CLONING AND EXPRESSION OF THE GENE FOR BACTERIOPHAGE-T7 RNA-POLYMERASE
CLONING AND EXPRESSION OF THE GENE FOR BACTERIOPHAGE-T7 RNA-POLYMERASE
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DOI：
10.1073/pnas.81.7.2035
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发表时间：
1984-01-01
期刊：
影响因子：
--
通讯作者：
STUDIER, FW
中科院分区：
文献类型：
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作者：
DAVANLOO, P;ROSENBERG, AH;STUDIER, FW
在质粒pBR322中克隆了噬菌体T7 RNA聚合酶基因（T7基因1）的完整编码序列。当克隆基因从lac UV5启动子转录时，大肠杆菌中可积累大量的活性酶。在纯化过程中，蛋白酶的活性显然可以在不使蛋白质分解的情况下切割蛋白质，这可能是一个问题，但本文描述了一种方法，该方法可以获得高产量的、本质上均匀的、高活性的酶，适用于生化和物理研究。T7 RNA聚合酶对其自身的启动子具有严格的特异性，并会选择性地转录与该启动子相关的DNA。这种特异性使得这种酶既可以在体外产生特定的RNA，也可以在细胞内指导选定基因的表达。克隆的基因也使得对T7 RNA聚合酶的功能进行详细的突变分析成为可能。
The complete coding sequence of the gene for bacteriophage T7 RNA polymerase (T7 gene 1) was cloned in the plasmid pBR322. Large amounts of active enzyme can be accumulated in Escherichia coli when the cloned gene is transcribed from the lac UV5 promotor. A protease activity that apparently can nick the protein without causing it to fall apart can be a problem during purification, but a procedure is described that gives good yields of essentially homogeneous, highly active enzyme suitable for biochemical and physical studies. T7 RNA polymerase has a stringent specificity for its own promoters and will selectively transcribe DNA that was linked to such a promoter. This specificity makes the enzyme useful both for producing specific RNA in vitro and for directing the expression of selected genes inside the cell. Having the cloned gene also makes possible a detailed mutational analysis of the functioning of T7 RNA polymerase.