Avtor/Urednik | Cencič, A; LeFevre, F; Koren, S; La Bonnardiere, C | |
Naslov | Tetracycline-controlled expression of glycosylated porcine interferon-gamma in mammalian cells | |
Tip | članek | |
Vir | Anim Biotechnol | |
Vol. in št. | Letnik 10, št. 1-2 | |
Leto izdaje | 1999 | |
Obseg | str. 63-79 | |
Jezik | eng | |
Abstrakt | 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. | |
Deskriptorji | GENE EXPRESSION REGULATION INTERFERON TYPE II TETRACYCLINE RABBITS CELLS, CULTURED GLYCOSYLATION SWINE TRANSFECTION |