In recent years, cell-free systems have advanced significantly in terms of protein yield, cost per milligram and purity. In spite of this, the use of such systems is not yet commonplace in most research labs. In this article, we explore some of the barriers to adoption of these systems. We subsequently demonstrate how our cell-free system overcomes these barriers through the synthesis of four common lab reagents at high titers and purities.
One of the most common misconceptions about cell-free protein synthesis is that for non-model proteins, the yields may be low. One factor contributing to this is that many researchers have only had experience with older systems, such as rabbit reticulocyte lysates, which were only capable of producing low-microgram quantities of protein per milliliter of reaction. This is no longer the case; in fact, most commercially available systems now boast of being able to produce more than 500 μg/mL of protein. Here we provide strong evidence to support our claim that producing high-microgram to low-milligram quantities of purified proteins is now entirely possible even for non-model proteins using our cell-free platform.
It is also noteworthy to highlight three factors often overlooked that can negatively affect yields in cell-free expression systems. The first is the quality of the DNA prep. Obtaining high yields requires pure template DNA; crude purification strategies such as the Miraprep method tend to result in significantly lower yields. The second is the template DNA architecture and optimization. For example, optimizing the N-terminal codons
can help robustly boost productivities. The third, and perhaps the most important factor, is lack of proper reaction scale-up. The yield of cell-free reactions under standard test tube conditions tend to drop as reaction sizes increase; to counteract this phenomenon, cell-free reactions must be carried out in specialized reaction formats
Another important misconception is that, outside of screening assays, using cell-free systems would be cost-prohibitive. This has historically been true; the price per milligram of protein until now for the commercially available systems has been relatively high, thus making the use of cell-free systems inaccessible to labs that did not prepare such systems in-house. This was exacerbated by the fact that lack of commercially available reaction scaling tools
meant that milligram scale protein production was virtually impossible due to high cost. As demonstrated using the examples here, this is no longer the case. Milligrams of purified cell-free-manufactured proteins can now be obtained for just a few hundred dollars (in the examples here we were able to obtain proteins in the ~$125-$250/mg range when using our 5 mL kit
3. Protein Size
The last common fallacy, particularly when it comes to bacterial cell-free systems, is that such systems are only capable of producing short peptides. Some mistakenly hold the view that anything larger than the commonly expressed fluorescent proteins falls outside of the capabilities of such systems. As demonstrated in the examples provided here, this view is patently false.
To support the claims provided here, we expressed Renilla luciferase, T4 ligase, SP6 RNA polymerase and deGFP in our cell-free system. Our cartridge-based cell-free reactions were capable of producing all four construct at high titers and purities. All four reactions and purifications were carried out in parallel in accordance with the protocol provided in our application note. The results are demonstrated below:
As we have demonstrated here, cell-free protein expression platforms can now be reliably used for small-scale protein expression in research labs. Given the advantages of cell-free systems, in many cases the use of time-consuming bacterial hosts is no longer the logical or the economical approach. This is because the perceived cost savings associated with cell-based expression strategies are often offset by the accompanying high labour costs demanded by them.
Experiments presented here were carried out by Taylor Sheahan. Special thanks to Alexander Klenov and Taylor Sheahan for their edits to this post.