Figure 1
From: Green fluorescent protein as a reporter of prion protein folding
Establishment of the Prp-GFP refolding assay. A. Fragments of the mouse prnp a allele whose structure is shown were amplified by PCR and positioned at the N terminus of GFP in a E.coli expression vector under transcriptional control of the T7 promoter. B. Purified PrP-GFP fusion proteins were analysed by 10% SDS-PAGE before (lanes 1 & 2) and after (lanes 3 & 4) the refolding reaction. The lanes are: M-Molecular weight markers as shown; 1&3-PrP23–231-GFP; 2&4-PrP76–156-GFP. The lower staining intensity of the refolded samples is due to dilution in the refolding buffer. C. Recovery of fluorescence with time following dilution of the solublised PrP-GFP fusion proteins into refolding buffer. In this experiment the increase in fluorescence units was 6 fold (□) and 27 fold (●) for PrP23–231-GFP and PrP76–156-GFP respectively. Assays were done in duplicate and the average fluorescent units plotted against time.