Characterization of Recombinant α-Galactosidase for Use in Seroconversion from Blood Group B to O of Human Erythrocytes

Abstract

α-Galactosidase (α-GAL) purified from green coffee bean cleaves the terminal galactose residues from the surface of group B erythrocytes, thereby converting these cells serologically to group O cells. Such enzymatically converted red cells have been transfused into group A and O recipients as part of the first phase of FDA-approved clinical trials. Recently we expressed the recombinant α-GAL (rα-GAL) in large quantities in a methylotrophic yeast strainPichia pastorisand purified the protein to apparent homogeneity by chromatography on a macro prep S50 column. Purified rα-GAL, migrating as a single band of 41 kDa on a SDS–PAGE, appears to be identical to its native counterpart in specific activity (32 U/mg) and kinetic parameters (Km= 0.363 mMandVmax= 46.9 U/mg). Both enzymes demonstrate the same pH profile in the pH range from 2 to 9, with an optimal pH at 6.4 when tested with the substratep-nitrophenol-α-D-galactopyranoside. Furthermore, as with its native counterpart, rα-GAL specifically cleaves α-linked terminal galactose residues from group B red cells without affecting other major antigens on the red cell surface. In addition, we developed a method for using RT-PCR to detect possible DNA contamination in the purified protein preparation, which is one of the concerns forin vivostudies. Thus, with a simple procedure for over-expression and purification of rα-GAL fromP. pastoriscul- ture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells.