Absence of cells makes usage of toxic or extreme environmental conditions like high pH or temperature feasible. Also not using cells makes the long process of cell culturing disappear which I think is one of the greatest advantage. Also its easier to add tagged amino acids or artificial components using efficient isotope or fluorescence tagging.
Two cases for me would be : using toxic and extreme environment and another would be no need of tedious cell culturing 1. Describe the main components of a cell-free expression system and explain the role of each component. A cell free expression system mainly consists of mixture of pure elements required for protein synthesis, such as enzymes, nucleotides, amino acids, and other molecular machinery. Because of the system's ability to replicate the biological environment, mRNA can be translated into protein.
Amino acids: synthesize proteins directly as there is no cell, they work on mRNA and form proteins. as amino acids are what make up proteins.
Transfer RNA: carry out translation of mRNA into protein by carrying specific amino acids to the ribosome and making sure that right amino acids are bring added to the elongating peptide
ATP /NTP sources for energy: three main steps of transcription ie initiation, elongation and termination require energy, hence energy sources are very essential in a cell free expression system.
Ribosomes : They catalyze the creation of peptide bonds between amino acids in cell-free systems, converting the mRNA template into protein.
Covalent ions like Mg2+, NH4+: for enzyme activation and functioning
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https://www.creative-biostructure.com/cell-free-expression-524.htm
Energy sources are extremely important for the transcription-translation system, for the efficient synthesis of protein from mRNA, the system requires constant supply of energy but this has to be one of the main limitations to the success of these systems is providing energy for processes involved in cell-free protein synthesis. A method that I would suggest would be employed to give constant ATP supply and which can be feasible and economical at the same time would be:
Dual energy system
Use of glucose as a cheaper energy source offers a prospect for the application of cell-free protein synthesis in the production of industrial proteins. During its conversion into pyruvate through the glycolytic pathway in the S30 extract, one molecule of glucose can generate 2 molecules of ATP, and there are sufficient evidences showing that at least part of pyruvate molecules are consumed for ATP regeneration through oxidative phosphorylation pathway.
| Prokaryotic cell free expression system | Eukaryotic cell free expression system |
|---|---|
| simple to work with | complex compared to prokaryotic system |
| has higher speed of production | relatively slower production of protein |
| system has a lower cost | higher cost associated with culturing of eukaryotic cells |
| I would produce Green fluorescent protein in prokaryotic cell free expression system . GFP has a high demand in the biology sector because of its nature and role, it serves as a biosensor. Also GFP is non glycosylated and has a simple structure that can be synthesized in a simple system like this. | I would produce Insulin in the eukaryotic cell free expression system. Eukaryotic systems, like yeast or mammalian cells, can express proinsulin, which includes the A, B, and C chains. These cells have the necessary proteases to cleave the peptide. Also insulin is a very important protein hormone required for the regulation of sugar |
For this we will have to mimic the nature of membrane of a cell, cell membrane is hydrophobic in nature so there have to be the attributes that make the system hydrophobic in nature Addition of lipid elements can do that. Also the machinery we will be using for the system has to be Membrane protein friendly. There should be energy regeneration components to maintain system activity.
I think in context of membrane protein production my main challenge would be replicating the natural level of hydrophobicity of the cell membrane Membrane proteins tend to stick together in water-based systems because they are hydrophobic. However, this problem can be solved by using membrane mimetics, like lipid nanodiscs, liposomes, or mild detergents, which act like natural membranes and help them fold and dissolve properly.
Other general problems will be there too like
misfolding leading to low activity of the produced protein is a typical issue, especially for complex or eukaryotic proteins. environment. Chaperone added to the procedure can also help in directing protein folding, avoiding aggregation, and increasing the yield.
toxicity of the produced protein , can lead to inactivity of enzyme even before it .continuous exchange cell free systems in this case , in which a semipermeable membrane connects the reaction chamber to a feeding chamber. This configuration keeps the expression system active and , even for toxic proteins, by constantly eliminating inhibitory byproducts. **
https://www.creative-biolabs.com/blog/index.php/membrane-protein-overview/
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.
Low yield can be a reason of various inputs or reactions taking place ina cell free expression system some of them might be as follows
A. Misfolding of protein: The protein produced might fold since there is absence of natural system and its factors like chaperons or different temperature than the system requires. this misfolding can eventually lead to poor yield
Troubleshooting: Addition of temperature, lowering the temperature according to the protein of interest that the proteins don't fold
B. Constant supply of energy may not sustain for long time, as the cell free machinery requires supply of ATP , non efficient resource system can lead to poor yield
Troubleshooting: a energy system that employs slow and stable supply of ATP energy should be employed, also we should try to get a system that makes the use of present machinery to regenerate energy than creating it from scratch. for ex. phosphoenolpyruvate system.
**C. Unstable/ poor quality of elements : This can lead to poor yield of protein as well as the its cell independent and these machinery components are the building blocks of this system.
Troubleshooting: the component of system must be freshly prepared and the source or manufacture must be validated. also the amount of these components must be accurate. About the stability, components with better stability must be checked first and then used to conserve resources
Unwanted enzyme activity in the environment can lead to reduced protein production too, there are no cells but the possibility of proteases like enzymes shouldn't be neglected. for this enzyme-immune versions can be used also use inhibitors is highly recommended
Here is my documentation for my Individual final project:
Individual Final Project Documentation
Lectures, recitations and slides
*Calhoun, K.A., Swartz, J.R. (2007). Energy Systems for ATP Regeneration in Cell-Free Protein Synthesis Reactions. In: Grandi, G. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology™, vol 375. Humana Press. https://doi.org/10.1007/978-1-59745-388-2_1
Kim, H.-C., & Kim, D.-M. (2009). Methods for energizing cell-free protein synthesis. Journal of Bioscience and Bioengineering, 108(1), 1–4. https://doi.org/10.1016/j.jbiosc.2009.02.007
Garenne, D., Haines, M.C., Romantseva, E.F. et al. Cell-free gene expression. Nat Rev Methods Primers 1, 49 (2021). https://doi.org/10.1038/s43586-021-00046-x
https://www.thermofisher.com/in/en/home/life-science/protein-biology/protein-expression/cell-free-protein-expression.html?ef_id=bf282f50a26c179638954765731c6d2b:G:s&s_kwcid=AL!3652!10!77240849382047!!!!2329040505681148!!401481115!1235851312539978&cid=bid_clb_pex_r01_co_cp0000_pjt0000_bid00000_0se_bng_dy_pur_con*
OpenAI ChatGPT (2025). explained concepts. OpenAI. Retrieved from https://openai.com/chatgpt
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