Programmed spatial organization of biomacromolecules into discrete, coacervate-based protocells

Abstract

The cell cytosol is crowded with high concentrations of many different biomacromolecules, which is difficult to mimic in bottom-up synthetic cell research and limits the functionality of existing protocellular platforms. There is thus a clear need for a general, biocompatible, and accessible tool to more accurately emulate this environment. Herein, we describe the development of a discrete, membrane-bound coacervate-based protocellular platform that utilizes the well-known binding motif between Ni²⁺-nitrilotriacetic acid and His-tagged proteins to exercise a high level of control over the loading of biologically relevant macromolecules. This platform can accrete proteins in a controlled, efficient, and benign manner, culminating in the enhancement of an encapsulated two-enzyme cascade and protease-mediated cargo secretion, highlighting the potency of this methodology. This versatile approach for programmed spatial organization of biologically relevant proteins expands the protocellular toolbox, and paves the way for the development of the next generation of complex yet well-regulated synthetic cells.

Fig. 1. Schematic overview of chemically programmed loading of His-tagged proteins into Ni2⁺-NTA-functionalized protocells.

Q-Am (red), Cm-Am (blue), and Ni-NTA-Am (teal) are mixed to form a coacervate droplet in which His-tagged proteins are loaded. Upon addition of the terpolymer, the droplets are stabilized. These stabilized coacervates can be loaded with functional cargo to allow for an enhanced enzymatic activity or protease-mediated release.

Fig. 2. Controlled, homogeneous uptake of sfGFP-His into NTA-functionalized protocells.

a False-colored confocal micrographs of protocells show the specific uptake of sfGFP-His (green) by Ni-NTA-Am. The membrane is stained with Nile Red (purple), scale bar: 10 µm. Uncropped images are to be found in Supplementary Fig. 7. b Flow cytometry analysis of sfGFP-His loaded protocells. For clarity of the graph, a 10th of all data points collected is shown. Gating parameters and population statistics are reported in Supplementary Fig. 8. c Box plot analysis of the measured concentration of sfGFP-His measured inside the coacervate interior with confocal microscopy. Reference curve Supplementary Fig. 9. n = 69.

Fig. 3. Detailed characterization of the Ni²⁺-NTA/His uptake mechanism.

a Analysis of the purified GFP variants by SDS-PAGE analysis. Left to right: marker (M), purified sfGFP-His, purified −30GFP-His, +36GFP-His, M. For all three GFP variants, a prominent band is observed at the expected molecular weight. b Analysis of coacervate populations containing either the His-tagged or No His variant of each supercharged protein. Charge is indicated by color; green, neutral; blue, negative; and red, positive. For all cases, n > 60. The membrane is stained with Nile Red, scale bar: 10 µm. Uncropped images are available in Supplementary Fig. 12c, d. FRAP analyses of protein-loaded coacervate protocells. c Confocal micrographs at different time points during the recovery experiments. Scale bar: 10 µm. d Analysis of the fluorescence recovery over time. Open blue circles, −30GFP without His-tag; closed blue circles, −30GFP-His; and green circles, sfGFP-His. Shaded areas represent the standard deviation of three individual measurements. The unbroken graph and data fits are available in Supplementary Figs. 14 and 15.

Fig. 4. Increased enzymatic activity by controlled loading of a synthetic two-enzyme cascade.

a Schematic overview of the reaction scheme to produce Indigo from l-Trp by TnaA and FMO. b Analysis of the purified enzyme variants by SDS-PAGE gel electrophoresis. Left to right: marker (M), TnaA, FMO, M. c Confocal micrographs of coacervates loaded with 100 nM Sulfo-Cy5-NHS-labeled TnaA + 200 nM unlabeled FMO (top) or 200 nM FMO + 100 nM unlabeled TnaA (bottom). Membrane stained with Nile red (purple), scale bar: 10 µm. Uncropped images are to be found in Supplementary Fig. 18. d Box plot analysis of the loading of fluorescently labeled enzymes inside the coacervates. For all cases, n > 50. e, f NADPH consumption measured by the absorbance at 340 nm. 1 mM l-Trp and 0.5 mM NADPH was added prior to the measurement. Orange circles represent enzymes inside the coacervates, gray circles show enzymes in solution. Three individual batches of coacervates were made for each condition, the standard deviation of three individual measurements is represented by the shaded area. For clarity of the graph, only 1/3rd of all collected data points are shown. e 250 nM TnaA + 500 nM FMO. f 125 nM TnaA + 250 nM FMO.

Fig. 5. Protein excretion induced by TEV protease-mediated His-tag removal.

a Schematic overview of the GFP construct design and the TEV protease-mediated release. b Box plot analysis of the fluorescence intensity inside the coacervate droplet incubated with (orange) and without (gray) TEV protease for both 100 nM of sfGFP (left) and −30GFP (right). n > 30. Membrane stained with Nile red (purple), scale bar: 10 µm. Uncropped images (Supplementary Fig. 27). c FRAP analysis of both the sfGFP and −30GFP after 1 h of incubation with TEV protease. Top: confocal images of the FRAP experiment. Scale bar: 10 µm. Bottom: Fluorescence recovery plotted over time. Left: sfGFP with TEV (orange), sfGFP-His without TEV (gray). Right: sfGFP with TEV (orange), −30GFP without TEV (gray). The standard deviation of three individual measurements is represented by the shaded area. Data fits available in Supplementary Fig. 29. d Release profile of 250 nM sfGFP determined by confocal microscopy. Top: Time series of representative protocells. Membrane stained with Nile Red (purple), scale bar: 10 µm. Uncropped images (Supplementary Fig. 30) Bottom: Normalized fluorescence intensity of n = 20 protocells extracted from confocal time series. The standard deviation is represented by the shaded area.