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.
   1. Some proteins are very difficult to express in traditional, cell-based systems. This could be because they would be toxic, such as with antimicrobial peptides, or because they have non standard amino acids. Cell free systems allow researchers to synthesise these otherwise difficult to produce proteins. In addition, batch-to-batch variation in cell culture may result in intolerable variation. Using cell free systems allows for more control, as you can have the same starting mixture experiment after experiment. Two cases where cell free expression is more beneficial than cell production is that they can allow you to build more robust genetic circuits, with less noise. In addition, since they are not alive, they can allow you to experiment with proteins that could otherwise pose a biological hazard.
2. Describe the main components of a cell-free expression system and explain the role of each component.
   1. The main components of a cell free system are
      1. Whole cell extract :
         1. ribosomes - machinery that drives translation
         2. cofactors - stabilise the ribosome and enzymes
         3. enzymes
      2. small molecules and adapters:
         1. energy- the power source used to drive all the reactions
         2. Nucle
         3. otides - drive transcription
         4. Amino Acids - drive translation
         5. tRNA - used in translation
      3. DNA genes - the gene you want to express
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.
   1. Energy provision regeneration is critical in cell-free systems because if we run out of ATP, the reaction halts. One method, outlined in Kraußer et, al. 2025, is to generate acetyl phosphate from low-cost d-fructose and inorganic phosphate substrates. This method allows for a theoretical yield of 3 mol ATP per mol of d-fructose.
4. Compare prokaryotic versus eukaryotic cell-free expression systems. Choose a protein to produce in each system and explain why.
   1. Eukaryotic expression systems have native folding machinery, microsomes for glycosylation, and supports post‑translational modifications. A proteint that can be produced in a eukaryotic expression system is a human kinase, as they require post translational modifications that cannot be accomplished in a prokaryotic expression system.
   2. Prokaryotic expression systems are often quicker than eukaryotic expression systems, but they lack the machinery to allow for post translational modifications. You can use it to produce GFP, since it is a s fairly basic protein that does not require post-translational modifications.
5. 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.

If I were to actually design a cell-free experiment to optimize the expression of a membrane protein, I would conduct a literature review, keeping in mind my particular protein of interest, to determine the range of possible systems, the common challenges faced, and the solutions that are most often used. As this is a homework exercise, I will outline my findings of a single paper of interest by Roos et al. 2014 titled “High-level cell-free production of membrane proteins with nanodiscs.”

Membrane proteins are inherently unstable when not embedded in a membrane. This is because of their hydrophobic domains that tend to clump together. Nanodiscs solve this problem by enveloping the membrane protein, stabilizing the transmembrane domains and preventing the proteins from forming aggregates.

Another challenge outlined by the paper is low expression efficiency. This is because membrane protein mRNAs often form strong secondary structures near the ribosome binding site, which can hinder ribosome access and impair the initiation of translation.

The method that Roos et al. 2014 used to overcome this challenge was a tag variation screen. A tag variation screen is where a small expression tags (6-9 codons) are “fused to the translational start site of the target coding sequence by an overlap PCR approach ( see Fig. 3). The expression tags are optimized in suppressing secondary structure formation which may prevent the initiation of translation”

Schematic illustration of a MSP nanodisc with a 7-transmembrane protein embedded. Diameter is about 10 nm. Picture from Sligar Lab

1. 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 transcription levels:

   To determine if low transcription levels is the cause of the low yield, you can run a Northern blotting, RT-qPCR, or a microarray. Since there are likely not that many proteins you are trying to express, RT-qPCR should be fine. Once you run the PCR, comparing the results to a control, if the levels are found to be low, you could add more RNA polymerase or add in RNase inhibitors. (If you suspect that active RNases are degrading your RNA, then you aught to do further testing to determine if this is the case. You could potentially alter your setup to avoid this problem in the future.)

   Low translation levels:

   If you have ruled out low transcription levels, an alternate explanation could be low translation levels. To assess translation efficiency, you could use a labeled amino acid (such as 35S0methionine or a fluorescent analog) and use that to track protein expression. If it is found to be the culprit, possible solutions would be to increase ribosome concentrations, ensure that there is high enough tRNAs and amino acids, and confirm that there is correct magnesium and potassium concentrations.

   Misfolding protein, or aggregation

   Depending on the structure of the protein you are trying to express, the culprit could be misfolding or aggregation. You should first determine if this protein is prone to misfolding/aggregation. If it is, you could add chaperones to assist folding and use mild detergents to solubilize hydrophobic domains.

Citation: Kraußer F, Rabe K, Topham CM, Voland J, Lilienthal L, Kundoch JO, Ohde D, Liese A, Walther T. Cell-Free Reaction System for ATP Regeneration from d-Fructose. ACS Synth Biol. 2025 Apr 18;14(4):1250-1263. doi: 10.1021/acssynbio.4c00877. Epub 2025 Mar 26. PMID: 40143462; PMCID: PMC12012885.

Roos C, Kai L, Haberstock S, Proverbio D, Ghoshdastider U, Ma Y, Filipek S, Wang X, Dötsch V, Bernhard F. High-level cell-free production of membrane proteins with nanodiscs. Methods Mol Biol. 2014;1118:109-30. doi: 10.1007/978-1-62703-782-2_7. PMID: 24395412.