Rachel J. Harding1, Peter Loppnau1, Suzanne Ackloo1, Alexander Lemak2, Ashley Hutchinson1, Brittany Hunt1, Alex S. Holehouse3, Jolene C. Ho1, Lixin Fan4, Leticia Toledo-Sherman5, Alma Seitova1, Cheryl H. Arrowsmith1,2 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 From proteins to people: an open toolkit to accelerate Huntington’s disease research Huntington’s disease research has been hampered by a paucity of openly available biochemical tools to facilitate research into this devastating disease and help develop new therapeutics. This project aimed to generate resources for the Huntington’s disease research community, available without restriction, with the hope of accelerating research of the huntingtin protein which functions aberrantly in patients with this disease. Summary General population 15, 19, 23, 24, 25, 30 15, 19, 23, 24, 25, 30 HD patients 36, 42, 48 Juvenile HD patients 51, 52, 54, 60, 66, 73, 78, 79, 85 Extreme expansions 109, 139, 142, 145 pBacMam2-DiEx-LIC Huntingtin constructs are unencumbered and were created for open distribution ~15,000 publications referencing Huntington’s disease <50 publications detailing experiments with purified full-length huntingtin protein samples Data from PubMed search March 2019 for “Huntington’s disease” & “purified huntingtin protein” The mechanism of Huntington’s disease remains incompletely understood Many therapies in development and most of the current clinical trials targetting Huntington’s disease, aim to lower the levels of the huntingtin protein to slow or stop disease progression. Aim: Design and characterise an open toolkit of Huntington’s disease research resources for biochemical investigation of huntingtin protein Huntington’s disease patients have an expansion mutation in a CAG-repeat region of the huntingtin gene. This mutation leads to neuronal cell dysfunction and progressive neurodegenerative decline with complex psychological, cognitive and physcial symptoms. There are no disease modifying therapies available for Huntington’s disease. General population huntingtin gene < 35 CAG repeats Functional huntingtin protein Healthy neuron Patient population huntingtin gene > 35 CAG repeats Aberrant huntingtin protein Neurodegeneration However, we still don’t know very much about the physiological function of this criticial protein molecule. An open-source toolkit to make huntingtin protein samples Huntingtin is in the top 1% of human genes, encoding a 3000+ amino acid protein. This creates many technical barriers to make huntingtin protein samples. Constructs with different expansions were generated to allow expression and purification of huntingtin proteins reflecting different populations: pBacMam constructs permit flexible expression in either insect or mammalian cells to suit the needs of different labs. Suspension eukaryotic culture systems allow scalable production of protein, facilitating purification of large quanitites of material, needed for subsequent biochemical analysis. 5 10 15 0 50 100 150 200 Elution Volume (mL) A280 mAU Gel Filtration of insect HTT1-3144 Q23 HTT1-3144 Q23 250 130 100 75 55 48 35 25 15 MW (kDa) 11.5 12 12.5 13 13.5 14 Elution Volume (mL) 10 4-20% SDS-PAGE of monomer peak fractions Huntingtin samples can be simply purified with a 2-step protocol using FLAG-affinity chromatography and gel filtration. This yields highly pure huntingtin samples. Protei