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Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
This content is Free Access.
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
Chapters
- 00:04Title
- 01:12Expression of FAHD Proteins in Competent E. coli
- 03:44Lysis of Bacterial Pellets and Filtration of Debris
- 04:33Purification of His-tagged FAHD Proteins using Ni-NTA Affinity Chromatography
- 05:59Purification of FAHD Proteins via Ion Exchange Chromatography
- 06:35Basic FAHD Activity Assays with Substrates Oxaloacetate and Acetylpyruvate
- 07:53Results: Expression and Purification of Fumarylacetoacetate Hydrolase Domain-containing Proteins
- 09:19Conclusion
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.
