Abstract
Expression of eukaryotic proteins in Escherichia coli is challenging, especially when they contain disulfide bonds. Since the discovery of the prion protein (PrP) and its role in transmissible spongiform encephalopathies, the need to obtain large quantities of the recombinant protein for research purposes has been essential. Currently, production of recombinant PrP is achieved by refolding protocols. Here, we show that the co-expression of two different PrP with the human Quiescin Sulfhydryl OXidase (QSOX), a human chaperone with thiol/disulfide oxidase activity, in the cytoplasm of E. coli produces soluble recombinant PrP. The structural integrity of the soluble PrP has been confirmed by nuclear magnetic resonance spectroscopy, demonstrating that properly folded PrP can be easily expressed in bacteria. Furthermore, the soluble recombinant PrP produced with this method can be used for functional and structural studies.
Highlights
Prion diseases, referred as transmissible spongiform encephalopathies (TSEs), are a family of rare progressive neurodegenerative disorders that affect both humans and animals [1]
We examined protein expression and solubility of MoPrP(89230) in the presence and absence of Quiescin Sulfhydryl OXidase (QSOX) at various time points after induction in standard LB broth. Cells expressing both proteins showed a reduced growth rate compared to the one expressing only MoPrP (89-230) (Figure 2A), which is probably an effect of the metabolic burden imposed by the double recombinant proteins over-expression [24]
In cells without QSOX, the total concentration of prion protein (PrP) decreased compared to cells with QSOX, where the PrP concentration remained constant over time (Figure 2B, C, D)
Summary
Referred as transmissible spongiform encephalopathies (TSEs), are a family of rare progressive neurodegenerative disorders that affect both humans and animals [1]. TSEs include, for instance, bovine spongiform encephalopathy (BSE) in cattle, and Creutzfeldt-Jakob disease (CJD) in humans. These disorders are characterized by long incubation periods and characteristic spongiform changes in the brain associated with neuronal loss. The causative agent of TSEs is an infectious protein known as prion ( denoted as PrPSc) [2]. This pathogenic betasheet-rich conformer derives from the normal, mostly alpha-helical isoform, cellular prion protein (PrP or PrPC), through a conformational conversion event which leads to aggregates in the brains of affected individuals leading to neurodegeneration [3].
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