Affinity Purification of GST Fusion Proteins

Enzyme purification was performed according to the methods described in previous reports [52 (link),53 (link)] with modifications. Cell cultures (1 L) were separated via centrifugation at 8000× g for 10 min. The cell pellets were resuspended in 40 mL binding buffer (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, pH 7.0) and subjected to 400 pulses of sonication (400 W, 3 s each with a 5 s interval) in an ice-water bath [54 (link)]. Following centrifugation (13,000× g) at 4 °C for 30 min, the supernatant was passed through a 0.22 μm filter and applied to a GSTrap (GE Healthcare, Marlborough, MA, USA) FF column of the liquid chromatography system, AKTAPure (GE Healthcare, Marlborough, MA, USA), and GST fusion proteins were eluted via a GSTrap FF column using 10 mM reduced glutathione according to standard affinity chromatography. GST fusion proteins were digested using PreScission protease (GE Healthcare, Marlborough, MA, USA) according to the manufacturer’s instructions.