J Biol Chem. 1987 Feb 25;262(6):2528-35.

Heterogeneity of Chinese hamster ovary cell-produced recombinant murine interferon-gamma.

The Journal of biological chemistry

R Dijkmans, H Heremans, A Billiau

PMID: 2434486
Free Article

Abstract

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).

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Substances

• RNA, Messenger
• Recombinant Proteins
• Poly A
• RNA
• Interferon-gamma
• Methionine
• Cysteine
• Methotrexate

MeSH terms

• Animals
• Cell Line
• Cricetinae
• Cricetulus
• Cysteine / metabolism
• Electrophoresis, Polyacrylamide Gel
• Female
• Glycosylation
• Interferon-gamma / analysis
• Methionine / metabolism
• Methotrexate / pharmacology
• Mice
• Molecular Weight
• Ovary / metabolism
• Poly A / metabolism
• RNA / metabolism
• RNA, Messenger
• Recombinant Proteins / analysis

Publication Types

• Journal Article
• Research Support, Non-U.S. Gov't

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