Abstract
Building a universal biochemical constructor, an autonomously self-replicating biochemical system, remains a challenge in synthetic biology. The PURE cell-free system is a suitable starting point for exploring self-regeneration, and its 36 non-ribosome proteins constitute the primary macromolecular components that must be regenerated. Here, we demonstrate that the PURE system can be reconstituted from proteins synthesized by PURE itself. We first show that each of the 36 non-ribosome proteins can be individually synthesized in PURE. We then purify the PURE synthesized proteins as pooled subsets and reconstitute a fully functional PURE system by combining the subsets. Finally, we show that all 36 non-ribosome PURE proteins can be synthesized simultaneously in a single PURE reaction and, after purification, can reconstitute a functional PURE system. Together, these results establish that the non-ribosome proteins components of the PURE system can be self-regenerated, representing an essential step toward the realization of a universal biochemical constructor.
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Acknowledgements
The authors would like to express their gratitude to Dr. Fanjun Li, Amogh Kumar Baranwal, and Pao-Wan Lee for helpful input and comments. We also thank Dr. Ragunathan Bava Ganesh for providing some of the purified PURE protein components and linear DNA templates. Graphical schematic representations were created with BioRender.com.
Funding
This work was supported by a Swiss National Science Foundation MINT grant (200020_214843, S.J.M).
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Mottaghi, S.S., Maerkl, S.J. PURE makes PURE: reconstitution of the PURE cell-free system from self-synthesized proteins. Nat Commun (2026). https://doi.org/10.1038/s41467-026-73337-0
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DOI: https://doi.org/10.1038/s41467-026-73337-0
