CHARACTERIZATION OF A BETA-GALACTOSIDASE FUSION PROTEIN CONTAINING THE STARCH-BINDING DOMAIN OF ASPERGILLUS GLUCOAMYLASE

Granular starch,vas used as a biospecific adsorbent to investigate the possible application of the starch-binding domain (SBD) as an affinit y tail for a one-step purification of target proteins from crude cell extracts. A beta-galactosidase (beta-gal) fusion protein containing th e C-terminal 119 amino-acids from GA-I (BSB119) was used as a model sy stem to study the starch binding and elution. Because of proteolysis, approximately 40% of initial beta-gal activity lacked the SBD, and the remaining fusion protein contained from to one to four SBDs per molec ule of beta-gal tetramer. The fusion protein forms containing at least one intact SBD adsorbed to starch. The bound fusion protein was elute d by using 10 mM solutions of various maltooligosaccharides and cyclod extrins. The best elutants were 10 mM maltodextrin with an average deg ree of polymerization (<(DP)over bar>) of 10 and 10 mM beta-cyclodextr in. The elution of BSB119 with maltooligosaccharides of increasing DP suggested that the starch-binding site of the SBD consists of at least five glucosyl binding sites. SDS-PAGE gels and Western blots showed t hat the purity of the fusion protein eluted from starch,vas as good as or better than that obtained by conventional affinity chromatography.