Patent ID: 8241860

Claim:
A method for detecting interactions of a ligand molecule with an interacting protein having a binding site for said ligand, using a split monomeric reporter protein system in a cell-free assay, said method comprising the steps of: (a) providing (i) a cell-free system comprising coupled or uncoupled transcription and translational machinery; (ii) a first nucleic acid molecule comprising at least one stabilizing element region and encoding a first interacting fusion protein, said first interacting fusion protein comprising a first fragment of a reporter protein covalently linked to a first interacting domain of the interacting protein, said first interacting domain comprising a binding site for said ligand, and (iii) a second interacting fusion protein, said second interacting fusion protein comprising a second fragment of a reporter protein covalently linked to a second interacting domain of the second interacting protein, wherein neither the first nor second fragment of the reporter protein are active alone but are active when reassembled to form an active reporter protein; (b) allowing the expression of the first nucleic acid molecule in the cell-free system in step (a) to produce the first fusion interacting protein and providing a second interacting fusion protein; (c) contacting the first and second interacting fusion proteins either (i) with a composition where the second interacting fusion protein comprises a ligand of the first interacting fusion protein such that when the ligand and the first interacting fusion protein are present, the first and second fragments of the reporter reassemble to form an active reporter protein; or (ii) with a composition that contains a ligand of both interacting fusion proteins such that when a ligand to both of the first and second interacting fusion proteins is present, the ligand binds to the first and second fusion interacting proteins and the first and second fragments of the reporter located within the first and second interacting fusion proteins reassemble to form an active reporter protein; (d) detecting the active reporter protein when the ligand is present in the composition and the first and second reporter fragments have reassembled to form an active reporter protein.