QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/jcs.00086

This Article Figures Only Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Gauczynski, S. Articles by Weiss, S. Search for Related Content PubMed PubMed Citation Articles by Gauczynski, S. Articles by Weiss, S.

Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance

¹ Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Feodor-Lynen Str. 25, D-81377 Munich, Germany
² Veterinary Medicine and Primate Husbandry, German Primate Center, Kellnerweg 4, D-37077 Goettingen, Germany

^* Author for correspondence (e-mail: Weiss{at}lmb.uni-muenchen.de)

Accepted 1 August 2002

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.

Key words: Prion protein, PrP mutants, Proteinase K resistance, Semliki-Forest virus (SFV) system, Processing, Glycosylation

This article has been cited by other articles:

				M. Eiden, G. J. Palm, W. Hinrichs, U. Matthey, R. Zahn, and M. H. Groschup Synergistic and strain-specific effects of bovine spongiform encephalopathy and scrapie prions in the cell-free conversion of recombinant prion protein J. Gen. Virol., December 1, 2006; 87(12): 3753 - 3761. [Abstract] [Full Text] [PDF]

				D. A Harris Trafficking, turnover and membrane topology of PrP: Protein function in prion disease Br. Med. Bull., June 1, 2003; 66(1): 71 - 85. [Abstract] [Full Text] [PDF]