Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammalian cells.
Tetracycline-controlled expression plasmids that allow inducible expression of proteins in mammalian cells (Gossen & Bujard, 1992), have been used to express porcine interferon-gamma in the RK-13 rabbit kidney cell line. Following neomycin selection, stable clones produced recombinant, glycosylated porcine interferon-gamma (rGPoIFN-gamma) only after removal of tetracycline (Tc). Southern blot analysis of one clone showed that approximately 50 copies of IFN-gamma cDNA were present in the cell genome. In the absence of Tc, stable clones secreted large amounts of rGPoIFN-gamma (up to 16 microg/ml) into the medium supplemented with 10% FCS and high glucose concentration. Molecular weight comparison of 35S-Methionine, labelled rGPoIFN-gamma with natural leukocytic IFN-gamma after immunoprecipitation, revealed 4 major glycoforms with apparent Mr of 27,000; 25,000; 20,000 and 18,500, that are almost identical in both IFN-gamma species. In both cases, all 4 glycoforms resolved into 2 polypeptide monomers with apparent Mr of 16,500 and 14,500 upon deglycosylation with N-glycosydase F. The biological activity of rGPoIFN-gamma was in the same range as that of natural leukocytic PoIFN-gamma (2 x 10(6) U/mg). Eventually, this recombinant mammalian IFN-gamma should constitute a very useful substitute for leukocyte PoIFN-gamma in in vitro or in vivo experiments.
Cencic
A
A
Unité de Virologie et d'Immunologie Moléculaires, I.N.R.A., Jouy-en-Josas, France.
LeFèvre
F
F
Koren
S
S
La Bonnardière
C
C
D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects*; D006031:Glycosylation; D007371:Interferon-gamma / Q000096:biosynthesis / Q000235:genetics*; D011817:Rabbits; D013552:Swine; D013752:Tetracycline / Q000494:pharmacology*; D014162:Transfection
C046697:glycosylated gamma interferon; D007371:Interferon-gamma; D013752:Tetracycline