A systematic investigation of production of synthetic prions from recombinant prion protein

Christian Schmidt,Sebastian Brandner,John Collinge,Parmjit Jat,Steffi Klier,Anjna Badhan,Francesca Properzi,Louise Afran,Jonathan D F Wadsworth,Sammy Ho,Laszlo L P Hosszu,Mark Batchelor,Jacqueline M Linehan,Graham S Jackson,M Howard Tattum,Anthony R Clarke,Peter-Christian Klöhn ,Malin Sandberg ,Jérémie Fizet ,Julie Ann Edgeworth

doi:10.1098/rsob.150165

Abstract

According to the protein-only hypothesis, infectious mammalian prions, which exist as distinct strains with discrete biological properties, consist of multichain assemblies of misfolded cellular prion protein (PrP). A critical test would be to produce prion strains synthetically from defined components. Crucially, high-titre ‘synthetic' prions could then be used to determine the structural basis of infectivity and strain diversity at the atomic level. While there have been multiple reports of production of prions from bacterially expressed recombinant PrP using various methods, systematic production of high-titre material in a form suitable for structural analysis remains a key goal. Here, we report a novel high-throughput strategy for exploring a matrix of conditions, additives and potential cofactors that might generate high-titre prions from recombinant mouse PrP, with screening for infectivity using a sensitive automated cell-based bioassay. Overall, approximately 20 000 unique conditions were examined. While some resulted in apparently infected cell cultures, this was transient and not reproducible. We also adapted published methods that reported production of synthetic prions from recombinant hamster PrP, but again did not find evidence of significant infectious titre when using recombinant mouse PrP as substrate. Collectively, our findings are consistent with the formation of prion infectivity from recombinant mouse PrP being a rare stochastic event and we conclude that systematic generation of prions from recombinant PrP may only become possible once the detailed structure of authentic ex vivo prions is solved.

Highlights

Prions are infectious agents responsible for the transmissible spongiform encephalopathies or prion diseases, lethal neurodegenerative conditions including Creutzfeldt–Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk [1,2]
RecPrP folded into a native alpha-helical conformation (a-prion protein (PrP)) of a beta-sheet-enriched conformation (b-PrP) [5] was exposed to differing solvent conditions varying with respect to pH, redox potential and ionic strength
We explored the effects of reaction surfaces, notably stainless steel, given previous findings that prions can be efficiently concentrated on steel wires, allowing greatly enhanced prion rsob.royalsocietypublishing.org Open Biol. 5: 150165

Summary

Introduction

Prions are infectious agents responsible for the transmissible spongiform encephalopathies or prion diseases, lethal neurodegenerative conditions including Creutzfeldt–Jakob disease in humans, scrapie in sheep and goats, bovine spongiform encephalopathy in cattle, and chronic wasting disease in deer and elk [1,2]. Prions are thought to consist of fibrillar polymers of misfolded cellular prion protein (PrPC) that propagate by recruitment of host PrPC leading to elongation and fission [3]. It is increasingly thought that prion-like processes, with the spread of propagating proteopathic seeds, underlie the pathogenesis of more common neurodegenerative diseases, such as Alzheimer’s and Parkinson’s, leading to a wider interest in understanding prion structure and strain diversity [4]. It has proved difficult to adequately purify ex vivo prions and obtain sufficiently homogeneous material for high-resolution structural analysis; the structure of infectious prions and the structural basis of prion strain diversity remain unresolved.

Methods

Results

Conclusion