Abstract
BackgroundThe infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a conformational transition in animal and human prion diseases. Studies have shown that the interspecies conversion of PrPC to PrPSc is largely swayed by species barriers, which is mainly deciphered by the sequence and conformation of the proteins among species. However, the bank vole PrPC (BVPrP) is highly susceptible to PrPSc from different species. Transgenic mice expressing BVPrP with the polymorphic isoleucine (109I) but methionine (109M) at residue 109 spontaneously develop prion disease.ResultsTo explore the mechanism underlying the unique susceptibility and convertibility, we generated soluble BVPrP by co-expression of BVPrP with Quiescin sulfhydryl oxidase (QSOX) in Escherichia coli. Interestingly, rBVPrP-109M and rBVPrP-109I exhibited distinct seeded aggregation pathways and aggregate morphologies upon seeding of mouse recombinant PrP fibrils, as monitored by thioflavin T fluorescence and electron microscopy. Moreover, they displayed different aggregation behaviors induced by seeding of hamster and mouse prion strains under real-time quaking-induced conversion.ConclusionsOur results suggest that QSOX facilitates the formation of soluble prion protein and provide further evidence that the polymorphism at residue 109 of QSOX-induced BVPrP may be a determinant in mediating its distinct convertibility and susceptibility.
Highlights
The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a confor‐ mational transition in animal and human prion diseases
Proper intramolecular folding can only be determined through additional characterization of the protein using circular dichroism (CD) or thioflavin T (ThT) fluorescence assays to validate the quality of the product [2]
We have previously demonstrated the ability of Quiescin sulfhydryl oxidase (QSOX) to introduce a disulfide bond to the human and mouse prion proteins and to facilitate the expression of soluble PrP in E. coli [13]
Summary
The infectious prion protein (PrPSc or prion) is derived from its cellular form (PrPC) through a confor‐ mational transition in animal and human prion diseases. To fully understand the key molecular event in the pathogenesis of prion diseases, the structural conversion of PrP, generation of a soluble monomeric recombinant PrPC in Escherichia coli that can be used for monitoring conformational conversion in vitro has been one of the important steps. Expression of recombinant prion proteins (rPrP) in the cytoplasm of E. coli often forms inactive aggregates (termed inclusion bodies) [10]. These inclusion bodies must be solubilized in harsh reducing and denaturing conditions and subsequently refolded in mild oxidizing conditions in order to restore the normal intramolecular disulfide bridge [11]. Proper intramolecular folding can only be determined through additional characterization of the protein using circular dichroism (CD) or thioflavin T (ThT) fluorescence assays to validate the quality of the product [2]
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