A BACTERIOPHAGE-T7 RNA-POLYMERASE PROMOTER SYSTEM FOR CONTROLLED EXCLUSIVE EXPRESSION OF SPECIFIC GENES
A BACTERIOPHAGE-T7 RNA-POLYMERASE PROMOTER SYSTEM FOR CONTROLLED EXCLUSIVE EXPRESSION OF SPECIFIC GENES
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DOI：
10.1073/pnas.82.4.1074
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发表时间：
1985-01-01
影响因子：
11.1
通讯作者：
RICHARDSON, CC
中科院分区：
文献类型：
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作者：
TABOR, S;RICHARDSON, CC
将噬菌体T7的RNA聚合酶基因克隆到质粒pBR 322中，在大肠杆菌的诱导控制下。PL启动子。诱导后，T7 RNA聚合酶构成大肠杆菌可溶性蛋白的20%，比T7感染细胞中发现的水平增加200倍。将过量产生的酶纯化至同质。在提取过程中，酶对特定的蛋白水解敏感，该反应可以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，结合用利福平选择性抑制宿主RNA聚合酶的能力，允许在T7 RNA聚合酶启动子的控制下的基因的排他性表达。描述了这样的偶联系统及其用于表达高水平的噬菌体T7基因5蛋白（T7 DNA聚合酶的亚基）的用途。
The RNA polymerase gene of bacteriophage T7 was 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 was 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. Such a coupled system and its use to express high levels of phage T7 gene 5 protein, a subunit of T7 DNA polymerase, are described.