Deceglie Stefania, Lionetti Claudia, Roberti Marina, Cantatore Palmiro, Polosa Paola Loguercio
Department of Biosciences, Biotechnologies and Biopharmaceutics, University of Bari Aldo Moro, Bari, Italy.
Methods Mol Biol. 2014;1129:169-80. doi: 10.1007/978-1-62703-977-2_15.
GST fusion proteins expressed in bacteria often tend to form aggregates and are inefficiently purified by standard procedures, which employ a mixture of detergents that compromise the binding efficiency to the affinity resin and the biological activity of the recombinant proteins. Moreover, the binding to the resin is negatively affected by the molecular weight of the fusion protein. Here we report a simple and efficient method to purify active large GST-tagged proteins, which uses high ionic strength buffer to solubilize the protein aggregates in a bacterial lysate. Affinity-chromatography purification is achieved by adopting two columns connected in series, which facilitate the binding of large GST fused molecules. This approach was applied to purify the 180-kDa GST-tagged mitochondrial RNA polymerase. We also report conditions for simple and efficient GST tag removal from the eluted protein. Finally we demonstrate that the recombinant enzyme is capable to catalyze RNA synthesis.
在细菌中表达的谷胱甘肽 - S - 转移酶（GST）融合蛋白常常倾向于形成聚集体，并且通过标准程序进行纯化效率低下。标准程序使用的去污剂混合物会损害与亲和树脂的结合效率以及重组蛋白的生物活性。此外，融合蛋白的分子量对与树脂的结合有负面影响。在此，我们报告一种简单有效的方法来纯化具有活性的大分子量GST标记蛋白，该方法使用高离子强度缓冲液溶解细菌裂解物中的蛋白聚集体。通过采用串联连接的两根柱子实现亲和层析纯化，这有利于大分子量GST融合分子的结合。此方法应用于纯化180 kDa的GST标记线粒体RNA聚合酶。我们还报告了从洗脱蛋白中简单高效去除GST标签的条件。最后，我们证明重组酶能够催化RNA合成。
