Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system.
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.
Blochberger
T C
TC
Department of Neurology, University of California San Francisco, 94143, USA.
Cooper
C
C
Peretz
D
D
Tatzelt
J
J
Griffith
O H
OH
Baldwin
M A
MA
Prusiner
S B
SB
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells / Q000378:metabolism*; D002130:Calcium Phosphates; D006224:Cricetinae; D019286:Endopeptidase K / Q000378:metabolism; D015870:Gene Expression; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry / Q000235:genetics; D008647:Mesocricetus; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D011328:Prions / Q000235:genetics*; D011994:Recombinant Proteins; D014162:Transfection; D010738:Type C Phospholipases / Q000378:metabolism
D002130:Calcium Phosphates; D017261:Glycosylphosphatidylinositols; D011328:Prions; D011994:Recombinant Proteins; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; D019286:Endopeptidase K; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D005974:Glutamate-Ammonia Ligase; C485829:calcium phosphate, dibasic, anhydrous