NR AMHV
AU Weiss,S.; Famulok,M.; Edenhofer,F.; Wang,Y.H.; Jones,I.M.; Groschup,M.H.; Winnacker,E.L.
TI Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems
QU Journal of Virology 1995 Aug; 69(8): 4776-83
PT journal article
AB This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.
IN Eine die Aminosäuren 23-231 des Goldhamsterprionproteins kodierende cDNA der Weissmann-Gruppe wurde in einem Expressionsvektor hinter die kodierende Sequenz für Glutathion S-Transferase ligiert. Damit wurde in E. coli sowie in Baculovirus-infizierten Insektenzellen mg-weise ein stabiles, leicht isolierbares Glutathion S-Transferase - Prionprotein - Fusionsprotein produziert, welches dem normalen zellulären Prionprotein entspricht. Hieraus kann das Prionprotein durch Thrombin-Abspaltung gewonnen werden. Es erwies sich dann allerdings als sehr unstabil. Hefe erwies sich als ein ungeeignetes System für die Prionprotein-Expression, da das Fusionsprotein anders als bei den Insektenzellen nicht exportiert wurde.
ZR 36
MH Animal; Baculoviridae/genetics; Cell Line; Cloning, Molecular; Escherichia coli/genetics; Glutathione Transferase/*genetics; Hamsters; Mesocricetus; Pichia/genetics; Prions/*genetics; Recombinant Fusion Proteins/genetics; Spodoptera; Support, Non-U.S. Gov't
AD Laboratorium für Molekulare Biologie-Genzentrum-Institut für Biochemie der LMU München, Germany.
SP englisch
PO USA
OR Prion-Krankheiten 8