Tabor S, Richardson C C
Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074-8. doi: 10.1073/pnas.82.4.1074.
The RNA polymerase gene of bacteriophage T7 has been cloned into the plasmid pBR322 under the inducible control of the lambda PL promoter. After induction, T7 RNA polymerase constitutes 20% of the soluble protein of Escherichia coli, a 200-fold increase over levels found in T7-infected cells. The overproduced enzyme has been purified to homogeneity. During extraction the enzyme is sensitive to a specific proteolysis, a reaction that can be prevented by a modification of lysis conditions. The specificity of T7 RNA polymerase for its own promoters, combined with the ability to inhibit selectively the host RNA polymerase with rifampicin, permits the exclusive expression of genes under the control of a T7 RNA polymerase promoter. We describe such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase.
噬菌体T7的RNA聚合酶基因已被克隆到质粒pBR322中，置于λPL启动子的诱导控制之下。诱导后，T7 RNA聚合酶占大肠杆菌可溶性蛋白的20%，比在T7感染细胞中发现的水平增加了200倍。过量产生的酶已被纯化至同质状态。在提取过程中，该酶对一种特定的蛋白水解敏感，这种反应可通过改变裂解条件来防止。T7 RNA聚合酶对其自身启动子的特异性，加上用利福平选择性抑制宿主RNA聚合酶的能力，使得在T7 RNA聚合酶启动子控制下的基因能够独家表达。我们描述了这样一个偶联系统及其用于高水平表达噬菌体T7基因5蛋白（T7 DNA聚合酶的一个亚基）的用途。
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