Methods of using a bacterial GlcNAc-6-P 2′- epimerase to promote sialylation of glycoconjugates

摘要

The present invention relates to new methods to promote sialylation of glycoconjugates, including recombinant glycoproteins, in glycoconjugate production systems. The invention relates to methods to promote efficient glycoconjugate sialylation in recombinant expression systems, by providing simpler and more economical ways to produce large intracellular pools of sialic acid precursors. The invention is directed to nucleic acids, vectors, and cells harboring vectors comprising nucleic acids encoding enzymes involved in the synthesis of sialic acid precursors, and cells harboring these nucleic acids in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins in bacterial, fungal, plant, and animal cell expression systems. The engineered cells can be used to produce glycosylated proteins in virally-infected, transiently-transformed, or stably-transformed host cells, including lepidopteran insects and cultured cell lines derived from Spodoptera frugiperda, Trichoplusia ni, and Bombyx mori that can be infected by baculovirus expression vectors.

主权项

1. An isolated eukaryotic cell which is modified to comprise at least one nucleic acid encoding a polypeptide, GlcNAc-6-P 2′-epimerase (GNPE), which is capable of converting N-acetyl-D-glucosamine-6-phosphate (GlcNAc-6-P) to N-acetyl-D-mannosamine-6-phosphate (ManNAc-6-P), wherein each nucleic acid encoding said polypeptide is operably-linked to a promoter functional in said cell;
wherein said nucleic acid is obtained from a source belonging to the domain Bacteria; and wherein said polypeptide converts intracellular GlcNAc-6-P to intracellular ManNAc-6-P in an intact cell; and wherein said polypeptide is selected from the group consisting of: (a) a polypeptide comprising SEQ ID NO: 6; (b) a variant of the polypeptide specified in (a) that is at least 90% identical to SEQ ID NO: 6 in which GNPE function is retained; (c) a variant of the polypeptide specified in (b) that contains conservative amino acid substitutions in which GNPE function is retained; (d) a truncated or fusion variant of the polypeptide specified in (a), (b), or (c) comprising one or more insertions or deletions of amino acids in which GNPE function is retained;
wherein each of said one or more insertions or deletions are located between regions that are conserved among(1) the polypeptides specified in (a), (b), or (c); and(2) a polypeptide capable of converting GlcNAc-6-P to ManNAc-6-P which is obtained from a source belonging to the domain Bacteria; (e) a truncated or fusion variant of the polypeptide specified in (a), (b), (c), or (d) comprising one or more insertions or deletions of amino acids in which GNPE function is retained;
wherein each of said one or more insertions or deletions are located atthe amino terminus, the carboxy terminus, or both the amino and carboxy termini ofthe polypeptide specified in (a), (b), (c), or (d); wherein said modified cell also comprises at least one nucleic acid encoding a polypeptide (f) sialic acid-9-phosphate synthase (SAS) [Neu5Ac-9P synthetase, NANS], which is capable of converting ManNAc-6-P to N-acetylneuraminate-9-P (Neu5Ac-9P);
wherein each nucleic acid encoding polypeptide (f) is operably-linked to a promoter functional in said cell.