Chapter title |
Purification of Recombinant Human PARP-3
|
---|---|
Chapter number | 24 |
Book title |
Poly(ADP-Ribose) Polymerase
|
Published in |
Methods in molecular biology, July 2017
|
DOI | 10.1007/978-1-4939-6993-7_24 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6992-0, 978-1-4939-6993-7
|
Authors |
Jean-Christophe Amé, Barbara Camuzeaux, Françoise Dantzer, Valérie Schreiber |
Abstract |
The purification of poly(ADP-ribose) polymerase-3 (PARP-3) from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible two chromatographic steps protocol. After cell lysis, PARP-3 protein from the crude extract is affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute PARP-3 from the previous affinity column are removed on the high-performance strong cations exchanger MonoQ™ matrix. This step allows also the concentration of the protein. The columns connected to an ÅKTA™ purifier system allow the purification of the protein in 3 days with a high-yield recovery. As described in the protocol, more than 3 mg of pure and active human PARP-3 can be obtained from 1.5 L of E. coli culture. |
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