Abstract
The Semliki-Forest virus (SFV) system was used to overexpress human wild-type and mutant prion proteins as well as FLAG-tagged human and bovine PrP in mammalian cells. The application of recombinant SFV vectors allowed a high-level production of highly glycosylated prion proteins with a molecular weight ranging from 25 to 30 kDa for recombinant wild-type human PrP and from 26 to 32 kDa for wild-type bovine PrP. Further, we report here the generation of recombinant mutant prion proteins that are associated with inherited human prion diseases such as fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Both mutated variants, the FFI-associated PrP carrying a mutation at amino acid position 178 and the CJD-linked form containing an insertion of nine additional octarepeats reveal proteinase K resistance, one of the typical biochemical properties of the infectious scrapie isoform of the prion protein. By contrast, recombinant wild-type PrP was completely proteinase K sensitive when expressed in SFV-transfected BHK cells. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP. In order to purify recombinant human and bovine PrP from cell lysates, a FLAG-tag was introduced either at the N-terminus behind the signal peptide or at the C-terminus close to the adhesion site of the GPI anchor. N-terminal insertion did not extensively influence the trafficking of the FLAG-tagged protein to the cell surface, whereas insertion close to the GPI attachment site clearly affected the transport of the majority of PrP to the cell membrane, probably resulting in their retention within the secretory pathway. All FLAG-tagged prion proteins were expressed efficiently in BHK cells and showed a typical glycosylation pattern, allowing their rapid and simple purification via anti-FLAG antibody chromatography.
Highlights
The causative agent of transmissible spongiform encephalopathies (TSEs) is a prion, which is defined as a proteinaceous infectious particle (Prusiner, 1982)
The plasmid DNAs pSFV1-boPrP1-264, pSFV1-huPrP1-253 (Krasemann et al, 1996), pSFV1-huPrP-fatal familial insomnia (FFI) and pSFV1-huPrP+9OR (Krasemann et al, 1995) were used. pSFV1huPrP22FLAG was generated by the QuikChangeTM site-directed mutagenesis method (Stratagene) employing pSFV1-huPrP1-253 DNA as a template
The transfection efficiency of BHK cells used throughout all our experiments was almost 100%, warranting the high-level production of glycosylated prion proteins
Summary
The causative agent of transmissible spongiform encephalopathies (TSEs) is a prion, which is defined as a proteinaceous infectious particle (Prusiner, 1982). Prion diseases (for reviews, see Lasmézas and Weiss, 2000; Prusiner, 1998; Weissmann and Aguzzi, 1997) belong to a new class of fatal neurodegenerative disorders affecting humans and animals. Human prion diseases include kuru, Creutzfeldt-Jakob disease (CJD), fatal familial insomnia (FFI) and Gerstmann-Sträussler-Scheinker syndrome (GSS) (for reviews, see Lasmézas and Weiss, 2000; Prusiner, 1998; Weissmann and Aguzzi, 1997). Whereas 85% of all CJD cases occur sporadically in the absence of any mutations, FFI, GSS and the remaining 15% of CJD are dominantly inherited diseases caused by defined mutations within the Prn-p gene on chromosome 20 (Masters et al, 1981; Sparkes et al, 1986). The spontaneous conversion of PrPc to PrPSc in the absence of exogenous prions has been suggested (Young, 1999), but the exact mechanism by which these mutations provoke conformational changes is still unknown. Destabilization of the PrPc structure or effects on the thermodynamic stability of PrP have been suggested (Liemann and Glockshuber, 1999; Riek et al, 1998; Swietnicki et al, 1998; Zhang et al, 2000)
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