NR AWQC
AU Waldron,S.; Rogers,M.
TI Characterisation of PrPsc using alternative proteases to proteinase K
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions CE-50
PT Konferenz-Poster
AB PrPsc is involved in transmission of prion disease. Routine detection of this aberrant form of PrP is based on the different sensitivities of PrPc and PrPsc to proteinase K (PK). PrPc is completely digested by PK while PrPsc shows variable resistance to digestion resulting in an N-terminally truncated protein of ~27-30kDa (PrP27-30) Using bioinformatic analysis and literature review proteases were identified that should fully digest PrPc but leave PrPsc intact, including the N-terminal. Of these proteases trypsin, subtilisin and thermolysin were further investigated. Protease digest concentration gradients were performed on control mouse brains and ME7 infected mouse brains to determine the optimum concentration for complete digestion of PrPc but not PrPsc. Antibodies SAF83 (SPI-Bio), SAF32 (SPI-Bio) and Ab167(Polyclonal anti-peptide antisera raised against peptide of Hamster PrP (29-54) were used to detect intact PrPsc after protease digestion. SAF83 recognises an epitope in PrP within the central core of PrP that is accessible in PrPc but becomes misfolded during PrPsc formation and therefore requires PrPsc to be denatured for detection. Both SAF32 and Ab167 are N-terminal antibodies, SAF32 detects the octapeptide repeat and Ab167 detects a.a. 29-54. We have shown that trypsin, subtilisin and thermolysin all leave PrPsc intact after digestion but fully digest PrPc. It should now be possible to characterise intact PrPsc in its native state using an N-terminal antibody following protease digestion.
AD Prion Research Group, School of Biology and Environmental Science, University College Dublin, Dublin, Ireland. E-mail: Sibeal.Waldron@ucd.ie
SP englisch
PO Italien