Compositions and methods for detecting molecule-molecule interactions

Patent metadata

Field	Value
Publication number	US-11467166-B2
Application number	US-201816756464-A
Country	US
Kind code	B2
Filing date	Oct 23, 2018
Priority date	Oct 23, 2017
Publication date	Oct 11, 2022
Grant date	Oct 11, 2022

Abstract

Compositions and methods for detecting molecule-molecule interactions are provided. The methods employ a prokaryotic ubiquitin-like protein (Pup) and a Pup ligase that is coupled to one of the molecules. When the Pup ligase is brought to proximity to the other molecule by virtue of the molecule-molecule interaction, the Pup ligase can conjugate the Pup to a lysine residue on the other molecule. As such conjugation can be easily detected, this method allows easy identification of the molecule-molecule interaction.

First claim

The invention claimed is:

1. A method of detecting binding between a protein having at least a lysine residue and a molecule that interacts with the protein, comprising:
incubating a sample that comprises (a) a prokaryotic ubiquitin-like protein (Pup), (b) the protein, and (c) the molecule coupled to a Pup ligase, under conditions to allow the protein to bind the molecule, thereby allowing the Pup ligase to conjugate the Pup to the lysine residue of the protein; and
detecting the conjugation of the Pup to the protein which indicates binding between the protein and the molecule.

2. The method of claim 1 , wherein the molecule is a second protein, a small molecule drug, a hormone, a lipid or a polysaccharide.

3. The method of claim 1 , wherein the Pup ligase is conjugated to the molecule.

4. The method of claim 1 , wherein the Pup ligase is coupled to the molecule through a pair of proteins capable of binding each other.

5. The method of claim 4 , wherein the binding is chemically induced dimerization (CID).

6. The method of claim 1 , wherein the molecule is a second protein and the Pup ligase is fused to the molecule.

7. The method of claim 1 , wherein the binding is through hydrophobic interaction, electro statistic interaction, or hydrogen bond.

8. The method of claim 1 , wherein at least one of the protein and the molecule is a membrane-bound or transmembrane protein.

9. The method of claim 1 , wherein the protein is present on the surface of a first cell and the molecule is present on a second cell.

10. The method of claim 9 , wherein the first cell is a tumor cell and the second cell is a CAR-T cell or vice versa.

11. A method of coupling a molecule to a protein having at least a lysine residue, comprising contacting the protein with (a) the molecule coupled to a prokaryotic ubiquitin-like protein (Pup) in the presence of (b) a Pup ligase coupled to a second protein capable of binding the protein, under conditions to allow the second protein to bind the protein thereby enabling the Pup ligase to conjugate the Pup to the lysine residue, thereby coupling the molecule to the protein.

12. The method of claim 11 , wherein the protein is an antibody.

13. The method of claim 11 , wherein the second protein is protein G or an antibody having specificity to the Fc fragment of the antibody.

14. The method of claim 11 , wherein the molecule is selected from the group consisting of a small molecule drug, a detectable label, a nucleotide, a protein, and combinations thereof.

15. The method of claim 1 where the prokaryotic ubiquitin-like protein (Pup) ligase is coupled to a transmembrane protein.

16. The method of claim 15 , wherein the Pup ligase is fused to the intracellular domain of the transmembrane protein.

17. The method of claim 16 , wherein the Pup ligase is fewer than 20 amino acid residues away from the intracellular terminus of the protein.

Assignees

• Univ Shanghai Technology

Inventors

• Zhuang Min
• Wang Haopeng
• Liu Qiang

Classifications

• G01N33/6845 Primary

  Methods of identifying protein-protein interactions in protein mixtures · CPC title

• G01N2333/9015

  Ligases (6) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C07K2319/70

  containing domain for protein-protein interaction · CPC title

• C07K2319/03

  containing a transmembrane segment · CPC title