Amé Jean-Christophe, Kalisch Thomas, Dantzer Françoise, Schreiber Valérie
Groupe Poly(ADP-ribosyl)ation et Intégrité du Génome, FRE3211 du CNRS, École Supérieure de Biotechnologie de, Strasbourg, France.
Methods Mol Biol. 2011;780:135-52. doi: 10.1007/978-1-61779-270-0_9.
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.
从过表达细胞（Sf9昆虫细胞、大肠杆菌）中纯化聚（ADP - 核糖）聚合酶的方法已更新为一种快速且可重复的三步色谱法方案。细胞裂解后，粗提物中的蛋白质在肝素琼脂糖™柱上进行分离。然后，含PARP的组分在3 - 氨基苯甲酰胺琼脂糖™色谱步骤中进行亲和纯化。最后，使用高性能强阳离子交换剂Source™ 15S基质去除上一步亲和柱洗脱PARP时使用的3 - 甲氧基苯甲酰胺及残留污染物。连接到ÄKTA™纯化系统的柱子可在3天内实现PARP的高产量纯化回收。如该方案所述，从1 L Sf9昆虫细胞培养物中可获得超过11 mg的纯且高活性的小鼠PARP - 2。
