Separation of native prion protein (PrP) glycoforms by copper-binding using immobilized metal affinity chromatography (IMAC)

The conformational conversion of the normal cellular prion protein (PrP^C) into the pathology-associated PrP^Sc isoform is a key event in TSEs (transmissible spongiform encephalopathies). The host PrP^C molecule contains two N-linked glycosylation sites and binds copper under physiological conditions. In contrast with PrP^C, PrP^Sc is insoluble in non-ionic detergents and does not bind to Cu²⁺ ions. Hence, we utilized copper binding to separate and characterize both PrP isoforms. Infected and uninfected murine brain and bovine stem brain specimens were treated with the mild non-ionic detergent n-octyl-β-D-glucopyranoside (octylglucoside) to maintain the native PrP conformations during isolation. The solubilized homogenates were loaded on to Cu²⁺-saturated IMAC (immobilized metal affinity chromatography) columns and eluted using the chelating agent EDTA. Fractions were separated by SDS/PAGE and analysed by immunoblotting using anti-PrP monoclonal antibodies for glycosylation profiling. Whereas native PrP^C and denatured PrP^Sc were retained by a Cu²⁺-loaded resin, native PrP^Sc and PrP^res [PK (proteinase K)-resistant PrP] passed through the column. We demonstrate here that the IMAC technique is appropriate to isolate and partially purify PrP^C from healthy brains in its native-like and biologically relevant glycosylated copper-binding forms. The IMAC technique is also well suited for the separation of native PrP^C from aggregated PrP^Sc in infected brains. Our results indicate that in contrast with PrP^Sc in uninfected as well as infected brains, PrP^C is predominantly present in the glycosylated forms.