If you have perform an in vitro transcription reaction, using a T7-RNA polymerase, the buffer/solution doesn't appear to contain any inhibitors or issues for translation. Why then must then the transcript-of-interest be purified before in vitro translation?
Many kits offer a coupled transcription/translation for plasmids containing a T7-promoter. This sounds advantageous since RNA is so short lived and many clean-up steps are eliminated. Why do I come across many protocols which emphasize mRNA cleanup and DNAse treatment? How do coupled transcription/translation reactions not have this same issue?
I would like to pass along my response from Promega's Technical support. While clean-up steps are not entirely necessary, if you have an appropriate nucleic acid purification column or time to precipitate/concentrate your transcript that would likely benefit in vivo translation.
If your protein is particular or has solubility/folding troubles, I imagine that performing transcription separately would be preferable. Then you can better tweak the translation portion.
"Promega has a pretty comprehensive Protein Expression Product Guide that includes a references section with interesting resources and review articles. Like you, I was unable to find a Promega protocol that puts anything but purified mRNA transcript into the Wheat Germ in vitro transcription. I did find a reference stating, “With modern protocols, the mRNA can be used directly after transcription without any prior purification”. See the Test Expression section of the following Review Article:
- Fogeron Marie-Laure, Lecoq Lauriane, Cole Laura, Harbers Matthias, Böckmann Anja. Easy Synthesis of Complex Biomolecular Assemblies: Wheat Germ Cell-Free Protein Expression in Structural Biology. Frontiers in Molecular Biosciences .8. 2021. https://doi.org/10.3389/fmolb.2021.639587
My understanding is that purification of transcript is typically required for two reasons:
In vitro translation systems can be sensitive to salt concentration. One of the ways to optimize a cell-free translation is to add salts like magnesium or potassium. Though the components of a that RNA polymerase reaction may not inhibit the translation, they also may not allow for optimal translation and may be harder to compensate for than if one simply has transcript in water."
The reason for separating transcription and translation in eukaryotic cell-free systems is the different optimum concentration of Mg for transcription and translation. Inside the cell, these processes are also separated in the nucleus and cytoplasm, each with their optimum conditions. So in cell-free coupled transcription/translation, you need to use conditions supporting both processes while not optimal for both steps. Purifying the mRNA helps to remove the by-products (mainly phosphates binding Mg) to avoid decreasing efficiency of the transation step, as stated above. In bacterial systems the translation is coupled anyway, so the cell-free system can be run in the same buffer conditions.
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