Name: directed assembly of elastin-like proteins into defined supramolecular structures and cargo encapsulation in vitro
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Description: Watch this Scientific Journal Video about Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro at JoVE.com

The authors thank the BMBF for financial support and the Center for Biological Systems Analysis (ZBSA) for providing the research facility. We are grateful to P. G. Schultz, TSRI, La Jolla, California, USA for providing the plasmid pEVOL-pAzF. We thank the staff of the Life Imaging Center (LIC) in the Center for Biological Systems Analysis (ZBSA) of the Albert-Ludwigs-University Freiburg for help with their confocal microscopy resources, and the excellent support in image recording.

1. Design and cloning of amphiphilic elastin-like proteins (ELPs)
<ol>
	<li>Clone and design the constructs as described elsewhere8,20. Plasmids are available upon request.</li>
</ol>
2. Protein expression, purification and preparation
<ol>
	<li>Expression of F20E20-mEGFP and F20E20-mCherry
	<ol>
		<li>Inoculate main expression culture from overnight pre-culture to an OD600 of 0.3. Incubate at 37 &#176;C, 200 rpm in sterile ...

<ol>
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A fault while following the described protocols for&#160;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&#176;C is optimal. For successful affinity based purification of the amphiphilic ELP an urea conce...

The assembly of supramolecular structures for biotechnological applications is becoming increasingly important1,2,3,4,5. 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 micelles4,10,12, fibers13, sheets14 and vesicles9,15,16. Methods involving organic solvents have been applied for the formation of dynamic protein based vesicles8,11,14. So far, applied protocols for vesicle formation often lack assembly control over micrometer sized assemblies16,17 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 references8,21. 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&#8211;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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">1 &#181;m and 0.2 &#181;m Steril Filter</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">1,4-Dithiothreitol</td>
			<td style="width:411px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">1-butanol. &#62;99.5% p.a.</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">2log DNA ladder</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">2-Mercaptoethanol</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">50 mL Falcon tubes</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">79249 Alkyne Mega Stokes dye</td>
			<td style="width:411px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Acetic acid glacial</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Acetonitrile, anhydrous, 99.8%</td>
			<td style="width:411px;">Sigma-Aldrich</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Ampicillin sodium-salt, 99%</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">BDP-FL-PEG4-DBCO</td>
			<td style="width:411px;">Jena Bioscience</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Biofuge</td>
			<td style="width:411px;">Heraeus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Bottle Top Filter with PES membrane (45 &#181;m, 22 &#181;m)</td>
			<td style="width:411px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Brillant Blue G250 (Coomassie)</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">BspQI</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Camera DS Qi1</td>
			<td style="width:411px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Centrifuge 5417r</td>
			<td style="width:411px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Centrifuge 5810r</td>
			<td style="width:411px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">CF-400-Cu square mesh copper grid</td>
			<td style="width:411px;">EMS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Chloramphenicol</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">CompactStar CS 4</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Dextran, Texas Red, 3000 MW, neutral</td>
			<td style="width:411px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital sonifier</td>
			<td style="width:411px;">Branson</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Dimethylsulfoxide (DMSO)</td>
			<td style="width:411px;">Applichem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Dnase I</td>
			<td style="width:411px;">Applichem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">EarI</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">EcoRI-HF</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Environmental shaker incubator ES-20</td>
			<td style="width:411px;">Biosan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Ethanol absolute</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Ethidium bromide solution</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Filter supports</td>
			<td style="width:411px;">Avanti</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Glass plates</td>
			<td style="width:411px;">Bio-Rad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Glycerol Proteomics Grade</td>
			<td style="width:411px;">Amresco</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Glycin</td>
			<td style="width:411px;">Applichem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">H4-Azido-Phe-OH</td>
			<td style="width:411px;">Bachhem</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Heat plate MR HeiTec</td>
			<td style="width:411px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">HindIII</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">HisTrap FF crude column</td>
			<td style="width:411px;">GE Life Sciences</td>
			<td style="width:119px;"></td>
			<td>Nickel column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Hydrochloride acid fuming, 37%, p.a.</td>
			<td style="width:411px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Illuminator ix 20</td>
			<td style="width:411px;">INTAS</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Illuminator LAS-4000</td>
			<td style="width:411px;">Fujifilm</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Imidazole</td>
			<td style="width:411px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Immersions oil for microscopy</td>
			<td style="width:411px;">Merck</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Incubators shakers Unimax 1010</td>
			<td style="width:411px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Inkubator 1000</td>
			<td style="width:411px;">Heidolph</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">IPTG, &#62;99%</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Kanamycinsulfate</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">L(+)-Arabinose</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Laboratory scales Extend ed2202s/224s-OCE</td>
			<td style="width:411px;">Sartorius</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">LB-Medium</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Lyophilizer Alpha 2-4 LSC</td>
			<td style="width:411px;">Christ</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Lysozyme, 20000 U/mg</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Microscope CM 100</td>
			<td style="width:411px;">Philips</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Microscope Eclipse TS 100</td>
			<td style="width:411px;">Nikon</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Microscopy cover glasses (15 x 15 mm)</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Microscopy slides</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Microwave</td>
			<td style="width:411px;">Studio</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Mini-Extruder Set</td>
			<td style="width:411px;">Avanti Polar Lipids</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">NaCl, &#62;99.5%, p.a.</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Natriumhydroxid pellets</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Ni-NTA Agarose, PerfectPro</td>
			<td style="width:411px;">5 Prime</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Nucleopore Track-Etch Membrane</td>
			<td style="width:411px;">Avanti</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">PH meter 766 calimatic</td>
			<td style="width:411px;">Knick</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Phenylmethylsulfonylflourid (PMSF)</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Polypropylene Columns (1 mL)</td>
			<td style="width:411px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">PowerPac basic</td>
			<td style="width:411px;">BioRad</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Propanol-2-ol</td>
			<td style="width:411px;">Emplura</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Protein ladder 10-250 kDa</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Recirculating cooler F12</td>
			<td style="width:411px;">Julabo</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Reinforcement rings</td>
			<td style="width:411px;">Herma</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">SacI HF</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">SDS Pellets</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Sodiumdihydrogen phosphate dihydrate, NaH2PO4</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Sterile syringe filter 0.2 mm Cellulose Acetate</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">T4 DNA Ligase</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">TEMED</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">TexasRed Dextran-Conjugate</td>
			<td style="width:411px;">MolecularProbes</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Thermomix comfort</td>
			<td style="width:411px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">THF, &#62;99.5% p.a.</td>
			<td style="width:411px;">Acros</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Triton X 100</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Trypton/Pepton from Casein</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Ultrasonic cleaner</td>
			<td style="width:411px;">VWR</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Urea p.a.</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">Vacuum pump 2.5</td>
			<td style="width:411px;">Vacuubrand</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">XbaI</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">XhoI</td>
			<td style="width:411px;">NEB</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:495px;">ZelluTrans regenerated cellulose tubular membrane (12.0 S/ 3.5 S/ 1.0 V)</td>
			<td style="width:411px;">Roth</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

directed assembly of elastin-like proteins into defined supramolecular structures and cargo encapsulation in vitro

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.

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...

Watch this Scientific Journal Video about Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro at JoVE.com

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

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.

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...

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.

Research

JoVE Journal

Chemistry

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Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...