Production of sialylated <i>N</i>-glycans in lower eukaryotes

摘要

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.

主权项

1. A method for producing a recombinant sialylated glycoprotein in a Pichia pastoris host cell, the host cell selected or engineered to produce glycoproteins comprising a GalGlcNAcMan5GlcNAc2 glycoform, the method comprising the step of transforming into the host cell:
a. a nucleic acid encoding an enzyme having sialyltransferase activity, wherein the nucleic acid encodes a sialyltransferase enzyme comprising the amino acid sequence of SEQ ID NO:104 or a catalytic domain thereof; and b. a nucleic acid encoding a CMP-sialic acid transporter; and c. one or more nucleic acids encoding a CMP-sialic acid pathway consisting of: a bifunctional UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine Kinase, an N-acetylneuraminate-9-phosphate synthase, and a CMP-Sialic acid synthase; wherein upon passage of the recombinant glycoprotein through the Golgi apparatus of the host cell, a recombinant glycoprotein comprising a NANAGalGlcNAcMan5GlcNAc2 glycoform is produced.