Multi-well plate, well plate kit, and protein crystallization method

Abstract

To provide a multiwell plate that makes it possible to easily search for the conditions of crystallization of protein in a reduced state and effect its crystallization.SOLUTION: A multiwell plate 10 has a frame 12 having a plurality of wells 11 formed inside thereof. The multiwell plate has a communication part to permit steam diffusion between the pair of adjacent wells and electrodes at the bottom of one of the pair of adjacent wells.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a multi-well plate, a well plate kit, and a method for crystallizing a protein.

Background Art

[0002] In the research on the expression of protein functions, the analysis of the crystal structure of proteins has become increasingly important. For such analysis of the crystal structure of proteins, although it is essential to obtain high-quality protein crystals, for proteins lacking information on crystallization conditions, even the search for such conditions can be very complicated, and the automation of the search and the development of methods and devices that can test various conditions in a short time are being promoted.

[0003] Conventionally, a plurality of protein crystallization methods are known, and the vapor diffusion method is often used in that a large number of crystallization conditions can be tested from a small amount of protein. As an instrument used in the vapor diffusion method, a multi-well plate that is easy to operate and highly compatible with automation may be used.

[0004] As such a well plate, Patent Document 1 describes "a multi-well plate having a frame in which a plurality of wells are formed inside, each well having a first well having a relatively large volume and a second well having a relatively small volume and arranged so as to be located above at least a part of the first well."

[0005] In addition, Patent Document 2 describes "a kit for use in conducting crystallization experiments, comprising a pre-filled crystallization plate having a plurality of depressions each with an open upper end for receiving a crystallization solution, and an individual seal recessed downward from the upper end of each of the depressions by a certain distance for temporarily hermetically sealing the crystallization solution in the depression so that the pre-filled crystallization plate can be safely transported and handled before use, a first-level seal including the individual seals, and a second-level seal including a sealing surface above the first-level seal on the plate for sealing the upper part of the first-level seal in the depression so that vapor diffusion occurs after the individual seal is broken."

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] In the analysis of the crystal structure of proteins, it is considered important to adjust the environment inside the crystal to be not significantly different from the in-vivo environment and maintain the activity of the protein. Among proteins, there are those whose activity is rapidly lost by oxidation, and in order to obtain high-quality crystals of such proteins, it was essential to search for crystallization conditions in a reducing state.

[0008] Conventionally, in searching for crystallization conditions in a reducing state as described above, it was necessary to place the necessary instruments and devices in an anaerobic glove box and conduct experiments. The equipment was large-scale and the working efficiency was poor, and there was room for improvement in quickly testing a large number of conditions.

[0009] Therefore, an object of the present invention is to provide a multi-well plate that can easily search for crystallization conditions of a protein in a reduced state and perform the crystallization thereof. Another object of the present invention is to provide a well plate kit and a method for crystallizing a protein.

Means for Solving the Problems

[0010] As a result of intensive studies to achieve the above problems, the present inventors have found that the above problems can be achieved by the following configuration.

[0011] [1] A multi-well plate having a frame with a plurality of wells formed therein, the multi-well plate having a communication portion for enabling vapor diffusion between a pair of adjacent wells and an electrode at the bottom of one of the pair of adjacent wells. [2] The multi-well plate according to [1], wherein the cross-sectional area of the well having the electrode gradually increases along the depth direction from the bottom to the opening of the well. [3] The multi-well plate according to [1] or [2], which is for crystallizing a protein. [4] A well plate kit including the multi-well plate according to any one of [1] to [3] and a seal for sealing the well. [5] The well plate kit according to [4], wherein at least one selected from the group consisting of the seal and the frame has translucency. [6] A pair of wells of a multi-well plate having a frame with a plurality of wells formed therein, a communication portion for enabling vapor diffusion between a pair of adjacent wells, and an electrode at the bottom of one of the pair of adjacent wells, wherein a protein solution is accommodated in one of the pair of wells, a reagent solution is accommodated in the other, the well is sealed, the potential of the electrode is adjusted negatively, and crystals of the protein are formed by vapor diffusion between the pair of wells. A method for crystallizing a protein, comprising:

Effects of the Invention

[0012] According to the present invention, it is possible to provide a multi-well plate that can search for crystallization conditions of a protein in a reduced state and easily perform the crystallization thereof. Further, according to the present invention, a protein crystallization method can also be provided.

[0013] One of the characteristic points of the multi-well plate of the present invention is that it has an electrode at the bottom of one of a pair of wells capable of vapor diffusion. In the multi-well plate configured in this way, a protein solution is accommodated in the well having the electrode, a reagent solution (typically a high-concentration salt solution) is accommodated in the other well, and a negative potential is applied to the electrode, whereby a crystallization experiment by the vapor diffusion method can be carried out while the protein is in a reduced state.

[0014] Further, when the cross-sectional area of the well having the electrode gradually increases along the depth direction from the bottom to the opening of the well, it is preferable in that the accommodated protein solution easily gathers at the bottom and easily contacts the electrode. In the search for protein crystallization conditions and the experiment of crystallization, the amount of the protein solution that can be used is often limited. Even with such a small amount of protein solution, the above configuration can surely bring it into contact with the electrode, and the more excellent effects of the present invention can be obtained.

[0015] The well plate kit of the present invention includes the multi-well plate and a seal for sealing the wells of the well plate. According to the multi-well plate configured in this way, it is possible to search for crystallization conditions of a protein in a reduced state and perform crystallization, which have conventionally been carried out in an anaerobic glove box, in a simpler device and a more easily operable environment.

[0016] Further, when the seal and / or the frame has translucency, it is preferable in that the progress of crystallization can be observed.

[0017] The protein crystallization method of the present invention includes: a frame in which a plurality of wells are formed inside; a communication part for enabling vapor diffusion between a pair of adjacent wells; and an electrode at the bottom of one of the pair of adjacent wells. A protein solution is accommodated in one of the pair of wells of a multi-well plate having the above, a reagent solution is accommodated in the other, the well is sealed, the potential of the electrode is adjusted to be negative, and protein crystals are formed by vapor diffusion between the pair of wells.

[0018] According to the above method, it is possible to search for crystallization conditions and perform crystallization more easily while always applying a negative potential to the protein solution.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Figure 4

Embodiments for Carrying Out the Invention

[0020] Hereinafter, the present invention will be described in detail. The description of the constituent elements described below may be made based on representative embodiments of the present invention, but the present invention is not limited to such embodiments. In addition, in this specification, the numerical range represented by "~" means a range including the numerical values described before and after "~" as the lower limit value and the upper limit value.

[0021] FIG. 1 is a plan view of a multi-well plate according to an embodiment of the present invention, FIG. 2 is an enlarged view of the portion A-A′, FIG. 3 is a bottom view of the portion A-A′, and FIG. 4 is a cross-sectional view taken along the line B-B′.

[0022] The multi-well plate 10 has an array of wells 11 for accommodating samples. The multi-well plates 10 are typically arranged in a matrix of rows and columns perpendicular to each other within a frame 12. The wells 11 of the multi-well plate 10 are arranged in an 8×12 matrix of 96 wells, but the arrangement of the wells is not limited to the above. In addition to the above, for example, arrangements such as 4×6 (24 wells), 16×24 (384 wells), and 32×48 (1536 wells) can be mentioned.

[0023] Each well 11 of the multi-well plate 10 is such that two adjacent wells 11 form a pair, with the left side being the protein well 13 and the right side being the reservoir well 14 in a plan view. In the multi-well plate 10, adjacent left and right wells 11 form a pair in a plan view, but the multi-well plate of the present invention is not limited to the above, and adjacent upper and lower wells 11 may form a pair in a plan view.

[0024] The protein well 13 has a tapered side wall whose cross-sectional area gradually increases along the thickness direction from the bottom to the opening, and an electrode set 18 is disposed at the bottom. The protein well 13 has a portion where the cross-sectional area gradually increases and a portion where the cross-sectional area is substantially the same along the thickness direction from the bottom to the opening. However, the form of the side wall of the protein well 13 of the multi-well plate 10 of the present invention is not limited to the above, and the entire side wall may have a taper, that is, a form in which the thickness increases throughout the thickness direction from the bottom to the opening (for example, a mortar-like form).

[0025] According to the protein well 13 in which the cross-sectional area is sequentially reduced from the opening toward the bottom in this way, even when the amount of the protein solution 43 is small, the pipetted protein solution 43 is likely to come into contact with the electrode set 18.

[0026] The reservoir well 14 is partitioned by a vertical side wall and a bottom, but the shape of the reservoir well 14 is not limited to the above.

[0027] Note that the bottom of the protein well 13 and the bottom of the reservoir well 14 are formed at substantially the same height in the thickness direction of the multi-well plate 10 in sectional view, but are not limited to the above, and the bottom of the protein well 13 may be disposed at a higher (above in sectional view) position than the bottom of the reservoir well 14.

[0028] The electrode set 18 is composed of three electrodes 15 to 17, and includes a working electrode 15 for applying a potential to the specimen, a counter electrode 16, and a reference electrode 17, respectively. Each of these electrodes is connected to connectors 33 to 35 on the back surface of the multi-well plate 10 through through holes (not shown) provided along the thickness direction of the multi-well plate 10. Note that the region 30 in FIG. 3 corresponds to the position of the protein well 13, and the region 31 corresponds to the position of the reservoir well 14. Each connector is typically individually connected to a potentiostat, and controls the potential of the working electrode 15 according to conditions determined for each well 11.

[0029] Note that the electrode set 18 included in the multi-well plate 10 has three electrodes 15 to 17, but the electrode set 18 included in the multi-well plate 10 of the present invention is not limited to the above, and may be formed of two or more electrodes, or may be formed of four or more electrodes. Generally, the number of electrodes is preferably 10 or less.

[0030] The protein well 13 and the reservoir well 14 are provided midway in the depth direction of the well 11, and a communication part 19 is provided to enable vapor diffusion between the protein well 13 and the reservoir well 14. The shape and position of the communication part 19 are not particularly limited, as long as the protein solution 43 and the reagent solution 44 do not mix with each other and the above-described interaction is possible.

[0031] Next, a method of using the multi-well plate 10 will be described. First, the reagent solution 44 is accommodated in the reservoir well 14. Typically, the reagent solution 44 is a high-concentration salt solution. Next, the protein solution 43 is accommodated in the 13 protein well. Next, the upper part of the multi-well plate 10 is sealed.

[0032] Next, the potential of the working electrode 15 is controlled, and a negative potential is applied to the protein solution 43. The applied potential is not particularly limited as long as it is less than 0V. For example, it is preferably a potential that exceeds the lower limit value of the potential window and is less than 0V with reference to a silver / silver chloride electrode. By controlling the potential of the working electrode 15 to a potential that exceeds the lower limit value of the potential window, the generation of hydrogen by the cathodic reaction is suppressed, the protein is easily maintained in a reduced state, and a more excellent effect of the present invention can be obtained.

[0033] In this way, through the vapor diffusion process, the protein solution 43 interacts with the reagent solution 44 to obtain protein crystals. Also in this case, by maintaining the potential of the working electrode 15 negative, crystallization of the protein is performed in a reduced state, and the crystallized protein can also be maintained in a reduced state.

Industrial Applicability

[0034] According to the multi-well plate of the present invention, it is possible to search for crystallization conditions of a protein in a reduced state without using a glove box. According to the multi-well plate of the present invention, since many crystallization conditions can be tested simultaneously in parallel, it is an excellent experimental instrument particularly for studying the functions of proteins derived from living organisms.

Explanation of Symbols

[0035] 10: Multi-well plate 11: Well 12: Frame 13: Protein well 14: Reservoir well 15: Working electrode 16: Counter electrode 17: Reference electrode 18: Electrode set 19: Communication part 33, 34, 35: Connector 43: Protein solution 44: Reagent solution

Claims

1. A multi-well plate for protein crystallization having a frame with a plurality of wells formed therein, a communication part for enabling vapor diffusion between a pair of adjacent wells, an electrode provided at the bottom of one of the pair of adjacent wells, a through-hole formed in the bottom, a connector connected to the electrode through the through-hole, and a back surface provided with the connector. A multi-well plate.

2. The electrode is provided at the center of the bottom in a plan view, and the height of the center is lower than the peripheral edge of the bottom in a cross-sectional view. The multi-well plate according to Claim 1.

3. The multi-well plate according to Claim 1 or 2, wherein the electrode is a working electrode.

4. A well plate kit comprising the multi-well plate according to any one of Claims 1 to 3 and a seal for sealing the well.

5. The well plate kit according to Claim 4, wherein at least one selected from the group consisting of the seal and the frame has translucency.

6. Among the pair of wells of the multi-well plate having a frame with a plurality of wells formed therein, a communication part for enabling vapor diffusion between a pair of adjacent wells, and an electrode provided at the bottom of one of the pair of adjacent wells, a protein solution is accommodated in the well provided with the electrode, a reagent solution is accommodated in the other, and the well is sealed. Applying a negative potential to a connector connected to the electrode through a through-hole formed in the bottom to bring the protein solution into a reduced state. A method for protein crystallization, comprising forming crystals of the protein by vapor diffusion between the pair of wells.

7. The electrode is provided at the center of the bottom in a plan view, and the height of the center is lower than the peripheral edge of the bottom in a cross-sectional view. The method for protein crystallization according to Claim 6.

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