Gst tag resin

Briefly, a pGEX‐4T‐2 construct containing GST‐fused FolSrpk1, a pET28 construct containing His‐fused FolArd1, and FolSir2 were expressed in BL21 E. coli, respectively. Transformed cells were induced by adding isopropyl β‐D‐1‐thiogalactopyranoside (IPTG) to a final concentration of 0.2 mM when the OD600 reached 0.6, and the culture was further grown at 37°C for 3 h. Cells were harvested by centrifugation and lysed by sonication in lysis buffer (50 mM Tris–HCl, pH 7.5, 300 mM NaCl, 1 mM PMSF, and Roche EDTA free protease inhibitor). For purification, the clarified lysate was added to GST‐tag resin (Beyotime) and nickel column (Beyotime), respectively, followed by incubation for 2 h at 4°C. GST beads were then washed with 5 column volumes of PBS. The nickel column was washed with five column volumes of wash buffer (lysis buffer with 20 mM imidazole). GST and His fusion proteins were gradually eluted in elution buffer supplemented with 10 mM reduced glutathione or 100 mM imidazole, respectively. All proteins were then dialyzed at 4°C overnight.