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 ...

Research

JoVE Journal

Chemistry

Origami Inspired Self-assembly of Patterned and Reconfigurable Particles

There are numerous techniques such as photolithography, electron-beam lithography and soft-lithography that can be used to precisely pattern two ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

In Situ SIMS and IR Spectroscopy of Well-defined Surfaces Prepared by Soft Landing of Mass-selected Ions

Soft landing of mass-selected ions onto surfaces is a powerful approach for the highly-controlled preparation of materials that are inaccessible using ...

Atomically Defined Templates for Epitaxial Growth of Complex Oxide Thin Films

Atomically defined substrate surfaces are prerequisite for the epitaxial growth of complex oxide thin films. In this protocol, two approaches to obtain ...

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

Encapsulation of Cancer Therapeutic Agent Dacarbazine Using Nanostructured Lipid Carrier

The only formula of dacarbazine (Dac) in clinical use is intravenous infusion, presenting a poor therapeutic profile due to the low dispersity of the drug ...

Synthesis and Characterization of Supramolecular Colloids

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 ...