This disclosure relates to the technical field of synthetic biology and metabolic engineering. More particularly, this disclosure relates to the technical field of fermentation of metabolically engineered microorganisms. This disclosure describes engineered micro-organisms that produce oligosaccharides that are free of KDO-lactose impurities and/or KDO-oligosaccharide impurities.

The purpose of the present invention is to provide a novel monoclonal antibody having high affinity and that strictly recognizes, as a sugar chain epitope, only a “Siaα2,6Galβ1,4GlcNAc (6′-Sialyl-LacNAc): CDw75” sugar chain structure, being a molecular target for diagnosis of the malignancy of tumors. An anti-CDw75 monoclonal antibody is provided that recognizes “CDw75” sugar chain structures but does not recognize similar sugar chain structures indicated by “Galβ1,4GlcNAc”, “Siaα2,3Galβ1,4GlcNAc”, or “Siaα2,6Galβ1,4Glc”, by using a glycolipid antigen bonding a carrier lipid compound “HOCH.sub.2CH(NH—CO—(CH.sub.2).sub.22—CH.sub.3)—(CH.sub.2).sub.9—CH.sub.3 (C12L)” developed by the inventors to a “CDw75” sugar chain. The obtained anti-CDw75 monoclonal antibody is an excellent detection drug for B-cell lymphoma, gastric cancer, or colorectal cancer, an excellent diagnostic agent for tumor malignancy, etc., an excellent treatment agent for B-cell lymphoma, gastric cancer, or colorectal cancer, and an excellent prevention/treatment drug for influenza.

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that suchs enzyme are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

[Problem to be Solved]

The importance of sugar chains having α2,3- or α2,6-linked sialic acid at their non-reducing ends is known. Industrial production has been demanded for these sugar chain compounds. Particularly, the production of glycoprotein drugs or the like inevitably requires producing in quantity sugar chains having homogeneous structures by controlling the linking pattern (α2,6-linkage or α2,3-linkage) of sialic acid. Particularly, a triantennary or tetraantennary N-type complex sugar chain having sialic acid at each of all non-reducing ends is generally considered difficult to chemically synthesize. There has been no report disclosing that such a sugar chain was chemically synthesized. Furthermore, these sugar chains are also difficult to efficiently prepare enzymatically.

[Solution]

The present inventors have newly found the activity of sialyltransferase of degrading sialic acid on a reaction product in the presence of CMP and also found that formed CMP can be degraded enzymatically to thereby efficiently produce a sialic acid-containing sugar chain. The present inventors have further found that even a tetraantennary N-type sugar chain having four α2,6-linked sialic acid molecules, which has previously been difficult to synthesize, can be prepared at high yields by one-pot synthesis comprising the elongation reaction of a biantennary sugar chain used as a starting material without performing purification after each enzymatic reaction.

The present invention relates to methods for reducing antennary fucosylation of complex N-glycans in recombinantly expressed glycoproteins, cell lines that can be used in said methods, respective recombinant glycoproteins, and methods for expressing the same in said cell lines.

The present invention relates to the finding of methods to shift the glycosylation profile of recombinant produced serum glycoproteins to the predominant bi-antennary form found in human plasma. This is accomplished by providing a mammalian cell line according to the invention with a series of gene disruptions and/or gene insertions that facilitate this shift.

α2-6-Sialyltransferase (2,6ST) variants having improved α2-6-specific sialidase activity as compared to the native 2,6ST enzymes are described. The variants include GT80 sialyltransferases such as P. damselae Pd2,6ST. Methods for making de-sialylated products and screening sialidase activity are also described.

Described herein are fusion proteins, e.g., fusion proteins comprising enzymatically active portion(s) of ST6Gall or B4GalT1 as well as methods for producing them, nucleic acid molecule(s) encoding the fusion protein(s), vectors comprising the nucleic acid molecule(s), and host cell(s) comprising the vector(s). Also described herein are methods of sialyating immunoglobulin G (IgG) antibodies.

The present disclosure is directed to the properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions. Any of the mutants disclosed in here exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site. A fundamental finding documented in the present disclosure is that such enzyme are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.

The properties of certain glycosyltransferase variants having N-terminal truncation deletions or internal deletions are disclosed. Particularly, mutants that exhibit α-2,6-sialyltransferase enzymatic activity in the presence of CMP-activated sialic acid as co-substrate, and in the presence of a suitable acceptor site, are disclosed. A fundamental finding documented in the present disclosure is that enzymes are not only capable of catalyzing transfer of a sialidyl moiety but they are also capable of catalyzing hydrolytic cleavage of terminally bound sialic acid from a glycan.