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 that comprises a nucleic acid encoding an FX protein, wherein the FX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and wherein the FX protein comprises a modification relative to SEQ ID NO: 1, the modification comprising a substitution with a serine at position 289.
2. A cell that comprises a nucleic acid encoding an FX protein, wherein the FX protein comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1, and wherein the FX protein comprises a modification relative to SEQ ID NO: 1, the modification comprising a substitution with a Serine at position 289 and a substitution with a lysine at position 90.
3. The cell of claim 1 , wherein the FX protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
4. The cell of claim 2 , wherein the FX protein comprises an amino acid identical to SEQ ID NO: 1 except for having a serine at position 289 and a lysine at position 90.
5. The cell of claim 1 , wherein the cell is a Chinese hamster ovary (CHO) cell.
6. The cell of claim 2 , wherein the cell is a Chinese hamster ovary (CHO) cell.
7. The cell of claim 1 , wherein the cell expresses said FX protein and a glycoprotein.
8. The cell of claim 7 , wherein the glycoprotein is an antibody.
9. The cell of claim 7 , wherein the ability of the cell to fucosylate said glycoprotein at 37° C. is no more than 10% of the ability to fucosylate the glycoprotein exhibited by a cell lacking said modification.
10. The cell of claim 2 , wherein the cell expresses said FX protein and a glycoprotein.
11. The cell of claim 10 , wherein the ability of the cell to fucosylate said glycoprotein at 37° C. is no more than 10% of the ability to fucosylate the glycoprotein exhibited by a cell lacking said modification.
12. The cell of claim 10 , wherein the glycoprotein is an antibody.
13. A method for making a glycoprotein with reduced fucosylation, the method comprising:
(a) culturing a cell of claim 1 , wherein the cell expresses said FX protein and a glycoprotein; and
(b) isolating the glycoprotein from said culture.
14. The method of claim 13 , wherein said culturing further comprises culturing the cell at a temperature of 37° C. in the absence of an external fucose source.
15. The method of claim 13 , wherein the glycoprotein is an antibody.
16. The method of claim 13 , wherein no more than 2% of said glycoprotein is fucosylated.
17. A method for making a glycoprotein with reduced fucosylation, the method comprising:
(a) culturing a cell of claim 2 , wherein the cell expresses said FX protein and a glycoprotein; and
(b) isolating the glycoprotein from said culture.
18. The method of claim 17 , wherein said culturing further comprises culturing the cell at a temperature of 37° C. in the absence of an external fucose source.
19. The method of claim 18 , wherein the glycoprotein is an antibody.
20. The method of claim 19 , wherein no more than 2% of said glycoprotein is fucosylated.