Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

The efficient assembly of supramolecular structures for minimal cell design and drug delivery remains challenging. Here, we demonstrate efficient protocols that yield supramolecular structures based on amphiphilic elastin-like proteins. The presented assembly protocols generate versatile supramolecular structures with adaptable physicochemical properties, such as vesicles, fibers, and coacervates.

Protein membrane-based compartments demonstrate phase separation behavior and allow for the encapsulation of chemically diverse fluorescent cargo molecules. For expression of F20E20-mEGFP and F20E20-mCherry, inoculate the main expression culture from the overnight pre-culture to an OD600 of 0.3. Incubate it in 400 milliliters of sterile LB medium supplemented with appropriate antibiotics at 37 degrees Celsius and 200 rpm.

When the expression culture reaches OD600 of 0.5 to 0.8, add IPTG to a final concentration of one millimolar, and reduce the incubation temperature to 20 degrees Celsius. For expression of amphiphilic ELP with the unnatural amino acid, inoculate the main expression culture from overnight E.coli pre-culture containing the appropriate plasmids to an OD of 0.3. Incubate it in 400 milliliters of sterile LB medium supplemented with kanamycin and chloramphenicol at 37 degrees Celsius and 200 rpm.

To prepare the 100-millimolar unnatural amino acid stock, add 206.2 milligrams of unnatural amino acid to eight milliliters of ultrapure water. Raise the pH of the solution with three-molar sodium hydroxide, and mix vigorously. When the unnatural amino acid is dissolved, lower the pH to approximately 10.5, and add ultrapure water to a volume of 10 milliliters.

Filter the solution with a sterile 0.22-micrometer filter, and aliquot it in two-milliliter reaction tubes. When the expression culture reaches OD600 of 0.5 to 0.8, add the unnatural amino acid to a final concentration of two millimolar. Incubate it for 10 more minutes.

Then induce expression of the target protein and the necessary tRNA synthetase via simultaneous addition of IPTG and arabinose. Reduce the incubation temperature to 20 degrees Celsius, and allow expression to continue for approximately 20 hours. After the incubation, harvest the cells by centrifugation at four degrees Celsius and 4, 000 times g for 40 minutes.

Resuspend the cell pellet in lysis buffer with lysozyme and PMSF. Incubate the solution for 30 minutes on ice. Then freeze and thaw it twice by submerging in liquid nitrogen.

Sonicate the suspension, and clear the lysate by centrifugation at 10, 000 times g and four degrees Celsius for 40 minutes. Then purify the protein using affinity chromatography. After protein elution, store it at four degrees Celsius until further use.

Add one microliter of fluorescent dye to 500 microliters of ELP solution, and incubate the reaction for about 10 hours at 15 degrees Celsius while shaking in a ThermoMixer, making sure to protect it from light. To remove excessive fluorescent dye, equilibrate a dialysis membrane in ultrapure water for 10 minutes. Cut the membrane into the correct size to be placed on top of the opening of the reaction tube with the clicked ELP solution.

Then fix the membrane to the opening by closing the tube with a lid that has no core. Fill the space between lid and membrane with buffer to prevent trapping of air. Then place the reaction tube in the chosen buffer, push it below the surface, and turn it upside down.

Perform dialysis for at least three hours at four degrees Celsius, allowing dialysis to continue for at least three more hours after each buffer change. For THF swelling, dialyze the homogenous ELP solution against phosphate or Tris buffer with stable pH 7.5. Prepare the lyophilizer, and cool it to starting temperature for freeze-drying.

Aliquot the dialyzed protein solution in 1.5-milliliter reaction tubes, and seal with caps with a small hole to avoid losing the solid sample due to pressure changes in the course of freeze-drying. Shock-freeze the samples in liquid nitrogen. Take the frozen protein samples out of the liquid nitrogen, and immediately place them in the lyophilizer to start freeze-drying.

After the sample is completely dry, ventilate it with dry nitrogen, and immediately close the reaction tube lids to avoid contact with air moisture. Add pure THF to the lyophilized samples, and place the solution in a water bath sonicator with ice water for 15 minutes. Preheat a thermocycler to 30 to 60 degrees Celsius for vesicle formation or up to 90 degrees Celsius for fiber formation, and prepare new reaction tubes with either ultrapure water or buffer.

After sonication, place the ELP/THF solution and the prepared water or buffer in the thermocycler for five minutes. Then stratify the ELP solution on top of the water or buffer, making sure that the separation of the two phases and a distinct interphase is visible, and place the mixture back in the thermocycler. Let the sample cool down at room temperature for 10 minutes, and proceed with dialysis against water or buffer or with fluorescence microscopy.

Prepare a one-to 50-micromolar ELP solution, and add 10 to 20%1-butanol. Immediately mix the solution by pipetting up and down. The turbidity of the solution should increase during mixing, indicating vesicle formation.

To achieve a narrow size distribution, extrude vesicles with a mini extruder through a membrane with a pore size of one micrometer. For dye encapsulation, mix approximately 40 microliters of ELP solution in 10-millimolar Tris-HCl with one microliter of Dextran Texas Red stock solution in DMSO. Add 10 microliters of 1-butanol, and extrude five to 10 times through a syringe equipped with a 0.25-by-25-millimeter needle.

The THF swelling method is composed of three successive steps and results in different supramolecular assemblies of the ELP depending on the temperature. The epifluorescence microscopy images show vesicles assembled from BDP-R20F20 and fibrillary structures assembled from BDP-R40F20. The 1-butanol extrusion method leads exclusively to the formation of ELP vesicles.

About two orders of magnitude more vesicles are produced compared to the THF swelling method. BDP-R40F20 was mixed with 10 to 15%butanol, and vesicles were prepared via extrusion of the mixture. Different supramolecular structures were assembled from BDP-R40F20 via the THF swelling protocol.

The pH of the buffer and the temperature of the assembly process was adjusted to form either coacervates, fibrils, or vesicles. Small mistakes in the assembly protocol can lead to the formation of aggregates. Positively charged dye ATTO Rho 13 and the polysaccharide dextran red 3000 were encapsulated into the lumen of vesicles assembled from F20R20-mEGFP via the 1-butanol extrusion method.

Confocal microscopy images show the vesicles in the green channel, the cargo in the red channel, and the successful encapsulation in the resulting merged channel. The phase separation and fusion behavior of ELP amphiphiles upon mixing of single PMBC building blocks versus assembled PMBC populations was also demonstrated. Mixing prior to PMBC assembly leads to homogenously distributed molecules within the assembled PMBC membrane, while mixing assembled vesicle populations leads to membrane patches of red or green fluorescence that are visible for at least 20 minutes.

When attempting this procedure, it is important to pay attention to the correct step order and for the differences between the THF swelling method and the butanol extrusion method. Following this protocol, the ELP vesicles can be used as drug delivery shuttles, as platform for enzymatic reactions, such as in vitro transcription and translation, or in the formation of synthetic minimal cells.