Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity

Biochem Biophys Res Commun. 1996 Feb 6;219(1):173-9. doi: 10.1006/bbrc.1996.0201.

Abstract

PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cloning, Molecular
  • Cricetinae
  • Endopeptidase K
  • Escherichia coli
  • Glutathione Transferase / biosynthesis
  • Mesocricetus
  • PrP 27-30 Protein / chemistry
  • PrP 27-30 Protein / metabolism*
  • Prions / chemistry
  • Prions / isolation & purification
  • Prions / metabolism*
  • Recombinant Fusion Proteins / biosynthesis
  • Recombinant Fusion Proteins / isolation & purification
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Serine Endopeptidases / metabolism*
  • Spodoptera
  • Substrate Specificity
  • Thrombin / metabolism
  • Transfection

Substances

  • Prions
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • PrP 27-30 Protein
  • Glutathione Transferase
  • Serine Endopeptidases
  • Thrombin
  • Endopeptidase K