J Bacteriol Virol.  2006 Dec;36(4):211-220. 10.4167/jbv.2006.36.4.211.

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

• Lee JS
• Paik TH
• Yoo YC
• Lee J
• Shin A
• Song CH
• Jo EK
• Kim HJ
• Park JK

• KMID: 2055029
• DOI: http://doi.org/10.4167/jbv.2006.36.4.211

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, (P)H 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.

Keyword

Mycobacterium tuberculosis; Protein antigen; Purification; Ag85 complex

MeSH Terms

Ammonium Sulfate
Chromatography
Hydrogen-Ion Concentration
Hydrophobic and Hydrophilic Interactions
Immunodiffusion
Indicators and Reagents
Mycobacterium tuberculosis*
Mycobacterium*
Tuberculosis
Ultrafiltration
Vaccines
Ammonium Sulfate
Indicators and Reagents
Vaccines

Figure

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  Figure 1. Protein concentration and SDS-PAGE analysis of culture filtrates from M. tuberculosis H37Rv. (A) The protein concentration of culture filtrates during different culture period (1∼8 week). The culture filtrates of M. tuberculosis H37Rv were isolated from growing tubercle bacilli in Sauton's synthetic medium. And the protein concentrations were measured by bicinchoninic acid (BCA) protein assay. (B) SDS-PAGE analysis of culture filtrates during various culture periods. The culture filtrates for SDS-PAGE analysis were concentrated 100-fold by ultrafiltration. The 70 μg of protein was loaded to gradient Bis-Tris gel (4∼12%), except culture filtrates for 1∼2 weeks (10 μg of protein), separated by SDS-PAGE, and Coomassie blue stained. Lane 1∼8; week 1∼8, respectively.

• 

  Figure 2. (A) SDS-PAGE analysis of M. tuberculosis culture filtrate protein concentration by anion-exchange chromatography. The unheated culture filtrate protein of M. tuberculosis H37Rv was concentrated by anion-exchange chromatography. The culture filtrate protein of M. tuberculosis was diluted with 2.5 volume of 20 mM Tris-HCl, pH 8.3 and loaded to a column (2.5 by 20 cm) of Macro-prep High Q support (Bio-Rad). The CFPs were eluted in 20 mM Tris-HCl, pH 8.3 with 100 mM NaCl, concentrated by ultrafiltration (1st elution; lane 1). All pass-through fractions produced in the procedure were pooled, and then reapplied to Macro-prep High Q column, eluted with 100 mM NaCl again and concentrated by ultrafiltration (2nd elution; lane 2). This process was repeated twice more (3rd and 4th elution; lane 3, 4) (B) SDS-PAGE analysis after ammonium sulfate fractionation of the culture filtrate proteins (CFPs). Lane 1, 0∼40% ammonium sulfate fraction; lane 2, 40∼80% ammonium sulfate fraction.

• 

  Figure 3. SDS-PAGE analysis of finally purified Ag85 complex, 38-kDa, and MTB12 antigen of M. tuberculosis culture filtrate. The Ag85 complex, 38-kDa, and MTB12 were purified from unheated culture filtrate of M. tuberculosis H37Rv by 1st anion-exchange chromatography (Macro-prep high Q supported column (Bio-Rad), ultrafiltration, ammonium sulfate fractionation, hydrophobic interaction chromatography and 2nd anion-exchange chromatography (UNO-Q6 column (Bio-Rad) (FPLC). Lane 1, purified Ag85 complex; Lane 2, purified 38-kDa Ag; lane 3, purified MTB12 Ag.

• 

  Figure 4. Two-dimensional gel electrophoresis analysis of the purified 38-kDa and MTB12 antigens purified from M. tuberculosis culture filtrate. The purified 38-kDa and MTB12 antigens were resolved by isoelectrofocusing (38-kDa Ag, pH 4.7∼5.9; MTB12, pH 4.0∼7.0) followed by SDS-PAGE (38-kDa Ag, 12% acrylamide gel; MTB12 Ag, 15% acrylamide gel). The gels were stained with Coomassie blue.

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