荧光蛋白MiCy、Citrine作为FRET传能对的研究  

Green Fluorescent Proteins MiCy and Citrine as a Novel Donor/Acceptor Pair for Fluorescence Resonance Energy Transfer

作　　者：刘德龙[1,2] 王晓静[1] 郭慧芳[2] 白娟[2] 孙大业[2] 

机构地区：[1]河北师范大学化学学院,河北石家庄050016 [2]河北师范大学生命科学学院,河北石家庄050016

出　　处：《分析测试学报》2009年第1期44-48,共5页Journal of Instrumental Analysis

基　　金：国家自然科学基金资助项目(20475013)

摘　　要：探索了2种新型荧光蛋白MiCy、Citrine作为荧光共振能量转移技术(FRET)传能对的可能性。首先通过基因工程方法表达纯化了这2种蛋白,并对其进行了光谱学研究,由于MiCy具有高的量子产率,且MiCy与Citrine的光谱重叠积分J较大,计算表明MiCy与Citrine的Frster距离为R0=5.81nm,比CFP-YFP的大(R0=5nm)。进一步以Ni2+-NTA-agarose为FRET模型,结果表明:无论在荧光分光光度计还是在激光共聚焦显微镜上,都可以明显地检测到MiCy与Citrine的能量转移。由于二者较文献报道的其它FRET对具有更大的Frster距离,而且更适合以激光作为激发光进行测量,因此该FRET对应有更加广泛的应用前景。Fluorescence resonance energy transfer（FRET） between mutant green fluorescent proteins （GFP） has been widely employed to detect protein - protein interactions in living cells. In this work, two GFP MiCy and Citrine were proposed as a novel donor/acceptor pair for FRET. The GFP, ex- pressed via the genetic engineering method were purified by histidine affinity chromatographic column and characterized by spectroscopic method. Based on the facts that MiCy has a high fluorescence quantum yield and there is a relative large spectral overlapping integration J between MiCy and Cit- fine, the F6rster distance between Micy and Citrine was calculated to be 5.81 nm, which is higher than that of CFP/YFP pair（5 nm）. By using Ni2＋ -NTA -agarose as the FRET model, the FRET between MiCy/Citrine pair was successfully detected by fluorescence spectrophotometer and confoeal microscope, respectively. Owing to the large F^rster distance between the Micy/Citrine pair and the maximum excitation wavelength of MiCy （472 nm） is close to that of Ar laser （457 nm）, the pro- posed FRET pair will have a promising application prospect.

关 键 词：绿色荧光蛋白 MiCy Citrine 荧光共振能量转移 Frster距离 

分 类 号：O657.39[理学—分析化学] Q51[理学—化学]