Week 9 homework
Cell-free systems 🧪
General homework questions
1. Explain the main advantages of cell-free protein synthesis over traditional in vivo methods, specifically in terms of flexibility and control over experimental variables. Name at least two cases where cell-free expression is more beneficial than cell production.
Compared to conventional in vivo methods, cell-free protein synthesis provides modularity and substantially higher experimental control, as all the system’s components can be readily added or removed, especially when the strategy employed is to separately produce or extract each cellular element required for the process and then combine them all together into a single reaction. Cell-free systems also offer the potential for precise control over reaction conditions, such as pH and ion concentration, while being more flexible and versatile since they allow the expression of proteins deleterious to living cells, support the integration of non-natural and non-canonical amino acids into peptide backbones, and are compatible with diverse DNA templates (linear or plasmid). Additionally, they eliminate constraints imposed by the existence of living cells. For instance, unlike traditional cell cultures, they do not need any monitoring, cultivating, or other interventions aimed at preservation, nor are they susceptible to issues of cell viability, growth limits, or stress responses. Similarly, since the cell-free apparatus exists outside of the context of a cellular platform, there are no cell-membrane barriers, facilitating access to biochemical reactions, while, at the same time, there is no interference or competition from other metabolic procedures or regulatory signals, enabling all the available resources to be channeled towards the synthesis of the desired protein. The absence of living cells can be translated into abolishing the need for cloning and cellular transformation as well, which, in turn, ensures safer handling, as no genetically modified organisms are involved in cell-free protein production. More generally, one of the method’s most significant advantages is that it is a highly efficient technique for rapid protein synthesis that can also withstand being transferred across larger distances for longer periods of time, as the entire system can be easily freeze-dried and stored for later use.
For more tangible examples, more specific cases where cell-free expression is more beneficial than cell-dependent protein production are presented below:
- In theranostic applications, where the system has to be implanted in close proximity or inside the human body. Since no living cells are implicated, whose parts could potentially be recognized as harmful agents, the probability for a toxic immune or allergic reaction is low.
- In experiments conducted to study the foundations of transcription and translation. The isolation of a cell-free platform ensures the appropriate conditions to investigate gene expression mechanisms without the background noise from other cellular processes.
- For remote field testing, as cell-free systems generally require far less infrastructure than traditional cell-based production installations. Because of this, cell-free platforms can very easily be converted into portable platforms, enabling carrying out experiments, for instance, even in space.
- For on-demand biomanufacturing, since, not only are all the system’s resources directed to the generation of the desired product, but also cell-free systems can achieve higher titers in considerably less time (minutes to hours instead of days). Apart from the efficiency, the desired product is less contaminated with unwanted cellular metabolites, allowing for higher purity and, therefore, for the implementation of less complex purification methods.
2. Describe the main components of a cell-free expression system and explain the role of each component.
The main input of cell-free systems is a circular or linear DNA sequence that contains the gene to be expressed (including an appropriate promoter), while the principal output is a desired protein. The first step for the expression of the gene involves its transcription, for which the enzyme RNA polymerase is required, along with Mg2+ ions, which act as essential co-factors. For the mRNA of the desired gene to be physically synthesized, the cell-free system should contain the needed building blocks too, namely nucleotides. To effectively translate the transcript into protein, the reaction should also have access to ribosomes and tRNA molecules, which will build the peptide sequence and carry the amino acids (found in the solution too) to the correct position of the nascent peptide respectively. Lastly, the energy required for this entire machinery to function can be obtained with the addition of ATP into the cell-free system.
3. Why is energy provision regeneration critical in cell-free systems? Describe a method you could use to ensure continuous ATP supply in your cell-free experiment.
Despite their many advantages, cell-free systems are also characterized by their inability to regenerate energy, mostly due to their lack of intricate cellular structures, such as intracellular compartments and membrane protein complexes. For this reason, it is critical to “recharge” cell-free platforms of protein production with the frequent addition of ATP.
The host cells are also still naturally producing their own proteins in order to stay alive. These other proteins can interfere with or delay the production of the target protein. Furthermore, once the protein has been made, it might be difficult to separate the target protein from all the other proteins and cellular components. In some cases, the target protein may itself be toxic to the host cell, making the host cells die before significant amounts of protein can be made. To help overcome all of these challenges, researchers have developed a method of expressing proteins that doesnât require living cells, called cell-free protein expression.
There are two major strategies currently used to make cell-free reactions. Some components, like nucleotides and amino acids, can be chemically synthesized. Other components, such as ribosomes and polymerases, still need to be produced by living cells and then separated from the cells. Since scientists have to individually create and purify each component, setting up this type of cell-free reaction is still complex and costly. However, because scientists are able to individually determine every molecule that is put into the reaction, they have tremendous control over the process which can result in high-quality proteins. The second method is to extract all the components directly from host cells all at once. Scientists grow up a large amount of cells and then break them open through a process called lysis. In doing so, scientists can extract the polymerases, ribosomes, and other biological components needed for transcription and translation, and then supplement it with chemically-synthesized nucleotides, amino acids, and an energy source. This makes the entire process much simpler and more cost-effective, but it also results in a less purified reaction, as the extract will still contain many unneeded cellular components.
Since cell-free reactions donât have cells membranes getting in the way, scientists can directly interact with and manipulate the different components in the reaction. This allows them to learn more and experiment with cellular processes that were previously too difficult to study in living cells. One example is to incorporate non-natural amino acids into the reaction. There are 20 naturally occurring amino acids, but scientists have been able to develop synthetic amino acids with unique chemical properties, and then use these non-natural amino acids to build new proteins in cell-free reactions that cannot be built in natural cells. In 2018, Kazutoyo Miura and their team used nonnatural amino acids to develop a new malaria antigen, which is a small protein that mimics a pathogen used in vaccines to âtrainâ immune systems to fight against specific diseases. The non-natural amino acids in this antigen allow it to bind strongly to immune cells, trigger an immune response, and train them to recognize similar pathogens in the future. With many parts of the world still suffering from malaria and other diseases, we need new vaccines and treatments; using non-natural amino acids may help us discover them. Cell-free reactions donât have cells that need to be kept alive, but they do contain sensitive molecules that require specific storage conditions. To get around this, scientists freeze-dry the reaction to make them last longer at room temperature. By freezing the reaction and then pulling all of the water out with a vacuum pump, they produce a dry solid that is stable outside of the freezerâsimilar to how beef left at room temperature will begin to rot, but beef jerky is stable for a long time. All the user has to do is rehydrate their reaction with water, add their DNA of interest, and transcription and translation will begin. Typically, pharmaceutical companies will produce medically-relevant proteins in large batches and ship them on ice to the patients who need them. However, the live-cell production and cold shipping processes are expensive. Freeze-dried, cell-free reactions could be shipped instead so therapeutic proteins can be produced directly in small batches on-demand, virtually anywhere in the world, at a fraction of the cost
Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why. How would you design a cell-free experiment to optimize the expression of a membrane protein? Discuss the challenges and how you would address them in your setup. Imagine you observe a low yield of your target protein in a cell-free system. Describe three possible reasons for this and suggest a troubleshooting strategy for each.