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bioRxiv · 10.64898/2026.10.06.757017

Releasing Protein Cargo from Synthetic Cells by Coupling Cell-Free Expression to Osmotic Actuation

Liu, H.·Marlhens, J.·Schubotz, S.·Koeppl, H.
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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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Liu, H., Marlhens, J., Schubotz, S., Koeppl, H.. 2026-10-07. Releasing Protein Cargo from Synthetic Cells by Coupling Cell-Free Expression to Osmotic Actuation. https://doi.org/10.64898/2026.10.06.757017

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