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. Author manuscript; available in PMC: 2015 Jun 15.
Published in final edited form as: Methods Mol Biol. 2011;680:29–43. doi: 10.1007/978-1-60761-901-7_2

Figure 2. Unimolecular constructs for luciferase PCAs.

Figure 2

The upper construct contains a single protein fragment for the human estrogen receptor with amino acids 281–549 encompassing helix 12 (H12) and the ligand binding domain (LBD) of the receptor, flanked by inactive fragments of Renilla luciferase (Nrluc and Crluc) (3). This sensor for estrogen agonists and antagonists produces strong luminescence activity upon ligand-induced interaction of H12 with the LBD.

The middle construct contains two interacting protein/peptide regions, an FHA2 phosphopeptide binding domain and an AKT substrate peptide, separated by short linkers and flanked by inactive fragments of firefly luciferase. This sensor for active (21) produces luminescence upon AKT phosphorylation of the substrate peptide, which binds to the FHA2 region of the protein and disrupts complementation of the luciferase fragments. (See diagram)

The third construct codes for a single protein which is cleaved to form a bimolecular product. The construct includes three functional regions: PepA and PepB are proteins with a strong constitutive interaction which is disrupted in the intact fusion protein, and DEVD is a substrate for Caspase-3, an enzyme which is activated early in apoptosis. This apoptosis sensor produces increased luminescence upon cleavage of the DEVD peptide by Caspase-3 and subsequent complementation of the luciferase fragments driven by association of PepA and PepB (22).