60 channels
61 Solenoid valve
70 Peristaltic pump
80 conductivity meter
90 Control PC
100 temperature chamber

Claims (14)

  1. 循環流路内に溶液を流し、
    該溶液中の生体高分子および溶液の物理化学的性質を変化させる少なくとも1種の物質(以下、試薬と呼ぶ)の少なくとも一つの濃度を連続的または断続的に、循環流路内の容量を一定に維持しつつ変化させ、かつ溶液中の結晶生成を連続的または断続的にモニタリングして、前記生体高分子の結晶が生成する条件を探査することを含み、
    前記循環流路内の容量は、循環流路内への溶液注入に伴って、流路内の溶液の一部を、半透膜を介して流路外に排出することで一定に維持される、生体高分子の結晶生成条件探査方法。     Pour the solution into the circulation channel,
    At least one concentration of the biopolymer in the solution and at least one substance (hereinafter referred to as a reagent) that changes the physicochemical properties of the solution is continuously or intermittently kept constant in the circulation channel. And continuously or intermittently monitoring crystal formation in solution to explore the conditions under which the biopolymer crystals are formed,
    The capacity in the circulation channel is maintained constant by discharging a part of the solution in the channel to the outside of the channel through the semipermeable membrane as the solution is injected into the circulation channel. , Method for exploring biopolymer crystal formation conditions.
  2. 循環流路は、生体高分子含有溶液の添加部位、半透膜を介しての排出部位、試薬含有溶液の添加部位、分散状態モニター部位及び結晶生成モニター部位をこの順に有し、前記結晶生成モニター部位の次に前記生体高分子含有溶液の添加部位を有する請求項1に記載の方法。 The circulation channel has an addition site for the biopolymer-containing solution, a discharge site through the semipermeable membrane, an addition site for the reagent-containing solution, a dispersion state monitoring site, and a crystal formation monitoring site in this order. 2. The method according to claim 1, further comprising a site for adding the biopolymer-containing solution next to the site.
  3. 結晶生成のモニタリングと並行して溶液に含有される生体高分子の分散状態が連続的または断続的にモニタリングされる、請求項1～2のいずれかに記載の方法。 The method according to claim 1, wherein the dispersion state of the biopolymer contained in the solution is monitored continuously or intermittently in parallel with the monitoring of crystal formation.
  4. 分散状態のモニタリングは、動的光散乱法による散乱光強度分布測定により行う、請求項5に記載の方法。 6. The method according to claim 5, wherein the dispersion state is monitored by measuring scattered light intensity distribution by a dynamic light scattering method.
  5. 溶液は流路中をペリスタルティックポンプで循環する、請求項1～4のいずれかに記載の方法。 The method according to any one of claims 1 to 4, wherein the solution is circulated in the flow path by a peristaltic pump.
  6. ペリスタルティックポンプによる送液は、半透膜を介しての排出部位と試薬含有溶液の添加部位の間で行われる、請求項5に記載の方法。 6. The method according to claim 5, wherein the liquid transfer by the peristaltic pump is performed between the discharge site through the semipermeable membrane and the addition site of the reagent-containing solution.
  7. 溶液中の生体高分子の濃度変化は、流路内の溶液に生体高分子含有溶液を注入することで行い、溶液中の試薬の濃度変化は、流路内の溶液に試薬含有溶液または試薬を含有しない溶液を注入することで行う、請求項1～6のいずれかに記載の方法。 The concentration of the biopolymer in the solution is changed by injecting the solution containing the biopolymer into the solution in the flow channel, and the concentration change of the reagent in the solution is changed by adding the reagent-containing solution or reagent to the solution in the flow channel. The method according to any one of claims 1 to 6, which is carried out by injecting a solution that does not contain.
  8. 溶液中の生体高分子の濃度変化及び試薬の濃度変化は、独立に行われる請求項1～7のいずれかに記載の方法。 The method according to any one of claims 1 to 7, wherein the concentration change of the biopolymer and the concentration change of the reagent in the solution are performed independently.
  9. 溶液中の生体高分子の濃度を一定に維持しつつ、溶液中の試薬の濃度を変化させて結晶生成のモニタリングを行う請求項1～8のいずれかに記載の方法。 9. The method according to claim 1, wherein the crystal formation is monitored by changing the concentration of the reagent in the solution while maintaining the concentration of the biopolymer in the solution constant.
  10. 循環溶液中の試薬濃度の変化は、濃度の上昇または減少である、請求項1～9のいずれかに記載の方法。 The method according to any one of claims 1 to 9, wherein the change in the reagent concentration in the circulating solution is an increase or decrease in the concentration.
  11. 生体高分子が、水溶性タンパク質、膜タンパク質、及び核酸－タンパク質複合体から成る群から選ばれる少なくとも1種である、請求項1～10のいずれかに記載の方法。 The method according to any one of claims 1 to 10, wherein the biopolymer is at least one selected from the group consisting of a water-soluble protein, a membrane protein, and a nucleic acid-protein complex.
  12. 請求項1～11のいずれかに記載の生体高分子の結晶化条件探査方法に用いられる装置であって、
    (1)生体高分子含有溶液貯蔵及び投入部、
    (2)半透膜を用いた連続透析膜、
    (3)物理化学的性質を変化させる物質(以下、試薬と呼ぶ)含有溶液貯蔵及び投入部、
    (4)生体高分子の分散状態観察用のモニター、及び
    (5)結晶化セル、
    上記(1)～(5)の間を連絡する容量一定の流路、並びに
    流路内に溶液を流通させるためのポンプを有し、
    前記(4)による生体高分子の分散状態のモニタリング及び(5)結晶化セル内を流れる溶液中の前記生体高分子濃度及び前記試薬の少なくとも一つの濃度を連続的または断続的に変化させて、生体高分子が結晶化する条件を探査するために用いられる前記装置。     An apparatus for use in the biopolymer crystallization condition exploration method according to any one of claims 1 to 11,
    (1) Biopolymer-containing solution storage and input section,
    (2) continuous dialysis membrane using semipermeable membrane,
    (3) Solution storage and input unit containing a substance (hereinafter referred to as a reagent) that changes physicochemical properties,
    (4) a monitor for observing the dispersion state of the biopolymer, and
    (5) crystallization cell,
    A constant-volume channel communicating between the above (1) to (5), and a pump for circulating the solution in the channel,
    Monitoring the dispersion state of the biopolymer according to (4) and (5) continuously or intermittently changing the concentration of the biopolymer and at least one of the reagents in the solution flowing in the crystallization cell, The apparatus used for exploring conditions for crystallization of biopolymers.
  13. (4)生体高分子の分散状態のモニタリングは、動的光散乱法による散乱光強度分布測定装置に結合された動的光散乱法による散乱光強度分布セルである請求項12に記載の装置。 14. The apparatus according to claim 12, wherein the dispersion state of the biopolymer is monitored by a scattered light intensity distribution cell by a dynamic light scattering method coupled to a scattered light intensity distribution measurement apparatus by a dynamic light scattering method.
  14. ポンプは、ペリスタルティックポンプである請求項12または13に記載の装置。 14. The apparatus according to claim 12 or 13, wherein the pump is a peristaltic pump.
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