Releasing Protein Cargo from Synthetic Cells by Coupling Cell-Free Expression to Osmotic Actuation
- 1TU Darmstadt
Abstract
Macromolecular release from synthetic cells often relies on membrane pores or channels. We explored whether internally generated osmotic stress could increase membrane permeability and promote release. Using double-emulsion microfluidics, we encapsulated an Escherichia coli cell-free expression system in Pluronic L121 polymersomes to co-express the fluorescent protein mScarlet3 and the dextranase PsDex1711 in the presence of dextran. PsDex1711-mediated dextran hydrolysis produced a greater increase in bulk osmolality than reactions without PsDex1711 DNA. Tracking hundreds of individual polymersomes showed that PsDex1711-containing populations expanded, whereas control populations contracted. The distribution of normalized fluorescence revealed a subset with lower residual fluorescence than the main population. In selected expanded polymersomes, integrated internal mScarlet3 fluorescence declined to 18-22% of peak levels, without visible membrane disruption, consistent with protein release. These findings support a proof of concept for using internal gene expression to promote macromolecular release through osmotic actuation without introducing dedicated membrane pores.
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BibTeX
@misc{liu2026releasing,
title = {{Releasing Protein Cargo from Synthetic Cells by Coupling Cell-Free Expression to Osmotic Actuation}},
author = {H. Liu and J. Marlhens and S. Schubotz and H. Koeppl},
howpublished = {bioRxiv},
year = {2026},
month = oct,
doi = {10.64898/2026.10.06.757017},
url = {https://www.biorxiv.org/content/10.64898/2026.10.06.757017v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Releasing Protein Cargo from Synthetic Cells by Coupling Cell-Free Expression to Osmotic Actuation
AU - H. Liu
AU - J. Marlhens
AU - S. Schubotz
AU - H. Koeppl
PY - 2026
DA - 2026/10/06
PB - bioRxiv
DO - 10.64898/2026.10.06.757017
UR - https://www.biorxiv.org/content/10.64898/2026.10.06.757017v1
AB - Macromolecular release from synthetic cells often relies on membrane pores or channels. We explored whether internally generated osmotic stress could increase membrane permeability and promote release. Using double-emulsion microfluidics, we encapsulated an Escherichia coli cell-free expression system in Pluronic L121 polymersomes to co-express the fluorescent protein mScarlet3 and the dextranase PsDex1711 in the presence of dextran. PsDex1711-mediated dextran hydrolysis produced a greater increase in bulk osmolality than reactions without PsDex1711 DNA. Tracking hundreds of individual polymersomes showed that PsDex1711-containing populations expanded, whereas control populations contracted. The distribution of normalized fluorescence revealed a subset with lower residual fluorescence than the main population. In selected expanded polymersomes, integrated internal mScarlet3 fluorescence declined to 18-22% of peak levels, without visible membrane disruption, consistent with protein release. These findings support a proof of concept for using internal gene expression to promote macromolecular release through osmotic actuation without introducing dedicated membrane pores.
ER -