Promega’s TnT Quick Coupled Transcription/Translation System allows users to quickly determine whether a stable protein can be translated (from RNA) from their DNA construct of interest. Results are generally obtainable within 1-2 days and the system removes time constraints associated with plasmid/RNA construct transfection and analysis. For successful translation, the gene of interest must be downstream of a T7 or Sp6 RNA polymerase promoter. In addition, PCR products can be used as templates, although the amplicon must contain the T7 or Sp6 promoter sequence and preferably, a poly-A tail for added RNA stability. This allows quick transcription of RNA and subsequent translation into protein, all within a single 90-minute incubation.
The principle is relatively simple – the rabbit reticulocyte lysate contains all the translational “machinery” required for protein synthesis. By using RNA as a template, adding an amino acid mixture (containing all amino acids EXCEPT methionine) and a radio-labeled (usually 35S) methionine, the radiolabel is incorporated into the protein allowing visualization of the radioactive signal when the protein is electrophoresed on a denaturing SDS-PAGE. This method also enables the user to determine the kDa size of the generated protein. An obvious caveat to this system is that the protein must contain sufficient methionine residues to allow ample incorporation of 35S signal and subsequent visualization.
Previous incarnations of the TnT Transcription/Translation system required multi-step addition of reagents, leading to possibilities of reagent omission or pipetting errors. The enormous advantage of the Quick Coupled Transcription/Translation system is that all required components (polymerase, reticulocyte lysate, buffer, RNAse degrading enzyme [RNAsin], and amino acid mix lacking methionine) are present in a single tube; 40ul is used per reaction, along with 10-40uCi of 35S -methionine, 1-2ug of DNA, and nuclease-free water to a final volume of 50ul. After incubation at 30oC for 90 minutes (a one-step process!), the protein has been translated. Two positive control vectors, containing the luciferase gene (with either an upstream T7 or Sp6 promoter) are included in the TnT Quick Coupled Transcription/Translation System, allowing the user to determine optimal conditions for in vitro transcription/translation.
I have used the TnT Quick system for in vitro translations using DNA constructs cloned into plasmids. This system could also be used to test the effect of gene mutations, particularly truncation mutants, as well as a host of protein/DNA/RNA binding/interaction assays. As the synthesized protein is readily detectable by radiography, anything interacting with it can be co-localized using specific antibodies, primers, tags, etc.
An obvious advantage for using DNA constructs in plasmids, is that the construct does not need to be in a mammalian expression vector therefore, any cloning vector with an upstream T7 or Sp6 RNA promoter can be used. A major drawback is that the current systems are not compatible with a T3 promoter, which may limit some applications. I have tried to use T3 polymerase with the Quick kits (by simply using a T3-promoted construct, T3 polymerase and the Sp6 quick kit), but this strategy was unsuccessful.
In summary, Promega’s TnT Quick Coupled Transcription/Translation System is a robust, easy to use system, allowing fast and accurate synthesis of proteins from DNA constructs. In vitro translation is a very good way to validate that constructs can make viable proteins, particularly if the construct is to be used for in vivo studies.
Dworkin Sebastian
Graduate Student
Peter MacCallum Cancer Centre
Melbourne, Australia