12 September 2003 Detection of protein-protein interactions using Aequorea victoria bioluminescence resonance energy transfer
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Abstract
Bioluminescence resonance energy transfer (BRET) is a naturally occurring phenomenon taking place in some marine coelenterates. Emission of light in these organisms involves the energy transfer between chromophores of donor luciferase and acceptor fluorescent protein. Due to the strict dependence of BRET efficiency on the inter-chromophore distance, the phenomenon has been applied to study protein-protein interactions by fusing interacting partners with either donor or acceptor proteins. Here we describe a BRET-based homogeneous protein-protein interaction assay exploiting novel donor-acceptor pair formed by photoproteins of jellyfish Aequorea victoria bioluminescent system, aequorin and green fluorescent protein enhanced variant (EGFP). Two known interacting proteins, streptavidin (SAV) and biotin carboxyl carrier protein (BCCP) were fused, respectively, with aequorin and EGFP. The fusions were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-Aequorin and BCCP-EGFP was followed by BRET between aequorin (donor) and EGFP (acceptor) resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-Aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.
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Leonid M. Vinokurov, Leonid M. Vinokurov, Andrey Yu. Gorokhovatsky, Andrey Yu. Gorokhovatsky, Natalia V. Rudenko, Natalia V. Rudenko, Victor V. Marchenkov, Victor V. Marchenkov, Vitaly S. Skosyrev, Vitaly S. Skosyrev, Maxim A. Arzhanov, Maxim A. Arzhanov, Mikhail V. Zakharov, Mikhail V. Zakharov, Nils Burkhardt, Nils Burkhardt, Gennady V. Semisotnov, Gennady V. Semisotnov, Yuli B. Alakhov, Yuli B. Alakhov, } "Detection of protein-protein interactions using Aequorea victoria bioluminescence resonance energy transfer", Proc. SPIE 4967, Genetically Engineered and Optical Probes for Biomedical Applications, (12 September 2003); doi: 10.1117/12.477881; https://doi.org/10.1117/12.477881
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