US 7,863,020 B2Granted

Production of sialylated N-glycans in lower eukaryotes

AssigneeGlycoFi, Inc.

InventorStephen R. Hamilton

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Abstract

The present invention relates to eukaryotic host cells which have been modified to produce sialylated glycoproteins by the heterologous expression of a set of glycosyltransferases, including sialyltransferase and/or trans-sialidase, to become host-strains for the production of mammalian, e.g., human therapeutic glycoproteins. Novel eukaryotic host cells expressing a CMP-sialic acid biosynthetic pathway for the production of sialylated glycoproteins are also provided. The invention provides nucleic acid molecules and combinatorial libraries which can be used to successfully target and express mammalian enzymatic activities (such as those involved in sialylation) to intracellular compartments in a eukaryotic host cell. The process provides an engineered host cell which can be used to express and target any desirable gene(s) involved in glycosylation.

Claims6

Dependent claims

A method for producing a recombinant sialylated glycoprotein in a Pichia pastoris host cell, the host cell selected or engineered to produce recombinant glycoproteins comprising a glycoform selected from the group consisting of Gal(1-4) GlcNAc(1-4) Man3GlcNAc2, the method comprising:

(a) transforming into the host cell one or more nucleic acid molecules encoding a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase, an N-acetylneuraminate-9-phosphate synthase, and a CMP-Sialic acid synthase;
(b) transforming into the host cell a nucleic acid molecule encoding a CMP-sialic acid transporter;
and
(c) transforming into the host cell a nucleic acid molecule encoding a 2,6-sialyltransferase catalytic domain fused to a cellular targeting signal peptide encoded by nucleotides 1-108 of the S. cerevisiae Mnn2 wherein nucleotides 1-108 encoding the S. cerevisiae Mnn2 have the nucleotide sequence shown by nucleotides 13 to 120 of SEQ ID NO:103;
wherein, upon passage of the recombinant glycoprotein through the secretory pathway of the host cell, a recombinant sialylated glycoprotein comprising a glycoform selected from the group consisting of NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2 glycoform is produced.

2 more elements

Dependencies are inferred from the claim text.

Facts

Application: 11/429,672Filed 5 May 2006 · continuation in part
Priority: 20 Feb 2003
Published: US 2006/0040353 A121 Dec 2006
Granted: 4 Jan 2011
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

C12P 21/005
Preparation of peptides or proteins: glycopeptides, glycoproteins
C12N 9/1081
Glycosyltransferases: transferring other glycosyl groups
C12N 9/2402
Acting on glycosyl compounds: hydrolysing O- and S- glycosyl compounds
C12Y 302/01018
Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds: exo-alpha-sialidase , i.e. trans-sialidase

Family

Application10/371,877Parent of this continuation-in-part · filed 20 Feb 2003
Application11/108,088Parent of this continuation-in-part · filed 15 Apr 2005
US 2006/0286637 A1Pre-grant publication · published 21 Dec 2006
US 7,795,002 B2Parent of this continuation-in-part
US 7,449,308 B2Parent of this continuation-in-part
US 2004/0018590 A1Published application
US 2006/0040353 A1Published application
US 7,863,020 B2This patent

From the US filing history. Foreign family members aren’t listed.