NR ATKE
AU Moonen,P.; Ruiter,S.; van den Burg,B.; Langeveld,J.P.M.; Bossers,A.
TI Differentiation between PrPsc isolates using thermostable proteases
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Diagnosis DIA-16
PT Konferenz-Poster
AB
A key feature in the diagnosis of transmissible spongiform encephalopathies (TSEs) is the immunological detection of accumulation of a proteinase K (pK) resistant isoform (PrPsc) of the host-encoded, normally protease sensitive, prion protein (PrP). The patterns seen in immunoblot after pK digestion of PrPsc derived from different species (e.g. human, bovine, ovine) show small but distinct differences in band size and number, sufficient for differentiation. The possibility to differentiate is especially important in the diagnosis of scrapie, considering the implications of a possible BSE infection of sheep.
To survey whether it would be possible to increase the differences between scrapie and/or BSE isolates, or even to determine differences between TSE strains, we studied the proteolytic effect of 24 different thermostable (thermolysin-like) proteases on bovine and ovine PrPsc. These thermolysin-like proteases are functional under non-physiological circumstances, in which protein folding may be less compact than in a physiological environment. This might result in the exposition of a protease cleavage site causing the degradation of the investigated protein to a distinctly smaller residue, or to complete degradation of the, under normal circumstances, protease resistant core.
We used the 24 proteases at several non-physiological conditions, such as a pH range (7-9) and a temperature range (37-90°). The remaining fractions were detected on Western blot using anti PrP antibodies.
Differences were observed in digestion efficiencies of purified bovine and ovine PrPsc and thus far not in size of the remaining protease resistant core or digestion products.
AD Peter Moonen, Saskia Ruiter, Jan Langeveld, Alex Bossers, CIDC, Lelystad, The Netherlands; Bertus van den Burg, IMEnz bioengineering, Groningen, The Netherlands
SP englisch
PO Deutschland