Engineering the prion protein using chemical synthesis

In recent years, the technology of solid-phase peptide synthesis (SPPS) has improved to the extent that chemical synthesis of small proteins may be a viable complementary strategy to recombinant expression. We have prepared s everal modified and wild-type prion protein (PrP) polypeptides, of up to 11 2 residues, that demonstrate the flexibility of a chemical approach to prot ein synthesis. The principal event in prion disease is the conformational c hange of the normal, alpha -helical cellular protein (PrPC) into a beta -sh eet-rich pathogenic isoform (PrPSc). The ability to form PrPSc in transgeni c mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletio ns 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (s PrP106HT) have been prepared successfully using a highly optimized Fmoc che mical methodology involving DCC/HOBt activation and an efficient capping pr ocedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reverse d-phase purification step gave homogeneous protein, in excellent yield. Wit h respect to its conformational and aggregational properties and its respon se to proteinase digestion, sPrP106 was indistinguishable from its recombin ant analog (rPrP106). Certain sequences that proved to be more difficult to synthesize using the Fmoc approach, such as bovine (Bo) PrP(90-200), were successfully prepared using a combination of the highly activated coupling reagent HATU and t-Boc chemistry. To mimic the glycosylphosphatidyl inosito l (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surf ace, where the conversion of PrPC is believed to occur, a lipophilic group or biotin, was added to an orthogonally side-chain-protected Lys residue at the C-terminus of sPrP sequences. These groups enabled sPrP to be immobili zed on either the cell surface or a streptavidin-coated ELISA plate, respec tively, in an orientation analogous to that of membrane-bound, GPI-anchored PrPC. The chemical manipulation of such biologically relevant forms of PrP by the introduction of point mutations or groups that mimic post-translati onal modifications should enhance our understanding of the processes that c ause prion diseases and may lead to the chemical synthesis of an infectious agent.