Characterization and polyanion-binding properties ef purified recombinant recombinant protein

Certain polysulphated polyanions have been shown to have prophylactic effec ts on the progression of transmissible spongiform encephalopathy disease, p resumably because they bind to prion protein (PrP). Until now, the difficul ty of obtaining large quantities of native PrP has precluded detailed studi es of these interactions. We have over-expressed murine recombinant PrP (re cPrP), lacking its glycophosphoinositol membrane anchor, in modified mammal ian cells. Milligram quantities of secreted, soluble and partially glycosyl ated protein were purified under non-denaturing conditions and the identiti es of mature-length aglycosyl recPrP and two cleavage fragments were determ ined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. rec PrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociatio n kinetics that were consistent with ligand-directed recPrP aggregation. Th e differences in the binding strengths of recPrP to pentosan polysulphate a nd to other sulphated polyanions were found to parallel their in vivo anti- scrapie and in vitro anti-scrapie-specific PrP formation potencies. When re cPrP was immobilized by capture on metal-ion chelates it was found, contrar y to expectation, that the addition of polyanions promoted the dissociation of the protein.