The prefix “bio” may mean life, but life is not essential for many biological processes. In 1897 the German researcher Eduard Buchner showed how the scraps and juice left over after yeast was mutilated and crushed could still drive the fermentation of sugar. In doing so, he won a Nobel Prize and finally defeated “vitalism”—the notion that there was something special about the activities of living organisms that defied biological and physical explanation.
Freeing biochemistry from the confines of the living cell has facilitated other breakthrough discoveries. Most notably, so-called cell-free systems that could shape amino acids into proteins helped Marshall Nirenberg and J. Heinrich Matthaei decipher the genetic code in 1961 at the US National Institutes of Health (1). And some 60 years later, the rise of synthetic biology has renewed scientific interest in liberating the machinery of life from living cells—from gene expression to protein synthesis. Researchers are again reveling in the freedom that Buchner first exploited.
With no cell membranes to traverse, no cell lines to sustain, and no need to worry about toxins that might kill or degrade cell activity, cell-free systems allow researchers to control, disrupt, and develop the biochemical tools of life. Modern cell-free systems can be used to help design and manufacture new proteins or design and test novel metabolic pathways. Expensive reagents can be replaced with cheap, portable versions that can be dried and reconstituted on demand in remote or low-resource settings. Vaccines and medicines can be made on the spot by using cell-free components.
“In the late 1990s and early 2000s, work on cell-free systems went through a bit of a Renaissance with the development of the field of synthetic biology. People started realizing this was a fantastic way into prototyping, design, testing, and all sorts of other applications,” says Paul Freemont, a synthetic biologist at Imperial College London in the United Kingdom. “There is a big drive to use cell-free in the future as a manufacturing platform for high-value components like antibodies.” Obstacles remain: Some cell-free processes are hard to scale up and many are expensive, so the approach is not going to sweep away existing practices any time soon. But it’s already making its mark in some niche and specialist applications and helping researchers push boundaries in the manufacture of proteins.
Smash and Grab
The simplest route to a cell-free system is to follow Buchner’s example and break open (lyse) living cells. That’s not as easy as it sounds. Whereas his rivals such as Louis Pasteur tried unsuccessfully to squash yeast cells with a pestle and mortar, Buchner found that rubbing them with a little sand worked better. Today’s researchers can choose from a range of options, including mechanical techniques like bombarding a cell suspension with tiny steel balls that tear into the cytoplasm, adding buffer chemicals to weaken membrane proteins by raising the pH, or using detergents to make proteins more soluble.
Once the cells have spilled their contents, researchers typically remove some of the cellular detritus. Centrifugation separates out genomic DNA and any surviving unlysed cells. Left behind in solution (the lysate) are useful compounds including metabolic enzymes and biological components needed for protein synthesis such as ribosomes.
For some cell-free applications, this crude mix of leftovers is enough. In 2018, researchers at the University of Texas at Austin showed that lysate could be used instead of expensive commercial enzymes as chemical reagents for lab reactions (2). Such enzymes, including those used to amplify DNA in the polymerase chain reaction (PCR), are usually produced in industrial processes by modified bacteria; then they’re extracted and purified. The University of Texas team found that the purification step was not necessary. Instead, they could simply lyse the cells and use the cell-free extract as the reagent.
In a series of common lab procedures, including PCR and plasmid synthesis, the researchers demonstrated that the lysed modified Escherichia coli bacteria performed as well as purified reagents. “We thought this would be a good approach, to have easily available reagents that anyone could make in their lab,” says Sanchita Bhadra, a biologist who led the Texas study.
These “cellular reagents” cost a fraction of the purified proteins to produce and are much less sensitive to temperature, which means they do not need to be kept in a fridge or freezer. This makes them an attractive option to researchers in many parts of the world who struggle to access commercial reagents, owing to either high costs or difficulties importing them from abroad. Bhadra’s group found they could even freeze-dry the lysed cells and store them at room temperature for several months, before rehydrating and using them as before.
Earlier this year, the researchers improved the process further, showing that the freeze-drying step was unnecessary and that the bacteria can be grown, lysed, and then dried in a standard lab incubator with simple desiccating chemicals. To demonstrate the flexibility that this method offers, they sent dried cell lysate by FedEx and then in personal luggage to collaborators in Cameroon and Ghana, where it was successfully used in lab reactions (3).
“We’ve taken the technology to a place where it actually becomes usable. People outside our lab were able to use this technology and it worked,” Bhadra says. The approach is not always suitable, she adds. The exact make-up of the processed cell-free mixture isn’t necessarily consistent, which means different batches could perform differently. “For regulated processes like clinical diagnostics, this is not yet approved.”
Still, for research purposes, cell-free systems can also make common proteins that are hard to express inside living cells. Membrane proteins such as receptors, channels, and transporters, for example, make up about a quarter of all human proteins and are targeted by almost half of drugs. But they are notoriously difficult to study. Natural sources don’t supply enough, so researchers instead rely on bacteria such as E. coli to overexpress these proteins in sufficient quantities to work with.
Even very low levels of many of these membrane proteins prove toxic to the bacterial cells that host them, resulting in poor yields. Cultures based on yeast, mammal, and insect cells suffer from the same problem. That’s one reason why membrane proteins only represent about 1% of solved protein structures.
Breaking open the bacterial cells allows their gene expression machinery to work even if the cell itself is not viable. By adding the necessary genetic instructions, researchers can put these cell-free systems to work to make select proteins on demand.
In 2016, biologists at the RIKEN Systems and Structural Biology Center in Yokohama, Japan, used a cell-free E. coli system to produce significant amounts of 19 mammalian membrane proteins. They found that the resulting proteins are often easier to access and purify than when bound inside intact cells, as in conventional production that’s based on live cultures (4)….
