Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

被引：16

作者：

Gorokhovatsky, AY

Rudenko, NV

Marchenkov, VV

Skosyrev, VS

Arzhanov, MA

Burkhardt, N

Zakharov, MV

Semisotnov, GV

Vinokurov, LM

Alakhov, YB

机构：

[1] RAS, Inst Bioorgan Chem, Branch, Pushchino 142290, Russia

[2] RAS, Inst Prot Res, Pushchino 142290, Russia

[3] Bayer AG, Pharmaforsch Zentrum, D-42096 Wuppertal, Germany

来源：

ANALYTICAL BIOCHEMISTRY | 2003年 / 313卷 / 01期

基金：

俄罗斯基础研究基金会;

关键词：

aequorin; biotin carboxyl carrier protein; green fluorescent protein; streptavidin; fusion proteins; bioluminescence resonance energy transfer;

D O I：

10.1016/S0003-2697(02)00514-6

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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 5 10 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. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：68 / 75

页数：8

共 31 条

[1] Quenching of biotinylated aequorin bioluminescence by dye-labeled avidin conjugates: Application to homogeneous bioluminescence resonance energy transfer assays [J].