pH-Responsive Elastin-Like Polypeptide Designer Condensates

Biomolecular condensates are macromolecular complexes formed by liquid–liquid phase separation. They regulate key biological functions by reversibly compartmentalizing molecules in cells, in a stimulus-dependent manner. Designing stimuli-responsive synthetic condensates is crucial for engineering compartmentalized synthetic cells that are able to mimic spatiotemporal control over the biochemical reactions. Here, we design and test a family of condensate-forming, pH-responsive elastin-like polypeptides (ELPs) that form condensates above critical pH values ranging between 4 and 7, for temperatures between 20 and at 37 °C. We show that the condensation occurs rapidly, in sharp pH intervals (ΔpH < 0.3). For eventual applications in engineering synthetic cell compartments, we demonstrate that multiple types of pH-responsive ELPs can form mixed condensates inside micron-sized vesicles. When genetically fused with enzymes, receptors, and signaling molecules, these pH-responsive ELPs could be potentially used as pH-switchable functional condensates for spatially controlling biochemistry in engineered synthetic cells.


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Table S1.PRE protein sequences.Trailer sequence WPC is added (tryptophan (W) for protein A 280 adsorption quantification, proline (P) as a spacer, and cysteine (C) for fluorescent dye conjugation using thiol-maleimide chemistry).

Figure S3 -
Figure S3-Turbidity measurements of PRE-h-43 at 25 μM in phosphate-saline buffers of approximately equal ionic strength, but varying in phosphate and NaCl concentration.A transition temperature (T t ) shift of approxiately 10 °C is observed, can be partly attributed to a higher Na + ion concentration.

Figure S4 -
Figure S4 -(a) Turbidity measurements of PRE-h-43 at various protein concentrations.Black dotted line are sigmoidal fits through the data.(b) Transition temperature (T t ) -defined as midpoints of the sigmoid plots -are plotted over the PRE-h-43 polymer concentration.

Figure S5 -
Figure S5-Turbidity measurement of 25 μM PRE-h-43 in presence of 14.3 mg/mL GDL at constant temperature of 21 °C.In blue y-axis the GDL calibration is plotted and the OD 350 is recorded in the red y-axis as a measure for turbidity (indicating condensation).From the data it can be observed that complete condensation can take >20 min indicating that the dynamics of pH-triggered condensation are relatively slow compared to temperature-driven condensation (where condensation typically completes within ~ 3 min with a 1 °C /min temperature ramp).

Figure S6 -
Figure S6 -Brightfield image of double emulsion (water-oil-water) droplet production using PDMS-based microfluidic setup.The inner aqueous stream composed of PREs flows through the flow-focusing junction, forming water-in-oil single emulsions.The single emulsions flow through the serpentine loop and get pinched-off at the second junction by a second cross-flow of surfactant-containing PBS solution, leading to water-in-oil-in-water double emulsion droplets.

Figure S7 -
Figure S7 -Full views of sequential (mixed) condensate formation in microcompartments water-oil-water droplets.Cyan colored highlights the square showing Figure 7b of the main text.(a) t 0 , (b) t 1 (c) t 2 , (d) t end .The droplets observed in the background are unwanted oil droplets formed as a by-product of double emulsion production.