tac promoter
The tac promoter ( tac promoter ) is a synthetically produced DNA fragment that is used in molecular biology , biochemistry and biotechnology as a tool in protein production. The DNA construct serves to increase the gene expression level in cells of the bacterium Escherichia coli and thus to overexpress recombinant proteins . The term tac is made up of trp and lac , the names of the operons that were used to produce the tac construct.
Bacterial promoters consist of two parts, the so-called −35 region and the −10 region - also known as the Pribnow box . The sigma factor of the RNA polymerase binds to these two regions , which then initiates the transcription of the gene behind it .
There are strong and weak promoters, which means that the sigma factor binds better to some promoters (consequently more mRNA is formed on these ) and weaker to some (here the amount of mRNA is lower). So-called consensus sequences for strong and weak promoters can be derived from the comparison of the DNA sequences of individual promoters . In protein overexpression experiments, one is interested in as large an amount of recombinant protein as possible, so strong promoters are used here.
An example of a strong promoter from Escherichia coli is the trp promoter. 1983 was -35 region of the trp promoter with the -10 region of another promoter - the lac promoter - in vitro combined. So a so-called hybrid promoter was constructed, the tac promoter. It was found that the tac promoter, like the lac promoter , can be regulated by IPTG and lactose , that is, the mRNA synthesis at this promoter is induced by the addition of these substances. This ability to regulate gene expression is important because many foreign proteins are toxic to the host cell.
It could also be shown that the tac promoter is 3 times stronger than the trp promoter and 10 times stronger than the lac promoter, an ideal prerequisite for experiments on protein overexpression.
literature
- ↑ de Boer, HA et al. (1983): The tac promoter: a functional hybrid derived from the trp and lac promoters. In: Proc. Natl. Acad. Sci. USA Vol. 80, pp. 21-25. PMID 6337371 PDF