de Marco Ario
IFOM-IEO Campus for Oncogenomics, via Adamello 16, I-20139, Milano, Italy.
Nat Protoc. 2006;1(3):1538-43. doi: 10.1038/nprot.2006.289.
The present purification protocol applies to target proteins that are fused to a double tag, such as NusA-His6, through a linker that includes a protease-recognition sequence. It involves two steps of immobilized metal ion affinity chromatography (IMAC). NusA stabilizes the passenger protein during translation, whereas the His-tag enables affinity purification of the fusion. The eluate resulting from the first IMAC is buffer-exchanged to remove the imidazole and to achieve optimal conditions for the enzymatic cleavage performed by a His-tagged recombinant protease. The digested sample is loaded directly for a second IMAC step and the target protein is selectively recovered in the flow-through. The resin binds residual non-digested fusion protein, double-tagged moiety, protease and any contaminant that bound the affinity resin and was eluted from the first IMAC. The purity of the target protein usually makes a further purification step unnecessary for most of the lab applications. It takes less than 5 hours to purify the protein from a 5 g pellet.
目前的纯化方案适用于通过包含蛋白酶识别序列的接头与双标签（如NusA-His6）融合的目标蛋白。该方案包括两步固定化金属离子亲和色谱（IMAC）。NusA在翻译过程中稳定目标蛋白，而His标签则使融合蛋白能够进行亲和纯化。第一次IMAC得到的洗脱液进行缓冲液置换，以去除咪唑，并为His标签重组蛋白酶进行的酶切反应创造最佳条件。消化后的样品直接用于第二次IMAC步骤，目标蛋白在流穿液中被选择性回收。树脂结合残留的未消化融合蛋白、双标签部分、蛋白酶以及任何与亲和树脂结合并从第一次IMAC中洗脱的污染物。对于大多数实验室应用而言，目标蛋白的纯度通常无需进一步纯化步骤。从5克沉淀中纯化该蛋白所需时间不到5小时。
