Synthesizing proteins in vitro through cell-free expression systems using rabbit reticulocytes, E. coli S30, or wheat germ extracts can be invaluable in studying protein function. If you only need a small amount (100s of nanograms), it’s also faster and easier than synthesizing vast quantities in bacterial or mammalian cells (~ 90 minutes for cell-free vs. long growth times and extraction steps after an initial optimization for protein synthesized in larger scale). There are many systems out there, and knowing which to use can sometimes be difficult. Many kits include components that combine transcription and translation in one-step, eliminating the need to provide your own RNA. But when you want to make your own RNA templates to add to lysates, then there are additional concerns.
Many people don’t want to work with RNA since the common lab lore suggests it’s a finicky molecule, and for good reason. Extracting it requires the utmost care in technique and elimination of nucleases. Failing to do so results in degradation of the molecule, and so with it your experiments (see our recent blog by Terri Sundquist on tips for isolating RNA with ease). Preparing RNA for cell-free expression is subject to the same concerns as extracted RNA, but with the proper care is not that much more of a challenge than using a DNA template.
The first step for using cell-free expression systems with RNA templates is to make the RNA. Here are some tips that will ensure success.
- The easiest way to generate RNA is to use a cloning vector with a DNA sequence of interest inserted downstream of a promoter for in vitro transcription. Most cloning vectors include a promoter (SP6, T7, T3) flanking a multi-cloning region that allows you to select the right promoter to generate the sense transcript of your mRNA of interest.
- You absolutely need to ensure your insert contains a start (AUG) codon and a stop codon so in vitro transcribed transcripts can be properly translated. Additional sequences such as the 5’ and 3’ UTRs, and a poly(A) sequence may be useful for optimizing translation of some mRNAs.
- Once you have prepared your vector, you will need to purify high-quality plasmid DNA. Column-based plasmid purification kits make this quick and painless.
- In vitro transcription works best when you linearize the template with a restriction enzyme 3´ to the end of your insert, but you should avoid doing so with restriction enzyme that leave 3´ 4-base overhangs. 3´ 4-base overhangs can be problematic in generating spurious transcripts. If there is no other choice, you can produce blunt ends by using DNA Polymerase I Large (Klenow) fragment.
- Once you linearize the template, you may want to run the fragment on a gel and subsequently gel purify with a column kit.
- Use a good quality in vitro transcription kit to get good yield and purity of RNA.
- You may notice a theme here (another purification step): purify the transcribed RNA. Again, column kits are your friend.
- Quantitate your RNA concentration. Why? Well, every mRNA and protein is different, and what works well for one transcript won’t necessarily work for all. In each case, it’s best to optimize the input concentration of mRNA. So knowing what concentration you have is critical.
- We have found that a range of 5–80 µg/ml for rabbit reticulocytes, 100–200 µg/ml for wheat germ extracts, and 10–100 µg RNA for S30 works for most cases. So yes, even though it’s a lot of work, you should titrate the amount of mRNA initially.
- Most eukaryotic mRNA molecules contain a cap (7-methyl guanosine 5´ cap) that is important for translation. A cap isn’t necessary in cell-free expression systems but in some cases, including a cap analog can improve protein yield. However, increasing the concentration of the uncapped mRNA can sometimes achieve the same goal.
- Again, every mRNA is translated different, and the optimal salt concentrations (potassium and magnesium) sometimes need to be optimized; some systems, such as the Flexi® Rabbit Reticulocyte Lysate System, allow you to do so.
- If your RNA has a lot of secondary structure, it may be necessary to denature the RNA prior to the reaction. You can heat the RNA to 65–67°C briefly (~ 10 minutes), then cool on ice prior to adding to a lysate.
While this may sound like a lot of work, the whole workflow can be done in a few days. The longest step will probably be cloning into a plasmid so you can transcribe RNA in vitro. But once you get to that point, there are straightforward steps to do everything else, and commercially supplied systems can make the work go by quickly. In no time, you’ll be expressing that protein.
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Can you please recommend a good quality in vitro transcription kit?
The Promega RiboMax™ kits are well-suited to generate in vitro transcribed RNA in large quantities suitable for in vitro translation.
We have two kits I’d suggest:
1) T7 RiboMAX™ Express Large Scale RNA Production System (http://www.promega.com/products/rna-analysis/in-vitro-transcription/t7-ribomax-express-large-scale-rna-production-system/) can be used to generate a large amount of RNA quickly using a DNA template with a T7 promoter.
2) RiboMAX™ Large Scale RNA Production Systems can be used to synthesize RNA from a T7 or SP6 promoter. http://www.promega.com/products/rna-analysis/in-vitro-transcription/ribomax-large-scale-rna-production-systems/
Please feel free to contact Promega Technical Services if you have additional questions:
email@example.com or you can give us a call at (800)-356-9526
If I have 20ng/microliter of mRNA, how much of this should I use for in vitro translation using promega RRL kit.
For Catalog Number L4960 or L4151 you can find the RNA template requirements on page 6 of the technical manual, linked here: https://www.promega.com/Resources/Protocols/Technical%20Manuals/0/Rabbit%20Reticulocyte%20Lysate%20Protocol/?fq=rabbit%20reticulocyte%20lysate (2µg/50µl reaction for a standard methione translation reaction);
In cases where there’s not much secondary structures with mRNAs, will it be a problem for translation if you still denature the mRNA sample before adding on translation reaction? Will degradation cause your translation to go awry?
Hi, We have forwarded your question to technical services and will post their reply here.
Here is the reply from our technical services scientist:
The heat step to denature mRNA secondary structure is typically short enough and at a low enough temperature that mRNA degradation is not a concern. However, the purity of the mRNA sample (e.g. presence of nuclease and divalent metal ions), alkaline pH, etc. can increase its susceptibility to degradation. Further, degraded mRNA can result in poor reaction efficiency and incomplete translation products. Therefore, if mRNA secondary structure is not a concern, it can be advantageous to omit the heat step.
If you require additional information, please feel free to reach out directly to our technical services group: https://www.promega.com/support/tech-support/
I’d like to translate proteins from mRNAs natively bound to a protein of interest (X) in the mouse brain. My plan is to immunoprecipitate X from brain tissue and use the IP as template for in vitro translation. The abundance of mRNA template is expected to be very low. Which cell-free IVT system do you recommend for this application?
We would suggest trying the Rabbit Reticulocyte Lysate, Nuclease-Treated (Cat.# L4960 – https://www.promega.com/products/protein-expression/cell-free-protein-expression/rabbit-reticulocyte-lysate-system_-nuclease-treated/). This would be a first choice for mRNAs from a cellular extract, from a mammalian source. Regarding the input amount, we recommend 1-2ug input in a standard reaction, although less has been successfully used. So, the input amount would be a challenge, but this would be so for any translation system. If you have more questions about your experiment, feel free to contact Technical Services via https://www.promega.com/support/tech-support/