Recombinant MIF Protein Purification

Recombinant MIF protein was purified from Escherichia coli, as previously described [23 (link),24 (link)]. Briefly, BL21 E. coli strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET28a-MIF in order to express and purify the fusion protein. Bacterial cultures were induced with 0.4 mM isopropyl β-d-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). Next, bacterial cultures were sonicated for 10 min. at 4 °C, and then centrifuged at 1600× g for 20 min. at 4 °C. Pellets containing His-tagged MIF (His-MIF) were resuspended in binding buffer (0.5 M NaCl, 20 mM Tris-HCl, 5 mM imidazole) containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). His-MIF was purified while using Ni-NTA His•Bind Resin (Merck, Darmstadt, Germany) and subsequently eluted using elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole) containing 4 M urea. Finally, MIF protein was dialyzed in order to remove imidazole, residual salts, and urea.