TRANSGENIC INSECT CELLS COMPRISING A BACTERIAL GLCNAC-6-P 2PRIME-EPIMERASE

摘要

The present invention relates to methods of facilitating the expression of recombinant polypeptides from cells, extracellular fluids, extracellular fibers, or any combination thereof, obtained from transgenic insect cells and larvae comprising a bacterial 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). The invention relates to methods to promote efficient glycoconjugate sialylation, by providing simpler ways to produce large intracellular pools of sialic acid precursors. The invention is also directed to nucleic acids, vectors, and cells comprising nucleic acids encoding polypeptides involved in the synthesis of sialic acid precursors, and cells in combination with nucleic acids encoding glycosyltransferases, including sialyltransferases, to facilitate the production of humanized recombinant glycoproteins. The engineered cells can be used to produce glycosylated proteins lepidopteran insects and cultured cell lines derived from Spodoptera frugiperda, Trichoplusia ni, and silkworms, such as Bombyx mori, particularly those that can be infected by baculovirus expression vectors.

主权项

1. A transgenic insect 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, and wherein said nucleic acid is derived from a source belonging to the domain Bacteria 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).