JPH0733796A - Method and container for crystallizing protein - Google Patents

Abstract

(57) [Summary] [Object] With regard to protein crystallization, it is an object to provide an apparatus configuration capable of realizing a plurality of crystallization methods in one container. [Structure] A screw-in portion that is screwed into a second container at a tip portion, an injection needle embedded at a centerline position of the screw-in portion, a storage portion that holds a protein solution in a central portion, and a rear portion A first container having a syringe structure having a piston part for pushing out the solution, a recessed part screwed into the screwed part of the first container at the tip, and a crystallization agent solution at the center part via silicon rubber. The second container has a storage part for holding and a second container having a piston part for pressurizing the crystallization agent solution at the rear part, and a screw-in part having an injection needle at the tip part of the first container is provided for the second container. A protein crystallization container is characterized by being screwed into a recess at the tip.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a container structure and a crystallization method thereof which can perform any of the protein crystallization methods such as a static batch method, a liquid-liquid interface diffusion method and a vapor diffusion method.

Biopolymers such as proteins and nucleic acids have been attracting attention as protagonists of biotechnology. In particular, proteins such as enzymes and antibodies have advanced molecular discrimination ability, and thus applied research in the form of biosensors has been promoted. Although it has been commercialized, the protein that exists naturally and is used by living organisms is unstable to heat, and it is denatured or inactivated by an organic solvent. There are many problems in using it. And such denaturation is caused by the change in the three-dimensional structure of the protein molecule, and the activation is greatly influenced by the slight change in the three-dimensional structure.

Therefore, in applied research such as protein engineering and drug design, it is necessary to obtain a single crystal of a protein containing no impurities in a complete form. The static batch method, liquid-liquid interface diffusion method, vapor diffusion method , Various crystallization methods such as dialysis method and temperature gradient method have been developed.

[0004]

2. Description of the Related Art In order to crystallize a protein, it is necessary to supersaturate a protein solution and reduce its solubility. As a crystallization agent, an inorganic salt such as ammonium sulfate, potassium chloride or sodium chloride or an organic solvent such as polyethylene glycol is used. It is used.

Here, the crystallization of protein is very delicate, the wall of the container such as a flask is an obstacle to obtaining a perfect crystal, and the convection of the solution and the precipitation of the crystal also cause the crystal growth. Is an obstacle and it is difficult to obtain good quality crystals. Therefore, various crystallizers have been developed to crystallize in an ideal environment such as outer space that is not affected by gravity, but the crystallizers up to now correspond to specific methods. No single device could perform the various methods.

[0006]

Various methods are known as a method for crystallizing a protein, but a crystallizing device corresponds to each method and there is no general-purpose one. Therefore, even when crystallization is performed in an ideal environment such as outer space that is not affected by gravity, it is difficult to change the crystallization method because the device configuration is bulky. Therefore, it is very effective if one apparatus can perform a plurality of crystallization methods.

[0007]

[Means for Solving the Problems] The above-mentioned problems include a threaded portion at which the first container is screwed into the second container at the tip portion, and an injection needle embedded at the center line position of this threaded portion. It has a first container for holding a protein solution in the central part and a piston for pushing out the protein solution in the rear part, and has a syringe structure, and the second container has a second part at the tip. No. 1 container has a hole to be screwed into the screwing portion, a second storage portion for holding the crystallization agent solution through the silicone rubber in the central portion, and a screw for pressing the crystallization agent solution in the rear portion. Use of a crystallization container configured by having a threaded portion having a syringe needle at the tip of the first container and a hole at the tip of the second container. Can be solved by.

[0008]

The present invention constitutes a crystallization container with a first container having a syringe structure and a second container containing a crystallization agent solution and screwed into the first container. The equipment configuration is shown when the batch method, liquid-liquid interface diffusion method, and vapor diffusion method are used separately.

That is, as shown in FIG. 1 (A), the basic structure of the crystallization container according to the present invention is such that the first container 1 can be screwed into the second container 2 at the tip of a syringe-shaped container. There is a threaded portion 3 and an injection needle 4 is embedded through the center of the threaded portion. A protein solution is put into a first storage portion 5 in the center of the first container 1, and a tip of the injection needle 4 is inserted by a piston 6. More protein solution can be supplied. In addition, the second container 2 has a hole 7 into which the threaded portion 3 of the first container is screwed at the tip portion, and a second container in which the crystallization agent solution is put in via the silicone rubber 8 in the center portion. There is a portion 9 and it is held by a screw-in portion 11 provided with a stirring rod 10 at the tip.

When the static batch method is performed, the first method is used.
The threaded portion 3 of the container is screwed into the hole 7 of the second container 2 so that the injection needle 4 penetrates the silicone rubber 8, and then the piston 6 is pressed and the screwed portion of the second container 2 is pressed. 11 is loosened, the protein solution in the first container is put in the second container 9 of the second container, and mixed with the crystallization agent solution to crystallize.

Further, when the liquid-liquid interface diffusion method is performed, the apparatus configuration shown in FIG. 1B is employed. The difference from FIG. 1A is that the second container 2 does not have the stirring rod 10. When this method is employed, the piston 6 is extremely slowly so that the protein solution in the first storage part 5 of the first container 1 does not mix with the crystallization agent solution in the second storage part 9 of the second container 2. It is necessary to move.

When the vapor diffusion method is used, the structure shown in FIG. 1C is adopted, and the structure of the first container 1 is the same as in the stationary batch method and the liquid-liquid interface diffusion method.
Since the amount of the protein solution used is small and several tens of μl, it is convenient to use a small first container and a piston for holding the protein solution in the first container 1. However, when performing the vapor diffusion method, the structure of the screw-in portion 13 of the second container 2 is different, and a porous material (for example, gauze) 14 in which a crystallization agent solution has been impregnated is placed inside the solution diffusion prevention network. The crystallization agent solution is prevented from migrating at 15 while the protein solution droplets 16 are made by the injection needle 4 at the position in contact with the silicone rubber 8 in the second storage section 9, and the second storage section 9 is closed. By saturating with the vapor of the crystallization agent, protein crystals are produced on the surface of the droplet 16.

Instead of silicon rubber, rubber having excellent chemical resistance and elasticity such as neoprene or butyl rubber may be used, and borosilicate glass or the like may be used as the constituent material of the first container and the second container. However, synthetic resins having excellent chemical resistance such as polymethylpentene, acrylic, and polycarbonate may be used.

[0014]

【Example】

Example 1: (Static batch method, corresponding to FIG. 2) A 0.1 M acetate buffer (pH 4.2) containing 4% lysozyme was used as a protein solution, and 9% sodium chloride was used as a crystallization agent solution. Using a 0.1 M acetate buffer (pH 4.2), the first container 5 of the first container 1 was filled with the protein solution by sucking it with the piston 6, and the second container 2 of the second container 2 was filled. After opening the screw-in part 11 in the storage part 9 and injecting the crystallization agent solution, a separate needle provided in the silicon rubber 8 is penetrated and pushed into the screw-in part 11 to the required position, and extra crystallization is carried out. The drug solution was discharged through the injection needle and then filled by removing the injection needle. Next, when the screwing portion 3 of the first container 1 is brought into contact with the hole portion 7 of the second container and screwed,
The injection needle 4 enters the silicone rubber 8. This is the starting point. (Above FIG. 2A) Next, the threaded portion 3 of the first container 1 is replaced with the hole 7 of the second container 2.
When it is screwed to the full, the injection needle 4 becomes the second container of the second container 2.
Into the crystallization agent solution in the storage part 9 of the. Next, the piston 6 of the first container 1 is pushed in, and at the same time, the screw-in portion 11 of the second container 2 is loosened and the whole protein solution is transferred to the second storage portion 9. At this time, as a result of the stirring rod 10 moving with the rotation of the screw-in portion 11, both liquids are stirred and mixed.
Next, as a result of standing for 24 hours in this state, a tetragonal crystal of protein having a side length of about 0.5 mm could be obtained in the solution. Example 2: (Liquid-liquid interfacial diffusion method, corresponding to FIG. 3) As a protein solution, a 0.1 mol acetate buffer (pH 4.2) containing 4% of lysozyme was used as a protein solution, and as a crystallization agent solution. A 0.1 molar acetate buffer (pH 4.2) containing 9% sodium chloride was used.

The structure of the apparatus differs from that of the first embodiment in that the screw-in portion 11 of the second container 2 is not provided with a stirring rod, and the pinton 6 is pressed so that the first storing portion 5 This is to keep the interface between the two liquids by extremely gradually performing the operation of transferring the protein solution in the second storage section 9 into the second storage section 9 so that the two do not mix. (As shown in FIG. 6C) As a result of allowing the solution to stand still for 24 hours while holding the interface between the two solutions, it was possible to obtain a tetragonal crystal of the protein having a side length of about 0.5 mm in the solution. (The above figure D) Example 3: (Vapor diffusion method, corresponding to FIG. 4) As the protein solution, ammonium sulfate 32% saturated containing ribonuclease S 3.75%, cesium chloride 2.4 M, 0.1 mol acetate buffer (pH 6.1) was used. Also, ammonium sulfate 40% saturated, cesium chloride 3.0 M, acetate buffer (pH 6.1) was used as the crystallization agent solution, and gauze was used as the porous material 14, and the tip recessed portion of the screw-in portion 13 was filled. Then, the crystallization agent solution was poured, and in the swollen state, the solution diffusion preventing mesh 15 was covered and fixed. (Above FIG. 4A) Next, when the screw part 3 of the first container 1 is screwed into the second container 2, the needle tip 20 of the injection needle 4 passes through the silicone rubber 8 and the space of the second storage part 9 is passed. A little goes out. Next, the piston 6 is pushed to form the droplet 16 of the protein solution on the exposed portion of the silicone rubber 8. (The above C in the same figure) Next, as a result of pulling the injection needle 4 back into the silicone rubber 8 and letting it stand for 2 weeks in this state, one side of the droplet 16 is about 0.
A 3 mm hexagonal crystal could be obtained.

[0016]

By using the crystallization container according to the present invention, three types of crystallization, a static batch method, a liquid-liquid interfacial diffusion method and a vapor diffusion method, can be carried out, thereby simplifying the structure of the apparatus. You can

[Brief description of drawings]

FIG. 1A is an apparatus configuration diagram for a static batch method, FIG. 1B is a liquid-liquid interface diffusion method, and FIG.

FIG. 2 is a process diagram of a static batch method.

FIG. 3 is a process diagram of a liquid-liquid interface diffusion method.

FIG. 4 is a process diagram of a vapor diffusion method.

[Explanation of Codes] 1 First container 2 Second container 3 Screw part 4 Injection needle 5 First storage part 6 Piston 7 Hole part 8 Silicon rubber 9 Second storage part 10 Stir bar 11, 13 Screw in Part 14 Porous material 16 Droplet 18 Interface

Claims (2)

[Claims] 1. An injection needle 4 is provided at the tip, a first container 1 for holding a protein solution is provided inside, and a hole 7 for screwing a screwing portion 3 of the first container 1 is provided at the tip, The first container 1 is screwed into the second container 2 which holds the crystallization agent solution therein.
1. A method for crystallizing protein, which comprises transferring the protein solution to the second container 2 for crystallization by the piston 6 provided in the above. 2. A first container 1 is provided with a second container 2 at its tip.
Has a threaded portion 3 to be screwed into the threaded portion, an injection needle 4 embedded at the center line position of the threaded portion 3, a first storage portion 5 for holding a protein solution in the central portion, and a rear portion And a piston 6 that pushes out the solution, and has a syringe structure.
In addition, the second container 2 has a hole portion 7 at the tip which is screwed into the screwing portion 3 of the first container 1, and a second portion for holding the crystallization agent solution in the central portion via the silicone rubber 8. It has a storage part 9 and a screw-in part 11 for pressurizing the crystallization agent solution in the rear part, and is arranged at the tip part of the first container 1 at the injection needle 4
A crystallization container for protein, characterized in that the screwing portion 3 having the above is screwed into the hole portion 7 at the tip portion of the second container 2.