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.