HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY OF PASTEURELLA-HAEMOLYTICA LEUKOTOXIN USING ANION-EXCHANGE PERFUSION COLUMNS

An exotoxin, called leukotoxin (LKT), from Pasteurella haemolytica, wh ich had previously proved difficult to purify, was purified by high-pe rformance liquid chromatography using rigid highly hydrophilic micropa rticulate anion-exchange columns. These anion-exchange stationary phas es were employed to overcome difficulties of the relatively hydrophobi c 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 LK T on a much larger scale. The purification of the LKT on the large por e strong anion-exchange perfusion column was best achieved when three consecutive linear gradients at increasing NaCl concentration in 20 mM Tris buffer, pH 8.0, containing 6.0 M urea and 0.25% Tween 20 were us ed. Under these conditions, a better separation was obtained for the t etrameric and aggregate peaks of LKT from the early eluting contaminan t peaks. This separation scheme allowed good recovery of activity and purification of the LKT to near homogeneity. (C) 1998 Elsevier Science B.V. All rights reserved.