High-performance liquid chromatography of Pasteurella haemolytica leukotoxin using anion-exchange perfusion columns

Abstract

An exotoxin, called leukotoxin (LKT), from Pasteurella haemolytica, which had previously proved difficult to purify, was purified by high-performance liquid chromatography using rigid highly hydrophilic microparticulate anion-exchange columns. These anion-exchange stationary phases were employed to overcome difficulties of the relatively hydrophobic LKT interacting with dextran or styrene-based resins. While a short non-porous DEAE column allowed the partial microscale purification of the leukotoxin at pH 7.0, a high capacity strong anion-exchange column of the perfusion chromatography type permitted the purification of LKT on a much larger scale. The purification of the LKT on the large pore strong anion-exchange perfusion column was best achieved when three consecutive linear gradients at increasing NaCl concentration in 20 m M Tris buffer, pH 8.0, containing 6.0 M urea and 0.25% Tween 20 were used. Under these conditions, a better separation was obtained for the tetrameric and aggregate peaks of LKT from the early eluting contaminant peaks. This separation scheme allowed good recovery of activity and purification of the LKT to near homogeneity.