Gribskov M, Burgess R R
Gene. 1983 Dec;26(2-3):109-18. doi: 10.1016/0378-1119(83)90180-4.
We have constructed a plasmid that overexpresses 100-fold the sigma subunit of Escherichia coli RNA polymerase. The plasmid was constructed by placing the pLoL promoter-operator of bacteriophage lambda 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.
我们构建了一种质粒，该质粒使大肠杆菌RNA聚合酶的σ亚基过表达100倍。通过将噬菌体λ的pLoL启动子 - 操纵子置于编码σ亚基的基因rpoD上游来构建该质粒。基于盐酸胍变性/复性、DEAE纤维素色谱和Sephacryl S - 200色谱，开发了一种简单的过表达蛋白纯化方法。已对纯化产物进行了表征，通过酶活性、热失活和部分蛋白酶解判断，发现其与先前方案纯化的正常表达的σ蛋白没有区别。
