Method and composition for detecting protein-protein and protein-nucleic acid interactions
The present invention provides methods and compositions for detecting protein-protein and/or protein-nucleic acid interactions in cells.
1. A method for detecting an interaction between a first polypeptide and a second polypeptide comprising:
providing a cell expressing the first polypeptide being foreign to and operably linked to a viral inclusion body protein or a biologically active portion thereof;
contacting the first polypeptide with the second polypeptide within the cell;
allowing formation of an inclusion body; and
detecting the second polypeptide in the inclusion body if the first polypeptide and the second polypeptide interact.
2. The method of claim 1 , wherein the viral inclusion body protein comprises a reoviral protein or a biologically active portion thereof.
3. The method of claim 2 , wherein the reoviral protein has an amino acid sequence corresponding to a protein or a biologically active portion of a protein from a reovirus selected from the group consisting of: Orthoreovirus, Orbivirus, Rotavirus, Coltivirus, Seadornavirus, Aquareovirus, Cypovirus, Entomoreovirus, Fijivirus, Phytoreovirus and Orzavirus.
4. The method of claim 3 , wherein the reoviral protein has an amino acid sequence corresponding to a protein or a biologically active portion of a protein from Orthoreovirus or Rotavirus.
5. The method of claim 2 , wherein the viral inclusion body protein is μNS or NSP5.
6. The method of claim 5 , wherein the viral inclusion body protein is a carboxy-terminal one-third of an orthoreoviral μNS protein.
7. The method of claim 1 , wherein detecting is by fluorescence microscopy.
8. The method of claim 1 , wherein detecting is performed by an automated system.
9. The method of claim 1 , wherein the second polypeptide is endogenously expressed by the cell.
10. The method of claim 1 , wherein the second polypeptide is expressed by an expression vector.
11. A method for identifying an agent that modulates binding of a first polypeptide to a second polypeptide comprising:
contacting a cell expressing the first polypeptide and the second polypeptide within the cell, wherein the second polypeptide being foreign to and is operably linked to a reoviral inclusion body protein or a biologically active portion thereof
contacting the cell with the agent;
allowing formation of an inclusion body; and
detecting the presence of the first polypeptide in the inclusion body, wherein the presence of the first polypeptide in the inclusion body is altered if the agent modulates binding of the first polypeptide to the second polypeptide.
12. The method of claim 11 , wherein the agent is a small molecule.
13. The method of claim 12 , wherein the small molecule is selected from a library.
14. The method of claim 11 , wherein the agent is a polypeptide.
15. The method of claim 11 , wherein the agent increases binding of the first polypeptide to the second polypeptide.
16. The method of claim 11 , wherein the agent decreases binding of the first polypeptide to the second polypeptide.