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Lee, Paik, Yoo, Lee, Shin, Song, Jo, Kim, and Park: Purification of Native Ag85 Complex, 38-kDa and MTB12 Protein Antigens from the Culture Filtrate of Mycobacterium tuberculosis
Original Article

J Bacteriol Virol 2006;36(4):211-220.

Published online: 18 February 2006

DOI: https://doi.org/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

Ji-Sook Lee1, Tae-Hyun Paik1, Yung-Choon Yoo1, Junglim Lee1, Arum Shin2, Chang-Hwa Song2, Eun-Kyung Jo2, Hwa-Jung Kim2, Jung-Kyu Park2

1Department of Microbiology, College of Medicine, Konyang University, Daejeon 302-718, Korea

2Department of Microbiology, College of Medicine, Chungnam National University, Daejeon 301-747, Korea

Received 1 September 2006       Accepted 17 October 2006

Copyright © 2006 Journal of Bacteriology and Virology

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Keywords: Mycobacterium tuberculosis, Protein antigen, Purification, Ag85 complex

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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.
jbv-36-211f1.tif
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.
jbv-36-211f2.tif
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.
jbv-36-211f3.tif
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.
jbv-36-211f4.tif
Table 1.
The protein and antigen concentration of culture filtrates during the different culture period of M. tuberculosis H37Rv
Duration of culture (weeks) 1 2 3 4 5 6 7 8
Protein concentration of culture filtrates (μg/ml) 2.6 5.4 25.5 74.5 264.0 331.0 479.0 549.0
Titer of specific antigena Ag85 complex 0 8 16 64 128 128 128 256
38-kDa 0 0 1 2 8 16 16 64
MTB12 2 4 32 128 256 512 512 512

a : Titer of specific antigen concentrated 100-fold by ultrafiltration was expressed as the reciprocal of the maximum antigen dilution that showed positive precipitin reaction with specific anti-serum by Ouchterlony immmunodiffusion

Table 2.
The purification procedures of Ag85 complex, 38-kDa and MTB12 antigens from the culture filtrate of Mycobacterium tuberculosis
Purification procedure Ag85 complex 38-kDa MTB12
Anion exchange chromatographya (Macro-prep high Q) 100 mM NaCl 100 mM NaCl 100 mM NaCl
Ammonium sulfate fractionation 0∼40% 40∼80% 40∼80%
Hydrophobic interaction chromatographyb 0.81∼0.45 M 1.35∼1.17 M 1.8∼1.62 M
Anion exchange chromatographyc (UNO-Q) 0∼20 mM NaCl 0∼20 mM NaCl 0∼10 mM NaCl

a : Bound proteins were eluted with 100 mM NaCl in 20 mM Tris-HCl, pH 8.3 contaning 0.02% NaN3, 1 mM EDTA.

b : Bound proteins were eluted with decreasing linear concentration of ammonium sulfate from 1.8 M to 0 M in 100 mM sodium phosphate buffer, pH 6.8 containing 0.02% NaN3 and 1 mM EDTA.

c : Concentration of NaCl in 20 mM Tris-HCl, pH 8.3 contaning 0.02% NaN3 and 1 mM EDTA

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