High Throughput Bacterial Screening Assay for Emission Properties Characterization of a Library of Aequorin Mutants

Luca Cevenini, Elisa Michelini, Laura Mezzanotte, Sylvia Daunert, Aldo Roda
Department of Pharmaceutical Sciences, University of Bologna; Department of Chemistry

The photoprotein aequorin, isolated from the jellyfish Aequorea victoria, is a calcium-sensitive bioluminescent protein formed with the apoprotein apoaequorin and the prosthetic group coelenterazine that emits light upon calcium binding. Aequorin belongs to the family of calcium-dependent luciferase/luciferin systems and because of its high sensitivity to calcium ions has been extensively used as a Ca2+ reporter in living cells or as a luminescent label in binding assays. In order to obtain aequorin variants with improved bioluminescence properties for bioanalytical application such as slower emission decay or red-shifted emission spectra, a library of aequorin mutants was generated by random mutagenesis and a rapid and low-cost screening was developed using the Varioskan® Flash multimode reader (Thermo Scientific).