Sialylation of N-linked glycoproteins in the baculovirus expression vector system
AssigneeBoyce Thompson Institute for Plant Research, Inc.
InventorH. Alan Wood
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Abstract
The present disclosure utilizes a novel approach to protein preparation in the baculovirus expression vector system (BEVS). Specifically, the present invention analyzes the effects of supplementing insect cell culture media with dexamethasone or N-acetylmannosarnine on complex glycosylation of proteins prepared via BEVS, including the addition of terminal sialic acid residues to N-linked oligosaccharides.
Claims13
A method of expressing a recombinant nucleic acid in an insect cell line, using a baculovirus expression system, such that said recombinant nucleic acid being expressed in said baculovirus expression system codes for a protein with N-linked glycosylation, comprising the steps of:
- a) culturing cells for use in said baculovirus expression system;
- b) infecting said cells with a recombinant baculovirus that expresses said recombinant nucleic acid encoding a recombinant protein that would normally have N-linked glycosylation if expressed in the source organism of said recombinant nucleic acid;
- and
- c) culturing said infected cells in media supplemented with serum and dexamethasone or N-acetylmannosamine, such that said recombinant protein is expressed.
A method of expressing a recombinant nucleic acid in an insect cell line, using a baculovirus expression system, such that said recombinant nucleic acid being expressed in said baculovirus expression system codes for a protein with N-linked glycosylation, comprising the steps of:
- a) culturing cells for use in said baculovirus expression system;
- b) infecting said cells with a recombinant baculovirus that expresses said recombinant nucleic acid encoding a recombinant protein that would normally have N-linked glycosylation oligosaccharides with terminal sialic acid residues, if expressed in the source organism of said recombinant nucleic acid;
- and
- c) culturing said infected cells in media supplemented with serum and dexamethasone or N-acetylmannosamine, such that said recombinant protein with N-linked glycosylation is expressed and obtains oligosaccharides having terminal sialic acid residues.
A method of culturing insect cells comprising the steps of:
- a) culturing insect cells in a medium that includes serum;
- and
- b) supplementing said medium with dexamethasone or N-acetylmannosamine, such that N-acetylglucosaminyl-transferase or galactosyl-transferase activities are induced.
A method of culturing insect cells comprising the steps of:
- culturing insect cells in a medium that includes serum and dexamethasone or N-acetylmannosamine, such that N-acetylglucosaminyl-transferase, galactosyl-transferase or sialic acid transferase activities are induced.
A cell line designated BTI-Tn-5B1-4h, characterized in that N-acetylglucosaminyl-transferase, galactosyl-transferase or sialic acid transferase activities are induced, when said cell line is cultured in a medium that includes serum and is supplemented with dexamethasone or N-acetylmannosamine.
A cell line having all the distinguishing characteristics of a cell line designated BTI-Tn-5B1-4h.
Dependencies are inferred from the claim text.