title: Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.
abstract: Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions 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.
authors.affiliation: Branch of the Institute of Bioorganic Chemistry, RAS, Nauki Pr 6, Pushchino 142290, Russia.
authors.forename: Andrey Yu
authors.initials: AY
authors.lastname: Gorokhovatsky
authors.forename: Natalia V
authors.initials: NV
authors.lastname: Rudenko
authors.forename: Victor V
authors.initials: VV
authors.lastname: Marchenkov
authors.forename: Vitaly S
authors.initials: VS
authors.lastname: Skosyrev
authors.forename: Maxim A
authors.initials: MA
authors.lastname: Arzhanov
authors.forename: Nils
authors.initials: N
authors.lastname: Burkhardt
authors.forename: Mikhail V
authors.initials: MV
authors.lastname: Zakharov
authors.forename: Gennady V
authors.initials: GV
authors.lastname: Semisotnov
authors.forename: Leonid M
authors.initials: LM
authors.lastname: Vinokurov
authors.forename: Yuli B
authors.initials: YB
authors.lastname: Alakhov
mesh_terms: D000331:Aequorin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D001710:Biotin / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D049452:Green Fluorescent Proteins; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037401:Scyphozoa / Q000737:chemistry*
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000331:Aequorin; D001710:Biotin