Efficient Constitutive Production of Human IFN-γ in Chinese Hamster Ovary Cells
| Autor: | D. Novick, D. Segev, Asher Zilberstein, J. Ben-Barak, Michel Revel, M. Rubinstein, M. Vigneron, Dina G. Fischer, S. Kargman, Batya Cohen, Y Mory |
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| Rok vydání: | 1986 |
| Předmět: |
Genes
Viral Antigens Polyomavirus Transforming viruses Genetic Vectors DNA Recombinant Simian virus 40 Biology Biochemistry Cell Line Interferon-gamma Cricetulus Cricetinae Gene duplication Genetics Animals Humans Cloning Molecular Antigens Viral Tumor Enhancer Molecular Biology Gene Selectable marker Base Sequence Chinese hamster ovary cell Ovary DNA Restriction Enzymes Oncogene Proteins Viral Transfection Molecular biology Long terminal repeat Genes Cell culture Female Protein Kinases |
| Zdroj: | DNA. 5:181-193 |
| ISSN: | 0198-0238 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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