| Summary: | <p>Abstract</p> <p>Background</p> <p>The amino terminal half of the cellular prion protein PrP<sup>c </sup>is implicated in both the binding of copper ions and the conformational changes that lead to disease but has no defined structure. However, as some structure is likely to exist we have investigated the use of an established protein refolding technology, fusion to green fluorescence protein (GFP), as a method to examine the refolding of the amino terminal domain of mouse prion protein.</p> <p>Results</p> <p>Fusion proteins of PrP<sup>c </sup>and GFP were expressed at high level in <it>E.coli </it>and could be purified to near homogeneity as insoluble inclusion bodies. Following denaturation, proteins were diluted into a refolding buffer whereupon GFP fluorescence recovered with time. Using several truncations of PrP<sup>c </sup>the rate of refolding was shown to depend on the prion sequence expressed. In a variation of the format, direct observation in <it>E.coli</it>, mutations introduced randomly in the PrP<sup>c </sup>protein sequence that affected folding could be selected directly by recovery of GFP fluorescence.</p> <p>Conclusion</p> <p>Use of GFP as a measure of refolding of PrP<sup>c </sup>fusion proteins <it>in vitro </it>and <it>in vivo </it>proved informative. Refolding <it>in vitro </it>suggested a local structure within the amino terminal domain while direct selection via fluorescence showed that as little as one amino acid change could significantly alter folding. These assay formats, not previously used to study PrP folding, may be generally useful for investigating PrP<sup>c </sup>structure and PrP<sup>c</sup>-ligand interaction.</p>
|
|---|