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Journal / Biochemistry / Expression, Purification, Crystallization, and…
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Biochemistry

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JoVE Journal Biochemistry
Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
 

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

Article DOI: 10.3791/59729-v • 10:21 min • June 20th, 2019
June 20th, 2019 •
1,2, 1,2, 1,2, 1, 1, 3, 3, 4, 1,2
1Research Institute for Biomedical Aging Research, University of Innsbruck Austria, 2Center for Molecular Biosciences Innsbruck (CMBI), University of Innsbruck Austria, 3Division of Genetic Epidemiology, Medical University of Innsbruck Austria, 4Faculty of Chemistry, Department of Organic Chemistry, University of Vienna Austria

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Expression and purification of fumarylacetoacetate hydrolase domain-containing proteins is described with examples (expression in E. coli, FPLC). Purified proteins are used for crystallization and antibody production and employed for enzyme assays. Selected photometric assays are presented to display the multi-functionality of FAHD1 as oxaloacetate decarboxylase and acylpyruvate hydrolase.

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Keywords: Expression Purification Crystallization Enzyme Assays Fumarylacetoacetate Hydrolase FHD Protein TCA Cycle Energy Metabolism Mitochondria Cellular Senescence IPTG Induction Affinity Chromatography FPLC BL21-DE3 PLysS E. Coli LB Agar NZCYM Medium
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