Background: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay. No harmonized method for producing the protein has been agreed upon, in part due to the variety of substrates that have been applied in RT-QuIC.
Methods: This study examines the effects of two different rPrP refolding protocols on the production, QuIC performance, and structure characteristics of two constructs of rPrP commonly used in QuIC: full length hamster and a sheep-hamster chimeric rPrP.
Results: Under the described conditions, the best performing substrate was the chimeric sheep-hamster rPrP produced by shorter guanidine-HCl exposure and faster gradient elution.
Conclusions: The observation that different rPrP production protocols influence QuIC performance indicates that caution should be exercised when comparing inter-laboratory QuIC results.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1016/j.pep.2017.10.007 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Recombinant ovine prion protein can be mutated at position 136 to improve its efficacy as an inhibitor of prion propagation.
Sci Rep
March 2023
School of Veterinary Medicine and Science, The University of Nottingham, College Rd., Sutton Bonington, Loughborough, LE12 5RD, Leicestershire, UK.
Prion diseases are progressive neurodegenerative disorders with no effective therapeutics. The central event leading to the pathology in the diseases is the conversion of PrP into PrP and its accumulation in the central nervous system. Previous studies demonstrated that recombinant PrP (rPrP) and PrP peptides can inhibit the formation of PrP.
View Article and Find Full Text PDFBank vole prion protein extends the use of RT-QuIC assays to detect prions in a range of inherited prion diseases.
Sci Rep
March 2021
MRC Prion Unit at UCL, Institute of Prion Diseases, Courtauld Building, 33 Cleveland Street, London, W1W 7FF, UK.
The cerebrospinal fluid (CSF) real-time quaking-induced conversion assay (RT-QuIC) is an ultrasensitive prion amyloid seeding assay for diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) but several prion strains remain unexplored or resistant to conversion with commonly used recombinant prion protein (rPrP) substrates. Here, bank vole (BV) rPrP was used to study seeding by a wide range of archived post-mortem human CSF samples from cases of sporadic, acquired and various inherited prion diseases in high throughput 384-well format. BV rPrP substrate yielded positive reactions in 70/79 cases of sporadic CJD [Sensitivity 88.
View Article and Find Full Text PDFPreserving prion strain identity upon replication of prions in vitro using recombinant prion protein.
Acta Neuropathol Commun
September 2018
Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, 111 S. Penn St, Baltimore, MD, 21201, USA.
Last decade witnessed an enormous progress in generating authentic infectious prions or PrP in vitro using recombinant prion protein (rPrP). Previous work established that rPrP that lacks posttranslational modification is able to support replication of highly infectious PrP with assistance of cofactors of polyanionic nature and/or lipids. Unexpectedly, previous studies also revealed that seeding of rPrP by brain-derived PrP gave rise to new prion strains with new disease phenotypes documenting loss of a strain identity upon replication in rPrP substrate.
View Article and Find Full Text PDFA Promising Antiprion Trimethoxychalcone Binds to the Globular Domain of the Cellular Prion Protein and Changes Its Cellular Location.
Antimicrob Agents Chemother
February 2018
Faculty of Pharmacy, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
The search for antiprion compounds has been encouraged by the fact that transmissible spongiform encephalopathies (TSEs) share molecular mechanisms with more prevalent neurodegenerative pathologies, such as Parkinson's and Alzheimer's diseases. Cellular prion protein (PrP) conversion into protease-resistant forms (protease-resistant PrP [PrP] or the scrapie form of PrP [PrP]) is a critical step in the development of TSEs and is thus one of the main targets in the screening for antiprion compounds. In this work, three trimethoxychalcones (compounds J1, J8, and J20) and one oxadiazole (compound Y17), previously identified to be potential antiprion compounds, were evaluated through different approaches in order to gain inferences about their mechanisms of action.
View Article and Find Full Text PDFAltered rPrP substrate structures and their influence on real-time quaking induced conversion reactions.
Protein Expr Purif
March 2018
National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada; Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address:
Background: Bacterially-produced recombinant prion protein (rPrP) has traditionally been used for in vitro fibrillation assays and reagent development for prion disease research. In recent years, it has also been used as a substrate for real-time quaking-induced conversion (RT-QuIC), a very sensitive method of detecting the presence of the misfolded, disease-associated isoform of the prion protein (PrP). Multi-centre trials have demonstrated that RT-QuIC is a suitably reliable and robust technique for clinical practice; however, in the absence of a commercial supplier of rPrP as a substrate for RT-QuIC, laboratories have been required to independently generate this key component of the assay.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!