Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells
Ligand-Activatable BRET9 Probes for Imaging Molecular Events in Living Mammalian Cells
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用于活体哺乳动物细胞中分子事件成像的配体激活 BRET9 探针
DOI：
10.1007/978-1-0716-1258-3_22
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
Paulmurugan Ramasamy
中科院分区：
文献类型：
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作者：
Kim Sung-Bae;Fujii Rika;Paulmurugan Ramasamy
生物发光共振能量转移（BRET）是研究蛋白质相互作用的常用分析系统。本方案引入了一种概念上独特的配体可激活的BRET系统（称为BRET 9），其中全长人工荧光素酶变体23（ALuc 23）作为能量供体，夹在感兴趣的蛋白质对FRB和FKBP之间，并进一步连接到作为能量受体的荧光蛋白，用于研究蛋白质-蛋白质相互作用。特异性配体雷帕霉素在探针内部引发FRB和FKBP的分子内相互作用，在夹心ALuc 23中产生分子应变以完成其折叠，因此，探针系统极大地增强了整个生物发光（BL）光谱和远红（FR）区域的BRET信号。这种新的BRET系统提供了一个强大的配体激活平台，有效地报告哺乳动物细胞中的FR-BL信号。
Bioluminescence resonance energy transfer (BRET) is a commonly used assay system for studying protein-protein interactions. The present protocol introduces a conceptually unique ligand-activatable BRET system (termed BRET9), where a full-length artificial luciferase variant 23 (ALuc23), acting as the energy donor, is sandwiched in between a protein pair of interest, FRB and FKBP, and further linked to a fluorescent protein as the energy acceptor for studying protein-protein interaction. A specific ligand, rapamycin, which initiates intramolecular interactions of FRB and FKBP inside the probe, which develops molecular strain in the sandwiched ALuc23 to complete its folding, thus, the probe system greatly enhances both the overall bioluminescence (BL) spectrum and the BRET signal in the far-red (FR) region. This new BRET system provides a robust ligand-activatable platform that efficiently reports FR-BL signals in mammalian cells.