Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system.
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.
Gorokhovatsky
Andrey Yu
AY
Branch of the Institute of Bioorganic Chemistry, RAS, Nauki Pr 6, Pushchino 142290, Russia.
Rudenko
Natalia V
NV
Marchenkov
Victor V
VV
Skosyrev
Vitaly S
VS
Arzhanov
Maxim A
MA
Burkhardt
Nils
N
Zakharov
Mikhail V
MV
Semisotnov
Gennady V
GV
Vinokurov
Leonid M
LM
Alakhov
Yuli B
YB
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*
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D000331:Aequorin; D001710:Biotin