Expression of a recombinant human glycosyltransferase from a synthetic gene and its utilization for synthesis of the human blood group B trisaccharide.
A 1034-bp synthetic gene encoding the human blood group B glycosyltransferase, which catalyzes the transfer of galactose from UDP-Gal to Fuc alpha(1-2)Gal beta-OR to give the blood group B determinant Gal alpha(1-3)[Fuc alpha(1-2)]Gal beta-OR (where R is a glycoprotein or glycolipid), has been expressed in Escherichia coli by replacing its membrane-anchoring domain with an ompA bacterial secretory signal. The active enzyme was purified from the periplasm using UDP-hexanolamine affinity chromatography and used in the synthesis of preparative amounts of the human blood group B trisaccharide antigen. The substrate specificity and kinetics of the recombinant enzyme were comparable to the enzyme from human sera. Thus we have achieved the construction of a completely synthetic glycosyltransferase gene and its successful expression.
Seto
N O
NO
Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada.
Palcic
M M
MM
Hindsgaul
O
O
Bundle
D R
DR
Narang
S A
SA
D000017:ABO Blood-Group System; D000595:Amino Acid Sequence; D001483:Base Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D005700:Galactosyltransferases / Q000235:genetics*; D005813:Genes, Synthetic; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014312:Trisaccharides / Q000096:biosynthesis*
D000017:ABO Blood-Group System; D004274:DNA, Recombinant; D011994:Recombinant Proteins; D014312:Trisaccharides; D005700:Galactosyltransferases; C023155:blood-group-substance alpha-D-galactosyltransferase