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 several modified and wild-type prion protein (PrP) polypeptides, of up to 112 residues, that demonstrate the flexibility of a chemical approach to protein synthesis. The principal event in prion disease is the conformational change of the normal, alpha-helical cellular protein (PrPc) into a beta-sheet-rich pathogenic isoform (PrP(Sc)). The ability to form PrP(Sc) in transgenic mice is retained by a 106 residue 'mini-prion' (PrP106), with the deletions 23-88 and 141-176. Synthetic PrP106 (sPrP106) and a His-tagged analog (sPrP106HT) have been prepared successfully using a highly optimized Fmoc chemical methodology involving DCC/HOBt activation and an efficient capping procedure with N-(2-chlorobenzyloxycarbonyloxy) succinimide. A single reversed-phase purification step gave homogeneous protein, in excellent yield. With respect to its conformational and aggregational properties and its response to proteinase digestion, sPrP106 was indistinguishable from its recombinant 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 inositol (GPI) anchor and target sPrP to cholesterol-rich domains on the cell surface, 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 immobilized on either the cell surface or a streptavidin-coated ELISA plate, respectively, 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-translational modifications should enhance our understanding of the processes that cause prion diseases and may lead to the chemical synthesis of an infectious agent.
Ball
H L
HL
Institute for Neurodegenerative Diseases and Department of Neurology, University of California at San Francisco, 94143-0518, USA. haydnb@itsa.ucsf.edu
King
D S
DS
Cohen
F E
FE
Prusiner
S B
SB
Baldwin
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin / Q000737:chemistry; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D019286:Endopeptidase K / Q000494:pharmacology; D004952:Esters / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011328:Prions / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D014407:Tumor Cells, Cultured
D004952:Esters; D017261:Glycosylphosphatidylinositols; D010455:Peptides; D011328:Prions; D011994:Recombinant Proteins; D001710:Biotin; D019286:Endopeptidase K