Simple two-step procedure for purification of cloned small sialidase from unclarified E. coli feedstocks

Abstract

An efficient process for the purification of cloned small sialidase (pCPN-1, small nanH cloned from Clostridium perfringens) from unclarified E. coli feedstocks has been developed. In contrast to conventional purification processes, expanded bed adsorption (EBA) technique allows clarification, product recovery, and concentration to be combined into a single step operation. The cloned NanH was collected from both intra- and extracellular fractions, and purified by expanded bed ion-exchange chromatography followed by affinity chromatography. The EBA technique using STREAMLINE DEAE was shown to be successful in purifying NanH with a purification factor of 12 and yield of 93%. Successive purification of the concentrated NanH purified by an EBA column was achieved by affinity chromatography. The elution of adsorbed NanH was performed with 0.1 m NaHCO 3 buffer pH 9.0 and the pH of the eluted NanH was adjusted to 7.0. An overall yield of 88% was obtained after the affinity chromatographic step. The enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis analysis. The molecular weight was measured as 43 KDa for cloned NanH. The procedure described is suitable for large-scale purification of cloned small NanH from unclarified E. coli feedstocks.