title: Highly efficient protein misfolding cyclic amplification.
abstract: Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrP(C) into PrP(Sc) in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrP(C) may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrP(C) into PrP(Sc) from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrP(Sc) by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 10¹²-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrP(C) susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrP(Sc)in vitro.
authors.affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America.
authors.forename: Nuria
authors.initials: N
authors.lastname: Gonzalez-Montalban
authors.forename: Natallia
authors.initials: N
authors.lastname: Makarava
authors.forename: Valeriy G
authors.initials: VG
authors.lastname: Ostapchenko
authors.forename: Regina
authors.initials: R
authors.lastname: Savtchenk
authors.forename: Irina
authors.initials: I
authors.lastname: Alexeeva
authors.forename: Robert G
authors.initials: RG
authors.lastname: Rohwer
authors.forename: Ilia V
authors.initials: IV
authors.lastname: Baskakov
mesh_terms: D000682:Amyloid / Q000138:chemical synthesis / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism / Q000473:pathology; D006224:Cricetinae; D004526:Efficiency; D008647:Mesocricetus; D051379:Mice; D008863:Microspheres; D018620:PrPSc Proteins / Q000138:chemical synthesis* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012608:Scrapie / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D013045:Species Specificity
chemical_list: D000682:Amyloid; D018620:PrPSc Proteins; D011994:Recombinant Proteins