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Lipid coated protein condensates as stable protocells with selective uptake abilities for biomolecules

To create cell-like synthetic systems, spatial confinement that is stable against environmental changes and selective uptake of diverse biomolecules into these compartments are key initial conditions. However, fabrication of protocells with these two features has been extremely difficult. Here, we used fully protein-based liquid condensates and a lipid coating on these condensates to construct highly stable protocells with an uptake ability for outside biomolecules. Condensates with an extremely high density of 6His-tagged proteins were coated with Ni(ii)-NTA(nitrilotriacetic acid)-modified lipids. High condensate rigidity and specific 6His-Ni-NTA interactions enabled the formation of lipid–protein protocells, which are stable even after centrifugations. In addition, immobile lipid coatings on condensates were permeable to outside biomolecules. When binding modules were fused into condensate-forming proteins, the resulting functionalized condensate-protocells could strongly and selectively uptake various outside proteins through specific protein interactions.

. Confocal images of lipid-coated PSH condensates before (left) and after (right) centrifugation with different OG concentrations. (a) PSH condensates were mixed with OG solubilized lipids (final OG concentration 0.67%) and centrifuged/resuspended without further OG dilution. (b, c) PSH condensates were mixed with OG solubilized lipids (final OG concentration 0.67%) and diluted to (b) 0.2% OG or to (c) 0.1% OG, followed by centrifugation/resuspension. Scale bars 20 μm.  The line scans of surface lipid intensities for boxed condensates were shown below. The same relative intensity and distance scales are applied for all scans. Scale bars 10 μm (2 μm for inserts). (b) Lipid coating at different PSH condensate formation time. Condensate formation was induced by metal addition, and the resulting mixtures were incubated for indicated time before lipid addition. (c) Lipid coating at different temperatures. Scale bars 10 μm (5 μm for inserts).  Confocal images of lipid-coated RL48 protocells with different DGS-NTA (Ni) portions. The line scans of surface lipid intensities for boxed condensates were shown below. The same relative intensity and distance scales are applied for all scans. Scale bars 10 μm (2 μm for inserts). (c) Fluorescence intensity profiles of lipids (red) and PSH/RL48 (cyan) across (white arrow) a lipid-coated PSH (left) or RL48 (middle) condensate. PSH profiles are identical to Figure 1c. Lipid intensity profiles of PSH (black) and RL48 (red) condensates are also shown (right) for comparison. Scale bars 5 μm.
Note I: All examined protocells are well coated with lipids. Compare to PSH protocells, fewer but larger RL48 protocells were produced, and thereby fewer protocells were examined from the same number of whole confocal images.
Note II: With high NTA-lipid concentrations, a large portion of lipids penetrated into RL48 condensates ( Figure S7b). Overlaid lipid intensity profiles for PSH and RL48 protocells also indicate more lipid penetration into RL48 condensates ( Figure S7c            Protein expression. Genes were cloned into the pET-21a expression vector (EMD Biosciences), and the plasmids were transformed to Escherichia coli BL21 (DE3) for protein expression. The transformed cells were grown at 37 °C until OD600 reaches 0.8 and induced with 1 mM IPTG followed by incubation for 18 h at 20 °C. The induced cells were collected by centrifugation for 5 min at 6000 rpm and washed once using PBS. Harvested cells were lysed by sonication in an equilibration buffer (500 mM NaCl and 50 mM Tris pH 8.0). Lysed cells were centrifuged at 12000 rpm for 15 min, and His-tagged proteins were purified using Ni-IDA columns (BioProgen). Purified proteins were dialyzed into PBS at 4 °C, and concentration was determined by measuring A280. Ferritin, mi3, and LAF were prepared as previously reported. [1][2][3] Fluorescent dye labeling of proteins. Proteins were labeled with the Cy5 N-hydroxysuccinimide (NHS) ester (Lumiprobe). Protein samples were mixed with the dye in a 10:1 protein-to-dye ratio and incubated for 1 h at RT. Dye-conjugated proteins were separated using PD-10 desalting columns (SephadexTM G-25 M, GE Healthcare).
GUV preparation. Giant Unilamellar vesicle (GUV) was produced based on the published protocel. 4 Briefly, 2 μL of a 2 mM lipid mix (94.5 mol% DOPC, 5 mol% DGS-NTA (Ni), 0.5 mol% Liss Rhod PE) dissolved in chloroform was spread on inidium-tin-oxide (ITO) coated glass (resistance ~ 10 Ωsq−1) slides, followed by overnight drying. Two ITO glass slides were spaced using a rubber ring and electectroformed with a sinusoidal voltage of 1 kHz with an amplitude of 2.5 V root mean square (RMS) for 3 h at 45 °C in PBS.
Lipid-coated protein condensate preparation. Protein (PSH) consendates were prepared by adding 100 μM NiCl2 to 100 μM PSH (final 50 μL) to induce phase separation. After 10 min (or indicated time) incubation, 10 μL of 2.5 mM lipid mix (94.5 mol% DOPC, 5 mol% DOGS-Ni NTA, 0.5 mol% DPPE-Lissamine Rhodamine B in 2% OG solution) was added to condensates (final protein:lipid concentration = 1:5 or to indicated ratios), where a final OG concentration was controlled to 0.67%. Following lipid-condensate binding for 10 min, the solution is diluted with 1xPBS to obtain a 0.2% final OG concentration. The resulting lipid-coated condensates were centrifuged at 3500 rpm for 5 min, followed by resuspension to 1xPBS. For RL48 condensate formation, reaction tubes were blocked with BSA to prevent condensate attachment to tube surfaces. Protein condensates of RL48 or RL48 with indicated amounts of PSH proteins (PSH, D7N-PSH, or ZE-PSH) were prepared by adding 800 μM NiCl2 to 300 μM RL48 proteins. Phase separation was conducted for 30 min before lipid coating. Again, 10 μL of 2.5 mM lipid mix was mixted with 50 μL of the RL48 condensate solution. For LAF droplet condensate formation, 100 μM NiCl2 was treated to 80 μM LAF, followed by 10 min incubation before lipid coating (10 μL of 2.5 mM lipid mix to 50 μL of the condensate solution).
Permeability and uptake assays. For permeability tests, protocells were prepared and isolated by centrifugation, and 10 μM of external proteins were treated to these protocells. For selective uptake tests, 0.1 μM of external proteins were treated to these protocells. The protein concentrations of external proteins that were recruited into protocells were estimated based on GFP intensity/concentration calibration curves. TEM imaging. Lipid-coated condensate samples were loaded onto a carbon grid for 10 sec and soaked using a filter paper. Liposomal giant unilamellar vesicles (GUV) were loaded onto a carbon grid for 30 sec and stainined for 30 sec using 2% uranyl acetate solution, followed by soaking using a filter paper. 200 kV Transmission Electron Microscope (Tecnai F20) (FEI Company) was used for observation.