Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors.
Fluorogen activating protein-affibody probes: modular, no-wash measurement of epidermal growth factor receptors.
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DOI：
10.1021/bc500525b
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发表时间：
2015-01-21
影响因子：
4.7
通讯作者：
Bruchez, Marcel P.
中科院分区：
文献类型：
--
作者：
Wang, Yi;Telmer, Cheryl A.;Schmidt, Brigitte F.;Franke, Josef D.;Ort, Stephan;Arndt-Jovin, Donna J.;Bruchez, Marcel P.
荧光对于动态活细胞成像至关重要，并且需要亲和试剂来定量内源蛋白。各种荧光染料可以报告生物运输的不同方面，但必须独立地与亲和试剂缀合，并针对特定的生物读数进行表征。在这里，我们介绍了用于研究受体池快速变化的小型抗 EGFR 亲和探针的新型模块化平台的特征。与孔雀石绿 (MG) 衍生物结合以进行荧光激活的蛋白结构域 (FAP dL5**) 表达为与紧凑 EGFR 结合亲和体 ZEGFR:1907 的一个或两个拷贝的重组融合物。这是一种针对天然 EGFR 分子的重组荧光标记试剂。体外荧光测定表明，这些染料与 FAP-亲和体融合体的结合产生了数千倍的荧光增强，具有高结合亲和力和快速关联率。流式细胞术测定和荧光显微镜证明，这些探针标记 A431 细胞上的内源 EGFR，而不破坏 EGFR 功能，并且使用双 ZEGFR:1907 构建体观察到低纳摩尔表面 Kd 值。光捕获荧光剂（染料）的应用显着改善了检测到的荧光信号。改变配体、探针和染料的添加顺序可以区分受体的表面池和内吞池，从而揭示内吞运输的快速动态。因此，FAP/亲和体偶联提供了一种构建紧凑和模块化亲和探针的新方法，该探针可标记活细胞上的内源蛋白，并可用于研究参与运输的受体池的快速变化。
Fluorescence is essential for dynamic live cell imaging, and affinity reagents are required for quantification of endogenous proteins. Various fluorescent dyes can report on different aspects of biological trafficking, but must be independently conjugated to affinity reagents and characterized for specific biological readouts. Here we present the characterization of a new modular platform for small anti-EGFR affinity probes for studying rapid changes in receptor pools. A protein domain (FAP dL5**) that binds to malachite-green (MG) derivatives for fluorescence activation was expressed as a recombinant fusion to one or two copies of the compact EGFR binding affibody ZEGFR:1907. This is a recombinant and fluorogenic labeling reagent for native EGFR molecules. In vitro fluorescence assays demonstrated that the binding of these dyes to the FAP–affibody fusions produced thousand-fold fluorescence enhancements, with high binding affinity and fast association rates. Flow cytometry assays and fluorescence microscopy demonstrated that these probes label endogenous EGFR on A431 cells without disruption of EGFR function, and low nanomolar surface Kd values were observed with the double-ZEGFR:1907 constructs. The application of light-harvesting fluorogens (dyedrons) significantly improved the detected fluorescence signal. Altering the order of addition of the ligand, probe, and dyes allowed differentiation between surface and endocytotic pools of receptors to reveal the rapid dynamics of endocytic trafficking. Therefore, FAP/affibody coupling provides a new approach to construct compact and modular affinity probes that label endogenous proteins on living cells and can be used for studying rapid changes in receptor pools involved in trafficking.
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DOI：
10.1093/protein/12.10.873
发表时间：
1999-10-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
Gunneriusson, E;Nord, K;Nygren, PÅ
通讯作者：
Nygren, PÅ
影响因子：
2.4
作者：
Kronqvist, Nina;Malm, Magdalena;Lofblom, John
通讯作者：
Lofblom, John
DOI：
10.1093/protein/8.6.601
发表时间：
1995-06-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
NORD, K;NILSSON, J;NYGREN, PA
通讯作者：
NYGREN, PA
影响因子：
5.2
作者：
Gostring, Lovisa;Chew, Ming Tsuey;Frejd, Fredrik Y.
通讯作者：
Frejd, Fredrik Y.
影响因子：
120.1
作者：
Nelson, Aaron L.;Dhimolea, Eugen;Reichert, Janice M.
通讯作者：
Reichert, Janice M.