The authors thank Aditya Iyer for the cloning of the pBAD-PercevalHR gene and Gea Schuurman-Wolters for aiding with protein production and purification. The research was funded by the NWO Gravitation program &#34;Building a Synthetic Cell&#34; (BaSyC).

Lipid Vesicle Encapsulation Protocol for Complex Protein Networks

<ol>
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The authors declare no competing financial interest.

We present a protocol for the synthesis of (membrane) protein containing sub-micrometer size lipid vesicles (proteoLUVs), and the conversion of proteoLUVs into giant-unilamellar vesicles (proteoGUVs). The protocol should be applicable for the reconstitution of other membrane proteins13,19,30,40 and the encapsulation of metabolic networks other than the L-arginine breakdown and glycerol 3-phosph...

The field of bottom-up synthetic biology focuses on constructing (minimal) cells1,2 and metabolic bioreactors for biotechnological3,4 or biomedical purposes5,6,7,8. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical functions of proteins and reaction networks9. As an intermediate step towards an autonomously functioning synthetic cell, modules are developed that capture essential features of living cells such as metabolic energy conservation, protein and lipid synthesis, and homeostasis. Such modules not only enhance our understanding of life but also have potential applications in the fields of medicine8 and biotechnology10.
Transmembrane proteins are at the heart of virtually any metabolic network as they transport molecules in or out of the cell, signal, and respond to the quality of the environment, and play numerous biosynthetic roles. Thus, the engineering of metabolic modules in synthetic cells requires in most cases the reconstitution of integral and/or peripheral membrane proteins into a membrane bilayer composed of specific lipids and high integrity (low permeability). The handling of these membrane proteins is challenging and requires specific knowledge and experimental skills.
Several methods have been developed to reconstitute membrane proteins within phospholipid vesicles, most often with the purpose of studying the function11,12, regulation13, kinetic properties14,15, lipid dependence15,16, and/or stability17 of a specific protein. These methods involve the rapid dilution of detergent-solubilized protein into aqueous media in the presence of lipids18, the removal of detergents by incubating detergent-solubilized protein with detergent-destabilized lipid vesicles and absorption of the detergent(s) onto polystyrene beads19, or the removal of detergents by dialysis or size-exclusion chromatography20. Organic solvents have been used to form lipid vesicles, for example, via the formation of oil-water interphases21, but the majority of integral membrane proteins are inactivated when exposed to such solvents.
In our laboratory, we mostly reconstitute membrane proteins by the detergent-absorption method to form large-unilamellar vesicles (LUVs)19. This method allows the co-reconstitution of multiple membrane proteins and the encapsulation in the vesicle lumen of enzymes, metabolites, and probes22,23. The membrane protein-containing LUVs can be converted into giant-unilamellar vesicles (GUVs) with/without encapsulation of water-soluble components, using either electroformation24 or gel-assisted swelling25 and specific conditions to preserve the integrity of the membrane proteins26.
This paper presents a protocol for the reconstitution in LUVs of an out-of-equilibrium metabolic network that regenerates ATP through the breakdown of L-arginine into L-ornithine27. The formation of ATP is coupled to the production of glycerol-3-phosphate (G3P), an important building block for phospholipid synthesis22,28. The metabolic pathway consists of two integral membrane proteins, an arginine/ornithine (ArcD) and a G3P/Pi antiporter (GlpT). In addition, three soluble enzymes (ArcA, ArcB, ArcC) are required for the recycling of ATP, and GlpK is used to convert glycerol into glycerol 3-phosphate, using the ATP from the breakdown of L-arginine, see Figure 1 for a schematic overview of the pathway. This protocol represents a good starting point for the future construction of even more complex reaction networks-for the synthesis of lipids or proteins or the division of cells. The lipid composition of the vesicles supports the activity of a wide variety of integral membrane proteins and has been optimized for the transport of diverse molecules into or out of the vesicles27,29,30.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66627/66627fig01v2.jpg" /> 
Figure 1: Overview of the pathway for ATP production and glycerol 3-phosphate synthesis and excretion. <a href="https://www.jove.com/files/ftp_upload/66627/66627fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
In short, purified membrane proteins (solubilized in dodecyl-&#946;-D-maltoside, DDM) are added to preformed lipid vesicles that have been destabilized with Triton X-100, which allows the insertion of the proteins into the membrane. The detergent molecules are subsequently (slowly) removed by the addition of activated polystyrene beads, resulting in the formation of well-sealed proteoliposomes. Soluble components can then be added to the vesicles and encapsulated via freeze-thaw cycles, which traps the molecules in the process of membrane fusion. The obtained vesicles are highly heterogeneous and many are multilamellar. They are then extruded through a polycarbonate filter with a pore size of 400, 200, or 100 nm, which yields more uniformly sized vesicles; the smaller the pore size, the more homogeneous and unilamellar the vesicles but at the price of a smaller internal volume. Non-incorporated proteins and small molecules are removed from the external solution by size-exclusion chromatography. The proteoLUVs can be converted into micrometer size vesicles by gel-assisted swelling, and these proteoGUVs are then collected and trapped in a microfluidic chip for microscopic characterization and manipulation. Figure 2 shows a schematic overview of the full protocol.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66627/66627fig02v2.jpg" /> 
Figure 2: Overview of the protocol for reconstituting membrane proteins and encapsulating enzymes and water-soluble components in lipid vesicles of sub-micrometer (LUVs) and micrometer size (GUVs). <a href="https://www.jove.com/files/ftp_upload/66627/66627fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The reconstitution and encapsulation protocols work well and the functionality of the proteins is retained, but the proteoLUVs and proteoGUVs are heterogeneous in size. Microfluidic approaches31,32 allow the formation of micrometer-sized vesicles that are more homogeneous in size, but functional reconstitution of membrane proteins is generally not possible because residual solvent in the bilayer inactivates the proteins. The proteoLUVs range in size from 100 to 400 nm, and at low concentrations of enzymes, the encapsulation may lead to vesicles with incomplete metabolic pathways (stochastic effects; see Figure 3). LUVs are ideal for constructing specific metabolic modules, as shown here for the production of ATP and building blocks like G3P. Such proteoLUVs can potentially be encapsulated in GUVs and serve as organelle-like compartments for the host vesicles.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66627/66627fig03v2.jpg" /> 
Figure 3: Number of molecules per vesicle with a diameter of 100, 200, or 400 nm. (A) When the encapsulated proteins (enzymes, probes) are in the range of 1-10 &#181;M. (B) The reconstitution is done at 1 to 1,000, 1 to 10,000, and 1 to 100,000 membrane proteins per lipid (mol/mol). We make the assumption that molecules are encapsulated at the indicated concentrations and incorporated in the membrane at these protein-to-lipid ratios. For some enzymes, we have seen that they bind to membranes, which can increase their apparent concentration in the vesicles. Abbreviation: LPR = Lipid-Protein-Ratio <a href="https://www.jove.com/files/ftp_upload/66627/66627fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma Aldrich</td>
			<td>A9414-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon cut-off filter</td>
			<td>Sigma Aldrich</td>
			<td>Milipore centrifugal filter units Amicon Ultra&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>BioBeads</td>
			<td>BioRad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>CHCl3</td>
			<td>Macron Fine Chemicals</td>
			<td>MFCD00000826</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Glucose</td>
			<td>Formedium</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>D(+)-Sucrose</td>
			<td>Formedium</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>DDM</td>
			<td>Glycon</td>
			<td>D97002 -C</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl Ether</td>
			<td>Biosolve</td>
			<td>52805</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>276855-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPC</td>
			<td>Avanti</td>
			<td>850375P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPE</td>
			<td>Avanti</td>
			<td>850725P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DOPG</td>
			<td>Avanti</td>
			<td>840475P-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Formedium&#160;</td>
			<td>DTT005</td>
			<td></td>
		</tr>
		<tr>
			<td>EtOH</td>
			<td>J.T.Baker Avantor</td>
			<td>MFCD00003568</td>
			<td></td>
		</tr>
		<tr>
			<td>Extruder</td>
			<td>Avestin Inc</td>
			<td>LF-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorimeter</td>
			<td>Jasco</td>
			<td>Spectrofluorometer FP-8300</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>BOOM</td>
			<td>51171608</td>
			<td></td>
		</tr>
		<tr>
			<td>Gravity flow column</td>
			<td>Bio-Rad</td>
			<td>732-1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 100 &#181;L</td>
			<td>Hamilton</td>
			<td>7656-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe 1000 &#181;L</td>
			<td>Hamilton</td>
			<td>81320</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld LCP dispenser</td>
			<td>Art Robbins Instruments</td>
			<td>620-411-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Handheld Sonicator</td>
			<td>Hielscher Ultrasound Technology</td>
			<td>UP50H</td>
			<td></td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>BOOM</td>
			<td>x76021889.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Roth</td>
			<td>X998.4-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Supelco</td>
			<td>1.05099.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>BOOM</td>
			<td>76028270.1</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Supelco</td>
			<td>1.04873.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipe</td>
			<td>Kimtech Science</td>
			<td>7552</td>
			<td></td>
		</tr>
		<tr>
			<td>Large Falcon tube centrifuge</td>
			<td>Eppendorf</td>
			<td>Centrifuge 5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Arginine</td>
			<td>Sigma-Aldrich</td>
			<td>A5006-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Leica</td>
			<td>DM LS2</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ornithine</td>
			<td>Roth</td>
			<td>T204.1</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Laser Scanning Confocal Microscope</td>
			<td>Zeiss</td>
			<td>LSM 710 ConfoCor 3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M2670-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Microfluidic chip</td>
			<td>Homemade&#160;</td>
			<td>PDMS based</td>
			<td>DOI: https://doi.org/10.1039/C8LC01275J</td>
		</tr>
		<tr>
			<td>Na-ADP</td>
			<td>Sigma-Aldrich</td>
			<td>A2754-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Supelco</td>
			<td>1.06404.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Nanodrop Spectrometer</td>
			<td>Isogen Life Science</td>
			<td>ND-1000 spectrophotometer NanoDrop</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Supelco</td>
			<td>1.06498.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Needles for GUVs</td>
			<td>Henke-Ject</td>
			<td>14-14575</td>
			<td>27 G x 3/4&#39;&#39; 0.4 x 20 mm</td>
		</tr>
		<tr>
			<td>Needles for microfluidics</td>
			<td>Henke-Ject</td>
			<td>14-15538</td>
			<td>18 G x 1 1/2&#39;&#39; 1.2 x 40 mm</td>
		</tr>
		<tr>
			<td>Ni2+ Sepharose</td>
			<td>Cytiva</td>
			<td>17526802</td>
			<td></td>
		</tr>
		<tr>
			<td>Nigericin</td>
			<td>Sigma-Aldrich</td>
			<td>N7143-5MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutator</td>
			<td>VWR</td>
			<td>83007-210</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmolality meter</td>
			<td>Gonotec Salmenkipp</td>
			<td>Osmomat 3000 basic freezing point osmometer</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmacleaner</td>
			<td>Plasma Etch</td>
			<td>PE-Avenger</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate filter</td>
			<td>Cytiva Whatman</td>
			<td>Nuclepor Track-Etch Membrane Product: 10417104</td>
			<td>0.4 &#181;m</td>
		</tr>
		<tr>
			<td>Polycarbonate ultracentrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>355647</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyranine</td>
			<td>Acros Organics</td>
			<td>H1529-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Quartz cuvette (black)</td>
			<td>Hellma Analytics</td>
			<td>108B-10-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Sephadex G-75 resin&#160;</td>
			<td>GE Healthcare</td>
			<td>17-0050-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics Sonics &#38; Materials INC</td>
			<td>Sonics vibra cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Sarstedt</td>
			<td>Filtropur S plus 0.2</td>
			<td>0.2 &#181;m</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Harvard Apparatus</td>
			<td>A-42467</td>
			<td></td>
		</tr>
		<tr>
			<td>Tabletop centrifuge</td>
			<td>Eppendorf</td>
			<td>centrifuge 5418</td>
			<td></td>
		</tr>
		<tr>
			<td>Teflon spacer</td>
			<td>Homemade&#160;</td>
			<td>Teflon based</td>
			<td>45 x 26 x 1.5 or 45 x 26 x 3 or 20 x 20 x 3 mm</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>PanReac AppliChem</td>
			<td>A1086.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma Aldrich</td>
			<td>T8787-100 ml</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima Max-E</td>
			<td></td>
		</tr>
		<tr>
			<td>UV lamp</td>
			<td>Spectroline</td>
			<td>ENB-280C/FE</td>
			<td></td>
		</tr>
		<tr>
			<td>UV/VIS Spectrometer</td>
			<td>Jasco</td>
			<td>V730 spectrophotometer</td>
			<td></td>
		</tr>
		<tr>
			<td>Valinomycin</td>
			<td>Sigma-Aldrich</td>
			<td>V0627-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Widefield fluorescence microscope</td>
			<td>Zeiss</td>
			<td>AxioObserver</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-Casein</td>
			<td>Sigma Aldrich</td>
			<td>C5890-500g</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of out&#45;of&#45;equilibrium metabolic networks in nano&#45; and micrometer&#45;sized vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

The reconstitution of solubilized membrane proteins in liposomes requires the destabilization of preformed vesicles. The addition of low amounts of Triton X-100 initially results in an increase of absorbance at 540 nm (A540) due to an increase in light scattering by the swelling of the vesicles (Figure 4). The maximum A540 value is the point where the liposomes are saturated with detergent (Rsat), after which any further addition of Triton X-100 will...

Watch this Scientific Journal Video about Author Spotlight: Tackling Challenges in Synthetic Cell Engineering at JoVE.com

Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

construction-out-equilibrium-metabolic-networks-nano-micrometer-sized

Research

JoVE Journal

Biochemistry

900 Views.  University of Groningen. We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Video: Author Spotlight: Tackling Challenges in Synthetic Cell Engineering

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique applications for OMV where traditional nanoparticles are proving too difficult to synthesize. To date, all Gram-negative bacteria have been shown to produce OMV demonstrating packaging of a variety of cargo that includes small molecules, peptides, proteins and genetic material. Based on their diverse cargo, OMV are implicated in many biological processes ranging from cell-cell communication to gene transfer and delivery of virulence factors depending upon which bacteria are producing the OMV. Only recently have bacterial OMV become accessible for use across a wide range of applications through the development of techniques to control and direct packaging of recombinant proteins into OMV. This protocol describes a method for the production, purification, and use of enzyme packaged OMV providing for improved overall production of recombinant enzyme, increased vesiculation, and enhanced enzyme stability. Successful utilization of this protocol will result in the creation of a bacterial strain that simultaneously produces a recombinant protein and directs it for OMV encapsulation through creating a synthetic linkage between the recombinant protein and an outer membrane anchor protein. This protocol also details methods for isolating OMV from bacterial cultures as well as proper handling techniques and things to consider when adapting this protocol for use for other unique applications such as: pharmaceutical drug delivery, medical diagnostics, and environmental remediation.

Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification

A protocol for the production, purification, and use of enzyme packaged outer membrane vesicles (OMV) providing for enhanced enzyme stability for implementation across diverse applications is presented.

An increasing interest in applying synthetic biology techniques to program outer membrane vesicles (OMV) are leading to some very interesting and unique ...

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

Automated Acoustic Dispensing for the Serial Dilution of Peptide Agonists in Potency Determination Assays

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. Screening peptides affords an additional layer of complexity as conventional tip-based sample handling methods expose peptides to a large surface area of plasticware, providing an increased opportunity for peptide loss via adsorption. Preventing excessive exposure to plasticware reduces peptide loss via adherence to plastics and thus minimizes inaccuracies in potency prediction, and we have previously described the benefits of non-contact acoustic dispensing for in vitro high-throughput screening of peptide agonists1. Here we discuss a fully integrated automation solution for non-contact acoustic preparation of peptide serial dilutions in microtiter plates utilizing the example of screening for peptide agonists at the mouse glucagon-like peptide-1 receptor (GLP-1R). Our methods allow for high-throughput cell-based assays to screen for agonists and are easily scalable to support increased sample throughput, or to allow for increased numbers of assay plate copies (e.g., for a panel of more target cell lines).

Peptide adsorption to plasticware during traditional tip-based serial dilutions can significantly impact potency determination and confound the understanding of structure-activity relationships used for lead identification and lead optimization phases of drug discovery. Here methods for automated acoustic non-contact serial dilution of peptide samples are described.

As with small molecule drug discovery, screening for peptide agonists requires the serial dilution of peptides to produce concentration-response curves. ...

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase solubility and facilitate excretion. On some occasions those structural modifications lead to the formation of new toxic products. In order to reduce animal testing, chemical risk can be assessed using metabolically competent cells. The expression of metabolic enzymes, however, is not stable over time in many in vitro primary culture systems and is often partial or absent in cell lines. Therefore, the study of medicines, additives, and environmental pollutants metabolism in vitro should ideally be conducted in cell systems where metabolic activity has been characterized. We explain here an approach to measure the activity of a class of metabolic enzymes (Human Phase I) in 2D cell lines and primary 3D cultures using chemical probes and their metabolic products quantifiable by UPLC mass spectrometry and luminometry. The method can be implemented to test the metabolic activity in cell lines and primary cells derived from a variety of tissues.

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Metabolic competency of in vitro systems is a key requirement for the biotransformation and disposition of drugs and toxicants. In this protocol, we describe the application of reference metabolic probes to assess phase I metabolism in cell cultures.

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

Identification of Functional Protein Regions Through Chimeric Protein Construction

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences in a structurally similar protein, in order to determine the functional importance of these regions. Such chimeras are generated by means of a nested PCR protocol using overlapping DNA fragments and adequately designed primers, followed by their expression within a mammalian system to ensure native secondary structure and post-translational modifications. 
The functional role of a distinct region is then indicated by a loss of activity of the chimera in an appropriate readout assay. In consequence, regions harboring a set of critical amino acids are identified, which can be further screened by complementary techniques (e.g. site-directed mutagenesis) to increase molecular resolution. Although limited to cases in which a structurally related protein with differing functions can be found, chimeric proteins have been successfully employed to identify critical binding regions in proteins such as cytokines and cytokine receptors. This method is particularly suitable in cases in which the protein's functional regions are not well defined, and constitutes a valuable first step in directed evolution approaches to narrow down the regions of interest and reduce the screening effort involved.

Structurally related proteins frequently exert distinct biological functions. The exchange of equivalent regions of these proteins in order to create chimeric proteins constitutes an innovative approach to identify critical protein regions that are responsible for their functional divergence.

The goal of this protocol encompasses the design of chimeric proteins in which distinct regions of a protein are replaced by their corresponding sequences ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil (Ers4tU)

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of the RNA produced during a brief period of labelling. This is done by attaching a biotin moiety to the incorporated thio group and affinity purifying, using streptavidin coated beads. Achieving a good yield of pure, newly synthesized RNA that is free of pre-existing RNA makes shorter labelling times possible and permits increased temporal resolution in kinetic studies. This is a protocol for very specific, high yield purification of newly synthesized RNA. The protocol presented here describes how RNA is extracted from the yeast Saccharomyces cerevisiae. However, the protocol for purification of thiolated RNA from total RNA should be effective using RNA from any organism once it has been extracted from the cells. The purified RNA is suitable for analysis by many widely used techniques, such as reverse transcriptase-qPCR, RNA-seq and SLAM-seq. The specificity, sensitivity and flexibility of this technique allow unparalleled insights into RNA metabolism.

Extremely Rapid and Specific Metabolic Labelling of RNA In Vivo with 4-Thiouracil Ers4tU

The use of thiolated uracil to sensitively and specifically purify newly transcribed RNA from the yeast Saccharomyces cerevisiae.

The nucleotide analogue, 4-thiouracil (4tU), is readily taken up by cells and incorporated into RNA as it is transcribed in vivo, allowing isolation of ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...