Lysis and purification of the E. coli replication termination protein, Tus
List reagents, supplies and equipment necessary to perform the protocol here. For those materials which have their own OWW pages, link to that page. Alternatively, links to the suppliers' page on that material are also appropriate.
- supply 1 (i.e. tubes of a certain size? spreaders?)
- reagent 1
- X μL reagent 2
- component A (reagent 2 is made up of multiple components)
- component B
- equipment 1
- equipment 2
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).
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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Relevant papers and books
- Cameron Neylon 15:14, 1 August 2008 (UTC):