Expression, purification, crystallization, and enzyme assays of fumarylacetoacetate hydrolase domain-containing proteins

Alexander K.H. Weiss, Max Holzknecht, Elia Cappuccio, Ilaria Dorigatti, Karin Kreidl, Andreas Naschberger, Bernhard Rupp, Hubert Gstach, Pidder Jansen-Dürr

Research output: Contribution to journalArticlepeer-review

7 Scopus citations


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 languageEnglish (US)
JournalJournal of Visualized Experiments
Issue number148
StatePublished - Jun 1 2019
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)
  • Immunology and Microbiology(all)
  • Chemical Engineering(all)
  • Neuroscience(all)


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