Various fusion tags are commonly employed to increase the heterologous expression and solubility of aggregation-prone proteins within Escherichia coli. Herein, we present a protocol for efficient recombinant expression and purification of the human RNA demethylases Alkbh5 and FTO. Our method incorporates a novel fusion tag (the N-terminal domain of bacterial enzyme I, EIN) that dramatically increases the solubility of its fusion partner and is promptly removed upon digestion with a protease. The presented protocol allows for the production of mg amounts of Alkbh5 and FTO in 1L of both rich and minimal media. We developed a liquid chromatography-mass spectrometry (LC-MS)-based assay to confirm that both proteins are enzymatically active. Furthermore, the LC-MS method developed here is applicable to other members of the AlkB family of Fe(II)/α-ketoglutarate-dependent dioxygenases. The superior protein yield, afforded by our expression and purification method, will facilitate biochemical investigations into the biological function of the human RNA demethylases and endorse employment of EIN as a broadly applicable fusion tag for recombinant expression projects.

中文翻译：

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细菌酶I的N末端结构域的N末端融合促进了人RNA脱甲基酶FTO和Alkbh5的重组表达和纯化。

通常使用各种融合标签来增加大肠杆菌中易于凝集的蛋白质的异源表达和溶解度。在这里，我们提出了有效重组表达和纯化人类RNA脱甲基酶Alkbh5和FTO的协议。我们的方法结合了一种新型融合标签（细菌酶I，EIN的N末端结构域），该标签可显着提高其融合伴侣的溶解度，并在用蛋白酶消化后迅速去除。提出的协议允许在1L的丰富和基本培养基中产生毫克量的Alkbh5和FTO。我们开发了一种基于液相色谱-质谱（LC-MS）的测定法，以确认这两种蛋白质均具有酶促活性。此外，这里开发的LC-MS方法适用于Fe（II）/α-酮戊二酸依赖性双加氧酶的AlkB家族的其他成员。我们的表达和纯化方法可提供优异的蛋白质产量，这将有助于对人RNA脱甲基酶的生物学功能进行生化研究，并支持EIN作为重组表达项目广泛适用的融合标签的使用。