Characterization of recombinant alpha-galactosidase for use in seroconversion from blood group B to O of human erythrocytes.
Alpha-Galactosidase (alpha-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 alpha-GAL (r)alpha-GAL) in large quantities in a methylotrophic yeast strain Pichia pastoris and purified the protein to apparent homogeneity by chromatography on a macro prep S50 column. Purified (r)alpha-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 (K(m) =0.363 mM and V(max) = 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 substrate p-nitrophenol-alpha-D-galactopyranoside. Furthermore, as with its native counterpart, (r)alpha-GAL specifically cleaves alpha-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 for in vivo studies. Thus, with a simple procedure for over-expression and purification of (r)alpha-GAL from P. pastoris culture, one can readily obtain the enzyme needed for large-scale sero-conversion of red cells.
Zhu
A
A
Lindsley F. Kimball Research Institute, The New York Blood Center, 10021, USA.
Leng
L
L
Monahan
C
C
Zhang
Z
Z
Hurst
R
R
Lenny
L
L
Goldstein
J
J
D000017:ABO Blood-Group System / Q000378:metabolism; D002850:Chromatography, Gel; D003069:Coffee / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004912:Erythrocytes / Q000276:immunology / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D000519:alpha-Galactosidase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*
D000017:ABO Blood-Group System; D003069:Coffee; D011994:Recombinant Proteins; D000519:alpha-Galactosidase