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

Authors

Liu, H., Marlhens, J., Schubotz, S., Koeppl, H.

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.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 08 Oct 2026.

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