Purification of r_EC by anion exchange chromatography. Crude fermentor supernatant was dialyzed and injected (10 mL) onto a 1-mL Mono Q HR 5/5 anion exchange column pre-equilibrated with the same buffer, as described in Materials and Methods. Bound protein was eluted with a 20-mL linear salt gradient (20 mmol/L Tris, pH 7.5, to 1 mol/L NaCl, 20 mmol/L Tris, pH 7.5) and fractions (1 mL) were collected. (A) Elution profile with absorbance plotted as a solid line (scale on the left) and the salt gradient (M NaCl) as the broken line (scale on the right). (B) Evaluation of fractions by SDS-PAGE. Fraction samples were run on 4% to 20% gradient SDS-PAGE under reducing conditions. S denotes crude fermentor supernatant. Lanes corresponding to sequentially collected fractions under the following peaks are indicated with lowercase letters: peak a, pooled flow-through; peak b, two fractions; peak c, four fractions; peak d, one fraction. Eluted material was deliberatedly overloaded on the gel to enhance detection of minor contaminants.