| Cells were harvested at 4°C by centrifugation at 9000 g for 15 min. The pellet was resuspended in ice-cold lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 1 mM EDTA, 0.1% Triton–X 100, 0.25 mg/ml of lysozyme and 1 mM phenylmethylsulfonyl fluoride (PMSF)) and stored at –80°C overnight. The pellet was thawed on ice for 1 hour and then 10 µg/ml DNAse and 20 mM MgSO4 (final concentration) were added to the cell suspension for 30 min. Cells were disrupted by ultrasonication (10 times with a 15s cycle) using a Branson Sonifier 450. Inclusions bodies were separated from the cell extract by centrifugation at 17000 g for 30 min. The pellet was then washed with 10 mM Tris–HCl pH 8.0 and 150 mM NaCl, sonicated (4 times with a 15s cycle) and collected by centrifugation at 17000 g for 20 min, this procedure was repeated three times. Inclusions bodies were solubilized by stirring at 4°C overnight in a 40 ml solution containing 10 mM Tris–HCl pH 8.0, 150 mM NaCl and 6 M guanidine hydrochloride. The solubilized protein was clarified from insoluble material at 4°C by centrifugation at 17000 g for 15 min. The supernatant was loaded (3 ml/min) onto a Ni2+-agarose column (1 ml resin for 5 mg of recombinant protein) previously equilibrated with buffer A (10 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM imidazole and 8 M urea). The column was then washed using 5 column volumes of 5% and 10% of buffer B (buffer A+500 mM imidazole). Enzyme was eluted with 50% of buffer B; fractions of eluted peaks containing purified Rv1399c were analyzed by SDS–PAGE, pooled and concentrated up to 4-5 mg/ml using an Amicon cell.
Refolding:
Purified Rv1399c was refolded by a dilution method consisting of diluting the concentrated protein in buffers with different pH and various compositions. The refolding conditions were determined by a refolding method based on the measurement of the turbidity at 340 nm using a 96-well plate. The final refolding conditions consisted of diluting Rv1399c 20 fold in 50 mM Tris buffer, pH 7, at 4°C for two days. Rv1399c was concentrated up to 2-3 mg/ml and traces of urea and imidazole were removed using a desalting column (HiPrepTM 26/10, Amersham Biosciences). Rv1399c was then concentrated up to 3 mg/ml and stored overnight at –20°C, and after thawing, the “active” refolded material was recovered by centrifugation at 17000 g for 15 min. Rv1399c is stable in the refolding buffer for at least 1 month at 4°C and can be stored at –20°C for several months. |