AI Article Synopsis
- The purification process for Poly(ADP-ribose) polymerases (PARPs) has been streamlined into a quick, three-step chromatographic protocol using overexpressing Sf9 insect cells and E. coli.
- After breaking down the cells, proteins are first sorted with a Heparine Sepharose™ column, followed by affinity purification with a 3-aminobenzamide Sepharose™ column.
- The final step uses a strong cations exchanger to eliminate remaining impurities, resulting in over 11 mg of pure and highly active mouse PARP-2 from just 1 L of culture, all achievable in three days with a high recovery yield.
Article Abstract
The purification of Poly(ADP-ribose) polymerases from overexpressing cells (Sf9 insect cells, Escherichia coli) has been updated to a fast and reproducible three chromatographic steps protocol. After cell lysis, proteins from the crude extract are separated on a Heparine Sepharose™ column. The PARP-containing fractions are then affinity purified on a 3-aminobenzamide Sepharose™ chromatographic step. The last contaminants and the 3-methoxybenzamide used to elute the PARP from the previous affinity column are removed on the high-performance strong cations exchanger Source™ 15S matrix. The columns connected to an ÄKTA™ purifier system allow the purification of PARPs in 3 days with a high-yield recovery. As described in the protocol, more than 11 mg of pure and highly active mouse PARP-2 can be obtained from 1 L of Sf9 insect cell culture.
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| http://dx.doi.org/10.1007/978-1-61779-270-0_9 | DOI Listing |
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