Week 3 HW: Cell-Free Protocols

CFPS REACTION DESIGN FOR SFGFP, PETASE, RETEPLASE

A. COMPARE CELL-FREE REACTION COMPOSITIONS

1. Why does the concentration of potassium glutamate and magnesium glutamate differ between compositions? Which is more sensitive?

Potassium glutamate and magnesium glutamate contribute K⁺ and Mg²⁺ ions, respectively, which provide the ionic strength and the cofactors essential for activating the transcription and translation machinery, whilst the Glu⁻ anion is the predominant natural physiological anion in the cytoplasm of E. coli (1).

K⁺ stabilises the structure of the ribosome, maintains the overall ionic strength, and facilitates the interaction between tRNA and mRNA, whilst Mg acts as a cofactor for T7 polymerase and is involved in the assembly of ribosomal subunits and complex formation with nucleotides such as ATP (1). The decrease in magnesium ions in the reaction due to the accumulation of inorganic phosphates causes premature stoppage of protein synthesis (2).

The optimum amount of Mg²⁺ depends on the energy system and the nucleotides used. Systems using PEP and NTPs require more Mg²⁺ because these compounds chelate it, while systems using pyruvate and NMPs release freer Mg²⁺ and require less of this ion (1). Furthermore, the optimum value may vary between batches of cell lysate. The amount of K⁺ also varies with the concentration of other charged reagents, as the demand for potassium to maintain the ideal ionic strength and isomolarity changes (3).

Finally, based on the ranges of variation in the glutamate concentrations, it can be deduced that Mg(Glu)₂ is the most sensitive, as its variation between protocols is the smallest; this indicates that even small variations in the optimal concentration can lead to a drastic drop in protein production or a complete halt to synthesis (4).

2. Explain how a PEP + NTP reaction formulation is different from a Ribose + NMP reaction formulation.

The PEP + NTPs-based formulation utilises phosphoenolpyruvate (PEP) as an energy source to regenerate ATP, together with triphosphate nucleotides (ATP, CTP, GTP and UTP), which possess three phosphate groups and represent one of the highest costs within the system (4); however, the rapid degradation of PEP leads to a high accumulation of free inorganic phosphate, which disrupts homeostasis and inhibits protein synthesis (5).

On the other hand, the ribose + NMPs-based formulation replaces the triphosphates with monophosphate nucleotides (AMP, CMP, GMP, and UMP) containing a single phosphate group. It utilizes ribose as a low-cost, non-phosphorylated sugar to activate the endogenous salvage pathways of the cell lysate, phosphorylating the NMPs in situ to convert them into active NTPs (6,7). Furthermore, it maintains free phosphate concentrations within stable physiological ranges (7), thereby prolonging protein synthesis.

3. Choose two cell-free reaction compositions from the literature, compare and contrast the choice of reagents and the concentration of reagents.

I chose the RFopt formulation from (7) and the PANOx-SP formulation. A comparison of the two formulations reveals a 98.5% reduction in cost and a 115% increase in protein yield for the RFopt formulation compared with the PANOx-SP formulation.

The main differences driving this cost reduction are the shift in energy source (ribose instead of PEP) and the replacement of expensive NTPs with NMPs. Crucially, this higher productivity goes hand-in-hand with an increased amino acid concentration (5.0 mM in RFopt vs. 2.0 mM in PANOx-SP); because the RFopt formulation sustains active protein synthesis over a significantly longer reaction time (20+ hours compared to 3–6 hours in PANOx-SP), a larger pool of amino acids is required as building blocks to prevent substrate exhaustion and achieve higher final titers.

Moreover, fewer cofactors (such as CoA, NAD, or folinic acid) need to be added explicitly because sufficient background levels are obtained directly from cell lysate carryover, while polyamines and oxalic acid were eliminated as they showed no significant effect on yield.

Finally, the changes in buffer concentrations aim to maintain pH stability during incubations while supplying the initial phosphate pool necessary for in situ NMP phosphorylation without reaching inhibitory levels.

B. DESIGN ONE REACTION COMPOSITION FOR EACH OF THE THREE PROTEINS

1. First unique 20 µL CFPS reaction composition for each of the following:

• sfGFP

I took the sfGFP ribose as a reference and incorporated findings from GPT-5 autonomous lab optimization. To lower overall reagent costs while boosting translation titers, AMP is partially substituted with adenosine, leveraging lysate nucleoside salvage pathways for in situ ATP regeneration.

The concentrations of potassium glutamate and magnesium glutamate fall well within the established functional range for cell-free systems, representing a testable condition to evaluate whether reducing solution viscosity improves acoustic dispensing accuracy while preserving optimal ionic strength for T7 RNA polymerase activity and ribosomal translation.

• PETase

I took the Ribose PETase formulation as the reference and mainly modified the concentrations of DsbC, GSSG, and GSH. The concentration of DsbC was reduced as a cost-saving strategy, while GSSG and GSH were added to maintain a favorable redox environment and compensate for the lower amount of disulfide isomerase.

This decision was based on Thames et al. (2023) (8), who successfully used 3 µM DsbC together with 4 mM GSSG and 1 mM GSH in an E. coli-based cell-free protein synthesis system and obtained soluble, functional proteins.

In addition, there is evidence that proteins containing only one disulfide bond can be produced in E. coli-based cell-free systems, as shown in the study of El-Baky et al. (2022) (9), where biologically active TNF-α, which contains a single intramolecular disulfide bond, was successfully expressed.

Therefore, since PETase also contains only one disulfide bond, it is expected that reducing DsbC may not significantly affect its activity if an adequate redox environment is maintained. Other parameters of the formulation were also modified, but the reasons for those changes were explained previously.

• Reteplase

For Reteplase, the ribose-based formulation was again used as the reference condition. DsbC, GSSG, and GSH were maintained because Reteplase contains multiple disulfide bonds and therefore requires a strong folding and redox support system.

Maltodextrin was added together with ribose as an experimental modification rather than mainly as a cost-reduction strategy, since the price difference between both substrates is not substantial. This modification was based on Garenne et al. (2021) (10), who reported a synergistic effect when 60 mM maltodextrin and 30 mM D-ribose were combined in an E. coli-based cell-free TXTL system, reaching protein yields of up to 4.05 mg/mL.

Maltose was also added, based on previous evidence showing that it can improve cell-free system performance under some formulations (11).

Therefore, these changes were introduced mainly to evaluate whether combining multiple carbohydrate sources could improve the energetic performance of the ribose-based Reteplase system rather than simply to reduce formulation costs.

2. As a HTBAA research team, we will have the opportunity to suggest new reagents to add to the cell-free reagent inventory. Contribute (or upvote) at least one reagent to the “Community Reagents” section of the website and provide a short rationale.

The reagent I decided to upvote in the list was FkpA because it is an E. coli peptidyl-prolyl isomerase and chaperone that helps protein folding and reduces aggregation.

In Groff et al. (2014) (12), it was shown that adding FkpA to an E. coli-based cell-free protein synthesis system significantly increased protein solubility and improved the folding and assembly of IgG, particularly when used together with DsbC.

3. Visit the cfps-coopetition forum page to ensure you will be able to take part in the discussion.

I can take part in the discussion.

References

1. Jewett MC, Swartz JR. Mimicking the Escherichia coli Cytoplasmic Environment Activates Long-Lived and Efficient Cell-Free Protein Synthesis. Biotechnol Bioeng. 2004 Apr 5;86(1):19–26. doi:10.1002/bit.20026. PubMed PMID: 15007837.

2. Harman J. Development of a cell-free strategy for the directed evolution of enzymes for high-value natural products. 2021.

3. Smith AA, Wong EL, Donovan RC, Chapman BA, Harry R, Tirandazi P, et al. Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis [Internet]. 2026. Available from: http://biorxiv.org/lookup/doi/10.64898/2026.02.05.703998
   doi:10.64898/2026.02.05.703998

4. Olsen ML, Copeland CE, Sundberg CA, Aw R, Shaver ZM, Rao G, et al. Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications. 2026 Dec 1;17(1). doi:10.1038/s41467-026-69605-8. PubMed PMID: 41786720.

5. Calhoun KA, Swartz JR. An Economical Method for Cell-Free Protein Synthesis using Glucose and Nucleoside Monophosphates. Biotechnol Prog. 2008 Sep 5;21(4):1146–53. doi:10.1021/bp050052y.

6. Smith AA, Wong EL, Donovan RC, Chapman BA, Harry R, Tirandazi P, et al. Using a GPT-5-driven autonomous lab to optimize the cost and titer of cell-free protein synthesis [Internet]. 2026. Available from: http://biorxiv.org/lookup/doi/10.64898/2026.02.05.703998
   doi:10.64898/2026.02.05.703998

7. Olsen ML, Copeland CE, Sundberg CA, Aw R, Shaver ZM, Rao G, et al. Design-driven optimization of low-cost reagent formulations for reproducible and high-yielding cell-free gene expression. Nature Communications. 2026 Dec 1;17(1). doi:10.1038/s41467-026-69605-8. PubMed PMID: 41786720.

8. Thames AH, Rische CH, Cao Y, Krier-Burris RA, Kuang FL, Hamilton RG, et al. A Cell-Free Protein Synthesis Platform to Produce a Clinically Relevant Allergen Panel. ACS Synth Biol. 2023 Aug 18;12(8):2252–61. doi:10.1021/acssynbio.3c00269.

9. El-Baky NA, EL-Fakharany EM, Sabry SA, El-Helow ER, Redwan EM, Sabry A. A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha. Biology (Basel). 2022 Jan 19;11(2):157. doi:10.3390/biology11020157.

10. Garenne D, Thompson S, Brisson A, Khakimzhan A, Noireaux V. The all-E. coli TXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth Biol. 2021 Oct 26;6(1). doi:10.1093/synbio/ysab017.

11. Guzman-Chavez F, Arce A, Adhikari A, Vadhin S, Pedroza-Garcia JA, Gandini C, et al. Constructing Cell-Free Expression Systems for Low-Cost Access. ACS Synth Biol. 2022 Mar 18;11(3):1114–28. doi:10.1021/acssynbio.1c00342.

12. Groff D, Armstrong S, Rivers PJ, Zhang J, Yang J, Green E, et al. Engineering toward a bacterial “endoplasmic reticulum” for the rapid expression of immunoglobulin proteins. MAbs. 2014 May 11;6(3):671–8. doi:10.4161/mabs.28172.

Use of AI

AI was used to identify sources that could guide changes in the compositions of cell-free systems.