Method of loading a crystallization device

Patent metadata

Field	Value
Publication number	US-10227378-B2
Application number	US-201615144246-A
Country	US
Kind code	B2
Filing date	May 2, 2016
Priority date	Sep 30, 2008
Publication date	Mar 12, 2019
Grant date	Mar 12, 2019

Abstract

The present invention pertains to a method for loading a crystallization device and for manufacturing a crystallization device comprising multiple receptacles with a pre-defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein, said method comprising the following steps: a) Modifying the state of aggregation of said at least one matrix-forming compound to a fluidic state which allows dispensing said at least one matrix-forming compound, and b) dispensing a defined amount of said at least one matrix-forming compound into at least one receptacle of the crystallization device, wherein said dispensed matrix-forming compound solidifies within said receptacle. Thereby prefilled crystallization devices are obtained which can be used as consumables in particular in automated crystallization processes. Also provided are protein crystallization methods using respectively prepared crystallization devices.

First claim

The invention claimed is:

1. A method of crystallizing biopolymers, comprising:
contacting a defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein with a liquid to form a crystallization matrix,
wherein said defined amount of the at least one matrix-forming compound is contained in at least one receptacle of a crystallization device,
wherein said crystallization device comprises multiple receptacles, wherein at least some of said receptacles comprise a defined homogeneous amount of at least one solid matrix-forming compound capable of forming a crystallization matrix for a membrane protein, and wherein said matrix-forming compound has not yet formed a crystallization matrix and does not comprise the membrane protein.

2. The method of claim 1 , comprising:
a) loading the crystallization device comprising multiple receptacles with a pre-defined amount of at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein to provide said crystallization device,
wherein loading the crystallization device comprises:
i) modifying the state of aggregation of said at least one matrix-forming compound from a solid to a fluidic state which allows dispensing said at least one matrix-forming compound;
ii) dispensing a defined amount of said at least one matrix-forming compound into at least one receptacle of the crystallization device, wherein the at least one matrix-forming compound does not comprise the membrane protein; and
iii) solidifying said at least one matrix-forming compound in said receptacle to form a solidified matrix-forming compound, wherein the solidified matrix-forming compound does not comprise the membrane protein and has not formed a crystallization matrix; and

b) contacting the at least one matrix-forming compound in said receptacle with a liquid to form a crystallization matrix.

3. The method of claim 1 , wherein said liquid comprises water, one or more additives and/or the biopolymer to be crystallized.

4. The method of claim 1 , wherein the crystallization matrix is contacted with a precipitating solution.

5. The method of claim 1 , wherein the crystallization device comprises at least one receptacle for receiving the at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein and at least one reservoir which is in communication with said at least one receptacle.

6. The method of claim 5 , wherein a precipitation solution is dispensed into the reservoir.

7. The method of claim 6 , wherein the precipitation solution in the reservoir is covered with a cover.

8. The method of claim 7 , wherein the precipitation solution in the reservoir is covered with a film which prevents evaporation.

9. The method of claim 1 , further comprising crystallizing the biopolymer.

10. The method of claim 1 , wherein the method is automated.

11. The method of claim 1 , wherein the biopolymer is a protein.

12. The method of claim 1 , wherein the biopolymer is a membrane protein.

13. The method of claim 12 , comprising:
contacting the defined amount of the at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein with an aqueous solution comprising the membrane protein to be crystallized to form a crystallization matrix comprising the membrane protein.

14. The method of claim 13 , wherein said aqueous solution further comprises one or more additives and/or detergents.

15. The method of claim 12 , comprising:
contacting the defined amount of the at least one matrix-forming compound capable of forming a crystallization matrix for a membrane protein with an aqueous liquid that does not comprise the membrane protein to be crystallized to form a crystallization matrix not comprising the membrane protein, and
contacting the crystallization matrix with an aqueous solution comprising the membrane protein to be crystallized.

16. The method of claim 15 , wherein said aqueous solution comprising the membrane protein further comprises one or more additives and/or detergents.

17. The method of claim 1 , wherein the biopolymer is a protein and wherein said crystallization device is sealed to protect the solid matrix-forming compound that has not yet formed a crystallization matrix and does not comprise the membrane protein from hydration.

18. The method of claim 1 , wherein said matrix-forming compound:
a) is capable of forming a meso phase; and/or
b) is capable of forming a cubic phase; and/or
c) is capable of forming a sponge phase; and/or
d) is a lipidic compound; and/or
e) is amphiphilic; and/or
f) comprises a saturated or unsaturated fatty-acid chain; and/or
g) it is an alcohol derivative from a fatty acid.

19. The method of claim 1 , wherein:
a) said matrix-forming compound is mixed with an additive thereby forming an additive composition; and/or
b) said matrix-forming compound or said additive composition comprises:
i) at least one compound selected from the group consisting of fatty acids, alcohol derivatives from fatty acids, monoglycerides, diglycerides, lipids and their derivatives, the corresponding compounds that have their acid group(s) replaced by a hydroxyl or thiol or ether or thioether group or ω-hydroxyalkenes or their ethers or homologous thiols or thioethers; monoacylglycerols, cis monounsaturated monoacylglycerols, monoolein (C18: c9), monopalmitolein (C16: c9) and monovacennin (C18: c7); medium-chain length alkyl glycosides; polyalkylenglycols, polyethylenglycols, diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof capable of forming a crystallization matrix for the membrane protein; and/or
ii) at least one compound selected from the group consisting of polyketides, saccharolipids, prenol lipids, sterol lipids, sphingolipids, glycerophospholipids and glycerolipids and/or derivates of lipids, phosphatidylcholine (PC), DOPC, phosphatidylethanolamine, DOPE, phosphatidylserine, DOPS, cardiolipin, lyso-phosphatidylcholine, 2-monoolein, oleamide, cholesterol, cell membrane components, and natural or synthetic compounds stabilizing the membrane protein in the crystallization matrix.

20. The method of claim 1 , wherein said matrix-forming compound is amphiphilic.

21. The method of claim 12 , wherein said matrix-forming compound is amphiphilic.

22. The method of claim 12 , wherein:
a) said matrix-forming compound is mixed with an additive thereby forming an additive composition; and/or
b) said matrix-forming compound or said additive composition comprises:
i) at least one compound selected from the group consisting of fatty acids, alcohol derivatives from fatty acids, monoglycerides, diglycerides, lipids and their derivatives, the corresponding compounds that have their acid group(s) replaced by a hydroxyl or thiol or ether or thioether group or ω-hydroxyalkenes or their ethers or homologous thiols or thioethers; monoacylglycerols, cis monounsaturated monoacylglycerols, monoolein (C18: c9), monopalmitolein (C16: c9) and monovacennin (C18: c7); medium-chain length alkyl glycosides; polyalkylenglycols, polyethylenglycols, diacylglycerophospholipids, monoacylglycerophospholipids and derivatives thereof capable of forming a crystallization matrix for the membrane protein; and/or
ii) at least one compound selected from the group consisting of polyketides, saccharolipids, prenol lipids, sterol lipids, sphingolipids, glycerophospholipids and g

Assignees

• Qiagen Gmbh
• Cube Biotech Gmbh

Inventors

• Kubicek Johann
• Schaefer Frank
• Labahn Joerg
• Bueldt Georg

Classifications

• C07K1/306 Primary

  by crystallization · CPC title

• B01D2009/0086

  Processes or apparatus therefor · CPC title

• B01L3/06

  Crystallising dishes · CPC title

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• B01D9/0077 Primary

  Screening for crystallisation conditions or for crystal forms · CPC title