Purification of Recombinant Human PARG and Activity Assays

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 1608; p. 395
• Main Authors: Amé, Jean-Christophe, Héberlé, Éléa, Camuzeaux, Barbara, Dantzer, Françoise, Schreiber, Valérie
• Format: Journal Article
• Language: English
• Published: United States 2017
• Subjects: Animals; Biological Assay - methods; Escherichia coli - enzymology; Glycoside Hydrolases - isolation & purification; Glycoside Hydrolases - metabolism; Humans; Poly Adenosine Diphosphate Ribose - metabolism; Protein Processing, Post-Translational; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Poly(ADP-ribose) metabolism; Poly(ADP-ribose) glycohydrolase; PARG purification
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-6993-7_25

The purification of Poly(ADP-ribose) glycohydrolase (PARG) from overexpressing bacteria Escherichia coli is described here to a fast and reproducible one chromatographic step protocol. After cell lysis, GST-PARG-fusion proteins from the crude extract are affinity purified by a Glutathione 4B Sepharose chromatographic step. The PARG proteins are then freed from their GST-fusion by overnight enzymatic cleavage using the preScission protease. As described in the protocol, more than 500 μg of highly active human PARG can be obtained from 1.5 L of E. coli culture.