JP3390874B2 - Protein crystal growth method - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for growing large protein crystals, and more particularly to a method for growing large protein crystals by forming a concentration gradient of a crystal precipitant. is there.

[0002]

2. Description of the Related Art Genetic engineering is carried out in order to improve medicines composed of proteins such as interferon and starch saccharifying enzyme, but in order to carry out genetic engineering effectively, information on the three-dimensional structure of the protein is required. is necessary. To analyze the three-dimensional structure of a protein, the only method at present is to form a protein crystal and analyze it by X-ray diffraction. A large single crystal is required for analysis by X-ray diffraction. is there. Conventionally, a powdery crystal precipitant was placed at the bottom of a container containing a solution containing a protein, and the precipitant was dissolved and diffused to form a concentration gradient of the precipitant to grow crystals (Japanese Patent Publication No. 58- 29958).

[0003]

When a salt that generates heat when dissolved in water, such as magnesium chloride or calcium chloride, is used as a crystal precipitant, when the aqueous solution of protein and the powder of the salt come into contact with each other to dissolve the salt. There is a drawback that the liquid temperature rises and the protein is denatured and large crystals cannot be formed. Therefore, there has been a need for means for preventing heat generation and forming a concentration gradient of the crystal precipitating agent.

[0004]

According to the present invention, a solution containing a protein is gently layered on a solution containing a crystal precipitating agent, and the crystal precipitating agent is diffused from a high concentration region to a low concentration region, whereby the concentration of the crystal precipitant is increased. A method for growing a protein crystal, which comprises forming a gradient and growing the protein crystal in the optimum concentration region.

In the present invention, instead of placing the powder of the crystal precipitating agent at the bottom of the container, first, the solution containing the crystal precipitating agent is placed in the container, and the solution containing the protein is gently overlaid on the container to add the crystal precipitating agent to the container. Localize on one side. The crystal precipitant spreads to the protein-containing solution by diffusion and forms a concentration gradient. In this method, a crystal precipitating agent that generates heat when dissolved in an aqueous solution such as magnesium chloride is completely dissolved in advance in the solution, and then cooled to a temperature for crystal growth after contact with the solution containing protein after the heat generated by the dissolution has subsided. Therefore, denaturation of protein due to heat can be prevented.

[0006]

Function: The crystal precipitant is dissolved in a small amount of liquid before contact with the protein. At this time, some crystal precipitants generate heat. When it generates heat, it is cooled to a temperature suitable for crystal growth. Next, the solution containing the crystal precipitant is placed in a container, and the solution containing the protein is gently overlaid thereon. Even a crystal precipitating agent that generates heat when dissolved does not generate heat even when it comes into contact with a solution containing a protein because the dissolution process has already passed at this stage, so the protein is not denatured by heat. The crystallizing precipitant provides a concentration gradient by forming upwardly diluted concentrations by diffusion.

Generally, there is a relationship between the protein solubility S and the crystal precipitating agent concentration γ as shown in the following equation. logS = logSo-Kγ where So and K are constants. Since protein crystals grow greatly when the initial protein concentration slightly exceeds the solubility, if the protein concentration in the container is constant and a salt concentration gradient is formed, crystal growth will occur at a specific point in the container. The optimum crystal precipitant concentration appears. Therefore,
A crystal grows at this point.

[0008]

[Examples] 1.0 g of magnesium chloride was placed in a test tube having an inner diameter of 13 mm, 3.0 ml of distilled water was added thereto, and the mixture was thoroughly stirred to dissolve it, and cooled to room temperature (about 20 ° C). Separately, 0.5 g of egg white lysozyme, which is a protein for forming crystals, was dissolved in 7.0 ml of distilled water, and the pH was adjusted to 4.65 with hydrochloric acid. This egg white lysozyme solution was added to a test tube containing a magnesium chloride solution. At this time, the layers were gently overlaid so that the interface between the magnesium chloride solution and the egg white lysozyme solution was not disturbed.

Next, the test tube was capped with a silicone and allowed to stand in a thermostat kept at 23 ° C. Over time, magnesium chloride molecules diffused upward, and a concentration gradient was formed in which the concentration of magnesium chloride gradually decreased from the lower side to the upper side. The largest crystal grew at a position 48 mm from the bottom, and after 21 days, the size was 1.4 × 1.4 × 2.9 mm. The growth curve of this crystal is shown in FIG.

[0010]

EFFECT OF THE INVENTION Magnesium chloride generates heat when dissolved in water. In the conventional method, when the protein solution and the magnesium chloride powder were brought into contact with each other, heat was generated in the process of dissolution, so that the protein was denatured and the crystals did not grow. As in the present invention, if the crystal precipitating agent is dissolved in a liquid in advance, the protein will not be exposed to the heat of dissolution and the protein will not be denatured by heat. It became possible to apply the concentration gradient method. Therefore, it can be suitably used for various industries targeting proteins and enzymes.

[Brief description of drawings]

FIG. 1 is a diagram showing a crystal growth curve when egg white lysozyme is used as a protein and magnesium chloride is used as a crystal precipitant according to an example of the present invention.

─────────────────────────────────────────────────── --Continued from the front page (56) References JP-A-1-308897 (JP, A) JP-A 64-56400 (JP, A) JP-A 63-282186 (JP, A) JP-A 53- 109554 (JP, A) (58) Fields surveyed (Int.Cl. ⁷ , DB name) C07K 1/02 C30B 7/00 C30B 29/54 JISST file (JOIS)

Claims (2)

(57) [Claims] 1. A crystal precipitating agent is previously dissolved in water to generate heat.
After that, the concentration of the crystal precipitant cooled to the temperature for crystal growth
Gently overlaid with an aqueous solution containing the protein on top of the aqueous solution,
A method of growing a protein crystal in the optimum concentration region by forming a concentration gradient of the crystal precipitant by diffusing the crystal precipitant from a high concentration region to a low concentration region. 2. The crystal precipitating agent is magnesium chloride.
A method for growing a protein crystal according to claim 1.