Abstract
Mammalian prions exist as multiple strains which produce characteristic and highly reproducible phenotypes in defined hosts. How this strain diversity is encoded by a protein-only agent remains one of the most interesting and challenging questions in biology with wide relevance to understanding other diseases involving the aggregation or polymerisation of misfolded host proteins. Progress in understanding mammalian prion strains has however been severely limited by the complexity and variability of the methods used for their isolation from infected tissue and no high resolution structures have yet been reported. Using high-throughput cell-based prion bioassay to re-examine prion purification from first principles we now report the isolation of prion strains to exceptional levels of purity from small quantities of infected brain and demonstrate faithful retention of biological and biochemical strain properties. The method’s effectiveness and simplicity should facilitate its wide application and expedite structural studies of prions.
Highlights
We show that the method works well with different prion strains from different hosts and yields extremely high-titre infectious prion preparations that contain full-length PrP of exceptional purity
proteinase K (PK) has been widely used in prion purification[15,16], not all prion strains show the same degree of PK-resistance and protease-sensitive prions are well documented[6,20,22,23]
Whilst complex separations on density gradients using ultra-centrifugation increase the purity of rodent prions[15,16,25,26], we found that once myelin-associated oligodendrocyte basic protein (MOBP), collagen fibres and intermediate filaments had been co-sedimented with RML prions they could not be differentially extracted or degraded without destroying a significant proportion of the infectious prion titre
Summary
We report the use of cell culture based prion bioassay to develop a rapid and simple protocol for isolating highly purified prions from mammalian brain. We show that the method works well with different prion strains from different hosts and yields extremely high-titre infectious prion preparations that contain full-length PrP of exceptional purity. As these methods utilise reagents that are readily commercially available and do not require specialised equipment they should be practicable in most existing prion laboratories
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