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Design and characterisation of mutant and wild-type huntingtin proteins produced from a toolkit of scalable eukaryotic expression systems

Harding, Rachel J.; Loppnau, Peter; Ackloo, Suzanne; Lemak, Alexander; Hutchinson, Ashley; Hunt, Brittany; Holehouse, Alex; Ho, Jolene; Fan, Lixin; Toledo-Sherman, Leticia; Seitova, Alma; Arrowsmith, Cheryl


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.

ACKNOWLEDGEMENTS We thank Dr. Xiaobing Zuo (Argonne National Laboratory) for expert support with SAXS measurements, and acknowledge the use of the SAXS Core facility of Center for Cancer Research (CCR), National Cancer Institute (NCI) which is funded by Frederick National Laboratory for Cancer Research under contract HHSN261200800001E and the intramural research program of the NIH, NCI, CCR. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products or organizations imply endorsement by the US Government. This research used 12-ID-B beamline of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. We also acknowledge Dr. Pravin Mahajan who constructed the pBMDEL vector. This research was supported by a Huntington's Disease Society of America Berman Topper Career Development Fellowship (RH), the Natural Sciences and Engineering Research Council of Canada (CHA) (grant RGPIN-2015-05939), Huntington Society of Canada (CHA), CHDI Foundation (LTS, CHA) and the SGC, a registered charity (number 1097737) that receives funds from AbbVie, Bayer Pharma AG, Boehringer Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada through Ontario Genomics Institute [OGI-055], Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck KGaA, Darmstadt, Germany, MSD, Novartis Pharma AG, Ontario Ministry of Research, Innovation and Science (MRIS), Pfizer, São Paulo Research Foundation-FAPESP, Takeda, and Wellcome. ASH is a postdoctoral fellow in the laboratory of R.V. Pappu at Washington University in St. Louis and is funded by the Human Frontiers Science Program (grant RGP0034/2017).
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