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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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Beads improve the amplification efficiency of SSLOW.SSLOW scrapie brain material was diluted 103-fold (lanes 1–8) or 104-fold (lanes 9–15) into 10% NBH, subjected to serial PMCA in the absence or presence of 3 small beads, as indicated, and digested with PK. Each PMCA round consisted of 48 cycles; the material amplified in each round was diluted 10-fold into 10% NBH for the next PMCA round. Undigested 10% NBH (lane 1) loaded at 1/10th the amount of the digested samples is provided as a reference.
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ppat-1001277-g005: Beads improve the amplification efficiency of SSLOW.SSLOW scrapie brain material was diluted 103-fold (lanes 1–8) or 104-fold (lanes 9–15) into 10% NBH, subjected to serial PMCA in the absence or presence of 3 small beads, as indicated, and digested with PK. Each PMCA round consisted of 48 cycles; the material amplified in each round was diluted 10-fold into 10% NBH for the next PMCA round. Undigested 10% NBH (lane 1) loaded at 1/10th the amount of the digested samples is provided as a reference.

Mentions: To test whether the positive effect of beads on prion amplification was limited to 263K, we used a synthetic prion strain, SSLOW, which was previously found to have a very peculiar amplification behavior in PMCA [25]. Previously we found that amplification efficiency of SSLOW varied significantly from preparation to preparation of NBH and that it had a much more unstable amplification behavior than 263K. For instance, SSLOW failed to amplify even in those preparations of NBHs, where 263K showed high amplification rates. In such preparations of NBHs, the amplification fold for SSLOW was found to be lower than the 10-fold dilution factor used for serial PMCA. Therefore, in the absence of beads, SSLOW PrPSc was no longer detectable by Western blotting after the first round of PMCA (Fig. 5, lanes 3–5). In the presence of beads, however, the amount of SSLOW PrPSc remained stable during serial PMCAb if the reactions were seeded with 103-fold diluted SSLOW brain homogenates (Fig. 5, lanes 6–8), or increased if 104-fold dilutions were used for seeding (Fig. 5, lanes 13-15). These results illustrate that the positive effect of beads is not limited to 263K and that beads improved the robustness of PMCA for a strain with poor amplification behavior.


Highly efficient protein misfolding cyclic amplification.

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

Beads improve the amplification efficiency of SSLOW.SSLOW scrapie brain material was diluted 103-fold (lanes 1–8) or 104-fold (lanes 9–15) into 10% NBH, subjected to serial PMCA in the absence or presence of 3 small beads, as indicated, and digested with PK. Each PMCA round consisted of 48 cycles; the material amplified in each round was diluted 10-fold into 10% NBH for the next PMCA round. Undigested 10% NBH (lane 1) loaded at 1/10th the amount of the digested samples is provided as a reference.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1001277-g005: Beads improve the amplification efficiency of SSLOW.SSLOW scrapie brain material was diluted 103-fold (lanes 1–8) or 104-fold (lanes 9–15) into 10% NBH, subjected to serial PMCA in the absence or presence of 3 small beads, as indicated, and digested with PK. Each PMCA round consisted of 48 cycles; the material amplified in each round was diluted 10-fold into 10% NBH for the next PMCA round. Undigested 10% NBH (lane 1) loaded at 1/10th the amount of the digested samples is provided as a reference.
Mentions: To test whether the positive effect of beads on prion amplification was limited to 263K, we used a synthetic prion strain, SSLOW, which was previously found to have a very peculiar amplification behavior in PMCA [25]. Previously we found that amplification efficiency of SSLOW varied significantly from preparation to preparation of NBH and that it had a much more unstable amplification behavior than 263K. For instance, SSLOW failed to amplify even in those preparations of NBHs, where 263K showed high amplification rates. In such preparations of NBHs, the amplification fold for SSLOW was found to be lower than the 10-fold dilution factor used for serial PMCA. Therefore, in the absence of beads, SSLOW PrPSc was no longer detectable by Western blotting after the first round of PMCA (Fig. 5, lanes 3–5). In the presence of beads, however, the amount of SSLOW PrPSc remained stable during serial PMCAb if the reactions were seeded with 103-fold diluted SSLOW brain homogenates (Fig. 5, lanes 6–8), or increased if 104-fold dilutions were used for seeding (Fig. 5, lanes 13-15). These results illustrate that the positive effect of beads is not limited to 263K and that beads improved the robustness of PMCA for a strain with poor amplification behavior.

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