METHODS FOR ASSAYING PROTEIN-PROTEIN INTERACTIONS

摘要

Provided herein is an assay for interrogating transient and dynamic protein-protein interactions and for screening and characterizing agents as agonists or antagonists of protein-protein interactions. The methods can provide a single assay for simultaneously assessing the bioavailability and efficacy of a test compound for increasing or decreasing a protein-protein interaction of interest.

主权项

1. A method of determining if a test agent modifies a protein-protein interaction between a first protein and a second protein, comprising:
inducing expression in a first host cell of a first fragment of a split-luciferase protein linked to the first protein and a second fragment of the split luciferase protein linked to the second protein, wherein the first and second fragments complement to form a functional split-luciferase protein, and wherein the first host cell comprises a nucleic acid sequence introduced by cre-recombinase mediated cassette exchange (RCME) that encodes the first fragment linked to the first protein and the second fragment linked to the second protein, each operably linked to an inducible promoter; contacting the first host cell with the test agent; detecting luciferase activity of the complemented split-luciferase protein; and identifying the test agent as an agent that modifies a protein-protein interaction if an increase or decrease in the luciferase activity as compared to a control is detected, and identifying the test agent as an agent that does not modify a protein-protein interaction if an increase or decrease in the luciferase activity as compared to the control is not detected, thereby determining if the test agent modifies the protein-protein interaction.