Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system

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 approximately 14-fold higher than that of PrPC found in Syrian hamster brain.

Association of Bcl-2 with Misfolded Prion Protein Is Linked to the Toxic Potential of Cytosolic PrP

“…Among those, addition of the GPI anchor seems to be of particular importance. Initial experiments in cell culture revealed that a mutant devoid of the C-terminal GPI anchor signal sequence (PrP⌬GPI) is imported into the ER but remains mainly unglycosylated, spontaneously adopts a misfolded conformation and is efficiently secreted (Rogers et al, 1993;Blochberger et al, 1997;Winklhofer et al, 2003c). The phenotype of anchorless PrP described in cultured cells was recently confirmed in transgenic mice.…”

“…To include a PrP conformer that misfolds in the secretory pathway, we used a mutant that contains the N-terminal ER- targeting signal but lacks the C-terminal GPI signal sequence (designated PrP⌬GPI). Targeting to and import into the ER of PrP⌬GPI is efficient, but in the secretory pathway PrP⌬GPI spontaneously adopts a misfolded conformation and is secreted (Rogers et al, 1993;Blochberger et al, 1997;Winklhofer et al, 2003c).…”

Structural changes of membrane-anchored native PrP C

“…Chinese hamster ovary (CHO) cell lines overexpressing PrP C of the Syrian Golden hamster sequence were established by Dr. S. B. Prusiner's group (23). PrP C derived from this cell line carries both the two Nglycosylations and the C-terminally attached GPI anchor.…”

“…Our preparation is much closer to the in vivo situation than those of the earlier studies. Native PrP C , carrying the two N-glycosylations and the natural GPI anchor, purified from an eukaryotic transgenic cell line (23,24), was anchored to a solidly supported, raft-like membrane in a buffered environment. In an earlier study, we exploited the alterations of surface plasmon resonance to analyze the lipid anchoring kinetics (25).…”

α-Helical Domains Promote Translocation of Intrinsically Disordered Polypeptides into the Endoplasmic Reticulum

“…(40). Deletion of the C-terminal GPI anchor signal sequence prevents membrane attachment of PrP-S230X but does not interfere with import into the ER or further trafficking through the secretory pathway (24,27,41). PrP-S230X is secreted both by cultured cells (Fig.…”