IN SITU ORIENTED IMMOBILIZATION OF PROTEINS ON A SUPPORT

• US 20110301057A1
• Filed: 06/03/2011
• Published: 12/08/2011
• Est. Priority Date: 06/03/2010
• Status: Active Grant

• Current Assignee: New York University
• Original Assignee: New York University
• Inventors: PROPHETER, Daniel C., MAHAL, Lara K.
• Granted Patent: US 9,567,378 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/31 from Staphylococcus (G)

Abstract

The present invention relates to a method of directing in situ oriented immobilization of a protein on a support. This method involves providing a support and contacting the support with a solution. The solution comprises (a) a protein comprising a coupling moiety and (b) a molecule comprising a first group reactive with the support and a second group reactive with the coupling moiety. The molecule binds (i) the support at the first group and (ii) the coupling moiety at the second group, thereby immobilizing and orienting, in situ, the protein on the support. Also described are a protein array and a method of screening compounds for protein interaction.

Claims

1. A method of directing in situ oriented immobilization of a protein on a support, said method comprising:

providing a support;

contacting the support with a solution comprising;

a protein comprising a coupling moiety and

a molecule comprising a first group reactive with the support and a second group reactive with the coupling moiety,

wherein the molecule binds (i) the support at the first group and (ii) the coupling moiety at the second group, thereby immobilizing and orienting, in situ, the protein on the support.

2. The method according to claim 1, wherein the support has a homogenous chemistry.

3. The method according to claim 1, wherein the support comprises N-Hydroxysuccinimide (NHS) or epoxide.

4. The method according to claim 1, wherein the protein is a recombinant fusion protein.

5. The method according to claim 4, wherein the coupling moiety comprises glutathione-S-transferase (GST).

6. The method according to claim 1, wherein the molecule is glutathione.

7. The method according to claim 6, wherein the solution has a pH of about 8.5.

8. The method according to claim 4, wherein the coupling moiety is located at the N-terminal region of the protein or the C-terminal of the protein.

9. The method according to claim 4, wherein the protein comprises lectin.

10. The method according to claim 9, wherein the protein is oriented to increase accessibility of a carbohydrate-binding site.

11. The method according to claim 1, wherein the support comprises glass or metal.

12. The method according to claim 1, wherein the protein comprises a polyhistidine tag.

13. The method according to claim 1, wherein said contacting is carried out by printing.

14. The method according to claim 5, wherein the solution further comprises an antibody to which the protein binds, and said method is carried out to direct in situ oriented immobilization of an antibody on a support.

15. The method according to claim 14, wherein the protein comprises Protein A (SpA), Protein G (SpG), or a fragment thereof.

16. The method according to claim 14, wherein the antibody is IgG, or a fragment thereof.

17. The method according to claim 14, wherein the protein binds the antibody via an Fc domain.

18. The method according to claim 14, wherein the protein is Peptostreptococcus magnus PpL.

19. The method according to claim 18, wherein the antibody is a multivalent antibody selected from IgM and IgA, or a fragment thereof.

20. The method according to claim 18, wherein the Peptostreptococcus magnus PpL binds the antibody at a light chain fragment of the antibody.

21. A protein array comprising:

a first protein immobilized at a first location on a support surface in an oriented manner, wherein the first protein is immobilized on the surface via a molecule comprising (i) a first group reactive with the protein and (ii) a second group reactive with the surface and

a second protein immobilized at a second location on the surface,

wherein the surface has a homogenous chemistry reactive with both the molecule at the second group and the second protein.

22. The protein array according to claim 21, wherein the homogenous chemistry comprises N-Hydroxysuccinimide (NHS) or epoxide.

23. The protein array according to claim 21, wherein the first protein is a recombinant fusion protein.

24. The protein array according to claim 23, wherein the fusion protein comprises a coupling moiety comprising glutathione-S-transferase (GST).

25. The protein array according to claim 21, wherein the molecule is glutathione.

26. The protein array according to claim 21, wherein the first and second proteins are lectins.

27. The protein array according to claim 26, wherein the first protein is oriented to increase accessibility of a carbohydrate-binding site.

28. The protein array according to claim 21, wherein the support comprises glass or metal.

29. The protein array according to claim 21, wherein the first protein comprises a polyhistidine tag.

30. The protein array according to claim 24 further comprising:

an antibody bound to the first protein in an oriented manner.

31. The protein array according to claim 30, wherein the first protein comprises Protein A (SpA), Protein G (SpG), or a fragment thereof

32. The protein array according to claim 30, wherein the antibody is IgG, or a fragment thereof.

33. The protein array according to claim 30, wherein the first protein is Peptostreptococcus magnus PpL.

34. The protein array according to claim 33, wherein the antibody is a multivalent antibody selected from IgM and IgA, or a fragment thereof.

35. The protein array according to claim 34, wherein the Peptostreptococcus magnus PpL binds the antibody at a light chain fragment of the antibody.

36. A method of screening compounds for protein interaction, said method comprising:

providing a protein array according to claim 21;

contacting the array with compounds to be screened; and

detecting compounds that interact with the first and/or second protein, thereby screening compounds for protein interaction.