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

Summary

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.

Abstract

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.

Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

Introduction

The assembly of supramolecular structures for biotechnological applications is becoming increasingly important^1,2,3,4,5. For the assembly of functional architectures such as coacervates, vesicles, and fibers with desired physicochemical properties it is important to understand and control the physicochemical and conformational properties of the components. Due to the molecular precision of molecules found in nature, building blocks for supramolecular structures are increasingly based on lipids, nucleic acids or proteins. Compared to synthetic polymers, proteinaceous building blocks allow for precise control over emergent supramolecular structures⁶ on the genetic level. The primary amino acid (aa) sequence of the individual protein building blocks intrinsically encodes the information for their assembly potential from the molecular up to the macroscopic level as well as the three dimensional shape and physical properties of the final supramolecular structure⁷.

Reported methods for the assembly of different supramolecular structures often involve amphiphilic proteins such as temperature sensitive elastin-like proteins (ELP)^5,8,9, recombinant oleosin¹⁰ and artificial protein amphiphiles¹¹. Temperature triggered methods have led to the assembly of micelles^4,10,12, fibers¹³, sheets¹⁴ and vesicles^9,15,16. Methods involving organic solvents have been applied for the formation of dynamic protein based vesicles^8,11,14. So far, applied protocols for vesicle formation often lack assembly control over micrometer sized assemblies^16,17 or have limited assembly yield⁵. In addition, some reported ELP based vesicles have impaired encapsulation potential¹² or limited stability over time⁹. Addressing these drawbacks, the presented protocols enable the self-assembly of micrometer and sub micrometer sized supramolecular structures with distinct physiochemical properties, good encapsulation potential and long-time stability. Tailored amphiphilic ELPs assemble into supramolecular structures, spanning the range from spherical coacervates and highly ordered twisted fiber bundles to unilamellar vesicles depending on the applied protocol and associated environmental conditions. Large vesicular Protein Membrane-Based Compartments (PMBC) reveal all main phenotypes such as membrane fusion and phase separation behaviour analogous to liposomes. PMBCs efficiently encapsulate chemically diverse fluorescent cargo molecules which can be monitored using simple epifluorescence microscopy. The repetitive ELP domains used in this study are attractive building blocks for protein based supramolecular architectures¹⁸. The ELP pentapeptide repeat unit (VPGVG) is known to tolerate different aa besides proline at the fourth position (valine, V), while preserving its structural and functional properties¹⁹. The design of amphiphilic ELPs containing distinctive hydrophilic and hydrophobic domains was realized by inserting aa guest residues (X) in the VPGXG repeat with distinct hydrophobicity, polarity, and charge²⁰. Amphiphilic ELP domains where equipped with hydrophobic phenylalanine (F) or isoleucine (I) while the hydrophilic domain contained charged glutamic acid (E) or arginine (R) as guest residues. A list of eligible amphiphilic ELP constructs and corresponding aa sequences can be found in the supplementary information and references^8,21. All building blocks where equipped either with small fluorescent dyes or fluorescent proteins for visualization via fluorescence microscopy. mEGFP and other fluorescent proteins were N-terminally fused to the hydrophilic domains of the ELP amphiphiles . Organic dyes were conjugated via copper-free strain promoted alkyne–azide cycloaddition (SPAAC) to a co-translationally introduced unnatural amino acid (UAA). The co-translational incorporation of the UAA para-azidophenylalanine (pAzF)²² permits the N-terminal modification of the hydrophilic ELP domain. In this way the green fluorescent dye BDP-FL-PEG₄-DBCO (BDP) or any small fluorescent molecule with a strained cyclooctyne can be used as fluorescent probe. Successful incorporation of the UAA pAzF and cycloaddition of the dye via SPAAC can be easily confirmed via LC-MS/MS due to efficient ionization of the corresponding tryptic peptides⁸. This small organic dye was applied to broaden the solvent choice for assembly protocols, since fluorescent proteins are incompatible with most organic solvents. The two most efficient assembly protocols for supramolecular structures developed in our lab are described below. The THF swelling method is only compatible with organic dye modified amphiphilic ELP. In contrast, the 1-butanol (BuOH) extrusion method is compatible with many proteins as fluorescent probe e.g. mEGFP, since the described method fully preserves the fluorescence of these fusion proteins. In addition, the encapsulation of small molecules and vesicular fusion behavior works best by employing the BuOH extrusion method.

Protocol

1. Design and cloning of amphiphilic elastin-like proteins (ELPs)

1. Clone and design the constructs as described elsewhere^8,20. Plasmids are available upon request.

2. Protein expression, purification and preparation

1. Expression of F20E20-mEGFP and F20E20-mCherry
   1. Inoculate main expression culture from overnight pre-culture to an OD₆₀₀ of 0.3. Incubate at 37 °C, 200 rpm in sterile 400 mL LB medium supplemented with appropriated antibiotics in a 2 L flask.
   2. Prepare IPTG stock solution (1 M) for induction of the expression culture in ultrapure water.
   3. When OD₆₀₀ 0.5–0.8 is reached, add IPTG to expression culture to a final concentration of 1 mM and reduce incubation temperature to 20 °C. Allow expression at 20 °C for approximately 20 h at 200 rpm.
2. Expression of amphiphilic ELP containing UAA pAzF
   1. Inoculate main expression culture from overnight E. coli pre-culture containing the two plasmids pEVOL pAzF and e.g. pET28-NMBL-(TAG)R40F20-his to an OD₆₀₀ of 0.3 (see supplementary information for amino acid sequences). Incubate at 37 °C, 200 rpm in sterile 400 mL LB medium supplemented with kanamycin and chloramphenicol in a 2 L flask.
   2. Prepare 100 mM pAzF stock solution in ultrapure water. For 10 mL of pAzF stock solution, weigh 206.2 mg pAzF and resuspend it in 8 mL of ultrapure water. To dissolve the pAzF raise the pH of the solution with 3 M NaOH and mix vigorously. When pAzF is dissolved, carefully lower the pH to 10.5 and add ultrapure water to a final volume of 10 mL. Use a sterile filter (0.22 µm) and aliquot the solution in 2 mL reaction tubes.
   3. Prepare 1 M IPTG stock solution in ultrapure water and 20% arabinose stock solution in ultrapure water.
   4. When OD₆₀₀ 0.5–0.8 is reached, add pAzF to the expression culture to a final concentration of 2 mM. Incubate culture for 10 min, 37 °C, 200 rpm to allow for pAzF uptake.
   5. Induce expression of target protein and expression of the necessary tRNA/t-RNA synthetase via simultaneous addition of IPTG (1 mM) and arabinose (2%) and reduce incubation temperature to 20 °C.
   6. Allow expression at 20 °C for approximately 20 h at 200 rpm. Harvest expression culture by centrifugation at 4 °C, 4000 x g, 40 min.
3. Cell lysis and protein purification
   1. Resuspend the E. coli pellet in lysis buffer (10 mL per liter of culture; 50 mM Tris-HCl pH 8, 500 mM NaCl, 4 M urea, 0.25% Triton X-100) containing lysozyme (0.1 mg/mL) and PMSF (0.1 mM). Incubate for 30 min on ice and freeze and thaw twice afterwards by submerging the sample in liquid nitrogen.
   2. Sonicate the suspension (30%, 15 times, 30 s: 10 s break) and clear the lysate via centrifugation (4 °C, 10,000 x g for 40 min).
   3. Purify protein using affinity chromatography (e.g. on a 1 mL nickel column using a FPLC system connected to a fraction collector; see Table of Materials). Elute the protein with elution buffer (50 mM Tris-HCl, 500 mM NaCl, 4 M urea, 250–500 mM imidazole) and store at 4 °C until further processing.
   4. Analyze the purification efficiency via SDS-PAGE.

3. Dye-modification of ELPs via SPAAC

1. Roughly estimate the concentration of the ELP solution.  A₂₈₀ absorption for concentration evaluation is not valuable since pAzF-R40F20 sequence is lacking amino acids absorbing in the UV range. Therefore, a previously lyophilized and weighted ELP amphiphile can be used as a reference for SDS PAGE band comparison. Through comparison of the summed gray value intesity of SDS PAGE bands from ELP solutions with known concentrations and your sample the rough concentration of your sample can be estimated.
2. Add 1 µL of fluorescent dye BDP-FL-PEG4-DBCO (10 mM stock solution; 20 µM final concentration) to 500 µL of ELP solution (~20 µM). Incubate the reaction for about 10 h at 15 °C, while shaking and protected from light.
3. For further use, dialyze the reaction to remove excessive BDP.
   1. Equilibrate a dialysis membrane (e.g. 12 kDa cutoff) in ultrapure water for 10 min. Cut the dialysis membrane into the correct size to be placed on top of the opening of an reaction tube containing the clicked ELP solution. To fix the dialysis membrane in the opening, place a reaction tube lid with punched out core on the opening, thus closing the tube.
   2. Place the reaction tube upside down in the chosen buffer. Exchange the buffer (2–5 L) twice after dialysis for at least 3 h every time. Remove any air bubbles trapped between the dialysis membrane and the buffer to ensure successful dialysis.

4. THF swelling protocol

1. Dialyze homogenous ELP solution against phosphate or tris buffer (10 mM) with stable pH 7.5 to remove salts and remaining compounds from his-tag purification.
2. Prepare the lyophilizer and cool down to starting temperature for freeze-drying.
3. Aliquot the dialyzed protein solution in 1.5 mL reaction tubes (50–500 µL per tube) and shock freeze in liquid nitrogen. To avoid unwanted mixing of different protein solutions during freeze-drying, caps with a small hole can be put on top of the reaction tube to seal it partially.
4. Take the frozen protein samples out of the liquid nitrogen and immediately place them in the lyophilizer to start freeze-drying. Freeze-drying is finished when the sample is completely dry (approximately 24–48 h). Subsequently, ventilate lyophilized amphiphilic ELPs with dry N₂, then immediately close the reaction tube lids to avoid contact with air moisture.
5. Add pure THF to the lyophilized samples (ELP, 5–10 µM) and place the solution in a water bath sonicator containing ice water for 15 min to allow for swelling of the ELP in THF.
6. Preheat a thermocycler to 30–60 °C for vesicle formation or up to 90 °C for fiber formation and prepare new reaction tubes containing either ultrapure water or buffer (50 mM NaH₂PO₄/Na₂HPO₄, 50 mM NaCl, pH 5–13). Spherical coacervates assemble predominantly at 20 °C within pH 9–13. Vesicle formation is favored at 50–60 °C between pH 7 and 9. Fiber formation is predominently induced above 60 °C between pH 5 and 12.
7. After the sonication step, place the ELP/THF solution as well as the prepared ultrapure water or buffer solution in the thermocycler and heat up to the desired temperature for 5 min. When temperature is reached the preheated ELP/THF solution should be carefully stratified on top of the preheated ultrapure water or buffer solution. A clear separation of the two phases with a distinct interface should be visible.
8. Place the mixture in the thermocycler again and incubate for 20 min to allow for vesicle or fiber formation at the interface. Afterwards, let the samples cool down to room temperature for 10 min before analysis via fluorescence microscopy or dialysis.
9. Dialyze solution containing the supramolecular structures against ultrapure water or buffer (50 mM NaH₂PO₄/Na₂HPO₄, 50 mM NaCl, pH 7–10).

5. BuOH extrusion protocol

1. Prepare a 1–50 µM ELP solution in ultrapure water or buffer (50 mM PB pH 7.5, 100 mM NaCl, may contain up to 4 M urea). The concentration of the amphiphilic ELP F20R20-mEGFP and F20R20-mCherry solution can be determined using the molar extinction coefficients (F20R20-mEGFP A₂₈₀ = 22015 M^-1 cm^-1 and F20R20-mCherry A₂₈₀ = 34380 M^-1 cm^-1) (see supplementary information for aa sequences).
2. Add 10%–20% (v/v) 1-butanol and immediately mix the solution by pipetting up and down or drawing it up through a syringe multiple times. A common 100 µL pipette or Hamilton syringe equipped with a 0.25 x 25 mm needle can be applied. The turbidity of the solution during mixing should increase, indicating vesicle formation. 1-octanol 5%–15% (v/v) can also be used for vesicle extrusion instead of 1-butanol.
3. In order to achieve a narrow size distribution, extrude vesicles using a mini extruder through a membrane with a pore size of 1 µm at room temperature. The membrane size used for extrusion determines the upper size cutoff of the vesicles.
4. Dialyze the vesicles as described above (step 3.3) to remove residual 1-butanol.

6. Dye encapsulation with the BuOH extrusion protocol

1. Mix approximately 40 µL ELP solution in 10 mM Tris-HCl, pH 8 with 1 µL Dextran Texas Red (0.0025 mg/mL final concentration).
2. Add 10 µL of BuOH to the solution and extrude 5–10 times through a syringe equipped with a 0.25 x 25 mm needle.

7. Analysis of supramolecular structures using fluorescence microscopy

1. Place a reinforcement ring on a glass slide and firmly press the adhesive side to the glass.
2. Add 5 µL of the sample to the inside of the reinforcement ring and place a cover slip on top.
3. Seal the sample with nail polish at the edges of the cover slip to avoid evaporation of the sample during analysis.
4. Carry out fluorescence microscopy as previously described⁸.

Results

Protocol development for vesicle production
Figure 1 outlines the two different vesicle preparation methods. The THF swelling method on the left side is composed of three successive steps and results in different supramolecular assemblies of the ELP depending on the temperature. In Figure 1A epifluorescence microscopy images show vesicles assembled from BDP-R20F20 and fibrillary structures assembled from BDP-R4...

Discussion

A fault while following the described protocols for the assembly of defined supramolecular structures mainly leads either to the formation of unspecific aggregates (Figure 2, IV) or to homogeneously distributed ELP-amphiphiles. Critical steps of the protocol are discussed below:

For high expression yield of the amphiphilic ELP, a relatively low temperature of 20°C is optimal. For successful affinity based purification of the amphiphilic ELP an urea conce...

Materials

Name	Company	Catalog Number	Comments
1 µm and 0.2 µm Steril Filter	VWR
1,4-Dithiothreitol	Merck
1-butanol. >99.5% p.a.	Roth
2log DNA ladder	NEB
2-Mercaptoethanol	Roth
50 mL Falcon tubes	VWR
79249 Alkyne Mega Stokes dye	Sigma Aldrich
Acetic acid glacial	VWR
Acetonitrile, anhydrous, 99.8%	Sigma-Aldrich
Ampicillin sodium-salt, 99%	Roth
BDP-FL-PEG4-DBCO	Jena Bioscience
Biofuge	Heraeus
Bottle Top Filter with PES membrane (45 µm, 22 µm)	Thermo Scientific
Brillant Blue G250 (Coomassie)	Roth
BspQI	NEB
Camera DS Qi1	Nikon
Centrifuge 5417r	Eppendorf
Centrifuge 5810r	Eppendorf
CF-400-Cu square mesh copper grid	EMS
Chloramphenicol	Roth
CompactStar CS 4	VWR
Dextran, Texas Red, 3000 MW, neutral	Life Technologies
Digital sonifier	Branson
Dimethylsulfoxide (DMSO)	Applichem
Dnase I	Applichem
EarI	NEB
EcoRI-HF	NEB
Environmental shaker incubator ES-20	Biosan
Ethanol absolute	Roth
Ethidium bromide solution	Roth
Filter supports	Avanti
Glass plates	Bio-Rad
Glycerol Proteomics Grade	Amresco
Glycin	Applichem
H4-Azido-Phe-OH	Bachhem
Heat plate MR HeiTec	Heidolph
HindIII	NEB
HisTrap FF crude column	GE Life Sciences		Nickel column
Hydrochloride acid fuming, 37%, p.a.	Merck
Illuminator ix 20	INTAS
Illuminator LAS-4000	Fujifilm
Imidazole	Merck
Immersions oil for microscopy	Merck
Incubators shakers Unimax 1010	Heidolph
Inkubator 1000	Heidolph
IPTG, >99%	Roth
Kanamycinsulfate	Roth
L(+)-Arabinose	Roth
Laboratory scales Extend ed2202s/224s-OCE	Sartorius
LB-Medium	Roth
Lyophilizer Alpha 2-4 LSC	Christ
Lysozyme, 20000 U/mg	Roth
Microscope CM 100	Philips
Microscope Eclipse TS 100	Nikon
Microscopy cover glasses (15 x 15 mm)	VWR
Microscopy slides	VWR
Microwave	Studio
Mini-Extruder Set	Avanti Polar Lipids
NaCl, >99.5%, p.a.	Roth
Natriumhydroxid pellets	Roth
Ni-NTA Agarose, PerfectPro	5 Prime
Nucleopore Track-Etch Membrane	Avanti
PH meter 766 calimatic	Knick
Phenylmethylsulfonylflourid (PMSF)	Roth
Polypropylene Columns (1 mL)	Qiagen
PowerPac basic	BioRad
Propanol-2-ol	Emplura
Protein ladder 10-250 kDa	NEB
Recirculating cooler F12	Julabo
Reinforcement rings	Herma
SacI HF	NEB
SDS Pellets	Roth
Sodiumdihydrogen phosphate dihydrate, NaH2PO4	VWR
Sterile syringe filter 0.2 mm Cellulose Acetate	VWR
T4 DNA Ligase	NEB
TEMED	Roth
TexasRed Dextran-Conjugate	MolecularProbes
Thermomix comfort	Eppendorf
THF, >99.5% p.a.	Acros
Triton X 100	Roth
Trypton/Pepton from Casein	Roth
Ultrasonic cleaner	VWR
Urea p.a.	Roth
Vacuum pump 2.5	Vacuubrand
XbaI	NEB
XhoI	NEB
ZelluTrans regenerated cellulose tubular membrane (12.0 S/ 3.5 S/ 1.0 V)	Roth