NR AOSN
AU Fimmen,L.; Redecke,L.; Basaiawmoit,R.V.; Koker,M.; Perbandt,M.; Bredehorst,R.; Betzel,C.; Notbohm,H.; Voelter,W.; Clos,J.
TI
Recombinant Expression and Conformational Analysis of Human and Hamster Prion Precursor Protein
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-91
PT Konferenz-Poster
AB
We have expressed human and hamster prion precursor protein in E. coli. Recombinantly expressed proteins were purified and refolded bound to a metal chelate resin. Following elution, the renatured protein could be concentrated up to 10 mg ml-1 and remained in solution at 200,000 x g.
Polyclonal antibodies (IgY) were purified from the eggs of laying hens that had been immunised with soluble human prion protein. In Western Blots, the antibody preparation is sensitive to PrP concentrations in the subnanogram range and detects prion protein in cell lysates of infected hamster brain. Its suitability for detection of prion aggregates in infected tissues will be evaluated, and the results will be presented.
The purified recombinant human PrP associated into higher order oligomers in a time- and temperature-dependent manner in the absence of seed aggregates. This was shown by Dynamic Light Scattering (DLS) analysis measuring the hydrodynamic radius in solution. We currently investigate the impact of bivalent metal ions, e.g. Cu2+, on the oligomerisation kinetics, secondary structure distribution, and the protein's ability to stimulate plasminogen activity in vitro.
AD L. Fimmen, L. Redecke, R. Basaiawmoit, M. Perbandt, C. Betzel, Institute of Medical Biochemistry and Molecularbiology, University Hospital Hamburg-Eppendorf, Hamburg, Germany; M. Koker, J. Clos, Bernhard Nocht Institute for Tropical Medicine, Hamburg, FRG; R. Bredehorst, Division of Biochemistry and Molecularbiology, University of Hamburg, Hamburg, Germany; H. Notbohm, Institute for Medical Molecularbiology, University of Lübeck, Lübeck, Germany; W. Voelter, Division of Physical Biochemistry, University Institute of Physiological Chemistry, Tübingen, Germany
SP englisch
PO Deutschland