Prion diseases are propagated by the conversion of the native form of the prion protein (PrPC) to an alternatively folded, disease causing form (PrPSc) catalyzed by the interaction of PrPSc with PrPC?. Therapeutic antibodies for prion diseases have focused on shielding PrPSc-PrPC interaction by directly binding to PrPC. We have developed a method for characterizing antibody-antigen interactions utilizing yeast surface display and next generation DNA sequencing to provide residue level epitope identification. A large random mutant prion protein library tethered to the yeast surface was incubated with soluble, fluorescently labeled antibodies and mutants with reduced antibody-antigen interaction were isolated by florescence-activated cell sorting (FACS). The isolated pool was sequenced using single molecule real time (SMRT) genetic sequencing to identify corresponding protein mutations responsible for ablated binding. Using this method we have identified residue specific epitopes for several common anti-prion antibodies providing additional insight into their molecular contacts.

Fine epitope determination by yeast surface display and next-generation sequencing

245th National Meeting (2013)

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