绿色荧光蛋白为传能对的供体光漂白法测定FRET效率的探讨

Determination of FRET Efficiency Using Donor Photobleaching Method with Green Fluorescent Proteins as Energy Transfer Pair

探讨以两种新荧光蛋白MiCy,mKo为传能对并应用供体光漂白法测量荧光共振能量转移(FRET)效率。首先通过基因工程方法表达纯化了这两种蛋白,并测量了荧光光谱及光漂白性质,表明MiCy极易光漂白而mKo抗光漂白。进一步以Ni-NTA-agarose为FRET模型,在Confocal上对MiCy进行光漂白时间常数的测量,并计算了FRET效率。结果表明MiCy-mKo传能对适合用供体光漂白法测量FRET效率,此传能对将在蛋白质相互作用研究中有广泛应用。

In this work, two new green fluorescence proteins, named MiCy and mKo, were used as the energy transfer pair and their FRET efficiency was measured by the donor photobleaching method. The excitation and emission spectra and the photobleaching curves of the purified MiCy and mKo proteins were determined. The results suggested that MiCy is the photobleaching donor and that mKo is a photostable acceptor. By using Ni -NTA -agarose as the FRET model, true transfer efficiency between MiCy and mKo was successfully detected on Confocal microscope. In conclusion, the MiCy - mKo is a proper FRET pair, and the donor photobleaching method can provide a very specific measurement of FRET efficiency.