WO2004019008A1

WO2004019008A1 - Methods of searching for crystallization conditions of biopolymer and searching apparatus

Info

Publication number: WO2004019008A1
Application number: PCT/JP2003/008765
Authority: WO
Inventors: Hideyuki Miyatake; Kazuki Takeda; Kunio Miki
Original assignee: Riken
Priority date: 2002-07-11
Filing date: 2003-07-10
Publication date: 2004-03-04
Also published as: JP3567988B2; JP2004045169A

Abstract

Methods of searching for the crystallization conditions of a biopolymer. Namely, the crystallization conditions of a biopolymer are searched for by adding, to a solution separated from another solution containing the biopolymer by a semipermeable membrane, a substance altering the physicochemical properties of the solution, thus altering the physicochemical properties of the biopolymer-containing solution and then monitoring the state of the biopolymer-containing solution. Further, the crystallization conditions of a biopolymer are searched for by decreasing or increasing the volume of a solution containing the biopolymer separated from another solution by a semipermeable membrane to thereby altering the concentration of the biopolymer in the biopolymer-containing solution and then monitoring the state of the biopolymer-containing solution. A method of producing biopolymer crystals by using the crystallization conditions searched for the above methods. An apparatus for searching for the crystallization conditions of a biopolymer which has a dialytic crystallization chamber and a solution-mixing chamber separated therefrom by a movable semipermeable membrane. The dialytic crystallization chamber has a window for monitoring the inside of the chamber while the solution-mixing chamber is connected to one or more reagent bottles in such a manner that a solution can be supplied from a reagent bottle.

Description

TECHNICAL FIELD The present invention relates to a method for exploring crystallization conditions of a biopolymer from a solution containing a biopolymer, and a biopolymer crystal under the searched conditions. The present invention relates to a method for producing a polymer, and an apparatus for searching for crystallization conditions of a biopolymer. '' Landscape technology The three-dimensional structure of biopolymers such as proteins is analyzed by crystallizing biopolymers and subjecting them to X-ray diffraction tests. However, there are various biopolymers, and crystallization is not always easy, and some biopolymers do not crystallize. Crystallization of currently employed biopolymers is performed by random trial and error of a large number of crystallization conditions, and is based on an irrational process that is likely to depend on chance. Evaporation-diffusion is the most widely used crystallization method for biopolymers. For biomacromolecules whose crystallization conditions are unknown, first the crystallization conditions are estimated by evaporative diffusion, and then refined by vapor diffusion, dialysis, patch, etc. In general, close examination of conditions is performed. When estimating the crystallization conditions using the evaporative diffusion method, an exploration method called the coarse matrix method is used. In the crude matrix method, hundreds of sample solutions having different parameters such as the pH, temperature, buffer, and precipitant of a solution containing a biopolymer are prepared, and the solution is allowed to stand. The crystallization conditions are investigated by periodically observing the formation of crystals while waiting for the precipitation of crystals. At present, kits for evaporative diffusion are available on the market that can prepare dozens of samples with varied parameters at once. The use of multiple such kits and the preparation of hundreds of samples requires significant amounts of biopolymer samples. Furthermore, the preparation of each sample itself and the observation of the presence or absence of crystal formation are usually performed by human hands and eyes, and when a large number of conditions are changed, the burden on the operator is large. In order to improve such situations, attempts have been made to automatically perform sample preparation and observation using robots. However, even with such a method, it is still necessary to prepare a large number of solutions with different parameters, and the problem that crystallization conditions cannot be explored with a small amount of a biopolymer sample has not been solved. In addition, there is a problem that the larger the search range, the more biopolymers are required. Furthermore, in the above method, since the crystallization conditions are searched discontinuously, there is an inherent problem that the discovery of the crystallization conditions themselves depends on chance, and the crystallization conditions may be essentially overlooked. . Furthermore, in the crystallization by the vapor diffusion method, it is necessary to pre-concentrate the initial concentration of the biopolymer to at least about half of the crystallization concentration. However, this concentration power often causes non-specific association of biopolymers that inhibits crystallization. There is also a problem. Crystallization of a biopolymer requires that the biopolymer be dissolved in a solution in a monodispersed state. Whether a biopolymer is in a monodispersed state can be measured nondestructively and quickly by, for example, dynamic light scattering measurement. However, it is time-consuming to determine the conditions under which several hundred types of biopolymer-containing solutions are in a monodispersed state. In the post-genome era, structural analysis of a large amount of proteins is awaited. High-performance X-ray diffractometers are also being developed for complicated structural analysis. However, the crystallization method of vital biopolymers depends on trial and error and contingency, which is the rate-limiting step in protein structural analysis. Therefore, an object of the present invention is to provide a method and an apparatus that can more reliably and easily find the crystallization conditions of a biopolymer even with a relatively small amount of a biopolymer. It is a further object of the present invention to provide a method for producing a biopolymer crystal under the crystallization conditions found by the above exploration method. DISCLOSURE OF THE INVENTION The present invention relates to a method for exploring the crystallization conditions of a biopolymer from a solution containing the biopolymer, the method comprising the steps of: The physicochemical properties of the biopolymer-containing solution are continuously or intermittently changed by continuously or intermittently adding a substance that changes the biological properties, during which the state of the biopolymer-containing solution is changed. To monitor the crystallization conditions of macromolecules of the living body by continuously or intermittently monitoring (hereinafter referred to as the first search method). Further, the present invention is a method for exploring crystallization conditions of a biopolymer from a biopolymer-containing solution, wherein the volume of the biopolymer-containing solution separated from the solution by a semipermeable membrane is continuously or intermittently measured. The concentration of the biopolymer in the solution containing the biopolymer is changed by decreasing or increasing the concentration of the biopolymer, and the state of the solution containing the biopolymer is monitored continuously or intermittently. (Hereinafter referred to as a second search method). In the second exploration method, the physicochemical property of the solution is changed in parallel with or independently of the change in the volume of the solution containing the biopolymer, to a solution separated by a semipermeable membrane from the solution containing the biopolymer. The substance to be added is added continuously or intermittently to change the physicochemical properties of the biopolymer-containing solution continuously or intermittently. During that time, the state of the biopolymer-containing solution is changed continuously or intermittently. It can be monitored continuously. Further, the present invention relates to a method for producing a biomolecular crystal using the crystallization conditions probed by the method of the present invention. In addition, the present invention comprises a dialysis crystallization chamber and a solution mixing chamber separated by a movable semipermeable membrane, wherein the dialysis crystallization chamber has a window for monitoring the room, and a solution mixing chamber. TECHNICAL FIELD The present invention relates to an apparatus for searching for crystallization conditions of a biopolymer, wherein one or more reagent bottles are connected so as to be able to supply a solution from the reagent bottles. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is an explanatory view schematically showing one embodiment of the crystallization condition detecting device of the present invention.

Figure 2 is a flowchart showing a control algorithm for searching for crystallization conditions.

FIG. 3 is a diagram showing one embodiment of the device of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION Both the first and second exploration methods of the present invention are methods for exploring conditions under which biomolecules crystallize from a biopolymer-containing solution.

In the present invention, examples of the biopolymer include a water-soluble protein, a membrane protein, a nucleic acid, and a protein-nucleic acid complex. The origin of biopolymers Although not particularly limited, the biopolymer used in the present invention can be, for example, a human-derived biopolymer.

[Exploration device of the present invention]

Hereinafter, first, the searching device of the present invention will be outlined.

FIG. 1 is a diagram schematically illustrating the device of the present invention.

In Fig. 1, 11 is a dialysis crystallization chamber, 1 2 is a semipermeable membrane, and 13 is a solution mixing chamber.Dialysis crystallization chamber 11 and a solution mixing chamber 13 are vertically movable semipermeable membranes 1 2 isolated. The dialysis crystallization chamber 11 and the solution mixing chamber 13 are arranged in a thermostat (not shown). The dialysis crystallization chamber 11 has a laser window 15 for monitoring the inside of the chamber and an injection valve 1 serving as a sample injection port. With 7. The dialysis crystallization chamber 11 is provided with a CCD camera 16 for observing crystals. The solution mixing chamber 13 is connected to one or more reagent potters 14 (14 a to 14 e) and, for example, via a discharge valve 18 (18 a to 18 e) using a piezo element. Connected so that solution from reagent bottle 14 can be supplied. In addition, one or both of the dialysis crystallization chamber and the solution mixing chamber are provided with a solution outlet (not shown in FIG. 1). The semipermeable membrane 12 is moved up and down by a step motor 32 connected via a plunger 31. FIG. 3 shows an embodiment of the apparatus of the present invention.

The device of FIG. 3 has the same basic structure and function as the device of FIG.

(a) shows a state in which the semipermeable membrane 12 has risen, and (b) shows a state in which the semipermeable membrane 12 has fallen. A to E are reagent bottles 14 (14a to 14e), F is a sample bottle, and G is a drain bottle for collecting waste liquid. In the state of (a), A to G are connected to the dialysis crystallization chamber 11 side. When the semipermeable membrane 12 is lowered to the state of (b), A to G are connected to the solution mixing chamber 13 side. The supply of the sample to the dialysis crystallization chamber 11 and the discharge of the waste liquid from the dialysis crystallization chamber 11 are performed in the state of (a), and the search for the crystallization conditions can be performed in the state of (b). The semipermeable membrane used in the apparatus of the present invention can be appropriately selected in consideration of the molecular weight of a biopolymer for which crystallization conditions are searched for in the apparatus of the present invention. 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 VIVASCIENCE can be used, but is not limited thereto. is not.

[First exploration method of the present invention]

Hereinafter, the first search method of the present invention will be described with reference to FIG. 1, which schematically shows the apparatus of the present invention.

In the first exploration method of the present invention, two solutions are prepared, one of which contains a biopolymer, and both are separated by a semipermeable membrane. In the apparatus shown in Fig. 1, a biopolymer-containing solution is put into the crystallization chamber 11, and a solution containing no biopolymer is put into the solution mixing chamber 13 separated from the dialysis crystallization chamber 11 by a semipermeable membrane 12. . At this point, the biopolymer-containing solution and the solution in the solution mixing chamber 13 separated from the biopolymer-containing solution by the semipermeable membrane 12 are, for example, aqueous solutions containing the same type and concentration of a pH buffer. Is a biopolymer The containing solution may be a solution further containing a biopolymer. In this state, since the composition of both solutions is the same except for the biopolymer, substantially no substance transfer occurs. Then, the physicochemical properties of the solution are added to the solution in the solution mixing chamber 13 from the reagent bottle 14 (one of 14a to 14e), for example, through a discharge pulp 18 (18a to 18e) using a piezo element. Is added continuously or intermittently. The substance 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 precipitant, salts, additives, and water. The pH buffer, precipitant, salts, and additives can be appropriately selected from substances conventionally used for crystallization of biopolymers. Examples of the pH buffer include sodium citrate, sodium acetate, sodium / potassium phosphate, MES, sodium dycodylate, HEPES, Tris-HC1, and the like. However, these are not limited. Examples of the precipitating agent include ammonium sulfate, sodium chloride, lithium chloride, polyethylene glycolone, 2-methinolay 2,4-pentanedionole, and the like. However, these are not limited. Salts include, for example, calcium chloride, magnesium chloride, zinc chloride, salt Examples include divan covanolate, lithium sulfate, ammonium acetate, magnesium acetate, zinc acetate, cesium chloride and the like. However, these are not limited.

Examples of the additives include ethylene glycol, glycerol, urea, ethanol, propanol, isopropanol, EDTA, DDT, hexanediol, and otatyl darcoside. However, these are not limited. When a substance that changes the physicochemical properties of the solution is added to the solution in the solution mixing chamber 13, a concentration difference occurs between the solution in the solution mixing chamber 13 and the biopolymer solution, and the semipermeable membrane 12 The substance is transferred to the biopolymer-containing solution via Thereby, the physicochemical properties of the biopolymer-containing solution can be changed continuously or intermittently. By continuously or intermittently monitoring the state of the biopolymer-containing solution during that time, the physicochemical properties of the solution in which the biopolymer crystallizes can be explored. Specifically, the state of a biopolymer-containing solution is monitored by, for example, a dynamic light scattering method, and by knowing the dispersion state of the biopolymer in the solution, the biopolymer tends to crystallize. It can be determined whether or not it is higher. Monitoring by the dynamic light scattering method can be performed by irradiating a laser through a laser window 15 provided in the dialysis crystallization chamber 11. Also, once crystallization occurs, the CCD force The crystal 16 makes it possible to monitor the state of crystal formation and growth. In addition to the above, the state of the biopolymer-containing solution can be monitored by, for example, measuring the ultraviolet absorption spectrum, the electric conductivity of the solution, and the differential heat absorption of the solution. Usually, it is difficult to search for crystallization conditions by one operation. Therefore, once the monitoring is completed, part or all of the solution containing the substance that changes the physicochemical properties of the solution is removed, a new solution is replenished, the conditions are changed, and the biological Explore molecular crystallization conditions. It is appropriate to repeat the process until the optimal crystallization conditions are obtained and to search for the crystallization conditions of the biopolymer. More specifically, according to the crystallization control algorithm as shown in the flowchart of FIG. 2, (1) a step of searching for conditions under which a biopolymer is stably present in a monodispersed state; It is appropriate to perform the step of searching for nucleation conditions and, if necessary, (3) the step of optimizing the crystallization conditions.

(1) In the step of searching for a condition in which the biopolymer is stably present in a monodispersed state, a solution in which the biopolymer is dispersed is prepared, and the dispersed state of the dissolved molecule is measured by a dynamic light scattering method. In addition, by gradually adding buffer, salt, etc. from the reagent bottle to the crystallization chamber, and by changing the concentration of the biopolymer by raising and lowering the semipermeable membrane, the biopolymer is stabilized. Exist monodisperse Search for a condition that If the above conditions can be searched, proceed to (2) Crystallization nucleation condition search step.If the conditions cannot be searched, discard the solution on the side containing no biopolymer, add a new solution, and retry. (1) A step of searching for a condition in which a biopolymer is stably present in a monodispersed state is performed. In the discarding step, first, the solution on the side containing no biopolymer is replaced by water, so that the solution on the side containing biopolymer passes through the semipermeable membrane, and as a result, the solution on the side of the biopolymer is removed. Replaced by water.

(2) In the step of searching for crystallization nucleation conditions, a substance that changes the physicochemical properties of the solution is added, but various substances can be used alone or in combination of two or more. That is, one substance (or a combination of substances) is supplied to the solution in the solution mixing chamber to increase the concentration of the solution in the solution mixing chamber, so that crystallization nucleation occurs or is likely to occur. Whether the state is reached or not is monitored by, for example, a light scattering method.

If nucleation does not occur even when the search for one substance (or a certain combination of substances) has progressed to the specified concentration, change the substance, add the substance, and discard the solution in the solution mixing chamber. Then, shift to the next condition, and perform the crystallization nucleation formation search step again. As this recycling process is repeated, the biopolymer itself may be denatured, making it impossible to withstand re-search. In that case, the solution containing the biopolymer should be discarded, and the inside of the crystallization chamber should be washed several times with water, etc., and the search should be restarted by injecting the biopolymer solution again. Can be done. The above solution disposal and the transition to the next solution are repeated until crystallization nuclei are formed. For example, by using a computer to link the monitoring of the crystallization nucleation by light scattering method with the addition of the substance and the control of the disposal of the solution and the transition to the next solution, the crystallization nucleation is performed. The crystallization nucleation condition search step can be performed repeatedly by changing the type and combination of the substances until the conditions under which the crystallization occurs can be reached. For example, a large number of solutions of substances that change the physicochemical properties of the solution are prepared by changing the type of substance, and the order and combination of the addition of these solutions to the solution mixing chamber (for example, when multiple solutions are added simultaneously). Combination) and the timing of suspension of solution addition and the timing of solution disposal are programmed in advance, and according to this programming, the operations of solution supply and monitoring are sequentially performed until crystallization nucleus formation is observed. And monitor the results and search for crystallization nucleation conditions.

The search progresses to a certain concentration for one substance (or a certain combination of substances), When nucleation occurs, the crystal is grown as it is. Or, if crystallization is performed but no good product is obtained, (3) a crystallization condition optimization step is performed. In the crystallization nucleus formation step, whether or not the obtained crystal nucleus is a good / defective initial crystal can be determined by observing a time-varying pattern of dynamic light scattering. That is, when a good crystal nucleus is formed, the dynamic light scattering pattern shows a dispersion value that fluctuates gradually near monodispersion. On the other hand, a sharp change in the dispersion state is observed in the defective crystal nuclei. From the good crystal nuclei obtained in this way, good quality single crystals grow. In any of the above steps (1) to (3), the temperature of the solution can be controlled to be constant, or the temperature can be changed in parallel with the addition of the solution. When the temperature at the time of adding the solution is kept constant, the search operation under the same conditions can be performed by changing the temperature. The solution temperature for carrying out the method of the present invention is not particularly limited. The solution temperature can be lowered as long as freezing does not occur, and can be raised as long as the biomolecule does not undergo denaturation. The solution temperature can be, for example, in the range of 0 to 40 ° C.

[Second exploration method of the present invention]

Next, a second search method of the present invention will be described with reference to FIG.

In the second exploration method of the present invention, similarly to the first method, two solutions are prepared, one of which contains a biopolymer, and both are separated by a semipermeable membrane 12. In the device of Figure 1, The biopolymer-containing solution is put into the dialysis crystallization chamber 11, and a solution containing no biopolymer is put into the solution mixing chamber 13 separated from the dialysis crystallization chamber 11 by a semipermeable membrane 12. At this point, the biopolymer-containing solution and the solution in the solution mixing chamber separated from the biopolymer-containing solution by a semipermeable membrane are, for example, aqueous solutions containing the same type and concentration of a pH buffer. It is a liquid, and the biopolymer-containing solution may be a solution further containing a biopolymer. In the second exploration method of the present invention, the volume of the biopolymer-containing solution is reduced or increased continuously or intermittently. The volume of the biopolymer-containing solution can be reduced or increased, for example, by making the semipermeable membrane 12 movable and moving it up and down in the figure. Alternatively, it is also possible to reduce or increase the volume of the biopolymer-containing solution by leaving the bottom or side wall of the dialysis crystallization chamber 11 partially movable, for example, and moving the movable bottom or side wall. it can. By decreasing or increasing the volume of the biopolymer-containing solution, the concentration of the biopolymer in the biopolymer-containing solution can be changed. That is, by reducing the volume, the solution other than the biopolymer moves from the solution in the dialysis crystallization chamber 11 to the solution mixing chamber 13, and as a result, the concentration of the biopolymer in the solution in the dialysis crystallization chamber 11 decreases. Increase. Conversely, increasing the capacity decreases the biopolymer concentration. The condition of the biopolymer-containing solution is monitored continuously or intermittently in response to changes in the biopolymer concentration, and the conditions of the solution where biopolymer crystals are generated are searched. Specifically, the state of the biopolymer-containing solution is monitored, for example, by dynamic light scattering, By knowing the dispersion state of the biopolymer in it, it can be determined whether or not the tendency of the biopolymer to crystallize has increased. Once crystallization has occurred, the CCD camera The state of formation and growth can be monitored. The method of monitoring the state of the biopolymer-containing solution is the same as the first search method. In the second exploration method of the present invention, in parallel with the change in the volume of the biopolymer-containing solution, or independently of the change in the volume of the biopolymer-containing solution, the biometric A substance that changes the physicochemical properties of the solution is continuously or intermittently added to the solution separated from the molecule-containing solution and the solution separated by the semipermeable membrane, so that the physicochemical properties of the biopolymer-containing solution are continuously changed. Alternatively, it can be changed intermittently and the state of the biopolymer-containing solution can be monitored continuously or intermittently. For example, by decreasing or increasing the volume of a solution containing a biopolymer, a biopolymer concentration at which a biopolymer is easily crystallized is found. Then, by adding a substance that changes the physicochemical properties of the solution at this biopolymer concentration, the conditions under which the biopolymer is crystallized can be further explored in the same way as the first exploration method. can do.

Even during the addition of a substance that changes the physicochemical properties of the solution, it is possible to increase or decrease the volume of the biopolymer-containing solution to change the solution to a state more suitable for crystallization. In the second exploration method, addition of substances that change the physicochemical properties of the solution When monitoring is once completed, remove part or all of the solution to which the substance that changes the physicochemical properties of the solution has been added, and replace with a new solution, as in the first method. Then, by changing the conditions, the crystallization conditions of the biopolymer can be searched again. The crystallization conditions of the biopolymer can be explored by repeatedly changing the conditions (including the concentration of the biopolymer) until the optimum crystallization conditions are obtained. In the first exploration method as well, the temperature of the solution can be controlled to be constant, or the temperature can be changed in parallel with the change in the concentration of the biopolymer or the addition of the solution. To keep the temperature constant when the concentration of the biopolymer changes or when the solution is added, the search operation under the same conditions can be performed by changing the temperature. The solution temperature for carrying out the method of the present invention is not particularly limited. The solution temperature can be lowered as long as freezing does not occur, and can be raised as long as the biopolymer does not undergo denaturation. The solution temperature can be, for example, in the range of 0 to 40 ° C. When the conditions for obtaining good quality crystals can be explored by the above method, biopolymer crystals are manufactured using the crystallization conditions. The biopolymer crystals to be produced preferably have a quality and a size suitable for structural analysis. The production of this biopolymer crystal can be carried out by a conventional method. For example, a vapor diffusion method, a dialysis method, and a batch method are known. The present invention includes a biopolymer crystal produced by the above production method. That Examples of such biopolymers include water-soluble proteins, membrane proteins, nucleic acids, nucleic acid-protein complexes, and high molecular hydrocarbons. Further, the biopolymer can be a human-derived biopolymer. Examples of the polymer hydrocarbon include a cellulose derivative and a xylose derivative. The obtained biopolymer crystal can be used for crystal structure analysis. The crystal structure analysis can be performed, for example, according to the MAD method. For example, X-ray diffraction data of the crystal is measured using synchrotron radiation. Synchrotron radiation that can be used for this purpose can be generated, for example, by the large synchrotron radiation facility SP ring — 8 / RIKEN (RI KEN) beamline IBL 44 B 2 (BL45XU). 'The disclosure of the present specification relates to the subject matter included in Japanese Patent Application No. 2002-202118 filed on July 11, 2002, the entire description of which is incorporated herein by reference in particular. Industrial Applicability The method of the present invention has the following advantages.

(1) Since the search is performed while continuously optimizing the crystallization conditions, the possibility of overlooking the crystallization conditions is lower than in the conventional method.

(2) —Because it can be performed if there is a fixed amount of biopolymer sample, prepare biopolymer The amount of sample is much smaller than the conventional method.

(3) In the second exploration method, biopolymers can be concentrated, and the biopolymer concentration can be lowered at the beginning of the crystallization process. As a result, nonspecific association of the biopolymer sample can be avoided.

Claims

The scope of the claims

1. A method for investigating the crystallization conditions of a biopolymer from a solution containing a biopolymer, which comprises a substance that changes the physicochemical properties of the solution into a solution separated by a semipermeable membrane from the solution containing the biopolymer. Is added continuously or intermittently to change the physicochemical properties of the biopolymer-containing solution continuously or intermittently, and during that, the state of the biopolymer-containing solution is changed continuously or intermittently. To monitor the crystallization conditions of biological macromolecules by monitoring

2. A method for exploring the crystallization conditions of a biopolymer from a biopolymer-containing solution, in which the volume of the biopolymer-containing solution separated from the solution by a semipermeable membrane is continuously or intermittently reduced. In addition, the concentration of the biopolymer in the solution containing the biopolymer is changed by increasing the concentration, and the state of the solution containing the biopolymer is continuously or intermittently monitored during the process, whereby the crystal of the biopolymer is crystallized. A method for exploring the condition of activation.

3. The method according to claim 2, wherein the volume of the biopolymer-containing solution is decreased or increased by moving a semipermeable membrane.

4. In parallel with or independently of the change in the volume of the biopolymer-containing solution, the substance that changes the physicochemical properties of the solution is continuously added to the solution separated by the semipermeable membrane from the biopolymer-containing solution. Or intermittently added to continuously or intermittently change the physicochemical properties of the biopolymer-containing solution, and continuously or intermittently monitor the state of the biopolymer-containing solution during that time. The method according to claim 2 or 3.

5. Before changing the conditions, the biopolymer-containing solution and the solution separated from the biopolymer-containing solution by the semipermeable membrane are aqueous solutions containing the same type and concentration of pH buffer. The method according to any one of claims 1 to 4, wherein

6. The method according to any one of claims 1 to 5, wherein the biopolymer is a water-soluble protein, a membrane protein, a nucleic acid, or a protein-nucleic acid complex.

7. The method according to claim 1, wherein the substance that changes the physicochemical properties of the solution is at least one substance selected from the group consisting of a pH buffer, a precipitant, a salt, an additive, and water. A method according to any one of the preceding claims.

8. The method according to claim 7, wherein at least 1'substance selected from the group consisting of a pH buffer, a precipitant, salts and additives is used as a solution containing this substance.

9. After the monitoring is completed, remove part or all of the solution containing the substance that changes the physicochemical properties of the solution, add a new solution, change the conditions, and again The method according to any one of claims 1, 3 to 8, wherein the crystallization conditions are searched.

10. The method according to claim 9, wherein the crystallization conditions of the biopolymer are explored by repeatedly changing the conditions until the optimum crystallization conditions are obtained.

1 1. The method according to any one of claims 1 to 10, wherein the state of the biopolymer-containing solution is monitored using a light scattering method and / or a CCD camera.

1 2. The method according to any one of claims 1 to 11, wherein the method is carried out with the temperature kept constant or fluctuated.

1 3. A method for producing a biopolymer crystal using the crystallization conditions probed by the method according to any one of claims 1 to 12.

14. A biopolymer crystal produced by the production method according to claim 13.

15. The biopolymer crystal according to claim 14, wherein the biopolymer is a water-soluble protein, a membrane protein, a nucleic acid, a protein mononuclear acid complex, or a polymer hydrocarbon.

16. The biopolymer crystal according to claim 15, wherein the biopolymer is derived from a human.

17. A dialysis crystallization chamber and a solution mixing chamber separated by a movable semi-permeable membrane, wherein the dialysis crystallization chamber has a window for monitoring the room, and

An apparatus for investigating crystallization conditions of a biopolymer, wherein the solution mixing chamber is connected to one or more reagent bottles so that the solution can be supplied from the reagent bottles.

18. The apparatus according to claim 17, wherein the window for monitoring the room is a laser window for monitoring by a dynamic light scattering method.

19. The apparatus according to claim 17 or 18, wherein the dialysis crystallization chamber has a sample inlet, and one or both of the dialysis crystallization chamber and the solution mixing chamber has a solution outlet.

20. The apparatus according to any one of claims 17 to 19, wherein the semipermeable membrane is moved up and down via a plunger.