Programme 8:  Development of a probe for prion protein ligand binding and propagation

Background
We have developed a fluorescently-labelled version of the prion protein (PrP) which is sensitive to the binding of both biological ligands (e.g. amyloid beta and anti-PrP antibodies) and small molecules (Risse et al., J. Biol Chem 2015 290 17020). These experiments used prion protein expressed in E. coli and therefore devoid of post-translational modification. We have now developed the over-expression and purification of the prion protein in HEK293 cells and plan to determine the effects of the post translational modifications upon ligand binding.

Rotation project
Using routine molecular biology techniques point mutations will be introduced into the construct used to over-express PrP. These constructs will then be transfected into HEK293 cells, the resulting protein purified and tested in the established fluorescence polarization assay for ligand binding.


PhD project
Once established the labelled fully-modified PrP will provide a valuable tool to monitor the association of PrP with a variety of putative ligands as well as to monitor changes in the PrP species during in vitro amplification processes, giving the student experience in a variety of biophysical and biochemical techniques. In collaboration with Programme 6 the labelling could be extended to include using nanogold to selectively label the PrP, enabling visualisation of its interactions with amyloid beta or with other PrP molecules by electron microscopy.