Editing Purification of Tth-Muts (section)

== Purification of T. thermophilus MutS protein ==

Frozen cells (100 g) were thawed, suspended in buffer I [50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 5 mM β-mercaptoethanol and 25% (w/v) sucrose] and disrupted by sonication on ice. Brij-58 was added to a final concentration of 0.5% (w/v) and then the cell extract was stirred for 30 min at 4°C. Following this, the cell lysate was incubated at 70° C for 10 min and centrifuged (15 000 g) for 40 min at 4°C. A DEAE-cellulose DE52 column (bed volume 500 ml) was equilibrated with buffer II [50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 5 mM β-mercaptoethanol and 10% (v/v) glycerol] before the supernatant was loaded. The column was then washed once with 150 ml of buffer II and eluted with a 3000 ml gradient of 0–500 mM NaCl in buffer II. Ammonium sulfate was added to the MutS fraction to give a final concentration of 15% (w/v) and then it was loaded onto a Phenyl-Toyopearl 650M column (bed volume 60 ml) previously equilibrated with 15% (w/v) ammonium sulfate in buffer II. The column was washed with 50 ml of the same buffer and then eluted with a 600 ml gradient of 15–0% (w/v) ammonium sulfate in buffer II. Fractions containing the protein were loaded onto a Sephacryl S-300 HR column (bed volume 400 ml) previously equilibrated with buffer III [50 mM Tris-HCl (pH 7.5), 100 mM KCl, 0.1 mM EDTA, 5 mM β-mercaptoethanol and 10% (v/v) glycerol]. The column was eluted with buffer III, the fractions containing MutS protein were concentrated and then stored at 4°C. At each step, the column fractionation pattern was analyzed by SDS-PAGE. The final amount of purified MutS protein was 40 mg and the yield was 0.4 mg protein/g cells.

The N-terminal amino acid sequence of the purified protein was analyzed using an automated protein sequencer (ABI, model 473A). The molar extinction coefficient of T.thermophilus MutS protein was calculated to be 51 000 M−1cm−1 at an absorption maximum of ∼278 nm