Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
8359241 | Protein Expression and Purification | 2018 | 36 Pages |
Abstract
An expansion of the polyglutamine (polyQ) tract within the deubiquitinase ataxin-3 protein is believed to play a role in a neurodegenerative disorder. Ataxin-3 contains a Josephin catalytic domain and a polyQ tract that renders it intrinsically prone to aggregate, and thus full-length protein is difficult to characterize structurally by high-resolution methods. We established a robust protocol for expression and purification of wild-type and expanded ataxin-3, presenting 19Q and 74Q, respectively. Both proteins are monodisperse as assessed by analytical size exclusion chromatography. Initial biophysical characterization was performed, with apparent transition melting temperature of expanded ataxin-3 lower than the wild-type counterpart. We further characterize the molecular envelope of wild-type and expanded polyQ tract in ataxin-3 using small angle X-ray scattering (SAXS). Characterization of protein-protein interactions between ataxin-3 and newly identified binding partners will benefit from our protocol.
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Authors
Miriam G.G. Contessotto, Luciana K. Rosselli-Murai, Maria Cristina C. Garcia, Cristiano L.P. Oliveira, Iris L. Torriani, Iscia Lopes-Cendes, Marcelo J. Murai,