Streamlined purification of fluorescently labeled Escherichia coli phosphate-binding protein (PhoS) suitable for...

Fluorescently labeled phosphate-binding proteins can be used as biomolecular tools to measure the release of inorganic phosphate (P_i) from enzymes in real time, enabling the detailed kinetic analysis of dephosphorylating enzymes using rapid-kinetics approaches. Previously reported methods to purify fluorescently labeled phosphate-binding proteins (PhoS) from Escherichia coli are laborious, and a simplified approach is needed. Here, we report the characterization of a cytosol-localized variant (A197C) of PhoS that allows a streamlined purification for subsequent covalent conjugation with a fluorescent dye. We show that export of PhoS into the periplasmic space is not required for the fluorescence-based detection of P_i binding. Furthermore, we report the addition of a C-terminal His-tag, simplifying the purification of PhoS from the cytosol via Ni²⁺-affinity chromatography, yielding a fully functional fusion protein (HC PhoS A197C). We demonstrate the utility of fluorescently labeled HC PhoS A197C for rapid-kinetics applications by measuring, using stopped-flow, the P_i release kinetics from LepA/EF4 following 70S ribosome-stimulated GTP hydrolysis. Altogether, the approach developed here allows for the high-yield and simplified in-house production of a P_i detection system suitable for rapid-kinetics approaches with comparable sensitivity to the commercially available Phosphate Sensor.