title: Affinity-tagged miniprion derivatives spontaneously adopt protease-resistant conformations.
abstract: An abridged PrP molecule of 106 amino acids designated PrP106 can form infectious miniprions in transgenic (Tg) mice (29). Addition of six-histidine (His(6)) affinity tags to selective sites within PrP106 resulted unexpectedly in new PrP proteins that spontaneously adopted protease-resistant conformations when expressed in neuroblastoma cells and Tg mice. Acquisition of protease resistance depended on the length, charge, and placement of the affinity tag. Introduction of the disease-linked mutation E200K into the sequence of PrP106(140/6His) increased the recovery of protease-resistant PrP fivefold, whereas introduction of the mutations C213A and Delta214-220 did not affect the recovery of protease-resistant PrP. Treatment of cultured cells expressing affinity-tagged PrP106 mutants with polypropyleneimine dendrimer rendered these proteins sensitive to protease digestion in a manner similar to wild-type PrP(Sc). We conclude that certain affinity-tagged PrP106 proteins spontaneously fold into conformations partially resembling, yet distinct from, wild-type PrP(Sc). These proteins might be useful tools in the identification of new disease-causing mutations as well as for screening compounds for therapeutic efficacy.
authors.affiliation: Institute for Neurodegenerative Diseases, University of California at San Francisco, San Francisco, California 94143, USA.
authors.forename: S
authors.initials: S
authors.lastname: Supattapone
authors.forename: H O
authors.initials: HO
authors.lastname: Nguyen
authors.forename: T
authors.initials: T
authors.lastname: Muramoto
authors.forename: F E
authors.initials: FE
authors.lastname: Cohen
authors.forename: S J
authors.initials: SJ
authors.lastname: DeArmond
authors.forename: S B
authors.initials: SB
authors.lastname: Prusiner
authors.forename: M
authors.initials: M
authors.lastname: Scott
mesh_terms: D000818:Animals; D010450:Endopeptidases / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D011328:Prions / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013329:Structure-Activity Relationship
chemical_list: D011328:Prions; D010450:Endopeptidases