TnT® Quick Coupled Transcription/Translation System From Promega

One method of studying proteins is to perform coupled transcription and translation reactions rather than using living organisms to produce them. Promega have developed a series of products based around production of gene products using rabbit reticulocyte lysates. The TnT® Quick Coupled Transcription/Translation System utilizes either T7 or SP6 polymerases to drive protein production for analysis. It is called the ‘Quick’ system as unlike the old transcription/translation kits, the important reaction components have been combined into a single master mix, containing RNA polymerase, nucleotides, buffers, amino acids and RNAse inhibitors as well as the reticulocyte lysates.

Both T7 and SP6 polymerase driven systems are available and each system is available in two sizes: 40 x 50 µl reactions and a handy trial size of 5 x 50µl reactions. Correct storage of the kit is important as the components are sensitive to heat, freeze thawing and carbon dioxide. Included in each kit is a control plasmid with luciferase under the appropriate promoter, as well as a luciferase assay reagent which can be used to determine luciferase activity.

It is important to read the manual for this system (available online through Promega) before starting any experiments as there are a number of options for detection of the final protein product, all of which require additional reagents and equipment to quantify the results. This System is really designed for using [³⁵S]methionine, as the master mix is methionine free. However, other non-radioactive methods of labeling the protein can be used including using biotinylated or fluorescently tagged lysines (methionine supplied with the kit can be used to supplement the master mix in this case). Other options include using a membrane mix (Canine Pancreatic Microsomal Membranes, Promega) to study membrane associated proteins.

Setting up a coupled reaction is straightforward. Either circular plasmid DNA or a linear fragment (T7 system only) is added to the master mix, along with the label of choice. The reaction is incubated at 30ºC for 60-90 minutes, and the product can then be analyzed by SDS PAGE and further steps according to the chosen label. The luciferase control reaction can also be tested for activity using a luminometer and the supplied Luciferase Assay Reagent.

We have used the TnT® Quick Coupled Transcription/Translation System from Promega with [³⁵S]methionine labeling to detect protein products. It is very simple to use, but care should be taken when handling the reagents to prevent RNAse contamination of the reaction or master mix (use RNase free tips and tubes at least). I recommend reading the manual thoroughly before beginning as there are a number of helpful suggestions for designing the vector that can influence the amount of protein produced. We normally use DNA purified using Promega Wizard® Plus Minipreps with no problem, although care should be taken to dry the excess alcohol from columns before eluting the DNA, as it can affect the yield if present during elution.