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 method for making a glycoprotein with reduced fucosylation, comprising:
culturing a mammalian cell that expresses a GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase (FX) protein comprising the amino acid sequence of SEQ ID NO: 1, except for an amino acid substitution of L289S, at a temperature of about 37° C. and in the absence of an external fucose source, wherein the cell expresses a glycoprotein; and, isolating the glycoprotein from the culture.
2. A method for making a glycoprotein with reduced fucosylation, comprising:
culturing a mammalian cell that expresses a FX protein that comprises the amino acid sequence of SEQ ID NO: 1, except for a first amino acid substitution of L289S and a second amino acid substitution selected from the group consisting of N79S, N90K, P136L, and G211R, at a temperature of about 37° C. and in the absence of an external fucose source, wherein the cell expresses a glycoprotein; and, isolating the glycoprotein from the culture.
3. The method of claim 1 , wherein said glycoprotein comprises an immunoglobulin CH2 region and an immunoglobulin CH3 region.
4. The method of claim 3 , wherein the glycoprotein is an antibody.
5. The method of claim 1 , wherein said cell is a Chinese hamster ovary (CHO) cell.
6. The method of claim 1 , wherein no more than 6% of said glycoprotein is fucosylated when said cell is cultured at a temperature of about 37° C.
7. The method of claim 1 , wherein no more than 2% of said glycoprotein is fucosylated when said cell is cultured at a temperature of about 37° C.
8. The method of claim 2 , wherein said second amino acid substitution is N90K.
9. The method of claim 8 , wherein said glycoprotein comprises an immunoglobulin CH2 region and an immunoglobulin CH3 region.
10. The method of claim 9 , wherein the glycoprotein is an antibody.
11. The method of claim 8 , wherein said cell is a Chinese hamster ovary (CHO) cell.
12. The method of claim 8 , wherein no more than 6% of said glycoprotein is fucosylated when said cell is cultured at a temperature of about 37° C.
13. The method of claim 8 , wherein no more than 2% of said glycoprotein is fucosylated when said cell is cultured at a temperature of about 37° C.