Dipartimento di Biochimica e Biologia Molecolare "Ernesto Quagliariello," Università degli Studi di Bari Aldo Moro, 70125 Bari, Italy.
Anal Biochem. 2012 Feb 15;421(2):805-7. doi: 10.1016/j.ab.2011.12.015. Epub 2011 Dec 13.
The glutathione S-transferase (GST) fusion protein system is widely used for high-level expression and efficient purification of recombinant proteins from bacteria. However many GST-tagged proteins are insoluble, and the existing procedures, which employ a mixture of detergents to solubilize the molecules, frequently compromise their functional activity. A further limitation is that large proteins (>80 kDa) are poorly isolated by the current methods and are contaminated by truncated forms. To overcome these problems, we provide here an improved method for efficient purification of active large GST-tagged enzymes such as the 180-kDa GST-fused mitochondrial RNA polymerase.
谷胱甘肽 S-转移酶 (GST) 融合蛋白系统广泛用于从细菌中高水平表达和有效纯化重组蛋白。然而，许多 GST 标记蛋白是不溶的，现有的采用混合去污剂溶解这些分子的程序常常会损害它们的功能活性。另一个限制是，目前的方法对大于 80 kDa 的大蛋白的分离效果较差，并且容易被截断形式污染。为了克服这些问题，我们在此提供了一种改进的方法，可有效纯化活性的大 GST 标记酶，如 180 kDa 的 GST 融合线粒体 RNA 聚合酶。
