Expression Using the T7 RNA Polymerase/Promoter System

作者：Stanley Tabor · 发表于：Current Protocols in Molecular Biology · 年份：1990 · DOI：10.1002/0471142727.mb1602s11 · 被引用次数：266 · 研究领域：Bacterial Genetics and Biotechnology、Bacteriophages and microbial interactions、RNA and protein synthesis mechanisms

This unit describes the expression of genes by placing them under the control of the bacteriophage T7 RNA polymerase. T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size. T7 RNA polymerase is also highly selective for initiation at its own promoter sequences and is resistant to antibiotics such as rifampicin that inhibit E. coli RNA polymerase. Consequently, the addition of rifampicin to cells that are producing T7 RNA polymerase results in the exclusive expression of genes under the control of a T7 RNA polymerase promoter (p(T7)). In the Basic Protocol, two plasmids are maintained within the same E. coli cell. One (the expression vector) contains p(T7) upstream of the gene to be expressed. The second contains the T7 RNA polymerase gene under the control of a heat-inducible E. coli promoter. Upon heat induction, the T7 RNA polymerase is produced and initiates transcription on the expression vector, resulting in turn in the expression of the gene(s) under the control of p(T7). If desired, the gene products can be uniquely labeled by carrying out the procedure in minimal medium, adding rifampicin to inhibit the E. coli RNA polymerase, and then labeling the proteins with [35S]methionine.