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J. Biol. Chem., Vol. 280, Issue 29, 26873-26879, July 22, 2005

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Protease-resistant Prion Protein Amplification Reconstituted with Partially Purified Substrates and Synthetic Polyanions^*

Nathan R. Deleault, James C. Geoghegan, Koren Nishina, Richard Kascsak, R. Anthony Williamson¶, and Surachai Supattapone||

From the Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755

Little is currently known about the biochemical mechanism by which induced prion protein (PrP) conformational change occurs during mammalian prion propagation. In this study, we describe the reconstitution of PrPres amplification in vitro using partially purified and synthetic components. Overnight incubation of purified PrP27–30 and PrP^C molecules at a molar ratio of 1:250 yielded 2-fold baseline PrPres amplification. Addition of various polyanionic molecules increased the level of PrPres amplification to 10-fold overall. Polyanionic compounds that stimulated purified PrPres amplification to varying degrees included synthetic, homopolymeric nucleic acids such as poly(A) and poly(dT), as well as non-nucleic acid polyanions, such as heparan sulfate proteoglycan. Size fractionation experiments showed that synthetic poly(A) polymers must be >0.2 kb in length to stimulate purified PrPres amplification. Thus, one possible set of minimal components for efficient conversion of PrP molecules in vitro may be surprisingly simple, consisting of PrP27–30, PrP^C, and a stimulatory polyanionic compound.

Received for publication, April 12, 2005 , and in revised form, May 16, 2005.

^* This work was supported by the Burroughs Wellcome Fund and by National Institutes of Health Grants AI058979 and NS046478. 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 Figs. S1–S3.

Both authors contributed equally to this work.

Current address: Institute for Basic Research, Staten Island, NY 10314.

^¶ Current address: The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.

^|| To whom correspondence should be addressed: Dept. of Biochemistry, 7200 Vail Bldg., Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650-1192; Fax: 603-650-1193; E-mail: supattapone{at}dartmouth.edu.

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