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University of Innsbruck Austria

3 ARTICLES PUBLISHED IN JoVE

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Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein
Ruth Greussing 1, Hermann Unterluggauer 1, Rafal Koziel 1, Andrea B. Maier 2, Pidder Jansen-Dürr 1
1Molecular and Cell Biology, Institute for Biomedical Aging Research, 2Department of Gerontology and Geriatrics, Netherlands Consortium for Healthy Aging, Leiden University Medical Center

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

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Biochemistry

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins
Alexander K. H. Weiss 1,2, Max Holzknecht 1,2, Elia Cappuccio 1,2, Ilaria Dorigatti 1, Karin Kreidl 1, Andreas Naschberger 3, Bernhard Rupp 3, Hubert Gstach 4, Pidder Jansen-Dürr 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

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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Biochemistry

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver
Andreas Andric 1, Eva Wagner 1, Anne Heberle 1, Max Holzknecht 1, Alexander K. H. Weiss 1
1Research Institute for Biomedical Aging Research, University of Innsbruck

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

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