Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

Journal of Visualized Experiments : JoVE
Andreas ReifHilal A Lashuel

Abstract

Huntington's Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (≥36) in the first exon of the HD gene, resulting in the expression of the Huntingtin protein (Htt) or N-terminal fragments thereof with an expanded polyglutamine (polyQ) stretch. The exon1 of the Huntingtin protein (Httex1) is the smallest Htt fragment that recapitulates many of the features of HD in cellular and animal models and is one of the most widely studied fragments of Htt. The small size of Httex1 makes it experimentally more amenable to biophysical characterization using standard and high-resolution techniques in comparison to longer fragments or full-length Htt. However, the high aggregation propensity of mutant Httex1 (mHttex1) with increased polyQ content (≥42) has made it difficult to develop efficient expression and purification systems to produce these proteins in sufficient quantities and make them accessible to scientists from different disciplines without the use of fusion proteins or other strategies that alter the native sequence of the protein. We present here a robust and optimized method for the production of milligram quantities of native, tag-free Httex1 based on the transient fusion of small ubiquitin ...Continue Reading

Methods Mentioned

BETA
electrophoresis
circular Dichroism

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