Expression and characterization of the product of a human immune interferon cDNA gene in Chinese hamster ovary cells.
Cotransformation with two plasmids, one [pSV2-IFN-gamma] encoding human immune interferon (Hu IFN-gamma) and the other [pAdD26SV(A)-3] encoding mouse dihydrofolate reductase, has been used to establish Chinese hamster ovary (CHO) cell lines that secrete high levels of Hu IFN-gamma. Hu IFN-gamma production by the transformed CHO cell lines E-10B and E-10C reached approximately 50,000 units/ml of culture medium, which compares favorably with that of stimulated lymphocytes. Furthermore, as the Hu IFN-gamma cDNA gene used in these studies is under the transcriptional control of the simian virus 40 early promoter, Hu IFN-gamma production is constitutive and thus does not require induction. CHO-produced Hu IFN-gamma migrates as two bands corresponding to molecular weights of 25,000 and 21,000 on NaDodSO4/polyacrylamide gels. These two species are shown to be the products of a single gene. As the molecular weight of native Hu IFN-gamma is around 55,000, it is likely to be a dimer. We have shown that the subunits of such a dimer cannot be linked by a disulfide bridge(s). Hu IFN-gamma from CHO cells is likely to be glycosylated and this should now permit comparison of the biological activities of glycosylated and nonglycosylated (bacterially produced) Hu IFN-gamma in animal studies.
D000818:Animals; D002460:Cell Line; D002471:Cell Transformation, Neoplastic; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D005260:Female; D005796:Genes; D006801:Humans; D007371:Interferon-gamma / Q000235:genetics* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008970:Molecular Weight; D009876:Operon; D010053:Ovary; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics
D046911:Macromolecular Substances; D007371:Interferon-gamma; D004247:DNA; D004262:DNA Restriction Enzymes