Heterogeneity of Chinese hamster ovary cell-produced recombinant murine interferon-gamma.
Chinese hamster ovary cells transformed with a hybrid expression plasmid containing both the murine interferon-gamma (MuIFN-gamma) and the murine dihydrofolate reductase-coding sequences were subjected to selection in stepwise increasing concentrations of methotrexate. By this procedure the production rate of MuIFN-gamma was increased from an initial level of approximately 20,000 to approximately 500,000 antiviral units per milliliter of culture supernatant. [35S]Methionine-labeled proteins secreted by these cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with or without prior immunoprecipitation with polyclonal or monoclonal antibodies against splenocyte-derived MuIFN-gamma. Besides two major components of Mr 19,000 and 38,000, a multiplicity of minor components were immunoprecipitated. Cells treated with tunicamycin, an inhibitor of N-glycosylation, secrete two major components of Mr 14,000 and 27,000 and only two minor components of Mr 12,000 and 13,000. When the proteins were labeled with [35S]cysteine, a residue that is only present at the carboxyl terminus of the mature MuIFN-gamma, no minor components could be detected in the growth medium of tunicamycin-treated cells. The presented results indicate that the heterogeneity of the recombinant Chinese hamster ovary-produced MuIFN-gamma is due to at least three cumulative modifications of the Mr 14,000 MuIFN-gamma peptide: carboxyl-terminal proteolytic processing (the Mr 13,000 and 12,000 components), variations in N-glycosylation (components ranging in size from Mr 12,000 to 26,500), and dimerization (components ranging from Mr 27,000 to 50,000).
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006031:Glycosylation; D007371:Interferon-gamma / Q000032:analysis*; D008715:Methionine / Q000378:metabolism; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D010053:Ovary / Q000378:metabolism; D011061:Poly A / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger; D011994:Recombinant Proteins / Q000032:analysis*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D011061:Poly A; D012313:RNA; D007371:Interferon-gamma; D008715:Methionine; D003545:Cysteine; D008727:Methotrexate