Methods and apparatus for rapid crystallization of biomolecules

• US 7,459,021 B2
• Filed: 09/02/2004
• Issued: 12/02/2008
• Est. Priority Date: 09/03/2003
• Status: Expired due to Fees

• Current Assignee: Shmuel Bukshpan
• Original Assignee: Shmuel Bukshpan
• Inventors: Bukshpan, Shmuel
• Primary Examiner(s): Kunemund; Robert M
• Application Number:
  US10/561,244
• Publication Number:
  US 20060137603A1
• Time in Patent Office: 1,552 Days
• Field of Search: 117/68, 117/69, 117/70, 422/245.1
• US Class Current: 117/68
• CPC Class Codes: C07K 1/306 by crystallization
• CPC Class Codes: C30B 29/58 Macromolecular compounds
• CPC Class Codes: C30B 7/00 Single-crystal growth from solutions using solvents which are liquid at normal temperature, e.g. aqueous solutions from molten solvents C30B9/00; by normal or gradient freezing C30B11/00; under a protective fluid C30B27/00

Abstract

The present invention relates to methods and apparatus for promoting rapid formation of biomolecule crystals from a solution of biomolecules, preferably proteins, wherein the protein solution undergoes rapid concentration according to its isoelectric point in an electric field. Protein crystallization according to the methods of the present invention takes place within a period of hours or less.

Claims

1. A method for rapid crystallization of biomolecules, comprising:

(a) providing at least one biomolecules species;

(b) providing at least one crystallization reactor comprising an isoelectric focusing buffer having a pH range, the pH range encompassing the pI of the at least one biomolecule species;

(c) bringing said at least one biomolecule species into contact with the at least one crystallization reactor;

(d) introducing an electric field at said at least one crystallization reactor thereby generating a concentrated solution of said at least one biomolecule species; and

(e) obtaining at least one crystal within said at least one crystallization reactor;

wherein the electric field is within the range of 50-2,000 V/cm.

2. The method according to claim 1, wherein step (c) further comprises depositing the at least one crystallization reactor and the at least one biomolecule species in running buffer.

3. The method according to claim 1, wherein step (e) further comprises extracting a biomolecule crystal from said at least one crystallization reactor.

4. The method according to claim 1, wherein the crystallization occurs within less than 12 hours.

5. The method according to claim 1, wherein the at least one biomolecule species is selected from the group consisting of peptides, proteins, glycoproteins, polypeptides, enzymes, antibodies, protein-DNA complexes, protein complexes comprising chemical entities, polynucleotides, DNA, RNA, antigens, antigenic epitopes and variants thereof, hormones, carbohydrates, lipids, phospholipids and biotinylated probes.

6. The method according to claim 5, wherein the biomolecule species is a protein.

7. The method according to claim 6, wherein the protein is recombinant.

9. The method according to claim 1, wherein the at least one crystallization reactor is provided within a capillary.

11. The method according to claim 1, wherein the isoelectric focusing buffer further comprises a polymer.

12. The method according to claim 11, wherein the polymer is selected from the group consisting of:

linear polymers, branched polymers, polyacrylamide, agarose, hydrogels, cellulose, modified cellulose, cross-linked polyvinyl alcohol, cross-linked polyethylene oxide and glycol polymer.

13. The method according to claim 1, wherein the isoelectric focusing buffer has a pH range of no more than 0.2 pH units.

14. The method according to claim 1, wherein in step (b) a plurality of crystallization reactors is provided, each crystallization reactor comprising an isoelectric focusing buffer.

15. The method according to claim 14, wherein a distinct protein species is crystallized in each crystallization reactor and wherein each distinct protein species has a distinct isoelectric point.

16. The method according to claim 14, wherein the isoelectric focusing buffers in the plurality of crystallization reactors are different from one another.

17. The method according to claim 14, wherein the crystallization reactors are isolated from one another.

18. The method according to claim 14, wherein the plurality of crystallization reactors are linked, joined, or substantially contiguous to a substrate.

19. The method according to claim 1, wherein the crystallization reactor is selected from the group consisting of:

immobilized pH gradient strips, pH membranes and pre-cast gels.

20. The method according to claim 1, further comprising prior to step (a) the step of sorting a solution comprising at least one biomolecule species.

8. A method for rapid crystallization of biomolecules, comprising:

(a) providing at least one biomolecules species;

b) providing at least one crystallization reactor comprising an isoelectric focusing buffer having a pH range, the pH range encompassing the pI of the at least one biomolecule species;

(c) bringing said at least one biomolecule species into contact with the at least one crystallization reactor;

(d) introducing an electric field at said at least one crystallization reactor thereby generating a concentrated solution of said at least one biomolecule species; and

(e) obtaining at least one crystal within said at least one crystallization reactor;

wherein the at least one biomolecule species in step (a) is immobilized onto a substrate comprising a porous web material.

10. A method for rapid crystallization of biomolecules, comprising:

(a) providing at least one biomolecules species;

(b) providing at least one crystallization reactor comprising an isoelectric focusing buffer having a pH range, the pH range encompassing the pI of the at least one biomolecule species;

(c) bringing said at least one biomolecule species into contact with the at least one crystallization reactor;

(d) introducing an electric field at said at least one crystallization reactor thereby generating a concentrated solution of said at least one biomolecule species; and

(e) obtaining at least one crystal within said at least one crystallization reactor;

wherein the at least one crystallization reaction is linked, joined, or substantially contiguous to a solid substrate comprising a porous web material.

21. A method for sorting a solution comprising a plurality of biomolecules and rapidly crystallizing at least one biomolecule species, comprising:

(a) providing a medium comprising a plurality of biomolecules;

(b) sorting the plurality of biomolecules on a substrate, thereby obtaining at least one locus on the substrate comprising at least one biomolecule species;

(c) recovering a portion from said substrate, the portion comprising the at least one locus;

(d) providing at least one crystallization reactor comprising an isoelectric focusing buffer having a pH range, the pH range encompassing the pI of the at least biomolecule;

(e) bringing the portion of (c) into contact with the at least one crystallization reactor;

(f) introducing an electric field at the at least one crystallization reactor thereby generating within said at least one crystallization reactor a concentrated solution of said at least one biomolecule species; and

(g) obtaining at least one crystal within said at least one crystallization reactor of (f).

22. The method according to claim 21, wherein step (b) is carried out by a method selected from the group consisting of:

isoelectric focusing, thin layer chromatography, including High Performance Liquid Chromatography (HPLC) techniques, and gel electrophoresis.

23. The method according to claim 22, wherein the method is performed under non-denaturing conditions.

24. The method according to claim 21, wherein sorting in step (b) is by the mass of the at least one biomolecule species.

25. The method according to claim 21, wherein step (e) further comprises depositing said portion and the at least one crystallization reactor in running buffer.

26. The method according to claim 21, wherein the at least one biomolecule species is a protein.

27. The method according to claim 21, wherein the electric field is within the range of 50-2,000 V/cm and the crystals are obtained within less than 12 hours.

28. The method according to claim 21, wherein the isoelectric focusing buffer comprises a polymer selected from the group consisting of:

polyacrylamide, agarose, hydrogels, cellulose, nitrocellulose, modified cellulose, cross-linked polyvinyl alcohol, cross-linked polyethylene oxide and glycol polymer.

29. The method according to claim 21, wherein the substrate in step (b) is a gel.

30. The method according to claim 21, wherein in step (d) a plurality of crystallization reactors comprising a plurality of isoelectric focusing buffers is provided, each isoelectric focusing buffer establishing a pH range, wherein at least one isoelectric focusing buffer establishes a pH range encompassing the pI of the at least one biomolecule.

31. The method according to claim 30, wherein each isoelectric focusing buffer comprises a polymer.

32. The method according to claim 30, wherein the plurality of crystallization reactors are isolated from one another.

33. The method according to claim 30, wherein the plurality of crystallization reactors are linked, joined, or substantially contiguous to a substrate comprising a porous web material.

34. The method according to claim 21, wherein the crystallization reactor is selected from the group consisting of:

immobilized pH gradient strips, pH membranes and pre-cast gels.

35. An apparatus suitable for inducing rapid formation of biomolecule crystals, comprising:

(a) a buffer chamber having an upper side and a lower side, the lower side being sealed with a bottom such that the buffer chamber encloses at least one buffer compartment capable of holding fluids;

(b) at least one crystallization reactor contained within said buffer chamber, the at least one crystallization reactor comprising an isoelectric focusing buffer;

(c) a device for generating an electrical field;

(d) two salt bridges having two ends, one end of each salt bridge being in contact with one end of the holder and one end of each salt bridge being contained within the at least one buffer chamber; and

, optionally,

(e) means for circulating fluids contained within the at least one buffer compartment.

36. The apparatus according to claim 35, wherein component (b) is a holder having two ends, an upper side and a lower side, the holder encompasses at least one crystallization reactor, the at least one crystallization reactor comprising an isoelectric focusing buffer, wherein the holder is contained within the buffer compartment.

37. The apparatus according to claim 35, comprising two buffer chambers, each buffer chamber enclosing one end of one salt bridge.

38. The apparatus according to claim 35, further comprising a temperature-controlled module enabling to manage the temperature at the at least one crystallization reactor.

39. The apparatus according to claim 35, wherein component (b) comprises a holder adapted for supporting a substrate and comprising at least one crystallization reactor.

40. The apparatus according to claim 39, wherein the holder is a capillary adapted for comprising at least one crystallization reactor.

41. The apparatus according to claim 39, wherein the holder encompasses at least one cavity wherein the at least one cavity is adapted for containing a crystallization reactor.

42. The apparatus according to claim 41, wherein the crystallization reactor is selected from the group consisting of:

immobilized pH gradient strips, ph membranes and pre-cast gels.

43. The apparatus according to claim 35, wherein the buffer chamber comprises a non-conductive material.

44. The apparatus to claim 39, wherein the holder comprises a material having a larger resistance than that of the polymer comprised within the crystallization reactor.

45. The apparatus according to claim 43, wherein the non-conductive material is selected from the group consisting of:

ply-N-methyl methacrylamide, acrylic, lucite, polystyrene, ceramic, glass and poly-methyl-methacrylate.

46. The apparatus according to claim 39, wherein the holder comprises a material that is impermeable to biomolecules.

47. The apparatus according to claim 35, wherein the device for generating an electrical field comprises a plurality of electrodes.

48. The apparatus according to claim 47, wherein the plurality of electrodes comprises a metal selected from the group consisting of:

platinum, titanium, chromium, gold, tantalum, palladium, palladium oxide, germanium, nickel and rhodium or alloys comprising same.

49. The apparatus according to claim 35, wherein the device for generating an electrical field supplies DC or AC currents and generates an electric field within the range of 50-2,000 V/cm.

50. The apparatus according to claim 35, wherein the buffer compartment is adapted for holding a solution comprising running buffer and at least one biomolecule dissolved with the running buffer.

51. The apparatus according to claim 35, further being automated.

52. The apparatus according to claim 35, wherein the biomolecule is selected from the group consisting of:

peptides, proteins, glycoproteins, polypeptides, enzymes, antibodies, protein-DNA complexes, protein complexes comprising chemical entities, polynucleotides, DNA, RNA, antigens, antigenic epitopes and variants thereof, hormones, carbohydrates, lipids, phospholipids and biotinylated probes.

53. The apparatus according to claim 52, wherein the biomolecule is a protein.

54. The apparatus according to claim 35, wherein crystallization occurs within less than 12 hours.

55. The apparatus according to claim 47, comprising two buffer chambers, each buffer chamber enclosing one electrode.