Development of a new largely scalable in vitro prion propagation method for the production of infectious recombinant prions for high resolution structural studies

Abstract

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions.

Structural features of an infectious recombinant PrPScprion using solid state NMR

“…We used PMSA to generate unlabeled and uniformly labelled (U-13 C, 15 N)-BVPrP Sc (109I)23-231. PMSA relies on shaking a PrP substrate in the presence of defined co-factors, zircone beads and a seed of preformed PrP Sc (14). Our procedure includes a PK-treatment step at the end of the conversion process to ensure elimination of any non-converted PrP substrate.…”

“…Our procedure includes a PK-treatment step at the end of the conversion process to ensure elimination of any non-converted PrP substrate. This material has been described in a previous publication (14) and is highly infectious, with attack rates of 100% and titers of 6.34·10 4 /μg of PrP in TgVole (1x). We carried out a full biochemical characterization of this material, to complete the partial one described in our previous study (14).…”

“…This material has been described in a previous publication (14) and is highly infectious, with attack rates of 100% and titers of 6.34·10 4 /μg of PrP in TgVole (1x). We carried out a full biochemical characterization of this material, to complete the partial one described in our previous study (14). We first analyzed the pattern of PK-resistant fragments using SDS-PAGE.…”

“…We have recently developed a method, protein misfolding shaking amplification (PMSA), which circumvents this limitation as it allows facile production of milligram quantities of bona fide, infectious recombinant PrP Sc (recPrP Sc ). This in turn opens for the first time the possibility of applying ssNMR to the study of the structure of PrP Sc (14). Given the complexity of ssNMR spectra of amyloids, typically featuring broad, overlapping signals, as a consequence of structural heterogeneity and peak crowding, solving the structure of recPrP Sc will likely advance piecemeal and will require diverse isotopic labelling strategies.…”

Pharmacological Protein Inactivation by Targeting Folding Intermediates

“…-231 was expressed by competent E. coli Rosetta (DE3) bacteria harboring pOPIN E expression vector containing the wild type I109 bank vole Prnp gene95 . Bacteria from a glycerolate maintained at -80 °C were grown in a 250 ml Erlenmeyer flask containing 50 ml of LB broth overnight.…”