JPH01111799A - Crystal preparation method - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION [Summary of the Invention] According to the present invention, when producing a single crystal by solution growth, conventionally, a concentration gradient solution was divided manually and crystallized. Therefore, there were problems such as poor reproducibility and a large number of man-hours. The present invention provides a method for producing a single crystal that can be simplified and improved in reproducibility by simply and automatically dividing a concentration gradient liquid using means such as a predetermined crimping jig.

[Industrial application field]

The present invention relates to a method for producing a single crystal, and more particularly to a method for producing a single crystal in which concentration gradient liquid can be automatically distributed when producing a single crystal by solution growth.

[Prior art and problems to be solved by the invention] In applied research such as protein engineering and drug design, it is essential to determine the higher-order structure of proteins by, for example, X-ray analysis. To this end, the problem is the production of single crystals of biopolymers that do not contain impurities.

Conventional methods for producing single crystals include ■Static batch method ■Free interface diffusion method ■Dialysis method ■Dialysis diffusion method ■Vapor diffusion method ■Concentration method (Biochemistry Experiment Course Vol. 1 ■Pages 6-17)
(1976). The invention particularly relates to static batch processes.

When crystallizing by this standing process, the aqueous solution of the substance to be crystallized includes ammonium sulfate, magnesium sulfate, sodium sulfate, sodium phosphate, sodium chloride, cesium chloride, methylbentanediol, ethanol, methanol, acetone, polyethylene glycol, etc. By adding an aqueous solution of and allowing the polymer to precipitate and stand, crystal nucleation and crystal growth are performed using the solution growth method.

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 succeeded in avoiding wasting valuable samples. We are proposing a method that can semi-automate the work (Figure 6).

In this method, a valve is switched to reach a predetermined concentration and the fluid is sent to a Frank John collector using a pump.

In this method, although the concentration gradient solution can be produced automatically to some extent, it has to be divided manually. Therefore, with this method, the results may vary depending on the skill level of the operator, and there are also problems in terms of reproducibility.

[Means and effects for solving the problems] The present invention has the following features:
This method was proposed to solve the above problem, and instead of relying on manual division to reach crystallization by continuously changing one factor of the crystallization conditions, it is possible to The aim is to save labor and improve reproducibility through automation.

That is, the present invention uses solution growth of biopolymer crystals to
In a method of dividing and individually crystallizing a liquid with a concentration gradient as one of the factors defining the crystallization conditions, the concentration gradient liquid is introduced into a soft hollow tube, and then the hollow tube is crimped. The method is characterized in that the method is divided into a plurality of small chambers, and each chamber is used as a crystallization solution (hereinafter referred to as Invention A method).

Furthermore, the present invention provides a method for dividing and individually crystallizing a liquid with a concentration gradient as one of the factors defining the crystallization conditions by solution growth of biopolymer crystals. Similarly, a crystal growth apparatus is assembled by alternately arranging a plurality of thin plates each having a hole to form a hollow lumen with communicating holes, and then filling the lumen with the solution to be crystallized. By disconnecting the lumen, the lumen is divided into small chambers, and each chamber is used as a crystallization container (hereinafter referred to as Invention B method).

For the crimping method in Method A of the invention, for example, as shown in FIGS. 1 and 2, a crimping jig 1 made of identical members having a rectangular cross section can be used.

For example, a soft tube 2 is held between this crimping jig 1,
Using the jig 1, it is divided into small chambers for crystal growth. For example, this device can implement method A of the present invention by operating as follows. First, one side of the crimping jig is fixed, four ball screws are attached to the other side (so that they are not cantilevered), and a timing pulley attached to one end of each screw is driven by a single motor using a timing belt. By doing so, one side of the crimping jig is pressed against the other. By using a photo ink converter or the like, the end point can be automatically detected and the power supply to the motor can be stopped. By providing a gear to the motor, reverse rotation after stopping can be prevented. FIG. 2 shows the state in which it is crimped with a crimping jig. A partially enlarged view is shown in FIG.

In this invention, the soft hollow tube may be transparent, but
If it is not necessary to observe the crystal growth process over time, it may be translucent or opaque. Also, it is preferable that the hollow tube is made of a material with low water permeability. For example, those made of Tygon or fluoropolymers such as FEP, TFE, TDA, PVDF can be used.

After introducing the sample into the soft hollow tube of the above-mentioned device from the concentration gradient preparation device, it is divided into small chambers using a crimping jig and crystal growth is performed. The crystals are recovered by removing the jig and cutting off one end of the small chamber of the soft tube.

In addition, in invention B, for example, a crystal is constructed by alternately arranging a plurality of blocks 4 having holes 3 at appropriate positions and a plurality of thin plates 6 having communicating holes 5 corresponding to the holes 3. Growth equipment can be used. This crystal growth apparatus can be used, for example, as follows. A hole is provided in the center of the upper end of the thin plate held between the blocks, and a rod for sliding the thin plate upward is passed through this hole. Next, ball screws are provided at both ends of this rod, and the thin plate 6 is simultaneously slid upward by driving the timing booth with a motor using a timing belt as in the above-described device.

This divides the lumen into small chambers, each of which serves as a crystallization vessel (see Figure 5).

The block material may be an inorganic material such as glass, or an organic polymer such as acrylic, polymethylpentene or polycarbonate.

The thin plate is preferably made of metal such as stainless steel or titanium.

Regarding the size of the block and thin plate, for example, a block made of a material with a thickness of 2 mm or more and a thin metal plate with a thickness of 200 μm or less can be used.

In producing the crystal growth apparatus according to the present invention, holes are provided that pass through the parts consisting of blocks and thin plates, and the parts are assembled using bolts and nuts. After crystal growth by a predetermined operation, the crystal can be collected, for example, as follows. That is, while standing the device vertically and tightening the entire block using a separately prepared disassembly jig, remove the bolts and nuts mentioned above (to prevent liquid leakage).
Remove the blocks one by one and collect the crystals in each chamber one by one.

The present invention will be further explained below with reference to Examples.

〔Example〕

Example 1 A transparent tube (made by Tygon in this example) with elasticity and low water vapor permeability as shown in Fig. 1 is placed at the end of the proposed liquid delivery piping shown in Fig. 6. . After creating a desired concentration gradient in the tube by sending a predetermined amount of liquid from the concentration gradient generator, the tube is divided into small parts by crimping at regular intervals, and crystal growth is performed on each part. I made it.

Specifically, ammonium sulfate solution (1) (80% saturated ammonium sulfate, 10mM sodium phosphate buffer, pH 7, o) and ammonium sulfate solution (2) (95% saturated ammonium sulfate, 10mM sodium phosphate buffer) were placed in a concentration gradient generator. pH7,
0), total liquid volume 3.34ml! linear gradient of 80-95% saturation of
) was created. Separately prepared protein solution (3% sperm whale myoglobin, 50% ammonium sulfate, 10%
IIIM sodium phosphate buffer pl+ 7.0 >
The flow rates of the two pumps were set to 0.5 ml/min and 1.0 ml/min, respectively, so that the above concentration gradient solution and the above concentration gradient solution were mixed at a volume ratio of 1:2. As a result, the final solution composition contained 1% myoglobin and 10 mM sodium phosphate buffer, and a concentration gradient was formed in which the ammonium sulfate concentration changed linearly from 70 to 80%.
It was guided into a Tygon tube 8c11 and divided into ten parts at 0.8 am intervals by sandwiching it between uneven metal plates. Thus each part is 70.5.71.5
．． ...to include ammonium sulfate concentration of 79.5% saturation?
After injecting the 8 liquids, keep the tubes in place at 20℃.
Crystal growth was performed in a constant temperature bath.

Crystals began to grow after one day and continued to grow for seven days. The most crystals produced were at 75.5% saturation, indicating that this concentration was optimal for crystallization in this example.

Example 2 In this example, instead of crimping and dividing a transparent tube with elasticity and low water vapor permeability into small parts, an acrylic resin block (length 0.8 Thin stainless steel plate (thickness 50 μm) with holes of 3 holes in diameter
A device (Figure 4) in which the resistors are sandwiched alternately is used.

First, the acrylic and stainless steel plates are sandwiched so that the holes match, and the solution is filled in the same manner as in Example 1, and then the stainless steel plate is slid 1 CI 11 to divide it into ten parts. Crystals were grown for 7 days in the same manner as in Example 1. The generated crystals were collected by dismantling the blocks using a dismantling jig as described above.

〔Effect of the invention〕

Since the present invention is configured as explained above,
It is possible to automate the sample, which was insufficient with the proposed method, eliminating the need for unskilled workers, improving reproducibility,
It is possible to reduce the number of man-hours.

In addition, compared to conventional automatic crystallization equipment, the method of the present invention is more compact and can perform systematic crystallization, and because there is no dead volume of piping, it is possible to avoid wasting valuable sample protein. play.

[Brief explanation of the drawing]

Fig. 1 is a schematic perspective view of the crimping jig used in the method of the present invention, Fig. 2 is an explanatory diagram showing the state of crimping with the crimping jig, and Fig. 3 is a partially enlarged view of Fig. 2. , FIG. 4 is an explanatory diagram of a crystal growth apparatus in another invention, FIG. 5 is an explanatory diagram showing an example of making a crystallization container, and FIG. 6 is a configuration diagram of the proposed crystallization production apparatus. It is. 1...Jig for crimping, 2...Soft tube, 3
．． 5...hole, 4...block, 6...
・Thin plate.

Claims (1)

[Claims] 1. In a method of solution growth of biopolymer crystals, in which a liquid with a concentration gradient as one of the factors regulating the crystallization conditions is divided and crystallized individually, A method for producing a crystal, which comprises introducing the concentration gradient liquid, and then dividing the hollow tube into a plurality of small chambers by crimping the tube, each chamber serving as a crystallization container. 2. The method according to claim 1, wherein the soft hollow tube is transparent. 3. The method according to claim 1, wherein the pipe is made of a material with low water permeability. 4. A method according to claim 1, characterized in that the tube is made of tygon. 5. In the solution growth of biopolymer crystals, in a method in which a solution is divided and individually crystallized with a concentration gradient as one of the factors that define the crystallization conditions, each block has a hole in each block. A crystal growth apparatus is assembled by alternately arranging a plurality of thin plates having holes in the pores to form a hollow lumen with communicating holes, and then filling the lumen with the solution to be crystallized, and then making the holes communicate with each other. A method as described above, characterized in that the lumen is divided into small chambers by release, each chamber serving as a crystallization vessel. 6. The method according to claim 5, characterized in that the block is made of a transparent material and has a thickness of 2 mm or more, and a metal thin film has a thickness of 200 μm or less. 7. The method according to claim 5, characterized in that the block is glass. 8. The method according to claim 5, wherein the block is acrylic, polymethylpentene, or polycarbonate. 9. The method according to claim 5, wherein the metal thin film is stainless steel.