Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein
Fluorescence resonance energy transfer between blue-emitting and red-shifted excitation derivatives of the green fluorescent protein
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DOI：
10.1016/0378-1119(95)00768-7
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发表时间：
1996-07-01
期刊：
影响因子：
3.5
通讯作者：
Youvan, DC
中科院分区：
文献类型：
--
作者：
Mitra, RD;Silva, CM;Youvan, DC
我们报告荧光共振能量转移（FRET）之间的两个连接的变体的绿色荧光蛋白（GFP）。GFP的红移变体（RSGFP 4）的C末端融合到含有因子X（a）蛋白酶切割位点的柔性多肽接头。该接头的C末端又与GFP的蓝色变体（BFP 5）的N末端融合。该基因产物具有光谱性质，表明能量转移发生在从BFP 5到RSGFP 4。在与因子X（a）孵育后，蛋白质被切割，并且两种荧光蛋白解离。这伴随着能量转移的显著减少。RSGFP 4：：BFP 5融合蛋白证明了在两个GFP衍生物之间使用FRET作为监测蛋白质-蛋白质相互作用的工具的可行性;此外，该构建体可以作为蛋白酶抑制剂的细胞内筛选找到应用。
We report fluorescent resonance energy transfer (FRET) between two linked variants of the green fluorescent protein (GFP). The C terminus of a red-shifted variant of GFP (RSGFP4) is fused to a flexible polypeptide linker containing a Factor X(a) protease cleavage site. The C terminus of this linker is in turn fused to the N terminus of a blue variant of GFP (BFP5), The gene product has spectral properties that suggest energy transfer is occurring from BFP5 to RSGFP4. Upon incubation with Factor X(a), the protein is cleaved, and the two fluorescent proteins dissociate. This is accompanied by a marked decrease in energy transfer. The RSGFP4::BFP5 fusion protein demonstrates the feasibility of using FRET between two GFP derivatives as a tool to monitor protein-protein interactions; in addition, this construct may find applications as an intracellular screen for protease inhibitors.