Maity Ranjan, Pauty Joris, Krietsch Jana, Buisson Rémi, Genois Marie-Michelle, Masson Jean-Yves
Genome Stability Laboratory, Oncology Axis, Hôtel-Dieu de Québec.
J Vis Exp. 2013 Oct 29(80):e50320. doi: 10.3791/50320.
Key assays in enzymology for the biochemical characterization of proteins in vitro necessitate high concentrations of the purified protein of interest. Protein purification protocols should combine efficiency, simplicity and cost effectiveness. Here, we describe the GST-His method as a new small-scale affinity purification system for recombinant proteins, based on a N-terminal Glutathione Sepharose Tag (GST) and a C-terminal 10xHis tag, which are both fused to the protein of interest. The latter construct is used to generate baculoviruses, for infection of Sf9 infected cells for protein expression. GST is a rather long tag (29 kDa) which serves to ensure purification efficiency. However, it might influence physiological properties of the protein. Hence, it is subsequently cleaved off the protein using the PreScission enzyme. In order to ensure maximum purity and to remove the cleaved GST, we added a second affinity purification step based on the comparatively small His-Tag. Importantly, our technique is based on two different tags flanking the two ends of the protein, which is an efficient tool to remove degraded proteins and, therefore, enriches full-length proteins. The method presented here does not require an expensive instrumental setup, such as FPLC. Additionally, we incorporated MgCl2 and ATP washes to remove heat shock protein impurities and nuclease treatment to abolish contaminating nucleic acids. In summary, the combination of two different tags flanking the N- and the C-terminal and the capability to cleave off one of the tags, guaranties the recovery of a highly purified and full-length protein of interest.
酶学中用于体外蛋白质生化特性表征的关键检测需要高浓度的目标纯化蛋白质。蛋白质纯化方案应兼顾效率、简便性和成本效益。在此，我们描述了一种GST-His方法，作为一种用于重组蛋白的新型小规模亲和纯化系统，该系统基于N端谷胱甘肽琼脂糖标签（GST）和C端10xHis标签，二者均与目标蛋白融合。后一种构建体用于产生杆状病毒，以感染Sf9细胞进行蛋白表达。GST是一个相当长的标签（29 kDa），用于确保纯化效率。然而，它可能会影响蛋白质的生理特性。因此，随后使用PreScission酶将其从蛋白质上切割下来。为了确保最大纯度并去除切割后的GST，我们基于相对较小的His标签添加了第二步亲和纯化。重要的是，我们的技术基于位于蛋白质两端的两个不同标签，这是去除降解蛋白的有效工具，因此能富集全长蛋白。本文介绍的方法不需要诸如快速蛋白质液相色谱（FPLC）等昂贵的仪器设备。此外，我们加入了MgCl2和ATP洗涤以去除热休克蛋白杂质，并进行核酸酶处理以消除污染的核酸。总之，位于N端和C端两侧的两个不同标签的组合以及切割其中一个标签的能力，保证了目标高纯度全长蛋白的回收。
