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...

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Bibliographic Details
Published in:Protein engineering 1997-12, Vol.10 (12), p.1465-1473
Main Authors: Blochberger, T C, Cooper, C, Peretz, D, Tatzelt, J, Griffith, O H, Baldwin, M A, Prusiner, S B
Format: Article
Language:English
Subjects:
Animals
Blotting, Western
Calcium Phosphates
CHO Cells - metabolism
Cricetinae
Endopeptidase K - metabolism
Gene Expression
Glutamate-Ammonia Ligase - metabolism
Glycosylphosphatidylinositols - chemistry
Glycosylphosphatidylinositols - genetics
Mesocricetus
Phosphatidylinositol Diacylglycerol-Lyase
Phosphoinositide Phospholipase C
Prions - genetics
Recombinant Proteins
Transfection
Type C Phospholipases - metabolism
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Summary: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.
ISSN:0269-2139
1741-0126
1741-0134
DOI:10.1093/protein/10.12.1465