title: Molecular cloning and high-level expression in Escherichia coli of fungal alpha-galactosidase from Absidia corymbifera IFO 8084.
abstract: A cDNA encoding the alpha-galactosidase of Absidia corymbifera IFO 8084 was cloned and sequenced. The cloned DNA has a single open-reading frame consisting of 2190 base pairs, and the deduced amino acid sequence revealed that the mature enzyme consisted of 730 amino acid residues with a molecular mass of 82,712 Da. The native structure of the alpha-galactosidase of A. corymbifera IFO 8084 was determined to be a tetramer. Comparison with amino acid sequences of other alpha-galactosidase showed high homology with sequences of members of family 36. An expression vector, pET32Trx/galalpha, was constructed by introducing the cDNA coding region into a thioredoxin fusion system, pET32-Ek/LIC. The resulting transformant, pET32Trx/galalpha, overproduced the active enzyme as a thioredoxin fused form in the host Escherichia coli. By using His-binding metal affinity chromatography, recombinant alpha-galactosidase was purified to homogeneity in a single step. The purified recombinant fusion alpha-galactosidase showed properties very similar to the native alpha-galactosidase from A. corymbifera IFO 8084.
authors.affiliation: Laboratory of Applied Microbiology, Graduate School of Agriculture, Hokkaido University, North 9 West 9, Kita-ku, Sapporo 060-8589, Japan.
authors.forename: S H
authors.initials: SH
authors.lastname: Baik
authors.forename: K
authors.initials: K
authors.lastname: Saito
authors.forename: A
authors.initials: A
authors.lastname: Yokota
authors.forename: K
authors.initials: K
authors.lastname: Asano
authors.forename: F
authors.initials: F
authors.lastname: Tomita