EXPRESSION AND CHARACTERIZATION OF RECOMBINANT ALPHA-GALACTOSIDASE INBACULOVIRUS-INFECTED INSECT CELLS

A cDNA encoding coffee bean alpha-galactosidase was subcloned into bac ulovirus expression vectors, pVL-1393 and pAc-GP67B, for intracellular and extracellular expression in Spodoptera frugiperda (Sf9) insect ce lls, respectively. The expressed protein (recombinant alpha-galactosid ase) was immunologically reactive with antisera raised against its nat ive counterpart isolated from coffee beans and was biologically active towards the substrate p-nitrophenyl alpha-galactopyranoside. The subc ellular distribution of recombinant alpha-galactosidase expressed from different vectors was analyzed by Western blotting, immunofluorescent labeling, and electron microscopy. In addition, recombinant alpha-gal actosidase was compared to the native enzyme with respect to glycosyla tion, thermostability, and pH profile. Furthermore, a recombinant alph a-galactosidase molecule with a His(6) tag at its C-terminus was const ructed by an overlap PCR method so that the enzyme expressed in Sf9 ce lls can be purified by a simple affinity chromatography procedure.