Chemistry and Biochemistry Faculty Works

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Browsing Chemistry and Biochemistry Faculty Works by Author "Blochberger, Thomas C."

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    Prion protein (PrP) synthetic peptides induce cellular PrP to acquire properties of the scrapie isoform
    (Proceedings of the National Academy of Sciences, 1995-11) Kaneko, K.; Peretz, David; Pan, K. K.; Blochberger, Thomas C.; Wille, H.; Gabizon, R.; Griffith, O. H.; Cohen, F. E.; Baldwin, Michael A.; Prusiner, Stanley B.
    Conversion of the cellular isoform of prion protein (PrPc) into the scrapie isoform (PrPSc) involves an increase in the /3-sheet content, diminished solubility, and resistance to proteolytic digestion. Transgenetic studies argue that PrPc and PrPSc form a complex during PrPScformation; thus, synthetic PrP peptides, which mimic the conformational pluralism of PrP, were mixed with PrPc to determine whether its properties were altered. Peptides encompassing two a-helical domains of PrP when mixed with PrPc produced a complex that displayed many properties of PrPSc. The PrPcpeptide complex formed fibrous aggregates and up to 65% of complexed PrPc sedimented at 100,000 x g for 1 h, whereas PrPc alone did not. These complexes were resistant to pro teolytic digestion and displayed a high /3-sheet content. Un expectedly, the peptide in a /3-sheet conformation did not form the complex, whereas the random coil did. Addition of 2% Sarkosyl disrupted the complex and rendered PrPc sensitive to protease digestion. While the pathogenic AllTV mutation increased the efficacy of complex formation, anti-PrP mono clonal antibody prevented interaction between PrPc and pep tides. Our findings in concert with transgenetic investigations argue that PrPc interacts with PrPSc through a domain that contains the first two putative a-helices. Whether PrPc-peptide complexes possess prion infectivity as determined by bioassays remains to be established.
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    Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system
    (Protein Engineering, 1997-09-05) Blochberger, Thomas C.; Cooper, Carl; Peretz, David; Tatzelt, Jorg
    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.
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    Prion protein expression in Chinese hamster ovary cells using a glutamine synthetase selection and amplification system
    (Protein Engineering, 1997) Blochberger, Thomas C.; Cooper, Carl; Peretz, David; Tatzelt, Jorg; Griffith, O. H.; Baldwin, Michael A.; Prusiner, Stanley B.