JP2721323B2 - Immobilization reagent for screening protein crystallization conditions - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an immobilization reagent for screening and determining conditions for crystallizing a desired protein from a protein-containing sample.

[0002]

2. Description of the Related Art Obtaining pure protein crystals of proteins having various biological functions is strongly required in the fields of biochemistry and protein engineering for structural analysis. I have. That is, while useful proteins such as enzymes, hormones, and antibodies are mass-produced by genetic recombination, and new proteins are created by artificially modifying natural proteins, elucidation of protein structures has become a particular issue. It is indispensable for exploring the relationship between the function and structure expressed by proteins and the mechanism of action. Further, based on the information, it becomes possible to design a protein having a further purpose function, and it is expected to greatly contribute to the development of the chemical industry, the pharmaceutical industry, the food and the material industry.

[0003] Many protein crystallization methods have hitherto been reported, and typical examples include the following methods 1) to 4). 1) A batch method in which a precipitant is directly added to a protein solution to an appropriate concentration and crystallized. 2) A dialysis method in which a dialysis membrane or a dialysis tube is used, and precipitation is performed by equilibrating the salt concentration (precipitation reagent) of the inner and outer liquids. 3) A liquid phase interface diffusion method in which two phases are formed by a difference in specific gravity between a protein sample solution and a salt solution, and diffusion is used at the interface. 4) Add the protein solution onto the spot plate and enclose it in a transparent box together with the precipitant in another container and precipitate by equilibration of both solutions (diffusion vapor method), or add the protein sample solution onto a cover glass. The cover glass is turned upside down, the droplets hang down from the glass plate, the container containing the precipitant solution is sealed with silicon grease or the like, and the solution is precipitated by equilibration of both solutions (hanging drop method).

[0004] Existing methods, including these methods, require very complicated operations and are inefficient. For example,
In 1), for example, ammonium sulfate is added to a protein in a test tube, but the allowable amount of addition is small, and a large amount of a high-concentration sample is required. Further, in the case of colored high-concentration proteins, it is difficult to determine the final point, the reproducibility is poor such as the precipitation of the amorphous substance, and it takes a long time to set the crystallization conditions. Operations 2) to 4) are complicated and require skill. In addition, it is necessary to know in advance the concentration of the precipitation reagent that produces a crystal. Therefore, there is a strong demand for the development of means for efficiently crystallizing proteins by effectively using samples. It is considered that the above-mentioned problems can be solved by screening a crystallization method suitable for the target protein in the sample using a small amount of the sample, and then crystallizing the protein by the established method. However, the conventional method requires complicated operations and is not suitable for screening, and cannot be a means for solving the problem.

[0005]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above-mentioned problems, and comprises a plurality of types of protein precipitants immobilized on a solid support in a distinguishable manner for screening protein crystallization conditions. A chemical reagent.

[0006] The solid support used in the present invention is not particularly limited as long as it has a light-transmitting property and can be fixed by applying a precipitant.
Synthetic resins such as quartz, polypropylene, and acrylic can be used. The protein precipitating agent to be immobilized is also applied to the selected support, dried, and then brought into contact with a protein-containing reagent, provided that the precipitated protein can be detected by an appropriate method, provided that it can be used in the art. It may be any precipitant used. As such a precipitant, polyethylene glycol (PEG) having a molecular weight of about 200 to 4,000,000, ammonium sulfate, a monovalent salt,
Examples thereof include divalent salts and organic solvents. In the present invention, it is preferable to use a plurality of PEGs having different molecular weights.

[0007] As used herein, the phrase "immobilize identifiably" means that the precipitants are immobilized so that they can be distinguished from each other. When different precipitants are used, their positions need only be identifiable. When PEG is used alone, it is preferable to apply PEGs having different molecular weights in multiple stages. These PEGs can be applied sequentially. “Immobilized in multiple stages” means immobilizing stepwise according to the molecular weight.
The immobilized sample of the present invention comprises a precipitant immobilized on a support,
It functions by contacting it with a minimum amount of a protein-containing sample solution and keeping it in a sealed state for a period sufficient for protein precipitation. Thus, the form of the immobilized sample is arbitrary, provided that it is itself sealable or can be held in a sealable container. Examples of the former include sticks, plates, small bottles, and the like, and examples of the latter include a support having a concave portion or a hollow portion.

However, a solid support in which a precipitant is immobilized on the inner wall of a concave portion or a hollow portion is more convenient for practical use, and an example of a support having a hollow portion has an inner diameter of 0.1. ５5 mm thin tube, preferably with an inner diameter of 0.
A capillary having an inner diameter of 3 to 2 mm, more preferably about 0.5 mm can be used. On the other hand, as an example of a support having a concave portion, a plate having a groove having a width and a depth corresponding to the inner diameter of the thin tube can be given. The size of such grooves is usually between 0.1 and 5 mm, preferably between 0.3 and 2 mm.
mm, more preferably about 0.5 mm and a depth of 0.1 to 5 mm, preferably 0.3 to 2 mm, more preferably about 0.5 mm. Another example is a plate having a plurality of wells. The size of the well is usually 0.1 to 20 mm in diameter, preferably 0.3 to 10 mm, more preferably about 3 mm, and 0.1 to 10 mm in depth, preferably 0.3 to 2 mm.
mm, more preferably about 1 mm.

[0009] The volume of the capillaries and grooves and the number and volume of the wells vary depending on the number and volume of the precipitant to be applied, but usually, an appropriate precipitant solution is 1 to 50 µl, preferably 1 to 10 µl, respectively. More preferably, about 2 μl is the volume that can be applied. A simple method of immobilizing the precipitant on these solid supports consists of applying the recognizable precipitant to the surface of the support at appropriate intervals and drying. In the case of using PEG alone, it is convenient to apply it in a band form in a multi-stage and continuous manner according to the molecular weight. When applying to the hollow portion or the concave portion of the support, the volume required for the hollow portion or the concave portion is determined as needed according to the number and amount of the precipitant used. However, in the case of a support having a hollow portion, the volume is in the range of the above inner diameter and the volume is 1 to 250 μl.
Capillary, preferably 5 to 50 μl, more preferably 5 to 50 μl
20 μl capillaries are suitable. In the case of a plate having a narrow groove, the size of the groove is set so as to be a volume corresponding to the volume of the above-mentioned thin tube. In the case of wells, the inner volume is about 1 to 250 μl, preferably about 10 to 50 μl, and more preferably about 30 to 50 μl. Therefore, the length of the narrow tube and the narrow groove is usually in the range of 1 to 15 cm.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, preferred embodiments of the present invention will be described in detail using PEG which is a well-known organic precipitant, but these do not limit the present invention. That is, the immobilization reagent of the present invention provides a means for efficiently screening protein crystallization conditions with a small number of samples by coating a protein precipitant on a solid support in a distinguishable manner. Is not limited to PEG. Similarly, the present invention is not limited to a particular solid support, but to improve the crystallization and to facilitate the observation of the crystals under a microscope, including the size of the capillaries and grooves, The shape of the support can be modified, and supports having different permeability can be arbitrarily selected.

PEG is a product of average molecular weight fraction from oily PEG 200 (molecular weight 200) to waxy PEG 4
000K (molecular weight 4,000,000) is available, but the immobilization reagent of the present invention includes PEG 1,000-70,000.
000, preferably PEG1000, PEG4000,
PEG6000, PEG8000, PEG10000,
Polyethylene glycol such as PEG 20000 can be used.

Generally, PEG used as a precipitant is several percent.
Crystallization of the protein is possible at concentrations of up to several tens of percent, and the actual crystallization is carried out under a low ionic strength, for example, 25 mM potassium phosphate buffer. PEG is soluble in organic solvents such as chloroform and acetone, and in the case of low molecular weight PEG, it is also soluble in water. In order to form a PEG layer of a segment of a certain length in multiple steps on the surface of the support, the solubility or P
In consideration of the volatilization and viscosity of the EG solution, it is necessary to select an appropriate solvent in order to prevent outflow to other segments.
Such solvents include water, chloroform, acetone,
Toluene is mentioned, but chloroform is preferred. The concentration of the precipitant solution is 5 to 20%, preferably 7 to 15%.
%, More preferably 10%. A 10% chloroform solution is most preferred. In addition, each PEG to each segment
Is adjusted to have a constant weight. The immobilized reagent of the present invention can be uniformly applied to the surface of the support (including the inner wall of a thin tube, a narrow groove, a well, etc.) in a fixed length segment, for example, in ascending order of molecular weight. can get.

[0013] The method of immobilization on the support is not particularly limited, and application by a brush or a spray method is possible. In the case of an inner wall of a thin tube or the like, a syringe or the like is used.
About 2 μl of a 10% chloroform solution of PEG is applied to each 0.5 cm to 3 cm, preferably about 1 cm long segment, and dried with nitrogen gas or the like. This operation is repeated based on the magnitude of the molecular weight, for example, sequentially from a PEG solution having a smaller molecular weight. In the case of immobilization on a flat surface of a plate or a groove provided in the plate, a solution other than the segment to be coated with a specific PEG is covered with a waterproof or organic solvent-resistant seal or the like, and then a solution of the PEG is applied. May be sequentially applied and dried. Furthermore, when applying to a well, the target PEG solution is applied to each well in order.
In the production of the present invention, as long as PEG is identifiably attached to the solid support, the method of application is optional,
Usually, it is preferable to apply to each segment continuously in a strip shape.

In order to screen the crystallization conditions of the protein in the sample, a part of the sample is brought into contact with the immobilization reagent of the present invention, and the required crystals are precipitated by equilibration with a precipitant. For example, it may be observed with a microscope. To bring the sample into contact with the immobilized reagent, the sample solution can be dropped or immersed in the sample solution.In the case of a thin tube or narrow groove, however, the sample can be filled manually or mechanically with a syringe or the like. It is developed using capillary action.

Next, the contact surface between the sample solution and the immobilized reagent is sealed by an appropriate method. For example, sealing with cover glass, paraffin, wax, vacuum compound, etc.,
Place in a closed container and allow to stand at a temperature of 4-45 ° C., usually at room temperature, for a period sufficient for crystallization. The crystals of each segment are observed with, for example, a polarizing microscope, and the segments where crystals suitable for structural analysis such as X-ray analysis are detected are examined. If the crystal is insufficient for X-ray analysis, further use the conditions in the segment to extract protein crystals from the original sample, using the hanging drop method, drop method (dialysis method, button method), sitting drop method, batch method, etc. By a conventional method.

Since the immobilizing reagent of the present invention has a plurality of types of protein precipitants immobilized thereon in a distinguishable manner, crystallization conditions suitable for the protein in the sample can be determined in one step. Since the solid support is a light-transmissive glass, quartz or synthetic resin thin tube, crystals can be easily detected by a polarizing microscope or the like. In the case of an immobilizing reagent in which a precipitant is immobilized on a thin tube or a narrow groove, a small amount of a sample can be used to screen for a crystallization condition of a very small amount of protein. In the case of an immobilization reagent in which PEGs having different molecular weights are successively applied in a multi-step manner, it is considered that the protein is less likely to be denatured than other precipitants and is less likely to affect the protein side. Very suitable for increasing the effective concentration of

[0017]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. Example 1 PEG1000, PEG2000, PEG4000, P
10% chloroform solutions of EG10000 and PEG20000 were respectively prepared. The support has an inner diameter of 0.9mm and a length of 1.
A 0 cm capillary glass tube was used. Using a syringe, apply 2.5 μl each to a segment of about 1.75 cm from polyethylene glycol of low molecular weight, that is, PEG 1000, from one end.
After drying for 2 hours, an immobilized reagent was obtained. FIG. 1 shows an immobilization reagent manufactured in the present embodiment.
G1000, PEG2000, PEG4000, PEG
10000 and PEG20000 are immobilized in a strip shape in order.

Example 2 PEG1000, PEG4000, PEG6000, P
A 10% chloroform solution of EG20000 was prepared, and a glass plate having a narrow groove of 0.7 to 0.8 mm in width and 4 cm in length was prepared.
10 μl of each of the approximately 1 cm segments was applied to each of the narrow grooves with a syringe in ascending order of molecular weight, and air-dried for about 2 hours to obtain an immobilized reagent. PEGs having different molecular weights are similarly immobilized on other narrow grooves. FIG. 2 shows a glass plate 1
2 shows an immobilization reagent in a state where PEG is immobilized on one narrow groove.

Using the immobilization reagent prepared in the examples, the crystallization conditions of the protein in the sample were screened. Experimental Example 1 Specific recognition of DNA / RNA hybrid and its RN
The crystallization conditions of RNase H1, an enzyme that cleaves and decomposes only the A chain, were examined. RNase H1 (3.0 mg / ml, pH
A portion of the protein solution of 8.2) was filled into the immobilization reagent obtained in Example 1 using a 20 μl syringe, and both ends were sealed with wax, and allowed to stand for 2 days. As a result of observation under a polarizing microscope, crystals were observed in each of the different PEG segments, but columnar crystals of RNase H1 most suitable for X-ray structural analysis were observed in the segments coated with PEG10000 (FIGS. 3 and 4). And 5). FIGS. 3, 4 and 5 are micrographs showing the state of crystallization in the segments of the capillary to which PEG of each molecular weight has been applied. The numbers at the bottom of the figure represent the molecular weight of PEG. Arrows indicate segments where the best crystals are deposited. Then, based on the above results, using PEG10000 as a precipitant,
0.5 × 0.5 × by the hanging drop method from the sample solution
RNase H1 crystals of about 0.6 mm in size were obtained.

Experimental Example 2 In the same manner as in Experimental Example 1, the immobilized reagent obtained in Example 2 was used, and fatty acid β-oxidases such as enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and acetyl-CoA were used. Acyltransferase (HD
T) An attempt was made to crystallize the complex enzyme. HDT (2.5 mg / ml,
50 μl of a protein solution (pH 5.5) was filled in the immobilizing reagent, and both ends were sealed with paraffin, and allowed to stand for 5 days for crystallization. Observation under a polarizing microscope showed that PEG200
In the segment coated with No. 00, suitable HDT crystals (0.2 × 0.2 × 0.3 mm) were observed. (FIG. 6
And 7). FIGS. 6 and 7 are photomicrographs showing the state of crystallization in the segments to which PEG of each molecular weight is applied in the narrow grooves of the glass plate. The numbers below the figure are PEG
Represents the molecular weight of Arrows indicate segments where the best crystals are deposited. Then, based on the above results, P
Using EG20000 as a precipitant, HDT crystals having a size of about 0.8 × 0.8 × 0.9 mm were obtained from the sample solution by a button method (dialysis method).

[0021]

According to the present invention, crystallization conditions suitable for proteins in a sample can be efficiently determined in one step.

[Brief description of the drawings]

FIG. 1 PEG1000, PEG on the inner wall of a glass tube 1
2000, PEG4000, PEG10000 and PE
FIG. 9 is a perspective view showing the inside of an immobilized reagent, in which G20000 is sequentially immobilized in a band shape, as seen through.

FIG. 2 shows PEG1000 in one narrow groove of the glass plate 1.
PEG 4000, PEG 6000 and PEG 20,000
FIG. 3 is a perspective view of an immobilized reagent obtained by immobilizing the reagent.

FIG. 3 shows RNs in the PEG-coated segments of molecular weights 1000 and 2000 in the capillary of FIG.
It is a microscope picture which shows the state of ase H1 crystallization. The numbers at the bottom of the figure represent the molecular weight of PEG.

FIG. 4 shows the R in the segment of the capillary of FIG. 1 coated with PEG having a molecular weight of 4000 and 10,000.
It is a microscope picture which shows the state of Nase H1 crystallization. The numbers at the bottom of the figure indicate the molecular weight of PEG, and the arrows indicate the segments where the best crystals precipitated.

FIG. 5 shows a molecular weight of 10,000 in the capillary of FIG.
It is a microscope picture which shows the state of RNase H1 crystallization in the segment which applied 20,000 and 20,000 PEG. The numbers at the bottom of the figure represent the molecular weight of PEG.

FIG. 6 shows a molecular weight of 1000 in a narrow groove of the glass plate of FIG.
7 is a micrograph showing a state of crystallization in a segment coated with PEG of No. 4000 and 4000. FIG. The numbers at the bottom of the figure represent the molecular weight of PEG.

FIG. 7 shows a molecular weight of 6000 in a narrow groove of the glass plate of FIG.
7 is a photomicrograph showing the state of crystallization in segments to which PEG of 10000 and 10000 were applied. The numbers at the bottom of the figure represent the molecular weight of PEG, and the arrows represent the segments where the best crystals precipitated.

Claims (11)

(57) [Claims] 1. An immobilizing reagent for screening protein crystallization conditions comprising a plurality of types of protein precipitants immobilized on a solid support in a distinguishable manner. 2. The immobilized reagent according to claim 1, wherein the solid support is a light-transmitting glass, quartz or synthetic resin. 3. The immobilized reagent according to claim 1, wherein the precipitant is immobilized on the inner wall of the concave portion or the hollow portion provided on the solid support. 4. The immobilized reagent according to claim 3, wherein the solid support is a tubular body, and a protein precipitant is immobilized on an inner wall thereof. 5. The immobilizing reagent according to claim 3, wherein the solid support is a plate in which one or more grooves are engraved, and a protein precipitant is immobilized on the inner wall of the grooves. 6. The immobilized reagent according to claim 3, wherein the solid support is a plate provided with two or more wells. 7. The immobilized reagent according to claim 1, wherein the protein precipitant is a plurality of polyethylene glycols having different molecular weights. 8. The polyethylene glycol having a molecular weight of 1,
The immobilized reagent according to claim 7, wherein the amount is in the range of 000 to 70,000. 9. The immobilizing reagent according to claim 7, wherein polyethylene glycols having different molecular weights are immobilized on the inner wall of a light-transmitting thin tube having an inner diameter of 0.1 to 5 mm in multiple stages. 10. The light transmissive plate has a width of 0.1 to 5 m.
9. Polyethylene glycols having different molecular weights are fixed in multiple steps in narrow grooves having a depth of 0.1 to 5 mm.
3. The immobilization reagent according to 1. 11. A method comprising contacting the immobilized reagent according to claim 1 with a sample containing a protein,
A protein crystallization condition screening method comprising detecting precipitated crystals after standing at 5 ° C.