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