Oturum Aç

Institute for Biomedical Aging Research

2 ARTICLES PUBLISHED IN JoVE

Biology

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Ruth Greussing¹, Hermann Unterluggauer¹, Rafal Koziel¹, Andrea B. Maier², Pidder Jansen-Dürr¹

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.

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 Dorigatti¹, Karin Kreidl¹, Andreas Naschberger³, Bernhard Rupp³, Hubert Gstach⁴, 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.