Enzyme fragment complementation assay

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

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. 

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. 

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... 

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. 

Remy 1, Campbell-Valois FX, Michnick SW. Detection of protein-protein interactions using a simple survival protein-fragment complementation assay based on the enzyme dihydrofolate reductase. Nat. Protoc. 2007 2 2120-2125. 

More recently Michnick and co-workers have introduced a dihydrofolate reductase complementation system, which seems to be particularly robust [61 - 65], They attribute the success of this system to the fact that the N-terminal (1 - 105) and C-terminal (106 - 186) DHFR fragments do not fold until they are dimerized. In addition to the obvious selection for essential metabolites dependent on the reduction of dihydrofolate to tetrahydrofolate, protein-protein interactions are detected based on the retention of a fluorescein-methotrexate conjugate. Several other enzymes have been employed for the design of complementation assays, including green fluorescent protein, which allows screens based on fluorescence or FRET [66 - 68]. As with the bacterial transcription assays, these complementation systems are new. It will be interesting to see if, as the selections are optimized, these systems prove competitive with the Y2H assay. 

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