Protein crystallization method and crystallization device

Patent metadata

Field	Value
Publication number	US-11110371-B2
Application number	US-201716489510-A
Country	US
Kind code	B2
Filing date	Dec 27, 2017
Priority date	Mar 2, 2017
Publication date	Sep 7, 2021
Grant date	Sep 7, 2021

Abstract

Provided are: an economically superior protein crystallization method capable of efficiently finding conditions for crystallization by using a small amount of protein; and a crystallization device used for the method. According to the present invention, a transparent sealed container 1 is filled with a solution of protein, a part of the transparent sealed container 1 being formed of a semipermeable membrane 2 with a molecular weight cut-off that inhibits passage of the protein while allowing passage of a precipitant, and then, a precipitant solution with changed concentration and/or pH of the precipitant is continuously supplied to the semipermeable membrane 2, to crystallize the protein with the precipitant that infiltrates from the semipermeable membrane 2 into the sealed container 1.

First claim

The invention claimed is:

1. A protein crystallization method comprising:
filling a transparent sealed container with a solution of protein, a part of the transparent sealed container being formed of a semipermeable membrane with a molecular weight cut-off that inhibits passage of the protein while allowing passage of a precipitant; and
continuously supplying a precipitant solution to the semipermeable membrane while changing concentration and/or pH of the precipitant in the precipitant solution, and circulating the precipitant solution at the same time, to change the concentration and/or pH of the precipitant infiltrating from the semipermeable membrane into the sealed container, and to crystallize the protein with the precipitant that infiltrates from the semipermeable membrane into the sealed container.

2. The protein crystallization method according to claim 1 ,
wherein dry air or liquid having hygroscopic properties is continuously supplied to a gas permeable membrane disposed in a position that faces the semipermeable membrane of the sealed container and having a molecular weight cut-off that inhibits passage of liquid while allowing passage of water vapor, to form gradients of the concentration and/or pH of the precipitant and a concentration of the protein in the sealed container.

3. The protein crystallization method according to claim 1 , wherein the solution with which the sealed container is filled contains, in addition to the protein, the precipitant having a concentration with which the protein is not crystallized.

4. A protein crystallization device comprising:
a transparent sealed container filled with a solution containing protein;
a semipermeable membrane that forms a part of the sealed container and inhibits passage of the protein while allowing passage of a precipitant; and
precipitant supply means that circulates and supplies a precipitant solution containing the precipitant to the semipermeable membrane,
wherein the precipitant supply means includes
a reservoir that stores the precipitant solution,
a circulation line that supplies the precipitant solution in the reservoir to the semipermeable membrane and returns the precipitant solution to the reservoir again,
a pump that is disposed on the circulation line and continuously supplies the precipitant solution to the semipermeable membrane, and
concentration adjustment means that changes the precipitant concentration in the precipitant solution.

5. The protein crystallization device according to claim 4 , wherein
the sealed container is a tubular member having one end closed and the other end formed with an opening,
the semipermeable membrane is formed in a bag-like shape integrated at the opening with a cylindrical narrow tube and is inserted into the sealed container via a sealing member,
the sealing member includes
a cylindrical elastic member having a hole formed in a center, the hole having a diameter smaller than outer diameters of the sealed container and the narrow tube, and
a cylindrical restraint tube having an inner diameter size smaller than an outer diameter size of the elastic member, and
the sealed container is fitted to one end of the hole in the elastic member while the narrow tube is fitted to the other end of the hole, and the compressed elastic member is fitted into the restraint tube.

6. The protein crystallization device according to claim 4 , comprising:
a gas permeable membrane disposed in a position that faces the semipermeable membrane of the sealed container and having a molecular weight cut-off that inhibits passage of liquid while allowing passage of water vapor;
second supply means that continuously supplies dry air or liquid having hygroscopic properties to the gas permeable membrane; and
discharge means for water vapor discharged out of the sealed container through the gas permeable membrane.

7. The protein crystallization device according to claim 4 , wherein the sealed container is made of glass or quartz.

8. The protein crystallization device according to claim 5 , wherein the elastic member is silicone rubber with a hardness of 15 degrees to 25 degrees.

Assignees

• Chiyoda Corp
• Confocal Science Inc

Inventors

• Tanigawa Naoki
• Tanaka Hiroaki
• Takahashi Sachiko
• Inaka Koji

Classifications

• B01D61/362

  Pervaporation · CPC title

• B01D9/0027

  by means of conveying fluid, e.g. spray-crystallisation (spray-drying F26B) · CPC title

• B01D2009/0086

  Processes or apparatus therefor · CPC title

• C12N9/2462 Primary

  Lysozyme (3.2.1.17) · CPC title

• C12Y302/01017

  Lysozyme (3.2.1.17) · CPC title