JPWO2013191173A1 - Sample dispensing apparatus, protein crystallization method, and microplate forming sheet - Google Patents

Abstract

In order to provide a sample dispensing apparatus suitable for the lipid mesophase method, the sample dispensing apparatus of the present invention includes a plate chamber (4), a sample dispensing microdispenser part (2), a crystallization solution dispensing microdispenser part (3 ) And a humidity control device (11) for adjusting the humidity in the plate chamber (4), and the humidity control device (11) supplies a mixed gas of water vapor and dry gas into the plate chamber (4). To do.

Description

  The present invention relates to a sample dispensing apparatus, a protein crystallization method, and a microplate forming sheet, and more specifically, a sample dispensing apparatus, a protein crystallization method, and a microplate forming sheet that can be used for crystallization of a membrane protein. About.

  Protein is an important molecule responsible for various life activities in the living body, and exerts a correct function by taking a correct three-dimensional structure. Therefore, analysis of the structure of proteins leads to a deep understanding of life phenomena, and the information obtained by structural analysis is useful for efficient development of drugs that control the action of proteins that cause diseases, for example. To do. However, some proteins are difficult to crystallize with high quality necessary for three-dimensional structure analysis. For example, membrane proteins called difficult-to-analyze proteins play an important role in biological functions such as various receptors and channels in cells. Therefore, the structural information has not only academic value but also industrial value such as drug development. However, membrane proteins are difficult to be prepared in large quantities, crystallized and structurally analyzed with the current state of the art while maintaining activity (function). Therefore, further technical advancement for obtaining a three-dimensional crystal essential for X-ray structure analysis of membrane proteins is desired from an academic and industrial viewpoint.

  As a technique for obtaining a three-dimensional crystal of a membrane protein, the lipid mesophase method (Non-patent Document 1) is considered to be one of the promising techniques. The lipid mesophase method enables X-ray crystallographic analysis of membrane proteins that have not been successful for many years, such as β2-adrenergic G protein coupled receptor (Non-patent Document 2) and bacteriorhodopsin (Non-patent Document 3). I have to. The lipid mesophase method is a technique in which a membrane protein is reconstituted in a lipid mesophase having a lipid bilayer as a constituent unit, and crystallized as it is in a lipid bilayer matrix.

  In a method often used in a conventional crystallization method such as a vapor diffusion method, a solution containing a solubilized membrane protein is dispensed into a well of a screening plate. After dispensing, a crystallization solution (buffer solution containing electrolyte and organic compound) having a different composition for each well is added here to start crystallization, and protein crystals grow Search for solution conditions. On the other hand, in the lipid mesophase method, as a commonly used method, first, an aqueous solution containing a membrane protein is obtained by mixing (i) a matrix lipid or (ii) a matrix lipid and an appropriate aqueous solution in advance. To prepare a sample of lipid mesophase containing membrane protein (protein-lipid mesophase sample or simply referred to as sample). A quantity of protein-lipid mesophase sample is then dispensed into the wells of the screening plate. After the protein-lipid mesophase sample is dispensed to each well, a crystallization solution having a different composition is added to each well to start crystallization, and a solution condition for crystal growth of the membrane protein is searched. In this sense, the lipid mesophase method uses a protein-lipid mesophase sample instead of a solution containing a solubilized membrane protein. Crystallization is proceeding with almost the same concept and procedure as the crystallization method (Non-Patent Document 4).

In addition, in order to perform the above search, it is necessary to repeat the dispensing operation of the protein-lipid mesophase sample and the crystallization solution many times (hundreds to several thousand times / day), which is extremely difficult for manual operation. Accompany. Therefore, an apparatus that automates some of these operations has been developed (Non-patent Document 5). A working example of the lipid mesophase method using this apparatus is as follows.
1) A protein-lipid mesophase sample is manually filled into a dispensing microsyringe (about 25-100 μl of microsyringe).
2) The dispensing microsyringe is manually set in the sample dispensing microdispenser part of the apparatus.
3) Manually set the 96-well screening plate and 96-well crystallization solution box in the instrument.
4) Position the dispensing microsyringe with the naked eye by visual observation so that the tip of the dispensing microsyringe is positioned at the center of an appropriate well (for example, A1 well) of the 96-well screening plate.
5) Whether the protein-lipid mesophase sample is stably discharged from the dispensing microsyringe by a certain amount is visually confirmed by visual observation.
6) In the apparatus, humidified air from an ultrasonic humidifier is blown onto the surface of a 96-well screening plate through a plastic tube.
7) Automatically dispense protein-lipid mesophase sample by instrument, then dispense crystallization solution automatically.
8) After dispensing, remove the 96-well screening plate from the device and apply the cover glass manually.

Lnadau, E.M. et al., Proc. Natl. Acad. Sci. USA, 1996, 93, 14532-14535 Cherezov, V. et al., Science, 2007, 318, 1258-1265. Pebay-Peyroula, E., et al., Science, 1997, 277, 1676-1681. Caffrey, M., et al., Nature Protocols, 2009, 4, 706-731. Cherezov, V., et al., Acta Cryst., 2004, D60, 1795-1807. Jeremiah S., et al., Method, 2011, 55, 342-349.

  However, the protein-lipid mesophase sample is significantly different from the protein solution solubilized with the surfactant, and the crystallization mechanism of the lipid mesophase method is also crystallized from the protein solution solubilized with the conventional surfactant. It is different from that of how to do Therefore, conventional techniques based on conventional biochemistry and molecular biology techniques accumulated by conventional crystallization using a surfactant-solubilized protein solution make the most of the advantages of the lipid mesophase method. It cannot be said that it is completely clear, and it is never sufficient as a method for obtaining a high-quality crystal.

  Therefore, the present invention has been made in view of the above problems, and an object thereof is to provide an apparatus and a method suitable for crystallization by a lipid mesophase method.

  In order to solve the above problems, the sample dispensing apparatus according to the present invention is a sample dispensing means for dispensing a sample of a lipid mesophase containing a plate chamber and a membrane protein to a microplate prepared in the plate chamber. , A solution dispensing means for dispensing a crystallization solution for crystallizing the protein to the microplate, and a humidity adjusting device for adjusting the humidity in the plate chamber. And a dry gas having a humidity lower than that of water vapor are supplied into the plate chamber.

  In this specification, the “lipid mesophase method” is prepared by mixing an aqueous solution containing a membrane protein with (i) a matrix lipid or (ii) a lipid mesophase obtained by previously mixing a matrix lipid and an appropriate aqueous solution. This refers to a technique in which a membrane protein is crystallized in a lipid bilayer matrix using a lipid mesophase sample (protein-lipid mesophase sample) containing the prepared membrane protein. In the present specification, the “lipid mesophase” means a substance or state having an intermediate property between a solid and a liquid appearing in a lipid / aqueous solution mixture system. Specific examples of lipid mesophases are “cubic liquid crystals” in which the lipid bilayer has a three-dimensional continuous structure, and “lamellar liquid crystals” and “cubic liquid crystals” in which the lipid bilayers are stacked in one direction. A mesophase having a lipid bilayer membrane as a constitutional unit such as a “sponge phase” in which order is lost can be mentioned. As shown in Non-Patent Document 5, the conventional lipid mesophase method suppresses changes in the concentration of each component in the crystallization solution due to evaporation of water during the dispensing of the protein-lipid mesophase sample and the crystallization solution. Therefore, approximately 85% R.D. H. Dispensing work is performed under the above humidity conditions. According to this humidity condition, an experimental result has been obtained that the concentration change of each component in the crystallization solution due to moisture evaporation within the dispensing operation time is within 10%. Therefore, in the conventional method, such a concentration change due to evaporation in the crystallization solution is considered to be an allowable range (Non-Patent Document 5). Moreover, in order to form the said humidity environment, the simple method of spraying the humidified air from a humidifier directly on the plate surface where crystallization solution is dispensed is used. Alternatively, a strategy of shortening the time required for evaporation of the crystallization solution by shortening the time required for dispensing by increasing the dispensing speed, and the like, etc., are taken. On the other hand, the influence of humidity on the protein-lipid mesophase sample is not fully considered.

  However, as a result of repeated studies by the present inventors, in the lipid mesophase method, the lipid mesophase method such that the quality of protein crystals (resolution of X-ray diffraction) greatly depends on the humidity conditions during the dispensing of the protein-lipid mesophase sample. I found a peculiar phenomenon. This is thought to be because the fine structure of the protein-lipid mesophase sample changes depending on the humidity at the time of dispensing, and in order to fully utilize the humidity effect peculiar to such a lipid mesophase method, more precise analysis is required. It was found that it was necessary to perform humidity control during injection.

  The humidity in the present specification means a relative humidity in which the ratio of the water vapor pressure of the gas to the saturated water vapor pressure at that temperature is expressed in%. H. It shall be expressed as Further, “strictly adjusting the humidity in the plate chamber” means that a desired humidity is set and the humidity inside the plate chamber is set to a set humidity value ± 1% R.D. H. , Preferably ± 0.5% R.D. H. It is intended to be within the scope of

  In addition, in order to solve the above problems, the protein crystallization method according to the present invention dispenses a sample of lipid mesophase containing a membrane protein into a microplate prepared in the plate chamber. A solution dispensing step of dispensing a crystallization solution for crystallizing the protein onto the microplate, a mixing step of combining the dispensed sample and the dispensed crystallization solution, A protein crystallization method comprising a pasting step of pasting a transparent cover on the upper surface of the microplate after the mixing step, and a crystal growth step of crystallizing the protein in the sample after the pasting step. In the sample dispensing step and the solution dispensing step, water vapor and a lower humidity than water vapor are dried inside the plate chamber. It has a configuration for adjusting the humidity of the inside of the plate chamber by supplying a mixed gas of scan.

  The sample dispensing process or the solution dispensing process may also serve as the mixing process. For example, first, a sample dispensing process is performed to dispense a sample into a microplate, and a crystallization solution is dispensed directly into the dispensed sample in the solution dispensing process, whereby the sample, the crystallization solution, and And a mixing step may be realized.

  The microplate forming sheet according to the present invention is a microplate forming sheet having one or more well-forming through-holes that are attached to a substrate to form a microplate, and an adhesive layer is formed on the surface. The well forming through hole has a structure formed by laser processing.

  As described above, the sample dispensing device according to the present invention includes a humidity adjusting device that adjusts the humidity in the plate chamber. This humidity adjusting device is configured to supply a mixed gas of water vapor and a dry gas having a lower humidity than water vapor. Supply into the plate chamber. Therefore, the humidity in the plate chamber can be adjusted more strictly.

It is a top view which shows schematic structure of the sample dispensing apparatus in this Embodiment. It is a front view which shows schematic structure of the sample dispensing apparatus in this Embodiment. It is a side view which shows schematic structure of a cover glass sticking part. It is a disassembled perspective view which shows schematic structure of a plate chamber. It is a figure which shows the result of evaluation of the controllability of humidity in an Example. It is a figure which shows the result of evaluation of the time stability of humidity control in an Example. It is a figure which shows the temperature change at the time of humidity control in an Example. It is a figure which shows the result of the evaluation of the water | moisture-content evaporation rate in an Example. It is a figure which shows the result of the evaluation of the repeated change response of humidity in an Example. It is a figure which shows the state of the crystal | crystallization grown in the bR mesophase in an Example. It is a figure which shows distribution of the magnitude | size and number of the crystal | crystallization grown in the bR mesophase in an Example. It is a figure which shows the relationship between the time (T) in which the crystal | crystallization in bR mesophase is recognized for the first time in an Example, and a crystallization solution density | concentration. It is a figure which shows the observation result of the structural change of the lipid mesophase by normal light and a polarization microscope observation in an Example. It is a figure which shows the measurement result of the structural change of the lipid mesophase by an SAXS measurement in an Example. It is a figure which shows the observation result by the confocal microscope in the vicinity of the through-hole of the microplate formation sheet in which the through-hole was formed by the mechanical punching process. It is a figure which shows the observation result of the state which affixed the cover glass on the microplate formation sheet | seat in which the through-hole was formed by mechanical punching. It is a figure which shows the observation result by the confocal microscope in the through-hole vicinity of the microplate formation sheet which formed the through-hole by laser processing. It is a figure which shows the observation result of the state which affixed the cover glass on the microplate formation sheet which formed the through-hole by laser processing.

(Sample dispensing device)
An embodiment of a sample dispensing apparatus according to the present invention will be described below with reference to FIGS.

  The sample dispensing apparatus according to the present embodiment dispenses a protein-lipid mesophase sample containing a target membrane protein into wells of a microplate when the membrane protein is crystallized by the lipid mesophase method. This is an apparatus suitably used for preparing a crystallization cell by dispensing and adding a crystallization solution for performing crystallization. In this embodiment, the case of using a 96-well microplate will be described. However, the sample dispensing apparatus according to the present invention can be used for a 24-well microplate or the like, or a commercially available plastic screening plate or the like. Available.

  FIG. 1 is a top view showing a schematic configuration of a sample dispensing apparatus in the present embodiment. FIG. 2 is a front view showing a schematic configuration of the sample dispensing apparatus. However, a portion corresponding to a portion indicated by a broken line A-A ′ in FIG. 1 is shown in a cross section in FIG. 2. As shown in FIG. 1 and FIG. 2, the sample dispensing apparatus 1 in this embodiment includes a sample dispensing microdispenser section (sample dispensing means) 2, a crystallization solution dispensing microdispenser section (solution dispensing means). 3, a cover pasting part (sticking means) 5, a plate chamber 4, a disposable chip rack 6, a crystallization solution rack 7, a dispensing observation part 8, and a humidity control device 11, and a sample dispensing microdispenser part 2 A personal computer (PC) (not shown) for controlling the operation of the crystallization solution dispensing microdispenser unit 3, the cover pasting unit 5, and the dispensing observation unit 8 is provided.

  The sample dispensing microdispenser unit 2 dispenses a protein-lipid mesophase sample into each well of a microplate such as a 96-well screening plate placed in the plate chamber 4. The sample dispensing microdispenser unit 2 has a detachable syringe 17 that discharges a protein-lipid mesophase sample.

  The sample dispensing microdispenser unit 2 dispenses protein-lipid mesophase samples in the designated order at designated locations on the microplate. The discharge amount of the protein-lipid mesophase sample from the syringe 17 is set to an arbitrary amount (1 nl unit) in the range of 10 nl to 200 nl, for example. Further, the discharge speed is set to an arbitrary speed (in units of 0.1 mm / sec) in the range of 1 mm / sec to 30 mm / sec, for example.

  The sample dispensing microdispenser unit 2 has a syringe chip cover that can store a syringe chip (discharge unit) that is the tip of the syringe 17. A melamine sponge etc. moistened with water is installed in the syringe tip cover, except when dispensing protein-lipid mesophase samples to prevent drying of the syringe tip and decontamination of the outer wall of the syringe tip, The tip of the syringe 17 is stored in a syringe tip cover. The tip of the syringe 17 is always held in the plate chamber 4 while the protein-lipid mesophase sample is being dispensed into the microplate.

  The crystallization solution dispensing microdispenser unit 3 dispenses the crystallization solution into each well of a microplate such as a 96-well screening plate placed in the plate chamber 4. In the present embodiment, the crystallization solution is dispensed and added to the protein-lipid mesophase sample dispensed to the well by the sample dispensing microdispenser unit 2. The crystallization solution dispensing microdispenser unit 3 includes eight dispensing heads 18 to which a disposable chip can be attached and detached. The crystallization solution dispensing microdispenser unit 3 attaches the disposable chips set in the disposable chip rack 6 to the dispensing head 18, and prepares the crystallization solution rack 7 in each of the dispensing heads 18 to which the disposable chips are attached. The crystallization solution is sucked from the crystallization solution box thus formed, held in the disposable chip, and discharged to a designated portion of the microplate. The discharge amount of the crystallization solution is set to an arbitrary amount (0.1 μl unit) in the range of 0.8 to 10 μl, for example. The suction amount, suction speed, and discharge speed of the crystallization solution are appropriately set according to the type of the crystallization solution. As the kind of crystallization solution, liquids having various viscosity ranges from low viscosity liquid to high viscosity liquid such as water, 50% PEG 8,000, and 30% PEG 20,000 are assumed. After the dispensing, the crystallization solution dispensing microdispenser unit 3 removes the attached disposable chip and discards it. The crystallization solution dispensing microdispenser unit 3 is not limited to eight dispensing heads, and may be eight or more or less, and further uses a fixed needle tip. Also good.

  The crystallization solution dispensing microdispenser unit 3 includes a chip sensor that detects the attachment of a disposable chip in each of the dispensing heads 18 and an attachment determination unit that determines whether the attachment is good or not based on detection data. When the attachment determination unit determines that the attachment is defective, the attachment operation is performed again.

  The cover affixing unit 5 affixes a transparent cover to the well forming surface of the microplate on which the protein-lipid mesophase sample and the crystallization solution have been dispensed. In this embodiment, since a cover glass is used as a transparent cover, a cover glass will be described below as an example, but the transparent cover is not limited to glass. The cover attaching part 5 can be moved on the plate chamber 4 and the cover storage part 27 by the guide rail 26 and the drive mechanism. That is, it can move in the direction indicated by the double-ended arrow B in FIG. FIG. 3 is a diagram schematically showing a side surface of the cover pasting portion 5. The cover sticking part 5 is comprised by the support part 5b which enables the movement to an up-down direction while supporting the cover glass sticking part main body 5a and the cover glass sticking part main body 5a. A suction pad 19 for sucking and holding the cover glass is provided on the lower side of the cover pasting portion main body 5a. Further, in order to enable even a thin cover glass to be transported without being damaged, the suction pad 19 is provided with a plurality (four) of soft rubber cover glass suction ports. The cover pasting unit body 5a has a transport unit that takes out one cover glass by the suction pad 19 from the cover storage unit 27 in which the cover glass is stored, and transports the cover glass vertically above the microplate. When the cover glass is transported vertically above the microplate, the cover pasting portion main body 5a is lowered, the cover glass is brought into contact with the adhesive surface of the upper surface (well forming surface) of the microplate, and a predetermined distance is further increased. Pressure is applied by lowering to complete the adhesion between the cover glass and the adhesive surface.

  Moreover, the cover sticking part main body 5a has a rotation-type protective cover removing claw 20. When the microplate is formed by adhering a double-sided adhesive plastic sheet having one or more through-holes to a glass substrate as will be described later, the cover adhering unit 5 adsorbs the cover glass. Before that, it moves onto the plate chamber 4 and peels off the protective cover of the double-sided adhesive plastic sheet using the protective cover removal claw (peeling means) 20 to expose the adhesive surface. Then, it moves on the cover storage part 27, adsorb | sucks one cover glass, moves again on the plate chamber 4, and affixes a cover glass on a microplate.

  The cover storage unit 27 is placed between the standby position of the cover pasting unit 5 and the plate chamber 4. On the bottom surface of the cover storage portion 27, a mat made of a resin tape or a soft and porous material such as paper, non-woven fabric, and sponge is laid. As a result, compared with a cover glass storage portion having a highly flat metal surface bottom surface, the adhesive force between the thin cover glass of 30 μm thickness and the bottom surface is weakened, suction becomes easier, and suction errors are prevented. be able to.

  The plate chamber 4 is a chamber for dispensing a protein-lipid mesophase sample and a crystallization solution into a microplate. FIG. 4 is an exploded perspective view of the plate chamber. As shown in FIG. 4, the plate chamber 4 is provided with a mounting table 21 for mounting the microplate and a glass plate mounting table 25 for performing a test shot of the sample. The plate chamber 4 is provided with a movable lid 12 that covers the upper portion of the plate chamber 4. The inside of the plate chamber 4 is adjusted to a desired humidity by a humidity adjusting device 11. In addition, the movable lid 12 is moved along a uniaxial direction (direction indicated by a double-ended arrow B in FIG. 1) by a lid driving unit 22 connected to the side surface of the lid 12. The operation is controlled by the PC.

  A slit (opening) 15 is formed in the movable lid 12 that covers the upper portion of the plate chamber 4. The sample dispensing microdispenser unit 2 introduces the tip of the syringe 17 into the plate chamber 4 through the slit 15 and dispenses a protein-lipid mesophase sample to each well of the microplate in the plate chamber 4. . In addition, before dispensing to the microplate, the dispensing observation unit indicates that a certain amount of sample is continuously dispensed by dispensing the sample to the glass plate for trial placement placed on the setting table 25. After confirming by 8, dispensing to the microplate is started. The crystallization solution dispensing microdispenser unit 3 introduces a disposable chip (containing the sucked crystallization solution) portion attached to the dispensing head 18 into the plate chamber 4 through the slit 15, A crystallization solution is dispensed into a predetermined well of a microplate in the plate chamber 4. The lid drive unit 22 dispenses the protein-lipid mesophase sample dispensing position and the crystallization solution dispensing position, that is, the position of the syringe 17 of the sample dispensing microdispenser unit 2 and the crystallization solution dispensing microdispenser unit 3. The lid 12 of the plate chamber 4 is moved so that the slit 15 overlaps the position of the head 18. Thereby, the protein-lipid mesophase sample and the crystallization solution can be dispensed through the slit 15 regardless of the dispensing position of the protein-lipid mesophase sample and the crystallization solution. Moreover, since the opening part in the plate chamber 4 is restrict | limited only to the slit 15 part, ie, the vicinity of the part to be dispensed, it can suppress that the humidity inside the plate chamber 4 changes. The lid driving unit 22 moves the lid 12 to a state where each operation can be performed when performing an operation of introducing and removing a microplate, an operation of attaching a cover glass, and the like.

  The lid 12 of the plate chamber 4 is made of a transparent material such as glass and acrylic so that a protein-lipid mesophase sample dispensed on the microplate placed in the plate chamber 4 and a droplet of the crystallization solution can be observed. It is made of material.

  The mounting table 21 is not particularly limited as long as it can mount and fix the microplate. For example, the mounting table 21 can be configured to circulate a Peltier element or constant temperature water to adjust the temperature of the microplate. It may be.

  The humidity adjusting device 11 supplies a mixed gas of water vapor and a dry gas having a lower humidity than water vapor into the plate chamber 4, and adjusts the mixing ratio of the water vapor and the dry gas in the mixed gas, thereby adjusting the inside of the plate chamber 4. The humidity is adjusted. In the sample dispensing apparatus 1 in the present embodiment, a mixed gas of water vapor and dry nitrogen gas is used as a mixed gas, but the mixed gas is a mixed gas of water vapor and dry gas as described above. Good. Dry gas does not affect the experiment, such as there is no chemical action such as oxidation and reduction on protein, lipid, protein-lipid mesophase sample, or crystallization solution, and no pH change occurs due to dissolution of dry gas. If there is no restriction in particular. For example, helium, argon, and nitrogen gas, dry air obtained by a known dehumidifier such as a compressor method or a desiccant method, and a mixed gas thereof can be used. The humidity value of the drying gas is not particularly limited as long as the inside of the plate chamber 4 can be set to a desired humidity value. However, the humidity value is low humidity (˜0% RH) to saturation humidity (100% RH). .)) For a wide humidity range control over 0% R.D. H. It is desirable to use a dry gas close to. As an example satisfying this requirement, it is clean and has 0% R.D. H. Nitrogen gas from which a dry gas close to 1 can be easily obtained can be suitably used. Further, the water vapor used to form the mixed gas only needs to be supplied as a gas containing water vapor. In addition to the case where it is a gas containing only water vapor, it also includes a gas containing fine water droplets (mist). It is. Further, in order to control a wide humidity range from low humidity (˜0% RH) to saturation humidity (100% RH), the gas containing water vapor has a relative humidity of 100% RH. H. It is preferable that the gas be close to. Incidentally, by using dry nitrogen gas as the dry gas, an unexpected effect of preventing mold from being generated in the flow path of the mixed gas and the plate chamber 4 was obtained. A mixed gas of water vapor and dry nitrogen gas is preferred.

  The plate chamber 4 is provided with a plurality of gas supply ports 24 on each of two opposing side surfaces. A gas supply pipe 23 for supplying the mixed gas into the plate chamber 4 is connected to each gas supply port 24 of the plate chamber 4. The gas supply pipe 23 is branched into a Y shape a plurality of times immediately before the gas supply port 24 to form eight tip portions. The eight tip portions are connected to the plate chamber 4 through different gas supply ports 24, respectively. Water vapor and dry nitrogen gas are uniformly mixed while passing through the Y-shaped branch channel.

  The humidity controller 11 includes a dry nitrogen gas generator 9 and a vaporizing humidifier 10. Water vapor included in the mixed gas is generated by the vaporizing humidifier 10. By generating water vapor with the vaporizing humidifier 10, it is possible to generate water vapor with minimal generation of mist.

  The humidity control device 11 includes a vaporization type humidifier 10. As the humidifier, a vaporization type (natural vaporization type, heaterless fan type, osmosis membrane type, dropping permeation type, capillary type, rotation type, etc.) is used. Water spray types (ultrasonic type, centrifugal type, high pressure spray type, two-fluid spray type, etc.), steam types, and composite types thereof can be used, and the humidifying mechanism is not particularly limited. However, as described below in Example 2, 96-98% R.D. is required to completely suppress water evaporation from the crystallization solution and the protein-lipid mesophase sample. H. Since high humidity conditions are required, it is desirable that water vapor close to saturation (100% RH) can be stably supplied. Also, when humidifying, the temperature rise that causes protein denaturation is small (preferably the temperature rise from the ambient temperature is within 1 ° C.), and the proportion of minute water droplets (mist) contained in water vapor is small Is preferred.

  If the ratio of mist present in the water vapor is large, water droplets of the mist adhere to the sample or the crystallization solution and dilute them, thereby greatly changing the component concentration. In addition, when mist adheres to the sensitive surface of the humidity sensor, wets the sensitive surface, and a thin water film is formed on the sensitive surface, the response of the sensor is delayed, and the accurate humidity value is unknown. As a result, there arises a problem that humidity control becomes unstable and reproducibility cannot be obtained. Therefore, when using a water spray type or steam type humidifier with a high mist generation rate, it is preferable to provide a mist removing mechanism using various filters, ceramic cylinders, eliminators, and the like in the water vapor channel. On the other hand, since the ratio of mist in the water vapor generated by the vaporizing humidifier is small, the humidity can be adjusted strictly without adding a mist removal device, etc., and the concentration of components in the sample and crystallization solution can be changed by dilution. Therefore, it can be preferably used.

  From the viewpoint of preventing components other than water from being mixed into the generated water vapor, it is preferable to use reverse osmosis membrane treated water, distilled water, or pure water obtained with a commercially available pure water production machine for the production of water vapor.

  Although there is no restriction | limiting in particular as the dry nitrogen gas generator 9, It has the structure which supplies the nitrogen gas obtained by vaporizing liquid nitrogen. In addition, a nitrogen gas cylinder or the like can be used.

Water vapor and dry nitrogen gas included in the mixed gas are supplied as follows, for example.
Water vapor: A flow rate value corresponding to the target humidity calibrated in advance (specifically, the scale of the flow valve) is set by the water vapor flow rate adjustment valve at the outlet of the vaporizing humidifier 10, and thereafter a constant flow rate of water vapor is continuously applied. Supply.
Nitrogen gas: Dry nitrogen gas obtained by vaporizing liquid nitrogen is reduced to zero under a low humidity condition such as 25% RH using a pressure reducer corresponding to the target humidity previously calibrated. .2 Mpa, 80% RH, etc., at a high humidity condition of 0.05 Mpa). Furthermore, using an area type (float type) flow meter, the flow rate is adjusted so that the actually measured humidity value in the plate chamber 4 becomes a predetermined humidity value. After the target humidity value is obtained, the dry nitrogen gas at the flow rate is continuously supplied during the sandwich plate making operation unless there is a large change in the outside air condition.

  The humidity adjusting device 11 adjusts the mixing ratio of water vapor and dry nitrogen gas in the mixed gas supplied into the plate chamber 4, thereby adjusting the inside of the plate chamber 4 by 5% R.D. H. ~ 100% R.V. H. It is possible to adjust to any relative humidity within the range.

  When the temperature or humidity of the outside air changes, the humidifying capacity of the vaporizing humidifier 10 changes. In that case, the measured humidity value in the plate chamber is, for example, 0.2% R.D. H. If the dry nitrogen gas flow rate is finely adjusted at the time of the change, a constant humidity value can be easily maintained. In the sample dispensing apparatus 1, in addition to the four humidity sensors 14 in the plate chamber, sensors for measuring the humidity and temperature of the outside air of the sample dispensing apparatus 1 are also installed. If the outside air humidity value is known, the setting of the water vapor flow rate and the setting value of the nitrogen gas flow rate can be easily adjusted.

  In the sample dispensing apparatus 1, a Y-shaped branch channel is provided in the gas supply pipe 23, a mixed gas is introduced into the chamber from two symmetrical directions, or a lid is provided on the plate chamber 4 to provide an opening portion. The humidity in the plate chamber 4 is made uniform by keeping the required amount to a minimum or by supplying appropriate amounts of water vapor and dry nitrogen gas.

  In order to confirm the uniformity of the humidity inside the plate chamber 4, humidity sensors 14 for detecting the humidity are attached to the four corners of the plate chamber 4 one by one. Data detected by the humidity sensor 14 can be transmitted to the PC, and the humidity fluctuation can be monitored in real time on the PC screen. In the present embodiment, the humidity sensor 14 is a humidity temperature sensor that can also measure the temperature.

  As described above, four humidity sensors 14 are installed in the present embodiment. However, if at least one humidity sensor 14 is installed, the humidity inside the plate chamber 4 can be measured, and based on the measurement data, that is, based on the actual humidity inside the plate chamber 4, mixing of water vapor and dry nitrogen gas The humidity in the plate chamber 4 can be adjusted by adjusting the ratio. However, it is preferable that two or more humidity sensors 14 are installed in order to confirm whether or not the humidity unevenness has occurred.

  The dispensing observation unit 8 images the state of dispensing of the protein-lipid mesophase sample. In the present embodiment, the dispensing observation unit 8 also performs imaging of the state of dispensing of the crystallization solution. The dispensing observation unit 8 includes a color CMOS camera as imaging means. The image captured by the color CMOS camera is taken into the attached storage device, and the captured image of the well of the microplate is displayed on the PC screen in almost real time.

  Protein-lipid mesophase samples are high viscosity materials with complex rheological properties. Therefore, even after the dispensing operation is stopped, spontaneous discharge or spontaneous contraction occurs in the syringe 17. Thus, at the start of dispensing, it often happens that even if a dispensing operation is performed, the sample is not discharged, or a larger amount of sample is discharged than set. In order to avoid this, it is possible to start trial dispensing of the sample before starting dispensing and after confirming that a certain amount of sample has been dispensed stably, dispensing to the microplate can be started. preferable. However, a protein-lipid mesophase sample is dispensed in an amount of, for example, 20 nl to 100 nl, but a small amount of a protein-lipid mesophase sample droplet of 20 nl to 100 nl is a microscopic body having a diameter of about 1 mm or less. Therefore, even if it is found that the protein-lipid mesophase sample is dispensed at the start of dispensing, it is accurately determined with the naked eye without the dispensing observation unit 8 whether or not a certain amount is continuously dispensed. It is impossible to judge. In this embodiment, before the protein-lipid mesophase sample is dispensed on the actual microplate used for crystal production, the test plate is subjected to test punching about 2 to 5 times in the plate chamber 4 and is fixed. Whether or not the amount is continuously dispensed is confirmed using the dispensing observation unit 8.

  The sample dispensing apparatus according to the present embodiment includes a dispensing observation unit 8 and observes the state of the protein-lipid mesophase sample droplet dispensed from the syringe 17 or the well into which it is dispensed on the screen of the PC. be able to. Thereby, since a defective dispensing well can be confirmed during the dispensing operation, re-dispensing is performed on the sample dispensing microdispenser unit 2 and the crystallization solution dispensing microdispenser unit 3 as necessary. It is possible to perform appropriate dispensing in each well.

  Further, initial setting (positioning) such that the tip of the syringe 17 is positioned at the center of the well is performed using the color CMOS camera. Thereby, the operation is less complicated than positioning with the naked eye, and positioning can be completed within a short time (for example, about 10 seconds).

  In addition, movement control and positioning control of the sample dispensing microdispenser unit 2 and the crystallization solution dispensing microdispenser unit 3 can be realized by a conventionally known technique by those skilled in the art. In addition, movement control and positioning control of the lid driving unit 22, the cover pasting unit 5, and the cover pasting unit main body 5a can also be realized by those skilled in the art with reference to conventionally known techniques.

[Protein crystallization method]
Next, an embodiment of the protein crystallization method according to the present invention will be described.

  The protein crystallization method in the present embodiment is a protein crystallization method using a lipid mesophase method, and is a sample dispensing method in which a protein-lipid mesophase sample is dispensed to a microplate prepared in a plate chamber. A solution dispensing step of dispensing a crystallization solution for crystallizing a protein onto the microplate, a mixing step of combining the dispensed sample and the dispensed crystallization solution. Then, after the mixing step, a pasting step of pasting a transparent cover on the upper surface of the microplate, and a crystal growth step of crystallizing the protein in the sample after the pasting step are included. In the sample dispensing process and the solution dispensing process, the humidity inside the plate chamber is adjusted by supplying a mixed gas of water vapor and dry nitrogen gas to the inside of the plate chamber. The humidity inside the plate chamber varies depending on the type of protein to be crystallized.

  The protein crystallization method in the present embodiment can be preferably carried out by using the sample dispensing apparatus in the above-described embodiment.

  As described above, in the lipid mesophase method, humidity control when dispensing a protein-lipid mesophase sample is very important in the success or failure of crystallization and the quality of crystals.

  As the lipid used in the lipid mesophase method, a lipid capable of forming a reverse cubic liquid crystal in water is used. For example, monoacylglycerols represented by 1-oleoyl-rac-glycerol (monoolein) (non-patent literature: M. Caffrey, Annu, Rev. Biophys. 2009, 38, 29-51.), 1-O -(3,7,11,15-tetramethylhexadecyl) -β-D-xyloside as a representative isoprenoid chain lipid (Non-patent literature: Hato, M. et al., Langmuir 2002, 18 (9), 3425-3429. Hato, M. et al., Langmuir 2004, 20 (26), 11366-11373 .; Yamashita, J. et al., J. Phys. Chem, B, 2008, 112, 12286-12296 .; Hato, M et al., J. Phys. Chem, B 2009.113, 10196-10209.) and the like.

  In the protein crystallization method according to the present embodiment, the microplate is a microplate formed by a substrate and a microplate forming sheet having one or more well-forming through holes attached to the substrate. . Transparent covers affixed to the substrate and microplate are used for microscopic observation of crystallization processes (normal light, polarization, fluorescence, UV, multiphoton excitation laser scanning, interference, etc.), circular dichroism spectrum, UV-visible spectrum In addition to measurements such as FRAP (Fluorescence Recovery After Photo-bleaching) and FCS (Fluorescence Correlation Spectroscopy), X-ray small angle scattering (SAXS), X-ray wide angle scattering (WAXS), and X-ray crystal diffraction etc. There is no particular limitation, and various types of glass, natural or artificial quartz, inorganic materials such as silicon nitride, and organic materials such as polymers can be used. Preferably, various glasses and natural or artificial quartz are used that are free of birefringence, are non-fluorescent and do not elute into crystallization solutions and protein-lipid mesophase samples.

  The lipid mesophase is a viscous substance having a viscosity comparable to that of silicon grease. Therefore, the lipid mesophase dispensed into the microplate does not naturally form a smooth surface like a liquid, but becomes a lump having a surface with irregular irregularities. Therefore, the lipid mesophase dispensed on the microplate causes light to be reflected or refracted in a complex manner. It is very difficult to observe the microcrystal inside the lipid mesophase as a clear image through such a surface having irregularities. Furthermore, the refractive index of the lipid mesophase (˜1.45) is closer to the refractive index of the protein (˜1.5) than the solution containing the solubilized membrane protein (˜1.33). Contrast decreases. Lipid mesophases are also often birefringent. For this reason, in plastic microplates that have been commonly used in other crystallization methods, it is difficult to observe fine protein crystals due to the birefringence of the plastic (particularly in observation with a polarizing microscope). For this reason, in the lipid mesophase method, a protein-lipid mesophase sample to which a crystallization solution has been added is generally sandwiched between two glass plates to form a glass sandwich cell (see above). Non-patent document 4). A glass sandwich cell generally includes a 1 mm thick glass plate of SBS (Society for Biological Screening) standard size, a double-sided adhesive plastic sheet (thickness of about 140 μm) provided with 96 through holes, and It is composed of 0.2 mm thick cover glass. Specifically, the plastic sheet having a through-hole is pasted on the glass plate, and after dispensing the protein-lipid mesophase sample and the crystallization solution into 96 wells formed thereby, the plastic sheet A glass sandwich cell sandwiched between samples is formed by covering and sealing the upper surface of the substrate with the above cover glass.

  However, in this embodiment, a thin glass plate (glass substrate) having a thickness of 30 μm to 150 μm is used as the glass plate in addition to the standard glass sandwich cell, and the cover glass has a thickness of 30 μm to 150 μm. A thin glass plate is used. That is, the formed glass sandwich cell is a thin glass sandwich cell sandwiched between two thin glass plates having a thickness of 30 μm to 150 μm. More preferably, it is a thin glass sandwich cell sandwiched between two thin glass plates having a thickness of 50 μm to 150 μm. Hereinafter, advantages of using a thin glass sandwich cell constituted by two thin glass plates having a thickness of 30 μm to 150 μm will be described.

  As a result of detailed examination of the crystallization process by the present inventors, the crystallization solution used for the purpose of inducing protein crystallization is not only the interaction between membrane proteins but also the lipid mesophase used as a crystallization field. It was found that the structure of the protein also changes, and that the structural change also determines the success or failure of protein crystallization. Therefore, in the crystallization of membrane protein by the lipid mesophase method, crystallization not only in optimizing the interaction between membrane proteins but also in the structure control of lipid mesophase is necessary. Since membrane-protein interaction and lipid mesophase structure are independent factors, there is no other way to optimize them simultaneously than by repeating many trials and errors beyond the conventional method, which is the bottleneck of the lipid mesophase method. It has become.

  In order to simultaneously optimize the independent factors of membrane protein-protein interaction and lipid mesophase structure, it is necessary to directly measure the fine structure of lipid mesophase and its changes simultaneously with the observation of crystal growth during the crystallization process. It is essential. The most reliable method for measuring the fine structure of lipid mesophase is the X-ray small angle scattering (SAXS) method. However, the standard glass sandwich cell used in the conventional lipid mesophase strongly absorbs X-rays, resulting in a significant decrease in X-ray intensity and is considered inappropriate for practical SAXS measurement.

  In recent years, a method for measuring a lipid mesophase structure using a SAXS beam line using synchrotron radiation has been proposed (Non-Patent Document 6). However, in this method, in order to avoid attenuation of X-rays, measurement is performed using a plastic cell with low X-ray absorption rather than a standard glass sandwich cell used in actual crystallization screening. Therefore, it is still difficult to directly measure the lipid mesophase structure in a standard glass sandwich cell in which actual crystallization screening is performed. Further, as described above, it is difficult to observe crystals in the plastic cell, and small crystals may be missed. Therefore, it is difficult to perform actual crystallization screening in a plastic cell. In addition, the use of synchrotron radiation facilities is limited, so measurement at the timing required for research is not guaranteed and information at the required time cannot be obtained accurately. Furthermore, the possibility of changes in the lipid mesophase structure cannot be ignored due to sample changes caused by temperature changes and mechanical stimuli that occur during transport of samples from the laboratory to the synchrotron radiation facility.

  On the other hand, in the thin glass sandwich cell formed by the crystallization method of the present embodiment, the absorption of X-rays in the glass is extremely less than that of the conventional standard glass sandwich cell. Therefore, it is possible to perform SAXS measurement by directly setting the formed thin glass sandwich cell on a device that can be used as needed in a normal laboratory. Therefore, it is possible to easily know the structure of lipid mesophase and its change at any point in the protein crystal growth process.

  Furthermore, the standard glass sandwich cell formed in the conventional lipid mesophase method has the following problems.

  When crystals are obtained in the screening process, it is important to quickly and accurately determine whether they are protein crystals or crystals other than proteins such as electrolytes. The identification of the protein crystal can also be performed by using, for example, a UV microscope. However, even if a protein crystal is obtained, it is very often a protein crystal from which an X-ray diffraction point necessary for structural analysis cannot be obtained. Therefore, it is further necessary to quickly and accurately determine whether or not the obtained protein crystal is a protein crystal from which an X-ray diffraction point can be obtained, and further, the resolution of the diffraction point. However, in the standard glass sandwich cell formed in the conventional lipid mesophase method, as described above, the X-ray absorption in the glass is large, so that it is difficult to perform X-ray diffraction measurement of the crystal. In order to avoid such difficulties and to perform X-ray diffraction measurement of crystals, when collecting crystals from a standard glass sandwich cell, the cover glass is mechanically broken (only the part covering the target well). The crystal inside the well must be collected by cutting the sample with a cutter. The mechanical breakage of the cover glass will cause mechanical damage to the crystal. In addition, reliable evaporation results cannot be obtained because many uncertain factors are caused by the evaporation of moisture during the sampling process, which causes crystal alteration and dissolution, and fine glass fragments are mixed when the cover glass is broken.

  However, in the thin glass sandwich cell formed by the crystallization method of the present embodiment, the absorption of X-rays in the glass is extremely less than that of the conventional standard glass sandwich cell. Therefore, the X-ray diffraction experiment of the protein crystal contained in the cell can be performed as it is with the thin glass sandwich cell without mechanically destroying the thin glass sandwich cell and taking out the crystal.

  Further, the through hole in the plastic sheet (microplate forming sheet) forming the standard or thin glass sandwich cell used in the crystallization method of the present embodiment is formed by laser processing.

As the laser processing, a processing machine using a diode laser, a yag laser, a CO2 laser, or the like can be used. As an example, when the MITSUBISHI CO2 LASER UNIT ML5036D equipped with a CO2 laser oscillator is used, the processing can be performed under optimum conditions by adjusting the processing conditions within a range of 20 W to 300 W. For example, a 96-well microplate under the conditions of output: 250 W, frequency: 500 Hz, duty: 100%, speed: 2500 mm / min, correction: 0.19 mm, gas pressure: 0.5 kgf / cm ² , assist gas: air Can be suitably performed.

  The plastic used in the conventional crystallization method is used for the components other than the through holes, such as the material forming the plastic sheet, the thickness of the plastic sheet, and the adhesive layer formed on both surfaces of the plastic sheet. Each configuration in the sheet can be used. Furthermore, since the through hole is formed by laser processing, the through hole can be easily formed even if the material and thickness of the plastic sheet, the type of adhesive, the size, shape, and layout design of the well are changed. Can do. In addition, by utilizing such laser processing characteristics, for example, a microplate-forming sheet that can easily implement new crystallization means that could not be carried out until now, such as vapor diffusion type lipid mesophase crystallization, can be obtained. Can be created.

  In a plastic sheet used in a conventional crystallization method, a through hole forming a well is formed by mechanical punching. In mechanical punching, burrs are formed at the edge of the processed hole, and unevenness is also generated in the adhesive layer between the holes due to mechanical stress during processing. When a thin cover glass having a thickness of 30 μm to 150 μm is attached to an uneven adhesive layer, a gap is formed between the cover glass and the adhesive layer, thereby forming a passage between adjacent wells. When a passage is formed between adjacent wells, water vapor moves between adjacent wells, and the independence of each well is lost. In addition, since the sealing performance as a whole is lowered, moisture evaporation from the crystallization solution in the well occurs during the crystallization period over several months, thereby reducing the reproducibility and reliability of the screening. In order to prevent this, it is necessary to repair each gap by hand. However, troubles such as breakage of the cover glass occur during the operation, and the experimental operation is remarkably complicated.

  In a plastic sheet in which through holes are formed by laser processing, no mechanical stress is applied around the processed holes. Therefore, it is possible to prevent unevenness from occurring in the adhesive layer. For this reason, a sandwich cell can be automatically created without causing the above-described problems during pasting.

  As described above, by using the sample dispensing apparatus and the protein crystallization method in the above-described embodiment, it becomes possible to precisely control the humidity, so that the standard glass sandwich cell handled by the prior art is remarkably produced. A new crystal that not only can be carried out reproducibly under strict conditions, but also grows higher quality crystals efficiently by controlling the crystal nuclei, their precursor formation rate, crystal size, etc. with humidity parameters. Proteins that have been difficult to crystallize by conventional crystallization methods such as vapor diffusion crystallization methods and conventional lipid mesophase methods are also increased.

  Furthermore, not only can the above-mentioned various measurements be performed with the highest performance, but also direct SAXS measurement of the lipid mesophase structure during crystallization and X-ray diffraction measurement of the crystals in the crystallization cell without breaking the cover glass. A thin glass sandwich cell can be automatically created. Thereby, rational selection of crystallization conditions, optimization, and host lipid screening can be performed efficiently.

[Summary]
As described above, the sample dispensing apparatus according to the present invention includes a plate chamber, a sample dispensing means for dispensing a lipid mesophase sample containing a membrane protein to a microplate prepared in the plate chamber, and a protein. A solution dispensing means for dispensing a crystallization solution for crystallization into the microplate, and a humidity adjusting device for adjusting the humidity in the plate chamber are provided. It has a configuration for supplying a mixed gas with a low-humidity dry gas into the plate chamber.

  According to the above configuration of the sample dispensing apparatus according to the present invention, the protein-lipid mesophase sample is dispensed to the microplate and the crystallization solution is dispensed to the microplate in the plate chamber in which the internal humidity is adjusted. Notes can be made. Further, the humidity in the plate chamber is controlled by supplying a mixed gas of water vapor and a dry gas having a lower humidity than water vapor into the plate chamber. Therefore, by changing the mixing ratio of water vapor and dry gas in the mixed gas to be supplied, the humidity in the plate chamber can be easily set to an arbitrary humidity value between the humidity value of water vapor and the humidity value of dry gas. Can be adjusted strictly. Thereby, the sample can be dispensed and the crystallization solution can be dispensed in a plate chamber whose humidity is strictly controlled.

  In the sample dispensing apparatus according to the present invention, the dry gas is preferably dry nitrogen gas. When dry nitrogen gas is used as the dry gas, it is possible to prevent mold and the like from being generated in the mixed gas flow path and the plate chamber.

  In the sample dispensing apparatus according to the present invention, it is preferable that the humidity adjusting device includes a humidifier that generates the water vapor. The humidifier is more preferably a vaporizing humidifier.

  In the sample dispensing apparatus according to the present invention, one or more humidity sensors are provided inside the plate chamber, and the humidity control apparatus is configured to use the mixed gas based on a measurement result of the one or more humidity sensors. It is preferable to adjust the mixing ratio of the water vapor and the dry gas.

  According to the above configuration, the humidity inside the plate chamber is measured, and the mixed gas in which the mixing ratio of the water vapor and the dry gas is adjusted based on the measurement result is supplied into the plate chamber. Therefore, the humidity can be adjusted based on the actual humidity inside the plate chamber. This mechanism can easily be automatically controlled by a computer.

  In the sample dispensing apparatus according to the present invention, it is preferable that two or more humidity sensors are provided.

  According to the said structure, the uniformity of the humidity in a plate chamber can be confirmed.

  In the sample dispensing apparatus according to the present invention, it is preferable that a movable lid provided with an opening is provided at an upper portion of the plate chamber.

  According to the said structure, the opening part of a lid | cover can perform the dispensing of the protein-lipid mesophase sample and the crystallization solution through the opening part simultaneously with the function as a discharge port of the said mixed gas. Further, the relative position of the opening with respect to the plate chamber can be changed by moving the lid in accordance with the change in the dispensing position in the microplate. Therefore, regardless of the dispensing position on the microplate, the protein-lipid mesophase sample and the crystallization solution can always be dispensed through the opening. Therefore, while the sample is being dispensed and the crystallization solution is being dispensed, the opening of the plate chamber is limited to the opening of the lid, so that the humidity in the plate chamber can be adjusted more precisely. it can.

  In the sample dispensing apparatus according to the present invention, there is provided imaging means for imaging at least one of a state of dispensing of the sample by the sample dispensing means and a discharge portion for discharging the sample in the sample dispensing means. Furthermore, it is preferable to provide.

  According to the said structure, it can be confirmed whether the protein-lipid mesophase sample was dispensed to the microplate by fixed amount. Further, it is possible to align the discharge unit with respect to the well.

  In the sample dispensing apparatus according to the present invention, it is preferable that the sample dispensing apparatus further includes an attaching means for attaching a transparent cover to the upper surface of the microplate.

  According to the said structure, after dispensing a protein-lipid mesophase sample and a crystallization solution, a transparent cover can be automatically affixed on the upper surface of a microplate. As a result, it is possible to minimize moisture evaporation at the time of pasting the transparent cover, and it is possible to realize the creation of a thin sandwich cell, which will be described later, and the efficiency of the work.

  In the sample dispensing apparatus according to the present invention, it is preferable that a protective cover is attached to the microplate, and further provided with a peeling means for peeling the protective cover from the microplate.

  According to the said structure, when a microplate is the structure by which the protective cover is affixed, it can peel off a protective cover automatically immediately before affixing a transparent cover. This avoids deterioration in well tightness due to a decrease in the adhesive force between the transparent cover and the adhesive layer due to water vapor adsorption on the surface of the adhesive layer on the top surface of the microplate during dispensing operations under high humidity conditions. And the efficiency of work can be realized.

  In addition, in order to solve the above problems, the protein crystallization method according to the present invention dispenses a sample of lipid mesophase containing a membrane protein into a microplate prepared in the plate chamber. A solution dispensing step of dispensing a crystallization solution for crystallizing the protein onto the microplate, a mixing step of combining the dispensed sample and the dispensed crystallization solution, A protein crystallization method comprising a pasting step of pasting a transparent cover on the upper surface of the microplate after the mixing step, and a crystal growth step of crystallizing the protein in the sample after the pasting step. In the sample dispensing step and the solution dispensing step, water vapor and a lower humidity than water vapor are dried inside the plate chamber. It has a configuration for adjusting the humidity of the inside of the plate chamber by supplying a mixed gas of scan.

  The sample dispensing process or the solution dispensing process may also serve as the mixing process. For example, first, a sample dispensing process is performed to dispense a sample into a microplate, and a crystallization solution is dispensed directly into the dispensed sample in the solution dispensing process, whereby the sample, the crystallization solution, and And a mixing step may be realized.

  According to the above configuration, the humidity inside the plate chamber is adjusted by supplying a mixed gas of water vapor and a dry gas having a humidity lower than that of the water vapor, so that the humidity inside the plate chamber can be strictly adjusted. . Therefore, in an environment in which the humidity is strictly controlled, the sample can be dispensed to the microplate and the crystallization solution can be added to the sample.

  In the protein crystallization method according to the present invention, the microplate includes a glass substrate having a thickness of 30 μm to 150 μm, and a microplate forming sheet having one or more well-forming through holes attached to the glass substrate. It is preferable that the cover is a cover glass having a thickness of 30 μm to 150 μm.

  According to the above configuration, a so-called sandwich cell is formed in which a protein crystallization field is sandwiched between a glass substrate and a cover glass. Since the thickness of the glass substrate and the cover glass are both 30 μm to 150 μm, the absorption of X-rays by the glass in this sandwich cell is small. Therefore, in the sandwich cell state, that is, without removing the cover glass, in addition to measurements including various microscopic observations, analysis using X-rays such as structural analysis of lipid mesophase by X-rays and confirmation of X-ray diffraction in protein crystals Can be applied directly.

  In the protein crystallization method according to the present invention, it is preferable that the well formation through-hole of the microplate forming sheet is formed by laser processing.

  According to the above configuration, when the adhesive layer is provided on the microplate forming sheet, the adhesive layer around the well-forming through hole formed by processing and the adhesive layer in the sheet portion other than the through hole are formed. Unevenness can be prevented from occurring. Therefore, when a cover glass is affixed, it can prevent that a space | gap arises between a cover glass and a well formation surface.

  The microplate forming sheet according to the present invention is a microplate forming sheet having one or more well-forming through-holes that are attached to a substrate to form a microplate, and an adhesive layer is formed on the surface. The well forming through hole has a structure formed by laser processing.

  According to the above configuration, the unevenness of the adhesive layer in the periphery of the through hole for forming a well of the microplate forming sheet and the adhesive layer in the sheet portion other than the through hole is compared with that in which the through hole is formed by mechanical punching. Therefore, when the cover glass is attached to the microplate forming sheet, the generation of voids can be suppressed.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims. That is, embodiments obtained by combining technical means appropriately modified within the scope of the claims are also included in the technical scope of the present invention.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail.

[Example 1: Evaluation of controllability of humidity]
The controllability of the humidity in the plate chamber of the sample dispensing apparatus in the present embodiment by mixing water vapor and dry nitrogen gas is expressed as a humidity value of 98% R.D. H. 70% R.V. H. , And 25% R.V. H. The case was evaluated. The results are shown in FIGS. 5A to 5C represents the time from the start of data acquisition of the experiment. The vertical axis | shaft in AC of FIG. 5 represents the humidity value in a plate chamber measured by each humidity temperature sensor installed in the four corners in a plate chamber. The humidity temperature sensor measures the humidity value in the plate chamber every 5 seconds. In AC of FIG. 5, the measured value from each of four humidity temperature sensors is shown by the solid line, the broken line, the thick solid line, or the thick broken line. In all cases, the water vapor flow rate was constant, and the humidity value was controlled by adjusting the flow rate of dry nitrogen gas. The upward arrow in the figure indicates that the dry nitrogen gas flow rate has been reduced to increase the humidity value at that time, and the downward arrow indicates the dry nitrogen gas flow rate to decrease the humidity value at that time point. It shows that increased. The humidity temperature sensor was corrected using an aqueous electrolyte solution with a known equilibrium gas phase humidity value (for example, non-patent literature: RH Stokes and RA Robinson, Ind. Eng. Chem., 1949, 2013).

  98% R.D. H. The case will be described in detail by taking the case (A in FIG. 5) as a specific example. Since the humidity value tended to decrease at the beginning, the nitrogen gas flow rate was decreased at the time indicated by arrow 1. As a result, the decreasing tendency of the humidity value was suppressed, but a slight increasing tendency was seen in the humidity value after 4 minutes. Therefore, the nitrogen flow rate was slightly increased at the time of arrow 2. As a result, a rapid decrease in humidity value was induced, so that the nitrogen flow rate was slightly decreased again at the time indicated by arrow 3. After that, almost 98% R.D. H. The constant value of was maintained. Furthermore, since all the sensor values are sensitively responding in the same direction due to increase / decrease in the dry nitrogen gas flow rate (a change in humidity value appears in about 5 to 10 seconds), the sample dispensing in this embodiment is performed. It was shown that humidity control is possible with sufficient response sensitivity. Such controllability is 98% R.D. H. 70% R.D. H. (FIG. 5B), and 25% R.D. H. In the case of (C in FIG. 5), similar responsiveness is shown.

  From the above, it was shown that the sample dispensing device in the present embodiment can obtain good controllability in a wide humidity range.

[Example 2: Evaluation of time stability of humidity control]
A microplate is installed in the plate chamber of the sample dispensing apparatus in the present embodiment, and the relative humidity value in the plate chamber is set to 98% R.D. H. (A in FIG. 6), 90% R.I. H. (FIG. 6B), 70% R.D. H. (C in FIG. 6), 25% R.D. H. (D in FIG. 6), or 5% R.D. H. The humidity value when set to (E in FIG. 6) was monitored over 30-40 minutes. The results are shown in FIGS. The horizontal axis in A to E in FIG. 6 represents the time from the start point of the experimental data acquisition. The vertical axis in A to E of FIG. 6 represents the humidity in the chamber obtained from each of the four humidity temperature sensors installed at the four corners in the plate chamber. The humidity temperature sensor measures the humidity value in the plate chamber every minute. Measurements from four different humidity temperature sensors are represented by four different symbols.

  As a representative example, 98% R.D. H. The result in the case (A in FIG. 6) will be described. As shown in FIG. 6A, each of the four sensor values over a 30 minute period was 98 ± 0.5% R.D. H. It was stably controlled within the range. That is, it was shown that the humidity in the plate chamber could be controlled uniformly and stably within 1%. Since the actual dispensing operation is about 10 minutes per microplate, the above results show that stable humidity control that is sufficient for the purpose of the present invention can be performed. Also, under other humidity value setting conditions, 98% R.D. H. As a result, essentially the same accuracy and time stability were confirmed, and it was shown that humidity control within 1% was possible.

  Note that the increase or decrease in the humidity value seen in C in FIG. 6 is due to fluctuations in the outside air temperature that occur when the room air conditioner in the room where the sample dispensing device is installed, etc. This is partly because of fluctuations. However, a constant humidity value can be easily maintained by finely adjusting the nitrogen flow rate as appropriate by continuously monitoring the humidity value. Also, the humidity control was hardly affected by the movement of the lid accompanying the dispensing operation or the entrance / exit of the sample dispensing microsyringe or the crystallization solution dispensing tip. In this embodiment, the control time is 30 to 40 minutes, but it is easy to extend this time.

  From the above, it was shown that the humidity in the plate chamber can be stably controlled for a long time at an arbitrary set value in a wide range from 5% to almost 100%.

  The composition of the crystallization solution is so complex that there is some variation for individual solutions, but the humidity of the gas phase in equilibrium with the crystallization solution estimated from the average composition and concentration of the crystallization solution is 96-99% R.D. H. It is estimated that On the other hand, the equilibrium water vapor pressure of the protein-lipid mesophase sample also depends on the composition and concentration of the aqueous solution contained in the protein-lipid mesophase, but is 96-99% R.S. like the crystallization solution. H. It is estimated that it is in the range. Therefore, humidity 98% R.D. H. These conditions are conditions in which there is almost no evaporation and condensation (see Example 4 below).

[Example 3: Temperature change during humidity control]
Set humidity value to 98% R.V. H. And 90% R.V. H. (A in FIG. 7), 70% R.V. H. And 25% R.V. H. (FIG. 7B), and 5% R.I. H. The temperature change in the plate chamber when the humidity was controlled in (C in FIG. 7) was measured. The results are shown in FIG. The horizontal axis in AC of FIG. 7 has shown the time from a data acquisition start. The vertical axis | shaft in AC of FIG. 7 has shown the temperature value from the humidity temperature sensor acquired every 5 seconds. T _out indicated by a broken line is an outside air temperature (temperature outside the plate chamber) measured by a humidity temperature sensor installed near the humidifier. T _ch indicated by a solid line is a temperature value in the plate chamber measured by four humidity temperature sensors installed at four corners in the plate chamber. Since the temperature values measured by the sensors were within 0.1 ° C., all the sensors were displayed as solid lines without distinguishing between the sensors.

The humidity in the plate chamber is 90% R.D. for about 40 minutes. H. And then 98% R.D. for 60 minutes. H. The results when the control is performed (A in FIG. 7) will be described. When the humidity control was started, the outside air temperature (T _out ) began to rise. This is because the outside air temperature has risen due to heat generated by components such as the motor and fan of the humidifier. The temperature in the plate chamber (T _ch ) started to rise as well. This is because for the same reason as above, heated water vapor (water vapor temperature at the outlet of the humidifier is about 27 to 29 ° C.) is supplied into the plate chamber. Thereafter, for 90 minutes, the temperature in the plate chamber gradually increased from the starting temperature (19 ° C.) and increased to 19.8 ° C. However, after that, the steady state (19.8 ° C.) was reached, and no further temperature increase was observed until the end of the 140-minute experiment.

  70% R.D. H. And 25% R.V. H. In the case where the humidity control was performed (B in FIG. 7), almost the same temperature increase was shown. In this case, the temperature in the plate chamber increased by about 0.5 ° C. by controlling the humidity for 110 minutes. On the other hand, 5% R.D. H. When the humidity control was performed (C in FIG. 7), almost no temperature increase was observed.

  70% R.D. H. And 25% R.V. H. When the humidity control is performed (B in FIG. 7), the temperature rise is small because 90% R.D. H. And 98% R.V. H. This is because the amount of water vapor introduced from the humidifier is smaller than when the humidity control is performed (A in FIG. 7). 5% R.V. H. When the humidity control was performed (C in FIG. 7), the reason why the temperature increase was hardly observed was that the humidifier was not operated.

  From the above results, it was shown that the temperature increase in the plate chamber by humidity control was within 1 ° C.

[Example 4: Evaluation of moisture evaporation rate]
A micro plate is installed in the plate chamber of the sample dispensing apparatus in the present embodiment, and the humidity in the plate chamber is set to 98% R.D. H. Set to. Next, after 1 μl of distilled water is dispensed (time zero) to each of the 8 wells in one row of the microplate by the crystallization solution dispensing microdispenser part, the time change of the volume of the dispensed water droplet is reduced approximately. Measured over 35 minutes. The same measurement was performed for water droplets dispensed to each well in another row, and the experiment was repeated three times in total. The volume of water droplets at each time was determined using the following method. At a predetermined time, a calibrated micro glass capillary (BLAUBRAND intre MARK 1/2/3/4/5 ul) was inserted from the upper opening of the plate chamber, and water droplets on each well were sucked into the capillary. From the change in capillary weight before and after the water droplet suction, the mass of the collected water droplet was determined. The volume was calculated on the assumption that the specific gravity of water was 1. The micro glass capillary was used after washing the inner wall of the capillary with 99% or more of absolute ethanol filtered through a filter having a hole diameter of 0.1 μm and then rapidly drying with a high-pressure nitrogen stream. Thereby, the blotting error was ± 3%. The results are shown in FIG.

  As shown in FIG. 8, a volume reduction of about 3% was measured in 35 minutes after dispensing. The time required for dispensing the sample and the crystallization solution to the microplate is approximately 10 minutes. Therefore, moisture evaporation during the production of the crystallization plate is suppressed to 1% or less. Compared with the humidity control in the conventional crystal production method that allows evaporation up to 10%, the evaporation suppression is realized by nearly 10 times.

[Example 5: Evaluation of repeated change response of humidity]
The mixing ratio between the amount of water vapor in the mixed gas and the dry nitrogen gas was adjusted, and the humidity (RH) in the plate chamber was repeatedly changed. Specifically, the humidity is 65% R.D. H. And 98% R.V. H. The humidity was measured with humidity temperature sensors installed at the four corners of the plate chamber. The results are shown in FIG. In FIG. 9, measured values from four different humidity temperature sensors are represented by four different symbols.

  As shown in FIG. 9, all the sensor values changed in humidity from 98% to 65% in about 1 to 3 minutes and from 65% to 98% in about 3 to 5 minutes with good reproducibility.

  In addition, the location dependence of the humidity in the plate chamber sometimes showed non-uniformity of about 2% until the steady state was reached. H. Was uniform within.

[Example 6: Comparison of crystal number, crystal size, and crystal quality by difference in humidity]
Bacteriorhodopsin (bR) (10 mg / ml) solubilized with octyl glycoside as a surfactant and 1-O- (3,7,11,15-tetramethylhexadecyl) -β-D-xyloside as a lipid (Β-XylOC _{16 + 4} ) was mixed at a ratio of 45/55 (w / w) to prepare a lipid mesophase reconstituted bR (referred to as bR mesophase). About this bR mesophase, it dispensed on the following two conditions using the sample dispensing apparatus which concerns on embodiment mentioned above, and implemented crystallization.
Condition A: Humidity 97 ± 1% R.D. H. Dispense 100 nl / well of bR mesophase under the following conditions of humidity. Immediately after dispensing, add 1 μl of crystallization solution (2.3 M sodium phosphate / potassium buffer (pH 5.6)), and attach a cover glass Crystallization started in the dark at 20 ° C.
Condition B: Humidity 65 ± 1% R.D. H. After dispensing 100 nl / well of bR mesophase under low humidity conditions, 65 ± 1% R.P. H. Incubate for 5 minutes to evaporate water from the bR mesophase, add 1 μl of crystallization solution (2.3 M sodium phosphate potassium buffer (pH 5.6)), apply cover glass, Crystallization started in the dark.

  The results are shown in FIG. 10 and FIG. FIG. 10A shows the state of the crystal grown in the bR mesophase one month after the start of crystallization in Condition A, and FIG. 10B shows the state in the bR mesophase one month after the start of crystallization in Condition B. The state of the grown crystal is shown. FIG. 11 shows the distribution of the size and number of crystals obtained under conditions A and B. The distribution under condition A is indicated by a broken line, and the distribution under condition B is indicated by black circles and black squares. The results of crystallization in two wells under all conditions are summarized.

  As shown in FIG. 11, in the operation under Condition A, a large number of relatively small crystals of about 35 μm at the maximum centered around 15 μm were obtained. On the other hand, in the operation under Condition B, the number of small crystals near 15 μm decreased to 1/2 or less of that in Condition A, and large crystals of 40 to 55 μm were obtained. That is, many relatively small crystals grew in the operation of condition A, whereas the number of small crystals decreased and a large crystal grew in the operation of condition B.

  The obtained crystal was subjected to X-ray diffraction measurement by the beamline BL1A of the Synchrotron Radiation Science Research Facility (PF). As a result, X-ray diffraction points up to about 2.3Å were confirmed for large crystals obtained by the operation under Condition B, and the lattice constants of P3, 61.3, 61.3, and 100.1Å could be determined. On the other hand, in the crystal obtained by the operation under the condition A, the X-ray diffraction points are not observed so much, and even when the diffraction points are obtained, the intensity is weak. There was a big difference in crystal quality.

  The above shows that the humidity conditions during dispensing have a significant effect on the crystal growth process, and the humidity control during dispensing effectively increases the crystal size and improves the X-ray diffraction power of the crystal. It was confirmed that

[Example 7: Comparison of crystallization speed due to difference in humidity]
As in Example 6, bR (15.5 mg / ml) solubilized with octyl glycoside and β-XylOC _{16 + 4} were mixed at a ratio of 45/55 (w / w) to reconstitute bR. (BR mesophase) was prepared. About this bR mesophase, it dispensed on the following two conditions using the sample dispensing apparatus which concerns on embodiment mentioned above, and implemented crystallization.
Condition C: Humidity 97 ± 1% R.D. H. 50 nl / well of bR mesophase was dispensed under the following humidity conditions. Immediately after dispensing, 0.8 μl of crystallization solution was added, a cover glass was attached, and crystallization was started at 20 ° C. in the dark. The crystallization solution is 1.0M, 1.2M, 1.5M, 1.7M, 2.0M, 2.3M, 2.5M or 3.0M sodium phosphate potassium buffer (pH 5.6). is there.
Condition D: Humidity 55 ± 1% R.D. H. After dispensing 50 nl / well of bR mesophase under low humidity conditions of 55 ± 1% R.P. H. Incubated for 5 minutes, then 0.8 μl of crystallization solution was added, a cover glass was applied, and crystallization was started at 20 ° C. in the dark. As in Condition C, the crystallization solution was 1.0 M, 1.2 M, 1.5 M, 1.7 M, 2.0 M, 2.3 M, 2.5 M, or 3.0 M sodium phosphate potassium buffer ( pH 5.6).

  Under both conditions C and D, crystallization under the same conditions was repeated three times, and the results were summarized. The results of observing the crystallization process over time are shown in FIG. FIG. 12 is a diagram showing the relationship between the time (T) at which crystals were first recognized and the crystallization solution concentration. The triangular symbol indicates the result under the condition C (97% RH condition), and the square symbol indicates the result under the condition D (50% RH condition).

  As shown in FIG. 12, in both conditions C and D, there was a tendency that the concentration of the crystallization solution increased and the time (T) for which crystals were first recognized was shortened. In addition, as compared with the case of Condition C, Condition D has a significantly shorter time (T) for which crystals are first recognized. For example, when the concentration of the crystallization solution is 2.0M, 2.3M, and 2.5M, the time (T) at which crystals are first observed is 20 days, 2 days, and 1 day in the case of Condition D, respectively. In contrast, in Condition C, they were 55 days, 25 days, and 20 days, respectively. That is, in the case of the condition C, compared with the condition D, the time (T) for which the crystal was first recognized was significantly delayed. Furthermore, when crystallization was continued for 3 months, under condition D, crystal growth could be confirmed under all the crystallization solution concentration conditions. On the other hand, under the condition C, the crystal grew only when the concentration of the crystallization solution was 2.0M or more. Thus, a large difference was observed in the crystal growth behavior between conditions C and D.

  Further, the crystal obtained under the condition D was a hexagonal crystal exhibiting polarized light, and an X-ray diffraction point up to 2.6 mm could be confirmed. However, under the condition C, only a crystal with a deformed shape was obtained.

  As described above, it was found that the humidity condition during dispensing greatly affects the crystal growth rate as well as the crystal quality.

[Example 8: Comparison of crystal growth probability depending on humidity]
Proteorhodopsin (pR) (30 mg / ml), a membrane protein solubilized with a surfactant, dodecyl maltoside, and 1-O- (5,9,13,17-tetramethyloctadecanoyl, a lipid) ) Erythritol (EROCOC _{17 + 4} ) was mixed at a ratio of 40/50 (w / w) to prepare a lipid mesophase reconstituted pR (referred to as pR mesophase). About this pR mesophase, it dispensed on the following three conditions using the sample dispensing apparatus which concerns on embodiment mentioned above, and implemented crystallization.
Condition E: Humidity 96 ± 1% R.D. H. After dispensing 50 nl / well of pR mesophase under the following humidity conditions, 0.8 μl of crystallization solution was added immediately after dispensing, a cover glass was attached, and crystallization was started at 20 ° C. in the dark. The crystallization solution is 0.1 M citrate buffer (pH 5.6), 8%, 7%, 6%, 5% or 4% (w / w) MPD, and 0.3M,. A solution containing 4M, 0.5M, 0.6M or 0.7M MgSO ₄ , systematically combining the concentrations of MPD and MgSO ₄ and using a total of 25 different types of crystallization solutions Yes.
Condition F: Humidity 52 ± 1% R.V. H. After dispensing 50 nl / well of pR mesophase under low humidity conditions of 52 ± 1% R.P. H. Incubated for an additional 5 minutes. Immediately thereafter, the humidity was 96 ± 1% R.D. H. The same 25 kinds of crystallization solutions used in condition E were added at 0.8 μl / well, a cover glass was attached, and crystallization was started at 20 ° C. in the dark.
Condition G: Humidity 52 ± 1% R.D. H. A crystallization plate was prepared under the same conditions as in Condition F except that the incubation time in was changed to 10 minutes (twice as long as Condition F), and crystallization was started at 20 ° C. in the dark.

  Crystallization was performed at 20 ° C. for one month in the dark, but no crystal growth was observed in any of the conditions E, F, and G. Thus, when the crystallization temperature was changed to 4 ° C. and crystallization was continued, crystal growth was observed. The number of crystallization solutions in which crystals were grown in 25 different crystallization solutions after changing to 4 ° C. for two months is summarized in Table 1.

  As shown in Table 1, the ratios of the crystallization solution in which crystals were grown under conditions F and G that were dispensed under low humidity conditions were 52% and 64%, respectively. Further, comparing the number of crystals appearing in the same crystallization solution, the number of crystals in the condition G is 2 to 5 times the number of crystals in the condition F, and the crystallization increases as the low humidity incubation time during dispensing increases. The tendency to promote was recognized. On the other hand, the number of crystallization solutions in which crystals under condition E were grown was reduced to less than half of conditions F and G. Accordingly, it was shown that the humidity condition during dispensing greatly affects the subsequent crystallization behavior even in the membrane protein / lipid system different from Examples 6 and 7.

[Example 9: Comparison of crystal growth probability depending on humidity]
40/60 (w / w) ratio of ARII (58 mg / ml) membrane protein solubilized with the surfactant dodecyl maltoside and lipid (monoolein containing 11% cholesterol) To prepare a lipid mesophase reconstituted with ARII (referred to as ARII mesophase). About this ARII mesophase, it dispensed on the following three conditions using the sample dispensing apparatus which concerns on embodiment mentioned above, and implemented crystallization.
Condition H: Humidity 97 ± 1% R.V. H. 50 nl / well of ARII mesophase was dispensed under the following humidity conditions, and 0.8 μl of crystallization solution was added immediately after dispensing, and a cover glass was attached, and crystallization was started at 20 ° C. in the dark. The crystallization solution is 12%, 14% or 16% PEG400 and 4%, 5%, 6% or 7% MPD in 0.1 M Tris-HCl buffer (pH 7.5 or pH 8.0). A total of 24 different types of crystallization solutions are used by systematically combining pH value, PEG 400 concentration and MPD concentration.
Condition I: Humidity 65 ± 1% R.D. H. 50 nl / well of ARII mesophase was dispensed under the humidity conditions of the following, and immediately after dispensing, the same 24 crystallization solutions used under condition H were added at 0.8 μl / well, and a cover glass was attached. Crystallization started in the dark at 20 ° C.
Condition J: Humidity 28 ± 2% R.V. H. 50 nl / well of ARII mesophase was dispensed under the humidity conditions of the following, and immediately after dispensing, the same 24 crystallization solutions used under condition H were added at 0.8 μl / well, and a cover glass was attached. Crystallization started in the dark at 20 ° C.

  As a result of observing the crystallization process over time for each of the conditions H to J, in the case of the condition H, no crystals were obtained. On the other hand, in the case of conditions I and J, growth of crystals could be confirmed with crystallization solutions of 15% and 33%, respectively. From the above, it was found that the crystal growth probability increases remarkably as the humidity during dispensing decreases. Thus, it was shown that the humidity conditions during dispensing greatly influence the crystallization behavior even in membrane protein / lipid systems different from Examples 6, 7 and 8.

[Example 10: Confirmation of X-ray diffraction on protein crystal]
A lysozyme crystal (50 μm × 50 μm) held in a well of a thin sandwich plate composed of a glass plate having a thickness of 150 μm and a cover glass having a thickness of 50 μm is subjected to the following conditions on a beam line BL-32XU of SPring-8. X-ray reflection measurement was performed: temperature 20 ° C., camera length 300 mm, attenuator 600 μm, beam size 10 μm × 10 μm. As a result, X-ray diffraction points up to about 3.1 to 3.2 mm were confirmed, and the lattice constants of P4, 80, 80, and 37 mm could be determined. Thus, it was confirmed that the X-ray diffraction ability could be checked as it was without collecting the protein crystals in the well of the glass sandwich plate to the outside.

[Example 11: Direct SAXS measurement and microscopic observation of lipid mesophase structure in protein crystallization process]
Bacteriorhodopsin (bR) (10 mg / ml) solubilized with octyl glycoside as a surfactant and 1-O- (3,7,11,15-tetramethylhexadecyl) -β-D-xyloside as a lipid (Β-XylOC _{16 + 4} ) was mixed at a ratio of 35/65 (w / w) to prepare a lipid mesophase (bR mesophase) in which bR was reconstituted. For this bR mesophase, using the sample dispensing apparatus according to the above-described embodiment, the humidity is 97 ± 1% R.P. H. The bR mesophase was dispensed into the wells of a microplate using 50 μm-thick substrate glass at 100 nl / well under the following humidity conditions. Then 1 μl of 1.0 M, 1.2 M, 1.5 M, 1.7 M, 2.0 M, 2.3 M, 2.5 M or 3.0 M sodium phosphate potassium buffer is added to the dispensed bR mesophase. After the addition, a cover cell having a thickness of 50 μm was pasted to prepare a sandwich cell, and bR was crystallized at 20 ° C.

  In order to analyze changes in lipid mesophase structure accompanying bR crystallization, 15 minutes, 1.5 hours, 2.5 hours, 18 hours, 42 hours and 6 days after crystallization start, SAXS measurement of lipid mesophase structure was performed using MicroMax-007HF (manufactured by Rigaku Corporation) under the conditions of 40 kV, 30 mA, X-ray irradiation time of 2 minutes, and measurement temperature of 20 ° C. Taking the crystallization system to which 2.3M sodium phosphate / potassium buffer solution is added as an example, the result of microscopic observation is shown in FIG. 13, and the result of SAXS measurement is shown in FIG.

  In FIG. 13, (A), (C), (E), and (F) show normal light micrographs after 15 minutes, 1.5 hours, 18 hours, and 6 days, respectively (B ) And (D) show polarized micrographs after 15 minutes and 1.5 hours, respectively. As shown in FIG. 13, the change in the dynamic mesophase morphology induced immediately after the start of crystallization can be clearly observed from the normal light micrograph and the polarization micrograph, and the birefringence of the lipid mesophase decreases with time. It was confirmed that the crystal was isotropic, and further 6 days after the start of crystallization, the formation of fine crystals of about 10 μm (some crystals were shown by arrows) was clearly confirmed. Further, from the SAXS measurement results shown in FIG. 14, the lipid mesophase gradually transitions from the birefringent lamellar phase to the isotropic Pn3m cubic phase as the bR crystallization progresses. Furthermore, it was confirmed that the lattice constant of the Pn3m cubic phase increased with time, and fine structure information of lipid mesophase that could not be obtained only by microscopic observation was obtained.

  As described above, it was confirmed that, together with microscopic observation, the lipid mesophase structure during crystallization can be directly measured using a SAXS apparatus that can be used at any time in a general laboratory.

[Example 12: Evaluation of microplate forming sheet]
About the microplate formation sheet | seat in which the through-hole was formed by the mechanical punching process, the shape of the edge vicinity of a through-hole was observed with the confocal microscope. The results are shown in FIG. As shown in FIG. 15, when the through hole was formed by mechanical punching, an adhesive burr having a height of about several tens of μm was remarkably observed at the edge portion of the through hole.

  Next, with respect to the microplate forming sheet in which the through holes were formed by mechanical punching, a cover glass having a thickness of 50 μm was applied, and the state thereof was observed. The results are shown in FIG. When a cover glass was applied to a microplate forming sheet having through holes formed by mechanical punching, smooth application was difficult due to the presence of burrs, and complete application could not be realized (FIG. 16). A). FIG. 16B shows an enlarged photograph of the adhesion surface between the wells, and a large number of bubbles remained between the adhesive layer between the wells and the cover glass.

  Next, the shape near the edge of the through hole was observed with a confocal microscope for the microplate forming sheet in which the through hole was formed by laser processing. The results are shown in FIG. As shown in FIG. 17, when the through hole is formed by laser processing, the height of the adhesive layer surface is low in a concentric circle portion having a width of around 100 μm at the edge portion of the through hole (maximum of about 20 μm). Although it was observed, the outside was hardly affected by the processing.

  Next, with respect to the microplate forming sheet in which the through holes were formed by laser processing, a cover glass having a thickness of 50 μm was applied, and the state thereof was observed. The results are shown in FIG. In FIG. 18, B in FIG. 18 shows an enlarged photograph of the adhesion surface between the wells. As shown in FIG. 18, when the cover glass is pasted on the microplate forming sheet in which the through holes are formed by laser processing, almost no residual bubbles are seen compared to the case of mechanical punching. It was confirmed that complete pasting with almost no voids could be realized.

  The present invention can be used for crystallization of membrane proteins and screening for crystallization conditions.

1 Sample dispensing device 2 Sample dispensing micro-dispenser section (sample dispensing means)
3 Crystallization solution dispensing micro dispenser part (solution dispensing means)
4 Plate chamber 5 Cover sticking part (sticking means)
5a Cover pasting part body (sticking means)
5b Support part (sticking means)
6 Disposable tip rack 7 Crystallization solution rack 8 Dispensing observation section (imaging means)
9 Dry Nitrogen Gas Generator 10 Vaporizing Humidifier 11 Humidity Control Device 12 Lid 14 Humidity Sensor 15 Slit (Opening)
20 Protective cover removal claw (peeling means)
25 Installation stand

Claims (14)

Plate chamber,
Sample dispensing means for dispensing a lipid mesophase sample containing a membrane protein into a microplate prepared in the plate chamber,
A solution dispensing means for dispensing a crystallization solution for crystallizing the protein to the microplate, and a humidity adjusting device for adjusting the humidity in the plate chamber;
The said humidity control apparatus supplies the mixed gas of water vapor | steam and dry gas of lower humidity than water vapor | steam in the said plate chamber, The sample dispensing apparatus characterized by the above-mentioned.   The sample dispensing apparatus according to claim 1, wherein the dry gas is dry nitrogen gas.   The sample dispensing apparatus according to claim 1, wherein the humidity control apparatus includes a humidifier that generates the water vapor.   The sample dispensing apparatus according to claim 3, wherein the humidifier is a vaporizing humidifier. One or more humidity sensors are provided inside the plate chamber,
The said humidity control apparatus adjusts the mixing ratio of the said water vapor | steam and the said dry gas in the said mixed gas based on the measurement result by the said 1 or more humidity sensor, The any one of Claims 1-4 characterized by the above-mentioned. The sample dispensing apparatus described in 1.   The sample dispensing apparatus according to claim 5, wherein two or more humidity sensors are provided.   The sample dispensing apparatus according to any one of claims 1 to 6, wherein a movable lid having an opening is provided at an upper portion of the plate chamber.   The imaging device for imaging at least one of a state of dispensing of the sample by the sample dispensing unit and a discharge unit that discharges the sample in the sample dispensing unit. The sample dispensing apparatus according to any one of 1 to 7.   The sample dispensing apparatus according to any one of claims 1 to 8, further comprising sticking means for sticking a transparent cover on an upper surface of the microplate. A protective cover is attached to the microplate.
The sample dispensing apparatus according to claim 1, further comprising a peeling unit that peels the protective cover from the microplate. A sample dispensing step of dispensing a sample of lipid mesophase containing membrane protein into a microplate prepared inside the plate chamber;
A solution dispensing step of dispensing a crystallization solution for crystallizing the protein onto the microplate;
A mixing step of combining the dispensed sample with the dispensed crystallization solution;
After the mixing step, a pasting step of pasting a transparent cover on the upper surface of the microplate;
A method of crystallizing a protein, comprising a crystal growth step of crystallizing the protein in the sample after the pasting step,
In the sample dispensing step and the solution dispensing step, the humidity inside the plate chamber is adjusted by supplying a mixed gas of water vapor and a dry gas having a lower humidity than water vapor into the plate chamber. A protein crystallization method characterized. The microplate is a microplate formed by a glass substrate having a thickness of 30 μm to 150 μm and a microplate forming sheet having one or more well-forming through holes attached to the glass substrate,
The protein crystallization method according to claim 11, wherein the cover is a cover glass having a thickness of 30 μm to 150 μm.   13. The protein crystallization method according to claim 12, wherein the well forming through-hole of the microplate forming sheet is formed by laser processing. A microplate forming sheet having one or more well-forming through-holes that are attached to a substrate to form a microplate,
An adhesive layer is formed on the surface,
The microplate forming sheet, wherein the well forming through hole is formed by laser processing.