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>

<ol>
	<li>Avila-Calderon, E. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+bacterial+membrane+vesicles.">Roles of bacterial membrane vesicles.</a> Arch. Microbiol. 197, 1-10 (2015).</li><li>Kulp, A., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+functions+and+biogenesis+of+secreted+bacterial+outer+membrane+vesicles.">Biological functions and biogenesis of secreted bacterial outer membrane vesicles.</a> Annu. Rev. Microbiol. 64, 163-184 (2010).</li><li>Beveridge, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structures+of+Gram+negative+cell+walls+and+their+derived+membrane+vesicles.">Structures of Gram negative cell walls and their derived membrane vesicles.</a> J. Bacteriol. 181, 4725-4733 (1999).</li><li>Kulkarni, H. M., Jagannadham, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+multifaceted+roles+of+outer+membrane+vesicles+from+Gram-negative+bacteria.">Biogenesis and multifaceted roles of outer membrane vesicles from Gram-negative bacteria.</a> Microbiology. 160, 2109-2121 (2014).</li><li>Ellis, T. N., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virulence+and+immunomodulatory+roles+of+bacterial+outer+membrane+vesicles.">Virulence and immunomodulatory roles of bacterial outer membrane vesicles.</a> Microbiol. Mol. Biol. Rev. 74, 81-94 (2010).</li><li>Berleman, J., Auer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+bacterial+outer+membrane+vesicles+for+intra-+and+interspecies+delivery.">The role of bacterial outer membrane vesicles for intra- and interspecies delivery.</a> Environ. Microbiol. 15, 347-354 (2013).</li><li>Alves, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+nanobioreactors-directing+enzyme+packaging+into+bacterial+outer+membrane+vesicles.">Bacterial nanobioreactors-directing enzyme packaging into bacterial outer membrane vesicles.</a> ACS Appl. Mater. Interfaces. 7, 24963-24972 (2015).</li><li>Kim, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+bacterial+outer+membrane+vesicles+with+enhanced+functionality.">Engineered bacterial outer membrane vesicles with enhanced functionality.</a> J. Mol. Biol. 380, 51-66 (2008).</li><li>Haurat, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+sorting+of+cargo+proteins+into+bacterial+membrane+vesicles.">Selective sorting of cargo proteins into bacterial membrane vesicles.</a> J. Biol. Chem. 286, 1269-1276 (2011).</li><li>Kesty, N. C., Kuehn, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+heterologous+outer+membrane+and+periplasmic+proteins+into+Escherichia+coli+outer+membrane+vesicles.">Incorporation of heterologous outer membrane and periplasmic proteins into Escherichia coli outer membrane vesicles.</a> J. Biol. Chem. 279, 2069-2076 (2003).</li><li>Zakeri, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide+tag+forming+a+rapid+covalent+bond+to+a+protein,+through+engineering+a+bacterial+adhesion.">Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesion.</a> Proc. Natl. Acad. Sci. U. S. A. 109, e690-e697 (2012).</li><li>Li, L., Fierer, J. O., Rapoport, T. A., Howarth, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+analysis+and+optimization+of+the+covalent+association+between+SpyCatcher+and+a+peptide+tag.">Structural analysis and optimization of the covalent association between SpyCatcher and a peptide tag.</a> J. Mol. Biol. 426, 309-317 (2014).</li><li>Dumas, D. P., Durst, H. D., Landis, W. G., Raushel, F. M., Wild, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+organophosphorus+nerve+agents+by+the+phophotriesterase+from+Pseudomonas-Diminuta.">Inactivation of organophosphorus nerve agents by the phophotriesterase from Pseudomonas-Diminuta.</a> Arch. Biochem. Biophys. 277, 155-159 (1990).</li><li>Bigley, A. N., Raushel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catalytic+mechanisms+for+phosphotriesterases.">Catalytic mechanisms for phosphotriesterases.</a> Biochimica et Biochim. Biophys. Acta, Proteins Proteomics. 1834, 443-453 (2013).</li><li>Minton, N. A., Murray, V. S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+organo-phosphate+poisoning.">A review of organo-phosphate poisoning.</a> Med. Toxicol. Adverse Drug Exper. 3, 350-375 (1988).</li><li>Bigley, A. N., Xu, C., Henderson, T. J., Harvey, S. P., Raushel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+neutralization+of+the+chemical+warfare+agent+VX:+Evolution+of+phosphotriesterase+for+phosphorothiolate+hydrolysis.">Enzymatic neutralization of the chemical warfare agent VX: Evolution of phosphotriesterase for phosphorothiolate hydrolysis.</a> J. Am. Chem. Soc. 135, 10426-10432 (2013).</li><li>Chatterjee, S. N., Chaudhuri, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gram-negative+bacteria:+the+cell+membranes.">Gram-negative bacteria: the cell membranes.</a> Outer Membrane Vesicles of Bacteria. SpringerBriefs in Microbiology. , 15-34 (2012).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+function+of+OmpA+in+Escherichia+coli.">The function of OmpA in Escherichia coli.</a> Biochem. Biophys. Res. Commun. 292, 396-401 (2002).</li><li>Danoff, E. J., Fleming, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+soluble,+periplasmic+domain+of+OmpA+folds+as+an+independent+unit+and+displays+chaperone+activity+by+reducing+the+self-association+propensity+of+the+unfolded+OmpA+transmembrane+beta-barrel.">The soluble, periplasmic domain of OmpA folds as an independent unit and displays chaperone activity by reducing the self-association propensity of the unfolded OmpA transmembrane beta-barrel.</a> Biophys. Chem. 159, 194-204 (2011).</li><li>Alves, N. J., Kline, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+the+inhibition+of+plasmin+and+delta-plasmin+via+benzamidine+derivatives.">Comparative study on the inhibition of plasmin and delta-plasmin via benzamidine derivatives.</a> Biochem. Biophys. Res. Commun. 457, 358-362 (2015).</li><li>Wang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lyophilization+and+development+of+solid+protein+pharmaceuticals.">Lyophilization and development of solid protein pharmaceuticals.</a> Int. J. Pharm. 203, 1-60 (2000).</li><li>Goormaghtigh, E., Scarborough, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Density-based+separation+of+liposomes+by+glycerol+gradient+centrifugation.">Density-based separation of liposomes by glycerol gradient centrifugation.</a> Anal. Biochem. 159, 122-131 (1986).</li><li>Alves, N. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalized+liposome+purification+via+Liposome+Extruder+Purification+(LEP).">Functionalized liposome purification via Liposome Extruder Purification (LEP).</a> Analyst. 138, 4746-4751 (2013).</li><li>Mayer, L. D., StOnge, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+free+and+liposome-associated+doxorubicin+and+vincristine+levels+in+plasma+under+equilibrium+conditions+employing+ultrafiltration+techniques.">Determination of free and liposome-associated doxorubicin and vincristine levels in plasma under equilibrium conditions employing ultrafiltration techniques.</a> Anal. Biochem. 232, 149-157 (1995).</li><li>Blakeley, B. D., Chapman, A. M., McNaughton, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-superpositive+GFP+reassembly+is+a+fast,+efficient,+and+robust+method+for+detecting+protein-protein+interactions+in+vivo.">Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo.</a> Mol. BioSyst. 8 (2036), (2012).</li><li>De Crescenzo, G., Litowski, J. R., Hodges, R. S., O'Connor-McCourt, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+the+interacation+of+two-stranded+de+novo+designed+coiled-coils:+effect+of+chain+length+on+the+kinetic+and+thermodynamic+constants+of+binding.">Real-time monitoring of the interacation of two-stranded de novo designed coiled-coils: effect of chain length on the kinetic and thermodynamic constants of binding.</a> Biochemistry. 42, 1754-1763 (2003).</li><li>Charalambous, A., Antoniades, I., Christodoulou, N., Skourides, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-Intein+for+simultaneous+site-specific+conjugation+of+quantum+dots+to+multiple+protein+targets+in+vivo.">Split-Intein for simultaneous site-specific conjugation of quantum dots to multiple protein targets in vivo.</a> J. Nanobiotechnol. 9, 1-14 (2011).</li><li>Lee, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+proteomic+profiling+of+native+outer+membrane+vesicles+derived+fromEscherichia+coli.">Global proteomic profiling of native outer membrane vesicles derived fromEscherichia coli.</a> Proteomics. 7, 3143-3153 (2007).</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=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><tbody><tr><td>IPTG</td><td>Any</td><td></td><td>Always prepare fresh or aliquot and freeze.</td></tr><tr><td>L-arabinose</td><td>Any</td><td></td><td>Can be prepared ahead of time and stored at 4 &amp;deg;C.</td></tr><tr><td>Ampicillin</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Chloramphenicol&amp;nbsp;</td><td>Any</td><td></td><td>Add immediately prior to use after media cools sufficiently from being autoclaved.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>TB/LB Culture Media</td><td>Any</td><td></td><td>Other growth medias will likely work similarly.</td></tr><tr><td>Triton X-100</td><td>Any</td><td></td><td>One of many potential suitable surfactants.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Baffled culture flasks</td><td>Any</td><td></td><td>The baffles promote higher levels of aeration.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>CHES</td><td>Fisher Bioreagents</td><td>BP318-100</td><td>Optimal buffer used for paraoxon degredation (pH &gt; 8).</td></tr><tr><td>Paraoxon</td><td>Chem Service</td><td>N-12816</td><td>Very toxic substance to be handled carefully and disposed of properly.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Syringe Filter 0.45 &amp;micro;m</td><td>Thermo Scientific</td><td>60183-221 (30 mm)</td><td>Filter diameter will depend on volume of sample. Low protein binding membrane is critical.</td></tr><tr><td>Shaker incubator</td><td>New Brunswick</td><td>Excella E24</td><td>Precise temperature and mixing is essential for reproducable bacterial growth.&amp;nbsp;</td></tr><tr><td>Sorvall Culture Centrifuge</td><td>Thermo Scientific</td><td>RC 5B PLUS</td><td>Large volume (500 ml) culture centrifuge capable of 7,000 x g.</td></tr><tr><td>Sorvall Ultracentrifuge&amp;nbsp;</td><td>Thermo Scientific</td><td>WX Ultra 90</td><td>Capable of centrifugal forces &amp;ge;150,000 x g.</td></tr><tr><td>Ultracentrifuge Rotor</td><td>Thermo Scientific</td><td>AH-629</td><td>Ensure the proper rotor and tubes are used and that everything is properly balanced.</td></tr><tr><td>Ultra-Clear Ultracentrifuge Tubes (25 x 89 mm)</td><td>Beckman Coulter</td><td>344058</td><td>Ensure no stress fractures are present prior to use and that tubes are presicely balanced.</td></tr><tr><td>Spectrophotometer&amp;nbsp;</td><td>Tecan</td><td>Infinite M1000</td><td>Necessary for enzyme kinetic assays.</td></tr><tr><td>DLS/particle tracking</td><td>NanoSight&amp;nbsp;</td><td>LM10</td><td>Necessary for OMV size distribution and concentration determination.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>BL21(DE3)</td><td>NEB</td><td></td><td>Suitable bacterial expression strain.</td></tr><tr><td>pET22</td><td>EMD Millipore</td><td>69744-3</td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>pACYC184</td><td>NEB</td><td></td><td>Other plasmids can be used in place of these.&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>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

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a target for future development of inhibitors to decrease the virulence of the pathogen. To perform biophysical and structural characterization as well as inhibitor screening, large amounts of pure and active protein will be needed. We have developed a protocol for efficient production and purification of PPEP-1 by the use of E. coli as the expression host yielding sufficient amounts and purity of protein for crystallization and structure determination. Additionally, using microseeding, highly intergrown crystals of PPEP-1 can be grown to well-ordered crystals suitable for X-ray diffraction analysis. The methods could also be used to produce other recombinant proteins and to study the structures of other proteins producing intergrown crystals.

Production, Crystallization and Structure Determination of C. difficile PPEP-1 via Microseeding and Zinc-SAD

Proline-proline endopeptidase-1 (PPEP-1) is a secreted metalloprotease and promising drug-target from the human pathogen Clostridium difficile. Here we describe all methods necessary for the production and structure determination of this protein.

New therapies are needed to treat Clostridium difficile infections that are a major threat to human health. The C. difficile metalloprotease PPEP-1 is a ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Chemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be greatly facilitated by the production of milligram amounts of pure, folded ligand binding domains. Attempts to heterologously express periplasmic ligand binding domains of bacterial chemoreceptors in Escherichia coli (E. coli) often result in their targeting into inclusion bodies. Here, a method is presented for protein recovery from inclusion bodies, its refolding and purification, using the periplasmic dCACHE ligand binding domain of Campylobacter jejuni (C. jejuni) chemoreceptor Tlp3 as an example. The approach involves expression of the protein of interest with a cleavable His6-tag, isolation and urea-mediated solubilisation of inclusion bodies, protein refolding by urea depletion, and purification by means of affinity chromatography, followed by tag removal and size-exclusion chromatography. The circular dichroism spectroscopy is used to confirm the folded state of the pure protein. It has been demonstrated that this protocol is generally useful for production of milligram amounts of dCACHE periplasmic ligand binding domains of other bacterial chemoreceptors in a soluble and crystallisable form.

A procedure is presented for the refolding of the dCACHE periplasmic ligand binding domain of Campylobacter jejuni chemoreceptor Tlp3 from inclusion bodies and the purification to yield milligram quantities of protein.

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

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 ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Bioengineering

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

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.

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Biology

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules derived from the parental cells. EVs function as intra- and inter-species communication vectors while also contributing to the interaction between bacteria and host organisms in the context of infection and colonization. Given the multitude of functions attributed to EVs in health and disease, there is a growing interest in isolating EVs for in vitro and in vivo studies. It was hypothesized that the separation of EVs based on physical properties, namely size, would facilitate the isolation of vesicles from diverse bacterial cultures. 
The isolation workflow consists of centrifugation, filtration, ultrafiltration, and size-exclusion chromatography (SEC) for the isolation of EVs from bacterial cultures. A pump-driven tangential flow filtration (TFF) step was incorporated to enhance scalability, enabling the isolation of material from liters of starting cell culture. Escherichia coli was used as a model system expressing EV-associated nanoluciferase and non-EV-associated mCherry as reporter proteins. The nanoluciferase was targeted to the EVs by fusing its N-terminus with cytolysin A. Early chromatography fractions containing 20-100 nm EVs with associated cytolysin A - nanoLuc were distinct from the later fractions containing the free proteins. The presence of EV-associated nanoluciferase was confirmed by immunogold labeling and transmission electron microscopy. This EV isolation workflow is applicable to other human gut-associated gram-negative and gram-positive bacterial species. In conclusion, combining centrifugation, filtration, ultrafiltration/TFF, and SEC enables scalable isolation of EVs from diverse bacterial species. Employing a standardized isolation workflow will facilitate comparative studies of microbial EVs across species.

Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria

Bacteria secrete nanometer-sized extracellular vesicles (EVs) carrying bioactive biological molecules. EV research focuses on understanding their biogenesis, role in microbe-microbe and host-microbe interactions and disease, as well as their potential therapeutic applications. A workflow for scalable isolation of EVs from various bacteria is presented to facilitate standardization of EV research.

Diverse bacterial species secrete ~20-300 nm extracellular vesicles (EVs), comprised of lipids, proteins, nucleic acids, glycans, and other molecules ...

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

Samenvatting

Meer video's verkennen