Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase.
Overexpression and purification of the sigma subunit of Escherichia coli RNA polymerase.
复制标题
大肠杆菌 RNA 聚合酶 sigma 亚基的过表达和纯化。
DOI：
10.1016/0378-1119(83)90180-4
复制
发表时间：
1983
期刊：
影响因子：
3.5
通讯作者：
Burgess,RR
中科院分区：
文献类型：
--
作者：
Gribskov,M;Burgess,RR
我们已经构建了一个质粒，过表达100倍的大肠杆菌RNA聚合酶的σ亚基。通过将λ噬菌体的P L O L启动子-操纵子置于编码σ亚基的基因rpoD的上游来构建质粒。基于盐酸胍变性/复性、DEAE纤维素层析和Sephacryl S-200层析，已经开发了用于纯化过表达蛋白的简单程序。纯化的产物已被表征，并发现通过酶活性、热灭活和部分蛋白水解判断，与通过先前方案纯化的正常表达的σ蛋白没有区别。
We have constructed a plasmid that overexpresses 100-fold the sigma subunit of Escherichia coli RNA polymerase. The plasmid was constructed by placing the P L O L promoter-operator of bacteriophage λ upstream from rpoD, the gene encoding the sigma subunit. A simple procedure for purification of the overexpressed protein has been developed based on guanidine hydrochloride denaturation/renaturation, DEAE cellulose chromatography, and Sephacryl S-200 chromatography. The purified product has been characterized and found to be indistinguishable from normally expressed sigma protein purified by previous protocols as judged by enzymatic activity, heat inactivation, and partial proteolysis.