Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system
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Date
1997-09-05
Authors
Blochberger, Thomas C.
Cooper, Carl
Peretz, David
Tatzelt, Jorg
Journal Title
Journal ISSN
Volume Title
Publisher
Protein Engineering
Abstract
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 - 14-fold higher than that of PrPc found in Syrian
hamster brain.
Description
10 pages
Keywords
Citation
Blochberger, T. C., Cooper, C., Peretz, D., Tatzlet, J., Griffith, O. H., Baldwin, M. A. and Prusiner, S. B. (1997). Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system, Protein Engineering, 10, 1465-1473.