JPH0524119B2 - - Google Patents

Description

[Detailed description of the invention]

[Summary] The present invention relates to an apparatus that automatically produces crystals of biopolymers such as proteins and nucleic acids according to preset procedures, and particularly relates to a device that automatically produces crystals of biopolymers such as proteins and nucleic acids according to preset procedures. The present invention relates to an apparatus that can realize a crystallization method (such as dialysis, dialysis, etc.) and can efficiently and reproducibly produce single crystals. [Industrial Application Field] The present invention relates to an apparatus for producing crystals of biopolymers such as proteins and nucleic acids. Atomic level structure elucidation by X-ray analysis of protein single crystals is being carried out in connection with molecular biology, protein engineering, etc. In order to understand the three-dimensional arrangement of the amino acid main chain involved in enzyme action and its relationship with action, rational experiments can be carried out by elucidating the crystal structure. When performing protein crystal structure analysis, (1) it is necessary to find the optimal protein crystal formation conditions using a small amount of protein sample; (2) X-ray diffraction requires crystals of a certain size (about several hundred microns). Factors that affect crystallization in (1) include protein concentration, type and concentration of neutral salt or organic solvent as a precipitant, pH, temperature, coexisting trace substances, etc. Related to (2), there is also an attempt to grow high-quality crystals under the weightless conditions of outer space. Genetic engineering methods, which use microorganisms to produce large amounts of physiologically active substances that exist in trace amounts in the bodies of higher animals, not only break the wall of quantitative constraints, but also break the wall of stability, and produce substances with the same biological activity. However, it can be made more stable than natural products. Any data on proteins in which arbitrary amino acids (residues) have been substituted is useful for understanding the structure, physical properties, and function of that protein. Genetic engineering allows for the purposeful substitution of arbitrary amino acids, increasing the reaction rate, changing the specificity of the reaction, and altering the polypeptide chain by heat, pH, or other enzymes. When designing an experiment to increase resistance to cleavage, if the three-dimensional structure of the protein is known, this can be carried out fairly rationally. Instruments and devices for collecting diffraction data are being developed. There are many factors that affect protein crystallization, and it is desirable to develop a crystallization process that essentially involves a series of trial and error processes. It is desired to develop an automatic machine for systematic observation and investigation. (Rigaku Denki Journal 161985, P2-5, etc.) Currently, the use of the space environment, zero gravity, no convection, high vacuum, heavy particle radiation, etc. is currently attracting attention. However, the use of the space environment has only just begun, and actual measured data is scarce, so we are still at the stage of verifying the extent to which the space environment is useful. In recent years, with the progress of genetic engineering, it has become relatively easy to modify proteins, with success stories such as modifying the substrate specificity of enzymes and improving heat resistance reported, and protein engineering is attracting attention. I've started collecting. The main approach up until now has been to randomly make changes by introducing mutations and select those that have acquired the desired function, but it is now possible to introduce changes at targeted positions using genetic engineering. It became. In order to take full advantage of this characteristic, the three-dimensional structure of the target protein is important. By the way, the current means of elucidating the three-dimensional structure of proteins has no choice but to rely on crystal X-ray diffraction. In this sense, there is great interest in the production of protein crystals. In the space environment, convection and sedimentation dissipation occur, and it is expected that large single crystals will be obtained. In addition, it has been reported that in space experiments that have already been conducted, crystal nuclei are formed early, resulting in 10 times as many crystals as on the ground. (Science, 225, P203-204, 1984) There are basically three types of protein crystal growth experimental devices that have been implemented or are being prepared for implementation, as shown in Figure 4. There is a review of Morita. (Journal of the Japanese Society of Crystallography 28, P47-49, 1986) W. Littke reported on the growth of protein single crystals using the free interface diffusion method in weightless space at the 17th AEROSPACE.
SCIENCE MEETING, New Orleans, La./
January 15−17, 1979, AIAA Paper 79−
0311, “THE GROWTH OF SINGLE
CRYSTALS FROM PROTEINS IN
GRAVITY-FREE SPACE" and the right column of page 3 states, "The details are not discussed here, but first, effective sealing and at the same time relatively slight protrusion loading were the main technical difficulties encountered. ”, pointing out the importance of sealing. Also, W.Littke is Z.Flugwiss.
Weltraumforsch.6 (1982), Heft5, 325-333
“Protein−Einkristallzuchtung in
"Microgravitationsfeld" [Protein single crystal growth under microgravity] discloses a protein crystallization apparatus for space experiments shown in FIGS. 5A, B, and C. Figure 5A is a system diagram including a cross section of a cell that grows protein crystals using the free interface diffusion method, and Figure 5B shows protein crystal growth using the free interface diffusion method using an aqueous protein solution, an aqueous buffer solution, and an aqueous precipitant solution. FIG. 5C is a front view and a side view showing the structure of the cell section and the effective sealing mechanism of the slider, and FIG. 5C is a plan view and a sectional view showing the separation slider. Littke sandwiched a separation slider to seal the aqueous solution, as shown in Figure 5B.
Dichtungsrige). Separation slider protrusion loading distance (stroke) is 15mm
It is. Sealing of the aqueous solution is performed using an O-shaped filling ring that seals the opening of the aqueous protein solution or the aqueous precipitant solution in each cell, and an oval-shaped filling ring that seals the opening of the aqueous buffer solution. In FIG. 5B, four O-shaped filling rings and four oval-shaped filling rings are in contact with one side of the separation slider, and four O-shaped filling rings and four oval-shaped filling rings are in contact with the other side of the separation slider. Total of 16 filling rings
The sealing of the aqueous solution is carried out with several filling rings. As shown in FIG. 5C, the separation slider has dimensions of 2 mm in thickness, 27.9 mm in width, and 167 mm in length, and is provided with two through holes each having a diameter of 3 mm and a diameter of 10 mm. Walter Littke used a thick plate with through-holes as a sliding plate, held a buffer solution in the through-holes, and moved the plate to perform crystal growth using the free interface diffusion method with protein solution/buffer solution/precipitant solution. However, it does not disclose an apparatus for simultaneously performing a plurality of different crystallization methods using a plurality of different types of cell groups according to the present invention. Fujita, the inventor of the present invention, also disclosed in Japanese Patent Application Laid-Open No. 62-106000 that a plurality of crystal production boxes (cell units) are arranged in a donut shape, and an optical path switching mirror is placed on the central axis of the donut to record images of the crystal growth process. Discloses an automatic biopolymer crystal production device. However, the apparatus disclosed in JP-A-62-106000 uses a pipeline to supply the desired liquid from four containers each containing a protein (myoglobin) solution, a precipitant (ammonium sulfate) solution, a buffer solution, and washing water. The solution is adjusted to suit the crystallization method and transported to each crystal production box to grow crystals of biopolymers. The crystal preparation box has an approximately inverted concave cross section, and is configured to distribute and store the solution into two small chambers separated by a weir that utilizes gravity according to the crystallization method.
The small rooms were not separated by bulkheads. or,
We will also describe the droplet method (free interface diffusion method, vapor diffusion method) that utilizes the surface tension of liquid under zero gravity conditions in space, but both of them will be used to grow biopolymer crystals according to the sequential crystallization method. This is a sequential method in which a plurality of different types of crystallization methods cannot be performed simultaneously as in the present invention. [Problems to be solved by the invention] Therefore, in the present invention, multiple crystal growth methods are executed simultaneously and differences in the growth process due to the different crystallization methods are investigated. The present invention aims to provide a biopolymer crystallization device that can clarify the following. Biopolymer crystal production in space has various constraints and requires complete automation, so in order to ensure reliable implementation, it is necessary to simplify the equipment and make it possible to set conditions. It is necessary to simplify it as much as possible. Experiment/Restraint Conditions (Equipment Constraints) A high degree of operational reliability must be ensured amidst the many constraints inherent to space-borne equipment. Among them, those that particularly affect device design are listed below. - There are limitations on device size and weight. - Energy usage is limited. - To withstand severe vibration during launch.・In the event of a malfunction or breakdown, other payloads should not be adversely affected. - Consider the optimal experimental conditions while satisfying all of these limiting conditions, and reflect them in the examination of equipment and methods. (Experimental conditions) (1) Time: The biggest constraint on producing biopolymer crystals in space is the need for a relatively long waiting period before launch. Therefore, it is necessary to design the crystallization sample solution so that it can withstand storage during that period. Further, it is desirable that the crystal growth time be as long as possible. This is because protein crystal growth takes several days, at least on earth, and in some cases several months or more. (2) Temperature: Conditions for protein crystallization on land are very delicate. This is because a slight temperature change affects the growth rate of the crystal and changes the crystal habit of the single crystal, and when crystals are produced at room temperature, the temperature change should normally be within ±1°C. However, since the amount of electricity used in space is generally limited, temperature control cannot be expected to be as good as on Earth. (3) Experimental methods There are many methods for protein crystallization, but in principle, a protein aqueous solution is slightly supersaturated to grow crystals. Among these methods, the present invention uses a crystal growth container (cell) that can carry out the three methods described below, since the crystallization method can be carried out using two liquids: a protein aqueous solution and a protein insolubilizer aqueous solution. ) structure. Stationary batch method: Mix the two liquids thoroughly to make a homogeneous mixture, and leave it in that state. It is expected that it will be possible to purely evaluate the nucleation process necessary for crystal growth and the superiority of a zero-gravity environment for protein supply during crystal growth. Free interface diffusion method: Bring the two solutions into contact while maintaining the interface between the aqueous protein solution and the insolubilizing agent solution, and the insolubilizing agent slowly permeates into the protein solution by diffusion, bringing the protein solution into a saturated state. . Since no convection phenomenon occurs in a zero gravity/vibration state,
Once formed, the free interface should be more stable than on the ground, and it is expected that we will be able to observe the crystallization phenomenon that occurs due to the increase in the concentration of the insolubilizing agent due to diffusion alone. Vapor diffusion method The aqueous protein solution (including the insolubilizing agent) and the insolubilizing agent are separated by air (or nitrogen gas), and the water in the protein solution is slowly removed to bring the protein into a supersaturated state. The influence of both moisture diffusion in the gas phase in the absence of thermal convection and diffusion of the insolubilizing agent in the solution can be considered. Equilibrium dialysis method An aqueous protein solution and an insolubilizing agent solution are separated by a dialysis membrane (such as cellulose nitrate) that allows insolubilizing agent molecules and solvent water molecules to pass through, but large protein molecules do not. The insolubilizing agent slowly diffuses into the protein aqueous solution through the dialysis membrane,
Protein precipitates and crystallizes. On the ground, crystal nuclei form near the dialysis membrane surface, but in microgravity, a gentler crystallization process is expected without being affected by convection. Therefore, when determining the cell structure, consideration must be given so that the above method can be carried out without difficulty. (Required Functions of the Cell) The following functions are required of the cell under the various restrictive conditions mentioned above. (1) Storing and preserving sample solutions during the standby period As mentioned above, sample solutions wait in the device for a relatively long period of time. Therefore, the sealing area should be as small as possible to prevent the solution from concentrating due to water evaporation.
It is necessary to prevent liquid leakage. In addition, sample proteins are inherently delicate and must be kept in a more unstable state (solution, room temperature) than normal conditions (dry powder, low temperature).
Therefore, at least due to physical and scientific affinity with the container, the solution structure must not change or proteins must not be denatured. (2) Starting crystal growth The best way to ensure operational reliability in an unmanned and fully automated device like the present invention is to simplify the starting mechanism. (3) Progress, observation, and recording of crystal growth The initiation mechanism must not cause any hindrance to the subsequent progress of crystal growth (e.g., generation of heat). Further, it is desirable that the structure be such that observation and recording can be performed during crystal growth. (Crystal production method) There are many methods for producing crystals. Basically, the protein is made slightly insoluble by mixing an aqueous protein solution with a protein insolubilizing agent solution (ammonium nitrate, methylpentanediol, etc.), and then allowed to stand. crystal growth. The purpose of the present invention is to be able to simultaneously perform four types of methods (static batch method, vapor diffusion method, free interface diffusion method, and equilibrium dialysis method) that have little similarity, and in order to make this possible, a detailed study of the equipment has been carried out. conduct. (Observation/Recording Department) Recording of protein crystal growth does not need to be a video. Further, the information obtained during the process is only images and temperature, and it is preferable to record high-quality images. [Means for Solving the Problems] As a means for solving the above problems, the present inventors have proposed the following: a first container made of a transparent material for containing a biopolymer solution; and a transparent material for containing a precipitant solution. a second container, the openings of which are arranged opposite to each other, and a slide-movable through hole that separates a first opening of the first container and a second opening of the second container; a partition wall without or having a through hole; and a first opening that seals the biopolymer solution contained in the first container and the precipitant solution contained in the second container; Seal means for sealing between one surface of the partition wall, between the second opening and the other surface of the partition wall, or between the first opening and the second opening when the partition wall is removed. , a crystal growth container in which the biopolymer solution and the precipitant solution are brought into contact with each other through the through-holes by removing the partition wall or by sliding the partition wall having the through-holes to grow crystals of the biopolymer ( A cell) is a unit, and a plurality of cells are arranged in a cylindrical shape such that the partition walls of each cell are tangent planes of the cylinder, and normal lines of the tangent planes intersect at the central axis of the cylinder. A biopolymer crystal production unit arranged radially with respect to a central axis, and a drive mechanism unit that synchronously pulls and removes or slides the partition walls of a plurality of cells in the biopolymer crystal production unit parallel to the central axis. and a control section for controlling the drive mechanism section; a first container made of a transparent material that accommodates a biopolymer solution; and a transparent container that accommodates a precipitant solution. a second container made of a material; and a partition wall that is slidably movable to separate a first opening of the first container and a second opening of the second container, the openings of which are arranged opposite to each other. and between one side of the partition wall and the first opening that seals the biopolymer solution contained in the first container and the precipitant solution contained in the second container; Seal means for sealing between the opening and the other surface of the partition wall or between the first opening and the second opening when the partition wall is removed; A biopolymer crystal growth container in which a partition wall having holes is slid and the biopolymer solution and the precipitant solution are brought into contact with each other through the through hole to grow crystals of the biopolymer, wherein the partition wall is penetrated. The sealing means is made of a thin plate having no hole or a through hole, and the sealing means seals between the first opening and one surface of the partition, and the second opening and the partition. a second O-ring sealing between the surfaces, and when the partition wall is removed, the first O-ring and the second O-ring further close the first opening and the second opening; We have discovered a biopolymer crystal growth container characterized by a seal between the spaces. [Operation] For the production of biopolymer crystals in space,
By reloading only the crystal growth containers for multiple crystallization methods, it is possible to freely select the crystallization method and conditions and produce crystals with good reproducibility and reliability. [Example] The present invention will be described below with reference to the drawings. (Observation/Recording System and Cell Arrangement) The arrangement of the cell largely depends on the observation/recording method used to produce biopolymer crystals. In principle, the object (cell) is fixed and the optical system is scanned and moved (optical system scanning method), the optical system is fixed and the cell is scanned and moved to the observation area (cell scanning method), the optical path is guided, and multiple Possible methods include recording cells simultaneously in divided images (divided recording method), and a combination of these methods. Table 1 shows these methods (~)
The results of the study are shown below.

【table】

[Table] In general, from the viewpoint of achieving both mechanical strength (vibration resistance) and measurement, and operation reliability, scanning and
Moving is problematic. It is also disadvantageous in terms of power consumption. Therefore, it is inappropriate to scan at least the entire optical system. Furthermore, when scanning a cell, the microgravity environment, which is a major premise for biopolymer crystal growth, is spoiled. Therefore, it is inevitable that the image quality and clarity of image recording will be inferior. As a result of considering the above, the present invention provides space equipment that satisfies the conditions necessary for observation by adopting a cylindrical arrangement as shown in Figure 2a, taking into account observation during crystal preparation. can meet the restrictions as follows. On the other hand, if observation is not important, the cells can be grouped together in blocks as shown in FIG. 2b, and a common slide plate can be used, allowing for a more space-saving arrangement. Of course, even with this arrangement, it is possible to observe and record some cells by placing an observation recording device (still camera, VTR, etc.) next to the cell. (Cell structure and experiment starting method) Since the basic structure of the cell largely depends on the starting method of the crystallization experiment, we devised a possible experiment starting method,
We examined whether each method satisfies the above-mentioned required characteristics. The comparative study results of each method are summarized in Tables 2 and 3, and the principle and cell structure are shown in Figure 3. The characteristics and problems of each are described below. (1) Membrane destruction method This is a method in which the experiment begins by separating two liquids with a thin rubber film, destroying it by reciprocating the blade, and opening it using rubber elasticity. The mechanical part is small and the three methods can be implemented relatively easily, but
The problem is whether a thin rubber film can be obtained that satisfies all the required properties (destructibility, scientific influence on sample proteins, water permeability, etc.). (2) Thick plate parallel sliding method This method is basically the same as Littke et al.'s method, but since the slide plate is pulled out and driven by a motor, etc., the mechanical part is slightly larger. The three methods are easy to implement and have a proven track record in space. (3) Thin plate parallel sliding method Instead of the thick plate in (2), use metal (stainless steel, etc.)
This method involves pulling out a thin plate. (2) Since there are fewer sealing parts, reliability is greatly improved. (4) Hollow cylinder slide method Pour the protein solution into the hollow cylinder and the insolubilizer solution at the bottom. The experiment begins by pushing in the cylinder. Although the cell seems to have good sealing properties, it is difficult to miniaturize it to suit the current observation method and cell arrangement, and the precision of cell manufacturing is also required. (5) Rotating stopcock method This is a variation of the slide method, allowing two liquids to come into contact with each other through rotational movement rather than straight movement. This also poses a problem in that it is difficult to miniaturize the cell. (6) Solution injection/filling method One liquid is poured into a cell sealed with a thick rubber sheet like a chemical ampoule. Insert the needle into the rubber sheet and inject the other liquid. The difficulty is that the movement of the injectate in zero gravity is unpredictable. (7) Solution injection/water bead formation method Slowly inject the two liquids into a cell with no liquid using a pump or syringe to form water beads. The formation state of the free interface cannot be predicted, and the crystals inside the water droplets may not be observed properly.

【table】

【table】

【table】

[Table] Among the above methods, two methods were found to be suitable for biopolymer crystal production equipment in space: the thick plate parallel slide method and the thin plate parallel slide method. (Cell material) The cell material needs to be inert to the sample protein solution during the long waiting period, and considering observation and sealing properties, it is recommended that the cell material be made of transparent resin and integrated with the observation window. is considered reasonable. A crystal growth container (cell) unit capable of realizing many types of crystallization methods has the same size, but has different structures depending on the crystallization method, as shown in FIG. Figure 1 shows free interface diffusion method a, stationary batch method b,
A conceptual diagram, a general method, and a method of the present invention are shown to explain different crystallization methods of vapor diffusion method (c) and equilibrium dialysis method (d). (1) By limiting the number of solutions to two and changing the shape of the precipitant side of the crystal preparation container and the partition wall, four types of crystallization methods that are normally carried out on the ground can be realized in a cell with the same external shape. (2) Each method shown in Figure 1 will be described. (a) Free interface diffusion method This method is the same as Littke et al.'s method, except that only two liquids are used and no buffer solution is used. (b) Stirring magnet (Teflon,
(coated with polypropylene, etc. to prevent direct contact with the solution) and stirred and mixed the two liquids by moving it back and forth using an external magnetic field. (c) Vapor diffusion method Using a perforated partition wall, the precipitant solution is impregnated with gauze, absorbent cotton, etc. The two liquids come into contact through the gas phase, and the precipitant concentration in the biopolymer solution containing the precipitant gradually increases. The temperature increases and crystals precipitate. (d) Equilibrium dialysis method Two liquids are brought into contact through a semipermeable membrane (which allows water and small molecules to pass through, but not biopolymers). Crystals can be produced with good reproducibility by an extremely simple operation of slowly pulling out the partition walls (stirring is performed only in the stationary batch method). The crystal growth container unit used in the static batch method and the free interface diffusion method is different from the crystal growth container unit used in the static batch method, which is used to mix the biopolymer solution and the precipitant solution in the initial stage. It is almost the same except that it has a small magnetic stirrer. The crystal growth container unit used in the vapor diffusion method can use a thick acrylic plate as a sliding plate to form a gas phase gap between the biopolymer solution holding chamber and the precipitant solution holding chamber. It is preferable to impregnate an absorbent material such as case or absorbent cotton with the precipitant solution to prevent movement of the liquid due to vibrations during launch, and to ensure that the two liquids come into contact through the gas phase rather than directly during production. can be maintained. FIG. 2a is a schematic cross-sectional view of a cell in a plane perpendicular to the rotation axis of a rotating mirror for realizing various crystallization methods arranged radially around the rotation axis of the rotating mirror. To observe the biopolymer crystal inside the cell, in order to block the scattered and reflected light from areas other than the cell part, a mask with circular holes that shields areas other than the cell part is installed in the crystal growth container as shown in Figure 2a. It is more preferable to provide a light-absorbing light-shielding plate on the mirror side of the unit, and on the back side of the crystal growth container unit. The biopolymer crystals are periodically photographed using a still camera equipped with a 250-frame film holder. An annular flash light emitter (ring strobe for close-up photography) located directly below the crystal growth container unit emits flash light in synchronization with the operation of the camera shutter. The lens is mounted on the cover plate of the device module. The solubility and crystal growth rate of biopolymer crystals generally depend on temperature, and it is desirable to control temperature as strictly as possible. For example, a 1% aqueous solution of Spine whale myoglobin (USA; Sigma product) was used as a biopolymer.
A saturated aqueous ammonium sulfate solution is used as the insolubilizer precipitant solution. Thereafter, the temperature of the crystal production box is adjusted by the temperature control section for a period necessary for crystal growth, and the crystal growth process is observed and recorded by the image recording section. An example of crystal production conditions for each crystal production method is shown in the table below.

[Table] Precipitating materials that can be used in the device of the present invention, ammonium sulfate, phosphate, magnesium sulfate,
Inorganic neutral salts such as sodium chloride and cesium chloride or 2-methylpentanediol (MPD),
Organic solvents that are miscible with water, such as acetone and alcohol, can be used. It is effective to use the following materials and conditions through trial production and crystallization experiments. Partition wall: Stainless steel plate (0.01 to 0.1 mm). However, for the vapor diffusion method, thick plates of transparent plastic such as acrylic, polycarbonate, and polymethylpentene are advantageous for observation, but they may react with proteins or precipitants, or dissolve. You can use anything that doesn't cause any damage. Container material: acrylic, polycarbonate,
Transparent materials such as polymethylpentene and various types of glass are good. O-rings to be used: O-rings with low water absorption and permeability, and containing no substances that can be eluted into the solution, Viton, fluorosilicone, or those coated with Teflon on the surface. Partition wall, sliding speed...0.05~10cm/min is suitable. An overall configuration diagram of the device to further clarify the present invention,
The control system will now be described with reference to a block diagram showing a growing crystal in a crystallization cell. FIG. 6A is a partially cutaway perspective view showing the overall configuration of the protein crystal growth apparatus of the present invention. This shows an experimental module in which 16 crystallization cell units each having a sliding plate are installed in a cylindrical and radial manner around a mirror rotated by a stepping motor in order to observe each cell unit in sequence. Each of the plurality of slide plates is connected to one triangular plate just above each cell, and the triangular plate is moved up and down by three ball screws that are driven by another stepping motor and rotated synchronously. , multiple slide plates can be pulled out at the same time. The cylindrical arrangement of the present invention provides a structure in which the partition removal mechanism is relatively compact and lightweight, yet resistant to vibrations. The left and right sides of FIG. 6A are rotated by 180°, and FIG. 6B is a plan view thereof, and FIG. 6C is a front view thereof. FIG. 6D is a block diagram of the control system. a, b,
Shown in c. The sixth still camera photographs the crystal growth process.
Although not shown in Figure A, it is installed on the optical axis of the lens (on the rotation axis of the mirror) attached to the cover plate of the experimental module. Since crystal solubility and crystal growth rate depend on temperature, it is desirable to control temperature as accurately as possible. Due to power supply limitations, passive temperature control is utilized consisting of foam insulation and heat storage components that utilize endothermic and exothermic reactions based on phase transitions of inorganic hydrates. This temperature control device protects the crystal from thermal fluctuations in the experimental environment and heat generation associated with battery discharge. The table below shows examples of inorganic hydrate compositions. The transition temperature can be adjusted by changing the composition of the inorganic hydrate.

〔Effect of the invention〕

Crystals can be produced with extremely simple operations, and container designs can be easily made interchangeable, so many types of crystallization methods can be performed simply by changing the container to the same operating machine. Additionally, this device can be designed to save energy and space, and can be used to create crystals in space. In the above example, an example of application to biopolymer crystal production in space was explained, but on earth, limitations such as power supply capacity are removed, and temperature control systems, recording optical systems, etc. Through various variations, it is possible to produce crystals by setting more precise conditions.

[Brief explanation of the drawing]

Figure 1 is a diagram illustrating different crystallization methods of the free interface diffusion method a, stationary batch method b, vapor diffusion method c, and equilibrium dialysis method d applied to the present invention. A cross-sectional view of cells arranged in a block shape to realize various types of crystallization methods,
Figure 3 is an explanatory diagram of the experiment starting method and cell structure for biopolymer crystal production, Figure 4 is an explanatory diagram of the published protein crystal growth experimental apparatus, and Figure 5 A, B,
C is an explanatory diagram of the conventional device; Fig. 6 A, B, C, D,
E is an explanatory diagram of the present invention.

Claims (1)

[Scope of Claims] 1. A first container made of a transparent material containing a biopolymer solution and a second container made of a transparent material containing a precipitant solution, the openings of which are arranged opposite to each other. a partition wall without or with a through hole that is slidable and capable of separating a first opening of the first container and a second opening of the second container; between the first opening that seals the biopolymer solution contained therein and the precipitant solution contained in the second container and one surface of the partition wall, and between the second opening and the other surface of the partition wall; and a sealing means for sealing between the first opening and the second opening when the partition wall is removed or the partition wall is removed, and the partition wall having the through hole is removed or the partition wall having the through hole is slid. A crystal growth container (cell) in which the biopolymer solution and the precipitant solution are brought into contact with each other through the through-hole to cause crystal growth of the biopolymer is used as a unit, and a plurality of cells are connected to the partition wall of each cell. are each tangential plane of the cylinder, and each normal line of the tangential plane intersects with the central axis of the cylinder. A drive mechanism unit that synchronizes and pulls the partition walls of a plurality of cells in the biopolymer crystal production unit in parallel to the central axis to remove or slide them, and a control unit that controls the drive mechanism unit. A device for producing biopolymer crystals. 2 an annular light source that illuminates a plurality of cells in the biopolymer crystal production unit; and a ring-shaped light source located at the center of the biopolymer crystal production unit that observes the inside of the cell illuminated by the illumination hole from the light source;
a rotating mirror for optical path switching, the mirror surface of which is arranged at an angle of 45 degrees to a rotation axis about the central axis; an image recording unit, which is arranged on the rotation axis of the rotating mirror; The biopolymer crystal production apparatus according to claim 1, comprising a control section for controlling an image recording section. 3. The shape of the cell is an isosceles trapezoid in cross-sectional shape in a plane perpendicular to the direction of movement of the partition, has a predetermined length in the direction of movement of the partition, and has its upper and lower surfaces parallel to the partition. It is a trapezoidal column, and the vicinity of the first opening and the second opening are cylindrical, and the biopolymer crystal producing section has the upper bottom surface of the cell facing the central axis, and 2. The biopolymer crystal production apparatus according to claim 1, wherein the wires are arranged cylindrically and radially so as to pass through the center of the first opening and the second opening of the cell. 4. The biopolymer crystal production apparatus according to claim 2, further comprising a mask having a circular hole that blocks light from areas other than those necessary for observation on the rotating mirror side of the cell. 5. The biopolymer crystal production apparatus according to claim 1, comprising a temperature adjustment section for temperature control. 6. Using a first partition wall made of a thin plate or a thin plate having through holes as the partition wall, remove the first partition wall, or move the first partition wall and pass the biopolymer through the through holes. a first cell in which a solution and the precipitant solution are brought into contact with each other to grow biopolymer crystals by a free-field diffusion method; and a stirrer provided in either the first container or the second container; A second partition wall made of a thin plate or a thin plate having a through hole is used as the partition wall, and the second partition wall is removed, or the second partition wall is
After the biopolymer solution and the precipitant solution are brought into contact with each other through the through-hole by moving the partition wall, the solution is mixed and stirred using the stirrer, and the crystal growth of the biopolymer is caused by a stationary batch method. and a third partition wall made of a thick plate having through holes as the partition wall, the biopolymer solution is moved through the third partition wall and passed through the gas held in the through holes. and the above-mentioned precipitant solution to cause crystal growth of the biopolymer by a vapor diffusion method, or the above-mentioned precipitation is carried out in the above-mentioned second container using a fourth partition consisting of a thin plate or a thin plate having through holes as the partition. At the same time, the precipitant solution impregnated with the water absorbing agent is accommodated so as to be in contact with the fourth partition wall through the gas phase, and the fourth partition wall is removed and the precipitant solution is impregnated into the water absorbing agent. The biopolymer solution and the precipitant solution are brought into contact with each other through the phase portion or through the through hole and the gas phase portion by moving the fourth partition, and crystal growth of the biopolymer is caused by a vapor diffusion method. using a fifth partition wall made of a thin plate having a through hole and a dialysis membrane provided in the through hole as the partition wall, move the fifth partition wall and pass the above through the dialysis membrane. A plurality of cell groups of different types selected from a fourth cell, which brings the biopolymer solution into contact with the precipitant solution and causes biopolymer crystal growth by an equilibrium dialysis method, is provided, The apparatus for producing biopolymer crystals according to claim 1, which simultaneously grows biopolymer crystals using a plurality of different crystallization methods selected from the batch method, the vapor diffusion method, and the equilibrium dialysis method. . 7. A stirrer made of a resin-coated magnetic material is provided in either the first container or the second container, and the stirrer is moved by an external magnetic field controlled by an external magnetic field control means to mix the solution. 7. The apparatus for producing biopolymer crystals according to claim 6, which grows biopolymer crystals by stirring and a stationary batch method. 8 The sealing means includes a first O-ring that seals between the first opening and one surface of the partition wall, and a second O-ring that seals between the second opening and the other surface of the partition wall.
The biopolymer crystal production device according to claim 1, having an O-ring. 9 The partition wall is made of a thin plate, and the sealing means is the first O-ring and the second O-ring that further seal between the first opening and the second opening after the partition wall is removed. A biopolymer crystal production device according to claim 8. 10 A first container made of a transparent material containing a biopolymer solution, a second container made of a transparent material containing a precipitant solution, and a first container of the first container whose openings are arranged opposite to each other. 1 opening and the second opening
a slidingly movable partition separating a second opening of the container and sealing the biopolymer solution contained in the first container and the precipitant solution contained in the second container; Between the first opening and one surface of the partition wall, between the second opening and the other surface of the partition wall, or further between the first opening and the second opening when removing the partition wall. and a sealing means for sealing,
Biopolymer crystal growth in which the biopolymer solution is brought into contact with the precipitant solution by removing the partition wall or by sliding the partition wall having the through hole to cause crystal growth of the biopolymer. In the container, the partition wall is made of a thin plate without or with a through hole, and the sealing means includes a first O-ring that seals between the first opening and one surface of the partition wall, and the first O-ring and the second O-ring. A second O-ring seals between the opening of No. 2 and the other surface of the partition wall, and when the partition wall is removed, the first O-ring and the second O-ring further seal between the opening of No. 2 and the other surface of the partition wall. A biopolymer crystal growth container characterized in that a seal is formed between the first opening and the second opening. 11 A stirrer made of a resin-coated magnetic material is provided in either the first container or the second container, and after the biopolymer solution and the precipitant solution are brought into contact, the mixture is stirred by an external magnetic field. 11. The biopolymer crystal growth container according to claim 10, wherein the stirrer is moved to mix and stir the solution to grow biopolymer crystals by a stationary batch method. 12 The partition wall is composed of a thin plate having a through hole and a dialysis membrane provided in the through hole, and the biopolymer solution and the precipitant solution are brought into contact with each other through the dialysis membrane, and the biopolymer is removed by an equilibrium dialysis method. 11. The biopolymer crystal growth container according to claim 10, wherein the biopolymer crystal growth container is used for crystal growth. 13 The shape of the cell is an isosceles trapezoid in cross-sectional outer diameter in a plane perpendicular to the direction of movement of the partition, has a predetermined length in the direction of movement of the partition, and has an upper and lower surface that corresponds to 11. The biopolymer crystal growth container according to claim 10, wherein the container is a trapezoidal column parallel to the partition wall, and the vicinity of the first opening and the second opening are cylindrical.