双分子荧光互补技术的应用与展望

Application and Prospects of Bimolecular Fluorescence Complementation Technique

双分子荧光互补技术是近年发展起来的用于体内或体外检测蛋白质相互作用的一项新技术。该技术是将荧光蛋白在合适的位点切开形成不发荧光的2个片段,这2个片段借助融合于其上的目标蛋白的相互作用,彼此靠近,重新构建成完整的具有活性的荧光蛋白分子,从而产生荧光。BiFC方法简单直观,具有可视性的特点,对温度敏感,荧光片段种类较多,被广泛地应用到不同的细胞中,既可以检测蛋白之间的相互作用,也可以定位蛋白质相互作用的位点。此外,BiFC还能在蛋白构型的确定以及RNA的检测方面发挥作用。经过若干年的发展,双色荧光互补技术已经发展成包括多色荧光互补技术,BiFC和FRET联用技术以及BiFC和YTH联用技术在内的多种技术,拓宽了BiFC的应用。

Bimolecular fluorescence complementation （BiFC） is a technique typically used to validate protein interactions. The principle of the BiFC assay is to split a fluorescent protein （FP） into two nonfluorescent fragments that are fused to two interacting proteins. Upon the interaction of the two proteins, the two non-fluorescent fragments are brought into close proximity so that an intact FP is reconstituted. Bimolecular fluorescence eomplementation assay is widely used because of its simplicity and high sensitivity. The BiFC assay can be used to visualize interactions between any proteins that can be fused to fluorescent protein fragments. The assay can be also used to detect interactions in any subcellular compartment in any aerobically-growing organism or cell that can be geneti- cally modified to express the fusion proteins. Moreover, the BiFC technique has been refined and expanded to include the abilities to simultaneously visualize multiple protein complexes in the same cell, RNA/protein interactions, and conformational changes of inte- rested proteins. Many different fluorescent protein fragments have been identified that can be used in muhicolor BiFC analysis. Recently, the BiFC-based FRET and YTH systems have been developed for visualization of ternary complexes, which have broadened the application of the BiFC assay.