In situ oriented immobilization of proteins on a support

• US 9,567,378 B2
• Filed: 06/03/2011
• Issued: 02/14/2017
• 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.
• Primary Examiner(s): LAM, ANN Y
• Application Number:
  US13/153,046
• Publication Number:
  US 20110301057A1
• Time in Patent Office: 2,083 Days
• Field of Search: None
• US Class Current: 1/1
• 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, wherein the protein is a recombinant fusion protein and the coupling moiety comprises glutathione-S-transferase (GST) and

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

wherein the glutathione 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 solution has a pH of about 8.5.

5. The method according to claim 1, wherein the coupling moiety is located at the N-terminal region of the protein or the C-terminal of the protein.

6. The method according to claim 1, wherein the protein comprises lectin.

7. The method according to claim 6, wherein the protein is oriented to increase accessibility of a carbohydrate-binding site.

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

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

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

11. The method according to claim 1, 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.

12. The method according to claim 11, wherein the protein comprises Protein A (SpA), Protein G (SpG), or a fragment thereof.

13. The method according to claim 11, wherein the antibody is IgG, or a fragment thereof.

14. The method according to claim 11, wherein the protein binds the antibody via an Fc domain.

15. The method according to claim 11, wherein the protein is Peptostreptococcus magnus PpL.

16. The method according to claim 15, wherein the antibody is a multivalent antibody selected from IgM and IgA, or a fragment thereof.

17. The method according to claim 15, wherein the Peptostreptococcus magnus PpL binds the antibody at a light chain fragment of the antibody.