Methods for capturing nascent proteins

• US 20100075374A1
• Filed: 10/31/2008
• Published: 03/25/2010
• Est. Priority Date: 11/08/2007
• Status: Abandoned Application

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Rothschild, Kenneth J., Lim, Mark J.
• Application Number:
  US12/290,783
• Publication Number:
  US 20100075374A1
• Time in Patent Office: Days
• US Class Current: 435/68.100
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione

Abstract

Methods of generating and capturing nascent proteins are described, including capturing nascent proteins on the same bead that comprises nucleic acid encoding the protein. The protein or nucleic acid can be sequenced before or after phototransfer to another surface.

Claims

20. A method of generating and capturing nascent proteins, comprising:

(a) providing i) nucleic acid encoding a protein or fragment thereof, ii) a plurality of beads, each bead comprising one or more amplification primers and one or more first binding molecules, and iii) a population of second binding molecules capable of binding to said protein and said first binding molecules;

(b) contacting said beads with said nucleic acid under conditions such that at least a portion of said nucleic acid is amplified to create treated beads comprising immobilized amplified nucleic acid;

(c) contacting said treated beads with said second binding molecules under conditions such that at least a portion of said first binding molecules bind to at least a portion of said second binding molecules so as to create capture beads; and

(d) producing nascent protein or fragments thereof from at least a portion of said immobilized amplified nucleic acid on said capture beads by cell free expression, at least a portion of said nascent protein or fragments thereof interacting with at least a portion of said second binding molecules so as to generate loaded beads comprising captured nascent protein or captured fragments thereof.

1. A method of generating and capturing nascent proteins, comprising:

(a) providing nucleic acid encoding a protein, and a plurality of beads, each bead comprising one or more amplification primers;

(b) contacting said beads with said nucleic acid under conditions such that at least a portion of said nucleic acid is amplified to create treated beads comprising immobilized amplified nucleic acid; and

(c) producing nascent protein from at least a portion of said immobilized amplified nucleic acid on said treated beads by cell free expression to create expressed beads, wherein at least a portion of said nascent protein is captured on said expressed beads.

2. The method of claim 1, wherein each of said beads, prior to step c), comprises a plurality of first binding agents on the bead surface, said first binding agents capable of binding said nascent protein.

3. The method of claim 2, wherein said first binding agents comprise chemical moieties.

4. The method of claim 3, wherein said chemical moieties are selected from the group consisting of amines, sulfhydryls, carboxyls, epoxy, and aldehyde moieties.

5. The method of claim 2, wherein said first binding agents are selected from the group consisting of antibodies, aptamers, streptavidin and avidin.

6. The method of claim 1, wherein each of said beads, prior to step c), comprises a plurality of first binding agents on the bead surface, said first binding agents capable of binding a plurality of second binding agents, said second binding agents capable of binding said nascent protein.

7. The method of claim 1, wherein each of said beads, prior to step c), comprises a plurality of first binding agents on the bead surface, said first binding agents capable of binding a plurality of second binding agents, said second binding agents capable of binding a plurality of third binding agents, said third binding agents capable of binding said nascent protein.

8. The method of claim 7, wherein said first binding agent comprises biotin, said second binding agent comprises avidin, and said third binding agent comprises biotinylated antibody.

9. The method of claim 8, further comprising, before or after step c), sequencing at least a portion of said immobilized amplified nucleic acid.

10. The method of claim 1, further comprising, after step c), sequencing at least a portion of said nascent protein.

11. The method of claim 1, wherein said contacting of step b) results in at least a portion of said nucleic acid annealing to said one or more amplification primers.

12. The method of claim 11, wherein after said annealing at least a portion of said primers are extended.

13. The method of claim 12, wherein said conditions comprise use of a polymerase.

14. The method of claim 13, wherein after said primers are extended, the beads are treated under denaturing conditions.

15. The method of claim 1, further comprising, after step c), determining whether said nascent protein comprises truncated protein.

16. The method of claim 1, further comprising, after step c), transferring at least a portion of said nascent protein to a non-bead solid support, so as to create transferred nascent protein.

17. The method of claim 16, further comprising the step of sequencing at least a portion of said transferred nascent protein.

18. The method of claim 1, further comprising, after step c), transferring at least a portion of said nascent protein and at least a portion of said amplified nucleic acid to a non-bead solid support, so as to create transferred nascent protein and transferred nucleic acid.

19. The method of claim 18, further comprising the step of sequencing at least a portion of said transferred nucleic acid.

21. The method of claim 20, wherein said first binding agents comprise chemical moieties.

22. The method of claim 21, wherein said chemical moieties are selected from the group consisting of amines, sulfhydryls, carboxyls, epoxy, and aldehyde moieties.

23. The method of claim 20, wherein said first binding agents comprise biotin.

24. The method of claim 20, wherein said second binding agents comprise antibody having affinity for said nascent protein or fragments thereof.

25. The method of claim 20, further comprising e) transferring at least a portion of said captured nascent protein to a non-bead solid support, so as to create transferred nascent protein.

26. The method of claim 25, further comprising f) sequencing at least a portion of said transferred nascent protein.

27. The method of claim 20, further comprising e) transferring at least a portion of said captured nascent protein and at least a portion of said amplified nucleic acid, so as to create transferred nascent protein and transferred nucleic acid.

28. The method of claim 27, further comprising f) sequencing at least a portion of said transferred nucleic acid.

29. The method of claim 20, further comprising after step d) determining whether said nascent protein comprises truncated protein.

30. The method of claim 23, wherein said biotin is linked to said beads via a photocleavable linker.

31. The method of claim 30, further comprising:

(e) treating said captured nascent protein so as to release at least a portion from said loaded beads so as to create free nascent protein.

32. The method of claim 31, wherein said treating of step e) comprises exposing said photocleavable linker to light.

33. The method of claim 20, wherein each bead of step (a) comprises a forward and a reverse PCR primer.

34. The method of claim 33, wherein prior to step (a) said forward and reverse PCR primers comprised 5′

amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups.

24-1. The method of claim 20, further comprising e) sequencing at least a portion of said nascent protein.

35. A method of generating and capturing nascent proteins, comprising:

(a) providing nucleic acid encoding a protein or fragment thereof, a plurality of beads, each bead comprising one or more amplification primers and one or more first binding molecules, a population of second binding molecules capable of binding to said first binding molecules, and a population of third binding molecules capable of binding to said second binding molecules and said protein or fragment thereof;

(b) contacting said beads with said nucleic acid under conditions such that at least a portion of said nucleic acid is amplified to create treated beads comprising immobilized amplified nucleic acid;

(c) contacting said treated beads with said second binding molecules under conditions such that at least a portion of said first binding molecules bind to at least a portion of said second binding molecules so as to create conjugated beads;

(d) contacting said conjugated beads with said third binding molecules under conditions such that at least a portion of said second binding molecules bind to at least a portion of said third binding molecules so as to create capture beads; and

(e) producing nascent protein or fragment thereof from at least a portion of said immobilized amplified nucleic acid on said capture beads by cell free expression, at least a portion of said nascent protein or fragment thereof interacting with at least a portion of said third binding molecules so as to generate loaded beads comprising captured nascent protein or captured fragment thereof.

36. The method of claim 35, further comprising f) sequencing at least a portion of said nascent protein.

37. The method of claim 35, further comprising f) transferring at least a portion of said captured nascent protein to a non-bead solid support, so as to create transferred nascent protein.

38. The method of claim 37 further comprising g) sequencing at least a portion of said transferred nascent protein.

39. The method of claim 35, further comprising f) transferring at least a portion of said captured nascent protein and at least a portion of said amplified nucleic acid, so as to create transferred nascent protein and transferred nucleic acid.

40. The method of claim 39, further comprising g) sequencing at least a portion of said transferred nucleic acid.

41. The method of claim 35, further comprising after step e) determining whether said nascent protein comprises truncated protein.

42. The method of claim 35, wherein said first binding molecules comprise biotin, said second binding molecules comprise streptavidin and said third binding molecules comprise biotinylated antibody, said antibody having affinity for said nascent protein or fragment thereof.

43. The method of claim 42, wherein said biotinylated antibody comprises biotin linked via a photocleavable linker to said antibody.

44. The method of claim 43, further comprising:

(f) treating said captured nascent protein or fragment thereof so as to release at least a portion from said loaded beads so as to create free nascent protein or free fragment thereof.

45. The method of claim 44, wherein said treating of step f) comprises exposing said photocleavable linker to light.

46. The method of claim 35, wherein each bead of step (a) comprises a forward and a reverse PCR primer.

47. The method of claim 46, wherein prior to step (a) said forward and reverse PCR primers comprised 5′

amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups.

48. A method of generating and capturing truncated protein, comprising:

(a) providing nucleic acid encoding a truncated protein, a plurality of beads, each bead comprising one or more amplification primers and one or more first binding molecules, a population of second binding molecules capable of binding to said first binding molecules, and a population of third binding molecules capable of binding to said second binding molecules and capturing said truncated protein;

(b) contacting said beads with said nucleic acid under conditions such that at least a portion of said nucleic acid is amplified to create treated beads comprising immobilized amplified nucleic acid;

(c) contacting said treated beads with said second binding molecules under conditions such that at least a portion of said first binding molecules bind to at least a portion of said second binding molecules so as to create conjugated beads;

(d) contacting said conjugated beads with said third binding molecules under conditions such that at least a portion of said second binding molecules bind to at least a portion of said third binding molecules so as to create capture beads; and

(e) producing truncated protein from at least a portion of said immobilized amplified nucleic acid on said capture beads by cell free expression, said third binding molecules capturing at least a portion of said truncated protein so as to generate loaded beads comprising captured truncated protein.

49. The method of claim 48, further comprising f) sequencing at least a portion of said nascent protein.

50. The method of claim 48, further comprising f) transferring at least a portion of said captured nascent protein to a non-bead solid support, so as to create transferred nascent protein.

51. The method of claim 50, further comprising g) sequencing at least a portion of said transferred nascent protein.

52. The method of claim 48, further comprising f) transferring at least a portion of said captured nascent protein and at least a portion of said amplified nucleic acid, so as to create transferred nascent protein and transferred nucleic acid.

53. The method of claim 52, further comprising g) sequencing at least a portion of said transferred nucleic acid.

54. The method of claim 52, further comprising, prior to step f), sequencing at least a portion of said amplified nucleic acid.

55. The method of claim 48, further comprising after step e) determining whether said nascent protein comprises truncated protein.

56. The method of claim 48, wherein said first binding molecules comprise biotin, said second binding molecules comprise streptavidin and said third binding molecules comprise biotinylated antibody.

57. The method of claim 56, wherein said biotinylated antibody comprises biotin linked via a photocleavable linker to said antibody.

58. The method of claim 57, further comprising:

(f) treating said captured truncated protein so as to release at least a portion from said loaded beads so as to create free truncated protein.

59. The method of claim 58, wherein said treating of step f) comprises exposing said photocleavable linker to light.

60. The method of claim 48, wherein each bead of step (a) comprises a forward primer encoding a first epitope and a reverse PCR primer encoding a second epitope.

61. The method of claim 60, wherein prior to step (a) said forward and reverse PCR primers comprised 5′

amine modifications and were attached to agarose beads comprising a plurality of primary amine reactive functional groups.

62. The method of claim 48, wherein at least a portion of said truncated protein is encoded by a portion of the APC gene.