US 11,788,108 B2
CMP-dependent sialidase activity
Harald Sobek, Biberach (DE); Michael Greif, Penzberg (DE); Marco Thomann, Penzberg (DE); and Sebastian Malik, Antdorf (DE)
Assigned to Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed by Roche Diagnostics Operations, Inc., Indianapolis, IN (US)
Filed on Jun. 23, 2021, as Appl. No. 17/355,962.
Application 17/355,962 is a division of application No. 15/629,040, filed on Jun. 21, 2017, granted, now 11,078,511.
Application 15/629,040 is a continuation of application No. PCT/EP2015/080741, filed on Dec. 21, 2015.
Claims priority of application No. 14199648 (EP), filed on Dec. 22, 2014.
Prior Publication US 2021/0340585 A1, Nov. 4, 2021
Int. Cl. C12P 21/00 (2006.01); C12N 9/10 (2006.01); C12P 19/44 (2006.01); C07K 16/00 (2006.01)
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.