N-terminal fusion of the N-terminal domain of bacterial enzyme I facilitates recombinant expression and purification of the human RNA demethylases FTO and Alkbh5.

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.

Identification of the binding between three fluoronucleoside analogues and fat mass and obesity-associated protein by isothermal titration calorimetry and spectroscopic techniques.

In this work, the interactions between fat mass and obesity-associated (FTO) protein and three fluoronucleoside analogues (three-member-ring compound (1a), five-member-ring compound (1b) and six-member-ring compound (1c)) have been investigated by fluorescence, ultraviolet-visible absorption spectroscopy, isothermal titration calorimetric (ITC) and molecular modeling. Analysis of fluorescence data showed that the binding between three analogues and FTO occurred via a static quenching mechanism. Both ITC and fluorescence results indicated that 1b is the strongest quencher. In contrast to spectroscopy techniques, ITC results suggested that there is no binding for 1c. ITC results showed that the binding between FTO and 1a (or 1b) were exothermic. Fluorescence results showed that the binding between three analogues and FTO were endothermic. Results of thermodynamic analysis and molecular modeling suggested that it was entropy driven event between FTO and 1a (or 1b).