Cell-Free Protein Synthesis Is Transforming Synthetic Biology

For bioengineer Michael Jewett, biology can sometimes feel like an obstacle.

Jewett studies how synthetic biology can improve human health and the planet. His work often relies on microorganisms to manufacture compounds or follow genetic instructions. But cells have evolved to prioritize their own survival and reproduction—not the goals of a researcher.

Microorganisms can inactivate foreign DNA segments, known as transgenes, that Jewett inserts into their genomes. They can also direct cellular resources toward their own needs instead of producing the molecules researchers want.

So Jewett changed his approach.

“Rather than fighting this tug of war that exists between what the cell wants to do and what we as engineers want to do, we kind of cut the rope,” says Jewett, who works at Stanford University in California. “We peel back their cell walls and collect their insides, which we use as molecular factories to carry out biological processes.”

This approach, known as cell-free protein synthesis, is not new. Systems based on cell-free protein expression helped researchers decipher the genetic code in the 1960s. The technology, however, continues to evolve.

“This is very much an ongoing trend,” says biophysicist Vincent Noireau, whose team at the University of Minnesota in Minneapolis developed the original system.

What is cell-free protein synthesis?

Although their composition varies, cell-free protein expression systems generally contain all the machinery needed to transcribe DNA into messenger RNA and translate that RNA into protein. These systems can be made from bacterial or eukaryotic cell lysates, or assembled from purified components.

The latter approach is called protein synthesis using recombinant elements, or PURE.

Cell-free systems allow protein designers to screen hundreds or thousands of options without modifying or growing cells. That can shorten the design-build-test-learn cycle from days or weeks to hours, says Wilson Wong, a synthetic biologist at Boston University in Massachusetts.

Researchers can use these systems to manufacture substances that are toxic to living cells or that would otherwise be broken down or transformed by cellular enzymes. Some scientists are developing freeze-dried formulations that could generate biological drugs such as vaccines on demand. Others are creating portable test kits for applications including water-quality testing and point-of-care diagnostics.

Meanwhile, synthetic biologists are studying PURE systems as a starting point for building artificial cells.

“There’s a lot going on,” says Noireau. “Many new applications have been demonstrated, and new cell-free gene-expression systems exhibit several new properties.”

Cell-free systems offer speed—but at a cost

Cell-free systems promise speed, customization and convenience, but they can be expensive. The reagents needed to run a liter-scale system can cost more than $4,000. Most studies also report producing less than 2 grams of protein, making these systems orders of magnitude more expensive than cell-based protein synthesis.

To keep high-throughput screening affordable, some researchers conduct experiments at the submicroliter scale.

Cell-free systems also have limited operating times. Bioengineers can continuously synthesize a product by growing bacteria or yeast, extracting the product and adding fresh growth medium. Cell-free systems, by contrast, quickly exhaust their energy supplies.

“Whatever we’re trying to do, it has to be done within a few hours,” Wong says.

Cell-free biosensors can detect water contaminants

The limited operating time of cell-free systems is less of a problem for cell-free sensors. These systems only need to detect a molecule of interest and produce a measurable signal within tens of minutes.

“Nature has already developed all these sensors,” says Jewett’s collaborator Julius Lucks, co-director of Northwestern University’s Center for Synthetic Biology in Evanston, Illinois. Cells need to recognize and respond to metals, antibiotics and other small molecules, making them a useful source of biological sensors.

Scientists can use living cells as biosensors, but doing so presents several challenges. The cells must remain alive, and the molecules being detected must pass through the cell wall and cell membrane. Researchers also risk releasing genetically modified organisms into the environment.

Cell-free systems allow researchers to build minimal biological sensors without using living organisms. In 2020, Lucks’s group described a chemically defined system called ROSALIND, which uses DNA-binding transcription factors as sensors.

Transcription factors block RNA synthesis until a specific contaminant or molecule of interest is detected.¹ When the target molecule is present, the proteins detach from the DNA, genes are transcribed and detectable signals are produced.

As a proof of principle, the research team freeze-dried the ROSALIND system and used it to detect copper and zinc in samples collected from a municipal water system contaminated by wildfires in California in 2018.

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Machine learning helps create a lead sensor

Cell-free synthesis is particularly useful for rapid, iterative protein engineering guided by machine learning.

Lucks, Jewett and their colleagues used this approach to fine-tune another transcription-factor-based biosensor—this time to detect lead.²

Lead is a challenging contaminant because many sensors that recognize it also bind to zinc, which is not generally considered harmful. To create a lead-selective sensor, the researchers fed data from a wide range of mutant proteins capable of detecting lead into a machine-learning model. They used the model’s results to design the next set of mutations, which they then tested in a cell-free system.

After a few rounds, the team developed a sensor that could detect lead at concentrations as low as 5.7 parts per billion.

“I think cell-free systems will play an increasingly important role in science because they have the advantage of facilitating accelerated expression of tens to hundreds of thousands of proteins,” Jewett says.

Some of Lucks’s sensors are available from Addgene, a nonprofit reagent repository. His ROSALIND paper also includes a Microsoft Excel file with detailed instructions for setting up the reaction.

“We try to be as user-friendly as possible,” Lucks says. “If they can put it together, get it set up and running, and it reacts at all, then you can use it.”

Cell lysates make efficient protein-production systems

To test the sensor, Jewett’s team cultivated laboratory bacteria, cracked open the cells and used their contents as a protein-expression mixture.

Compared with PURE systems, such cell lysates often provide higher yields at lower cost, making them useful for protein production, says Emily Chen, a production scientist at New England Biolabs (NEB) in Ipswich, Massachusetts. NEB’s NEBExpress protein-synthesis system, for example, uses an Escherichia coli lysate.

Purchasing cell lysates can be advantageous, especially for start-ups and laboratories that do not have all the equipment needed for cell-based protein production, Chen says.

Other commercial Escherichia coli systems include Noireau’s myTXTL, sold by Daicel Arbor Biosciences of Ann Arbor, Michigan; the CFPS kit from Ginkgo Bioworks of Boston; and CFXpress from GenScript of Piscataway, New Jersey.

Some providers also offer formulations designed to improve the production of difficult-to-express compounds. Ginkgo’s CFPS Premium Kit, for example, includes solubility enhancers for especially large or insoluble proteins, as well as proteins that require additional assistance with folding, says Cynthia Collins, general manager of Ginkgo’s reagent division.

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Researchers who expect to use large amounts of lysate can reduce costs and customize reagents by producing their own. However, Noireau suggests that scientists who are new to cell-free protein expression begin with standardized commercial products.

Lysates, whether homemade or commercially available, must be supplemented with components such as glucose and nucleotides. These reagents recreate aspects of the cellular environment, provide energy and supply raw materials for protein translation, says Jewett.

His laboratory found ways to reduce costs by identifying which components were essential. The researchers tested 1,231 different formulations using laboratory-produced lysates and optimized the recipe to just 12 reagents. This reduced costs by 95%, to less than $100 per gram of protein produced.³

Human-cell lysates can support complex proteins

Lysates made from other cell types are also available. They can offer advantages such as the ability to complete post-translational modifications, including the addition of sugars and ubiquitin molecules—processes that are not possible with standard bacterial systems.

Historically, researchers often used lysates derived from immature red blood cells, or reticulocytes, obtained from anemic rabbits. This model raises ethical concerns about animal use as well as scientific concerns about its translation process.

Reticulocytes do not require a “cap” on messenger RNA to protect the molecule and facilitate translation. They also contain enzymes that degrade RNA transcripts. Kalsis had to abandon a three-year project after discovering that the rabbit reticulocyte lysate he was studying used a non-standard approach to RNA degradation.⁴

Kalsis suggests that researchers studying translation should choose lysate systems that closely match the biology they are investigating, rather than selecting a system solely for its ability to produce proteins.

To support that approach, the research group he previously led at the University of Bern developed a technique for creating lysates from several human cell lines. They generated cell-free translation systems using cells derived from cervical cancer (HeLa), embryonic kidney (HEK-293), neuroblastoma (SH-SY5Y) and osteosarcoma (U2OS).⁵

Of these systems, lysates from HeLa cells attached to culture dishes provided the most efficient translation, he says.

Plant lysates could accelerate chloroplast engineering

Plants also provide useful cell-free systems. Lysates from chloroplasts—the light-harvesting organelles in plant cells—are particularly valuable, says synthetic biologist Henrike Niederholtmeyer of the Technical University of Munich in Straubing, Germany.

Plant bioengineers sometimes prefer to express transgenes in chloroplasts rather than in the nucleus. Modifications to nuclear DNA can make transgenes silent or cause their expression to be unpredictable. Biological containment is another concern because nuclear DNA can be transported in pollen.

Using artificial chloroplast systems could help researchers avoid these problems.

Chloroplast engineering is not easy, says Niederholtmeyer, and it takes time. To test genes and proteins, scientists must wait for plants to grow. Testing transgenes or regulatory sequences in chloroplast lysates can provide results much sooner.

Niederholtmeyer, Jewett and their colleagues compared chloroplast extracts from wheat, spinach (Spinacia oleracea) and poplar (Populus × canescens). They found that the spinach system could predict expression in the distantly related wheat plant.⁶

A large number of leaves are needed to obtain sufficient extract from chloroplasts alone. Niederholtmeyer says her students bought spinach from a local supermarket each week and scaled the reaction down to 400 nanoliters.

The study suggests that spinach chloroplast lysate could provide a reliable alternative for testing genetic designs in other plant species.