Huntington’s Disease: Neurodegeneration
Huntington’s disease (HD) is an autosomal dominant disorder caused by an elongated CAG repeat in the huntingtin gene. Symptoms are not only dominated by progressive chorea but also include psychiatric signs and progressive dementia. Postmortem brains of late-stage patients show extensive atrophy of the striatum (caudate nucleus and putamen), cerebral cortex, and pallidum, with corresponding neuronal loss primarily in the striatum. The mutation causes profound neuronal dysfunction independent of, or prior to, neurodegeneration, suggesting that pathophysiological mechanisms of dysfunction may be more pertinent therapeutic targets than neurodegeneration itself. Drugs targeting many cellular processes have shown some efficacy in animal models, but no neuroprotective treatments are presently available for humans.
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Expression and purification of ataxin-1 protein
2010, Journal of Neuroscience MethodsAtaxin-1 is part of a larger family of polyglutamine-containing proteins that is linked to nine distinct neurodegenerative disorders. There are no known effective therapies for any of these expanded polyglutamine tract disorders. One possible reason for this is the lack of sufficient amounts of pure polyglutamine-containing proteins suitable for biochemical and conformational studies. Here, we show that we were able to successfully purify a non-pathological, wild-type human ataxin-1 protein containing a 30-glutamine repeat sequence. This ataxin-1 protein was expressed in Escherichia coli as a fusion protein with a GST tag at the N-terminus and a double (His)6 tag at the C-terminus. The devised dual affinity tag strategy allowed successful purification of the full-length ataxin-1 fusion protein to 90% homogeneity as confirmed by Western blot analysis using the two monoclonal ataxin-1 antibodies developed in our laboratory. In addition, the GST tag was successfully removed from the purified ataxin-1 fusion protein by treatment with Tobacco etch virus (TEV) protease. Since polyglutamine-containing proteins tend to aggregate, solvents/buffers that minimize aggregation have been used in the purification process. This dual affinity purification protocol could serve as a useful basis for purifying aggregation-prone proteins that are involved in other neurodegenerative diseases.