Gst accept resin

All proteins for crystallizations and in vitro experiments were expressed in E. coli BL21 (DE3). After cultivating bacteria at 37°C until OD₆₀₀ reached 0.8 to 1.2, overnight culturing with 100 μM IPTG was performed at 16°C. After centrifugation, the bacteria were resuspended to PBS with 5 mM EDTA and lysed by sonication for 10 min. After centrifugation, the supernatants were incubated with affinity resin column: GST accept resin (Nacalai Tesque) for GST-fused proteins and Amylose Resin High Flow resin for MBP-fused proteins (New England Biolabs). After washing the resin with PBS three times, the proteins were eluted with glutathione buffer (10 mM glutathione and 50 mM Tris-HCl pH 8.0) for GST-fused proteins or maltose buffer (10 mM maltose, 20 mM Tris-HCl pH 8.0, 200 mM sodium chloride) for MBP-fused proteins. The eluates were then digested by HRV 3C protease at 4°C for overnight to remove the affinity tag. The proteins were further subjected to size exclusion chromatography (SEC) with 20 mM HEPES pH 6.8 and 150 mM sodium chloride by using Superdex 75 26/60 or Superdex 75 10/300 column (GE Healthcare). Synthesized SpHfl1(386-398) peptide (purchased from Bex Co.) was dissolved in water and purified by SEC with 20 mM HEPES pH 6.8 and 150 mM sodium chloride by using Superdex peptide 10/300 column (GE Healthcare).