Preparation of lyophilized recombinant prion protein for TSE diagnosis by RT-QuIC

Soyoun Hwang,Eric M Nicholson,Semakaleng Lebepe-Mazur,Trudy Tatum

doi:10.1186/s13104-018-3982-5

Soyoun Hwang, Eric M Nicholson + Show 2 more

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https://doi.org/10.1186/s13104-018-3982-5

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Abstract

ObjectiveTransmissible spongiform encephalopathies (TSEs) are a group of fatal neurodegenerative diseases, often referred as prion diseases. TSEs result from the misfolding of the cellular prion protein (PrPC) into a pathogenic form (PrPSc) that accumulates in the brain and lymphatic tissue. Amplification based assays such as real-time quaking induced conversion allow us to assess the conversion of PrPC to PrPSc. Real-time quaking induced conversion (RT-QuIC) can be used for the detection of PrPSc in a variety of biological tissues from humans and animals. However, RT-QuIC requires a continuous supply of freshly purified prion protein and this necessity is not sustainable in a diagnostic laboratory setting.ResultsIn this study, we developed a method to dry and preserve the prion protein for long term storage allowing for production of the protein and storage for extended time prior to use and room temperature shipping to appropriate diagnostic laboratory destinations facilitating widespread use of RT-QuIC as a diagnostic method.

Highlights

Prion diseases are a group of fatal neurologic diseases that result from the misfolding of the monomeric, cellular prion protein (PrPC) into an oligomeric, pathogenic form (PrPSc)
These diseases are referred to as transmissible spongiform encephalopathies (TSEs) and include bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goat, chronic wasting disease (CWD) in deer and elk, and Creutzfeldt–Jakob disease (CJD), fatal familial insomnia, Gerstmann–Sträussler–Scheinker syndrome, and kuru in humans. pathogenic prion protein (PrPSc) accumulates in the central nervous system in all TSEs, and in cases of scrapie in sheep and CWD in cervids, P rPSc accumulates in the lymphoid tissues [1–3]
Secondary structure and thermal denaturation of Bank vole (BV) recombinant prion protein (rPrP) with and without lyophilization In order to investigate whether lyophilization influenced the folding or stability, BV rPrP was analyzed with regard to secondary structure and thermal denaturation

Summary

Results

Protein recovery after lyophilization and resolubilization In order to investigate protein loss due to the lyophilization and resolubilization, following purification and refolded BV rPrP in 10 mM potassium phosphate pH 7.0 was lyophilized. Based upon the absorbance at 280 nm and the extinction coefficient of 62005 M−1 cm−1 it was determined that protein recovery following lyophilization and resolubilization was greater than 95%. Thermal denaturation curves of lyophilized and freshly prepared BV protein were measured to establish any changes in the cooperative folding that resulted from the lyophilization and resolubilization. Lyophilized BV rPrP is suitable for RT‐QuIC based detection of PrPSc To evaluate whether lyophilized BV protein can be used as a substrate for RT-QuIC reactions, assays containing lyophilized or freshly prepared recombinant BV protein were seeded with dilutions of a 10% (w/v) brain homogenate from 2 different scrapie positive sheep (Fig. 3a, b, d, e) or a confirmed negative control (Fig. 3c, d). Neither substrate produced fibril when seeded with brain homogenate from a non-inoculated control based on the absence of an increase in ThT fluorescence (Fig. 3c, f )

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