Expression and characterization of recombinant alpha-galactosidase in baculovirus-infected insect cells.
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.
Zhu
A
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The Lindsley F. Kimball Research Institute of The New York Blood Center, New York, USA.
Wang
Z K
ZK
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D003069:Coffee / Q000201:enzymology / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D005822:Genetic Vectors; D007120:Immunochemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009598:Nitrophenylgalactosides; D018411:Spodoptera; D013379:Substrate Specificity; D000519:alpha-Galactosidase / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D003069:Coffee; D018076:DNA, Complementary; D009598:Nitrophenylgalactosides; C059200:4-nitrophenylgalactoside; D000519:alpha-Galactosidase