Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive...
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| Veröffentlicht in: | Journal of visualized experiments 2019-06 (148) |
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| creator | Weiss, Alexander K H Holzknecht, Max Cappuccio, Elia Dorigatti, Ilaria Kreidl, Karin Naschberger, Andreas Rupp, Bernhard Gstach, Hubert Jansen-Dürr, Pidder |
| description | Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this superfamily generally display multi-functionality, involving mainly hydrolase and decarboxylase mechanisms. This article presents a series of consecutive methods for the expression and purification of FAHD proteins, mainly FAHD protein 1 (FAHD1) orthologues among species (human, mouse, nematodes, plants, etc.). Covered methods are protein expression in E. coli, affinity chromatography, ion exchange chromatography, preparative and analytical gel filtration, crystallization, X-ray diffraction, and photometric assays. Concentrated protein of high levels of purity (>98%) may be employed for crystallization or antibody production. Proteins of similar or lower quality may be employed in enzyme assays or used as antigens in detection systems (Western-Blot, ELISA). In the discussion of this work, the identified enzymatic mechanisms of FAHD1 are outlined to describe its hydrolase and decarboxylase bi-functionality in more detail. |
| doi_str_mv | 10.3791/59729 |
| format | Article |
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| subjects | Chromatography, Liquid - methods Crystallization Crystallography, X-Ray Enzyme Assays - methods Escherichia coli - metabolism Hydrolases - chemistry Hydrolases - isolation & purification Hydrolases - metabolism Protein Domains |
| title | Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins |
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