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Highly efficient protein misfolding cyclic amplification.

Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenk R, Alexeeva I, Rohwer RG, Baskakov IV - PLoS Pathog. (2011)

Bottom Line: 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.The increase in the amplification efficiency did not come at the expense of prion replication specificity.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America.

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.

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Related in: MedlinePlus

AFM imaging of rPrP fibril fragmentation.AFM imaging of intact rPrP fibrils (A) and fibrils sonicated for 30 sec in the absence (B) or presence of 5 small beads (C) using sonication conditions identical to those used in PMCA. Scale bars  = 0.5 µm. (D) Analysis of length, width and height for intact rPrP fibrils (green circles) and fibrils sonicated in the absence (orange circles) or presence of 5 small beads (red circles).
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ppat-1001277-g009: AFM imaging of rPrP fibril fragmentation.AFM imaging of intact rPrP fibrils (A) and fibrils sonicated for 30 sec in the absence (B) or presence of 5 small beads (C) using sonication conditions identical to those used in PMCA. Scale bars  = 0.5 µm. (D) Analysis of length, width and height for intact rPrP fibrils (green circles) and fibrils sonicated in the absence (orange circles) or presence of 5 small beads (red circles).

Mentions: To gain insight into the effect of beads on prion amplification, we tested whether beads affect the fragmentation efficiency of PrP aggregates during sonication. Amyloid fibrils produced from rPrP were sonicated in the presence or absence of beads, and the size of fibrillar fragments was analyzed using atomic force microscopy (AFM) imaging. Consistent with our previous studies [26], sonication was found to break fibrils into smaller fragments (Fig. 9A,B). Sonication in the presence of beads, however, reduced the size of fibrillar fragments even more producing smaller particles (Fig. 9C,D). In fact, AFM imaging revealed that after sonication with beads, the fibrillar fragments appeared as small oligomers.


Highly efficient protein misfolding cyclic amplification.

Gonzalez-Montalban N, Makarava N, Ostapchenko VG, Savtchenk R, Alexeeva I, Rohwer RG, Baskakov IV - PLoS Pathog. (2011)

AFM imaging of rPrP fibril fragmentation.AFM imaging of intact rPrP fibrils (A) and fibrils sonicated for 30 sec in the absence (B) or presence of 5 small beads (C) using sonication conditions identical to those used in PMCA. Scale bars  = 0.5 µm. (D) Analysis of length, width and height for intact rPrP fibrils (green circles) and fibrils sonicated in the absence (orange circles) or presence of 5 small beads (red circles).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3037363&req=5

ppat-1001277-g009: AFM imaging of rPrP fibril fragmentation.AFM imaging of intact rPrP fibrils (A) and fibrils sonicated for 30 sec in the absence (B) or presence of 5 small beads (C) using sonication conditions identical to those used in PMCA. Scale bars  = 0.5 µm. (D) Analysis of length, width and height for intact rPrP fibrils (green circles) and fibrils sonicated in the absence (orange circles) or presence of 5 small beads (red circles).
Mentions: To gain insight into the effect of beads on prion amplification, we tested whether beads affect the fragmentation efficiency of PrP aggregates during sonication. Amyloid fibrils produced from rPrP were sonicated in the presence or absence of beads, and the size of fibrillar fragments was analyzed using atomic force microscopy (AFM) imaging. Consistent with our previous studies [26], sonication was found to break fibrils into smaller fragments (Fig. 9A,B). Sonication in the presence of beads, however, reduced the size of fibrillar fragments even more producing smaller particles (Fig. 9C,D). In fact, AFM imaging revealed that after sonication with beads, the fibrillar fragments appeared as small oligomers.

Bottom Line: 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.The increase in the amplification efficiency did not come at the expense of prion replication specificity.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Biomedical Engineering and Technology, University of Maryland, Baltimore, Maryland, United States of America.

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.

Show MeSH
Related in: MedlinePlus