Expression and characterization of recombinant alpha-galactosidase in baculovirus-infected insect cells.

ABSTRACT

A cDNA encoding coffee bean alpha-galactosidase was subcloned into baculovirus expression vectors, pVL-1393 and pAc-GP67B, for intracellular and extracellular expression in Spodoptera frugiperda (Sf9) insect cells, respectively. The expressed protein (recombinant alpha-galactosidase) was immunologically reactive with antisera raised against its native counterpart isolated from coffee beans and was biologically active towards the substrate p-nitrophenyl alpha-galactopyranoside. The subcellular distribution of recombinant alpha-galactosidase expressed from different vectors was analyzed by Western blotting, immunofluorescent labeling, and electron microscopy. In addition, recombinant alpha-galactosidase was compared to the native enzyme with respect to glycosylation, thermostability, and pH profile. Furthermore, a recombinant alpha-galactosidase molecule with a His6 tag at its C-terminus was constructed by an overlap PCR method so that the enzyme expressed in Sf9 cells can be purified by a simple affinity chromatography procedure.