A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein–Protein Interaction,ACS Sensors

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A Bright Monomeric Near-Infrared Fluorescent Protein with an Excitation Peak at 633 nm for Labeling Cellular Protein and Reporting Protein–Protein Interaction
ACS Sensors ( IF 8.2 ) Pub Date : 2022-07-01 , DOI: 10.1021/acssensors.2c00286
Feng Liu ₁ , Huimin Hu _{2,

3} , Mengying Deng ₁ , Zongqin Xiang ₄ , Yuting Guo ₅ , Xinmeng Guan ₁ , Dong Li ₅ , Qinxue Hu _{2,

6} , Wenliang Lei ₇ , Hongjuan Peng ₈ , Jun Chu _{1,

9,

10}

Affiliation

Guangdong Provincial Key Laboratory of Biomedical Optical Imaging Technology & Center for Biomedical Optics and Molecular Imaging & CAS Key Laboratory of Health Informatics, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan 430071, China.
Savaid Medical School, University of Chinese Academy of Sciences, Beijing 100049, China.
Department of Neurosurgery, the First Affiliated Hospital, Jinan University, Guangzhou, Guangdong 510630, China.
National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Institute for Infection and Immunity, St George's, University of London, London SW17 0RE, United Kingdom.
Guangdong-Hong Kong-Macau Institute of CNS Regeneration, Jinan University, Guangzhou, Guangdong 510630, China.
Department of Pathogen Biology, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health, Southern Medical University, Guangzhou 510515, China.
Shenzhen Institute of Synthetic Biology, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
Shenzhen-Hong Kong Institute of Brain Science, Shenzhen Fundamental Research Institutions, Shenzhen 518055, China.

Bright monomeric near-infrared fluorescent proteins (NIR-FPs) are useful as markers for labeling proteins and cells and as sensors for reporting molecular activities in living cells and organisms. However, current monomeric NIR-FPs are dim under excitation with common 633/635/640 nm lasers, limiting their broad use in cellular/subcellular level imaging. Here, we report a bright monomeric NIR-FP with maximum excitation at 633 nm, named mIFP663, engineered from Xanthomonas campestris pv Campestris phytochrome (XccBphP). mIFP663 has high molecular brightness with a large extinction coefficient (86,600 M^–1 cm^–1) and a decent quantum yield (19.4%), and high cellular brightness that is 3–6 times greater than those of spectrally similar NIR-FPs in HEK293T cells in the presence of exogenous BV. Moreover, we demonstrate that mIFP663 is able to label critical cellular and viral proteins without perturbing subcellular localization and virus replication, respectively. Finally, with mIFP663, we engineer improved bimolecular fluorescence complementation (BiFC) and new bioluminescent resonance energy transfer (BRET) systems to detect protein–protein interactions in living cells.

中文翻译：

一种明亮的单体近红外荧光蛋白，在 633 nm 处具有激发峰，用于标记细胞蛋白和报告蛋白-蛋白相互作用

明亮的单体近红外荧光蛋白 (NIR-FP) 可用作标记蛋白质和细胞的标记物以及报告活细胞和生物体中分子活动的传感器。然而，目前的单体 NIR-FP 在普通 633/635/640 nm 激光的激发下会变暗，限制了它们在细胞/亚细胞水平成像中的广泛应用。在这里，我们报告了一种明亮的单体 NIR-FP，在 633 nm 处具有最大激发，命名为 mIFP663，由Xanthomonas campestris pv Campestris光敏色素 ( Xcc BphP) 设计。mIFP663 分子亮度高，消光系数大（86,600 M ^–1 cm ^–1) 和不错的量子产率 (19.4%)，以及在存在外源性 BV 的情况下，比 HEK293T 细胞中光谱相似的 NIR-FP 高 3-6 倍的高细胞亮度。此外，我们证明 mIFP663 能够分别标记关键的细胞和病毒蛋白，而不会干扰亚细胞定位和病毒复制。最后，使用 mIFP663，我们设计了改进的双分子荧光互补 (BiFC) 和新的生物发光共振能量转移 (BRET) 系统，以检测活细胞中的蛋白质-蛋白质相互作用。

更新日期：2022-07-01

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