For production of milligram quantities of the recombinant protein, optimized conditions were scaled up accordingly. The E. coli BL21 harboring pET23a/rv1733 construct was grown in 200 ml of Terrific-Broth in 1 L baffled flask at 37°C to an OD
600
of 0.6, followed by induction with 0.4 mM IPTG for 10 h. The cells were then pelleted by centrifugation and stored at −20°C before being lysed.
The cell pellets were resuspended in the selected lysis buffer (20 ml/g cell pellet, 20 mM NaH
2
PO
4
, 500 mM NaCl and 6 M urea, pH 8) (Merck, Darmstadt, Germany) and incubated for 15 min on ice. The suspension was sonicated six times for 20 s and cell debris was removed by centrifugation (13000 g, 30 min, 4°C). The supernatant was passed through 0.45 μm filter before loading on complete His-Tag Purification Column (Roche, Mannheim, Germany). The column was washed extensively with washing buffer (20 mM NaH
2
PO
4
, 500 mM NaCl, pH 8) until the OD
280
of the buffer reached below 0.01. The proteins were then eluted using a column volume of 500 mM imidazol (Sigma-Aldrich), pH 8 containing complete protease inhibitor (Sigma-Aldrich). The eluted fractions were combined and dialyzed against PBS (pH 7.4), and quantified by Bradford total protein assay kit (ZellBio GmbH, Ulm, Germany). LPS contamination was evaluated by the Limulus amoebocyte lysate (LAL) assay (Cambre, USA).
600
of 0.6, followed by induction with 0.4 mM IPTG for 10 h. The cells were then pelleted by centrifugation and stored at −20°C before being lysed.
The cell pellets were resuspended in the selected lysis buffer (20 ml/g cell pellet, 20 mM NaH
2
PO
4
, 500 mM NaCl and 6 M urea, pH 8) (Merck, Darmstadt, Germany) and incubated for 15 min on ice. The suspension was sonicated six times for 20 s and cell debris was removed by centrifugation (13000 g, 30 min, 4°C). The supernatant was passed through 0.45 μm filter before loading on complete His-Tag Purification Column (Roche, Mannheim, Germany). The column was washed extensively with washing buffer (20 mM NaH
2
PO
4
, 500 mM NaCl, pH 8) until the OD
280
of the buffer reached below 0.01. The proteins were then eluted using a column volume of 500 mM imidazol (Sigma-Aldrich), pH 8 containing complete protease inhibitor (Sigma-Aldrich). The eluted fractions were combined and dialyzed against PBS (pH 7.4), and quantified by Bradford total protein assay kit (ZellBio GmbH, Ulm, Germany). LPS contamination was evaluated by the Limulus amoebocyte lysate (LAL) assay (Cambre, USA).