JP2006124238A - Protein crystallization device and protein crystallization method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a protein crystallization device which can perform the crystallization experiment or crystallization condition screening of protein rapidly, economically and with high reliability. <P>SOLUTION: The protein crystallization device has a microarray 18 for protein crystallization having two or more crystallizing agent holding sections 16 holding a protein crystallizing agent and a plate 24 superimposed upon the microarray 18. The plate 24 has crystallization compartments 32 corresponding to the crystallizing agent holding sections 16 and capable of being filled with a protein-containing sample and dent parts 34 installed between the crystallization compartments 32. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an apparatus and method for crystallization of proteins or screening of crystallization conditions.

In recent years, research called structural genomics has been conducted in an attempt to elucidate the function of a gene that encodes a protein by clarifying the relationship between the three-dimensional structure of the protein and the function of the protein.
The analysis of the three-dimensional structure of a protein usually involves screening the crystallization conditions of the protein to be analyzed, then performing crystallization under optimal crystallization conditions, and then subjecting the obtained crystals to X-ray structural analysis. It is done by. Here, in the process of screening the protein crystallization conditions, a lot of time and a large amount of protein samples are spent to determine the conditions for obtaining sufficiently good crystals for conducting the three-dimensional structure analysis. It is a bottleneck in structural analysis.
On the other hand, various protein crystallization apparatuses, crystallization methods, and crystallization screening methods have been proposed in order to perform screening of protein crystallization conditions or to perform crystallization rapidly and in a smaller amount of sample.
Some of the present inventors have also conducted a crystallization experiment on a microarray chip for the purpose of quickly and economically screening protein crystallization conditions with a small amount of sample. (For example, refer to Patent Document 1).
In the invention described in Patent Document 1, a microarray is used, and the microarray holds gel-like substances in each section formed by through holes, and each gel-like substance has a plurality of types and concentrations. Contains a protein crystallization agent. By bringing the microarray into contact with a protein-containing sample, a plurality of crystallization conditions can be screened at one time. Furthermore, it can be carried out with a very small amount of protein sample and is very efficient.
WO03 / 053998 pamphlet

However, in the above-described protein crystallization apparatus, the protein-containing sample and / or the crystallization agent is moved and mixed (contaminated) between the sections of the microarray, and the conditions in which the crystals are precipitated are included in the gel-like material in the sections in advance. There is a possibility that the concentration and type of the crystallization agent to be inconsistent.
Further, in such an apparatus, it is necessary to manually supply the protein-containing sample to the crystallization agent holding portion, and the operation becomes complicated. Also, an automatic device that automatically provides a protein-containing sample is expensive.
The present invention has been made to solve the above-described problems, and provides a protein crystallization apparatus capable of quickly and economically performing protein crystallization experiments or screening of crystallization conditions with high reliability. For the purpose.

  The protein crystallization apparatus of the present invention includes a protein crystallization microarray having two or more crystallization agent holding portions holding a protein crystallization agent, and a plate laminated with the protein crystallization microarray, The plate has a crystallization section corresponding to the crystallization agent holding section and capable of being filled with a protein-containing sample, and a recess provided between the crystallization sections.

  According to the protein crystallization apparatus of the present invention, protein crystallization experiments or crystallization conditions can be screened quickly and economically with high reliability.

Embodiments of the present invention will be described below with reference to the drawings.
FIG. 1 is a schematic view showing an example of the protein crystallization apparatus of the present invention.
In this example, as shown in FIG. 1, a support 20, a silicone rubber packing 22, a protein crystallization microarray 18, and a plate 24 are provided.
The packing 22 has a hollow portion 23 having the same shape and area as the protein crystallization microarray 18, and the support 20 is provided with a microarray support portion 26 having the same shape and size as the outer periphery of the packing 22. The microarray support 26 is provided with a marking 28 having the same shape as the packing 22.
The packing 22 is placed on the microarray support part 26 in alignment with the marking 28, and the protein crystallization microarray 18 is stored and installed in the hollow part 23 of the packing 22 on the microarray support part 26. Yes. Here, in order to accurately contact the crystallization section 32 provided on the plate 24 and the crystallization agent holding portion 16 on the protein crystallization microarray 18, the upper surface of the protein crystallization microarray 18 and the support It is preferable that the upper surface of 20 is the same height.

FIG. 3 is an enlarged cross-sectional view of the central portion of the plate 24. FIG. 4 shows a partial cross-sectional view of the support 20, the protein crystallization microarray 18, and the plate 24 assembled.
As shown in FIGS. 3 and 4, the plate 24 is provided between the crystallization sections 32 and the crystallization sections 32 having an arrangement corresponding to each of the crystallization agent holding portions 16 of the protein crystallization microarray 18. And a recess 34. Each crystallization section 32 is supported at the tip of a crystallization section support portion 31 that is a portion that is convex with respect to the recess 34 in the plate 24.
The plate 24 is stacked on the protein crystallization microarray 18 supported by the support 20. That is, as shown in FIG. 4, the crystallization section 32 is sealed by the crystallization agent holding unit 16 and is supported on the crystallization section support 31, and similarly to the crystallization section support 31. Recesses 34 are located between each supported crystallization section 32.
Hereinafter, the surface of the support 20 that supports the protein crystallization microarray 18 is the microarray support surface 100, the surface of the plate 24 that is in contact with the protein crystallization microarray 18 is the reaction surface 102, and the opposite surface is the outer surface 104. Called.
In the plate 24, the outer edge of the region in which the crystallization sections 32 are arranged becomes a concave portion, so that a region having a certain area which is concave in the plate 24 is formed. Hereinafter, a concave region in which the crystallization sections 32 are arranged in the reaction surface of the plate 24 is referred to as a seal portion 30.

  In this example, the protein crystallization microarray 18 is installed on the microarray support portion 26 of the support 20. However, in the protein crystallization microarray 18, the crystallization agent holding portion 16 corresponds to the crystallization section 32. The crystallization section 32 may be installed so as to be sealed, and may be adhered to the support 20, or the microarray support 26 may be formed in a concave shape and adhered to the bottom of the microarray support. You may laminate | stack.

The material and shape of the support 20 are not particularly limited as long as the protein crystallization microarray 18 can be fixed. As the material of the support 20, it is preferable to use a light-transmitting material because the generated crystal can be confirmed with a microscope quickly and easily with the crystallization apparatus, and the crystal growth process can be observed over time. Examples of the light transmissive material include acrylic resin, polycarbonate resin, polystyrene resin, polydimethylsiloxane (PDMS), and glass.
The support 20 is provided with markings for determining the fixing position of the protein crystallization microarray 18. The marking method is not particularly limited as long as the protein crystallization microarray 18 can be accurately fixed. For example, the marking method has a shape and an area similar to that of the protein crystallization microarray 18, and the protein crystallization microarray. The point etc. which were painted in the position on the support body 20 corresponding to 18 predetermined positions are mentioned.
In this example, as shown in FIG. 7, the support 20 is further provided with a plate positioning hole 44 for corresponding the crystallization section 32 and the crystallization agent holding portion 16 when the plate 24 and the support 20 are laminated. It has been.

The protein crystallization microarray 18 has two or more crystallization agent holding portions 16 each holding a protein crystallization agent.
As the crystallization agent holding portion 16, a porous body (hereinafter referred to as a holding phase) that can hold a protein crystallization agent such as a gel can be used, and the holding phase can be used. As shown in FIG. 4, when the protein-containing sample filled in the crystallization section 32 comes into contact with the crystallization agent holding section 16, the protein crystallization agent moves from the holding phase to the crystallization section 32. Good.
As the retention phase, it is preferable to use a gel. By using the gel, the movement of the protein crystallization agent from the crystallization agent holding unit 16 to the protein-containing sample packed in the crystallization section 32 is controlled at a moderately moderate speed, thereby realizing stable crystallization. it can.
The material of the gel is not particularly limited. For example, acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl One or more monomers such as methacrylate, (meth) acrylic acid, and allyl dextrin, and a polyfunctional monomer such as methylene bis (meth) acrylamide, polyethylene glycol di (meth) acrylate, etc. A gel copolymerized in a medium can be used. In addition, as a gel, for example, a gel such as agarose, alginic acid, dextran, polyvinyl alcohol, polyethylene glycol, or a gel obtained by crosslinking these can be used.
In order to retain the gel as described above in the protein crystallization microarray 18, for example, a liquid containing a monomer such as acrylamide, a polyfunctional monomer, and an initiator, which are components of the gel, is placed in the container. It may be injected, polymerized, and gelled. As a method of gelling, in addition to a method of copolymerization in the presence of a polyfunctional monomer, a method using a crosslinking agent after copolymerization in the absence of a polyfunctional monomer may be used. . When agarose is used as the gel material, gelation may be performed by a temperature drop.
When the protein crystallizing agent is retained on the gel, the method is not particularly limited. For example, the protein crystallizing agent and the above polymerizable monomer are mixed in advance and introduced into an appropriate container, and then the gel is formed by performing a polymerization reaction to hold the protein crystallizing agent in the gel. Can be made. Here, the protein crystallizing agent can be impregnated with porous particles or the like, and the particles can be included in the gel.

The material, shape, and the like of the protein crystallization microarray 18 are not particularly limited as long as a large number of reaction substances are arranged and do not hinder the observation of crystal precipitation.
Examples of the material for the protein crystallization microarray 18 include glass, resin, metal, and the like, and a composite formed by combining these materials may be used. However, in order to easily confirm the presence or absence of protein crystal precipitation, it is preferable to use a material having high light transmittance.
Examples of the shape of the protein crystallizing microarray 18 include a circular shape, a square shape, and a rectangular shape. The thickness can be arbitrarily selected in consideration of improvement in crystallization efficiency and ease and speed of observation of crystal precipitation. For example, the thickness is 0.1 to 5 mm, preferably 0.2 to 2 mm. can do.

FIG. 2 shows a microarray in which a plurality of hollow tubular bodies are arranged as a suitable example of the protein crystallization microarray used in the protein crystallization apparatus of the present invention. As shown in FIG. 2, two or more hollow fibers 12 are sectioned and fixed as a hollow tubular body on the substrate 10, and these hollow fibers 12 have hollow portions 14, and these hollow portions 14. Furthermore, the gel holding the protein crystallizing agent is filled to form the crystallizing agent holding part 16. That is, two or more crystallizing agent holding portions 16 are sectioned on the substrate 10 to form a protein crystallization microarray 18.
Is configured.
The hollow fiber 12 may be, for example, a methacrylate polymer such as methyl methacrylate, ethyl methacrylate, or butyl methacrylate, a homopolymer of an acrylate monomer such as methyl acrylate or ethyl acrylate, or a copolymer thereof, polystyrene, polyethylene, polypropylene, nobornene / Examples thereof include those formed from an ethylene copolymer, polyethylene terephthalate, polycarbonate, glass and the like.
The inner wall surface of the hollow fiber 12 may be used in an untreated state, but may be subjected to a plasma treatment, or a radiation treatment such as a γ ray or an electron beam, if necessary. Furthermore, the hollow fiber 12 may have a reactive functional group introduced as necessary.
The outer diameter of the hollow fiber 12 is preferably 2 mm or less, and more preferably 0.7 mm or less in order to increase the number of crystallization agent holding portions 16 per unit area. Further, the inner diameter of the hollow fiber 12 is appropriately selected within the range of the outer diameter.
The following method can be used as a method of fixing the two or more hollow fibers 12 on the substrate 10 in a partitioned arrangement. That is, first, the hollow fibers 12 are arranged in parallel with a predetermined interval. These hollow fibers 12 are converged and then bonded to form a fiber array (three-dimensional array). The obtained three-dimensional array is cut in a direction intersecting the fiber axis, preferably in a direction perpendicular to the fiber axis, using a device for preparing a section such as a microtome, so that it has a hollow fiber array cross section. A microarray consisting of flakes (FIG. 2) can be obtained. The thickness of the flakes can be usually used as 100 to 5000 μm, preferably 200 to 2000 μm.
At this time, by arranging the hollow fibers 12 regularly and bonding them with a resin adhesive or the like, for example, a hollow fiber array in which the hollow fibers 12 are regularly and regularly arranged vertically and horizontally can be obtained. The shape of the hollow fiber array is not particularly limited, but is usually formed into a square, a rectangle, a circle, or the like by regularly arranging the fibers.
“Regularly” means that the fibers are arranged in order so that the number of fibers contained in a frame of a certain size is constant. For example, in the case where fibers having a diameter of 1 mm are bundled and arranged so as to form a square having a cross section of 10 mm in length and 10 mm in width, the number of fibers contained in one side in the square frame (1 cm ² ) is determined. The number of fibers is 10 and the 10 fibers are bundled in a row to form a sheet of one layer, and then the sheets are stacked so as to form 10 layers. As a result, a total of 100 fibers can be arranged, 10 vertically and 10 horizontally. However, the method of regularly arranging the fibers is not limited to the method of stacking sheets as described above.
By using the microarray formed by arranging a plurality of hollow tubular bodies as described above as the protein crystallization microarray 18, the thickness of the protein crystallization microarray 18 and the volume of the crystallization agent holding unit 16 are held. A protein crystallizing apparatus can be produced efficiently by controlling the type and concentration of the protein crystallizing agent well.

  The protein crystallization microarray 18 is preferably stored by being sealed with a hermetically sealed container or seal that prevents contact with the outside air. As a container with good airtightness, for example, a polymer material having a low gas or water permeability, glass, metal or the like can be used. In addition, such a microarray is preferably stored at a low temperature, and may be stored frozen especially in the case of long-term storage.

Furthermore, it is preferable that each crystallizing agent holding unit 16 is made of a gel prepared under different protein crystallization conditions. This makes it possible to perform screening on a single microarray when screening crystallization conditions. Can be done quickly.

Here, the protein crystallization conditions are the type and concentration of the protein crystallization agent, the retention phase that retains the protein crystallization agent, for example, the pH of the gel after the protein crystallization agent is retained and solidified, and the gel composition The degree of crosslinking, the temperature of the crystallization agent holding part 16 and the crystallization section 32, the time, the cooling profile, and the like.
Examples of the protein crystallizing agent include a precipitant, a pH buffer, and any combination thereof.
Examples of the precipitant include sodium chloride, polyethylene glycol, 2-methyl-2,4-pentanediol, and ammonium sulfate. Examples of the pH buffering agent include sodium acetate trihydrate, potassium phosphate, imidazole, sodium citrate, sodium cacodylate and the like. These may be used alone or in any combination of two or more. Furthermore, as a protein crystallizing agent, “WIZARD II” manufactured by Emerald Biostructures, “Crystal screen” manufactured by Hampton Research, “Grid Screen”, or the like can be used as a protein crystallization agent.
The concentration of the protein crystallizing agent depends on the type of the protein crystallizing agent to be used. For example, in the case of a precipitant made of polyethylene glycol, the concentration is 5 to 50% by volume, preferably 10 to 35% by volume, and pH In the case of a buffer, it is 0.05 to 0.5 mol / L, preferably 0.1 to 0.2 mol / L.

As described above, by preparing each crystallization agent holding unit 16 under different protein crystallization conditions, screening of the crystallization conditions for the target protein can be performed quickly. Here, for example, it is preferable to set the concentration of the protein crystallizing agent in multiple stages. Specifically, in the case of using a precipitating agent consisting of sodium chloride, in the range of 0.5 to 4.0 mol / L, Dilution trains can be prepared as in steps 5, 10, and 20, and protein crystallizing agents having respective concentrations can be filled in the crystallizing agent holding unit 16.
The protein crystallization microarray 18 preferably has 10 to 1000 crystallization agent holding portions 16 in order to collectively perform crystallization under a larger number of crystallization conditions. For example, in normal protein crystallization condition screening, it is necessary to examine crystallization conditions of about 800, so the number of crystallizing agent holding parts is preferably 10 or more, while crystals exceeding 1000 When the agent holding part 16 is provided, the interval between the crystallizing agent holding parts 16 becomes very narrow, and the handling efficiency is poor.
The protein crystallization microarray 18 is preferably made of a highly light-transmitting material in order to easily confirm protein precipitation.

  In this example, the packing 22 is made of silicon rubber, but the packing material is used in combination with other components in the protein crystallization apparatus (for example, the protein crystallization microarray 18, the plate 24, and the support 20). There is no particular limitation as long as it is suitable.

The plate 24 has a crystallization section 32 corresponding to the crystallization agent holding section 16 and capable of being filled with a protein-containing sample, and a recess 34 provided between the crystallization sections 32.
That is, the same number of crystallization sections 32 as the crystallization agent holding portions 16 are provided on the plate 24.
The plate 24 is preferably formed with, for example, a sample filling auxiliary tool positioning hole 50 as a positioning portion for aligning the position with a sample filling auxiliary tool described later.
In this example, a plate positioning member 44 is further provided on the support 20 so that the crystallization section 32 and the crystallization agent holding portion 16 correspond to each other when the plate 24 and the support 20 are stacked. A plate positioning hole 46 corresponding to the plate positioning member 44 provided on the support 20 is formed on the plate 24.
In this example, as shown in FIGS. 1 and 5, a sealing agent injection port 48 penetrating from the outer surface 104 of the plate 24 to the reaction surface 102 is further bored. As shown in FIG. 5, in this example, two sealing agent injection ports 48 are formed on opposite sides of each other via the seal portion 30. As a result, when the sealant is injected into the seal part 30 from the one sealant injection port 48, the air initially present in the seal part 30 is discharged from the other sealant injection port 48. The crystallization section 32 located between the recesses 34 at 30 is more reliably sealed.
The material and shape of the plate 24 are not particularly limited as long as they can be superimposed on the protein crystallization microarray 18, and any material can be used in consideration of the shape of the protein crystallization microarray 18. Examples of the material for the plate include glass, resin, metal, and the like. Any of these materials can be selected and used alone or in combination of two or more. In addition, in order to observe the crystallization state of the protein in the crystallization section 32, the portion overlapping with each crystallizing agent holding portion 16 when being superposed on the protein crystallization microarray 18 has light transmittance. Since it is preferable, it is preferable to use a light-transmitting material such as a transparent resin or glass as the material of the plate 24.

The plate 24 preferably further has a crystal recovery mechanism for recovering crystals precipitated in the crystallization section 32. By using such a crystal recovery mechanism, a crystal deposited in the crystallization section 32 is recovered and used as a seed crystal, and a crystal that can withstand X-ray structure analysis can be rapidly obtained by performing a crystal growth process subsequently. . Of course, if the precipitated crystal is a crystal that can withstand X-ray structure analysis as it is, it is recovered and used for analysis.
As such a crystal recovery mechanism, for example, the seal portion 30 is made of a member independent of the plate 24, and is connected to the plate 24 by a hinge mechanism so that the seal portion 30 can be opened to the outer surface 104 side as desired. Etc.

The shape of the crystallization section 32 is not particularly limited as long as it can be filled with a protein-containing sample and is sealed when it comes into contact with the crystallization agent holding unit 16.
The volume of the crystallization section 32 is less than 0.5 μl in order to reduce the amount of protein required for screening the crystallization condition when the purpose is to screen the crystallization condition for the target protein. Is preferred. On the other hand, when it is intended to use the precipitated crystal for subsequent X-ray structural analysis or as a seed crystal for producing a crystal for structural analysis, X-ray structural analysis can be performed. In order to obtain a large crystal of 0.1 mm or more, the volume of the crystallization section 32 is preferably 0.5 μl or more.

The relationship between the area of the crystallization agent holding part 16 and the crystallization section 32 is that the area of the surface where the crystallization agent holding part 16 and the crystallization section 32 are in contact is held in the crystallization agent holding part 16. It is sufficient that the agent moves to the crystallization section 32 and can react with the protein-containing sample filled in the crystallization section 32. The crystallization section 32 is more preferable than the crystallization agent holding unit 16. It can be large.
However, it is preferable that the crystallization section 32 does not contact the substrate 12 constituting the protein crystallization microarray 18.

In this example, as shown in FIG. 4, the sealing agent 35 is filled in all of the recesses 34 provided between the crystallization sections 32.
The sealant 35 may be any material that does not dissolve in the protein-containing sample and does not corrode the members such as the plate 24, the packing 22, and the substrate 12 constituting the protein crystallization microarray 18. For example, paraffin-based, silicon Oils such as those based on mineral, mineral oil, ester, synthetic oil (including fluorine), lithium, sodium, calcium, complex base, non-soap, and molybdenum can be used.
Even if the sealant 35 is not used, the recess 34 can prevent the protein-containing sample from entering the adjacent compartments. However, if the sealant 35 is used, the protein-containing samples can be mixed more reliably. In addition, it is possible to prevent intrusion into the adjacent crystallization section 32 and to prevent evaporation of the protein-containing sample.

  In this example, as a mechanism for press-contacting the protein crystallization microarray 18 supported by the support 20 and the plate 24, a first screw hole 40 penetrating the support 20 as shown in FIG. A second screw hole 42 penetrating the plate 24 corresponding to the one screw hole 40 is provided. A screw 41 is screwed into the first screw hole 40 and the second screw hole 42 as shown in FIG. By using such a mechanism, the crystallization section 32 can be more stably held in a sealed state.

In the protein crystallization apparatus of this example, a detection mechanism for monitoring protein crystallization in the crystallization section 32 can be further provided.
Examples of the detection mechanism include a detection mechanism including a microscope installed on the outer surface 104 of the plate 24 and a CCD camera mounted on the microscope. In such a detection mechanism, it is possible to quickly determine the success or failure of crystallization by photographing and recording the state of crystal precipitation with a CCD camera mounted on a microscope and processing the recorded image data. Therefore, protein crystallization conditions can be determined quickly.

Here, a preferred method of using the protein crystallization apparatus of this example is illustrated.
[Packing of protein-containing sample]
In this example, the protein-containing sample can be filled into the crystallization section 32 using a sample filling aid.
(Crystallization section)
When the present invention is used, when the purpose is to crystallize a protein, the volume of the crystallization section 32 is preferably 0.5 μl or more. For the purpose of quicker screening of protein crystallization conditions, the volume of the crystallization section 32 is preferably less than 0.5 μl.
(Protein-containing sample)
In the present invention, the protein-containing sample is a sample containing a protein to be crystallized or to specify crystallization conditions (hereinafter referred to as a target protein). Examples of proteins include natural or synthetic peptides, polypeptides, proteins, and protein complexes. These substances are extracted and isolated from natural or synthetic materials, or produced by genetic engineering techniques or chemical synthesis techniques, and then combined with conventional purification methods such as solvent extraction, column chromatography, and liquid chromatography. It is preferable to purify the protein of interest by using it.
Since the protein concentration and purity in the protein-containing sample also contribute to protein crystallization conditions, a protein-containing sample having several levels of concentration and purity may be prepared and reacted with the protein crystallization agent. For example, the protein concentration is preferably changed in several steps within the range of 1 to 50 mg / ml. The amount of the protein-containing sample to be reacted with the protein crystallization agent can be appropriately changed according to the volume and number of the crystallization compartments 32 used. The viscosity of the protein-containing sample is not particularly limited as long as the protein-containing sample does not leak from the crystallization section 32 when the plate 24 is held with the crystallization section 32 holding the protein-containing sample facing downward.
In addition to the target protein, the protein-containing sample may further contain a stabilizing agent such as a protein solubilizing agent or a reducing agent that assists in dissolving the protein. Examples of protein solubilizers include surfactants that dissolve membrane proteins. When a surfactant is used, proteins with low water solubility, such as membrane proteins, can be dispersed well in protein-containing samples, and the protein crystallization apparatus of this example can be applied to efficiently crystallize proteins. It can be carried out.

First, the plate 24 as shown in FIG. 1 is allowed to stand with the reaction surface 102 as shown in FIG. A cylindrical guide pin 52 having the same diameter as the sample filling auxiliary tool positioning hole 50 and the plate positioning hole 46 is inserted into the sample filling auxiliary tool positioning hole 50 and the plate positioning hole 46 formed in the plate 24 one by one. Fix it.
Next, on this reaction surface 102, as shown in FIG. 6, the array of perforations 56 corresponding to the crystallization sections 32 on the plate 24, and the positions where the perforations 56 correspond to the crystallization sections 32 on the plate 24. A thin plate-like sample filling auxiliary tool 54 having two alignment holes 58 is installed as an alignment mechanism. At this time, the alignment holes 58 drilled in the sample filling aid 54 are inserted into the guide pins 52 fixed to the sample filling aid positioning hole 50 and the plate positioning hole 46 of the plate 24, respectively. 54 is placed on the plate 24. Thus, the sample filling aid 54 is installed so that the crystallization section 32 of the plate 24 and the perforation 56 of the sample filling aid 54 correspond.
Thereafter, an amount of protein-containing sample that reaches the entire region where the perforations 56 are arranged in the sample filling aid 54 is placed on the sample filling aid 54 installed on the plate 24.
Further, the protein-containing sample is filled into the crystallization section 32 corresponding to the perforations 56 by rubbing the surface of the sample filling aid 54 using a spatula or the like.
Thereafter, the guide pin 52 is pushed out from the reaction surface 102 side of the plate 24 toward the outer surface 104 side, and the sample filling aid 54 is removed from the plate 24. As a result, all the crystallization compartments 32 are filled with the protein-containing sample.

As described above, by placing the sample filling aid 54 on the reaction surface 102 of the plate 24 and adding the protein-containing sample thereon, the protein-containing sample can be accurately filled into the crystallization section 32. it can.
The sample filling aid 54 is preferably made of stainless steel from the viewpoints of ease of molding, strength, salt resistance, and the like. Further, the sample filling aid 54 is preferably provided with a part to be handled by tweezers or the like so that the sample filling aid 54 can be easily removed after filling the crystallization section 32 with the protein-containing sample. It is preferable that the bent portion 59 is provided by being bent upward.
Here, the guide pins 52 are fixed to the sample filling assisting tool positioning hole 50 and the plate positioning hole 46, respectively. However, in the plate 24, a plurality of sample filling assisting tool positioning holes 50 are formed independently of the plate positioning hole 46. May be.

As a method for filling the protein-containing sample into the crystallization section 32, the protein-containing sample can be filled in the same amount in all the crystallization sections 32, and adhesion to components other than the crystallization section 32 can be suppressed. Any method can be used. For example, the protein-containing sample can be filled in each of the crystallization sections 32 using a pipetter or the like without using the sample filling aid 54, and an automatic device for sample addition is used. be able to.
However, in order to quickly and economically fill the protein-containing sample, the method using the sample filling aid 54 is preferable.
When a pipetter is used for filling a protein-containing sample, a small amount of liquid droplets adhering to the tip of the pipetter is removed from the vicinity of the crystallization section 32 without using a mechanism for discharging the protein-containing sample sucked and held by the pipetter. It is preferable to make it contact.

[Assembly of protein crystallization equipment]
As shown in FIG. 1, a packing 22 is installed in accordance with the marking 28 on the support 20, and the protein crystallization microarray 18 is placed on the support 20 so as to be stored in the hollow portion 23 of the packing 22. Install.
The plate 24 filled with the protein-containing sample in the crystallization section 32 is held with the reaction surface 102 facing down, and is laminated on the protein crystallization microarray 18 supported by the support 20. At this time, the plate positioning member 44 provided on the support 20 passes through the plate positioning hole 46 formed in the plate 24.
In the experimental operation, when the gel held in the crystallization section 32 is dried, there are cases where bubbles are mixed in the crystallization section 32 when the plates 24 are stacked as they are. In order to prevent air bubbles from being mixed, it is also possible to provide a protein sample in the crystallization section 32 in advance and laminate the plate 24 after the gel is sufficiently swollen. Further, the mist generated by the ultrasonic humidifier may be brought into contact with the crystallization section of the microarray to swell the gel.
In this example, the plate 24 is laminated on the protein crystallization microarray 18 supported by the support 20 with the reaction surface 102 facing downward, but the plate in which the crystallization section 32 is filled with a protein-containing sample. 24 may be allowed to stand with the reaction surface 102 facing upward, and the support 20 supporting the protein crystallization microarray 18 may be laminated on the plate 24 with the microarray support surface 100 facing down. It is preferable to install the plate 24 with the outer surface 104 facing upward because it is easy to inject the sealing agent from the sealing agent injection port 48 or to observe the crystals deposited in the crystallization section 32.

The plate 24 is pressed against the support 20 and held, and the screw 41 is screwed into the first screw hole 40 and the second screw hole 42 while holding the plate 24, and the protein crystallization supported by the plate 24 and the support 20. The microarray 18 for use. The pressure welding is preferably completed over several minutes after the support 20 contacts the crystallization section 32. According to such a pressure-bonding method, the gel held in the crystallization section 32 can be sufficiently swollen with the protein sample. Thereafter, the filling state of the protein-containing sample is confirmed using a microscope.
After confirming that the protein-containing sample is filled in each crystallization section 32, the sealant is removed from the sealant inlet 48 formed in the plate 24 using a pipette or the like as shown in FIGS. The seal portion 30 and the concave portion 34 are filled. As a result, the protein-containing sample leaked to a position other than the crystallization agent holding portion 16 in the protein crystallization microarray 18 can be replaced with the sealant, and the protein-containing sample moves between adjacent crystallization sections 32. It is possible to prevent contamination and change of conditions due to the above, and to prevent evaporation of the protein-containing sample. The amount of the sealing agent injected is such that when the sealing agent spreads in the recess 34 over the entire area of the seal portion 30 and the sealing agent is injected from one sealing agent injection port 48 as shown in FIGS. It is preferable that the maximum amount of the sealing agent does not leak from the sealing agent injection port 48 of the above.

[Screening of protein crystallization conditions]
Using the assembled protein crystallization apparatus, screening of protein crystallization conditions can be performed to determine the optimum crystallization conditions for the target protein. A crystal suitable for structural analysis can also be produced using the precipitated crystal.
After the protein crystallization agent and the protein-containing sample are reacted in the protein crystallization apparatus, the protein crystallization apparatus is kept in a sealed state or in the atmosphere under a certain temperature condition for a time sufficient for the protein to precipitate. Leave still.
The time sufficient for the protein to precipitate varies depending on the specific protein, concentration, crystallization conditions, etc., but is about 1 hour to 10 days. If no crystals are precipitated after about 30 days, The crystallization conditions are considered inappropriate. Further, since the temperature condition is one factor of the protein crystallization condition, crystallization may be performed by changing the temperature condition. The temperature condition is preferably set in a plurality of stages such as 4 ° C., 15 ° C., 18 ° C., 22 ° C., and the like.
After a sufficient time has elapsed for the protein to precipitate, the state of protein crystal precipitation is observed. At this time, it is preferable to observe with an optical microscope etc. from the outer surface 104 side of the plate 24, for example.
In this way, the optimal crystallization conditions for the protein of interest can be determined.

[Preparation of crystal for structural analysis]
For the purpose of protein crystallization, when the volume of the crystallization section 32 is 0.5 μl or more, among all the crystallization sections 32, the crystal is continuously grown in the crystallization section 32 where the crystals are deposited. Alternatively, the precipitated crystal is recovered from the crystallization section 32 and used as a seed crystal, and a crystal for structural analysis is produced by a known protein crystallization method under the same crystallization conditions as the crystallization section 32 from which the seed crystal is recovered. can do. Known protein crystallization methods include, for example, a hanging drop method, a sitting drop method, a dialysis method, and a batch method.
For the purpose of quick screening of protein crystallization conditions, when the volume of the crystallization section 32 is less than 0.5 μl, the target protein is crystallized under the optimum crystallization conditions obtained, and the crystal for structural analysis Can be obtained. At this time, as a method for obtaining a crystal for structural analysis, a known method such as a vapor diffusion method or a hanging drop method can be used.
By collecting the obtained crystal for structural analysis and subjecting the collected crystal to X-ray structural analysis by a known method, the three-dimensional structure of the target protein can be determined.

  In the protein crystallization apparatus of this example, after the protein-containing sample is filled in the crystallization section 32, the plate 24 and the protein crystallization microarray 18 are stacked, whereby the crystallization agent holding unit 16 and the protein-containing sample are separated. The filled crystallization sections 32 overlap correspondingly. As a result, the crystallization agent held in the crystallization agent holding part 16 moves into the crystallization section 32 and reacts with the protein. When each crystallization agent holding part 16 is set to different crystallization conditions, a crystal | crystallization precipitates in the crystallization division 32 set to the appropriate crystallization condition in order for the protein of interest to crystallize.

As described above, in the protein crystallization apparatus of this example, the protein-containing sample is held in the crystallization section 32 and sealed by the crystallization agent holding section 16, and each crystallization section 32 is isolated by the recess 34. Therefore, the movement of the protein-containing sample between the crystallization sections 32 and / or the mixing of the protein crystallization agent between the crystallization agent holding portions 16 can be prevented, and the solution from the protein-containing sample can be prevented. Evaporation is suppressed and a sample containing a minute amount of protein at the nl level can be stably held. Therefore, the protein can be crystallized with high reliability, and the suitability of the crystallization conditions can be determined with high reliability.
Furthermore, when the concave portion 34 is filled with a sealant, it is possible to more efficiently prevent mixing of the protein-containing samples and intrusion into the adjacent crystallization section 32 and to prevent evaporation of the protein-containing sample. Therefore, the crystallization conditions can be well controlled even if the amount of the protein-containing sample is very small.
Further, when the support 20 supporting the protein crystallization microarray 18 and the plate 24 are pressed against each other, the crystallization section 32 can be stably sealed, so that crystallization can be performed with higher reliability. .
By appropriately selecting the capacity of the crystallization section 32, both screening of protein crystallization conditions and preparation of crystals for structural analysis can be performed quickly and accurately.
Furthermore, when a sample filling aid is used, protein crystallization conditions can be screened simply and quickly.

  The protein crystallization apparatus of the present invention can be suitably used for both screening of protein crystallization conditions and preparation of crystals for protein structure analysis, for example, in research and development in the pharmaceutical field and the like.

(Production of hollow fiber array)
A hollow fiber array was produced as a component for supporting the crystallization agent holding part.
A perforated plate having 0.32 mm diameter holes and a distance between the centers of the holes of 0.42 mm, and stacking two 0.1 mm thick perforated plates arranged in a total of 100 rows in 10 rows. 100 polyethylene hollow fibers (manufactured by Mitsubishi Rayon Co., Ltd., outer diameter: about 500 μm, inner diameter: about 300 μm, length: about 100 cm) were passed through each hole of the perforated plate. Next, with the hollow fiber stretched, two locations of 10 cm and 40 cm from one end of the hollow fiber were fixed, and the distance between the two perforated plates was 30 cm.
Next, three sides of the space between the two perforated plates were surrounded with a silicon plate. From one side opened, a mixture of a resin composed of a polyurethane resin adhesive and 2.5% by mass of carbon black as a resin material with respect to the total mass of the resin material was poured between the two perforated plates. Subsequently, the resin was allowed to stand at room temperature for 1 week to cure the resin. Thereafter, the porous plate and the plate-like material installed between the porous plates were removed to obtain a hollow fiber array.
(Introduction and fixation of polymer gel in hollow fiber array)
A mixed solution having the following composition was prepared.
Acrylamide: 3.7 parts by mass Methylenebisacrylamide: 0.3 parts by mass 2,2′-azobis (2-amidinopropane) dihydrochloride: 0.1 parts by mass The above mixed solution and the hollow fiber obtained above The hollow body of the hollow fiber is placed in a desiccator and the inside of the desiccator is decompressed while the longer end of each hollow fiber is immersed in the mixed solution. The mixed solution was introduced into. Subsequently, this hollow fiber array was transferred to a sealed glass container whose interior was saturated with water vapor, and a polymerization reaction was carried out at 80 ° C. for 4 hours. As a result, a hollow fiber array in which the acrylamide gel was fixed to the hollow part of the hollow fiber was obtained.

(Preparation of gel-filled hollow fiber array flakes)
The acrylamide gel-containing hollow fiber array obtained above is cut into a thickness of about 2 mm using a microtome in a direction perpendicular to the longitudinal direction of the hollow fibers, whereby the hollow portions of the hollow fibers are filled with the gel. An array flake in which a total of 100 hollow fibers were regularly arranged in a square was obtained. FIG. 2 shows the gel-containing hollow fiber array flakes obtained by the above process. As shown in FIG. 2, the hollow portion 14 of the hollow fiber 12 is filled with the acrylamide gel produced above.
(Preparation of microarray for protein crystallization)
In the gel-containing hollow fiber array flakes produced above, the following A, B, C, D, E, F, G, H, I, J of acrylamide gel filled in the hollow portion 14 of the hollow fiber 12 By adding 1 μl of protein crystallization agent comprising an aqueous solution to the gel held in each hollow portion, the protein crystallization agent was held in the acrylamide gel, and a protein crystallization microarray 18 was produced.
A: 2.0 mol / L sodium chloride aqueous solution B: 0.5 mol / L sodium chloride aqueous solution C: 10% by volume Polyethylene glycol (molecular weight 400) aqueous solution D: 20% by volume Polyethylene glycol (molecular weight 400) aqueous solution E: 10% by volume Polyethylene Glycol (molecular weight 6000) aqueous solution F: 20% by volume Polyethylene glycol (molecular weight 6000) aqueous solution G: 20% by volume 2-methyl-2,4-pentanediol aqueous solution H: 20% by volume 2-methyl-2,4-pentanediol aqueous solution I: 0.5 mol / L ammonium sulfate aqueous solution J: 1.5 mol / L ammonium sulfate aqueous solution Hereinafter, the crystallization agent holding portions 16 holding the protein crystallization agents A to J are referred to as spots A to J, respectively.

[Example 1] Screening of protein crystallization conditions The crystallization conditions of lysozyme (manufactured by Sigma-Aldrich) were screened using the protein crystallization apparatus shown in FIG. The protein crystallization microarray 18 produced above was used as the protein crystallization microarray, and acrylic resin was used as the material for the support 20 and the plate 24.
(Addition of protein-containing sample)
Using the sample filling aid 54 shown in FIG. 6, the crystallization section 32 was filled with the protein-containing sample. As the sample filling auxiliary tool 54, one having perforations 56 of an array corresponding to the crystallization agent holding part 16 of the protein crystallization microarray 18 obtained above was used.
The crystallization section 32 had a cylindrical shape with one end closed having a diameter of 0.7 mm and a height of 0.15 mm, and the capacity of the crystallization section 32 was 105 nl.
A lysozyme-containing aqueous solution (80 mg / ml) was used as the protein-containing sample.
First, the sample filling aid 54 is placed on the plate 24 as shown in FIG. 6, and then 1.5 to 2 μl of protein-containing sample is added over all of the perforations 56 with a 20 μl autopipette. did. Then, the protein-containing sample was pressed onto the plate 24 with a spatula from above the sample filling aid 54, so that the protein-containing sample was filled in all the crystallization sections 32 on the plate 24.

(Assembly of protein crystallization equipment, screening of protein crystallization conditions)
Subsequently, the plate 24 filled with the protein-containing sample in the crystallization section 32 was held with the reaction surface 102 facing down, and was laminated on the protein crystallization microarray 18 supported by the support 20. After confirming that the crystallization section 32 is filled with the protein-containing sample, the sealant is injected into one sealant inlet 48 formed in the plate 24 from the outer surface 104 side of the plate 24 using a pipette. Paraffin oil (hereinafter referred to as oil) was injected. The amount of oil injected was until immediately before the oil spilled over the entire seal portion 30 or the oil overflowed from the other sealant injection port 48.
Thereafter, the protein crystallizer was allowed to stand at 20 ° C. for 3 hours. Subsequently, as a result of observing the inside of the crystallization section 32 with an optical microscope, no lysozyme crystals were observed in the spot B in which a polyethylene glycol aqueous solution was retained as a protein crystallization agent, and a 2.0 mol / L sodium chloride aqueous solution was retained. In the spot A, columnar crystals most suitable for X-ray structural analysis were observed.

(Preparation of crystals for structural analysis)
Further, based on the above results, lysozyme crystals were prepared from a lysozyme-containing sample solution by a hanging drop method using a 2.0 mol / L sodium chloride aqueous solution as a protein crystallization agent. At this time, the size of the obtained lysozyme crystal was 0.3 × 0.3 × 0.5 mm.

From the above results, as a result of examining the type and concentration of the protein crystallization agent among the crystallization conditions of lysozyme, it became clear that 2.0 mol / L sodium chloride aqueous solution is the optimal crystallization condition, and Crystals suitable for X-ray structural analysis could be obtained using the optimum crystallization conditions that were revealed.
That is, protein crystallization conditions could be screened quickly and economically with high reliability and with a small amount of protein-containing sample.

It is a schematic diagram which shows the use condition of the protein crystallization apparatus of this invention. The example of the microarray for protein crystallization used for this invention is shown. It is a schematic diagram which shows an example of the protein crystallization apparatus of this invention. It is sectional drawing of a part of plate used for this invention. It is a top view of the plate used for this invention. It is a top view which shows the use condition of the plate in this invention, and a sample filling auxiliary tool. It is a top view of the support body used for this invention. It is a top view which shows the use condition of the protein crystallization apparatus of this invention.

Explanation of symbols

12 hollow fiber 16 crystallizing agent holding part 18 microarray 20 for protein crystallization support 24 plate 32 crystallization section 34 recess

Claims (14)

  A protein crystallization microarray having two or more crystallization agent holding parts holding a protein crystallization agent; and a plate laminated with the protein crystallization microarray, wherein the plate includes the crystallization agent holding part A protein crystallization apparatus comprising: a crystallization section that can be filled with a protein-containing sample, and a recess provided between the crystallization sections.   The protein crystallization apparatus according to claim 1, wherein the crystallization agent holding unit is made of a gel prepared under different protein crystallization conditions.   The protein crystallization apparatus according to claim 1 or 2, wherein the protein crystallization microarray is a microarray formed by arranging a plurality of hollow tubular bodies.   4. The protein crystallization apparatus according to claim 1, further comprising a mechanism for press-contacting the protein crystallization microarray and the plate.   The protein crystallizing apparatus according to any one of claims 1 to 4, wherein the recess is filled with a sealant.   6. The protein crystallization apparatus according to claim 1, wherein the crystallization section has a volume of less than 0.5 μl.   6. The protein crystallization apparatus according to claim 1, wherein the crystallization section has a volume of 0.5 μl or more.   The protein crystallization apparatus according to any one of claims 1 to 7, wherein the protein crystallization microarray has 10 to 1000 crystallization agent holding portions. The protein crystallization apparatus according to any one of claims 1 to 8, wherein the plate further includes a crystal recovery mechanism for recovering crystals precipitated in the crystallization section.   The protein crystallization apparatus according to any one of claims 1 to 9, further comprising a detection mechanism for monitoring crystallization of the protein in the crystallization section. A sample filling aid for filling a protein-containing sample into the crystallization section of the protein crystallization apparatus according to any one of claims 1 to 10,
A sample filling aid comprising: an array of perforations corresponding to the crystallization compartments; and an alignment mechanism for causing the perforations to correspond to the crystallization compartments on the plate. The protein crystallization apparatus according to any one of claims 1 to 10, wherein a positioning portion for aligning the position with the sample filling aid according to claim 11 is formed on the plate. A protein crystallization condition screening method using the protein crystallization apparatus according to claim 12 and the sample filling aid according to claim 11,
A sample filling aid according to claim 11 is installed on the plate such that the perforations of the sample filling aid correspond to the crystallization section,
After the protein-containing sample is added to the perforation from above the sample filling aid and filled into the crystallization compartment, the sample filling aid is removed,
Laminating the plate and the protein crystallization microarray so that the crystallization section and the crystallization agent holding part are brought into contact with each other.
A protein crystallization condition screening method characterized by the above. A protein crystallization condition screening method using the protein crystallization apparatus according to claim 12 and the sample filling aid according to claim 11,
A protein-containing sample is added to each crystallization section of the protein crystallization apparatus according to claim 12,
A sample filling aid according to claim 11 is installed on the plate such that the perforations of the sample filling aid correspond to the crystallization section,
After a protein-containing sample is added to the perforation from above the sample filling aid and filled into the crystallization compartment,
Remove the sample filling aid,
Laminating the plate and the protein crystallization microarray so that the crystallization section and the crystallization agent holding part are brought into contact with each other.
A protein crystallization condition screening method characterized by the above.

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