Enzyme fragment complementation

There are cAMP assays that can be measured using a variety of techniques [35], including HTRF (CisBio) [36], bioluminescence (cAMP-Glo, Promega) [37], TR-FRET (CisBio) [38], LANCE [39], Alphascreen (Perkin Elmer) [40] and Enzyme Fragment Complementation (EFC) [41] (Hithunter, DiscoverX). This review will focus on the LANCE, the TR-FRET and ECF assays as they are the most used in our laboratories. 

Fig. 7 Hithunter (CisBio) enzyme fragment complementation assay for the detection of cAMP. See text for detailed explanation of the assay components...

Protein fragment complementation assays are based on an enzyme reassembly strategy whereby a protein-protein interaction promotes the efficient refolding and complementation of enzyme fragments to restore an active enzyme. The approach was initially developed using the reconstitution of ubiquitin as a sensor for protein-protein interactions (Johnsson and Varshavsky, 1994). Ubiquitin is a 76 amino acid protein that... 

Weber, M., Ferrer, M., Zheng, W., Inglese, J., Strulovici, B., and Kunapuli, P., A 1536-well cAMP assay for Gs- and Gi-coupled receptors using enzyme fragmentation complementation, Assay Drug Dev. Technol., 2, 39, 2004. 

Golla, R. and Seethala, R., A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists, /. Biomol. Screen., 7, 515, 2002. 

FIGURE 4.1 Assays commonly used in GPCR research. SPA = scintillation proximity assay FP = fluorescence polarization TR-FRET = time-resolved fluorescence resonance energy transfer FCS = fluorescence correlation spectroscopy SeAP = secreted alkaline phosphate TF = transcription factor EFC = enzyme fragment complementation DMR = dynamic mass redistribution CDS = cellular dielectric spectroscopy. 

Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments, Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3469-3474. 

Mevel-Ninio and co-workers (1977) and Gervais and co-workers (1980) succeeded in splitting cytochrome 62 into different fragments, one with the heme group, the other with the flavin group, and to obtain reconstitution of the enzyme by complementation of fragments. 

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