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J. Biol. Chem., Vol. 281, Issue 46, 35245-35252, November 17, 2006

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 281/46/35245    most recent M603964200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Saá, P. Articles by Soto, C. Search for Related Content PubMed PubMed Citation Articles by Saá, P. Articles by Soto, C. Social Bookmarking                      What's this?

Ultra-efficient Replication of Infectious Prions by Automated Protein Misfolding Cyclic Amplification^*

Paula Saá, Joaquín Castilla, and Claudio Soto¹

From the George and Cynthia Mitchell Center for Alzheimer Disease and Related Neurodegenerative Disorders, Departments of Neurology, Neuroscience and Cell Biology, and Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, Texas 77555 and Centro de Biología Molecular, Universidad Autónoma de Madrid, Madrid 28049, Spain

Prions are the unconventional infectious agents responsible for transmissible spongiform encephalopathies, which appear to be composed mainly or exclusively of the misfolded prion protein (PrP^Sc). Prion replication involves the conversion of the normal prion protein (PrP^C) into the misfolded isoform, catalyzed by tiny quantities of PrP^Sc present in the infectious material. We have recently developed the protein misfolding cyclic amplification (PMCA) technology to sustain the autocatalytic replication of infectious prions in vitro. Here we show that PMCA enables the specific and reproducible amplification of exceptionally minute quantities of PrP^Sc. Indeed, after seven rounds of PMCA, we were able to generate large amounts of PrP^Sc starting from a 1 x 10^-12 dilution of scrapie hamster brain, which contains the equivalent of 26 molecules of protein monomers. According to recent data, this quantity is similar to the minimum number of molecules present in a single particle of infectious PrP^Sc, indicating that PMCA may enable detection of as little as one oligomeric PrP^Sc infectious particle. Interestingly, the in vitro generated PrP^Sc was infectious when injected in wild-type hamsters, producing a disease identical to the one generated by inoculation of the brain infectious material. The unprecedented amplification efficiency of PMCA leads to a several billion-fold increase of sensitivity for PrP^Sc detection as compared with standard tests used to screen prion-infected cattle and at least 4000 times more sensitivity than the animal bioassay. Therefore, PMCA offers great promise for the development of highly sensitive, specific, and early diagnosis of transmissible spongiform encephalopathy and to further understand the molecular basis of prion propagation.

Received for publication, April 25, 2006 , and in revised form, August 21, 2006.

^* This work was supported in part by National Institutes of Health Grants AG0224642 and NS049173. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Neurology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555. Tel.: 409-747-0017; Fax: 409-7470020; E-mail: clsoto{at}utmb.edu.

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