Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems
- 1. Structural Genomics Consortium, University of Toronto, MaRS South Tower, 101 College Street, Toronto, Ontario M5G 1L7, Canada
- 2. Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada
- 3. Department of Biomedical Engineering and Center for Biological Systems Engineering, Washington University in Saint Louis, Saint Louis, Missouri 63130, USA
- 4. Basic Science Program, SAXS Core facility of National Cancer Institute, Frederick National Laboratory for Cancer Research , Frederick, MD 21701, USA
- 5. CHDI Foundation, 6080 Center Drive, Suite 700, Los Angeles, CA 90045, USA
- 6. Structural Genomics Consortium, University of Toronto, MaRS South Tower, 101 College Street, Toronto, Ontario M5G 1L7, Canada and Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 1L7, Canada
The pathogenic Huntington’s disease (HD) mutation causes polyglutamine (polyQ) tract expansion of the 348 kDa HTT protein above a critical threshold of ~35 glutamines. HD mutation effect on HTT is poorly understood, partly due to difficulties in performing biochemical studies with this large protein. To facilitate such studies, we generated resources for HTT production in multiple eukaryotic expression systems, comprising constructs with polyQ lengths representing general population, HD patients, juvenile HD patients and the more extreme expansions used in some tissue and animal models. These reagents yield milligram quantities of pure HTT protein. We biophysically characterised HTT samples produced using this HD resource, gleaning insight into the nature of full-length HTT in its apo form and when bound to its binding partner HAP40. Work outlined in this manuscript and the tools generated, lay a foundation for further biochemical study of the HTT protein and its functional interactions with other biomolecules.