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US 11,237,174 B2Granted
Method for detecting protein-protein interactions in a cell utilizing particle-forming polypeptide-bait fusion proteins and virus-like particles
AssigneesVib Vzw, Universiteit Gent
InventorsJan Tavernier, Sven Eyckerman
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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.
Claims11
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.
3 more elements
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.
3 more elements
Dependencies are inferred from the claim text.