Interaction-activated, circularly permutated proteins

An interaction-dependent enzyme activation system, said system comprising:

• a nucleic acid sequence encoding a first oligopeptide and a second oligopeptide, each fused in frame through a first and a second break-point terminus, respectively, to a circularly permutated marker protein, wherein said circularly permutated marker protein reassembles to form a functionally reconstituted marker protein that produces a detectable signal upon the association of said first oligopeptide with said second oligopeptide or upon simultaneous association of said first oligopeptide and said second oligopeptide with a third oligopeptide.

An interaction-dependent enzyme activation system, said system comprising:

• a first nucleic acid sequence encoding a first oligopeptide and a second oligopeptide, each fused in frame through a first and a second break-point terminus, respectively, to a circularly permutated β-lactamase, wherein said circularly permutated β-lactamase reassembles to form a functionally reconstituted marker protein that produces a detectable signal upon the association of said first oligopeptide with said second oligopeptide or upon simultaneous association of said first oligopeptide and said second oligopeptide with a third oligopeptide encoded by a second nucleic acid sequence.

An intracellular signal transduction biosensor, said biosensor comprising:

• a first nucleic acid sequence encoding a first intracellular polypeptide and a second intracellular polypeptide, each fused in frame through a first and a second break-point terminus, respectively, to a circularly permutated β-lactamase, wherein said circularly permutated β-lactamase reassembles to form a functionally reconstituted marker protein that produces a detectable signal upon the association of said first oligopeptide with said second oligopeptide or upon simultaneous association of said first oligopeptide and said second oligopeptide with a third intracellular polypeptide encoded by a second nucleic acid sequence.

A DNA sequence comprising as operably linked components in the direction of transcription, nucleic acid sequences encoding a first interactor domain, a circularly permutated marker protein, and a second interactor domain, wherein said circularly permutated marker protein functionally reassembles upon binding of said first and said second interactor domains to each other or simultaneously to a third polypeptide.

A circularly permutated marker protein fused in frame through each of its N- and C-termini to a first interactor domain and a second interactor domain, wherein said circularly permutated marker protein functionally reassembles upon the binding of said first and said second interactor domains to each other or simultaneously to a third polypeptide.

A method of high-throughput identification of compounds that inhibit phosphorylation-regulated cell signal transducers, said method comprising:

• expressing from a plasmid in a host cell an oligopeptide comprised of in the direction of translation, a first interactor domain, a circularly permutated β-lactamase, and a second interactor domain, wherein said first interactor domain comprises a phosphorylation regulated cell signal transducer protein and said second interactor domain comprises an immunoglobulin variable region that binds to said first interactor domain only under the required state of phosphorylation, wherein the binding of said first interactor domain with said second interactor domain results in the functional reconstitution of said circularly permutated β-lactamase to produce a colored host cell in the presence of chromogenic β-lactamase substrate,
• whereby production of a colored host cell is indicative of a compound that inhibits phosphorylation-regulated cell signal transduction.