WO2020170321A1

WO2020170321A1 - Biopolymer crystallization device

Info

Publication number: WO2020170321A1
Application number: PCT/JP2019/005996
Authority: WO
Inventors: 広明田仲; 浩治伊中; 斌厳; 幸子高橋
Original assignee: 株式会社コンフォーカルサイエンス
Priority date: 2019-02-19
Filing date: 2019-02-19
Publication date: 2020-08-27

Abstract

The biopolymer crystallization device according to the present invention comprises: a first tubular member which is filled with a solution containing a biopolymer; a second tubular member into which a precipitant is supplied; and a dialysis membrane which allows the passage of the precipitant but not the biopolymer therethrough. The second tubular member has an end face wherein an opening is formed inside the first tubular member. Inside the first tubular member, the precipitant is supplied from the inside of the second tubular member to the biopolymer-containing solution through the opening and the dialysis membrane. Further, this biopolymer crystallization device comprises: an elastic body which is fitted inside the first tubular member; and a reservoir which houses the second tubular member within the inside thereof filled with the precipitant so that the precipitant is supplied to the inside of the second tubular member. The dialysis membrane is in contact with the end face in such a manner as to close the opening. At the same time, the dialysis membrane is fixed in a state of being in contact with the outer peripheral face of the second tubular member and the inner peripheral face of the elastic body.

Description

Biopolymer crystallizer

The present invention relates to a crystallization device for crystallizing a biopolymer such as a protein.

As a method of developing a new drug, a method of devising an inhibitor drug that binds to a biopolymer such as a protein associated with a disease with high affinity and synthesizing it newly is known. At this time, highly accurate three-dimensional structural coordinate data of the target biopolymer such as a disease-related protein or a complex in which the biopolymer is bound to the inhibitor drug is useful information for devising or improving the inhibitor drug. Such new drug development based on structural data of biopolymers is called SBDD (Structure Based Drug Discovery/Design) and is becoming an indispensable technology for pharmaceutical companies.

By the way, in order to analyze the three-dimensional structural coordinate data of the protein by X-ray diffraction, neutron diffraction or the like, it is necessary to obtain a high quality biopolymer crystal such as the protein (see the following Patent Documents). 1). Therefore, in recent years, it is increasingly necessary to add a precipitating agent to a solution of a biopolymer such as protein to reduce the solubility of the solution and crystallize the biopolymer. One of them is dialysis. It is adopted by some.

For example, FIG. 9(a) shows a conventional dialysis button container manufactured by Hampton Research Co., which is used in the above dialysis method. The dialysis button container 20 has a chamber 21 having an opening on the upper surface at the center thereof. At the same time, an O-ring fitting groove 22 is formed on the lower periphery.

Using this dialysis button container 20, for example, the protein to be crystallized, first filled with sample solution L ₁ of the protein in the chamber 21, from above the solution L ₁ which raised somewhat domed surface tension The membrane is covered with a dialysis membrane 23 that does not allow proteins to permeate, and is fixed by an O-ring 24.

Then, the dialysis button container 20 is placed in a container 25 filled with a precipitant L ₂ as shown in FIG. 9( b ), and the dialysis button container 20 is dissolved by the precipitant L ₂ passing through the dialysis membrane 23. The protein in the chamber 21 is crystallized by reducing the solubility of the protein.

However, in the dialysis method using the dialysis button container 20, it is difficult to prepare a large amount of protein as a sample, and biopolymers such as proteins associated with diseases are generally difficult to prepare. It is necessary to handle a small amount of sample such as several μL to several tens μL. Further, when the dialysis membrane 23 is set, bubbles are likely to be present between the solution and the dialysis membrane 23, which requires a skill in the work. In addition, when the chamber 21 is filled with a small amount of sample such as several μL, there is a problem that valuable sample is likely to be lost.

Furthermore, when the dialysis button container 20 is placed in the container 25, a sudden osmotic pressure difference is generated between the dialysis button container 20 and the precipitating agent L ₂ , so that air bubbles are easily generated in the chamber 21, and crystallization for avoiding this occurs. There is also a problem that it is not easy to set the conditions.

Japanese Patent Publication No. 2002-526745

The present invention has been made in view of the above circumstances, and when crystallizing a biopolymer such as a protein by a dialysis method, a drastic osmotic pressure difference does not occur from the precipitant to the biopolymer solution side, and It is an object of the present invention to provide a biopolymer crystallization apparatus which can avoid the loss of a valuable sample solution and is excellent in workability.

In order to solve the above-mentioned problems, in the first aspect of the present invention, a first tubular member having a solution containing a biopolymer filled therein, and a second tubular member having a precipitant supplied therein are provided. A dialysis membrane that allows the precipitant to pass without passing the biopolymer, wherein the second tubular member has an end face in which an opening is formed inside the first tubular member, In the inside of the first tubular member, the precipitant passes through the opening and the dialysis membrane from the inside of the second tubular member and is added to the solution containing the biopolymer. A device, wherein an elastic body fitted inside the first tubular member and the second tubular member housed inside the precipitating agent are filled into the interior of the second tubular member. And a reservoir supplying the precipitant to the dialysis membrane, the dialysis membrane contacting the end face so as to close the opening, and the outer peripheral surface of the second tubular member and the inner peripheral surface of the elastic body. It is characterized in that it is fixed in a state of abutting against.
The above-described present invention may further include an introduction tube inserted into the inside of the first tubular member.

Further, in the second aspect of the present invention, a transparent first tubular member filled with a solution containing a biopolymer, a sealing member that closes one end of the first tubular member, and the above first A second tubular member that is inserted into the other end of the tubular member via a seal member, a dialysis membrane that closes one end opening of the second tubular member that is located inside the first tubular member, and the above inside A reservoir for accommodating the first and second tubular members and filled with the precipitant solution to supply the precipitant solution into the second tubular member; and the seal member has an outer diameter. The inner diameter of the first tubular member is tightly fitted into the other end of the first tubular member by forming the inner diameter of the second tubular member larger than the inner diameter of the second tubular member. The second tubular member is tightly fitted to the second tubular member, and the permeable membrane is made of a material that allows the precipitant to pass through but does not allow the biopolymer to pass through. The outer peripheral portion is sandwiched between the outer peripheral surface of the second tubular member and the inner peripheral surface of the seal member.
In the present invention, the closed container may be made of glass or quartz.

In the first aspect of the present invention, the state in which the dialysis membrane is in contact with the end surface (the end surface having the opening) of the second tubular member is maintained by the elastic body, so that the second tubular shape is opened from the opening. Instead of passing through the dialysis membrane in the process in which the precipitating agent that has flowed out to the outside of the member diffuses "in multiple directions," the precipitating agent that is filled inside the second tubular member does not "pass through the opening. It will pass through the dialysis membrane as it flows. Therefore, it is possible to maintain a small area where the precipitating agent filled inside the second tubular member passes through the dialysis membrane, and to prevent a large amount of precipitating agent from passing through the dialysis membrane in a short time. Therefore, it is possible to suppress the occurrence of a sudden osmotic pressure difference.
Therefore, in the present invention, it is not necessary to adjust the concentration and flow rate of the precipitating agent supplied to the inside of the second tubular member in order to suppress the occurrence of a sudden osmotic pressure difference. Therefore, the second tubular member can be accommodated in the reservoir filled with the precipitant, and the state in which the precipitant is naturally supplied to the inside of the second tubular member can be maintained. Therefore, the precipitant can be added to the solution containing the biopolymer without requiring complicated operations.

In the second aspect of the present invention, a transparent first tubular member having one end closed and filled with a solution containing a biopolymer, and a sealing member on the other end of the first tubular member. And a second tubular member to which a precipitant solution is supplied by being inserted through the double tubular structure, and a dialysis membrane is fixed to an opening of one end of the second tubular member located in the first tubular member. Therefore, it is possible to secure a flow path in the first tubular member through which the precipitant solution diffuses through the dialysis membrane, and thus it is possible to avoid applying a rapid osmotic pressure difference.
In addition, when filling the biopolymer solution into the first tubular member, a polyimide tube having a diameter smaller than that of the first tubular member or the like can be used to easily add the solution to the first tubular member. Since it can be injected into the tubular member, it is possible to avoid the loss of a valuable biopolymer solution or the inclusion of air bubbles.

FIG. 3 is a vertical cross-sectional view showing a main part in one embodiment of the present invention. It is a longitudinal section showing the whole one embodiment of the present invention. It is a longitudinal cross-sectional view which shows the state which assembled the 1st and 2nd tubular member of FIG. FIG. 5 is a vertical sectional view showing a state in which the first and second tubular members of FIG. 4 are stored in a container. It is a longitudinal cross-sectional view showing a state where the precipitant solution is filled in the second tubular member. FIG. 6 is a vertical cross-sectional view showing a state in which the biopolymer solution is filled in the first tubular member of FIG. 5. FIG. 7 is a vertical cross-sectional view showing a state in which one end of the first tubular member is sealed after being filled with the biopolymer solution of FIG. 6. It is a microscope picture which shows the result of the Example of this invention. (A) is a front view showing a conventional dialysis button container used in a dialysis method, and (b) places the dialysis button container in a container filled with a precipitating agent to crystallize the protein therein. It is the front view which carried out the partial cross section view which shows a state.

1 and 2 show an embodiment of a biopolymer crystallization apparatus according to the present invention, in which reference numeral 1 is a first tubular member filled with a solution L ₁ containing a biopolymer. ..
The first tubular member is made of a transparent glass tube, and in the present embodiment, it has an outer diameter of 2.5 mm, an inner diameter of 1.9 mm, and a length of 18 mm. Then, an introduction tube 3 made of a transparent glass tube is inserted into the one end portion 1a of the first tubular member 1 via a cylindrical silicon tube 2.

Here, the silicon tube 2 is formed so that the outer diameter dimension thereof is larger than the inner diameter dimension of the first tubular member 1, so that the silicon tube 2 is tightly fitted into the one end portion 1a of the first tubular member 1 and the inner diameter dimension is Since the introduction pipe 3 is formed to have a diameter smaller than the outer diameter of the introduction pipe 3, the introduction pipe 3 is closely fitted.

By the way, in this embodiment, the silicon tube 2 is formed to have an outer diameter of 2.0 mm, an inner diameter of 1.0 mm, and a length of 5 mm, and the introduction tube 3 has an outer diameter of 1.2 mm, an inner diameter of 0.7 mm, and a length of 0.7 mm. Is formed to 8 mm. Then, the end portion of the introduction tube 3 is filled with the solution L ₁ containing the biopolymer inside and then closed by the nontoxic clay 4 (sealing member).

A second tubular member 6 made of a transparent glass tube is inserted into the other end portion 1b of the first tubular member 1 via a cylindrical silicon tube (sealing member, elastic body) 5. The second tubular member 6 is supplied with the precipitant solution L ₂ therein, as will be described later, and has an open end 6a extending outward from the first tubular member 1.

The silicone tube 5 also has an outer diameter dimension larger than the inner diameter dimension of the first tubular member 1, so that the silicon tube 5 is closely fitted into the other end portion 1b of the first tubular member 1 and has an inner diameter dimension. Is smaller than the outer diameter of the second tubular member 6, so that the second tubular member 6 is closely fitted.

In this embodiment, the silicon tube 5 is formed to have an outer diameter of 2.0 mm, an inner diameter of 1.0 mm and a length of 5 mm, and the second tubular member 6 has an outer diameter of 1.2 mm, an inner diameter of 0.5 mm and a long length. Is formed to be 10 mm.

The one end opening in the first tubular member 1 of the second tubular member 6 is closed by the dialysis membrane 7. The dialysis membrane 7 has a molecular weight cut-off in the range of 2,000 to 100,000 Da, and is formed into a bag shape with a material that allows the precipitant to pass through but does not allow the biopolymer to pass through. It is sandwiched between the outer peripheral surface of the second tubular member 6 and the inner peripheral surface of the silicon tube 5.

Here, the dialysis membrane 7 is a regenerated cellulose membrane (RC) such as cupra ammonium rayon (CR) or saponified cellulose (SCA), a hemofan membrane, a PC membrane, a surface-modified regenerated cellulose membrane such as a vitamin E coating membrane, Cellulose acetate (CA) such as cellulose diacetate (CDA) and cellulose triacetate (CTA), polyacrylonitril (PAN), polymethylmethacrylate (PMMA), ethylene vinyl alcohol copolymer (EVAL), polysulfone (PS), A synthetic polymer film such as poamide (PA) or polyester polymer alloy (PEPA) can be used.

In the biopolymer crystallization apparatus, the first and second

tubular members

1 and 6 are filled with the precipitant solution L _{2 in} the second tubular member 6 as shown in FIG. It is used for crystallization of the biopolymer in the first tubular member 1 by being arranged in the reservoir 8 supplying the precipitant solution L ₂ .

Next, a procedure for assembling the first and second

tubular members

1 and 6 and performing the crystallization will be described with reference to FIGS. 3 to 7.
First, as shown in FIG. 3, the silicon tube 5 is pushed into the other end 1b of the first tubular member 1 by about 1 mm. Next, the dialysis membrane 7 is placed in the one end opening of the second tubular member 6, and after these are inserted into the silicon tube 5, they are pushed into the first tubular member 1 together with the silicon tube 5.

On the other hand, also at the one end portion 1a of the first tubular member 1, the silicon tube 2 is similarly pushed in by about 1 mm, and then the introduction tube 3 is inserted into the silicon tube 2 and then the first tubular member 1 together with the silicon tube 2 is inserted. Push it in.

The thus assembled first and second

tubular members

1 and 6 and the introduction pipe 3 are stored in a reservoir 8 filled with pure water L ₃ , for example, as shown in FIG.

Then, in order to crystallize a biopolymer such as a protein with the biopolymer crystallization device having the above-described configuration, first, as shown in FIG. 5, the first and second

tubular members

1 and 6 and the introduction are introduced. The tube 3 was taken out from the storage reservoir 8 shown in FIG. 4, and a polyimide tube (outer diameter 0.38 mm, inner diameter 0.30 mm) was connected to the micropipette tip inside from the open end of the second tubular member 6. Insert the filling jig 10. Then, the precipitant solution is supplied from the filling jig 10 to fill the inside of the second tubular member 6 with the precipitant solution L ₂ .

Here, the precipitant may be any salt or polymer capable of condensing the protein without denaturing it, and sodium chloride, ammonium sulfate, polyethylene glycol, etc. can generally be used.

Then, as shown in FIG. 6, a filling jig 11 in which a polyimide tube (outer diameter 0.62 mm, inner diameter 0.50 mm) is connected to a micropipette tip is inserted from the opening end of the introduction tube 3 and the filling treatment is performed. After supplying and filling the biopolymer solution L ₁ into the first tubular member 1 from the tool 11, the open end of the introduction tube 3 is sealed with a nontoxic clay 4 as shown in FIG. 7. ..

Then, as shown in FIG. 2, the first and second

tubular members

1 and 6 and the introduction pipe 3 are housed in a reservoir 8 filled with a precipitant solution L ₂ , to start crystallization. ..

As described above, in the biopolymer crystallizing apparatus having the above-described configuration, the transparent _first 1a in which the one end 1a is closed by the sealing member 4 and the solution L ₁ containing the biopolymer is filled inside And a second tubular member 6 into which the precipitant solution L ₂ is supplied by being inserted into the other end portion 1b of the first tubular member 1 via the silicon tube 5 Since the dialysis membrane 7 is fixed to the one end opening of the second tubular member 6 located in the first tubular member 1, the precipitant solution L is introduced into the first tubular member 1 through the dialysis membrane 7. It is possible to secure a flow path in which ₂ diffuses, and thus it is possible to avoid applying a sudden osmotic pressure difference.

In addition, when filling the biopolymer solution L ₁ into the first tubular member 1, a polyimide tube or the like having a smaller diameter than the first tubular member 1 is used to facilitate the solution L ₁ Since it can be injected into the first tubular member 1, it is possible to avoid the loss of a valuable biopolymer solution or the inclusion of air bubbles.

In addition, in the said embodiment, although the case where nontoxic clay 4 was used as a sealing member which closes the one end part 1a of the 1st tubular member 1 was demonstrated, this invention is not limited to this. However, various sealing members can be used as long as the one end 1a of the first tubular member 1 can be hermetically sealed.

In addition, the end portion of the glass introduction tube 3 is melted by a microburner, or the introduction tube 3 is formed of a material having elasticity and the end portion is subjected to consolidation treatment to form a sealing member. Is also possible. Further, the end of the first tubular member 1 is directly sealed with the harmless clay 4 or the like without providing the silicon tube 2 and the introduction pipe 3 described above on the one end 1a of the first tubular member 1. You may do it.

Further, as the material of the first and second

tubular members

1 and 6, besides the glass shown in the present embodiment, quartz can be preferably used, and a transparent synthetic resin such as polycarbonate may also be used. It is possible.

A protein crystallization experiment was conducted using the biopolymer crystallization apparatus shown in the above embodiment.
In this experiment, 25 mg/ml lysozyme, 5% PEG 4000, 0.3 M NaCl, 50 mM acetate buffer pH 4.5 was used as the protein sample solution (biopolymer solution L ₁ ), and the initial precipitant solution L ₂ was used. As the solution, a solution having a composition of 5% PEG 4000, 0.5M NaCl, 50 mM acetate buffer pH 4.5 was used.

Then, the above-mentioned protein sample solution L ₁ was filled in the first tubular member 1 and left standing horizontally in the reservoir 8 filled with the above-mentioned precipitant solution L ₂ to start crystallization.

Then, 12 days after the start of the experiment, the first tubular member 1 and the like were placed in a reservoir 8 filled with a precipitant solution L ₂ having a composition of 5% PEG 4000, 0.7 M NaCl, 50 mM acetate buffer pH 4.5. The crystals were transferred and similarly left to stand to produce crystals.

As a result, 14 days after the start of the experiment, crystals were formed as shown in FIG. 8(a), and one month after the start of the experiment, the growing crystals were about 450 μm in size, as shown in FIG. 8(b). It was

1 First tubular member 4 Non-toxic clay (sealing member)
5 Silicon tube (seal member, elastic body)
6 Second tubular member 7 Dialysis membrane 8 Reservoir L ₁ Biopolymer solution L ₂ Precipitant solution

Claims

A first tubular member whose inside is filled with a solution containing a biopolymer;
A second tubular member having a precipitating agent supplied therein;
A dialysis membrane that allows the precipitant to pass without passing the biopolymer,
The second tubular member has an end surface in which an opening is formed inside the first tubular member,
The precipitant is added to the solution containing the biopolymer through the opening and the dialysis membrane from the inside of the second tubular member inside the first tubular member. A device,
An elastic body fitted inside the first tubular member;
And a reservoir for supplying the precipitant to the interior of the second tubular member by accommodating the second tubular member in the interior filled with the precipitant.
The dialysis membrane is fixed in a state of abutting on the end surface so as to close the opening and abutting on an outer peripheral surface of the second tubular member and an inner peripheral surface of the elastic body. A biopolymer crystallizing device.
The biopolymer crystallization apparatus according to claim 1, further comprising an introduction tube inserted inside the first tubular member.
A transparent first tubular member filled with a solution containing a biopolymer, a sealing member that closes one end of the first tubular member, and a seal member at the other end of the first tubular member. A second tubular member to be inserted, a dialysis membrane that closes one end opening of the second tubular member located inside the first tubular member, and the first and second tubular members are housed inside. And a reservoir that is filled with the precipitant solution and supplies the precipitant solution into the second tubular member.
The outer diameter dimension of the sealing member is larger than the inner diameter dimension of the first tubular member, so that the sealing member is closely fitted into the other end portion of the first tubular member, and the inner diameter dimension is the first dimension. The second tubular member is formed into a smaller diameter than the outer diameter of the second tubular member, and is made of a tubular elastic body into which the second tubular member is tightly fitted.
The permeable membrane is made of a material that allows the precipitating agent to pass through but does not allow the biopolymer to pass through. The outer peripheral portion of the permeable membrane is sandwiched between the outer peripheral surface of the second tubular member and the inner peripheral surface of the seal member. A biopolymer crystallization device characterized by being
The biopolymer crystallization apparatus according to claim 3, wherein the closed container is made of glass or quartz.