期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 294, 期 17, 页码 6986-7001出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.RA118.007204
关键词
Huntington disease; neurodegeneration; protein expression; protein purification; biophysics; small-angle X-ray scattering (SAXS); molecular dynamics
资金
- National Institutes of Health, NCI [HHSN26120080001E]
- Intramural Research Program of the National Institutes of Health, NCI, Center for Cancer Research
- Huntington's Disease Society of America Berman/Topper Family Career Development Fellowship
- Natural Sciences and Engineering Research Council of Canada
- Huntington Society of Canada [RGPIN-2015-05939]
- CHDI Foundation
- SGC
- AbbVie [1097737]
- 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 [115766]
- Janssen
- Merck KGaA, Darmstadt, Germany
- MSD
- Novartis Pharma AG
- Ontario Ministry of Research, Innovation and Science (MRIS)
- Pfizer
- Sao Paulo Research Foundation (FAPESP)
- Takeda
- Wellcome Trust
The gene mutated in individuals with Huntington's disease (HD) encodes the 348-kDa huntingtin (HTT) protein. Pathogenic HD CAG-expansion mutations create a polyglutamine (polyQ) tract at the N terminus of HTT that expands above a critical threshold of approximate to 35 glutamine residues. The effect of these HD mutations on HTT is not well understood, in part because it is difficult to carry out biochemical, biophysical, and structural studies of this large protein. To facilitate such studies, here we have generated expression constructs for the scalable production of HTT in multiple eukaryotic expression systems. Our set of HTT expression clones comprised both N- and C-terminally FLAG-tagged HTT constructs with polyQ lengths representative of the general population, HD patients, and juvenile HD patients, as well as the more extreme polyQ expansions used in some HD tissue and animal models. Our expression system yielded milligram quantities of pure recombinant HTT protein, including many of the previously mapped post-translational modifications. We characterized both apo and HTT-HTT-associated protein 40 (HAP40) complex samples produced with this HD resource, demonstrating that this toolkit can be used to generate physiologically meaningful HTT complexes. We further demonstrate that these resources can produce sufficient material for protein-intensive experiments, such as small-angle X-ray scattering, providing biochemical insight into full-length HTT protein structure. The work outlined and the tools generated here lay a foundation for further biochemical and structural work on the HTT protein and for studying its functional interactions with other biomolecules.
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