- UniProt: P00573
T7 RNA polymerase, the RNA polymerase from phage T7, a virus which infects the intestinal Escherichia coli. The enzyme plays an important role in biotechnology in the expression of recombinant proteins, especially in E. coli as well as in Bacillus subtilis and Pseudomonas exist applications.
The first RNA transcript of the phage T7 are carried out with the RNA polymerase of the host. Including information for the translation of the very specific promoter of T7 RNA polymerase. The T7 polymerase recognizes only own its own promoter and is therefore a very good method to assimilate the metabolism of the host and to use for their own reproduction. T7 RNA polymerase is considered in combination with the T7 promoter of its own as a particularly "strong " expression system. This means that it is often much protein per copy can be produced on a DNA promoter that much RNA, and as a result. T7 polymerase during the elongation phase of transcription has a Nukleotidadditionsgeschwindigkeit of 200-260 nucleotides per second (the normal E. coli RNA polymerase reaches about 40 nucleotides per second).
The T7 promoter
The T7 promoter has a specific sequence:
The transcription with GGGAGA starts. The promoter has almost no Basaltranskription. That is, in the absence of T7 RNA polymerase is he as good as not read.
Application in biotechnology
The strength of the T7 expression system make the T7 promoter and the T7 polymerase very interesting for the expression rekombinater proteins. Therefore, since there are a variety of vectors for protein expression using the T7 - system, such as the pET- series of the company Merck / Novagene.
Usually located on a vector, the T7 promoter sequence, and behind it ( before the required RBS) the coding region for a " protein of interest". ( As is produced by the expression organism no T7 polymerase), since such an expression system because of the above reasons would not be functional, the T7 polymerase must be artificially introduced into the system. The most common method for this is the genomic anchoring a T7 expression cassette in the production organism. The most commonly used for this organism is E. coli. When it is referred to the corresponding genotype of the genomic T7 polymerase displays with (DE3 ) as the T7 polymerase has been integrated by means of the DE3 prophage into the genome. Here, the expression of the T7 polymerase is under the control of the lacUV5 promoter can be induced by IPTG or lactose.
Problems in the use of T7 expression systems
There are several problems that arise from the use of the T7 expression system:
The used genomically anchored expression cassette for the T7 RNA polymerase is not " tight" one speaks of a so-called Basaltranskription. The result, therefore always a certain amount of T7 polymerase is present to ensure that the production organism expresses proteins also already before the actual induction. This is very detrimental especially in toxic products and also it is an unnecessary metabolic burden of the production organism. Also, the addition of a lac operator on the vector between the promoter and protein-coding sequence here shows little success.
Another method is the genomic anchoring a lysozyme expression cassette ( genotype designation E. coli pLys ) or the addition of an appropriate plasmid. Lysozyme binds to the T7 - polymerase and inhibits it. However, this method also means an additional burden on the production organism and does not provide optimal results with respect to the Basaltranskription.
The latest developments are T7 strains that no longer have the (DE3 ) genotype, ie carrying the T7 polymerase under control of the lacUV5 promoter, but under much tighter promoter systems such as the rhamnose or Arabinoseoperon. In these strains, the T7 polymerase will be produced only after the addition of the corresponding sugar. This leads to a substantially lower Basaltranskription. Since these sugars are relatively expensive compared to IPTG, such systems do not, however, apply to be economically in the production of low-cost recombinant products.
A second problem with the use of the T7 system is strong overexpression of the " protein of interest". The consequence is a high product loss due to inclusion bodies.