Sharrocks A D
Department of Biochemistry and Genetics, Medical School, University of Newcastle upon Tyne, UK.
Gene. 1994 Jan 28;138(1-2):105-8. doi: 10.1016/0378-1119(94)90789-7.
The pGEX system for protein production in E. coli is widely used in molecular biology. A bacterial expression vector, pETGEXCT, which incorporates features of the pGEX and pET expression systems was designed. pETGEXCT allows the production of N- and C-terminal fusions to glutathione S-transferase (GST) under the tight control of the T7 promoter. Use of this vector can circumvent problems associated with unstable or inactive fusions to the N terminus of GST. Indeed, it is demonstrated that fusions to the N terminus of the eukaryotic DNA-binding protein, RSRFC4, cannot be tolerated. Fusion of RSRFC4 to the N terminus of GST in the pETGEXCT vector is a prerequisite to purify the RSRFC4 DNA-binding domain in an active form using glutathione-agarose affinity chromatography.
用于在大肠杆菌中生产蛋白质的pGEX系统在分子生物学中被广泛使用。设计了一种细菌表达载体pETGEXCT，它整合了pGEX和pET表达系统的特性。pETGEXCT允许在T7启动子的严格控制下产生与谷胱甘肽S-转移酶（GST）的N端和C端融合蛋白。使用该载体可以规避与GST N端不稳定或无活性融合相关的问题。事实上，已证明真核DNA结合蛋白RSRFC4与GST N端的融合是不能被容忍的。在pETGEXCT载体中将RSRFC4与GST的N端融合是使用谷胱甘肽-琼脂糖亲和色谱法以活性形式纯化RSRFC4 DNA结合结构域的前提条件。
