JP2010030811A - Treatment device for protein solution and preparation method of protein crystal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a treatment device for a protein solution, efficiently attaining crystallization conditions of proteins with high accuracy and preparing a protein crystal, and to provide a preparation method of a protein crystal. <P>SOLUTION: In a stimulus application step (STA), after starting to apply stimulus for accelerating crystallization to a mixture liquid 3 in a specified container 2 in a plurality of containers 2 that store a mixture solution 3 containing a protein solution, application of stimulus to the mixture solution 3 in other containers 2 is sequentially started at a predetermined time interval; and when changes associated with the crystallization are detected in the specified container 2, application of stimulus to the mixture solution 3 in the all containers 2 is stopped so as to prepare mixture solutions 3 having different stimulus application times as samples for selecting crystallization conditions. These samples are stored under predetermined conditions in a storage step (STB) to grow crystal nuclei produced in the mixture solutions 3. In an observation step (STC), degrees of crystallization acceleration are observed, by which an optimal stimulus application time for crystallization acceleration can be found. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a protein solution processing apparatus for performing a predetermined process for generating crystal nuclei in a protein solution in the production of a protein crystal, and a protein crystal production method for producing a protein crystal from a protein solution.

  In recent years, efforts to make effective use of genetic information in fields such as medicine have become active, and efforts have been made to analyze the structure of proteins, which are gene products, as the basic technology. This protein structure analysis specifies the three-dimensional structure of the protein and is performed by methods such as NMR (nuclear magnetic resonance) and X-ray crystal structure analysis. Such protein structural analysis can be carried out in the form of a solution using a NMR apparatus for proteins having a molecular weight of 20,000 or less, but proteins having a molecular weight of 20,000 or more, which occupy the majority, are required to be crystallized first. Various crystallization methods and crystallization condition screening methods have been proposed in addition to conventionally known crystallization methods such as vapor diffusion methods and microbatch methods (see, for example, Patent Documents 1 and 2).

Patent Document 1 discloses the knowledge that a nucleus of a macromolecular crystal can be formed and grown by irradiating a solution of a macromolecule such as a protein with light. Moreover, in patent document 2, based on the knowledge that it is possible to produce | generate the crystal nucleus of a protein by irradiating a low supersaturated solution of a protein solution with a laser beam, the crystal nucleus produced | generated by laser beam irradiation is used. There are disclosed a method for obtaining a protein crystal by growing, a method for obtaining desirable crystallization conditions by determining the presence or absence of generation of crystal nuclei, and the like. By exploring protein crystallization based on this knowledge, screening of protein crystallization conditions, which previously had to rely on the intuition and experience of skilled researchers, can be performed more efficiently. It is expected that.
JP 2003-306497 A International Publication No. 2004/018744 Pamphlet

  However, in the above-mentioned patent document examples, the level of knowledge is that protein crystallization is possible by light irradiation or laser light irradiation, and protein crystallization and crystallization conditions are only achieved by the techniques disclosed in these documents. It was still difficult to perform screening efficiently. In other words, in order to reach the conditions of light irradiation and laser light irradiation that give good protein crystallization results, trials and errors such as determining the presence or absence of protein crystal nuclei in various cases In the practical sense, it has not yet reached a practical level of technology that can efficiently reach crystallization conditions that are reasonably accurate and can produce protein crystals with good reproducibility.

  Therefore, an object of the present invention is to provide a protein solution processing apparatus and a protein crystal preparation method capable of efficiently reaching protein crystallization conditions with high accuracy and preparing a protein crystal.

The protein solution processing apparatus of the present invention is a protein solution processing apparatus for performing a predetermined process for producing crystal nuclei in a protein solution in the production of a protein crystal, and comprises a solution containing a liquid containing the protein solution. A plurality of container holders that can be held; stimulus applying means for applying a stimulus for promoting the generation of crystal nuclei to the liquid in the container held in the container holder; and stimulation by the stimulus applying means The stimulus application starting means for starting the application of the stimulus from a specific container among the plurality of containers, and thereafter sequentially starting the application of the stimulus to the liquid in the other container at a predetermined time interval; and A stimulus application stopping unit that stops applying stimulus to the liquid in the containers for all of the plurality of containers based on a detection result of detecting the change of the liquid in a specific container among the plurality of containers; With was.

  The protein crystal production method of the present invention is a protein crystal production method for producing a protein crystal by growing a crystal nucleus generated by performing a predetermined treatment on a protein solution, the protein solution and the crystallization Preparing a plurality of containers containing a liquid containing a solution, starting to give a stimulus for promoting the formation of crystal nuclei with respect to the liquid in the specific container among the plurality of containers; After the application of the stimulus to the liquid in the container is started, the application of the stimulus to the liquid in the other container is sequentially started at a predetermined time interval to change the liquid in the specific container. If the change is detected in the monitoring process, the application of the stimulus to the liquid in all the containers is stopped, and the liquid in the plurality of containers to which the stimulus is applied is stored under a predetermined condition. The plurality Grown crystal nuclei said generated in the liquid in the container to produce the protein crystals.

  The protein crystal production method of the present invention is a protein crystal production method for producing a protein crystal by growing crystal nuclei generated by performing a predetermined treatment on a protein solution, including the protein solution. Preparing a plurality of containers containing liquid, starting to give a stimulus for promoting the generation of crystal nuclei to the liquid in a specific container among the plurality of containers, In the process of starting the application of the stimulus to the liquid and then sequentially starting the application of the stimulus to the liquid in the other container at a predetermined time interval, and monitoring the change of the liquid in the specific container If the change is detected, the application of the stimulus to the liquid in all the containers is stopped, and the liquid in the plurality of containers to which the stimulation is applied is mixed with the liquid containing the crystallization solution. Under the specified conditions By tube, the plurality of crystal nuclei generated in the liquid mixture in the container is grown to produce the protein crystals.

  According to the present invention, after starting to give a stimulus for crystallization promotion to a liquid in a specific container among a plurality of containers containing a liquid containing a protein solution, the liquid in another container is started. Stimulation is initiated sequentially at predetermined time intervals, and if changes in the liquid associated with crystallization are detected in the process of monitoring changes in the liquid in a particular container, By stopping the application of the stimulus to the liquid, a plurality of containers containing liquids having different stimulus application times are prepared as samples for crystallization condition selection. Crystallization is promoted by storing the liquids in a plurality of containers to which these stimuli are applied under predetermined conditions and growing the crystal nuclei generated in the mixed liquid in the plurality of containers to produce protein crystals. Therefore, it is possible to find the optimal stimulus application time for the protein, and to efficiently reach the protein crystallization conditions with high accuracy and produce the protein crystal.

(Embodiment 1)
FIG. 1 is a block diagram showing the configuration of a protein solution processing apparatus according to Embodiment 1 of the present invention, FIG. 2 is a process explanatory diagram of the protein crystal production method according to Embodiment 1 of the present invention, and FIG. FIG. 4 is a flow chart showing a crystal nucleus generation condition in the first embodiment of the present invention, and FIGS. 5, 6, and 7 are the present invention. The block diagram which shows the structure of the processing apparatus of the protein solution of Embodiment 1 of this, FIG. 8 is explanatory drawing of the test result performed for determination of the crystallization promotion degree in Embodiment 1 of this invention.

First, the structure of the protein solution processing apparatus 1 will be described with reference to FIG. The protein solution processing apparatus 1 is used in a series of test operations for screening crystallization conditions in the production of protein crystals for the purpose of protein structure analysis, and a predetermined process for generating crystal nuclei in the protein solution. It has the function to perform.

  In FIG. 1, containers 2A, 2B, 2C, 2D, and 2E are all sample storage containers made of a transparent material such as glass or resin, and the containers 2A, 2B, 2C, 2D, and 2E are treated. The target mixed liquid 3 (liquid) is stored. The mixed solution 3 is a mixture of the protein solution 3a and the crystallization solution 3b, and the containers 2A, 2B, 2C, 2D, and 2E are held by the container holders 4A, 4B, 4C, 4D, and 4E, respectively. In the following description, the containers 2A to 2E and the container holders 4A to 4E are simply referred to as the container 2 and the container holder 4 when it is not necessary to distinguish them individually. That is, the assembly of the container holders 4 constitutes a container holder that can hold a plurality of containers 2 that contain a mixed solution 3 (liquid) containing the protein solution 3a.

  The container holders 4A, 4B, 4C, 4D, and 4E are mounted on the vibrators 5A, 5B, 5C, 5D, and 5E, respectively, and the vibrators 5A, 5B, 5C, 5D, and 5E are operated by the control unit 7. By doing so, mechanical vibrations having a predetermined frequency and amplitude are applied to the liquid mixture 3 stored in each of the containers 2A, 2B, 2C, 2D, and 2E. The application of such mechanical vibration to the mixed solution 3 serves as a physical stimulus to the protein solution 3a contained in the mixed solution 3, and has the effect of promoting the generation of crystal nuclei indispensable for protein crystallization by this stimulus. . Therefore, the vibrators 5A, 5B, 5C, 5D, 5E and the control unit 7 for operating them are for accelerating the generation of crystal nuclei with respect to the liquid mixture 3 in the container 2 held in the container holding part. It is a stimulus applying means for applying a stimulus.

  Among these plural containers 2, a specific turbidity container 2 (here, the container 2A located at the left end) is used as a detection target, and a white turbidity detection sensor 6 is provided. Communicated. Here, the white turbidity detection sensor 6 detects a white turbidity phenomenon, which is an optical change that occurs in the liquid mixture 3 inside the container 2 </ b> A with the application of vibration. This white turbidity is caused by the generation of protein crystal nuclei in the mixed solution 3, and the generation thereof is promoted by physical stimulation such as mechanical vibration.

  The white turbidity in the mixed solution 3 does not directly indicate that crystal nuclei have grown and protein crystals have been produced, and changes due to crystal nucleation denaturation or aggregation at a stage where the crystals have not yet been reached are optical. Indicates that detection is possible. In other words, the mixed solution 3 in which white turbidity is detected indicates that there is a high possibility that protein crystal nuclei are generated in the mixed solution 3 at any time prior to the detection of white turbidity. Therefore, the white turbidity detection sensor 6 serves as a detection means for detecting a change in the liquid mixture 3, that is, a change caused by the generation of protein crystal nuclei, for a specific container 2A.

  The control unit 7 controls four vibration timers DT1, DT2, DT3, DT4, a time limit timer LT1, and five time measurement timers in order to temporally control the vibration applying operation by the vibrators 5A, 5B, 5C, 5D, 5E. RT1, RT2, RT3, RT4, RT5 are built in. The delay timers DT1, DT2, DT3, and DT4 have a function of measuring the operation delay time for sequentially operating the vibrators 5A, 5B, 5C, 5D, and 5E at predetermined time intervals. That is, the timing of the delay timer DT1 starts simultaneously with the start of the operation of the vibrator 5A that starts the operation first. Thereafter, when the delay timer DT1 times out, the next vibrator 5B starts to operate and the time measurement of the delay timer DT2 starts. Similar operation control is sequentially executed for the other vibrators 5C to 5E and the delay timers DT3 and DT4. Accordingly, the vibrators 5A, 5B, 5C, 5D, and 5E are sequentially turned on every time when the delay time defined by the delay timers DT1, DT2, DT3, and DT4 elapses after the vibrator 5A starts operating. Start operation.

  The time timer 9 measures whether or not the processing duration time of the protein solution processing apparatus 1 has reached a time set in advance as an upper limit value. That is, when the vibrators 5A, 5B, 5C, 5D, and 5E start operating sequentially and it is confirmed by the time-up of the timed timer 9 that the preset processing duration has elapsed, the control unit 7 Stops the operation of all the vibrators 5A to 5E. The time measurement timers RT1, RT2, RT3, RT4, and RT5 are applied with actual vibrations in which vibrations are applied to the containers 2A, 2B, 2C, 2D, and 2E by the vibrators 5A, 5B, 5C, 5D, and 5E, respectively. It has a function of measuring time and storing measurement results.

  In the above-described configuration, the control unit 7 including the four delay timers DT1 to DT4 applies the stimulation by the vibrators 5A, 5B, 5C, 5D, and 5E, which are stimulation applying means, to a specific one of the plurality of containers 2. It is a stimulus application starting means that starts with the container 2A and thereafter sequentially starts applying the stimulus to the mixed solution 3 in the other containers 5B, 5C, 5D, and 5E with a predetermined time interval. Based on the detection result of detecting the change of the mixed liquid 3 in the container 2A, it functions as a stimulus application stopping unit that stops applying stimulus to the mixed liquid 3 in the containers 2 for all the plurality of containers 2. The stimulus application stopping unit is configured to stop applying the stimulus based on the detection result of the white turbidity detection sensor 6 which is a detection unit that detects a change in the liquid mixture 3 for the specific container 2A.

  The operation / input unit 8 is an input device such as a keyboard or a mouse, and performs operations such as commands for operating the protein solution processing apparatus 1 and input of delay times set in the delay timers DT1 to DT4. The display unit 9 is a display panel such as a liquid crystal device, and displays a guidance screen during operation by the operation / input unit 8 and time measurement results by the time measurement timers RT1 to RT5. Instead of detecting the white turbidity in the container 2A by the white turbidity detection sensor 6, an operator who performs the processing operation by the protein solution processing apparatus 1 visually observes the inside of the container 2A, and white turbidity is generated in the mixed liquid 3. The stimulus may be stopped by visually recognizing the fact and inputting the fact from the operation / input unit 8. In this case, the stimulus application stopping unit is configured to stop applying the stimulus based on a predetermined operation input performed by an operator observing the container 2A, which is a specific container, by detecting a change in the liquid. ing.

  FIG. 2 shows a process flow of a protein crystal manufacturing method performed using the protein solution processing apparatus 1 shown in FIG. In the protein crystal manufacturing method shown in the present embodiment, a physical stimulus such as mechanical vibration is first applied to the mixed solution 3 (mixed solution of the protein solution 3a and the crystallization solution 3b) stored in the container 2. The stimulus imparting step (STA) for imparting is performed to generate crystal nuclei in the mixed solution 3. Thereafter, for the purpose of growing crystal nuclei generated in the stimulating step, a storage step (STB) for storing the container 2 containing the mixed solution 3 in a predetermined environment in an incubator or the like is executed, An observation step (STC) is performed for observing the mixed solution 3 whose storage time has elapsed to determine the degree of crystallization promotion. At this time, a crystal nucleus suitable for crystallization is generated by applying an appropriate stimulus in the stimulus applying step, and the crystallization of this crystal nucleus is favorably promoted in the mixed solution 3a. With the passage of time, crystal nuclei grow into protein crystals in the mixed solution 3, and the formation of protein crystals 10 is confirmed in the observation step.

As shown in Patent Documents 1 and 2, it has been obtained as qualitative knowledge from the past that the application of physical stimulation to the mixed solution 3 contributes to the promotion of the formation of crystal nuclei. It was. However, it has not yet been elucidated at present how the strongest vibration and how long it is applied for the generation of crystal nuclei has not been elucidated. There is a fact that there is no effective way to find out the appropriate stimulus application conditions by trial. For this reason, in the protein crystal manufacturing method shown in the present embodiment, in order to find the optimum conditions for generating crystal nuclei, a plurality of samples with different vibration application times are prepared as described below. Then, by storing these plural samples under a predetermined condition and observing the degree of acceleration of crystallization, the optimum condition for imparting the stimulus for generating the crystal nucleus is estimated.

  Next, detailed steps of the stimulus applying step (STA) shown in FIG. 2 will be described with reference to FIG. In the following description, a stimulus application step using the protein crystal solution processing apparatus 1 shown in FIG. 1 is shown, and mechanical vibration application using a vibrator is used as a physical stimulus application method. However, the specific form of stimulus application is not limited to mechanical vibration, but a method of applying stimulus by light irradiation as described later, or a method of combining mechanical vibration and light irradiation. It may be.

  First, when an operation input for starting a processing operation is performed from the operation / input unit 8, an operation command is output from the control unit 7 to the vibrator 5A serving as a stimulus applying unit, thereby starting to give a stimulus to the container 2A. (ST1). At the same time, time measurement by the time measurement timer RT1, time limit timer LT and time measurement by the delay timers DT1 to DT4 are started. Next, it is confirmed whether or not the delay timer DT1 has timed up (ST2). If NO here, the next step is skipped and proceeds to (ST4), and if YES, the control unit 7 controls the vibrator 5B. By outputting the operation command, the stimulus application to the container 2B is started (ST3). At the same time, the time measurement by the time measurement timer RT2 starts.

  Thereafter, it is confirmed whether or not the delay timer DT2 has timed up (ST4). If NO in this case, the next step is skipped and the process proceeds to (ST6). By outputting an operation command to 5C, stimulus application to container 2C is started (ST5). At the same time, the time measurement by the time measurement timer RT3 starts. Next, it is confirmed whether or not the delay timer DT3 has timed up (ST6). If NO in this case, the next step is skipped (ST8), and if YES, the control unit 7 controls the vibrator 5D. By outputting an operation command to, stimulus application to the container 2D is started (ST7). At the same time, the time measurement by the time measurement timer RT4 starts.

  Thereafter, it is confirmed whether or not the delay timer DT4 has timed up (ST8). If NO here, the next step is skipped (ST10), and if YES, the control unit 7 causes the vibrator to vibrate. By outputting an operation command to 5E, stimulus application to container 2E is started (ST9). At the same time, the time measurement by the time measurement timer RT5 starts. Subsequently, it is confirmed whether the cloudiness in the liquid mixture 3 in the container 2A is detected by the cloudiness detection sensor 6. If YES here, the stimulus application to all the containers 2 is stopped and all the timers are stopped (ST12). Then, the measurement result of the stimulus application time to each container 2 measured by the time measurement timers RT1 to RT5 is displayed on the display unit 9, and all the processes are completed.

  If (NO) in (ST10), it is confirmed whether or not the time limit timer LT has timed up (ST11). If the time has not yet expired, the process returns to (ST2) and repeats the same processing steps. To do. When the time is up in (ST11), it is determined that the processing duration time of the protein solution processing apparatus 1 has reached a predetermined upper limit time, and the stimulus application to all the containers 2 is stopped, and all The timer is stopped (ST14). Next, after the predetermined upper limit time has elapsed, a message indicating that no cloudiness has been detected is displayed on the display unit 9, and all the processes are terminated.

In this way, by applying the stimulus to the mixed liquid 3 stored in the five containers 2A to 2E, as shown in FIG. 4, five types of samples S1 having different application times when the stimulus is applied are shown in FIG. -S5 are prepared. That is, each of the samples S1 to S5 is given stimulation by mechanical vibration for different application times Ts1 to Ts5, and the application time Ts1.
There is a time difference corresponding to the delay time set by the delay timers (DT1 to DT4) between -Ts5. These samples S1 to S5 are stored under predetermined environmental conditions by an apparatus such as an incubator in a storage step (STB). And after passing through this storage process, in the observation process (STC), the presence or absence of crystals in the mixed liquid 3 is observed for the samples S1 to S5.

  Here, the protein crystal 10 is not observed in the sample S1 having the longest application time (application time Ts1), and in the samples S2, S3 (application times Ts2, Ts3) corresponding to the second and third longest application times, Protein crystals 10 are observed. In the samples S4 and S5 corresponding to the fourth and fifth longest application times (application times Ts4 and Ts5), the protein crystal 10 is not observed. From this, it can be seen that the optimum application time for protein crystal formation is shorter than the application time Ts1 until the cloudiness is detected in the mixed solution 3.

  That is, the protein crystal production method shown in FIGS. 2 and 4 is a protein crystal production method for producing a protein crystal by growing crystal nuclei generated by performing a predetermined treatment on a protein solution, A plurality of containers 2 containing a mixture 3 containing a protein solution 3a and a crystallization solution 3b are prepared, and the generation of crystal nuclei is promoted with respect to the mixture 3 in a specific container 2A among the plurality of containers 2. After the start of giving the stimulus for the liquid mixture 3 in the specific container 2A, the stimulus is given to the liquid mixture 3 in the other container 2 at a predetermined time interval. It is supposed to start sequentially. In the process of monitoring the change of the liquid mixture 3 in the specific container 2A, if a change in the liquid mixture 3 is detected, the application of the stimulus to the liquid mixture 3 in all the containers 2 is stopped and the stimulus is applied. By storing the mixed liquid 3 in the plurality of containers 2 under a predetermined condition, a crystal nucleus generated in the mixed liquid 3 in the plurality of containers 2 is grown to produce a protein crystal. . In the present embodiment, as described above, the change in the liquid mixture 3 is white turbidity of the liquid mixture 3, and this white turbidity is optically detected by the white turbidity detection sensor 6.

  In addition to the configuration example of the protein solution processing apparatus 1 shown in FIG. 1, the configurations of the above-described stimulus applying means, stimulus applying start means, and stimulus applying stop means include the protein solution processing apparatus 1A shown in FIG. A configuration may be used. In FIG. 5, the protein solution processing apparatus 1 </ b> A includes a container storage unit 14, a container vibration unit 16, and a container transfer mechanism 20, and these are controlled by the control unit 7. The control unit 7 includes four delay timers DT1 to DT4, a time limit timer LT1, and five time measurement timers RT1 to RT5, as in the control unit 7 shown in the protein solution processing apparatus 1. It performs the same function as in the device 1.

  The container storage portion 14 is configured by placing a container holding portion 15 on a base portion 14a. The plurality of holding holes 15a provided in the container holding portion 15 store the mixed liquid 3 before the vibration applying process. A plurality of containers 2 are held. The container vibration unit 16 has a structure in which a container holding unit 17 is coupled to a vibrator 18, and the liquid mixture 3 to be subjected to vibration is placed in a plurality of holding holes 17 a provided in the container holding unit 17. A plurality of stored containers 2 are held. In a state where the vibrator 18 is operated by the control unit 7, the container 2 is held by the container holding unit 17, whereby mechanical vibration is applied to the container 2. In the container oscillating unit 16, a white turbidity detection sensor 6 similar to the protein solution processing apparatus 1 is disposed at a position where the container 2A to be transferred first is held, and the mixing contained in the container 2A. The white turbidity that is the change of the liquid 3 can be optically detected.

A container transfer mechanism 20 configured to horizontally move the transfer head 22 along the moving table 21 is disposed above the container storage unit 14 and the container excitation unit 16. The transfer head 22 has a configuration in which a gripping mechanism 25 is attached to a lower end portion of a lifting shaft 24 that is lifted and lowered by a lifting mechanism 23. The gripping mechanism 25 can grip the container 2 by two gripping chucks 25 a, and the container transfer mechanism 20 having such a configuration is controlled by the control unit 7, whereby the container 2 held in the container storage unit 14. Can be transferred to the container excitation unit 16. In FIG. 5, among the five containers 2 </ b> A, 2 </ b> B, 2 </ b> C, 2 </ b> D, and 2 </ b> E initially held in the container storage unit 14, the containers 2 </ b> A and 2 </ b> B are transferred to the container vibration unit 16 and are transferred to the container holding unit 17. The held state is shown.

  When the transfer operation of the container transfer mechanism 20 is controlled by the control unit 7, an operation command is output from the control unit 7 to the container transfer mechanism 20 by the start of the processing operation of the protein solution processing apparatus 1A. First, the container 2A is transferred from the container storage unit 14 to the container vibration unit 16. Next, each time the delay timers DT1 to DT4 time up, the containers 2B, 2C, 2D, and 2E are sequentially transferred from the container storage unit 14 to the container vibration unit 16. At this time, due to the continued application of vibration by the vibrator 18, the delay times set in the delay timers DT <b> 1 to DT <b> 4 for the containers 2 sequentially transferred to the container exciting unit 16. Sequential vibrations are started at intervals. Thereby, it is possible to apply vibration to the plurality of containers 2 with different application times by the single vibrator 18.

  In other words, in this configuration, the control unit 7 and the container transfer mechanism 20 including the delay timers DT1 to DT4 apply the stimulus by the vibrator 18 that is the stimulus applying means to the specific container 2A among the plurality of containers 2. Then, the stimulus application starting means for sequentially starting the application of the stimulus to the mixed liquid 3 in the other container 2 after a predetermined time interval is provided. Moreover, the control part 7 gives the irritation | stimulation with respect to the liquid mixture 3 in the container 2 about all the some containers 2 based on the detection result which detected the change of the liquid mixture 3 in specific container 2A among the some containers 2. FIG. It functions as a stimulus application stopping means for stopping. The stimulus application stopping unit is configured to stop applying the stimulus based on the detection result of the white turbidity detection sensor 6 which is a detection unit that detects a change in the liquid mixture 3 for the specific container 2A.

  Further, in the protein solution processing apparatus 1 and the protein solution processing apparatus 1A, an example in which mechanical vibration by a vibrator is applied as a physical stimulus for generating crystal nuclei in the mixed solution 3 is shown. However, the physical stimulus is not limited to vibration, and the same effect can be obtained by irradiating light. As a light source for light irradiation, an ultraviolet light source, a laser light source, a mercury lamp, a xenon lamp, a halogen lamp, or the like is used. In this way, a configuration example in which physical stimulation is applied by light irradiation will be described with reference to FIGS.

  First, the protein solution processing apparatus 1B shown in FIG. 6 includes a container holding unit 30 in which a plurality of holding holes 30a for holding the container 2 are provided in a planar arrangement, and the container holding unit 30 includes a plurality of containers 2. Can be held. Above the container holding part 30, a plurality of light source parts 32A, 32B, 32C, and 32E that can be individually controlled for lighting are arranged in correspondence with the positions of the holding holes 30a. In a state where 2A, 2B, 2C, 2D, and 2E are held, the light source units 32A, 32B, 32C, and 32E are positioned above the containers 2A, 2B, 2C, 2D, and 2E.

  In the container holding part 30, light shielding parts 31 are erected on both sides of the holding hole 30a. The containers 2A, 2B, 2C, 2D, and 2E are shielded by the light shielding part 31 on both sides. Thereby, the light irradiated from the adjacent light source and the light from the outside are prevented from being irradiated to each container 2. Among these containers 2, a specific container 2A is provided with a white turbidity detection sensor 6 similar to the protein solution processing apparatus 1, and a mixed liquid such as a white turbidity in the mixed liquid 3 stored in the container 2A. The change of 3 can be detected optically.

  The light sources 32A, 32B, 32C, and 32E are individually controlled to be turned on by the controller 7, and the detection result of the cloudiness detection sensor 6 is transmitted to the controller 7. The configuration of the control unit 7 includes four delay timers DT1 to DT4, a time limit timer LT1, and five time measurement timers RT1 to RT5, as in the control unit 7 shown in the protein solution processing apparatus 1. When the light source units 32A, 32B, 32C, and 32E are turned on by the control unit 7, first, the light source unit 32A is turned on to start light irradiation on the container 2A. Next, each time the delay timers DT1 to DT4 time up, the light source units 32B, 32C, 32D, and 32E are sequentially turned on in this order. Thereby, the irradiation of light by the corresponding light source units is sequentially started with a predetermined time interval with respect to the plurality of containers 2A, 2B, 2C, 2D, 2E. Then, when the white turbidity detection sensor 6 detects the white turbidity in the mixed liquid 3 in the container 2A, the control unit 7 stops the lighting of all the light source parts 32B, 32C, 32D, 32E, thereby the containers 2A, 2B, Light irradiation to 2C, 2D, and 2E is stopped.

  That is, in the above configuration, the light source units 32A, 32B, 32C, 32D, and 32E and the control unit 7 that operates them promote the generation of crystal nuclei with respect to the liquid mixture 3 in the container 2 held in the container holding unit 30. It is a stimulus imparting means for performing light irradiation as a stimulus imparting to do. The control unit 7 including the delay timers DT1 to DT4 starts the light irradiation by the light source units 32A, 32B, 32C, 32D, and 32E as the stimulus applying means from a specific container 2A among the plurality of containers 2, and thereafter It is a stimulus application starting means for sequentially starting light irradiation with respect to the mixed liquid in the other container 2 at predetermined time intervals. Further, the control unit 7 stops light irradiation on the mixed liquid 3 in the containers 2 for all the plurality of containers 2 based on the detection result of detecting the change of the mixed liquid 3 in the specific container 2A among the plurality of containers 2. It functions as a stimulus application stop means. The stimulus application stopping unit is configured to stop the light irradiation based on the detection result of the white turbidity detection sensor 6 which is a detecting unit for detecting the change of the mixed liquid 3 for the specific container 2A.

  In addition, the protein solution processing apparatus 1C shown in FIG. 7 replaces the light source units 32A, 32B, 32C, 32D, and 32E that are individually controlled in the protein solution processing apparatus 1B of FIG. A common light source unit 32 is provided above 2C, 2D, and 2E. Between the light source unit 32 and the containers 2A, 2B, 2C, 2D, and 2E, shutters 33A, 33B, 33C, 33D, and 33E that can be individually controlled to open and close are disposed, and the shutters 33A, 33B, 33C, and 33D and 33E are individually controlled to be opened and closed by the control unit 7, so that the light from the light source unit 32 can be transferred to the containers 2A and 2A only through the shutters that are opened among the shutters 33A, 33B, 33C, 33D, and 33E. 2B, 2C, 2D, and 2E can be selectively irradiated.

  In the processing operation by the protein solution processing apparatus 1C, first, in a state where the light source unit 32 is turned on, the control unit 7 opens the shutter 33A to start light irradiation on the container 2A. Next, every time the delay timers DT1 to DT4 time up sequentially, the shutters 33B, 33C, 33D, and 33E are sequentially opened in this order. Thereby, the light irradiation by the light source unit 32 is sequentially started at predetermined time intervals with respect to the plurality of containers 2A, 2B, 2C, 2D, and 2E. Then, when the white turbidity detection sensor 6 detects white turbidity in the mixed liquid 3 in the container 2A, the control unit 7 closes all the shutters 33A, 33B, 33C, 33D, 33E and blocks the light from the light source unit 32. . Thereby, the light irradiation with respect to the containers 2A, 2B, 2C, 2D, and 2E is stopped.

That is, in the above configuration, the light source unit 32 serves as a stimulus applying unit that performs light irradiation as a stimulus for promoting the generation of crystal nuclei with respect to the mixed solution 3 in the container 2 held in the container holding unit 30. ing. The control unit 7 that includes delay timers DT1 to DT4 and controls the opening and closing of the shutters 33A, 33B, 33C, 33D, and 33E performs light irradiation by the light source unit 32, which is a stimulus applying unit, among a plurality of containers 2. It is a stimulus application starting means that starts from the container 2A and thereafter sequentially starts light irradiation on the mixed liquid 3 in the other container 2 at predetermined time intervals. Moreover, the control part 7 is the stimulus which stops light irradiation with respect to the liquid mixture 3 in the container 2 about all the some containers 2 based on the detection result which detected the change of the liquid in specific container 2A among the some containers 2. It functions as a grant stop means. The stimulus application stopping unit is configured to stop the light irradiation based on the detection result of the white turbidity detection sensor 6 which is a detecting unit for detecting the change of the mixed liquid 3 for the specific container 2.

  In each of the above-described embodiments, the protein solution processing apparatus 1 and the protein solution processing apparatus 1A provide mechanical vibration as a method of applying stimulation to the mixed solution 3, and also process the protein solution. In the apparatus 1B and the protein solution processing apparatus 1C, stimulation is applied to the mixed solution 3 by light irradiation. However, in the method for producing a protein crystal shown in FIGS. Stimulation may be applied to the liquid mixture 3 by combining light irradiation.

  Next, a test example carried out to demonstrate the relationship between the stimulus application time and the degree of crystallization promotion in the protein crystal production method shown in the first embodiment will be described with reference to FIG. Here, the protein crystallization is performed in the mixed solution by applying stimulation by light irradiation to a mixed solution obtained by mixing a lysozyme (lysozyme) solution that is a protein solution and a crystallization solution. .

  The experimental procedure will be described. First, 100 μl of paraffin oil was poured into each well of a microplate, and 1.4 M NaCl · 0.2% PEG 8000 (50 mM acetate buffer pH 4.3) as a crystallization solution and 14 mg / ml as a protein solution in the well oil. An equal amount of ml Lysozyme (50 mM acetate buffer pH 4.3) was mixed. (Final concentration: 7 mg / ml Lysozyme, 0.7 M NaCl.0.1% PEG8000). Here, seven types of samples S1 to S7 were prepared in order to perform tests for seven different light irradiation times.

  Next, the mixed solution in the well of the microplate was irradiated with light by an xenon lamp through oil. The irradiation time is 0, 0.5, 1, 1.5, 2, 3, 4 minutes for samples S1 to S7, respectively. And as a result of visually judging the presence or absence of the cloudiness in the well after light irradiation, the cloudiness was recognized from about 3 to 4 minutes of irradiation time. Thereafter, each well was vibrated at a rotational speed of 2000 rpm, and then stored in an environment of a temperature of 20 ° C. and a humidity of 50% for 1 day to generate crystals. Then, the degree of crystallization promotion in the mixed solution was determined by observing the inside of the well. Here, one having a short side of 20 μm or more is defined as a protein crystal.

  The determination of cloudiness was determined based on whether the visual result corresponds to any of the three stages of X, Δ, and ◯ shown in the following criteria. That is, X was determined when no cloudiness was observed at all, Δ determination was made when doubtful as to cloudiness, and ○ determination was made when visible cloudiness was observed. In addition, the degree of crystallization promotion was determined depending on whether the visual result corresponds to one of three stages 0, 1, and 2 shown in the following criteria. That is, when the light irradiation time is equal to sample 1 with no vibration at 0 minutes, 0 is determined, and when both the crystal appearance frequency and the number of crystals are slightly increased with respect to sample 1, 1 is determined. On the other hand, when both the crystal appearance frequency and the number of crystals were clearly increased, 2 judgments were made.

As shown in FIG. 8, white turbidity is detected in samples S6 and S7 having a long light irradiation time, and it can be seen that a certain period of stimulus application time is required for the occurrence of white turbidity. On the other hand, the degree of crystallization promotion after one day is determined as 1 in samples S2 and S5, and samples S3 and S4.
2 is determined. In other words, the degree of crystallization promotion is not directly related to the occurrence of white turbidity in relation to the stimulus application time, and the degree of crystallization promotion is better if the application of stimulus is stopped before the occurrence of white turbidity. I know that there is. In other words, the white turbidity detected by visual and optical detection is not a change indicating that the protein crystal has grown, but the white turbidity corresponds to the occurrence of turbidity in order to effectively generate crystal nuclei with a high probability of crystallization. The stimulus application may be stopped before the stimulus application time elapses. For example, the experimental example shown in FIG. 8 shows that a good crystallization promoting effect can be obtained by setting the stimulus applying time to 1/2 to 1/4 of the stimulus applying time at which white turbidity can be visually observed. .

(Embodiment 2)
FIG. 9 is a process explanatory diagram of a method for producing a protein crystal according to the second embodiment of the present invention, and FIG. 10 is an explanatory diagram of test results performed for determining the degree of crystallization promotion in the second embodiment of the present invention. is there.

  In the first embodiment, as shown in FIG. 2, an example in which stimulation is applied to the mixed solution 3 in which the protein solution 3a and the crystallization solution 3b are mixed in advance has been described. As shown in FIG. 9, the liquid containing the protein solution 3a accommodated in the container 2 is a target for stimulation, and the liquid containing the protein solution 3a after the stimulation is applied by light irradiation or mechanical vibration. The crystallization solution 3b is mixed. That is, in the second embodiment, there is a crystal between the stimulus applying step and the storage step as compared to the processing flow including the stimulus applying step (STA), the storage step (STB), and the observation step (STC) shown in FIG. This is a process configuration to which a chemical solution mixing process (STD) is added.

  In the crystallization solution mixing step (STD), the protein solution 3a after the stimulus is applied for a predetermined time in the stimulus applying step (STA) to the container 102 in which the liquid 3d containing the crystallization solution 3b is previously stored. A liquid 3c containing is mixed to prepare a liquid mixture 3 to be crystallized. And the storage process (STB) and the observation process (STC) are performed for the liquid mixture 3 in the container 102.

  That is, the protein crystal production method shown in FIG. 9 is a protein crystal production method for producing a protein crystal by growing crystal nuclei generated by performing a predetermined treatment on a protein solution. Preparing a plurality of containers 2 containing the liquid 3c to be included, and starting applying a stimulus for promoting the generation of crystal nuclei to the liquid 3c in the specific container 2A among the plurality of containers 2; After the application of the stimulus to the liquid 3c in the container 2A is started, the application of the stimulus to the liquid 3c in the other container 2 is started sequentially at predetermined time intervals, and the liquid 3c in the specific container 2A is started. If a change is detected in the process of monitoring the change, the application of the stimulus to the liquid 3c in all the containers 2 is stopped.

  Then, by storing the liquid mixture 3 obtained by mixing the liquid 3c in the plurality of containers 2 to which the stimulus is applied with the liquid 3d including the crystallization solution 3b under a predetermined condition, the liquid mixture 3 in the plurality of containers 102 is stored in the liquid mixture 3 in the plurality of containers 102. The produced crystal nucleus is grown to produce a protein crystal. In addition, the stimulus applied to the liquid 3c is either light or mechanical vibration or a combination thereof, and the change that occurs in the liquid 3c is cloudiness of the liquid 3c, and this cloudiness is optically detected. This is also the same as in the first embodiment.

  Next, a test example conducted for demonstrating the relationship between the stimulus application time and the degree of crystallization promotion in the protein crystal production method shown in the second embodiment will be described with reference to FIG. Here, after stimulating light irradiation to a Lysozyme (lysozyme) solution that is a protein solution, the protein is crystallized in a mixed solution obtained by mixing the protein solution and the crystallization solution.

  The experimental procedure will be described. First, Lysozyme was adjusted to 0.3 mg / ml with a buffer solution (100 mM CAPS pH 10.5) near the isoelectric point. 800 μl of the Lysozyme solution obtained by this operation was added to a quartz cell having an optical path length of 2 mm, and irradiated with light using a xenon lamp. Here, in order to perform tests for seven different light irradiation times, seven samples S1 to S7 are prepared, and 0, 1, 2, 4, 6, 8, and 16 minutes for samples S1 to S7, respectively. The light irradiation was performed for the light irradiation time of. And as a result of visually judging the presence or absence of white turbidity about each sample after light irradiation, white turbidity was recognized by irradiation time 8 minutes or more.

  Next, the 33.11 mg / ml Lysozyme solution (50 mM acetate buffer pH 4.3) was mixed with the Lysozyme solution after the light irradiation at a ratio of 1/10 volume to obtain a Lysozyme solution having a concentration of about 29.8 mg / ml. . Then, the obtained Lysozyme solution and the crystallization solution of 1.4 M NaCl · 0.2% PEG 8000 (50 mM acetate buffer pH 4.3) in an equal amount in a well of a microplate previously filled with paraffin oil Mixed to prepare a crystallization plate.

  Thereafter, the produced crystallization plate was stored for 3 days in an environment of a temperature of 20 ° C. and a humidity of 50% to generate crystals. Then, the degree of crystallization promotion in the mixed solution was determined by observing the inside of the well. Here too, a crystal having a short side of 20 μm or more is defined as a crystal. Further, the determination standard for white turbidity and the determination standard for the degree of crystallization promotion are the same as those shown in FIG.

  As shown in FIG. 10, white turbidity is detected in samples S6 and S7 having a long light irradiation time. In this case as well, it is necessary that a certain period of stimulus application time elapses in order to generate white turbidity. I understand. On the other hand, the degree of crystallization promotion after 3 days is 2 in sample S3 and 1 in sample S4. That is, in this case as well, as in the example shown in FIG. 8, the degree of crystallization promotion is not directly related to the occurrence of cloudiness in relation to the stimulus application time, and the stimulus application is stopped before the occurrence of cloudiness. However, it can be seen that the degree of crystallization promotion is better. In other words, the white turbidity detected by visual or optical detection is not a change indicating that the protein crystal has grown, but it is a stimulus that causes white turbidity to effectively generate crystal nuclei with a high probability of crystallization. Stimulation may be stopped before the passage of time. For example, the experimental example shown in FIG. 10 shows that a good crystallization promoting effect can be obtained by setting the stimulus applying time to about ¼ of the stimulus applying time at which white turbidity can be visually observed.

  As described above, in the first and second embodiments, the application of the stimulus for promoting crystallization to the liquid in the specific container 2A among the plurality of containers 2 containing the liquid containing the protein solution is started. Thereafter, stimulus is sequentially applied to the liquid in the other container 2 at predetermined time intervals, and the liquid is generated in connection with crystallization in the process of monitoring the change of the liquid in the specific container 2A. If a change is detected, the application of the stimulus to the liquid in all the containers 2 is stopped. Thereby, the several container 2 and the container 102 which accommodated the liquid from which irritation | stimulation provision time differs are prepared as a sample for crystallization condition selection.

  These samples are stored under predetermined conditions, and crystal nuclei generated in the mixed liquid in the plurality of containers 2 are grown to produce protein crystals, thereby providing optimum stimulation for promoting crystallization. It is possible to find time. Therefore, protein crystals can be produced by efficiently reaching high-accuracy protein crystallization conditions as compared with the prior art in which crystallization conditions are inevitably searched for a large amount of samples.

The apparatus for treating a protein solution and the method for producing a protein crystal according to the present invention have the effect that the protein crystal can be produced by efficiently reaching a highly accurate protein crystallization condition. This is useful in protein crystallization treatment performed prior to the three-dimensional structure analysis.

The block diagram which shows the structure of the processing apparatus of the protein solution of Embodiment 1 of this invention. Process explanatory drawing of the manufacturing method of the protein crystal of Embodiment 1 of this invention Flow chart of the stimulus imparting step in the protein crystal production method of Embodiment 1 of the present invention Flow chart showing crystal nucleus generation conditions in Embodiment 1 of the present invention The block diagram which shows the structure of the processing apparatus of the protein solution of Embodiment 1 of this invention. The block diagram which shows the structure of the processing apparatus of the protein solution of Embodiment 1 of this invention. The block diagram which shows the structure of the processing apparatus of the protein solution of Embodiment 1 of this invention. Explanatory drawing of the test result performed for determination of the crystallization promotion degree in Embodiment 1 of this invention Process explanatory drawing of the manufacturing method of the protein crystal of Embodiment 2 of this invention Explanatory drawing of the test result performed for determination of the crystallization promotion degree in Embodiment 2 of this invention

Explanation of symbols

1, 1A, 1B, 1C Protein solution processing device 2, 2A, 2B, 2C, 2D, 2E Container 3 Mixed solution 3a Protein solution 3b Crystallized solution 3c, 3d Liquid 4A, 4B, 4C, 4D, 4E Container holding part 5A, 5B, 5C, 5D, 5E Exciter 6 Cloudiness detection sensor 10 Protein crystal 14 Container storage unit 16 Container excitation unit 18 Exciter 20 Container transfer mechanism 30 Container holding unit 32 Light source unit 32A, 32B, 32C, 32D, 32E Light source unit 33A, 33B, 33C, 33D, 33E Shutter

Claims (7)

A protein solution processing apparatus for performing a predetermined process for producing crystal nuclei in a protein solution in the production of a protein crystal,
A container holding unit capable of holding a plurality of solutions containing a liquid containing the protein solution;
Stimulus applying means for applying a stimulus for promoting the generation of crystal nuclei to the liquid in the container held in the container holding unit;
Stimulation in which stimulation is applied by the stimulation applying means from a specific container among the plurality of containers, and then the stimulation is sequentially applied to liquids in other containers at predetermined time intervals. Grant starting means;
Stimulation application stopping means for stopping the application of stimulation to the liquid in the containers for all the plurality of containers based on the detection result of detecting the change in the liquid in a specific container among the plurality of containers. A protein solution processing apparatus.   2. The protein solution according to claim 1, wherein the stimulus application stopping unit stops applying the stimulus based on a detection result of a detection unit that detects a change in the liquid for the specific container. Processing equipment.   2. The stimulus application stopping unit stops application of the stimulus based on a predetermined operation input performed by an operator observing a specific container by detecting a change in the liquid. Protein solution processing equipment. A protein crystal production method for producing a protein crystal by growing a crystal nucleus generated by performing a predetermined treatment on a protein solution,
Preparing a plurality of containers containing a liquid containing the protein solution and the crystallization solution;
Starting to give a stimulus for promoting the formation of crystal nuclei to the liquid in a specific one of the plurality of containers,
After starting to give a stimulus to the liquid in the specific container, sequentially start giving the stimulus to the liquid in another container at a predetermined time interval,
If the change is detected in the process of monitoring the change in the liquid in the specific container, stop applying the stimulus to the liquid in all the containers;
Storing the liquid in the plurality of containers provided with the stimulus under a predetermined condition to grow crystal nuclei generated in the liquid in the plurality of containers to produce the protein crystal. A method for producing a characteristic protein crystal. A protein crystal production method for producing a protein crystal by growing a crystal nucleus generated by performing a predetermined treatment on a protein solution,
Preparing a plurality of containers containing a liquid containing the protein solution;
Starting to give a stimulus for promoting the formation of crystal nuclei to the liquid in a specific one of the plurality of containers,
After starting to give a stimulus to the liquid in the specific container, sequentially start giving the stimulus to the liquid in another container at a predetermined time interval,
If the change is detected in the process of monitoring the change in the liquid in the particular container, stop applying the stimulus to the liquid in all the containers;
Crystals generated in the mixed liquid in the plurality of containers by storing a mixed liquid obtained by mixing the liquid in the plurality of stimulated containers with a liquid containing a crystallization solution under a predetermined condition. A method for producing a protein crystal, comprising growing a nucleus to produce the protein crystal.   6. The method for producing a protein crystal according to claim 4, wherein the stimulus is either light or mechanical vibration, or a combination thereof.   The method for producing a protein crystal according to any one of claims 4 to 6, wherein the change is cloudiness of the liquid, and the cloudiness is optically detected.

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