NR AOUG
AU Hundt,C.; Müller,S.; Besir,H.; Fernholz,E.; Hloch,P.; Eberle,W.
TI Prion protein expression in the Rapid Translation System (RTS)
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-95
PT Konferenz-Poster
AB
Rapid Translation System (RTS) is a scalable in vitro transcription/translation expression platform from Roche Diagnostics. RTS offers the advantages of a cell-free protein expression system using an Escherichia coli lysate and eliminating the need for laborious up- and down-stream steps (e.g., host cell transformation, culturing, or lysis).
Here we show the expression of a non- and a HIS-tagged bovine prion protein in the RTS 100. Moreover scale up experiments employing the RTS 500 kit and the ProteoMaster instrument were also successful. The expressed PrP showed Proteinase K sensitivity and the main fraction was insoluble. Using different disulfide bond forming expression conditions for the RTS 500 format we were able to increase the solubility of the expressed prion protein.
The RTS is a very convenient tool not only for the expression of the prion protein but also for studies regarding the influence of different drugs during the synthesis of PrP. To investigate the formation of PrPsc in status nascendi we analysed the effect of different copper concentrations on the PrP expression and observed no significant difference. Nevertheless it should be possible to trigger the generation of PrPsc in the RTS without the addition of pathogenic prion proteins. This could lead to new ideas regarding the in vivo mechanism of the conversion of PrPc to PrPsc.
AD Christoph Hundt, Silke Mueller, Hueseyin Besir, Erhard Fernholz, Peter Hloch, Walter Eberle, Roche Diagnostics, Germany
SP englisch
PO Deutschland