Recombinant scrapie-like prion protein of 106 amino acids is soluble.
The N terminus of the scrapie isoform of prion protein (PrPSc) can be truncated without loss of scrapie infectivity and, correspondingly, the truncation of the N terminus of the cellular isoform, PrPC, still permits conversion into PrPSc. To assess whether additional segments of the PrP molecule can be deleted, we previously removed regions of putative secondary structure in PrPC; in the present study we found that deletion of each of the four predicted helices prevented PrPSc formation, as did deletion of the stop transfer effector region and the C178A mutation. Removal of a 36-residue loop between helices 2 and 3 did not prevent formation of protease-resistant PrP; the resulting scrapie-like protein, designated PrPSc106, contained 106 residues after cleavage of an N-terminal signal peptide and a C-terminal sequence for glycolipid anchor addition. Addition of the detergent Sarkosyl to cell lysates solubilized PrPSc106, which retained resistance to digestion by proteinase K. These results suggest that all the regions of proposed secondary structure in PrP are required for PrPSc formation, as is the disulfide bond stabilizing helices 3 and 4. The discovery of PrPSc106 should facilitate structural studies of PrPSc, investigations of the mechanism of PrPSc formation, and the production of PrPSc-specific antibodies.
Muramoto
T
T
Department of Neurology, University of California, San Francisco 94143, USA.
Scott
M
M
Cohen
F E
FE
Prusiner
S B
SB
D001483:Base Sequence; D003001:Cloning, Molecular; D016296:Mutagenesis; D009838:Oligodeoxyribonucleotides; D016366:Open Reading Frames; D018620:PrPSc Proteins / Q000096:biosynthesis / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012521:Sarcosine / Q000031:analogs & derivatives; D017384:Sequence Deletion; D012995:Solubility
D009838:Oligodeoxyribonucleotides; D018620:PrPSc Proteins; D011994:Recombinant Proteins; C025231:sarkosyl; D012521:Sarcosine