ABSTRACT
Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.
Subject(s)
Chymotrypsin/chemistry , Glycoconjugates/chemical synthesis , N-Acetyllactosamine Synthase/chemistry , Oligosaccharides/chemical synthesis , Recombinant Proteins/chemistry , Chymotrypsin/metabolism , Glycosylation , N-Acetyllactosamine Synthase/metabolism , Oligosaccharides/chemistry , Polymers , Recombinant Proteins/metabolism , Sepharose/chemistry , Sialyltransferases/chemistry , Sialyltransferases/metabolism , Solubility , Uridine Diphosphate Galactose/metabolism , Water , beta-D-Galactoside alpha 2-6-SialyltransferaseABSTRACT
An active and soluble human beta1,4-galactosyltransferase (beta-GT) was produced in Escherichia coli using a maltose-binding protein fusion system. The purified recombinant beta-GT has a K(m) value of 0.035 mM for UDP-galactose and a V(max) of 643 x 10(3) nmol/mg/h. The enzyme catalyzes the transfer of galactose from UDP-galactose to N-linked oligosaccharides. The properties of the purified enzyme were identical to those of bovine milk beta-GT.
