METHODS TO SELECT FOR AGENTS THAT STABILIZE PROTEIN COMPLEXES

• US 20170160285A1
• Filed: 07/17/2015
• Published: 06/08/2017
• Est. Priority Date: 07/22/2014
• Status: Active Grant

• Current Assignee: VIB VZW, Vrije Universiteit Brussel
• Original Assignee: VIB VZW, Vrije Universiteit Brussel
• Inventors: Steyaert, Jan, Wohlknig, Alexandre, Triest, Sarah
• Granted Patent: US 10,641,779 B2
• Time in Patent Office: Days
• CPC Class Codes: G01N 33/4915 using flow cells flow cytometry G01N15/14
• CPC Class Codes: G01N 33/492 Determining multiple analytes
• CPC Class Codes: G01N 33/6845 Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6854 Immunoglobulins
• CPC Class Codes: G01N 33/6857 Antibody fragments

Abstract

The application relates to the field of structural biology. More specifically, the disclosure relates to methods for the identification and characterization of biomolecular tools allowing the selective recognition and/or stabilization of distinct conformational states of protein complexes, including transient protein-protein interactions and protein-nucleic acid complexes. Such tools can then be used for purification purposes, crystallization and structure determination of these stabilized protein complexes, for drug discovery, as research tools, as well as for diagnosis and treatment of diseases.

Claims

1. A method for selecting a conformation-selective binding agent of a protein complex, the method comprising the steps of:

a) displaying a collection of binding agents at the extracellular surfaces of a population of cells; and

b) utilizing cell sorting to separate, from said population of cells, cells displaying binding agents that

i. specifically bind to a protein complex conformation and not to the individual members of the complex, and/or

ii. specifically bind to one of the individual members of a protein complex and not to the protein complex conformation nor to the other individual member(s) of the complex, and/or

iii. specifically bind to one of the individual members of a protein complex and to a protein complex conformation and not to the other individual member(s) of the complex.

2. The method of claim 1, wherein the individual members of the protein complex are distinguishably tagged and step b) comprises the steps of:

a) incubating a mixture of distinguishably tagged individual members of a protein complex with the population of cells under suitable conditions to allow binding to the cells; and

b) utilizing cell sorting to select, from said population of cells, cells displaying binding agents that

i. specifically bind to a protein complex conformation and not to the individual members of the complex,

ii. specifically bind to one of the individual members of a protein complex and not to the protein complex conformation nor to the other individual member(s) of the complex, and/or

iii. specifically bind to one of the individual members of a protein complex and to a protein complex conformation and not to the other individual member(s) of the complex.

3. The method of claim 1, wherein at least one of the individual members of the protein complex is labeled with a fluorescent label.

4. The method of claim 3, wherein the individual members of the protein complex are each labeled with a distinguishable fluorescent label.

5. The method claim 1, wherein the cell sorting in step b) is done utilizing fluorescence-activated cell sorting (“

FACS”

).

6. The method of claim 1, wherein said protein complex is a protein-protein complex comprising at least two interacting monomeric proteins.

7. The method of claim 1, wherein said protein complex is a protein-nucleic acid complex comprising at least one monomeric protein interacting with at least one nucleic acid molecule.

8. The method claim 1, wherein said method comprises isolating the binding agent from the cell sorted in step b).

9. The method of claim 8, further comprising:

measuring the binding specificity and/or affinity of the binding agent for the protein complex conformation as compared to the individual members of the protein complex by any suitable technique.

10. The method of claim 1, wherein said plurality of binding agents is a library of antibodies or antibody fragments.

11. The method of claim 10, wherein said antibody fragments are immunoglobulin single-domain antibodies.

12. The method of claim 10, wherein said antibodies or antibody fragments are obtained from an animal that has been immunized with the protein complex in a cross-linked form.

13. The method of claim 12, wherein said animal is a camelid.

14. The method of claim 1, wherein said population of cells is a population of yeast cells.

15. A composition comprising a binding agent produced by the method according to claim 1.

16. A composition comprising a protein complex and a binding agent produced by the method according to claim 1, wherein the protein complex is stabilized by the binding agent.

17. The method according to claim 9, wherein the suitable technique is a biophysical method.

18. The method according to claim 9, wherein the suitable technique is fluorescence-activated cell sorting (“

FACS”

).

19. The method according to claim 11, wherein the immunoglobulin single-domain antibodies are VHH.