Method for mass production of factor VII/VIIA
A method for the mass production of human coagulation Factor VII. The method includes a) providing an expression vector carrying i) a dihydrofolate reductase promoter devoid of one or more CCGCC repeat sequences from the GC-rich region thereof and a dihydrofolate reductase (DHFR) gene operably linked thereto and ii) a cytomegalovirus (CMV) promoter and a human coagulation Factor VII gene operably linked thereto; b) obtaining a transformed host cell line containing the expression vector; and c) culturing the transfected host cell in the presence of a dihydrofolate reductase inhibitor to select cells which express human coagulation Factor VII with high efficiency; and d) adding sodium butyrate to the selected host cells.
1. A method for mass production of human coagulation
Factor VII, comprising:
a) providing a Chinese hamster ovarian carcinoma cell line transfected with an expression vector, said expression vector carrying i) a dihydrofolate reductase promoter devoid of one or more CCGCCC repeat sequences from the GC-rich region and a dihydrofolate reductase gene operably linked thereto and ii) a cytomegalovirus promoter and a human coagulation Factor VII gene operably linked thereto;
b) culturing the transfected cell line of step a) in the presence of a dihydrofolate reductase inhibitor to select cells which express human coagulation Factor VII;
c) culturing the selected cells in a medium containing sodium butyrate in concentration from 1 mM to 1.5 mM to produce human coagulation Factor VII; and
d) harvesting the produced human coagulation Factor VII, wherein the human coagulation Factor VII gene has the nucleotide sequence of SEQ ID NO: 3.
2. The method of claim 1 , wherein the GC-rich region contains as many as or less than one CCGCCC repeating sequence.
3. The method of claim 1 , wherein the expression vector is pXOGC-FVII.
4. The method of claim 1 , wherein the Chinese hamster ovarian carcinoma cell line before transfection in step a), is devoid of dihydrofolate reductase gene.
5. The method of claim 1 , wherein the selected cells of step c) is HMF708, which has Accession Number KCTC 11779BP.
6. The method of claim 1 , further comprising purifying the human coagulation Factor VII.
7. The method of claim 1 , further comprising activating the human coagulation Factor VII.
8. A cell line for producing human coagulation Factor VII, wherein said cell line is HMF708 having Accession Number KCTC 11779BP.