Bead-ligand-nascent protein complexes

• US 20100062451A1
• Filed: 11/06/2008
• Published: 03/11/2010
• Est. Priority Date: 11/06/2007
• Status: Active Grant

• Current Assignee: Ambergen Inc.
• Original Assignee: Ambergen Inc.
• Inventors: Rothschild, Kenneth J., Lim, Mark J.
• Granted Patent: US 8,481,263 B2
• Time in Patent Office: Days
• US Class Current: 435/7.100
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Bead-ligand-nascent protein complexes, and method of creating and detecting a bead-ligand-nascent protein complexes, are described. PCR-amplified product which is attached to a surface, e.g. of a bead, is used to generate nascent protein, which in turn is captured on the bead and detected, e.g. by fluorescence.

Claims

1. A method, comprising:

a) providing i) a population of template molecules, each template molecule encoding a nascent protein or protein fragment, and ii) at least one surface comprising forward and reverse PCR primers attached to said surface;

b) amplifying at least a portion of said population of template molecules so as to create amplified product attached to said surface;

c) generating nascent protein or protein fragment from said amplified product, said nascent protein or protein fragment comprising an affinity tag or first epitope, and d) capturing said nascent protein or protein fragment on said surface via a first ligand, said first ligand attached to said bead and reactive with said affinity tag or first epitope.

2. The method of claim 1, wherein said at least one surface is on a bead.

3. The method of claim 1, wherein said first ligand is attached to said bead after step b) and prior to step c).

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

5. The method of claim 1, wherein said first ligand comprises a metal chelator.

6. The method of claim 1, wherein said affinity tag comprises biotin and said first ligand is selected from the group consisting of avidin and streptavidin.

7. The method of claim 4, wherein said antibody is attached to said bead through a biotin-streptavidin linkage.

8. The method of claim 1, wherein said amplifying of step b) comprises i) mixing a plurality of beads in solution with said template under conditions such that at least a portion of said template hybridizes to at least a portion of said PCR primers on at least a portion of said beads to create hybridized primers, ii) extending at least a portion of said hybridized primers to created treated beads, iii) washing said treated beads so as to create washed beads, said washed beads being substantially free of template, and iv) thermally cycling said washed beads in the presence of amplification reagents.

9. The method of claim 8, wherein said amplification reagents comprise a thermostable polymerase.

10. The method of claim 1, wherein the nascent protein or fragment thereof generated in step c) is generated in a cell-free translation reaction.

11. The method of claim 10, wherein said affinity tag is introduced into said nascent protein during said translation reaction.

12. The method of claim 4, wherein said antibody reacts with said first epitope on said nascent protein.

13. The method of claim 1 wherein nucleic acid encoding said first epitope is introduced during amplification in step b).

14. The method of claim 1, wherein said first epitope is encoded by a nucleic acid sequence of one of said PCR primers.

15. The method of claim 1, wherein said forward PCR primer comprises i) a sequence corresponding to a promoter, ii) a sequence corresponding to a ribosome binding site, iii) a start codon, and iv) said sequence coding for said first epitope.

16. The method of claim 15, wherein said forward PCR primer further comprises v) a sequence complementary to at least a portion of said template molecules.

17. The method of claim 1, wherein said template sequence comprises at least a region of a gene, said gene selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, the p53 gene, and the BCR-able gene.

18. The method of claim 1, wherein said reverse PCR primer comprises i) at least one stop codon, and ii) a sequence coding for a second epitope.

19. The method of claim 1, wherein said first ligand is attached via a photocleavable linker.

20. The method of claim 19, wherein said captured nascent protein or protein fragment of step d) is photoreleased.

21. The method of claim 18, wherein said captured nascent protein of step d) comprises a second epitope.

22. The method of claim 21, wherein said first epitope is an N-terminal epitope and said second epitope is a C-terminal epitope.

23. The method of claim 21, further comprising e) reacting said captured nascent protein with a second ligand, said second ligand having affinity for said second epitope.

24. The bead-ligand-nascent protein complex created by the method of claim 3.

25. The method of claim 20, wherein said nascent protein or protein fragment is photoreleased onto a non-bead surface.

26. The method of claim 25, wherein said non-bead surface is compatible with mass spectrometry.

27. The method of claim 26, wherein the mass of said nascent protein or protein fragment is measured by mass spectrometry.

28. The method of claim 24, wherein said bead-ligand-nascent protein complex is detected by flow cytometry.

29. The method of claim 24, wherein said bead-ligand-nascent protein complex is fluorescent.

30. The method of claim 29, wherein said fluorescent bead-ligand-nascent protein complex is analyzed under a microscope capable of detecting fluorescence.

31. The method of claim 29, wherein said fluorescent bead-ligand-nascent protein complex is analyzed by a fluorescent activated cell sorter.

32. The method of claim 29, wherein said fluorescent bead-ligand-nascent protein complex is analyzed under a microarray reader capable of detecting fluorescence.

33. The method of claim 29, wherein said fluorescent bead-ligand-nascent protein complex is detected by microfluidics.

34. A method, comprising:

a) providing

     1) a template sequence encoding a nascent protein or fragment thereof and

     2) a surface comprising a PCR primer, said PCR primer comprises i) a promoter sequence, ii) a ribosome binding site sequence, iii) a start codon sequence, iv) a sequence coding for a first epitope, and v) a sequence complementary to at least a portion of said a template sequence;

b) amplifying said template so as to create amplified product immobilized on said surface, said amplified product encoding a nascent protein or fragment thereof, and encoding said first epitope;

c) attaching a first ligand capable of capturing said nascent protein or fragment thereof by reacting with said first epitope;

d) generating said nascent protein or fragment thereof comprising said first epitope from said amplified product, and e) capturing said nascent protein or fragment thereof on said surface via said first ligand, thereby generating a surface comprising captured nascent protein, or fragment thereof, and amplified product coding said nascent protein or fragment thereof.

35. The method of claim 34, wherein said surface is a bead surface.

36. The method of claim 34, wherein said first ligand comprises an antibody.

37. The method of claim 34, wherein said first ligand comprises a metal chelator.

38. The method of claim 34, wherein said template sequence comprises at least a region of a gene, said gene selected from the group consisting of the APC gene, the NF1 gene, the NF2 gene, the BRCA1 gene, the BRCA2 gene, the Kras gene, the p53 gene, and the BCR-able gene.

39. The method of claim 34, wherein said first ligand is attached via a photocleavable linker.

40. The method of claim 34, wherein said captured nascent protein or fragment thereof of step e) is photoreleased.

41. The method of claim 34, wherein said captured nascent protein of step e) further comprises a second epitope.

42. The method of claim 41, wherein said first epitope is an N-terminal epitope and said second epitope is a C-terminal epitope.

43. The method of claim 41, further comprising f) reacting said captured nascent protein with a second ligand, said second ligand having affinity for said second epitope.

44. The bead-ligand-nascent protein complex created by the method of claim 35.

45. A method of creating and detecting a bead-ligand-nascent protein fluorescent complex, comprising:

a) providing

     1) a template sequence encoding a nascent protein or fragment thereof and

     2) a bead comprising first and second PCR primers, said first PCR primer comprising i) a promoter sequence, ii) a ribosome binding site sequence, iii) a start codon sequence, iv) a sequence coding for a first epitope, and v) a sequence complementary to at least a portion of said a template sequence, and said second PCR primer comprising i) at least one stop codon, and ii) a sequence coding for a second epitope;

b) amplifying said template so as to create amplified product immobilized on said bead, said amplified product encoding a nascent protein or fragment thereof, and encoding said first and second epitopes;

c) attaching to said bead a first ligand capable of capturing said nascent protein or fragment thereof by reacting with said first epitope;

d) generating said nascent protein or fragment thereof comprising said first epitope from said immobilized amplified product, e) capturing said nascent protein or fragment thereof on said bead via said first ligand, thereby generating a bead-ligand-nascent protein complex;

f) contacting said bead-ligand-nascent protein complex with a second ligand capable of binding to said second epitope, said second ligand comprising a fluorescent moiety, thereby creating a bead-ligand-nascent protein fluorescent complex; and

g) detecting said fluorescent moiety of said bead-ligand-nascent protein fluorescent complex.

46. The method of claim 45, wherein said first ligand comprises an antibody.

47. The bead-ligand-nascent protein fluorescent complex created by the method of claim 45.

48. The method of claim 45, wherein said bead-ligand-nascent protein complex is detected by flow cytometry.

49. The method of claim 45, wherein said fluorescent moiety of said bead-ligand-nascent protein fluorescent complex comprises Cy3.

50. The method of claim 45, wherein said fluorescent bead-ligand-nascent protein complex is detected under a microscope capable of detecting fluorescence.

51. The method of claim 45, wherein said fluorescent bead-ligand-nascent protein complex is detected by a fluorescent activated cell sorter.

52. The method of claim 45, wherein said fluorescent bead-ligand-nascent protein complex is detected under a microarray reader capable of detecting fluorescence.

53. The method of claim 45, wherein said fluorescent bead-ligand-nascent protein complex is detected by microfluidics.