This research was funded by the Office of Naval Research through Core funds provided to the Naval Research Laboratory.

1. Preparation of Plasmids
<ol>
	<li>Prepare a plasmid (e.g., pET22) containing the anchor protein (OmpA) fused to a biorthogonal linkage domain (referred to in this protocol as anchor-ST), epitope tag (such as 6xHis, myc or FLAG tags for purification and identification), periplasmic localization tag, antibiotic resistance, and appropriate origin of replication based on the selected bacterial strain7.
	<ol>
		<li>Extract plasmid DNA from overnight cultures using a commercially available DNA isolation kit following the manufacturer&#39;s protocol.</li>
	</ol>
	</li>
	<li>Prepare a plasmid (e.g., pACYC184) containing the enzyme/protein (PTE) to be packaged within the OMV fused to the complementary bioorthogonal linkage domain to that of the anchor protein (referred to in this protocol as enzyme-SC), epitope tag, periplasmic localization tag, antibiotic resistance that is different than the anchor protein plasmid, and appropriate origin of replication based on the selected bacterial strain7.
	<ol>
		<li>Extract plasmid DNA from overnight cultures using a commercially available DNA isolation kit following the manufacturer&#39;s protocol7.</li>
	</ol>
	</li>
</ol>
2. Generation of an OMV Packaging E. coli Culture
<ol>
	<li>Plasmid Isolation
	<ol>
		<li>Purify plasmids encoding the enzyme-SC construct and anchor-ST constructs from separate maintenance cell lines using a commercial plasmid isolation kit as per manufacturer&#39;s protocol7. Determine DNA concentration and purity by measuring absorbance at 260 nm and 280 nm.</li>
	</ol>
	</li>
	<li>Co-transformation
	<ol>
		<li>Select an E. coli strain that is commercially available to facilitate protein expression. 
		NOTE: BL21(DE3) cells were utilized here. The T7 lysogen is required for expression of the anchor-ST construct, which relies on the T7 promoter and T7 RNA polymerase for protein expression. Use of other bacterial strains requires either the presence of the T7 lysogen gene or the use of a plasmid other than pET227.</li>
		<li>Dilute purified plasmid DNA to equivalent molar concentrations then transform them into competent bacterial cells via either heat shock transformation or electroporation following the manufacturer&#39;s protocol for the competent cells chosen.</li>
		<li>Plate transformed cells onto antibiotic-containing Luria-Bertani (LB) agar plates with both ampicillin (pET22) and chloramphenicol (pACYC184); both present at final concentrations of 25 &#181;g/ml.</li>
		<li>Place the culture dish at 37 &#176;C overnight to isolate only clones containing both plasmids. 
		NOTE: Colonies that survive antibiotic selection will be used for all future OMV preparations. Antibiotics (ampicillin and chloramphenicol) will be added to all liquid cultures to maintain selective pressure and ensure the presence of both plasmids.</li>
	</ol>
	</li>
</ol>
3. OMV Production
<ol>
	<li>Colony Expansion
	<ol>
		<li>Inoculate 5 ml of culture media (typically Terrific Broth (TB) or LB) containing antibiotics at the aforementioned concentrations (step 2.2.3) with a single colony from the selection plates described in 2.2.4.</li>
		<li>Perform a 1:100 fold dilution of the overnight starter culture into baffled culture flasks. 
		NOTE: Use a volume of media between 250-500 ml.</li>
		<li>Maintain the culture at 37 &#176;C shaking at 250 rpm to ensure that sufficient aeration of the culture is attained.</li>
	</ol>
	</li>
	<li>Induction of Each Plasmid
	<ol>
		<li>Allow the bacterial culture to grow to an OD600 of 0.6-0.8, approximately 3 hr of growth after inoculation of the primary growth flask.</li>
		<li>To improve the final yield of PTE-SC loaded vesicles, centrifuge the entire culture at 7,000 x g for 15 min. Resuspend the cell pellet in pre-warmed, fresh culture media. 
		NOTE: This optional step helps to remove any empty OMV that are present in the culture media prior to PTE-SC induction.</li>
		<li>Make a 20% stock solution of L-arabinose in water and sterile filter using a 0.22 &#181;m filter. Store L-arabinose solution at 4 &#176;C for 2-4 weeks.</li>
		<li>Add the L-arabinose solution to the culture flask to a final 0.2% concentration to initiate PTE-SC production. 
		NOTE: This helps to preload the periplasmic space with PTE-SC prior to promoting increased vesiculation through induction of the mutant OmpA-ST.</li>
		<li>Make a 1 M Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG) solution in water and sterile filter using a 0.22 &#181;m filter. 
		NOTE: IPTG must be used immediately once hydrated. Aliquots of IPTG can be stored frozen for 3-6 months and can be thawed immediately prior to use.</li>
		<li>Add IPTG after an additional 3 hr incubation period to a final concentration of 0.5 mM to initiate production of the OmpA-ST.</li>
		<li>Allow the culture to grow for an additional 8-14 hr at 37 &#176;C while shaking.</li>
	</ol>
	</li>
</ol>
4. OMV Purification
<ol>
	<li>Removal of Intact Bacteria
	<ol>
		<li>Centrifuge the bacterial culture media for 15 min at 7,000 x g at 4 &#176;C to pellet intact bacteria.</li>
		<li>Decant, and save, the media off the top of the cell pellet post centrifugation being careful to not disturb the cell pellet.</li>
		<li>Repeat steps 4.1.1 and 4.1.2 for one additional centrifugation cycle to ensure all intact bacteria are removed from the culture media being sure to decant and save the media.</li>
	</ol>
	</li>
	<li>Removal of Protein Aggregates
	<ol>
		<li>Further purify the bacterial culture media using a 0.45 &#181;m membrane filter to remove residual bacteria and undesired large cellular material. 
		NOTE: Use a biologically compatible membrane material for higher recoveries and lower protein adsorption such as cellulose acetate (CA) or polyvinylidene difluoride (PVDF). Syringe filter volumes less than 100 ml and vacuum filter volumes greater than 100 ml.
		<ol>
			<li>To syringe filter, draw culture medium into sterile 10-50 ml syringe. Attach 0.45 &#181;m filter to Luer end of syringe. Depress plunger and collect flow-through in a new vessel.</li>
			<li>To vacuum filter, transfer culture medium to a sterile filtration apparatus. Attach unit to vacuum pump or sink aspirator to generate suction. Transfer filtered medium to new vessel.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Isolation of OMV via Ultracentrifugation
	<ol>
		<li>Add the filtered culture media into centrifugation tubes being careful to balance opposing tubes in the centrifuge.</li>
		<li>Pellet at ~150,000 x g for 3 hr at 4 &#176;C in an ultracentrifuge using the corresponding rotor matching both the instrument and tubes being utilized.</li>
		<li>Decant the OMV depleted culture media from the OMV pellet, being careful not to disturb the OMV pellet, immediately at the completion of centrifugation as allowing the OMV pellet to remain in the media will soften the pellet reducing OMV recovery. 
		NOTE: This pellet will be slightly brown and depending on the starting amount of bacterial culture may or may not be visible by eye. Aspirating the media directly from the top of the centrifugation tube is also an option keeping in mind that the more media removed from the OMV pellet the more pure the final OMV sample will be.</li>
		<li>Save the centrifuged supernatant for later dynamic light scattering (DLS) testing to verify that the OMV particles were fully depleted from the media.</li>
		<li>Add 0.5-1 ml of sterile filtered Phosphate Buffered Saline (PBS) pH 7.4 or 100 mM N-Cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer at pH 8.0 to the OMV pellet allowing it to incubate for 30 min at RT. Ensure that the pellet does not dry. 
		NOTE: Extremes in solution ionic strength, pH, and temperature may result in reduced OMV solubility and promote aggregation.</li>
		<li>Carefully pipet the purified OMV solution from the centrifuge tube, mixing gently to ensure the entire pellet has been solubilized.</li>
	</ol>
	</li>
</ol>
5. OMV Characterization
<ol>
	<li>Use any commercially available DLS or nanoparticle tracking software following the provided manufacturer&#39;s protocols unique to each instrument to determine OMV size distribution and particle concentration. 
	NOTE: Here, OMV size distribution and concentration was determined utilizing NanoSight LM10 Nanoparticle Tracking and NTA 2.3 Analysis software.
	<ol>
		<li>Open the software.</li>
		<li>Turn on microscope and clean all surfaces on the microscope and nanoparticle tracking unit.</li>
		<li>Add ~0.5 ml of OMV sample directly to the viewing window of the particle tracking device through the Luer fitting with a 1 ml sterile syringe being sure to cover the entire viewing window without injecting any air bubbles into the system.</li>
		<li>Place the nanoparticle tracking unit on the microscope stage and turn on the laser.</li>
		<li>Click &#34;Capture&#34; in the software.</li>
		<li>Adjust the microscope focus and stage to visualize the particles on the preview screen present in the software window.</li>
		<li>Adjust the &#34;Camera Shutter&#34; and &#34;Camera Gain&#34; until bright particles are clearly visualized on a dark background.</li>
		<li>Adjust capture duration to 60 sec.</li>
		<li>Click &#34;Record&#34;.</li>
		<li>When prompted, input the sample/device temperature, label the sample, and save the video data at the completion of the each run into a user designated folder.</li>
		<li>Keep all analysis parameters (Detection Threshold, Blur, Min Track Length, Min Expected Particle Size) on auto detect mode and click &#34;Process Sequence.&#34; 
		NOTE: Consult the manual for how each parameter can be adjusted independently to fine tune analysis results.</li>
		<li>Record particle size distribution and particles/ml concentration as determined by the software. 
		NOTE: A typical OMV hydrodynamic size range is between 30-200 nm with a particle/ml concentration of ~10 x 108.</li>
	</ol>
	</li>
	<li>Determining OMV Protein Content
	<ol>
		<li>Use standard SDS-PAGE and Western blot protocols to assess OMV purity, enzyme production, and the SC-ST crosslinking efficiency7. 
		NOTE: Crosslinking efficiency will not be 100%7.</li>
	</ol>
	</li>
</ol>
6. Verification of Enzyme Packaging
<ol>
	<li>PTE Activity Assay
	<ol>
		<li>Perform all enzymatic assays in a suitable buffer such as 100 mM CHES buffer (pH 8) at 25 &#176;C in either a cuvette or multiplexed in a 96/384 well plate.</li>
		<li>Add 5 &#181;l of a 1:1,000 dilution of paraoxon in CHES buffer to 90 &#181;l of CHES and 5 &#181;l of purified OMV (~36-fold concentrated compared to the OMV concentration present in the culture media). 
		Caution: Paraoxon is a toxic pesticide and must be handled per manufacturer and institutional guidelines.</li>
		<li>Take absorbance readings at regular intervals (every 20 sec for ~2 hr total reaction time) at 405 nm (&#949; = 18.1 mM-1cm-1) and at 348 nm (isosbestic point &#949; = 5.4 mM-1cm-1) to monitor the reaction progression of the chromogenic paraoxon breakdown product, p-nitrophenol. 
		NOTE: At pH values above 8 the dominant deprotonated form of p-nitrophenol is present allowing for accurate monitoring at 405 nm. In all other complex or lower pH solutions it is necessary to utilize the isosbestic point of 348 nm as multiple forms of the p-nitrophenol will be present.</li>
		<li>Calculate initial reaction velocities by determining the slope of the linear portion of the progress curves from step 6.1.3 to compare the relative quantity of PTE in each sample, packaging efficiency, and total PTE production.</li>
		<li>Perform standard enzymatic assay protocols to determine KM, Vmax and kcat for the OMV encapsulated PTE. 
		NOTE: A Lineweaver-Burk analysis can be utilized for determination of these kinetic parameters where the y-intercept = 1/Vmax and slope = KM/Vmax20.</li>
	</ol>
	</li>
	<li>Transmembrane Substrate/Product Diffusion.
	<ol>
		<li>Perform the same kinetic assay analysis as described in step 6.1 utilizing increasing concentrations of Triton X-100 in CHES buffer from 0-3% by volume. 
		NOTE: The addition of Triton X-100 to the solubilized OMV functions to disrupt the lipid bilayer allowing for the contents of the OMV to be freely accessible to the reaction solution.</li>
		<li>Calculate and compare the initial velocities at each Triton X-100 level to verify transmembrane passage of the substrate/product indicated by minimal change in enzyme activity (initial velocities) compared to enzyme activity in the absence of Triton X-100. If a substantial increase in enzyme activity is observed in the presence of Triton X-100 it is likely that the substrate does not freely diffuse into the OMV.</li>
		<li>Assay free enzyme activity (as described above in section 6.1) in the presence of Triton X-100 to verify that enzyme activity is not inhibited by the surfactant itself. 
		NOTE: If activity is greatly impacted by Triton X-100, alternative additives, such as saponins or various polysorbate/tween surfactants may be more suitable to rupture the OMV membrane.</li>
	</ol>
	</li>
</ol>
7. OMV Storage
<ol>
	<li>Freezing
	<ol>
		<li>Place ~100 &#181;l aliquots of purified OMV directly into liquid nitrogen to snap freeze the aliquots being sure not to fully submerge the vials or contact any liquid nitrogen with the sample itself. 
		NOTE: Purified OMV can be snap frozen directly in the buffer that was selected for solubilizing the OMV pellet during the purification process with no additional cryoprotectants necessary7.</li>
		<li>Store the snap frozen aliquots at -80 &#176;C.</li>
		<li>Thaw frozen aliquots from step 7.1.1 by placing them at RT being sure not to heat the samples.</li>
		<li>Prior to preparing all samples for storage in this manner perform the enzyme assay described in step 6.1 comparing initial velocities before and after freezing to verify that there is no significant loss in enzyme activity post freeze-thaw. 
		NOTE: Store purified OMV at 4 &#176;C if a reduction in enzyme activity is observed due to freezing.</li>
	</ol>
	</li>
	<li>Lyophilization
	<ol>
		<li>Lyophilize the snap frozen purified OMV aliquots utilizing commercially available lyophilization equipment21.</li>
		<li>Store lyophilized aliquots at RT for weeks or at -80 &#176;C for many months.</li>
		<li>Add the same volume of purified water to the lyophilized OMV as prior to lyophilization and let stand at RT for 30 min to rehydrate the sample. Mix gently when necessary being sure not to vortex or sonicate the OMV.</li>
		<li>Add lyophilized OMV powder directly to point-of-care for applications where prior dilution/solubilization is not necessary.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protecting+enzymatic+function+through+directed+packaging+into+bacterial+outer+membrane+vesicles.">Protecting enzymatic function through directed packaging into bacterial outer membrane vesicles.</a> Sci. Rep. 6, 24866 (2016).</li><li>Alves, N. J., Turner, K. B., Medintz, I. L., Walper, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+therapeutic+delivery+capabilities+and+challenges+utilizing+enzyme/protein+packaged+bacterial+vesicles.">Emerging therapeutic delivery capabilities and challenges utilizing enzyme/protein packaged bacterial vesicles.</a> Ther. Delivery. 6, 873-887 (2015).</li></ol>

The authors have nothing to disclose.

This protocol functions to demonstrate a representative directed packaging technique in which an enzyme of interest is produced and packaged into OMV by E. coli. As with many complex techniques there are multiple areas in which the protocol can be modified to accommodate for use in different unique applications, some of which are detailed below. While the mechanism of OMV packaging and enzyme encapsulation can be adapted to specific needs there are several steps within this protocol which are critical to its suc...

Presented here is a method for the design, production, and purification of enzyme-loaded bacterial outer membrane vesicles (OMV). OMV are small, primarily unilamellar, proteoliposomes that range in size from 30-200 nm1,2. All Gram-negative and Gram-positive bacteria that have been studied to date have demonstrated release of either OMV or extracellular vesicles (EV) from their surface3,4. The precise mechanism by which OMV are produced have yet to be fully elucidated due to the diverse bacterial populations that secrete them as well as the varying functions that they serve. OMV have been shown to transport a wide range of cargo from small molecules, peptides, and proteins to genetic material serving a variety of complex signaling, gene translocation, and virulence functions5,6.
The exact mechanisms of OMV biogenesis are not well characterized, and appear to differ between bacterial species. Despite this fact, we have developed a method for enhancing the packaging efficiency of a recombinant protein into OMV by creating a synthetic linkage between a protein of interest and a highly abundant protein endogenous to the bacterial outer membrane and subsequent OMV. In the absence of a synthetic linkage, or artificially incorporated affinity, between the recombinantly expressed protein and the OMV the observed packaging efficiency is very low7. This result is to be expected as incorporation of the proteins within the OMV either occurs through random chance encapsulation at the precise moment of OMV formation at the bacterial surface or through directed packaging by mechanisms that are not well understood. Some success has been observed in packaging proteins simply through over expression within the periplasmic space which relies on random chance encapsulation but effective packaging is highly protein dependent with some proteins packaging at high efficiency compared to others that do not package at all8-10. By utilizing common synthetic biology techniques we sought to engineer Escherichia coli (E. coli) to simultaneously produce, package and secrete an active enzyme of interest into OMV that circumvents current knowledge limitations regarding how OMV are formed and how cargo is selected by the bacteria for packaging.
For the purposes of this application a split protein bioconjugation system was selected as the synthetic linkage of choice to facilitate directional packaging into the OMV. As the name suggests a split protein bioconjugation system is comprised of two complementary subunit domains that interact with one another. The split protein domains selected for the purposes of this protocol are referred to as the SpyCatcher (SC) and SpyTag (ST) domain and are derived from the Streptococcus pyogenes fibronectin-binding protein (FbaB)11. This split protein system is unusual in that when the two subunits are within proximity an isopeptide bond spontaneously forms between the proximal aspartic acid and lysine amino acid residues creating a covalent linkage. Isopeptide bond formation does not require the addition of chaperone proteins, catalytic enzymes, or cofactors and can readily occur at room temperature (RT) and over a wide range of physiologically relevant conditions12.
As a proof of concept, phosphotriesterase (PTE) (EC 3.1.8.1) from Brevundimonas diminuta was selected to be packaged into E. coli derived OMV13. PTE contains a binuclear Zn/Zn active site and has the ability to break down organophosphates through a hydrolysis reaction converting aryldialkylphosphates into dialkylphosphates and aryl alcohols14. Exposure to organophosphates impairs proper neurotransmitter function through inhibiting the hydrolysis of acetylcholine by acetylcholinesterase at neuromuscular junctions making organophosphate derived compounds extremely dangerous15. Prolonged or significant exposure to organophosphates commonly results in uncontrollable convulsions and typically causes death via asphyxiation. While PTE exhibits the highest catalytic activity toward paraoxon, a very potent insecticide, it is also capable of hydrolyzing a broad range of other pesticides and V/G type chemical nerve agents16. To facilitate OMV packaging, a bacterial plasmid was designed that encodes a gene construct that contains an inducible promoter, a periplasmic localization sequence, and a short multiple cloning site upstream of the SC gene sequence. Insertion of the PTE gene between the leader and SC sequence allowed for creation of a genetic switch that targets the PTE-SC fusion protein to the periplasmic space for OMV packaging. While the efforts described here focus on PTE, the enzyme gene is interchangeable and could readily be replaced with another gene sequence to facilitate packaging of an alternate enzyme or protein.
As the second part of the synthetic linkage, an abundant outer membrane protein (OmpA) is chosen to present the ST peptide sequence. While the choice of anchor protein can vary, it is essential that the protein has a permissive domain that presents within the periplasmic space, tolerates the fusion construct without inducing cytotoxicity, is known to be present in OMV, and does not aggregate when it is recombinantly produced. OmpA is a 37.2 kDa transmembrane porin protein that is known to be highly expressed in the bacterial outer membrane and subsequent OMV17. It is implicated in the transport of small molecules, &#60;2 nm in size, across the bacterial membrane18. Native OmpA has two structurally unique domains, a transmembrane beta barrel motif and a periplasmically soluble C-terminal portion known to interact with the peptidoglycan19. In the mutant OmpA-ST fusion designed here the C-terminal portion of OmpA was deleted and the ST was fused to the periplasmically facing N- or C-termini. Deleting the periplasmic portion of OmpA decreases the number of interactions between the outer membrane and the peptidoglycan resulting in membrane destabilization leading to hyper-vesiculation7. Genomic OmpA was maintained in addition to the recombinantly expressed OmpA-ST construct to mitigate gross membrane destabilization.

<table border="0" cellpadding="0" cellspacing="0" width="617">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">IPTG</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Always prepare fresh or aliquot and freeze.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">L-arabinose</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Can be prepared ahead of time and stored at 4C.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Ampicillin</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Add immediately prior to use after media cools sufficiently from being autoclaved.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Chloramphenicol&#160;</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Add immediately prior to use after media cools sufficiently from being autoclaved.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">TB/LB Culture Media</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Other growth medias will likely work similarly.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Triton X-100</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">One of many potential suitable surfactants.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Baffled culture flasks</td>
			<td style="width:93px;">Any</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">The baffles promote higher levels of aeration.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">CHES</td>
			<td style="width:93px;">Fisher Bioreagents</td>
			<td style="width:96px;">BP318-100</td>
			<td style="width:273px;">Optimal buffer used for paraoxon degredation (pH &#62; 8).</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Paraoxon</td>
			<td style="width:93px;">Chem Service</td>
			<td style="width:96px;">N-12816</td>
			<td style="width:273px;">Very toxic substance to be handled carefully and disposed of properly.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Syringe Filter 0.45 &#181;m</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">60183-221&#160;&#160;&#160;&#160;&#160;&#160; (30 mm)</td>
			<td style="width:273px;">Filter diameter will depend on volume of sample.&#160; Low protein binding membrane is critical.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Shaker incubator</td>
			<td style="width:93px;">New Brunswick</td>
			<td style="width:96px;">Excella E24</td>
			<td style="width:273px;">Precise temperature and mixing is essential for reproducable bacterial growth.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Sorvall Culture Centrifuge</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">RC 5B PLUS</td>
			<td style="width:273px;">Large volume (500 mL) culture centrifuge capable of 7,000 x g.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Sorvall Ultracentrifuge&#160;</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">WX Ultra 90</td>
			<td style="width:273px;">Capable of centrifugal forces &#8805;150,000 x g.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Ultracentrifuge Rotor</td>
			<td style="width:93px;">Thermo Scientific</td>
			<td style="width:96px;">AH-629</td>
			<td style="width:273px;">Ensure the proper rotor and tubes are used and that everything is properly balanced.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:155px;">Ultra-Clear Ultracentrifuge Tubes&#160;&#160;&#160;&#160;&#160;&#160;&#160; (25 x 89 mm)</td>
			<td style="width:93px;">Beckman Coulter</td>
			<td style="width:96px;">344058</td>
			<td style="width:273px;">Ensure no stress fractures are present prior to use and that tubes are presicely balanced.&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">Spectrophotometer&#160;</td>
			<td style="width:93px;">Tecan</td>
			<td style="width:96px;">Infinite M1000</td>
			<td style="width:273px;">Necessary for enzyme kinetic assays.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">DLS / particle tracking</td>
			<td style="width:93px;">NanoSight&#160;</td>
			<td style="width:96px;">LM10</td>
			<td style="width:273px;">Necessary for OMV size distribution and concentration determination.&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">BL21(DE3)</td>
			<td style="width:93px;">NEB</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Suitable bacterial expression strain.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">pET22</td>
			<td style="width:93px;">EMD Millipore</td>
			<td style="width:96px;">69744-3</td>
			<td style="width:273px;">Other plasmids can be used in place of these.&#160;&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:155px;">pACYC184</td>
			<td style="width:93px;">NEB</td>
			<td style="width:96px;"></td>
			<td style="width:273px;">Other plasmids can be used in place of these.&#160;&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:155px;">Gel Extraction Kit</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:96px;">28704</td>
			<td style="width:273px;">Example kit.</td>
		</tr>
	</tbody>
</table>

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.

Simultaneous expression of two recombinant proteins, as is required for the OMV packaging strategy detailed in this protocol, can be accomplished through a number of different avenues. Here, a two vector system was utilized with compatible origins of replication and separate inducible gene cassettes. For the expression of the PTE-SC construct a commercial plasmid backbone (pACY184) was engineered to include an arabinose inducible gene cassette and a twin arginine periplasmic localization ...

Watch this Scientific Journal Video about Directed Protein Packaging within Outer Membrane Vesicles from Escherichia coli: Design, Production and Purification at JoVE.com

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.

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 ...

directed-protein-packaging-within-outer-membrane-vesicles-from

Research

JoVE Journal

Biochemistry

13.3K Views.  Naval Research Laboratory. 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. 

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

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Tandem Affinity Purification of Protein Complexes from Eukaryotic Cells

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo identification of novel subunits and post-translational modifications. Bacterial systems allow for the expression and purification of a wide variety of single polypeptides and protein complexes. However, this system does not enable the purification of protein subunits that contain post-translational modifications (e.g., phosphorylation and acetylation), and the identification of novel regulatory subunits that are only present/expressed in the eukaryotic system. Here, we provide a detailed description of a novel, robust, and efficient tandem affinity purification (TAP) method using STREP- and FLAG-tagged proteins that facilitates the purification of protein complexes with transiently or stably expressed epitope-tagged proteins from eukaryotic cells. This protocol can be applied to characterize protein complex functionality, to discover post-translational modifications on complex subunits, and to identify novel regulatory complex components by mass spectrometry. Notably, this TAP method can be applied to study protein complexes formed by eukaryotic or pathogenic (viral and bacterial) components, thus yielding a wide array of downstream experimental opportunities. We propose that researchers working with protein complexes could utilize this approach in many different ways.

We describe here a novel, robust, and efficient tandem affinity purification (TAP) method for the expression, isolation, and characterization of protein complexes from eukaryotic cells. This protocol could be utilized for the biochemical characterization of discrete complexes as well as the identification of novel interactors and post-translational modifications that regulate their function.

The purification of active protein-protein and protein-nucleic acid complexes is crucial for the characterization of enzymatic activities and de novo ...

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

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 ...

Creating Highly Specific Chemically Induced Protein Dimerization Systems by Stepwise Phage Selection of a Combinatorial Single-Domain Antibody Library

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the dissection and manipulation of biological pathways. Currently, only a limited number of chemically induced dimerization (CID) systems exist and engineering new ones with desired sensitivity and selectivity for specific small-molecule ligands remains a challenge in the field of protein engineering. We here describe a high throughput screening method, combinatorial binders-enabled selection of CID (COMBINES-CID), for the de novo engineering of CID systems applicable to a large variety of ligands. This method uses the two-step selection of a phage-displayed combinatorial nanobody library to obtain 1) &#34;anchor binders&#34; that first bind to a ligand of interest and then 2) &#34;dimerization binders&#34; that only bind to anchor binder-ligand complexes. To select anchor binders, a combinatorial library of over 109 complementarity-determining region (CDR)-randomized nanobodies is screened with a biotinylated ligand and hits are validated with the unlabeled ligand by bio-layer interferometry (BLI). To obtain dimerization binders, the nanobody library is screened with anchor binder-ligand complexes as targets for positive screening and the unbound anchor binders for negative screening. COMBINES-CID is broadly applicable to select CID binders with other immunoglobulin, non-immunoglobulin, or computationally designed scaffolds to create biosensors for in vitro and in vivo detection of drugs, metabolites, signaling molecules, etc.

Creating chemically induced protein dimerization systems with desired affinity and specificity for any given small molecule ligand would have many biological sensing and actuation applications. Here, we describe an efficient, generalizable method for de novo engineering of chemically induced dimerization systems via the stepwise selection of a phage-displayed combinatorial single-domain antibody library.

Protein dimerization events that occur only in the presence of a small-molecule ligand enable the development of small-molecule biosensors for the ...

Mechanical Separation and Protein Solubilization of the Outer and Inner Perivitelline Sublayers from Hen's Eggs

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. It is formed by two proteinaceous sublayers that are tightly associated and formed by distinct female reproductive organs. Both structures are assumed to have their own functional specificities, which remain to be defined. To characterize the function of proteins composing each sublayer, the first challenge is to establish the conditions that would allow for the mechanical separation of these two intricate layers, while limiting any structural damage. The second step is to optimize the experimental conditions to facilitate protein solubilization from these two sublayers, for subsequent biochemical analyses. The efficiency of this approach is assessed by analyzing the protein profile of each sublayer by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), which is expected to be distinct between the two structures. This two-step procedure remains simple; it requires classical biochemical equipment and reagents; and is compatible with further in-depth proteomics. It may also be transposed to other avian eggs for comparative biology, knowing that the structure and the composition of the perivitelline layer has been shown to have species-specific features. In addition, the non-denaturing conditions developed for sublayers separation (step 1) allow their structural analyses by scanning and transmission electron microscopy. It may also constitute the initial step for subsequent protein purification to analyze their respective biological activities and 3D structure, or to perform further immunohistochemical or functional analyses. Such studies would help to decipher the physiological function of these two sublayers, whose structural and functional integrities are determinant criteria of the reproductive success.

This article reports a simple method to isolate the outer and inner perivitelline sublayers of chicken eggs while minimizing structural alteration and to optimize protein solubilization of each sublayer for proteomic analyses.

The perivitelline layer that surrounds the egg yolk plays a fundamental role in fertilization, in egg defense, and in the development of the avian embryo. ...