Purification of Native Ag85 Complex, 38-kDa and MTB12 Protein Antigens from the Culture Filtrate ofMycobacterium tuberculosis

Abstract

The purification of immunodominant native protein antigens from the culture filtrates of Mycobacterium tuberculosis is needed for the development of new vaccines and immunodiagnostic reagents against tuberculosis. In the present study, we conducted large scale purification of well-known secreted antigens, Ag85 complex, 38-kDa, and MTB12, from the culture filtrate proteins (CFPs) prepared from M. tuberculosis H37Rv grown as a surface pellicle on synthetic Sauton medium. The protein and antigen concentrations of culture filtrates were sufficiently increased after 6 week of culture. The MTB12 antigen was detected as early as 1 week of culture, and Ag85 complex and 38-kDa antigen were detected after 2 and 3 week of culture, respectively, by immunodiffusion with specific antiserum against 100-fold concentrated culture filtrates. For large-scale purification, the six-week-culture filtrates of M. tuberculosis H37Rv diluted 2.5-fold with 20 mM Tris-HCl, pH 8.3 were subjected to anion-exchange chromatography. The CFPs were eluted with 100 mM NaCl-20 mM Tris-HCl, pH 8.3 and concentrated by ultrafiltration. The concentrated CFPs were fractionated with ammonium sulfate, and followed by hydrophobic interaction chromatography and anion-exchange chromatography (FPLC). Eventually, 10 mg of Ag85 complex, 0.56 mg of 38-kDa, and 1.81 mg of MTB12 antigens were purified from 1 liter of the six-week-culture filtrates of M. tuberculosis H37Rv which contained 307.81 mg of protein of culture filtrate.

Therapeutic Effects of Mycobacterial Secretory Proteins Against Established Asthma in BALB/c Mice

“…Ag85 (0.632 mg/mL) and 38-kDa protein (0.95 mg/mL) were purified from the culture supernatants of M. tuberculosis , as described previously 15,19,20. MPB70 protein (1.8 mg/mL) from BCG culture supernatants was purified by a two-step process: hydroxylapatite chromatography followed by anion-exchange chromatography.…”

“…Ag85 (0.632 mg/mL) and 38-kDa protein (0.95 mg/mL) were purified from the culture supernatants of M. tuberculosis , as described previously. 15 , 19 , 20 MPB70 protein (1.8 mg/mL) from BCG culture supernatants was purified by a two-step process: hydroxylapatite chromatography followed by anion-exchange chromatography. Briefly, a 50%-80% ammonium sulfate precipitate of BCG culture filtrate was dissolved and dialyzed against 1 mM potassium phosphate buffer (PB, pH 7.0), loaded to a column of hydroxylapatite equilibrated with 1 mM PB, and eluted with 1 mM PB (because MPB70 was not retained on the column).…”