The research described above was supported by NIH TL1 TR002549-03 training grant. We thank Drs. John C. Tilton and Zachary Troyer (Case Western Reserve University) for facilitating access to the particle size analyzer instrument; Lew Brown (Spectradyne) for technical assistance with analysis of the particle size distribution data; Dr. David Putnam at Cornell University for providing pClyA-GFP plasmid14; and Dr. Mark Goulian at the University of Pennsylvania for providing us with the E. coli MP113.

<ol>
	<li>Yanez-Mo, M., 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=Biological+properties+of+extracellular+vesicles+and+their+physiological+functions.">Biological properties of extracellular vesicles and their physiological functions.</a> Journal of Extracellular Vesicles. 4, 27066 (2015).</li><li>Chatterjee, S. N., Das, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+microscopic+observations+on+the+excretion+of+cell-wall+material+by+Vibrio+cholerae.">Electron microscopic observations on the excretion of cell-wall material by Vibrio cholerae.</a> Journal of General Microbiology. 49 (1), 1-11 (1967).</li><li>Ciofu, O., Beveridge, T. J., Kadurugamuwa, J., Walther-Rasmussen, J., Hoiby, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+beta-lactamase+is+packaged+into+membrane+vesicles+and+secreted+from+Pseudomonas+aeruginosa.">Chromosomal beta-lactamase is packaged into membrane vesicles and secreted from Pseudomonas aeruginosa.</a> Journal of Antimicrobial Chemotherapy. 45 (1), 9-13 (2000).</li><li>Yonezawa, H., 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=Outer+membrane+vesicles+of+Helicobacter+pylori+TK1402+are+involved+in+biofilm+formation.">Outer membrane vesicles of Helicobacter pylori TK1402 are involved in biofilm formation.</a> BMC Microbiology. 9, 197 (2009).</li><li>Mashburn, L. M., Whiteley, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+traffic+signals+and+facilitate+group+activities+in+a+prokaryote.">Membrane vesicles traffic signals and facilitate group activities in a prokaryote.</a> Nature. 437 (7057), 422-425 (2005).</li><li>Kato, S., Kowashi, Y., Demuth, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outer+membrane-like+vesicles+secreted+by+Actinobacillus+actinomycetemcomitans+are+enriched+in+leukotoxin.">Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin.</a> Microbial Pathogenesis. 32 (1), 1-13 (2002).</li><li>Petousis-Harris, H., 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=Effectiveness+of+a+group+B+outer+membrane+vesicle+meningococcal+vaccine+against+gonorrhoea+in+New+Zealand:+a+retrospective+case-control+study.">Effectiveness of a group B outer membrane vesicle meningococcal vaccine against gonorrhoea in New Zealand: a retrospective case-control study.</a> Lancet. 390 (10102), 1603-1610 (2017).</li><li>Kim, O. 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=Bacterial+outer+membrane+vesicles+suppress+tumor+by+interferon-gamma-mediated+antitumor+response.">Bacterial outer membrane vesicles suppress tumor by interferon-gamma-mediated antitumor response.</a> Nature Communications. 8 (1), 626 (2017).</li><li>Thery, C., 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=Minimal+information+for+studies+of+extracellular+vesicles+2018+(MISEV2018):+a+position+statement+of+the+International+Society+for+Extracellular+Vesicles+and+update+of+the+MISEV2014+guidelines.">Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.</a> Journal of Extracellular Vesicles. 7 (1), 1535750 (2018).</li><li>Consortium, E. -. T., 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=EV-TRACK:+transparent+reporting+and+centralizing+knowledge+in+extracellular+vesicle+research.">EV-TRACK: transparent reporting and centralizing knowledge in extracellular vesicle research.</a> Nature Methods. 14 (3), 228-232 (2017).</li><li>Watson, D. C., 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=Efficient+production+and+enhanced+tumor+delivery+of+engineered+extracellular+vesicles.">Efficient production and enhanced tumor delivery of engineered extracellular vesicles.</a> Biomaterials. 105, 195-205 (2016).</li><li>Watson, D. C., 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=Scalable,+cGMP-compatible+purification+of+extracellular+vesicles+carrying+bioactive+human+heterodimeric+IL-15/lactadherin+complexes.">Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes.</a> Journal of Extracellular Vesicles. 7 (1), 1442088 (2018).</li><li>Lasaro, M., 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=Escherichia+coli+isolate+for+studying+colonization+of+the+mouse+intestine+and+its+application+to+two-component+signaling+knockouts.">Escherichia coli isolate for studying colonization of the mouse intestine and its application to two-component signaling knockouts.</a> Journal of Bacteriology. 196 (9), 1723-1732 (2014).</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> Journal of Molecular Biology. 380 (1), 51-66 (2008).</li><li>Beveridge, T. J. <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> Journal of Bacteriology. 181 (16), 4725-4733 (1999).</li><li>Reimer, S. L., 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=Comparative+analysis+of+outer+membrane+vesicle+isolation+methods+with+an+Escherichia+coli+tolA+mutant+reveals+a+hypervesiculating+phenotype+with+outer-inner+membrane+vesicle+content.">Comparative analysis of outer membrane vesicle isolation methods with an Escherichia coli tolA mutant reveals a hypervesiculating phenotype with outer-inner membrane vesicle content.</a> Frontiers in Microbiology. 12, 628801 (2021).</li></ol>

The authors have no conflicts of interest to declare.

In the protocol above, a method is described that is scalable and reliably isolates EVs from various gram-negative/positive and aerobic/anaerobic bacteria. It has several potential stopping points throughout the procedure, although it is better to avoid taking longer than 48 h to isolate EVs from conditioned bacterial culture media.
First, it consists of culturing bacteria to generate conditioned bacterial culture medium. It was found that increasing the culture time to at least 48 h and using...

Extracellular vesicles (EVs) are nanometer-sized, liposome-like structures comprised of lipids, proteins, glycans, and nucleic acids, secreted by both prokaryotic and eukaryotic cells1. Since the early studies visualizing the release of EVs from gram-negative bacteria2, the number of biological functions attributed to bacterial EVs (20-300 nm in diameter) has constantly been growing in the past decades. Their functions include transferring antibiotic resistance3, biofilm formation4, quorum sensing5, and toxin delivery6. There is also growing interest in the use of bacterial EVs as therapeutics, especially in vaccinology7 and cancer therapy8.
Despite the growing interest in EV research, there are still technical challenges regarding methods of isolation. Specifically, there is a need for isolation methods that are reproducible, scalable, and compatible with diverse EV-producing organisms. To create a unified set of principles for planning and reporting EV isolation and research methods, the International Society for Extracellular Vesicles publishes and updates the MISEV position paper9. Moreover, the EV-TRACK consortium provides an open platform for reporting detailed methodologies for EV isolation used in published manuscripts to enhance transparency10.
In this protocol, previous methodologies used for the isolation of EVs from mammalian cell culture were adapted11,12 to enable the isolation of EVs from bacterial cell culture. We sought to employ methods that enable EV isolation from a variety of microbes, which can be scalable, and balance EV purity and yield (as discussed in the MISEV position paper9). After removing bacterial cells and debris by centrifugation and filtration, the culture medium is concentrated either by centrifugal device ultrafiltration (for a volume of up to ~100 mL) or pump-driven TFF (for larger volumes). EVs are then isolated by SEC using columns optimized for the purification of small EVs.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63155/63155fig01.jpg" /> 
Figure 1: Bacterial EV isolation workflow schematic overview. Abbreviations: EV = extracellular vesicle; TFF = tangential flow filtration; SEC = size exclusion chromatography; MWCO = molecular weight cut-off. <a href="https://www.jove.com/files/ftp_upload/63155/63155fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A mouse-commensal strain of Escherichia coli (i.e., E. coli MP113) was used as a model organism and modified to express EV-associated nanoluciferase by fusion to cytolysin A, as previously reported14. The methods used here can process at least up to several liters of bacterial cultures and effectively separate EV-associated from non-EV-associated proteins. Finally, this method can also be used for other gram-positive and gram-negative bacterial species. All relevant data of the reported experiments were submitted to the EV-TRACK knowledgebase (EV-TRACK ID: EV210211)10.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 mL flat cap, thin-walled PCR tubes</td>
			<td>Thermo Scientific</td>
			<td>3430</td>
			<td>it is important to use thin-walled PCR tubes to obtain accurate readings with Qubit</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde (formaldehyde) aqueous solution</td>
			<td>Electron microscopy sciences</td>
			<td>15700</td>
			<td></td>
		</tr>
		<tr>
			<td>250 mL Fiberlite polypropylene centrifuge bottles</td>
			<td>ThermoFisher</td>
			<td>010-1495</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Fiberlite polypropylene centrifuge bottles</td>
			<td>ThermoFisher</td>
			<td>010-1493</td>
			<td></td>
		</tr>
		<tr>
			<td>65 mm Polypropylene Round-Bottom/Conical Bottle Adapter</td>
			<td>Beckman Coulter</td>
			<td>392077</td>
			<td>Allows Vivacell to fit in rotor</td>
		</tr>
		<tr>
			<td>Akkermansia mucinophila</td>
			<td>ATCC</td>
			<td>BAA-835</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon-15 (100 kDa MWCO)</td>
			<td>MilliporeSigma</td>
			<td>UFC910024</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-20 XPI centrifuge</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td>No longer sold by Beckman. Avanti J-26XP is closest contemporary model.</td>
		</tr>
		<tr>
			<td>Bacteroides thetaiotaomicron VPI 5482</td>
			<td>ATCC</td>
			<td>29148</td>
			<td></td>
		</tr>
		<tr>
			<td>Bifidobacterium breve</td>
			<td>NCIMB</td>
			<td>B8807</td>
			<td></td>
		</tr>
		<tr>
			<td>Bifidobacterium dentium</td>
			<td>ATCC</td>
			<td>27678</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart infusion (BHI) broth</td>
			<td>Himedia</td>
			<td>M2101</td>
			<td>After autoclaving, Both BHI broth and agar were introduced into the anaerobic chamber, supplemented with Menadione (1 &#181;g/L), hematin (1.2 &#181;g/L), and L-Cysteine Hydrochloride (0.05%). They were then incubated for at least 24 h under anaerobic conditions before inoculation with the anaerobic bacterial strains.</td>
		</tr>
		<tr>
			<td>C-300 microfluidics cartridge</td>
			<td>Spectradyne</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>MP Biomedicals</td>
			<td>ICN19032105</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli HST08 (Steller competent cells)</td>
			<td>Takara</td>
			<td>636763</td>
			<td></td>
		</tr>
		<tr>
			<td>Escherichia coli MP1</td>
			<td>Dr. Mark Goulian (gift)</td>
			<td></td>
			<td>commensal bacteria derived from mouse gut</td>
		</tr>
		<tr>
			<td>Fiberlite 500 mL to 250 mL adapter</td>
			<td>ThermoFisher</td>
			<td>010-0151-05</td>
			<td>used with Fiberlite rotor to enable 250 mL bottles to be used for smaller size of starting bacterial culture</td>
		</tr>
		<tr>
			<td>Fiberlite fixed-angle centrifuge rotor</td>
			<td>ThermoFisher</td>
			<td>F12-6x500-LEX</td>
			<td>fits 6 x 500 mL bottles</td>
		</tr>
		<tr>
			<td>Formvar Carbon Film 400 Mesh, Copper</td>
			<td>Electron microscopy sciences</td>
			<td>FCF-400-CU</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde (EM-grade, 10% aqeous solution)</td>
			<td>Electron microscopy sciences</td>
			<td>16100</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematin</td>
			<td>ChemCruz</td>
			<td>207729B</td>
			<td>Stock solution was made in 0.2 M L-histidine solution as&#160; 1.2 mg/mL</td>
		</tr>
		<tr>
			<td>Infinite M Nano+ Microplate reader</td>
			<td>Tecan</td>
			<td></td>
			<td>This equibment was used to measure the mCherry fluorescence</td>
		</tr>
		<tr>
			<td>In-Fusion&#160; HD Cloning Plus</td>
			<td>Takara</td>
			<td>638909</td>
			<td>For cloning of the PCR fragements into the PCR-lineraized vectors</td>
		</tr>
		<tr>
			<td>JS-5.3 AllSpin Swinging-Bucket Rotor</td>
			<td>Beckman Coulter</td>
			<td>368690</td>
			<td></td>
		</tr>
		<tr>
			<td>Lauria Bertani (LB) broth, Miller</td>
			<td>Difco</td>
			<td>244620</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine Hydrochloride</td>
			<td>J.T. Baker</td>
			<td>2071-05</td>
			<td>It should be weighed and added directly to the autoclaved BHI media inside the anaerobic chamber</td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polypropylene, Straight, Female Luer to Hose Barb Adapter, 1/8&#34; ID; 25/PK</td>
			<td>cole-parmer - special</td>
			<td>HV-30800-08</td>
			<td>connection adapters for filtration tubing circuit</td>
		</tr>
		<tr>
			<td>Masterflex Fitting, Polypropylene, Straight, Male Luer to Hose Barb Adapter, 1/8&#34; ID; 25/PK</td>
			<td>cole-parmer - special</td>
			<td>HV-30800-24</td>
			<td>connection adapters for filtration tubing circuit</td>
		</tr>
		<tr>
			<td>Masterflex L/S Analog Variable-Speed Console Drive, 20 to 600 rpm</td>
			<td>Masterflex</td>
			<td>HV-07555-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Easy-Load Head for Precision Tubing, 4-Roller, PARA Housing, SS Rotor</td>
			<td>Masterflex</td>
			<td>EW-07514-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Masterflex L/S Precision Pump Tubing, PharmaPure, L/S 16; 25 ft</td>
			<td>Cole Palmer</td>
			<td>EW-06435-16</td>
			<td>low-binding/low-leaching tubing</td>
		</tr>
		<tr>
			<td>Menadione (Vitamin K3)</td>
			<td>MP</td>
			<td>102259</td>
			<td>Stock solution was made in ethanol as 1 mg/mL</td>
		</tr>
		<tr>
			<td>MIDIKROS 41.5CM 100K MPES 0.5MM FLL X FLL 1/PK</td>
			<td>Repligen</td>
			<td>D04-E100-05-N</td>
			<td>TFF device we have used to filter up to 2 L of E. coli culture supernatant</td>
		</tr>
		<tr>
			<td>Nano-Glo Luciferase Assay System</td>
			<td>Promega</td>
			<td>N1110</td>
			<td>This assay kit was used to measure the luminescence of the nluc reporter protein</td>
		</tr>
		<tr>
			<td>NanoLuc (Nluc) Luciferase Antibody, clone 965808</td>
			<td>R&#38;D Systems</td>
			<td>MAB10026</td>
			<td></td>
		</tr>
		<tr>
			<td>nCS1 microfluidics resistive pulse sensing instrument</td>
			<td>Spectradyne</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>nCS1 Viewer</td>
			<td>Spectradyne</td>
			<td></td>
			<td>Analysis software for particle size distribution</td>
		</tr>
		<tr>
			<td>OneTaq 2x Master Mix with Standard Buffer</td>
			<td>NEB</td>
			<td>M0482</td>
			<td>DNA polymerase master mix used to perform the routine PCR reactions for colony checking</td>
		</tr>
		<tr>
			<td>Protein LoBind, 2.0 mL, PCR clean tubes</td>
			<td>Eppendorf</td>
			<td>30108450</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 High-Fidelity 2x Master Mix</td>
			<td>NEB</td>
			<td>M0492</td>
			<td>DNA polymerase master mix used to perform the PCR reactions needed for cloning</td>
		</tr>
		<tr>
			<td>qEV original, 35 nm</td>
			<td>Izon</td>
			<td></td>
			<td>maximal loading volume of 0.5 mL</td>
		</tr>
		<tr>
			<td>qEV rack</td>
			<td>Izon</td>
			<td></td>
			<td>for use with the qEV-original SEC columns</td>
		</tr>
		<tr>
			<td>qEV-2, 35 nm</td>
			<td>Izon</td>
			<td></td>
			<td>maximal loading volume of 2 mL</td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>ThermoFisher</td>
			<td></td>
			<td>Item no longer available. Closest available product is Qubit 4.0 Fluorometer (cat. No. Q33238)</td>
		</tr>
		<tr>
			<td>Qubit protein assay kit</td>
			<td>ThermoFisher</td>
			<td>Q33211</td>
			<td>Store kit at room temperature. Standards are stored at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Sorvall Lynx 4000 centrifuge</td>
			<td>ThermoFisher</td>
			<td>75006580</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x Microplate reader</td>
			<td>Molecular Devices</td>
			<td></td>
			<td>This equipment was used to measure the nanoluciferase bioluminescence</td>
		</tr>
		<tr>
			<td>Stericup Quick-release-GP Sterile Vacuum Filtration system (150, 250, or 500 mL)</td>
			<td>MilliporeSigma</td>
			<td>S2GPU01RE 
			S2GPU02RE 
			S2GPU05RE</td>
			<td>One or multiple filters can be used to accommodate working volumes. In our experience, you can filter twice the volume listed on the product size.</td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron microscopy sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
		<tr>
			<td>Vinyl anaerobic chamber</td>
			<td>Coy Lab</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vivacell 100, 100,000 MWCO PES</td>
			<td>Sartorius</td>
			<td>VC1042</td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman Anotop 10 Plus syringe filters (0.02 micron)</td>
			<td>MilliporeSigma</td>
			<td>WHA68093002</td>
			<td>to filter MRPS diluent</td>
		</tr>
	</tbody>
</table>

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.

To assess which SEC chromatography fractions were enriched for EVs, the SEC column was loaded with 2 mL of E. coli MP1-conditioned culture medium that had been concentrated 1,000-fold by TFF, and sequential fractions were collected. Using MRPS, it was found that Fractions 1-6 contained the most EVs (Figure 2A). Subsequent fractions contained very few EVs, comprising instead of EV-free proteins (Figure 2B). EVs were primarily &#60;100 nm in diameter (<st...

Watch this Scientific Journal Video about Scalable Isolation and Purification of Extracellular Vesicles from Escherichia coli and Other Bacteria at JoVE.com

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.

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 ...

scalable-isolation-purification-extracellular-vesicles-from

Research

JoVE Journal

Biology

3.5K Views.  Cleveland Clinic. 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.

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

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

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 ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular vesicles (EVs) function outside the cell to regulate global physiological processes by transferring proteins, nucleic acids, metabolites, and lipids between tissues. EVs reflect the physiological state of their cells of origin. EVs are implicated to have fundamental roles in virtually every aspect of human health. Thus, EV protein and genetic cargos are being increasingly analyzed for biomarkers of health and disease. However, the EV field still lacks a tractable invertebrate model system that permits the study of EV cargo composition. C. elegans is well suited for EV research because it actively secretes EVs outside of its body into its external environment, permitting facile isolation. This article provides all the necessary information for generating, purifying, and quantifying these environmentally secreted C. elegans EVs including how to work quantitatively with very large populations of age-synchronized worms, purifying EVs, and a flow cytometry protocol that directly measures the number of intact EVs in the purified sample. Thus, the large library of genetic reagents available for C. elegans research can be tapped into for investigating the impacts of genetic pathways and physiological processes on EV cargo composition.

Purification and Analysis of Caenorhabditis elegans Extracellular Vesicles

This article presents methods for generating, purifying, and quantifying Caenorhabditis elegans extracellular vesicles.

The secretion of small membrane-bound vesicles into the external environment is a fundamental physiological process of all cells. These extracellular ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon bactericidal antibiotic treatment, the bulk of the bacterial population is rapidly killed. This first rapid phase of killing is followed by a substantial decrease in the rate of killing as the persister cells remain viable. Classically, persistence is determined at the population level by time/kill assays performed with high doses of antibiotics and for defined exposure times. While this method provides information about the level of persister cells and the killing kinetics, it fails to reflect the intrinsic cell-to-cell heterogeneity underlying the persistence phenomenon. The protocol described here combines classical time/kill assays with single-cell analysis using real-time fluorescence microscopy. By using appropriate fluorescent reporters, the microscopy imaging of live cells can provide information regarding the effects of the antibiotic on cellular processes, such as chromosome replication and segregation, cell elongation, and cell division. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

Antibiotic persistence describes the ability of small subpopulations within a sensitive isogenic population to transiently tolerate high doses of bactericidal antibiotics. The present protocol combines approaches to characterize the antibiotic persistence phenotype at the molecular and cellular levels after exposing Escherichia coli to lethal doses of ofloxacin.

Antibiotic persistence refers to the capacity of small bacterial subpopulations to transiently tolerate high doses of bactericidal antibiotics. Upon ...