LARGE-SCALE EXPRESSION OF RECOMBINANT SIALYTTRANSFERASES AND COMPARISON OF THEIR KINETIC-PROPERTIES WITH NATIVE ENZYMES

Values of K-m were determined for three purified sialyltransferases an d the corresponding recombinant enzymes. The enzymes were Gal beta 1-4 ,GlcNAc alpha 2-6sialyltransferase and Gal beta 1-3(4)GlcNAc alpha 2-3 sialyltransferase from rat liver; these enzymes are responsible for th e attachment of sialic acid to N-linked oligosaccharide chains; and th e Gal beta 1-3GalNAc alpha 2-3sialyltransferase from porcine submaxill ary gland that is responsible for the attachment of sialic acid to O-l inked glycoproteins and glycolipids. A procedure for the large scale e xpression of active sialyltransferases from recombinant baculovirus-in fected insect cells is described. For the liver enzymes values of K-m were determined using rat and human asialo(alpha 1) acid glycoprotein and N-acetyllactosamine as variable substrates; lacto-N-tetraose was a lso used with the Gal beta 1-3(4)GlcNAc alpha 2-3sialyltransferase. An tifreeze glycoprotein was used as the macromolecular acceptor for the porcine enzyme. Values for K-m were also determined using CMP-NeuAc as the variable substrate.