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.