Scientists express proteins for many experimental and clinical needs. With cell-free kits, expression can be used in even more ways. A variety of vendors offer such kits, which makes it easier than ever to take this approach. Despite the benefits of cell-free expression, making the transition takes some work.
“Cell-free protein expression provides flexibility not available with in vivo expression systems,” says Javin Oza, assistant professor of chemistry and biochemistry at California Polytechnic State University. “This flexibility starts with a choice of linear versus circular DNA, enabling rapid screening and discovery.”
Oza notes other benefits. “There is flexibility in the chemical environment of cell-free expression,” he says. “Researchers can choose from reducing to oxidizing conditions to produce traditionally intractable proteins.” He also points out “flexibility in the post-production workflow, enabling researchers to assay without the need for isolation or purification of their protein.” The last benefit that he mentions is flexibility in the scale of a reaction; this, he says, lets researchers “go from high-throughput discovery all the way to manufacturing.”
From Dublin City University, Alan Costello, a postdoctoral researcher in the mammalian cell engineering group, adds, “If you are using commercial cell-free kits for both mammalian and bacterial cell-free systems, you don’t require dedicated cell culture facilities, such as shakers, incubators, biological safety cabinets, etcetera.” Plus, he says, “Speed is another major advantage along with the ability to use substances that impact cell proliferation and viability.”
From Oza and Costello’s comments, other scientists will see a range of reasons to consider going cell-free in expression. Like all methods, though, cell-free expression includes some challenges.
It’s not all free
Even with kits, cell-free expression isn’t necessarily simple. Still, Oza says, “Kits obviate many of the specialized technical skills and instrumentation required to implement cell-free.” Nonetheless, what works the best in one lab might not be the best choice in another.
Part of the inconsistencies between labs comes from variations in the technology. “As cell-free continues to gain traction in the research community,” Oza says, “there is an impetus within the community and support from the National Institute for Standards and Technology to standardize the platform.”
Although Oza notes that cell-free methods are not difficult, he does call them nuanced. “So, researchers interested in converting to a cell-free approach should be prepared for some trial-and-error until the methods become reproducible in their hands,” he says.
That means that scientists need to spend some time getting the hang of the cell-free approach, but it’s not too bad. Although Costello points out that he’s pretty new to the cell-free method, when asked what it takes to go from cell-based to cell-free expression, he says, “If going with commercial kits, then not much has to change in terms of your inputs.” He continues, “For example you can use plasmid DNA expression vectors.” For anyone planning to go cell-free and kit-free, it’s a little more complicated. “If making your own cell-free lysate,” Costello explains, “you will be replacing culture media with defined supplements for a generic reaction buffer, instrumentation to facilitate mechanical cell distribution, and storage of the lysate.”
Easier options
To get started as easily as possible, many labs turn to kits for cell-free expression. Thermo Fisher Scientific offers many cell-free kits for expressing proteins in various systems: including Escherichia coli, rabbit reticulocyte, and HeLa cells. The company recommends the bacterial system for high yield, and the mammalian systems, Thermo Fisher notes, are “more likely to produce proteins with native post-translational modifications.”
Arbor Biosciences also makes kits to consider. For example, myTXTL is advertised as an “easy-to-use cell-free protein expression platform for protein synthesis and synthetic biology in industry and academia.” This kit uses an E. coli system, and gene transcription and translation take place in a single tube. “Gene expression is initialized by simply adding the nucleotide template to the myTXTL Master Mix and recombinant proteins can be detected after only a few minutes,” the company notes.
Other companies around the world also make cell-free kits. One example is AMS Biotechnology’s Alice. This system uses tobacco-cell lysates. The company notes that this kit produces 3 milligrams of protein/milliliter in 48 hours, compared to “0.1 mg/ml of other eukaryotic [cell-free protein expression] systems.” This company’s list of suggested applications is long: from expression analysis, mutant screening and target-protein characterization to biopharmaceuticals, herbicide screening, and metabolomics.
DIY lives
Even with so many commercial kits available for cell-free expression, some scientists still want to take a do-it-yourself approach. Even there—maybe even more so there—teamwork counts. Oza and his colleagues, for example, hope to make cell-free expression available and accessible to new users and ones who work in new fields.
“In one effort, we have completely redefined the upstream processing steps required to generate cell extracts, making the process faster, cheaper, and—importantly—easier and more reproducible,” Oza says.1 In describing the approach, he points out that Cal Poly is primarily an undergraduate institution. “So if my freshmen can implement these methods,” Oza says, “so can graduate students and post-docs around the world.”
That upstream track, however, makes up only part of Oza’s plan. He adds, “In another effort, we have developed downstream-processing steps to significantly enhance the shelf-life of cell-extracts to enable field applications.” Oza believes that this “innovation is going to unlock cell-free’s potential in point-of-care applications.”
To make it as easy as possible for other scientists to take advantage of these approaches, Oza and his colleagues created a user’s guide and a video tutorial.
However a scientist decides to take on cell-free expression—with commercial kits or DIY techniques—it could open new research opportunities. Plus, some of the applications could end up improving healthcare, maybe in ways that remain to be seen. Like most advances in technology, though, cell-free expression is not without a bit of a learning curve. To make the curve less of a challenge, kits can help. Plus, Oza’s tools also make it easier even without a kit. As more scientists go cell-free, the transition could get even easier.
References
1. Levine, MA; So, B; Mullin, AC; et al. Redesigned upstream processing enables a 24-hour workflow from E. coli cells to cell-free protein synthesis. bioRxiv. 2019.