Revision as of 06:53, 21 July 2008
The stored frozen cells Rosetta 2 pLysS/pCM862 were thawed at 4 ºC and resuspended in 15 mL.g-1 of ice-cold lysis buffer (50 mM Imidazole, pH 6.8, 1 mM EDTA 10% w/v sucrose, 0.05 M NaCl, 2 mM DTT, 10 mM spermidine-3HCl). Lysozyme was added to a concentration of 0.2 mg.mL-1 and the suspension was stirred on ice for 30 minutes. The cells were then subjected to three freeze-thaw cycles, by direct addition of liquid nitrogen, whilst stirring until frozen and then fully defrosting at room temperature, whilst stirring. Cell debris was removed from the suspension by centrifugation (30 100 g, 10 minutes, 4 ºC).
The volume of the supernatant was measured. The NaCl concentration was adjusted to 1M and solid ammonium sulfate to a final concentration of 15% slowly added. The resulting suspension was stirred on ice for 1 hour and then centrifuged again (30 100 g, 20 minutes, 4ºC). The supernatant was retained and a sample was taken for SDS-PAGE analysis. The volume of the supernatant was again measured and solid ammonium sulfate was slowly added to a final concentration of 0.36g.mL-1. The resulting suspension was stirred on ice for 2 hours before further centrifugation (30 100 g, 20 minutes, 1º C). A small sample of the supernatant was retained for SDS-PAGE, but the remainder was discarded and the pellet was resuspended in a minimal volume (~1 mL) of Buffer C (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The solution was dialysed against two changes of 1 litre of Buffer C.
The dialysed solution was diluted to ~2 mg.mL-1 this was loaded onto a Pharmacia DEAE FastFlow (1mL or 5mL) pre-packed column equilibrated in Buffer C. The column was washed with at least three column volumes of Buffer C and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl over 10-100mL in Buffer C. The fractions containing Tus were combined and were loaded directly onto a SO3 monolithic column (BIA separations) or a 5mL Phosphocellulose column equilibrated in Buffer C. The column was washed with 5 column volumes of Buffer C and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer C as before.
The resultant protein was exchanged into storage buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 20% w/v glycerol, 1 mM DTT)by dialysis or using an Amicon 15mL spin concentrator. Following exchange the protein was concentrated to ~20μM using a spin concentrator. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquotted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.