Keding Cheng1, Angela Sloan2, Brooks Waitt2, Robert Vendramelli2, Debra Godal2, Sharon L R Simon2, Joe O'Neil3, Michael Carpenter4, Dave Jackson5, Jane Eastlake5, Gary Mallinson5, J David Knox6. 1. National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada; Department of Human Anatomy and Cell Sciences, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada. Electronic address: Keding.Cheng@phac-aspc.gc.ca. 2. National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada. 3. Department of Chemistry, University of Manitoba, Winnipeg, Manitoba, Canada. 4. National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada; Department of Medical Microbiology, Faculty of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada. 5. Bristol Institute for Transfusion Sciences (BITS), National Blood Service, Bristol, UK. 6. 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: David.Knox@phac-aspc.gc.ca.
Abstract
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 (PrPd). 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. Crown
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 (PrPd). 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-hamsterrPrP 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. Crown