ISISBio:Protocols/Purification/Tus

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Cell Lysis

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). 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 freezing at -80 ºC and then thawing under cold running water. 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 0.078 g.mL-1 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 add a further 0.29 g.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 (~1-5 mL) of Buffer A (50 mM imidazole-HCl, pH 6.8, 1 mM EDTA, 1 mM DTT). The suspension was then transferred to 10-12 kDa cut off dialysis tubing and dialysed against two changes of Buffer A (at least 1 litre each for at least one hour).

Purification

The dialysed solution was diluted in Buffer A to an OD280 of ~2 and loaded onto a Pharmacia DEAE FastFlow (5mL) pre-packed column equilibrated in Buffer A. The column was washed with at least three column volumes of Buffer A and the Tus protein was eluted in a gradient of 0.00 to 1.50 M NaCl in 10 column volumes in Buffer A ####TUS-LPETGG-His6 MAY NOT ELUTE WHERE WE EXPECT - MAY BE IN FLOW THROUGH####. The fractions containing Tus were combined and loaded directly onto a 5-15 mL Whatmann P11 phosphocellulose column equilibrated in Buffer A. The column was washed with 5 column volumes of Buffer A and the bound protein was eluted by the application of a gradient from 0.00 to 1.50 M NaCl in Buffer A 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 (at least three times >100 volumes) or with a desalting column equilibrated in Storage buffer. Following exchange the protein was concentrated to ~10-100 μM using a spin concentrator or Amicon. The protein concentration was estimated from the UV Absorbance spectrum of the protein solution, and the samples were aliquoted and snap-frozen in liquid nitrogen before moving to store at –80 ºC.

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