Artificial cells with liquid–liquid phase separation–regulated cell-free protein synthesis

Dongdong Fan, Kaini Liang, Bingjie Wu, Michael W. Chen, Chengyu Sun, Lei Sun, Yan Zhang, and Yanan Du 

PNAS; November 21 2025; 122 (47) e2511283122; https://doi.org/10.1073/pnas.2511283122

Significance

While artificial cells offer exciting prospects in synthetic biology for mimicking life and enabling sophisticated functions, achieving dynamic control over their internal processes remains challenging. Here, we engineer artificial cells with responsive protein expression capabilities by harnessing liquid–liquid phase separation (LLPS) to precisely control protein synthesis. We further demonstrate the utility of these responsive artificial cells as in vivo disease sensors in a pathological model. This research proposes a strategy for controllable protein synthesis within artificial cells and significantly advances their potential for in vivo biomedical applications, particularly in diagnostics.

Abstract

The rapid advancement of synthetic biology has enabled the construction of artificial cells that closely mimic the morphology and functionality of their natural counterparts. However, significant limitations remain in engineering artificial cells capable of regulated protein expression. Here, we demonstrate that engineered polymers containing multivalent association motifs can reversibly regulate translational activity through liquid–liquid phase separation (LLPS)–induced protein aggregation, enabling precise temporal control of cell-free protein synthesis (CFPS) activity. This aggregation mechanism exerts a broad inhibitory effect on various enzymes and facilitates the construction of artificial cells with controllable reaction processes. Leveraging this phenomenon, we have developed a microfluidic platform to fabricate giant unilamellar vesicles (GUVs) that encapsulate CFPS systems, thereby constructing artificial cells with finely tunable protein expression. By incorporating targeted DNA templates, these artificial cells can selectively express specific proteins in response to pH adjustments. Furthermore, in vivo studies using a bile duct ligation mouse model with liver injury further confirmed significant differences in protein expression under alkaline conditions compared to neutral conditions. Our findings highlight the potential of leveraging aggregate dynamics for precise, in situ modulation of protein synthesis within artificial cells, thereby opening avenues for their advanced biomedical applications.

See https://www.pnas.org/doi/10.1073/pnas.2511283122

Figure 1:

Engineered dextran-regulated phase separation in CFPS. (A) CE-Dex was used to induce LLPS in the CFPS system. (B) Carboxyethyl dextran (CE-Dex) was synthesized via a Michael addition reaction between dextran side chains and acrylamide, introducing carboxyethyl groups. (C) Visualization of LLPS using Cy5-labeled CFPS proteins. CE-Dex induced aggregate formation, whereas pure lysate, unmodified dextran, and other polymers (e.g., PEI, PEG) did not induce phase separation. (Scale bar, 50 μm.) (D) Quantitative analysis indicated that CE-Dex induced approximately 72% protein aggregation, whereas control molecules (dextran, PEI, PEG, PAM) had no significant effect. (E) ITC results demonstrated decreases in both the Gibbs free energy (ΔG) and entropy change (ΔS) of the system. (F) CFPS proteins were labeled with Cy3 and Cy5 to assess the occurrence of FRET within the aggregates. (Scale bar, 50 μm.) (G) Under a fixed excitation wavelength of 550 nm, a pronounced FRET signal peak was observed in the aggregate group. (H) Quantitative analysis revealed that the aggregates exhibited a FRET efficiency of approximately 24.5%, compared to only 4.5% in the non-LLPS group. (I) The formation of aggregates resulted in a reduction of the Cy3 fluorescence lifetime by approximately 0.19 ns. Data are presented as the mean ± SD (n = 3).