CPC C12P 21/005 (2013.01) [C07K 16/00 (2013.01); C12N 9/1081 (2013.01); C12P 19/44 (2013.01); C12Y 204/99001 (2013.01); C12Y 301/03 (2013.01); C07K 2317/41 (2013.01)] | 7 Claims |
1. A method of producing in vitro a sialylated target molecule comprising:
(a) providing an aqueous composition comprising
(i) a soluble human β3-galatoside-α-2,6-sialyltransferase I comprising an amino acid motif from position 90 to position 108 in SEQ ID NO:1;
(ii) cytidine-5′-monophospho-N-acetylneuraminic acid;
(iii) a glycosylated target molecule selected from the group consisting of a glycoprotein and a glycolipid, the target molecule comprising one or more antenna(e), at least one antenna having as a 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;
(iv) an aqueous solution permitting glycosyltransferase enzymatic activity comprising a buffer salt;
wherein the aqueous composition further comprises an enzyme selected from the group consisting of an alkaline phosphatase, an acid phosphatase and a 5′ nucleotidase that hydrolyzes the phosphoester bone in 5′-cytidine-monophosphate under conditions permitting glycosyltransferase enzymatic activity;
(b) forming one or more terminal antennal N-acetylneuraminyl-α2,6-β-D-galactosyl-1,4-N-acetyl-β-D-glucosamine residue(s) by incubating the aqueous composition of step (a), thereby reacting cytidine-5′-monophospho-N-acetylneuraminic acid as co-substrate, thereby forming 5′-cytidine-monophosphate; and
(c) hydrolyzing the phosphoester bond of the 5′-cytidine-monophosphate formed in step (b), thereby reducing 5′-cytidine-monophosphate-mediated inhibition, thereby maintaining the activity of the soluble human β-galactoside-α-2,6-sialyltransferase I;
thereby producing in vitro the sialylated target molecule.
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