Recombinant Ag85B and p24 Purification

Recombinant Ag85B and p24 proteins were purified from E. coli as previously described45 (link) with minor modification. For the expression and purification of fusion protein, E. coli BL21 strains (RBC Bioscience, Taipei City, Taiwan) were transformed with pET23a-Ag85B or -p24. Protein expression was induced by adding 0.4 mM isopropyl β-D-thiogalactoside (IPTG, Duchefa Biochemie, Haarlem, Netherlands). Bacterial cells were harvested and disrupted by sonication on ice for 10 min. Sonicated lysates were centrifuged at 1600 ×g for 20 min at 4 °C, and the pellets containing Ag85B and p24 proteins were resuspended in binding buffer containing 4 M urea (Sigma Aldrich, St. Louis, MO, USA). The proteins were purified using Ni-NTA His binding resin (Merck, Darmstadt, Germany), and eluted with elution buffer (300 mM NaCl, 50 mM sodium phosphate buffer, 250 mM imidazole) containing 4 M urea. Purified proteins were dialyzed serially against the elution buffer to remove imidazole, urea and residual salts.