Optimizing Protein Production in the One-Pot PURE System: Insights into Reaction Composition and Expression Efficiency
Creators
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1.
California Institute of Technology
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2.
Imperial College London
Abstract
The One-Pot PURE (Protein synthesis Using Recombinant Elements) system simplifies the preparation of traditional PURE systems by coculturing and purifying 36 essential proteins for gene expression in a single step, enhancing accessibility and affordability for widespread laboratory adoption and customization. However, replicating this protocol to match the productivity of traditional PURE systems can take considerable time and effort due to uncharacterized variability. In this work, we observed unstable PURE protein expression in the original One-Pot PURE strains, E. coli M15/pREP4 and BL21(DE3), and addressed this issue using glucose-mediated catabolite repression to minimize burdensome background expression. We also identified several limitations making the M15/pREP4 strain unsuitable for PURE protein expression, including coculture incompatibility with BL21(DE3) and uncharacterized proteolytic activity. We showed that consolidating all expression vectors into a protease-deficient BL21(DE3) strain minimized proteolysis, led to more uniform coculture cell growth at the time of induction, and improved the stoichiometry of critical translation initiation factors in the final PURE mixture for efficient cell-free protein production. In addition to optimizing the One-Pot PURE protein composition, we found that variations in commercial energy solution formulations could compensate for suboptimal PURE protein stoichiometry. Notably, altering the source of E. coli tRNAs in the energy solution alone led to significant differences in the expression capacity of cell-free reactions, highlighting the importance of tRNA codon usage in influencing protein expression yield. Taken together, this work systematically investigates the proteome and biochemical factors influencing the One-Pot PURE system productivity, offering insights to enhance its robustness and adaptability across laboratories.
Copyright and License
Copyright © 2025 American Chemical Society.
Acknowledgement
We sincerely thank the Murray Lab members, particularly Dr. Zoila Jurado and Miryong (Miki) Yun, who contributed to the project’s early setup and discussions. We also thank b.next, a company working on open-source synthetic cell protocols (https://bnext.bio), for insightful discussions on the 36-Pot PURE preparation. We also thank Dr. Jurado, Manisha Kapasiawala, and Dr. John Marken for their review and feedback on this manuscript. Dr. Jurado prepared the PT7-MGA-UTR1-deGFP plasmid28 used in this work. We thank Prof. Lulu Qian for access to the ChemiDoc Imager used for protein gel image acquisition. We thank the Caltech Protein Expression Center (PEC) for preparing the purified T7 RNAP used in this work. The chemical, protein, and tRNA icons used in Figures 6A and 7A are obtained from BioRender under the Academic licensing agreement ZF282CDXBI. The authors made all the graphics in Adobe Illustrator.
Funding
R.M.M. and Y.Z. are supported by the National Science Foundation, award MCB-2152267, and Schmidt Futures, grant number 2023-3-2-1. Y.Z. is also supported by the Caltech Presidential Postdoctoral Fellowship. P.S.F. and M.D. are supported by the Engineering and the Physical Sciences Research Council (EPSRC), award EP/T013788/1. M.D. is also supported by the EPSRC Centres for Doctoral Training in Biodesign Engineering at Imperial College London. L.B. is supported by the Caltech Summer Undergraduate Research Fellowship (SURF).
Contributions
Y.Z., R.M.M., P.S.F., and T.C.: Conceptualization; Y.Z., M.D., Y.Q., L.B., and Z.A.M.: Investigation; Y.Z., M.D., Y.Q., L.B., and Z.A.M.: Analysis; Y.Z., M.D., L.B., and Z.A.M.: Writing–manuscript; Y.Z., M.D., R.M.M., P.S.F., L.B., Z.A.M., Y.Q., and T.C.: Writing–review and editing; Y.Z., M.D., L.B., and Z.A.M.: Visualization; R.M.M., P.S.F., and T.C.: supervision.
Conflict of Interest
The authors declare the following competing financial interest(s): R.M.M. has a financial stake in Tierra Biosciences, a private company that uses bacterial lysate-based cell-free technologies for protein expression and screening.
Supplemental Material
Sequence information for all plasmids used in this work, including their Addgene catalog numbers where applicable (PDF).
Extended methods for One-Pot PURE protein purification, homemade PURE energy solution preparation, T7 RNA polymerase and deGFP purification, and the mass spectrometry workflow for proteomics analysis (PDF).
Supplementary figures and tables referenced throughout the main text, providing additional analysis and characterizations to support the paper’s conclusion (PDF).
Files
sb4c00779_si_001.pdf
Additional details
Identifiers
- PMID
- 40209036
Related works
- Describes
- Journal Article: 40209036 (PMID)
Funding
- National Science Foundation
- MCB-2152267
- Schmidt Futures
- 2023-3-2-1
- California Institute of Technology
- Engineering and Physical Sciences Research Council
- EP/T013788/1
Dates
- Submitted
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2024-11-09
- Updated
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2025-03-25
- Accepted
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2025-03-25
- Available
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2025-04-10Published online