Quantitative control of sialylation

A method of producing in vitro a sialylated target molecule with a controlled quantity of sialyl residues, the method comprising the steps of

• (a) providing a glycosylated target molecule in an aqueous solution and under conditions permitting glycosyltransferase enzymatic activity, the target molecule being selected from a glycoprotein and a glycolipid, the target molecule comprising a plurality of antennae, at least two of the antennae each having as terminal structure a β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine moiety with a hydroxyl group at the C6 position in the galactosyl residue;
• (b) forming one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s) [=α2,6 sialylated terminal antennal residue(s)] by incubating the target molecule of step (a) for a first pre-determined time with N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I having the amino acid sequence of SEQ ID NO:2 and in the presence of cytidine-5′-monophospho-N-acetylneuraminic acid, or a functional equivalent thereof, as donor compound thereby providing a sialylated target molecule;
• (c) hydrolyzing the α2,6 glycosidic bond in one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl- 1,4-N-acetyl-β-D-glucosamine residues by incubating the sialylated target molecule of step (b) for a second pre-determined time with the N-terminally truncated human β-galactoside-α-2,6-sialyltransferase I having the amino acid sequence of SEQ ID NO:2;
• thereby producing in vitro the sialylated target molecule with a controlled quantity of sialyl residues.