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By borrowing life machines from broken cells, researchers are producing new proteins, cheap laboratory reagents, and vaccines that only require water

The prefix "biology" may mean life, but life is not essential to many biological processes. In 1897, German researcher Eduard Buchner showed how the residue and juice left after yeast was cut and crushed can still promote sugar fermentation. In doing so, he won the Nobel Prize and finally defeated the "vitality theory"-that is, the activities of living organisms have some special things that cannot be explained by biological and physical explanations.

Freeing biochemistry from the limitations of living cells facilitated other breakthrough discoveries. Most notably, the so-called cell-free system that can shape amino acids into proteins helped Marshall Nirenberg and J. Heinrich Matthaei decipher the genetic code at the National Institutes of Health in 1961 (1). About 60 years later, the rise of synthetic biology has rekindled scientific interest, liberating life machines from living cells-from gene expression to protein synthesis. The researchers were once again fascinated by the freedom that Buchner first exploited.

Since there is no need to cross cell membranes, maintain cell lines, and worry about toxins that may kill or reduce cell viability, cell-free systems enable researchers to control, destroy, and develop biochemical tools for life. Modern cell-free systems can be used to help design and manufacture new proteins or design and test new metabolic pathways. Expensive reagents can be replaced with cheap portable reagents that can be dried and reconstituted on demand in remote or resource-poor environments. Cell-free ingredients can be used to make vaccines and drugs on the spot.

"In the late 1990s and early 2000s, with the development of synthetic biology, the work on cell-free systems experienced some renaissance. People began to realize that this was an excellent way to prototype, design, test, and various other applications. ," said Paul Fremont, a synthetic biologist at Imperial College London, UK. "The future use of cell-free as a manufacturing platform for high-value components such as antibodies will have a lot of impetus." Obstacles still exist: some cell-free processes are difficult to scale up, and many processes are expensive, so this method will not quickly eliminate the current situation. There are ways. But it has been successful in some niche and professional applications, and has helped researchers push the boundaries of protein manufacturing.

Cell-free biology will not replace traditional practice anytime soon, but it has already achieved success in some niche applications, while helping researchers push the boundaries of protein manufacturing. Image source: Dave Cutler (artist).

By far, one of the most useful applications of cell-free biology may be portable biosensors. About 5 years ago, researchers developed a paper-based diagnostic method for Zika virus and deployed it as a research tool and monitoring tool in multiple countries/regions. Reprinted with permission from reference. 12.

The easiest way to a cell-free system is to follow Buchner's example and open (lyse) living cells. This is not as easy as it sounds. Although his competitors, such as Louis Pasteur, tried unsuccessfully to flatten the 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 such as bombarding cell suspensions with tiny steel balls that are torn into the cytoplasm, adding buffer chemicals to weaken membrane proteins by raising pH, or using detergents Make the protein more soluble.

Once a cell overflows its contents, researchers usually remove some cell debris. The genomic DNA and any surviving unlysed cells are separated by centrifugation. What remains in the solution (lysate) are useful compounds, including metabolic enzymes and biological components required for protein synthesis, such as ribosomes.

For some cell-free applications, this coarse mixing of the residue is sufficient. In 2018, researchers at the University of Texas at Austin showed that lysates can be used instead of expensive commercial enzymes as chemical reagents for laboratory reactions (2). These enzymes, including those used to amplify DNA in polymerase chain reaction (PCR), are usually produced by modified bacteria in an industrial process; they are then extracted and purified. The University of Texas team found that the purification step was unnecessary. Instead, they can simply lyse the cells and use the cell-free extract as a reagent.

In a series of common laboratory procedures, including PCR and plasmid synthesis, the researchers demonstrated that the lysed modified E. coli performed as well as the purification reagents. "We think this will be a great way for anyone to make readily available reagents in their laboratory," said Sanchita Bhadra, a biologist who leads the Texas research.

The production cost of these "cell reagents" is only a small part of the purified protein, and the sensitivity to temperature is much lower, which means that they do not need to be stored in the refrigerator or freezer. This makes them an attractive choice for researchers in many parts of the world who have difficulty obtaining commercial reagents due to high costs or difficulty in importing from abroad. Bhadra's team discovered that they can even freeze-dry the lysed cells and store them at room temperature for several months, then rehydrate and use them as before.

Earlier this year, the researchers further improved the process, showing that the freeze-drying step is unnecessary. The bacteria can grow, lyse, and then be dried using simple drying chemicals in a standard laboratory incubator. To demonstrate the flexibility provided by this method, they sent the dried cell lysate to personal luggage via FedEx, and then successfully used it for laboratory reactions with collaborators in Cameroon and Ghana (3).

"We have brought this technology to where it is truly usable. People outside of our laboratory can use this technology and it works," Bhadra said. She added that this approach is not always appropriate. The exact composition of the processed cell-free mixture is not necessarily the same, which means that the performance of different batches may be different. "For regulated processes such as clinical diagnosis, this has not yet been approved."

Nevertheless, for research purposes, cell-free systems can also make common proteins that are difficult to express in living cells. For example, membrane proteins such as receptors, channels, and transporters account for about a quarter of all human proteins, and almost half of the drug targets. But as we all know, they are difficult to study. The supply of natural sources is insufficient, so researchers turned to bacteria such as Escherichia coli to overexpress these proteins in sufficient quantities for research.

Even very low levels of many of these membrane proteins prove to be toxic to the host's bacterial cells, leading to low yields. Cultures based on yeast, mammalian and insect cells also face the same problem. This is one of the reasons why membrane proteins only represent about 1% of the resolved protein structure.

Even if the cells themselves cannot survive, turning on the bacterial cells allows their gene expression mechanisms to work. By adding the necessary genetic instructions, researchers can use these cell-free systems to make specific proteins on demand.

In 2016, biologists at the RIKEN System and Structural Biology Center in Yokohama, Japan used the cell-free E. coli system to produce a large number of 19 mammalian membrane proteins. They found that the resulting protein is usually easier to obtain and purify than when combined in intact cells, as in traditional production based on live cultures (4).

Removing cells from the picture—and the need to keep them alive—also allows researchers to make complex or foreign proteins, including proteins that don't exist in nature. One method is to force the cell to convert the amino acids it uses when translating genetic information into protein. For example, the amino acid canavanine (naturally found in the seeds of certain plants to defend against insects) is considered to have useful medicinal and medical properties (5). But as a non-protein forming amino acid, canavalinine does not exist in natural protein, so it is difficult to study its biological effects.

The standard way to incorporate this non-classical amino acid into proteins in living cell culture is to trick the cell’s genetic machinery and replace the normally incorporated amino acid with the desired intruder. Canavanine is an analogue of the classic amino acid arginine, so it can theoretically be substituted for it in proteins. But canavalinine is lethal to many animal cells (making it an effective plant defense). Proteins that carry canavalinine instead of arginine are less stable and block the pathways that regulate and catalyze reactions. This toxicity effectively prevents the production of potentially useful canavalinine-based proteins in living cell cultures.

However, by using a cell-free E. coli system, German researchers showed in 2015 that they can successfully express and collect a large amount of green fluorescent protein, in which each arginine molecule is replaced by a canavalinine molecule (6).

And canavanine is not the only troublesome protein that can be produced more efficiently in a cell-free system. Others include a candidate anticancer drug called onconase, which is only found in the eggs of the northern leopard frog. Due to a common problem of recombinant DNA technology, Onconase is difficult to express in cell lines. High levels of protein expression can overwhelm the cell's ability to ensure that they form and fold correctly. As a result, a large number of partially folded and misfolded protein molecules aggregate into so-called inclusion bodies in living cells. These unwanted aggregates are difficult to handle, and they reduce the efficiency of the gene expression system.

One reason for the failure of cell quality control is that the accumulation of oncogene enzymes in the cytoplasm degrades the circulating chains of transfer RNA (tRNA), which play a key role in assembling the amino acid chains that will form the protein. Increasing tRNA levels in living cells is impractical because they do not pass through the cell membrane. But in a cell-free system, this is a trivial task. By adding additional doses of tRNA every 15 minutes, chemical engineers at Brigham Young University in Provo, Utah, created a cell-free system based on E. coli that can produce a large number of oncogenes (7).

This cell-free protein synthesis system (also known as the transcription-translation system or TX-TL) cannot rely solely on the natural components remaining in the cell lysate. These components need to be supplemented with a buffer mixture that contains all the other original components needed to regulate gene expression responses, including enzyme cofactors, energy sources, nucleotides, substrates, amino acids, and tRNA. This started to become expensive.

The high price of raw materials means that the price of cell-free synthesis will rise rapidly as the process expands to produce commercial quantities of protein.

An economic evaluation conducted by process engineers at Imperial College London last year compared the relative cost-effectiveness of monoclonal antibody preparation using a traditional Chinese hamster ovary (CHO) cell line and a CHO cell-free system (8).

Assuming that 200 kg of antibodies are produced on an industrial scale each year, the evaluation concluded that the production cost using standard cell line technology is approximately US$85 per gram of protein. However, using the existing cell-free technology to produce the same amount of product, the cost per gram will be US$2,700, which is more than 30 times that of uncompetitive. For small-scale production (25 kg/year, which may be required for early analysis, for example), the difference is small but still significant: standard cell lines are $958/g, and cell-free lines are $3,230/g.

There is a key challenge: Researchers still need to use expensive fermentation processes to produce cells, and then lyse the cells to make cell-free extracts. "So your cost is the same as for a cell-based process. Most importantly, you have the additional cost of a cell-free reaction," said Cleo Kontoravdi, a chemical engineer who led the evaluation at Imperial College.

"I have not studied the economics of cell-free and standard cell culture production, but I am not surprised," said Allen Liu, a mechanical engineer who works with cell-free systems at the University of Michigan in Ann Arbor. "Traditional protein production efficiency will be higher because cells have their own metabolic capacity and can produce large amounts of required raw materials."

Although for many commercial proteins, cell-free manufacturing may not provide a more cost-effective method than existing methods, it may be competitive for specialty proteins. At least one company is already doing business. Headquartered in San Francisco, California, Sutro Biopharma operates a production facility based on cell-free protein synthesis, focusing on specialized, difficult-to-manufacture proteins for the pharmaceutical industry.

Cell-free has another value: portability. "You have a distributed manufacturing opportunity," Fremont pointed out. "If the cell-free extract can be freeze-dried and you have the genetic code to make the product, then you can distribute this material anywhere in the world and make the product." Or indeed, away from the world. He added that NASA has a large-scale project on cell-free systems. The agency is exploring cell-free extracts for space applications.

Using principles similar to cell-free reaction reagents that can be dried and transported at room temperature, researchers are exploring how to do the same for therapies and vaccines. For example, earlier this year, researchers at Northwestern University in Evanston, Illinois, showed that they can use a cell-free process to produce conjugate vaccines. Usually used to prevent bacterial diseases in children, such as meningitis C, conjugate vaccines combine complex sugars from the surface of infectious bacteria with proteins known to stimulate a strong immune response, such as toxins from another bacteria. This makes them more challenging and more expensive to manufacture than traditional vaccines.

The researchers worked in Escherichia coli and created a lysate containing bacterial sugars. Then they added the DNA needed to make the protein and freeze-dried the mixture. When they later added water, the cell-free system expressed the protein and combined it with sugar to make a conjugate vaccine (9).

The study showed that a usable vaccine against Francisella tularensis (Francisella tularensis) can be produced within one hour of adding water to a dry mixture, which is a highly toxic substance classified as a category A bioterrorism agent. Power bacteria. Tested on mice, the instant vaccine protects the animals from bacteria.

This work builds on earlier experiments that showed how particles of freeze-dried cell components can be combined with similar particles containing specific DNA and recombined to produce a series of useful proteins. These include small proteins and antibacterial compounds that can be used as diphtheria vaccines (10).

James Collins, a synthetic biologist at the Massachusetts Institute of Technology in Cambridge who participated in the study, said that one of the most useful applications of the technology so far is the manufacture of rugged and portable biosensors. In 2014, he participated in a project to integrate gene switches into paper-based cell-free sensors that change color when they encounter Ebola virus DNA (11). A year and a half later, the team developed a paper-based Zika virus diagnosis method and deployed it as a research tool and monitoring tool in many countries/regions (12).

He envisioned that doctors, soldiers and even astronauts could carry acellular particles. "Then when you need a specific drug or therapeutic molecule, you just add the cell-free particle to the water, along with the particle that encodes the molecule you are interested in, and it will be produced," Collins said.

In practice, some challenges beyond cost must be overcome before cell-free systems can be widely used. One is that many complex proteins need to be significantly modified by specific enzymes after expression. Researchers have only just begun to understand how this post-translational modification of many proteins occurs in living cells. Over millions of years, living cells have evolved all the ingredients they need to work. Attempting to perform the same complex metabolic dance in a streamlined cell-free system is much more difficult.

"We are not fully in place yet," Collins said. "As a field, it is exciting to find the instructions, ingredients, and manufacturer materials needed to get everything you need in a cell-free system."

"As a field, it is exciting to find the instructions, ingredients, and manufacturer materials needed to get everything you need in a cell-free system."

Although the cell-free system offers a series of compelling future applications, Fremont emphasizes the strengths of basic researchers who basically follow Buchner's method-trying to understand the basic principles of how living cells work and what they can produce . "The cell-free system has an element of discovery," he said.

For example, the Fremont laboratory is repairing cell-free gene expression in bacteria of the genus Streptomyces, which is the source of most known antibiotics. "You can start expressing different gene clusters and see what new products you can make," he said. "We are at the beginning of exploring the huge opportunities in the entire region."

In fact, there are many opportunities. In order to see the possibilities, researchers only need to push the boundaries of cells. As Buchner said in his Nobel speech in 1907, to study the contents of a container, it must first be opened (13).

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