JPH0244089A - Method for growing biopolymer crystal - Google Patents

Abstract

PURPOSE:To prevent changes in the composition of solution and the generation of bubbles and to stably carry out the crystallization reaction of biopolymers by carrying out crystal growth after previously forming a plastic vessel into water-saturated state. CONSTITUTION:A vessel for crystal growth made of plastic is previously formed into water-saturated state by means, e.g., of immersion into water. Biopolymer crystal growth is carried out in the above state. By this method, the vessel for crystal formation can be prevented from absorbing sample solution at the time of growing crystals over a long period and, as a result, changes in the composition of the solution and the denaturation if the biopolymers are prevented and the conditions of crystal growth can be prevented from being disturbed. Accordingly, a single crystal of the biopolymer free from impurities can be produced.

Description

[Detailed Description of the Invention] [Summary] Regarding a biopolymer crystal growth method, crystal growth is performed for the purpose of preventing a plastic container in which crystal growth is performed from absorbing water and disturbing crystal growth conditions. The plastic container is configured to be saturated with water before use.

[Industrial application field]

The present invention relates to a method for growing biopolymer crystals.

In applied research such as protein engineering and drug design, elucidation of the higher-order structure of proteins by, for example, X-ray analysis is essential. For this purpose, it is important to produce single crystals of biopolymers that do not contain impurities.

[Conventional technology]

Conventional methods for producing single crystals include ■static batch method, ■free interface diffusion method, ■dialysis method, ■dialysis diffusion method, ■vapor diffusion method,
■There are concentration methods etc. (Biochemistry Experiment Course Volume 1 ■6-17
Page, 1976).

When crystallizing by this method, the basic idea is to add ammonium sulfate, magnesium sulfate, sodium sulfate,
By adding an aqueous solution of sodium phosphate, sodium chloride, cesium chloride, methylbentanediol, ethanol, methanol-Jk, acetone, polyethylene glycol, etc., and allowing the polymer to precipitate and stand, crystal nucleation can be achieved using a solution growth method. It causes crystal growth.

However, since the setting of crystallization conditions is delicate, crystallization is often performed under multiple conditions in parallel. Normally, this work is done manually, but the applicant has already considered and disclosed the following automated apparatus and a crystal growth container suitable for the apparatus (Japanese Patent Application No. 213,729/1982). This method consists of two containers with openings facing each other (each holding a protein solution and a precipitant solution), a retractable or movable sliding plate separating these two containers, and an O-ring sealing the openings. A mechanism that enables experiments to be started by arranging the containers in a circular radial pattern and pulling out the slide plate all at once, and a rotatable mirror placed in the center that allows the camera above to record the crystal growth process inside each container. A crystal manufacturing apparatus equipped with a mechanism can automatically start crystal growth and record the growth process.

[Problem to be solved by the invention]

The above-mentioned crystal growth container must not have an adverse effect on the biopolymer to be crystallized, and must not be corroded by the precipitant inside the container. From this point of view, acrylic, polycarbonate, polymethylpentene, glass, etc. can be used as the container material.

Among these, glass containers have many advantages such as being unaffected by organic solvents, but they have the disadvantage of being extremely expensive to manufacture.

On the other hand, transparent plastics such as acrylic are much easier to process than glass, and have the advantage that containers can be manufactured at low cost. However, plastics generally have water absorption or water permeability, and as the crystallization reaction progresses over several months, they absorb water from the sample solution inside, so biopolymer crystals generally have a small sample solution capacity. Sample solutions for growth have the disadvantage that the liquid composition changes significantly or that the biopolymer is denatured at the air-liquid interface due to the inclusion of air bubbles, which hinders the crystal growth reaction.

Therefore, an object of the present invention is to solve such problems.

[Means to solve the problem]

As a result of intensive study on this phenomenon, we found that in the case of thick-walled containers like the container mentioned above, the biggest cause of the problem was water absorption by the material itself rather than water permeability. It has been found that if the crystal growth is performed after the crystal is saturated with water, the above-mentioned obstacles to the crystallization reaction can be prevented.

Thus, according to the present invention, in a method for growing biopolymer crystals using a plastic container, the biopolymer crystal growth is performed after the plastic container is saturated with water in advance. A method for growing molecular crystals is provided.

The material of the plastic container used is not particularly limited, but transparent resins such as acrylic resin, polycarbonate resin, and polytylpentene resin are preferably used.

To bring a plastic container to a state of water saturation, typically, the plastic container is immersed in water (it can be determined that the state of water saturation has been reached by the increase in weight of the plastic container reaching saturation). Carbohydrate-saturated containers were observed to retain water absorption for several months.

In the present invention, biopolymer crystal growth itself can be performed as conventionally known or as previously illustrated by the applicant.

[For production]

Since the plastic container is saturated with water in advance, the container is prevented from absorbing the sample solution during crystal growth.
As a result, changes in liquid composition, generation of bubbles, etc. are prevented.

〔Example〕

FIG. 1 shows the weight change when an acrylic resin plate (thickness: 3 mm or 6 mm) is immersed in water and when it is left in an environment with a relative humidity of 30 to 85%. According to the figure, it can be seen that when immersed in water, the weight gradually increases over 100 to 150 days and reaches a steady state.

Therefore, a biopolymer crystal growth apparatus (Japanese Patent Application No. 62-213729) as shown in FIG. 2 was fabricated using acrylic resin. In the figure, 1 and 2 are containers made of transparent acrylic resin blocks, 3 is a spacer, 4.5 is an O-ring, 6 is a slide plate, 7 is a space for containing a biopolymer solution, 8 is a space for containing a precipitant, 9 and 10 are holes for filling with a solution.

After thoroughly cleaning the device in its disassembled state, it was placed in a vat filled with deionized water so that it was completely immersed in water. The vat was sealed and placed in a constant temperature bath at 20°C for 150 days. At this time, if the temperature was set at 50° C. to promote equilibration, the objective could be achieved with immersion treatment for 30 days.

Note that processing at a higher temperature is inappropriate because it causes deformation of the acrylic resin.

After the above treatment was completed, the container was thoroughly washed and assembled as shown in Figure 2.
One side was filled with an aqueous solution (containing a phosphate buffer solution of mole/I) and the other was filled with a 95% saturated ammonium sulfate aqueous solution, the slide plate was pulled out, and the generation of bubbles was observed for 100 days in a constant temperature bath at 20°C. The diameter and number of bubbles in the container were measured, and the total volume of bubbles was calculated.

The results are shown in Figure 3. It can be clearly seen that the water immersion treatment significantly suppresses the generation of bubbles.

〔Effect of the invention〕

According to the present invention, when growing a biopolymer crystal over a long period of time, it is possible to prevent the crystal growth container from absorbing water, thereby preventing changes in the liquid composition and denaturation of the biopolymer, thereby preventing the crystallization reaction. It can be executed stably.

[Brief explanation of the drawing]

Figure 1 is a graph showing the water absorption characteristics of acrylic resin, Figure 2 is a graph showing the water absorption characteristics of acrylic resin.
Figures (A) and (B) are respectively a perspective view and a sectional view of the biopolymer crystal growth apparatus, and FIG. 3 is a diagram showing the results of the example. 1.2... Container made of transparent plastic block, 3... Spacer, 6... Slide plate, 7... Space for biopolymer solution, Death... Space for precipitant, 9.10. ...Liquid filling hole. (A) (B) Main polymer crystal growth apparatus Figure 2

Claims (1)

[Claims] A method for growing biopolymer crystals using a container made of plastic, the method comprising growing the biopolymer crystals after bringing the plastic container into a water-saturated state in advance.