Highly Efficient Protein Misfolding Cyclic Amplification

被引:80
|
作者
Gonzalez-Montalban, Nuria [1 ]
Makarava, Natallia [1 ]
Ostapchenko, Valeriy G. [1 ]
Savtchenk, Regina [1 ]
Alexeeva, Irina [2 ]
Rohwer, Robert G. [2 ,3 ]
Baskakov, Ilia V. [1 ,4 ]
机构
[1] Univ Maryland, Ctr Biomed Engn & Technol, Baltimore, MD 21201 USA
[2] Vet Affairs Maryland Hlth Care Syst, Med Res Serv, Baltimore, MD USA
[3] Univ Maryland, Sch Med, Dept Neurol, Baltimore, MD 21201 USA
[4] Univ Maryland, Sch Med, Dept Anat & Neurobiol, Baltimore, MD 21201 USA
关键词
RECOMBINANT PRION PROTEIN; IN-VITRO; INFECTIOUS PRIONS; PHYSICAL-PROPERTIES; BLOOD; PRPSC; CONVERSION; REPLICATION; DISEASE;
D O I
10.1371/journal.ppat.1001277
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 similar to 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 10(12)-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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页数:10
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