Controlled nucleation of protein crystals

• US 6,596,077 B2
• Filed: 07/31/2001
• Issued: 07/22/2003
• Est. Priority Date: 07/31/2001
• Status: Expired due to Fees

• Current Assignee: Intellectual Ventures Holding 19 LLC (Intellectual Ventures LLC)
• Original Assignee: Illinois Institute of Technology
• Inventors: Myerson, Allan S.
• Primary Examiner(s): Kunemund, Robert M
• Application Number:
  US09/918,935
• Publication Number:
  US 20030024470A1
• Time in Patent Office: 721 Days
• Field of Search: 117/68, 117/69, 117/935, 117/936, 117/937
• US Class Current: 117/68
• 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

A method for the non-photochemical laser induced nucleation in which short high-intensity laser pulses are used to induce nucleation in supersaturated solutions including protein solutions. The laser induces nucleation only in the area where the beam is focused or passes through, resulting in fewer nuclei than would be achieved by spontaneous nucleation. In addition, the laser reduces nucleation time significantly.

Claims

1. A method of inducing the nucleation of protein crystals comprising the steps of:

a. preparing a supersaturated solution of a protein in a solvent;

b. selecting a wavelength, power and polarization state of light necessary to induce the crystallization of a protein crystal from the supersaturated solution of the protein in the solvent; and

c. subjecting the supersaturated solution to the light for a period of time so as to induce nucleation of the protein crystal.

2. The method as claimed in claim 1, wherein the supersaturated solution is subjected to laser light until at least a portion of the protein in the supersaturated solution has crystallized into the protein crystal.

3. The method as claimed in claim 2, wherein the laser light is at such a wavelength that the supersaturated solution will not absorb the laser light.

4. The method as claimed in claim 2, wherein supersaturation is achieved by a method selected from the group consisting of cooling, heating, solvent evaporation, and altering solvent composition.

5. The method as claimed in claim 1, wherein the solvent is selected from the group consisting of organic solvents, inorganic solvents, and supercritical solvents.

6. The method as claimed in claim 2, wherein the laser light is pulsed.

7. The method as claimed in claim 6, wherein the laser light pulses at between 1 pulse and 100 pulses per second.

8. The method as claimed in claim 7, wherein the laser light pulses at approximately 10 pulses per second.

9. The method as claimed in claim 1, wherein the supersaturated solution is subjected to the laser light for a period of between 0.01 second and 60 seconds.

10. The method as claimed in claim 6, wherein the laser light is in the near infrared range.

11. The method as claimed in claim 1, wherein the supersaturated solution further comprises a buffer.

12. The method as claimed in claim 11, wherein the buffer is selected from the group consisting of acetate buffer, citrate buffer, phosphate buffer, sodium acetate buffer, sodium phosphate buffer, and potassium phosphate buffer.

13. The method as claimed in claim 1, wherein the precipitant is selected from the group consisting of MES,2-(N-morpholino)-ethanesulfonic acid, ammonium acetate, glycerol, spermine, cacodylate, potassium chloride, ethanol, phosphate, HEPES, potassium phosphate, sodium citrate, sodium phosphate, calcium chloride, EDTA, dithiothreitol, sodium acetate, sodium cacodylate, magnesium chloride, sodium azide, 2-methyl-2,4-pentanediol, sodium chloride, tris-HCI, ammonium sulfate, PEG (200-35000) (with PEG 4000, PEG 6000 and PEG 8000 being used most often out of the PEGs).

14. The method as claimed in claim 1, wherein the supersaturated solution has a pH of between 2 and 12.

15. The method as claimed in claim 14, wherein the supersaturated solution has a pH of approximately 4.0.

16. The method as claimed in claim 1, wherein the supersaturated solution is maintained at a temperature of between 0 C. and 80 C.

17. The method as claimed in claim 16, wherein the supersaturated solution is maintained at a temperature of approximately 25 C.

18. The method as claimed in claim 1, wherein the supersaturated solution comprises between 0.1% and 50% protein.

19. The method as claimed in claim 1, wherein the laser light is linearly polarized.

20. The method as claimed in claim 1, wherein the laser light is circularly polarized.

21. A method of inducing the nucleation of protein crystals comprising the steps of:

a. preparing a supersaturated solution comprising a protein and a solvent;

b. selecting a state of laser light necessary to induce nucleation of a protein crystal from the supersaturated solution; and

c. subjecting the supersaturated solution to the light for a period of time so as to induce nucleation of the protein crystal.

22. The method as claimed in claim 21, wherein the supersaturated solution is subjected to laser light until at least a portion of the protein in the supersaturated solution has crystallized into the protein crystal.

23. The method as claimed in claim 22, wherein the laser light has a wavelength in the near infrared range and has a polarization selected from the group consisting of linear polarization and circular polarization.

24. The method as claimed in claim 23, wherein the laser light is pulsed.

25. The method as claimed in claim 24, wherein the laser light pulses at between 1 pulse per second and 100 pulses per second.

26. A method for controlling the nucleation of protein crystals comprising the steps of:

a. preparing a supersaturated solution of a protein in a solvent;

b. selecting a wavelength, power and polarization state of laser light necessary to induce the crystallization of a protein crystal from the supersaturated solution of the protein in the solvent;

c. subjecting the supersaturated solution to the laser light for a period of time so as to induce nucleation of the protein crystal; and

d. varying the wavelength, power and polarization state of the laser light so as to control the parameters of the nucleation of the protein crystals.

27. The method as claimed in claim 26, wherein the supersaturated solution is subjected to laser light until at least a portion of the protein in the supersaturated solution has crystallized into at least one protein crystal.

28. The method as claimed in claim 27, wherein the laser light is pulsed.

29. The method as claimed in claim 28, wherein the laser light pulses at between 1 pulse and 100 pulses per second.

30. The method as claimed in claim 26, wherein the supersaturated solution is subjected to the laser light for a period of between 0.01 second and 60 seconds.

31. The method as claimed in claim 26, wherein the laser light is in the near infrared range.

32. The method as claimed in claim 26, wherein the laser light is linearly polarized.

33. The method as claimed in claim 26, wherein the laser light is circularly polarized.