The Machado-Joseph disease-associated expanded form of ataxin-3: Overexpression, purification, and preliminary biophysical and structural characterization.

Publisher: Academic Press Country of Publication: United States NLM ID: 9101496 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1096-0279 (Electronic) Linking ISSN: 10465928 NLM ISO Abbreviation: Protein Expr Purif

Publication: Orlando, FL : Academic Press
Original Publication: San Diego : Academic Press, c1990-

Ataxin-3/*chemistry ; Machado-Joseph Disease/*genetics ; Peptides/*chemistry ; Recombinant Proteins/*chemistry ; Repressor Proteins/*chemistry; Ataxin-3/biosynthesis ; Ataxin-3/genetics ; Ataxin-3/isolation & purification ; Chromatography, Gel/methods ; Cloning, Molecular ; Escherichia coli/genetics ; Escherichia coli/metabolism ; Gene Expression ; Genetic Vectors/chemistry ; Genetic Vectors/metabolism ; Humans ; Leukocytes, Mononuclear/metabolism ; Leukocytes, Mononuclear/pathology ; Machado-Joseph Disease/metabolism ; Machado-Joseph Disease/pathology ; Models, Molecular ; Peptides/metabolism ; Protein Domains ; Protein Folding ; Protein Structure, Secondary ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/genetics ; Recombinant Proteins/isolation & purification ; Repressor Proteins/biosynthesis ; Repressor Proteins/genetics ; Repressor Proteins/isolation & purification ; Scattering, Small Angle ; X-Ray Diffraction

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.
(Copyright © 2018. Published by Elsevier Inc.)

Keywords: Expanded ataxin-3; Machado-Joseph disease; Small angle X-ray scattering; Thermal shift assay

0 (Peptides)
0 (Recombinant Proteins)
0 (Repressor Proteins)
26700-71-0 (polyglutamine)
EC 3.4.19.12 (ATXN3 protein, human)
EC 3.4.19.12 (Ataxin-3)

Date Created: 20180724 Date Completed: 20190822 Latest Revision: 20190822

20221213

10.1016/j.pep.2018.07.005

30036587