The plasmids were transformed into Rosetta (DE3) Escherichia coli cells. The single clones were cultured in 2YT medium containing antibiotic at 37°C to an optical density of 0.6 nm and then induced with 0.3 mM Isopropyl-β-d-1-thiogalactopyranoside (IPTG) for 12 hours at 16°C. After the cells were harvested and sonicated, the supernatant was incubated with glutathione resin (GenScript) or Ni-charged resin (GenScript) at 4°C for 2 hours. The resins were washed with washing buffer [50 mM tris (pH 7.5), 200 mM NaCl, and 0.1% Tween 20] and then eluted with washing buffer containing 20 mM reduced glutathione for GST fusion proteins or 100 mM imidazole for His fusion proteins.

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