Back to results
US 11,136,558 B2Granted
Compositions and methods for reducing fucosylation of glycoproteins in insect cells and methods of use thereof for production of recombinant glycoproteins
AssigneeUniversity of Wyoming
InventorsHideaki Mabashi-Asazuma, Donald L. Jarvis
Life of this patent
Estimating the term…
Abstract
Compositions for reducing fucosylation of glycoproteins in insect cells are provided. Also disclosed are methods of use of such compositions for the production of recombinant humanized proteins.
Claims21
A method for production of a non-fucosylated protein in insect larvae, comprising
- a) providing insect larvae;
- b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins, wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector;
- c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose;
- and
- d) isolating said protein of interest,
- wherein said RMD encoding nucleic acid is the reverse complement of nucleotides 10034-10948 of SEQ ID NO:
- 1.
4 more elements
A method for producing at least one protein of interest lacking fucose, comprising
- a) providing insect cells;
- b) introducing a baculovirus comprising at least one nucleic acid molecule encoding the enzyme GDP-4-dehydro-6-deoxy-D-mannose reductase (RMD) operably driven by an immediate early expression control sequence for expression immediately after infection or an inducible promoter, thereby stabilizing inhibition of fucosylation, and at least one additional nucleic acid molecule encoding at least one protein of interest driven by an expression control sequence active later in infection, thereby producing non-fucosylated proteins, wherein said additional nucleic acid is present on the same baculovirus encoding RMD or is present on a second baculovirus vector;
- c) incubating under conditions wherein GDP-4-dehydro-6-deoxy-D-mannose reductase blocks the production of GDP-L-fucose, and said at least one protein of interest is produced lacking fucose;
- and
- d) isolating said at least one protein of interest,
- wherein said RMD encoding nucleic acid is the reverse complement of nucleotides 10034-10948 of SEQ ID NO:
- 1.
4 more elements
Dependencies are inferred from the claim text.