JP2650274B2 - Crystal preparation method - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Summary of the Invention] The present invention relates to the production of a single crystal by solution growth,
Conventionally, the concentration gradient liquid has been divided manually and crystallized. For this reason, there were problems such as poor reproducibility and many man-hours. SUMMARY OF THE INVENTION The present invention provides a single crystal manufacturing method capable of simplifying and automatically dividing a concentration gradient liquid by using a predetermined pressure-bonding jig or the like, thereby enabling omission and improvement of reproducibility.

[Industrial applications]

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 a concentration gradient liquid can be automatically distributed when a single crystal is produced by solution growth.

[Problems to be solved by the prior art and the invention]

In applied research such as protein engineering and drug design, it is essential to determine the higher-order structure of a protein by, for example, X-ray analysis. For this purpose, the production of a single crystal of a biopolymer containing no impurities becomes a problem.

Conventional methods for producing single crystals include the conventional stationary batch method, the free interface diffusion method, the dialysis method, the dialysis diffusion method, the vapor diffusion method, and the enrichment method (Biochemical Experiment Course, Vol. 1, III 6-1)
7 pages (1976). The invention particularly relates to the stationary batch method.

When crystallizing by this stationary batch method, ammonium sulfate, magnesium sulfate, sodium sulfate, sodium phosphate, sodium chloride, cesium chloride, methyl pentane, diol, ethanol, methanol, acetone, polyethylene glycol, etc. An aqueous solution is added to precipitate a polymer, and the mixture is allowed to stand, whereby crystal nucleation and crystal growth are performed by a solution growth method.

However, since the setting of crystallization conditions is delicate, crystallization is often performed under a plurality of conditions in parallel. Usually, this work is manually performed, but the present applicant has already proposed a method capable of semi-automating the work without wasting valuable samples (FIG. 6). In this method, a valve is switched to a predetermined concentration to send it to a fraction collector by a pump. According to this method, the concentration gradient liquid can be produced to some extent automatically, but when it is divided, it has to be manually performed. Therefore, in this method, the result may vary depending on the skill of the operator, and there is a problem in reproducibility.

[Means and actions for solving the problems]

The present invention has been proposed in order to solve the above-mentioned problems, instead of relying on humans to continually change one factor of the crystallization conditions and splitting, instead of reaching crystallization. It is intended to save labor and improve reproducibility by automating the division.

That is, the present invention provides a method of dividing a liquid having one of the factors defining crystallization conditions into a concentration gradient by solution growth of a biopolymer crystal and individually crystallizing the solution. The liquid is introduced, and then the hollow tube is press-bonded to divide the chamber into a plurality of small chambers, and each chamber is formed as a crystallization solution (hereinafter, referred to as invention A method).

Further, the present invention, by the solution growth of biopolymer crystals,
In a method in which a liquid having a concentration gradient as one of the factors defining the crystallization conditions is divided and crystallized separately, a plurality of blocks each having a hole and a plurality of thin plates each having a hole are alternately formed. A crystal growth apparatus is assembled by arranging and forming a hollow lumen in which holes communicate with each other, and then the solution to be crystallized is filled in the lumen, and then the lumen is divided into small chambers by releasing communication of the holes. At the very least, each room is used as a crystallization vessel (hereinafter referred to as the invention B method).

In the crimping method in the invention A method, for example, as shown in FIG. 1 and FIG. 2, a crimping jig 1 made of the same member having a rectangular cross section can be used. For example, a soft tube 2 is sandwiched between the crimping jigs 1 and divided into small rooms for crystal growth using the jigs 1. This apparatus can carry out the method A of the present invention, for example, by operating as follows. First, fix one of the crimping jigs, attach four ball screws to the other (so as not to be cantilevered), and drive a timing pulley attached to one end of each screw with a single motor using a timing belt. By doing so, one of the crimping jigs is pressed against the other. By using a photo interrupter or the like, the end point can be automatically detected and the power supply to the motor can be stopped. By rotating the motor through a gear, reverse rotation after stopping can be prevented. FIG. 2 shows a state of being crimped by the crimping jig. FIG. 3 is a partially enlarged view of FIG.

In the present invention, the soft hollow tube may be transparent, but may be translucent to opaque if it is not necessary to observe the crystal growth process over time. The material of the hollow tube is preferably a material having low water permeability. For example, Tygon (polyvinyl chloride; Norton Performance in the United States)
Plastics Corporation trademark) or FEP, TF
E, TDA, PVDF and other fluoropolymers can be used.

After the sample is introduced into the soft hollow tube of the above-mentioned device by the concentration gradient creating device, the sample is divided into small rooms by a jig for crimping, and the crystal is grown. The crystal is recovered by removing one end of the small chamber of the soft tube after removing the jig.

In the invention B, for example, a crystal in which a plurality of blocks 4 having holes 3 at appropriate positions and a plurality of thin plates 6 having holes 5 communicating with the holes 3 are alternately arranged. A growth device 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 sandwiched between the blocks, and a rod for sliding the thin plate upward is passed through the hole. Next, ball screws are provided at both ends of the rod, and the thin plate 6 is simultaneously slid upward by driving a timing pulley by a motor with a timing belt in the same manner as in the above-described apparatus. As a result, the lumen is divided into small rooms, and each room is used as a crystallization container (see FIG. 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.

The size of the block and the thin plate is, for example, 2 m in thickness.
A block made of a material of m or more and a thin metal plate having a thickness of 200 μm or less can be used.

In making the apparatus for crystal growth in the present invention,
A hole is formed through a block and a thin plate to assemble it with bolts and nuts. Then, after the crystal is grown by a predetermined operation, the crystal can be recovered, for example, as follows. That is, stand the device vertically, and tighten the whole block with a dismantling jig prepared separately (to prevent liquid leakage), remove the bolts and nuts described above,
The blocks are removed one by one, and the crystals in each chamber are sequentially collected.

 Hereinafter, the present invention will be further described with reference to examples.

〔Example〕

Example 1 At the end of the liquid feeding pipe of the proposed example as shown in FIG. 6, an elastic, low water vapor permeable and transparent tube shown in FIG. 1 (Tygon (polyvinyl chloride; Made by Norton Performance Plastics Corporation of the United States). After a predetermined amount of liquid has been sent from the concentration gradient maker to form an intended concentration gradient in the tube, the tube is divided into small portions by crimping at regular intervals, and crystal growth is performed for each portion. I let you.

Specifically, the ammonium sulfate solution (1) (80% saturated ammonium sulfate, 10 mM sodium phosphate buffer pH
7.0) and ammonium sulfate solution (2) (95% saturated ammonium sulfate, 1
0 mM sodium phosphate buffer (pH 7.0).
34 ml of 80-95% saturation linear gradient
t) was prepared. Separately made protein solution (3% sperm whale myoglobin, 50% ammonium sulfate, 10 mM
The pumping rates of the two pumps were set at 0.5 ml / min and 1.0 ml / min, respectively, so that the sodium phosphate buffer (pH 7.0) and the concentration gradient solution were mixed at a volume ratio of 1: 2. Thus, the final solution composition contains 1% myoglobin, 10 mM sodium phosphate buffer, and ammonium sulfate concentration.
A concentration gradient was formed that varied linearly from 70 to 80%. This was guided into an 8 cm tube made of Tygon having an inner diameter of 3 mm, and divided into ten parts at 0.8 cm intervals by being sandwiched between metal plates having irregularities. Thus, each part is 70.5, 71.5, ... 79.5
After injecting the solution so as to contain a concentration of ammonium sulfate at a% saturation, crystal growth was performed in a constant temperature bath at 20 ° C. while sandwiching the solution.

One day later, the crystals began to grow and continued to grow for 7 days. Most of the generated crystals were at 75.5 saturation, and this example shows that the same concentration was optimal for crystallization.

Example 2 In this example, instead of crimping a transparent tube having elasticity and low water vapor permeability and dividing it into small parts, an acrylic resin block having a hole with a diameter of 3 mm (length 0.8 mm)
And a stainless steel plate with a hole with a diameter of 3mm (thickness 50μm)
(FIG. 4). First, a solution is filled in the same manner as in Example 1 in a state where the holes of the acrylic and the stainless steel are sandwiched so as to coincide with each other, and then the stainless steel plate is slid by 1 cm to be divided into ten parts. Crystals were grown for 7 days as in Example 1. The generated crystals were disassembled into blocks using a dismantling jig as described above to collect crystals.

〔The invention's effect〕

Since the present invention is configured as described above, it is possible to automate a sample which was insufficient with the proposed method, work by unskilled people, improvement of reproducibility, reduction of man-hours, etc. Can be achieved.

In addition, compared to the conventional automatic crystallization apparatus, it is possible to perform a small and systematic crystallization than in the case of the method of the present invention,
Since there is no dead volume in the piping, it is possible to avoid wasting valuable sample proteins.

[Brief description of the drawings]

FIG. 1 is a schematic perspective view of a crimping jig used in the method of the present invention, FIG. 2 is an explanatory view showing a state of being crimped by the crimping jig, and FIG. 3 is a partially enlarged view of FIG. FIG. 4 is an explanatory view of an apparatus for crystal growth in another invention, FIG. 5 is an explanatory view showing an example of producing a crystallization vessel, and FIG. 6 is a structural view of a proposed crystallization producing apparatus. It is. 1 ... crimping jig, 2 ... soft tube, 3,5 ... hole, 4 ... block, 6 ... thin plate.

Claims (9)

(57) [Claims] 1. A method for dividing a liquid having one of the factors defining crystallization conditions into a concentration gradient by solution growth of a biopolymer crystal and individually crystallizing the solution, the method comprising the steps of: A method for producing a crystal, comprising: introducing a liquid; and thereafter, compressing the hollow tube to divide it into a plurality of small chambers, and forming each room as a crystallization container. 2. The method according to claim 1, wherein the flexible hollow tube is transparent. 3. The method according to claim 1, wherein the tube is made of a material having low water permeability. 4. The method according to claim 1, wherein the tube is made of polyvinyl chloride. 5. A method in which a solution having a concentration gradient defined as one of the factors defining crystallization conditions is divided and individually crystallized by solution growth of a biopolymer crystal, wherein a plurality of blocks each having a hole are provided. A plurality of thin plates each having a hole are alternately arranged to form a hollow lumen in which the holes communicate with each other to assemble an apparatus for crystal growth, and then a solution to be crystallized is filled in the lumen, The method described above, wherein the lumen is divided into small chambers by releasing the communication with each other, and each room is a crystallization container. 6. The method according to claim 5, wherein the block is made of a transparent material having a thickness of 2 mm or more and a metal thin film having a thickness of 200 μm or less. 7. The method according to claim 5, wherein 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.