Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli
Specific modification at the C-terminal lysine residue of the green fluorescent protein variant, GFPuv, expressed in Escherichia coli
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大肠杆菌中表达的绿色荧光蛋白变体 GFPuv C 端赖氨酸残基的特异性修饰
DOI：
10.1038/s41598-019-41309-8
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发表时间：
2019
影响因子：
4.6
通讯作者：
Issei Tamura & Atsuko Yamashita
中科院分区：
文献类型：
--
作者：
Takahiro Nakatani;Norihisa Yasui;Issei Tamura & Atsuko Yamashita
绿色荧光蛋白（Green fluorescent protein， GFP）具有在多种细胞中重组表达的特性，在生命科学研究中有着广泛的应用。我们发现，GFP的一种常用突变体GFPuv的重组表达，inE。通过凝胶内荧光SDS-PAGE检测，大肠杆菌产生了两个不同的分子种。通过阴离子交换色谱法可以分别纯化出I型和II型分子，在荧光光谱上没有明显差异。质谱分析表明，形式I的分子质量与计算值几乎相同，而形式II的分子质量比形式I大约1da。进一步的质谱自上而下测序确定了GFPuv形式II的修饰，其中c端Lys238残基的ε-氨基被转化为羟基。在水母（equorea victoria）的原生GFP中未观察到相应的修饰，表明这种修饰与生理无关。两种植物的晶体结构分析证实了主链和发色团附近的结构相同。本研究中发现的修饰也可能在其他GFP变体以及其他重组表达系统中产生。
Green fluorescent protein (GFP) is amenable to recombinant expression in various kinds of cells and is widely used in life science research. We found that the recombinant expression of GFPuv, a commonly-used mutant of GFP, inE. coliproduced two distinct molecular species as judged by in-gel fluorescence SDS-PAGE. These molecular species, namely form I and II, could be separately purified by anion-exchange chromatography without any remarkable differences in the fluorescence spectra. Mass spectrometric analyses revealed that the molecular mass of form I is almost the same as the calculated value, while that of form II is approximately 1 Da larger than that of form I. Further mass spectrometric top-down sequencing pinpointed the modification in GFPuv form II, where the ε-amino group of the C-terminal Lys238 residue is converted into the hydroxyl group. No equivalent modification was observed in the native GFP in jellyfishAequorea victoria, suggesting that this modification is not physiologically relevant. Crystal structure analysis of the two species verified the structural identity of the backbone and the vicinity of the chromophore. The modification found in this study may also be generated in other GFP variants as well as in other recombinant expression systems.