Spehr V, Frahm D, Meyer T F
Max Planck Institute for Infection Biology, Department of Molecular Biology, Schumannstr. 21/22, 10117, Berlin, Germany.
Gene. 2000 Oct 31;257(2):259-67. doi: 10.1016/s0378-1119(00)00400-5.
One of the most efficient systems for the high-level expression of cloned genes in Escherichia coli makes use of a phage T7 late promoter whose activity depends on a regulated transcription unit supplying the specific T7 RNA polymerase. Various T7 RNA polymerase/T7 promoter-based vector host systems with differential control on expression of the T7 RNA polymerase are in use. Most of them show high levels of expression in non-induced cells, low factor of induction or impaired growth of host cells. We describe a novel and efficient control system in which basal level expression of T7 RNA polymerase is suppressed by the use of the genes for the Lac repressor and T7 lysozyme, integrated on the expression vector. T7 lysozyme expression is probably down-regulated in the induced expression system by antisense RNA. This overcomes the inhibitory effect of T7 lysozyme on T7 RNA polymerase as shown by SDS PAGE and flow cytometry analysis of expressed GFP. The main features of the expression vector compared with other systems are low background, high factor of induction and unaffected growth of non-induced cells.
在大肠杆菌中用于高效表达克隆基因的最有效系统之一，利用了噬菌体T7晚期启动子，其活性取决于提供特定T7 RNA聚合酶的调控转录单元。各种基于T7 RNA聚合酶/T7启动子的载体宿主系统对T7 RNA聚合酶的表达具有不同的控制。其中大多数在未诱导的细胞中表现出高水平表达、诱导系数低或宿主细胞生长受损。我们描述了一种新型高效控制系统，其中通过使用整合在表达载体上的Lac阻遏物和T7溶菌酶基因来抑制T7 RNA聚合酶的基础水平表达。在诱导表达系统中，T7溶菌酶的表达可能通过反义RNA而下调。如通过SDS-PAGE和表达的绿色荧光蛋白的流式细胞术分析所示，这克服了T7溶菌酶对T7 RNA聚合酶的抑制作用。与其他系统相比，该表达载体的主要特点是背景低、诱导系数高且未诱导细胞的生长不受影响。
