Materials and Reagents

NaH₂PO₄
EDTA
DTT
Tris
NaCl
Glycine
Ethylene glycol
HCI
QAE Sepharose Column Starting buffer (see Recipes)
QAE Sepharose Column Elution buffer (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)
Buffer C (see Recipes)

Equipment

High Performance Liquid Chromatography (HPLC) system (Millenium V2.00, Waters, USA)
SDS-PAGE (Mini-PROTEAN^® Tetra Cell, catalog number: 165-8000 )
Phenyl Sepharose HP column (3.5 x 1 cm) (GE Healthcare, catalog number: 17-1082-01 )
QAE sepharose column (Amersham Pharmacia – anion exchange chromatography).

Procedure

Purification of 30 kDa antigen:
The secreted Antigen 85 complex of mycobacteria consists of three proteins Ag 85A, B and C. Among these Antigen 85B otherwise known as 30 kDa antigen is one of the major immunogenic antigen.
The 30 kDa antigen was purified from the whole culture filtrate of M. tuberculosis by first passing through QAE column followed by passing the 30 kDa enriched fraction through a Phenyl Sepharose hydrophobic interaction column.
1. Anion exchange chromatography-QAE sepharose (purification of Ag85 complex)
  (PURIFICATION OF AG 85 COMPLEX)
  1. Culture filtrate antigen (CFA) between 10 to 30 mg was prepared as mentioned in the 38 kDa antigen purification protocol (Hyperlink here) was separated into multiple fractions by passing through a prepacked anion exchange column of QAE Sepharose. 0.05 M Tris-HCI buffer (pH 8.1) was used as the starting buffer and 0.05 M Tris-HCI, pH 8.1, with 1.0 M sodium chloride was used as the elution buffer. The separation was carried out in the High Performance Liquid Chromatography (HPLC, Millenium V2.00, Waters, USA) system under standard running conditions. The eluted fractions were analyzed on 12.5% SDS-PAGE which separates proteins based on its molecular weight, was run under standard conditions (120 V, 1.5 A). The Ag85 complex is identified as a 30/31 kDa doublet band on the gel. The fractions containing Antigen 85 complex were pooled and concentrated by Amicon centrifuge filters with the molecular cut off of 3 kDa.
  2. Further purification of the A, B and C components from the antigen 85 complex was done by passing it through the hydrophobic interaction chromatography (HIC) column namely the Phenyl Sepharose HP column.
2. Hydrophobic interaction chromatography-phenyl sepharose (separation of the individual components of the antigen 85 complex)
  1. Purification of the antigen 85 complex into its individual components Ag 85A; 85B and 85C was done by passing it through the Phenyl Sepharose HP column using the Biologic Chromatography system (FPLC, Bio-Rad, USA). Three different buffers were used during the run. The buffers A, B, C used are mentioned under recipes section.
  2. The run conditions were such that the column was subjected to an isocratic flow of Buffer A for 10 min (flow rate 1 ml/min), followed by the injection of the sample (2 ml) which was dialysed against buffer A at the rate of 0.5 ml/min. Then an isocratic flow of Buffer B for 10 min at the rate of 1 ml/min was used for washing the unbound material.
  3. Elution of the individual components of Antigen 85 complex namely Ag85A, B and C were carried out with a linear gradient of Buffer C (from 0 -100%) for 30 min with the flow rate of 1 ml/min. Subsequently, the protein containing fractions were identified by optical density measured at 280 nm on a spectrophotometer. The fractions containing Ag 85B were pooled, concentrated and checked on 12.5% SDS PAGE for purity.

Recipes

QAE Sepharose Column Starting buffer
0.05 M Tris-HCI (pH 8.1)
QAE Sepharose Column Elution buffer
0.05 M Tris-HCI (pH 8.1)
1 M NaCl
Buffer A
0.01 M NaH₂PO₄
1 mM EDTA
1 mM DTT (pH 6.8)
Buffer B
0.01 M Tris
100 mM Glycine
1 mM EDTA
1 mM OTT(pH 8.9)
Buffer C
0.01 M Tris
100 mM Glycine
1 mM DTT
50% (v/v) Ethylene Glycol (pH 8.9)

Acknowledgments

This protocol was extracted from the original publication by Devi et al. (2002). This work was supported by Indo US VAP & DBT India granted for Dr. Alamelu Raja.

References

Devi, K. R., Kumar, K. S., Ramalingam, B. and Alamelu, R. (2002). Purification and characterization of three immunodominant proteins (38, 30, and 16 kDa) of Mycobacterium tuberculosis. Protein Expr Purif 24(2): 188-195.
Raja, A., Uma Devi, K. R., Ramalingam, B. and Brennan, P. J. (2004). Improved diagnosis of pulmonary tuberculosis by detection of free and immune complex-bound anti-30 kDa antibodies. Diagn Microbiol Infect Dis 50(4): 253-259.