Method for detecting protein-protein interactions in a cell utilizing particle-forming polypeptide-bait fusion proteins and virus-like particles

Patent metadata

Field	Value
Publication number	US-11237174-B2
Application number	US-201916600672-A
Country	US
Kind code	B2
Filing date	Oct 14, 2019
Priority date	May 24, 2012
Publication date	Feb 1, 2022
Grant date	Feb 1, 2022

Abstract

The disclosure relates to a virus-like particle in which a protein complex is entrapped, ensuring the formation of the protein complex under physiological conditions, while protecting the protein complex during purification and identification. The disclosure further relates to the use of such virus-like particle for the isolation and identification of protein complexes.

First claim

What is claimed is:

1. A method of detecting a protein-protein interaction in a cell, the method comprising:
expressing in the cell a recombinant fusion protein comprising a viral particle-forming polypeptide and a bait polypeptide of interest, wherein the expressed fusion protein forms a protein-protein complex with an endogenously-expressed prey polypeptide within a virus-like particle;
isolating the virus-like particles from the cell; and
undertaking a mass spectrometry (MS) analysis of the isolated virus-like particles to detect specific bait-prey protein-protein interactions as compared to background protein-protein interactions from a reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest;
thereby detecting the protein-protein interaction in the cell;
wherein the viral particle-forming polypeptide is an adenovirus particle-forming polypeptide, a HIV particle-forming polypeptide, or a HCV particle-forming polypeptide.

2. The method according to claim 1 , wherein the fusion protein comprises a linker between the viral particle-forming polypeptide and the bait polypeptide of interest.

3. The method according to claim 2 , wherein the viral particle-forming polypeptide comprises one or more modifications relative to the naturally occurring viral particle-forming protein, wherein the modification is a deletion, mutation, or functional fragment not inhibiting viral particle formation, or is a fusion protein.

4. The method according to claim 1 , wherein the endogenously-expressed prey polypeptide is untagged.

5. The method according to claim 1 , wherein the recombinant fusion protein and/or the virus-like particle comprises a purification tag.

6. The method according to claim 5 , wherein isolating virus-like particles from the cell occurs via the purification tag prior to detecting the presence of the prey polypeptide.

7. The method according to claim 1 , wherein the protein-protein interaction was previously unknown or was previously uncharacterized.

8. The method according to claim 1 , further comprising forming virus-like particles comprising the protein-protein complex prior to isolating the virus-like particles from the cell.

9. The method according to claim 1 , wherein the reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest comprises a reference set of virus-like particles comprising viral particle-forming polypeptides to a bait polypeptide different from the bait polypeptide of interest.

10. A method of detecting protein-protein interactions in a cell, the method comprising:
expressing in the cell recombinant fusion proteins comprising a viral particle-forming polypeptide and a bait polypeptide of interest, wherein the expressed fusion proteins form a plurality of different protein-protein complexes with a plurality of different endogenously-expressed prey polypeptides within virus-like particles;
isolating the virus-like particles from the cell; and
undertaking a mass spectrometry (MS) analysis of the isolated virus-like particles to detect specific bait-prey protein-protein binding interactions as compared to background protein-protein interactions from a reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest;
thereby detecting the protein-protein interactions in the cell;
wherein the viral particle-forming polypeptide is an adenovirus particle-forming polypeptide, a HIV particle-forming polypeptide, or a HCV particle-forming polypeptide.

11. The method according to claim 1 , wherein the reference set of virus-like particles comprising viral particle-forming polypeptides not linked to the bait polypeptide of interest comprises a reference set of virus-like particles comprising viral particle-forming polypeptides to a bait polypeptide different from the bait polypeptide of interest.

Assignees

• Vib Vzw
• Univ Gent

Inventors

• Tavernier Jan
• Eyckerman Sven

Classifications

• C07K2319/43

  containing a FLAG-tag · CPC title

• C12N7/045

  Pseudoviral particles; Non infectious pseudovirions, e.g. genetically engineered · CPC title

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• G01N2570/00

  Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes · CPC title

• C07K2319/20

  containing a tag with affinity for a non-protein ligand · CPC title