Purification of Recombinant Human PARG and Activity Assays.
Fiche publication
Date publication
janvier 2017
Journal
Methods in molecular biology (Clifton, N.J.)
Auteurs
Membres identifiés du Cancéropôle Est :
Dr DANTZER Françoise, Dr SCHREIBER Valérie
Tous les auteurs :
Amé JC, Héberlé É, Camuzeaux B, Dantzer F, Schreiber V
Lien Pubmed
Résumé
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.
Référence
Methods Mol. Biol.. 2017 ;1608:395-413