Abstract
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.
Original language | English (US) |
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Journal | Journal of Visualized Experiments |
Volume | 2019 |
Issue number | 148 |
DOIs | |
State | Published - Jun 1 2019 |
Externally published | Yes |
Bibliographical note
Generated from Scopus record by KAUST IRTS on 2023-02-15ASJC Scopus subject areas
- General Biochemistry, Genetics and Molecular Biology
- General Medicine
- General Immunology and Microbiology
- General Chemical Engineering
- General Neuroscience