Recombinant Tuberculosis Antigen Purification

Recombinant Ag85B, CFP10, ESAT-6, and TBCM proteins were purified from Escherichia coli as previously described [18 (link),19 (link)]. Briefly, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET28a-Ag85B, pET28a-CFP10, pET28a-ESAT-6, or pET28a-TBCM for the expression and purification of each fusion protein. Protein expression was induced by adding 0.4 mM isopropyl β-D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, The Netherlands). Cultured bacterial cells were disrupted by sonication (10 min, 4 °C), and the resultant lysates were centrifuged (1600× g, 20 min, 4 °C). The pellets containing each protein (Ag85B, CFP-10, ESAT-6, or TBCM) were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). Each protein was purified with Ni-NTA His binding resin (Merck, Darmstadt, Germany) and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, and 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea, and residual salts.