NR ABKI
AU Blochberger,T.C.; Cooper,C.M.; Peretz,D.; Tatzelt,J.; Griffith,O.H.; Baldwin,M.A.; Prusiner,S.B.
TI Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system
QU Protein Engineering 1997 Dec; 10(12): 1465-73
IA http://peds.oxfordjournals.org/cgi/reprint/10/12/1465
PT journal article
AB Syrian hamster prion protein (PrPc) and a truncated Syrian hamster prion protein lacking the glycosylphosphatidylinositol (GPI) anchor C-terminal signal sequence (GPI-) were expressed in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system. The CHO cell clones expressing the GPI- PrP secreted the majority of the protein into the media, whereas most of the PrP produced by clones expressing the full-length protein with the GPI anchor was located on the cell surface, as demonstrated by its release upon treatment with phosphatidylinositol-specific phospholipase C (PIPLC). A cell clone that expressed the highest levels of full length PrP was subcloned to obtain clone 30C3-1. PrP from clone 30C3-1 was shown to be sensitive to proteolysis by proteinase K and to react with monoclonal and polyclonal antibodies that recognize native PrPc. The recombinant PrP migrated as a diffuse band of 19-40 kDa but removal of the N-linked oligosaccharides with peptide N-glycosidase F (PNGase F) revealed three protein species of 19, 17 and 15 kDa. The 19 kDa band corresponding to deglycosylated full-length PrP was quantified and found to be expressed at a level approximately 14-fold higher than that of PrPc found in Syrian hamster brain.
MH Animals; Blotting, Western; CHO Cells/*metabolism; Calcium Phosphates; Cricetinae; Endopeptidase K/metabolism; *Gene Expression; Glutamate-Ammonia Ligase/*metabolism; Glycosylphosphatidylinositols/chemistry/genetics; Mesocricetus; Phosphatidylinositol Diacylglycerol-Lyase; Phospholipase C/metabolism; Prions/*genetics; Recombinant Proteins; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S.; Transfection
AD
Thomas C. Blochberger, Carol Cooper, David Peretz, Jörg Tatzelt (Current address: Max-Planck-Institut für Biochemie, Dept. of Cellular
Biochemistry, 82152 Martinsried, Germany), Michael A. Baldwin, Stanley B. Prusiner, Department of Neurology, University of California San Francisco, San Francisco, CA 94143, USA; O. Hayes Griffith, Institute of Molecular Biology and Department of Chemistry, University of Oregon, Eugene, OR 97403, USA
SP englisch
PO England