RESUMO
Recombinant (rec) prion protein (PrP) is an extremely useful resource for studying protein misfolding and subsequent protein aggregation events. Here, we report mass production of high-purity rec-polypeptide encoding the C-terminal globular domain of PrP; (90-230) for human and (89-231) for murine PrP. These proteins were expressed as His-tagged fusion proteins in E. coli cultured by a high cell-density aerobic fermentation method. RecPrPs recovered from inclusion bodies were slowly refolded under reducing conditions. Purification was performed by a sequence of metal-affinity, cation-exchange, and reverse-phase chromatography. The current procedure yielded several dozens of milligrams of recPrP per liter with ï¼95% purity. The purified recPrPs predominantly adopted an α-helix-rich conformation and were functionally sufficient as substrates to measure the seeding activity of human and animal prions. Establishment of a procedure for high-level production of high-purity recPrP supports the advancement of in vitro investigations of PrP including diagnosis for prion diseases.
Assuntos
Proteínas Priônicas/biossíntese , Proteínas Priônicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Animais , Cromatografia Líquida , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Humanos , Corpos de Inclusão , Camundongos , Proteínas Priônicas/química , Proteínas Priônicas/isolamento & purificação , Dobramento de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Prions composed of pathogenic scrapie prion protein (PrPSc) are infectious pathogens that cause progressive neurological conditions known as prion diseases or transmissible spongiform encephalopathies. Although these diseases pose considerable risk to public health, procedures for early diagnosis have not been established. One of the most recent attempts at sensitive and specific detection of prions is the real-time quaking-induced conversion (RT-QuIC) method, which measures the activity of PrPSc aggregates or amyloid formation triggered by PrPSc seeds in the presence of recombinant PrP. In this review, we summarize prions, prion diseases, and current approaches to diagnosis, including the principle, conditions for assay performance, and current diagnostic applications of RT-QuIC.
