Scaling up Cell-Free Protein Expression Reactions
Cell-free protein expression has the potential to displace traditional cell-based protein production for making small quantities of proteins. This is because cell-free reagents eliminate the need for many time-consuming steps and much of the infrastructure involved with cell-based expression such as transformation, culturing, harvesting and lysis. However, most labs, including those with expertise in the cell-free domain, still use cells when it comes to small-scale protein production; cell-free systems have, to date, been mainly used as screening tools rather than production chassis. Here we explore some challenges to scaling up cell-free reactions from droplet-sized volumes to the milliliter scale and outline strategies to get the most out of your cell-free.
When it comes to scaling, the devil is in the details
Standard test-tube cell-free reactions do not scale. While many cell-free experts would agree with this statement, they will also point out that scaling issues can be solved through one of two ways. The first is to sparge a gas into the reaction, as in a stirred tank bioreactor. The second is to use a thin-film reaction format, such as pipetting your sample into shallow volumes in a petri dish or 24-well plate.
Despite these apparent solutions, most cell-free manuscripts continue to use reaction sizes of 15 μL or below. “The greatest trick the devil ever played was convincing the world that he did not exist.” Perhaps we need to give the devil its due by acknowledging the fact that in spite of these great technical demonstrations, scaling remains a significant problem for small-scale protein production.
Now, one may argue that the lack of adoption of these techniques is a function of the high cost of cell-free protein expression reactions; commercially available systems are generally costly and thus carrying out cell-free protein expression reactions is simply not economically viable. This view is incorrect for two reasons. Inexpensive cell-free protein expression reagents have been available on the market for quite some time. More importantly, many cell-free labs are more than capable of making inexpensive reagents but yet do not routinely perform large scale reactions. It is therefore worthwhile delving into some of the underlying reasons and addressing them from a practical viewpoint.
Issues with current scale-up methods
Cell-free reaction scale-up through sparging of gases can be quite robust and has been in use by large commercial enterprises, such as Sutro Biopharma. However, due to fairly complex equipment requirements and longer set-up times, the technology has not been widely adopted by cell-free labs.
Thin-film reactions seem to be more popular among cell-free labs owing to their simplicity, but many problems such as evaporation and sample loss (e.g. due to adhesion to the container) remain. Hybrid methods have also been developed by enterprising researchers, such as running a reaction within a tube while it is placed on its side and shaken in an incubator. While these ad-hoc methods can be used for a small range of volumes under favourable conditions (e.g. using a humidity chamber), they often fail to do so in a consistent and reliable manner. There is a significant and urgent need for a platform that can help consistently and reproducibly scale up cell-free reactions.
The scale-up problem: an example
Before we talk about any potential solutions, it is worthwhile discussing how severe scaling issues can be. The figure below shows the effect of reaction volume on specific yield of mature deGFP expressed in cell-free (here fluorescence is used as a proxy for specific yield since the relationship between florescence to deGFP protein concentration is approximately linear).
It is important to note that deGFP requires oxygen for maturation. Therefore, one could argue that the effects observed is simply due to lack of oxygenation. However, looking at cell-free expressed deGFP bands on a 12% SDS-PAGE Coomassie-stained gel, we can observe a similar trend.
As noted above, this phenomenon has also been previously observed with other proteins. For example, specific yields for cell-free production of the E. coli chloramphenicol acetyl transferase (CAT) and a GCSF/scFv fusion (GLH) have been shown to drop as reaction volumes increase. The large magnitude of this scaling problem makes milligram-scale production of proteins virtually inaccessible due to high cost.
A simplified solution to the scale-up problem: thin-film gas exchange bioreactor cartridges
Recognizing the need, we have created easy-to-use cartridges that can be operated using standard laboratory equipment. These cartridges have been designed to solve evaporation, consistency and reproducibility issues associated with the conventional methods.
The resulting product, called Feather, is a spin-column that provides easy reaction set-up and sample collection. Importantly, Feather can also be used without any modification for purifying cell-free generated target proteins using conventional chromatography reagents such as Nickel-NTA beads.
Not only do larger reactions in these cartridges outperform those in traditional test tubes, they also outperform the smaller 15 μL reactions. The broad applicability of these cartridges has now been confirmed using a range of target proteins, including single-chain antibodies, enzymes and fluorescent proteins.
Consistency and Reproducibility
Our results to date also suggest that these cell-free expression cartridges can boost production consistently across different volume scales. This enables seamless linear scaling of reactions as work moves from construct screening through optimization and validation. Furthermore, we have now created a suit of cartridges that enable reactions at various volumes, while optimizing for high oxygenation, low evaporation and minimal sample loss.
As noted above, Feathers can also be used for purifying the protein of interest directly after cell-free production. The following is a Coomassie-stained gel for an enzyme expressed at the 200 μL scale and purified within the cartridge using Nickel-NTA beads (target contained a hexa-histidine tag). This experiment has now been done with three enzymes using our cell-free protein expression reagents. Nanodrop eluate concentration estimates for three different enzymes expressed in our thin-film bioreactors and purified in the cartridges (eluted in the same volume as the reaction) were 0.775 mg/mL, 2.73 mg/mL and 1.72 mg/mL.
Here, we share an early look at our new high-yield, proprietary thin-film gas exchange bioreactors that can be used to consistently scale cell-free production from microgram to low-milligram quantities of purified proteins. We envision this technology will enable and encourage more labs to produce their own custom proteins, and that cell-free protein production can now be carried out with little upfront infrastructure or cost. To learn more about our cell-free protein expression cartridges, see our product page here. Our intellectual property portfolio also includes gas sparging technologies that can enable higher throughput applications; if you are interested in more information, please reach out to us.
The work presented here is the fruit of tireless work by Dr. Alexander Klenov and Taylor Sheahan. Their ingenuity and hard work has made the technology presented here possible. These individuals have also provided significant feedback and edits to this post.