Methods for enriching subpopulations

• US 20090264298A1
• Filed: 11/04/2008
• Published: 10/22/2009
• Est. Priority Date: 11/06/2007
• Status: Abandoned Application

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Rothschild, Kenneth J., Lim, Mark J.
• Application Number:
  US12/290,801
• Publication Number:
  US 20090264298A1
• Time in Patent Office: Days
• US Class Current: 506/1
• CPC Class Codes: C12N 15/1062 mRNA-Display, e.g. polypeptide and encoding template are connected covalently
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms

Abstract

Methods of enriching a subpopulation of beads are described. In one embodiment, first beads comprise immobilized first amplified product, said first amplified product encoding a truncated version of a first protein, and second beads comprise immobilized second amplified product, said second amplified product encoding an untruncated version of said first protein. Both first and second beads are exposed to a translation system under conditions such that said truncated and untruncated versions of said first protein are generated from at least a portion of said first and second immobilized amplified products, and these protein products are captured on the first and second beads, respectively. Using a ligand (e.g. with affinity for the untruncated version of said first protein), a portion of the second beads is separated from the mixture, thereby enriching a subpopulation of beads comprising truncated protein.

Claims

1. A method of enriching a subpopulation of beads in a mixture, comprising:

a) providing a mixture comprising i) a plurality of first beads, said first beads comprising immobilized first amplified product, said first amplified product encoding a first nascent protein or fragment thereof, and ii) a plurality of second beads, said second beads comprising immobilized second amplified product, said second amplified product encoding a second nascent protein or fragment thereof;

b) exposing said mixture to translation system under conditions such that said first and second nascent proteins or fragments thereof are generated from at least a portion of said first and second immobilized amplified products, c) capturing at least a portion of said first nascent protein or fragment thereof on said first bead and capturing at least a portion of said second nascent protein or fragment thereof on said second bead, so as to create a mixture of beads comprising captured proteins or fragments thereof; and

d) contacting said mixture of beads comprising captured proteins or fragments thereof with a ligand with affinity for said first nascent protein or fragment thereof, said contacting performed under conditions such that at least a portion of said first beads are separated from said mixture, thereby enriching a subpopulation of beads.

2. The method of claim 1, wherein each of said beads, prior to step c), comprises a plurality of reactive chemical moieties on the bead surface, said moieties capable of reacting with said nascent protein.

3. The method of claim 1, wherein said ligand comprises an antibody.

4. The method of claim 3, wherein said antibody is attached to magnetic beads.

5. The method of claim 4, wherein said conditions of step d) comprise exposure of said mixture to a magnet.

6. The method of claim 5, wherein said exposure to a magnet creates precipitated beads and a supernatant.

7. The method of claim 6, wherein said conditions of step d) further comprise removing said supernatant, so as to create an isolated precipitate.

8. The method of claim 6, wherein said conditions of step d) further comprise removing said precipitated beads, so as to create a depleted supernatant.

9. The method of claim 1, wherein the ratio of first beads to second beads in said mixture of step a) is 50:

50.

10. The method of claim 7, wherein said isolated precipitate is contaminated with less than 30% of second beads.

11. The method of claim 7, wherein said isolated precipitate is contaminated with less than 10% of second beads.

12. The method of claim 7, wherein said isolated precipitate is contaminated with less than 1% of second beads.

13. The method of claim 1, wherein said mixture of step a) further comprises a plurality of third beads.

14. The method of claim 13, wherein said third beads lack immobilized amplified product.

15. The method of claim 13, wherein said third beads comprise immobilized third amplified product, said third amplified product encoding a third nascent protein or fragment thereof.

16. The method of claim 1, wherein said first immobilized amplified product comprises at least a portion of a cancer-related gene.

17. The method of claim 16, wherein said cancer-related gene is selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, and the p53 gene.

18. The method of claim 1, wherein the number of second beads in said mixture is less than the number of said first beads.

19. A method of enriching a subpopulation of beads in a mixture, comprising:

a) providing a mixture comprising i) a plurality of first beads, said first beads comprising immobilized first amplified product, said first amplified product encoding a first nascent protein or fragment thereof, and ii) a plurality of second beads, said second beads comprising immobilized second amplified product, said second amplified product encoding a second nascent protein or fragment thereof;

b) exposing said mixture to a translation system under conditions such that said first and second nascent proteins or fragments thereof are generated from said first and second immobilized amplified products, c) capturing said first nascent protein or fragment thereof on said first bead and capturing said second nascent protein or fragment thereof on said second bead, so as to create a mixture of beads comprising captured proteins or fragments thereof;

d) contacting said mixture of beads comprising captured proteins or fragments thereof with a first ligand with affinity for said first nascent protein or fragment thereof, so as to create a mixture of treated beads;

e) contacting said mixture of treated beads with a second ligand, said second ligand having affinity for said first ligand, said contacting performed under conditions such that at least a portion of said first beads are separated from said mixture, thereby enriching a subpopulation of beads.

20. The method of claim 19, wherein each of said beads, prior to step c), comprises a plurality of reactive chemical moieties on the bead surface, said moieties capable of reacting with said nascent protein.

21. The method of claim 19, wherein said first ligand comprises a first antibody.

22. The method of claim 21, wherein said second ligand comprises a second antibody.

23. The method of claim 22, wherein said second antibody is attached to magnetic beads.

24. The method of claim 23, wherein said conditions of step e) comprise exposure of said mixture to a magnet.

25. The method of claim 24, wherein said exposure to a magnet creates precipitated beads and a supernatant.

26. The method of claim 25, wherein said conditions of step e) further comprise removing said supernatant, so as to create an isolated precipitate.

27. The method of claim 25, wherein said conditions of step e) further comprise removing said precipitated beads, so as to create a depleted supernatant.

28. The method of claim 19, wherein the ratio of first beads to second beads in said mixture of step a) is 50:

50.

29. The method of claim 26, wherein said isolated precipitate is contaminated with less than 30% of second beads.

30. The method of claim 26, wherein said isolated precipitate is contaminated with less than 10% of second beads.

31. The method of claim 26, wherein said isolated precipitate is contaminated with less than 1% of second beads.

32. The method of claim 19, wherein said mixture of step a) further comprises a plurality of third beads.

33. The method of claim 32, wherein said third beads lack immobilized amplified product.

34. The method of claim 32, wherein said third beads comprise immobilized third amplified product, said third amplified product encoding a third nascent protein or fragment thereof.

35. The method of claim 19, wherein said first immobilized amplified product comprises at least a portion of a cancer-related gene.

36. The method of claim 35, wherein said cancer-related gene is selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, and the p53 gene.

37. The method of claim 19, wherein the number of second beads in said mixture is less than the number of said first beads.

38. A method of enriching a subpopulation of beads, comprising:

a) providing a mixture comprising i) a plurality of first beads, said first beads comprising immobilized first amplified product, said first amplified product encoding a truncated version of a first protein, and ii) a plurality of second beads, said second beads comprising immobilized second amplified product, said second amplified product encoding an untruncated version of said first protein, wherein the number of first beads in said mixture is less than the number of said second beads;

b) exposing said mixture to a translation system under conditions such that said truncated and untruncated versions of said first protein are generated from at least a portion of said first and second immobilized amplified products, c) capturing said truncated version of said first protein on said first bead and capturing said untruncated version of said first protein on said second bead, so as to create a mixture of beads comprising captured proteins or truncated fragments thereof; and

d) contacting said mixture of beads comprising captured proteins or truncated fragments thereof with a ligand with affinity for said untruncated version of said first protein, so as to create a mixture of treated beads, said contacting performed under conditions such that at least a portion of said second beads are separated from said mixture, thereby enriching a subpopulation of beads comprising truncated protein.

39. The method of claim 38, wherein each of said beads, prior to step c), comprises a plurality of reactive chemical moieties on the bead surface, said moieties capable of reacting with and capturing said nascent protein.

40. The method of claim 38, wherein said ligand comprises an antibody.

41. The method of claim 40, wherein said antibody has affinity for a region of said untruncated version of said first protein that is lacking in said truncated protein.

42. The method of claim 41, wherein said antibody is attached to magnetic beads.

43. The method of claim 42, wherein said conditions of step d) comprise exposure of said mixture to a magnet.

44. The method of claim 43, wherein said exposure to a magnet creates precipitated beads and a supernatant.

45. The method of claim 44, wherein said conditions of step d) further comprise removing said supernatant, so as to create an isolated precipitate.

46. The method of claim 44, wherein said conditions of step d) further comprise removing said precipitated beads, so as to create a depleted supernatant.

47. The method of claim 38, wherein the ratio of first beads to second beads in said mixture of step a) is less than 1:

10.

48. The method of claim 45, wherein said isolated precipitate is contaminated with less than 5% of said first beads.

49. The method of claim 45, wherein said isolated precipitate is contaminated with less than 2% of said first beads.

50. The method of claim 45, wherein said isolated precipitate is contaminated with less than 1% of said first beads.

51. The method of claim 38, wherein said mixture of step a) further comprises a plurality of third beads.

52. The method of claim 51, wherein said third beads lack immobilized amplified product.

53. The method of claim 51, wherein said third beads comprise immobilized third amplified product, said third amplified product encoding a third nascent protein or fragment thereof.

54. The method of claim 38, wherein said first immobilized amplified product comprises at least a portion of a cancer-related gene.

55. The method of claim 54, wherein said cancer-related gene is selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, and the p53 gene.

56. A method of enriching a subpopulation of beads, comprising:

a) providing a mixture comprising i) a plurality of first beads, said first beads comprising immobilized first amplified product, said first amplified product encoding a truncated version of a first protein, and ii) a plurality of second beads, said second beads comprising immobilized second amplified product, said second amplified product encoding an untruncated version of said first protein, wherein the number of first beads in said mixture is less than the number of said second beads;

b) exposing said mixture to a translation system under conditions such that said truncated and untruncated versions of said first protein are generated from at least a portion of said first and second immobilized amplified products, c) capturing said truncated version of said first protein on said first bead and capturing said untruncated version of said first protein on said second bead, so as to create a mixture of beads comprising captured proteins or truncated fragments thereof;

d) contacting said mixture of beads comprising captured proteins or truncated fragments thereof with a first ligand with affinity for said untruncated version of said first protein, so as to create a mixture of treated beads; and

e) contacting said mixture of treated beads with a second ligand, said second ligand having affinity for said first ligand, said contacting performed under conditions such that at least a portion of said first beads are separated from said mixture, thereby enriching a subpopulation of beads comprising truncated protein.

57. The method of claim 56, wherein each of said beads, prior to step c), comprises a plurality of reactive chemical moieties on the bead surface, said moieties capable of reacting with said nascent protein.

58. The method of claim 56, wherein said first ligand comprises a first antibody.

59. The method of claim 58, wherein said antibody has affinity for a region of said untruncated version of said first protein that is lacking in said truncated protein.

60. The method of claim 59, wherein said second ligand comprises a second antibody.

61. The method of claim 60, wherein said second antibody is attached to magnetic beads.

62. The method of claim 61, wherein said conditions of step e) comprise exposure of said mixture to a magnet.

63. The method of claim 62, wherein said exposure to a magnet creates precipitated beads and a supernatant.

64. The method of claim 63, wherein said conditions of step e) further comprise removing said supernatant, so as to create an isolated precipitate.

65. The method of claim 63, wherein said conditions of step e) further comprise removing said precipitated beads, so as to create a depleted supernatant.

66. The method of claim 56, wherein the ratio of first beads to second beads in said mixture of step a) is less than 1:

10.

67. The method of claim 64, wherein said isolated precipitate is contaminated with less than 5% of said first beads.

68. The method of claim 64, wherein said isolated precipitate is contaminated with less than 2% of said first beads.

69. The method of claim 64, wherein said isolated precipitate is contaminated with less than 1% of said first beads.

70. The method of claim 56, wherein said mixture of step a) further comprises a plurality of third beads.

71. The method of claim 70, wherein said third beads lack immobilized amplified product.

72. The method of claim 70, wherein said third beads comprise immobilized third amplified product, said third amplified product encoding a third nascent protein or fragment thereof.

73. The method of claim 56, wherein said first immobilized amplified product comprises at least a portion of a cancer-related gene.

74. The method of claim 73, wherein said cancer-related gene is selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, and the p53 gene.