Characterization of a β-galactosidase fusion protein containing the starch-binding domain of Aspergillus glucoamylase

Abstract

Granular starch was used as a biospecific adsorbent to investigate the possible application of the starch-binding domain (SBD) as an affinity tail for a one-step purification of target proteins from crude cell extracts. A β-galactosidase (β-gal) fusion protein containing the C-terminal 119 amino-acids from GA-I (BSB119) was used as a model system to study the starch binding and elution. Because of proteolysis, approximately 40% of initial β-gal activity lacked the SBD, and the remaining fusion protein contained from to one to four SBDs per molecule of β-gal tetramer. The fusion protein forms containing at least one intact SBD adsorbed to starch. The bound fusion protein was eluted by using 10 m m solutions of various maltooligosaccharides and cyclodextrins. The best eluants were 10 m m maltodextrin with an average degree of polymerization ( DP ) of 10 and 10 m m β-cyclodextrin. 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 that the purity of the fusion protein eluted from starch was as good as or better than that obtained by conventional affinity chromatography.