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Cloning and test expression of HTT fragment clones - 2019/02/28

Research datakeyboard_double_arrow_right Dataset 28 Feb 2019Publisher:Zenodo

Authors: Harding, Rachel; Hutchinson, Ashley; Seitova, Alma; Arrowsmith, Cheryl; Lee, Matt;

doi: 10.5281/zenodo.2630830 , 10.5281/zenodo.2600051 , 10.5281/zenodo.2600050

Cloning and test expression of HTT fragment clones - 2019/02/28

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Abstract

Project: High resolution structural analysis of purified HTT proteins Experiment: Cloning and test expression of HTT fragment clones Date completed: 2019/02/28 Rationale: I aim to complement our previous work on purifying stable regions of huntingtin by cloning further regions of huntingtin corresponding to discrete HEAT repeats for eukaryotic expression. Constructs will initially be screened by BVES in insect sf9 cells. High quality constructs will undergo extensive crystallization experiments and structure determination by X-ray crystallography using well established high-throughput and systematic protocols in place at the SGC. Any solved structures may allow generation of a pseudoatomic resolution (<4 Å) model of the HTT protein using the cryoEM model as a guide.

Dr. Harding is the recipient of the Huntington's Disease Society of America Berman Topper Career Development Fellowship which funds and supports this research, in addition to generous funding from the CHDI Foundation and the Huntington Society of Canada. The SGC is 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.

Related Organizations

University of Toronto
Canada
CHDI Foundation
United States

Keywords

Protein purification, Protein expression, Huntingtin, X-ray crystallography

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Preparation of dephosphorylated HTT samples for cryoEM analysis 2019/03/20
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Analysis of dephosphorylated HTT samples for cryoEM analysis and further sample preparation 2019/04/15
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Coexpression and purification of HTT domains with HAP40 - 2018/12/03
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Large-scale purification of HTT domain constructs 2019/04/01
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Analysis of dephosphorylated HTT samples by cryoEM analysis 2019/05/01
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Purification of HTT Q23 and Q54 from EXPI293F for mass spectrometry analysis 2019/09/30
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