A cell-free system to unlock the Sec translocon for research and biotechnological applications
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Abstract
Membrane proteins equip cells with diverse and crucial functions. Most of them are integrated into the membrane via the Sec translocation machinery, a universally conserved membrane complex found across all kingdoms of life. Due to its essentiality but also because of implications for synthetic biology and biotechnology, Sec has been a popular research target for decades. However, both research and applications have been limited by toxicity issues in vivo and the lack of efficient analysis methods.
In this work, I describe a powerful in vitro system to study and engineer Sec, which enables the collection of large amounts of quantitative data. I use cell-free protein synthesis (CFPS) to synthesize and integrate functional E. coli SecYEG into blank synthetic vesicles. CFPS is combined with a luminescence assay to quantitatively probe translocation and insertion of model Sec substrates in real time, enabling direct testing of Sec variants produced from DNA libraries in high-throughput, circumventing longstanding viability constraints.
Screening ~300 SecY variants in a single experiment, I consolidate three decades of Sec research, while vastly expanding structure-function insights. Mapping over 30 functionally critical regions that modulate Sec activity across three orders of magnitude, I uncover dozens of highly active variants with up to eightfold increased translocation, and one mutant with improved insertion efficiency.
The purely genetic design allows one to easily add or remove auxiliary components of the Sec machinery, like SecB, SecA, the signal recognition particle pathway (SRP/SR), LepB and YidC, which I leveraged to study their complex interaction network and even engineer their activity. The system further improved the quality of a cell-free synthesized model membrane protein setting the foundation for the future use as a platform for simple membrane protein production in CFPS.
Using CFPS produced SecYEG as a model for the secretion of recombinant proteins, I engineered the secreteability of a nanobody improving its translocation more than sevenfold. Lastly, I specifically optimized the Sec system for maximum in vitro activity reaching about twelve times better nanobody translocation than the WT machinery making not only a powerful system to study SecYEG but also a tool to open up SecYEG for advancing applications in synthetic biology and biotechnology.
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Issued: 2026-06-12
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Other relations: https://doi.org/10.64898/2025.12.09.688994
Faculty
FB17:Biologie
Language
en
Keywords
membrane proteincell-freetranslocationsecretionSecYEG
DFG-subjects
2.11-01 - Biochemie
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Meier, Markus: A cell-free system to unlock the Sec translocon for research and biotechnological applications. : 2026-06-12. DOI: https://doi.org/10.17192/openumr/878.
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Except where otherwised noted, this item's license is described as Attribution-NonCommercial-NoDerivatives 4.0 International