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Altered rPrP substrate structures and their influence on real-time quaking induced conversion reactions
Keding Cheng1, Angela Sloan2, Brooks Waitt2
1National 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.
Protein Expression and Purification
|October 17, 2017
Summary
Different recombinant prion protein (rPrP) production methods impact real-time quaking-induced conversion (RT-QuIC) assay performance. The chimeric sheep-hamster rPrP refolded with shorter guanidine-HCl exposure and faster elution showed superior results.
Area of Science:
- Neuroscience
- Biochemistry
- Molecular Biology
Background:
- Bacterially-produced recombinant prion protein (rPrP) is crucial for prion disease research, including in vitro fibrillation assays and reagent development.
- Recombinant prion protein (rPrP) serves as a substrate in real-time quaking-induced conversion (RT-QuIC), a sensitive diagnostic assay for prion diseases.
- The lack of a commercial supplier necessitates independent rPrP production, leading to varied laboratory protocols and no standardized method.
Purpose of the Study:
- To investigate the impact of two distinct rPrP refolding protocols on rPrP production.
- To evaluate the QuIC performance and structural characteristics of two common rPrP constructs (full-length hamster and sheep-hamster chimeric).
Main Methods:
- Two different recombinant prion protein (rPrP) refolding protocols were applied.
- The study utilized full-length hamster and sheep-hamster chimeric rPrP constructs.
- Assessed production yield, QuIC assay performance, and structural properties of the refolded rPrP.
Main Results:
- The chimeric sheep-hamster rPrP, produced with shorter guanidine-HCl exposure and faster gradient elution, demonstrated the best performance as an RT-QuIC substrate.
- Variations in rPrP production protocols significantly influenced the efficacy of the QuIC assay.
Conclusions:
- The method of producing recombinant prion protein (rPrP) directly affects its performance in real-time quaking-induced conversion (RT-QuIC) assays.
- Researchers must exercise caution when comparing inter-laboratory RT-QuIC results due to differing rPrP production protocols.