Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity.
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.
Weiss
S
S
Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Germany.
Rieger
R
R
Edenhofer
F
F
Fisch
E
E
Winnacker
E L
EL
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D019286:Endopeptidase K; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000096:biosynthesis; D008647:Mesocricetus; D018622:PrP 27-30 Protein / Q000737:chemistry / Q000378:metabolism*; D011328:Prions / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism*; D018411:Spodoptera; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014162:Transfection
D011328:Prions; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018622:PrP 27-30 Protein; D005982:Glutathione Transferase; D012697:Serine Endopeptidases; D013917:Thrombin; D019286:Endopeptidase K