Efficient constitutive production of human IFN-gamma in Chinese hamster ovary cells.
A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-gamma was fused through its 5'-untranslated region to the corresponding region of the simian virus 40 (SV40) T-antigen gene. The SV40 early promoter used contained a modified transcriptional enhancer element with a 93-bp repeat. Supercoiled plasmid DNA was used to transfect Chinese hamster ovary (CHO) cells, the selectable marker being a SV40-dihydrofolate gene construct. Constitutive expression of the IFN-gamma gene in primary transformants was high, especially if a Harvey murine sarcoma virus long terminal repeat (LTR) was present in addition to the SV40 promoter. After gene amplification by methotrexate selection, CHO-gamma cell lines were obtained that produce 1.5-2 million units of IFN-gamma per million cells and per day (200,000 molecules per cell per minute). Metabolic labeling showed that over 90% of the protein secreted by such cells is human IFN-gamma. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-gamma with 1-2 X 10(8) units/mg protein. Like IFN-gamma from human white blood cells, the IFN-gamma from CHO-gamma cells is a mixture of two glycoproteins of 26,000 and 20,000 daltons with traces of the unglycosylated 17,000-dalton polypeptide. Large-scale cultures in 1% serum routinely yield over 600,000 units of human IFN-gamma/ml culture per day.
D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes; D004274:DNA, Recombinant / Q000378:metabolism; D005260:Female; D005796:Genes; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics*; D009856:Oncogene Proteins, Viral / Q000235:genetics; D010053:Ovary; D011494:Protein Kinases / Q000235:genetics; D013539:Simian virus 40 / Q000235:genetics
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D004274:DNA, Recombinant; D009856:Oncogene Proteins, Viral; D007371:Interferon-gamma; D011494:Protein Kinases; D004262:DNA Restriction Enzymes