Fucosylation-deficient cells
An isolated nucleic acid encoding an FX protein having a serine at position 79, a lysine at position 90, a leucine at position 136, an arginine at position 211, a serine at position 289, and a combination thereof is provided. Cells having a gene encoding a modified FX protein are provided, wherein the cells exhibit a reduced ability to fucosylate a glycoprotein at a first temperature, but exhibit the ability to fucosylate the glycoprotein at a second temperature. Methods and compositions for making glycoproteins with reduced fucosylation are provided.
1. A cell capable of fucosylating a glycoprotein, wherein the cell comprises a modified GDP-4-keto-6-deoxy-mannose-3,5-epimerase-4-reductase (FX) gene that codes for an FX protein comprising the amino acid sequence SEQ ID NO:2 and wherein no more than about 10% of the glycoprotein is fucosylated when the cell is grown at a temperature of about 37° C. in the absence of an external fucose source.
2. The cell of claim 1 , wherein the cell is a Chinese hamster ovary (CHO) cell.
3. The cell of claim 1 , wherein the cell expresses a glycoprotein that comprises an immunoglobulin CH2 region and an immunoglobulin CH3 region.
4. The cell of claim 3 , wherein the glycoprotein is an antibody.
5. The cell of claim 3 , wherein no more than about 6% of the glycoprotein is fucosylated when the cell is grown at a temperature of about 37° C. in the absence of an external fucose source.
6. The cell of claim 5 , wherein no more than about 2% of the glycoprotein is fucosylated when the cell is grown at a temperature of about 37° C. in the absence of an external fucose source.
7. The cell of claim 1 , wherein the cell fucosylates at least 70% of the glycoprotein when the cell is grown at a temperature of about 34° C. in the absence of an external fucose source.
8. The cell of claim 1 , wherein the cell fucosylates at least 90% of the glycoprotein when the cell is grown at a temperature of about 34° C. in the absence of an external fucose source.
9. The cell of claim 1 , wherein the cell fucosylates at least 70% of the glycoprotein when the cell is grown at a temperature of about 37° C. in the presence of an external fucose source.
10. The cell of claim 1 , wherein the cell fucosylates at least 90% of the glycoprotein when the cell is grown at a temperature of about 37° C. in the presence of an external fucose source.
11. An isolated nucleotide sequence encoding an FX protein, wherein the FX protein comprises the amino acid sequence SEQ ID NO:2.
12. The cell of claim 1 , further comprising a nucleotide sequence encoding an immunoglobulin gene.