Novel BRET-based biosensors with a new bioluminescent donor, Gaussia luciferase, for estrogen receptor ligands

BRET is a natural non-radiative energy transfer phenomenon occurring in marine organisms between a bioluminescent donor protein and a fluorescent acceptor protein. In order to study an interaction between two target proteins, labelled proteins are generated by genetic fusion with either a bioluminescent donor or fluorescent acceptor. Recently, several in vivo and in vitro applications of BRET assays have been reported, ranging from membrane-bound and cytosolic receptors dimerization studies to nuclear factors interactions monitoring. The majority of BRET assays use Renilla luciferase (Rluc) as energy donor; in the presence of coelenterazine, Rluc instead of emitting blue light, emits blue light which is able to excite a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) acting as fluorescent acceptor. The recent cloning of several reporter proteins with different spectral properties has enlarged the number of possible bioluminescent donor proteins to be used in BRET assays. In particular, the use of a new bioluminescent donor, Gaussia luciferase, has been investigated as an alternative to Rluc. This protein has the advantages of smaller size, higher bioluminescent signal emission when it is codon humanized and flash kinetic (max 480 nm), thus suitable for efficient energy transfer to EYFP. Novel bioluminescence resonance energy transfer (BRET) homogeneous assays for estrogen- and androgen-like compounds have been developed and optimized based on estrogen and androgen receptor homo- and hetero-dimerization. The developed assays fulfil all the standard requirements of accuracy and intra- assay and inter-assay precision with a limit of detection comparable to those of whole-cell biosensors or analogues bioassays, with the peculiar advantages of homogeneous assays.