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Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation
Published on: January 16, 2019
Assays of proteasome-dependent cleavage products
Stefan Tenzer1, Hansjörg Schild
1Institute of Immunology, University of Mainz, Germany.
Abstract:
The degradation of misfolded, aged, or no longer needed cytosolic proteins depends largely on the ubiquitin-proteasome system. Proteasomes degrade their substrates into fragments of 3-20 amino acids. Human 20S proteasomes can be purified from human erythrocytes by batch adsorption to DEAE-cellulose, ammonium sulfate precipitation, anion-exchange fast protein liquid chromatography (FPLC), and glycerol density gradient ultracentrifugation. 20S proteasomes purified by this method are suitable for the in vitro digestion of synthetic peptides as well as full-length proteins. The degradation products produced by proteasomes are separated by reversed-phase HPLC using an acetonitrile gradient. The obtained fractions are further analyzed by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and Edman degradation, which allows a quantitative analysis of the digestion products.
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