Summary Akanes are fluorescent proteins that have several fluorescence maxima. In this report, Akane1 and Akane3 from Scleronephthya gracillima were selected, successfully overexpressed in Escherichia coli, and purified by affinity chromatography. Fluorescence spectra of the recombinant Akanes matured in darkness, or ambient light were found to have several fluorescence peaks. SDS-PAGE analysis revealed that Akanes matured in ambient light have two fragments. MS/MS analysis of Akanes digested with trypsin showed that the cleavage site is the same as observed for the photoconvertible fluorescent protein Kaede. The differences between the calculated masses from the amino acid sequence of Akane1 and the measured masses of Akane1 fragments obtained under ambient light coincided with those of Kaede. In contrast, a mass difference between the measured N-terminal Akane3 fragment and the calculated mass indicated that Akane3 is modified in the N-terminal region. These results indicate that numerous peaks in the fluorescent spectra of Akanes partly arise from isoproteins of Akanes and photoconversion. Photoconversion of Akane1 caused a fluorescence change from green to red, which was also observed for Akane3; however, the fluorescent intensity decreased dramatically when compared with that of Akane3.

中文翻译：

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重组光转换绿色荧光 Akane 的表征

摘要 Akane 是具有多个荧光最大值的荧光蛋白。在本报告中，选择了来自 Scleronephthya gracilima 的 Akane1 和 Akane3，在大肠杆菌中成功过表达，并通过亲和层析纯化。发现在黑暗或环境光下成熟的重组Akanes的荧光光谱具有多个荧光峰。SDS-PAGE分析显示在环境光下成熟的Akanes有两个片段。用胰蛋白酶消化的 Akane 的 MS/MS 分析表明，切割位点与光转换荧光蛋白 Kaede 中观察到的切割位点相同。根据 Akane1 的氨基酸序列计算出的质量与在环境光下获得的 Akane1 片段的测量质量之间的差异与 Kaede 的质量一致。相反，测量的 N 末端 Akane3 片段与计算的质量之间的质量差异表明 Akane3 在 N 末端区域被修饰。这些结果表明，Akanes 荧光光谱中的许多峰部分源自 Akane 的同种蛋白和光转换。Akane1 的光转换导致荧光从绿色变为红色，Akane3 也观察到这种情况；然而，与Akane3相比，荧光强度急剧下降。