Efficient Constitutive Production of Human IFN-γ in Chinese Hamster Ovary Cells

Abstract

A human genomic DNA segment of 5.6 kb containing the entire gene for immune interferon-γ 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-γ 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-γ cell lines were obtained that produce 1.5–2 million units of IFN-γ 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-γ. A one-step immuno-affinity chromatography on monoclonal antibodies yielded pure IFN-γ with 1–2 × 10⁸ units/mg protein. Like IFN-γ from human white blood cells, the IFN-γ from CHO-γ 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-γ/ml culture per day.