Hodgson Louis, Pertz Olivier, Hahn Klaus M
Department of Pharmacology, Lineberger Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
Methods Cell Biol. 2008;85:63-81. doi: 10.1016/S0091-679X(08)85004-2.
This chapter details the design and optimization of biosensors based on a design used successfully to study nucleotide loading of small GTPase proteins in living cells. This design can be generalized to study many other protein activities, using a single, genetically encoded chain incorporating the protein to be studied, an "affinity reagent" which binds only to the activated form of the targeted protein, and mutants of the green fluorescent protein (GFP) that undergo fluorescence resonance energy transfer (FRET). Specific topics include procedures and caveats in the design and cloning of single-chain FRET sensors, in vitro and in vivo validation, expression in living cell systems for biological studies, and some general considerations in quantitative fluorescence imaging.
本章详细介绍了基于一种成功用于研究活细胞中小GTPase蛋白核苷酸负载的设计的生物传感器的设计与优化。这种设计可以推广到研究许多其他蛋白质活性，使用一条单一的、基因编码的链，其中包含要研究的蛋白质、仅与靶向蛋白质的活化形式结合的“亲和试剂”，以及经历荧光共振能量转移（FRET）的绿色荧光蛋白（GFP）突变体。具体主题包括单链FRET传感器的设计与克隆、体外和体内验证、在活细胞系统中用于生物学研究的表达以及定量荧光成像中的一些一般考虑因素的程序和注意事项。
