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

Summary

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.

Abstract

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.

Introduction

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 nm^1,2. 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 surface^3,4. 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 functions^5,6.

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 all^8-10. 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, <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.

Protocol

1. Preparation of Plasmids

1. 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.
   1. Extract plasmid DNA from overnight cultures using a commercially available DNA isolation kit following the manufacturer's protocol.
2. 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.
   1. Extract plasmid DNA from overnight cultures using a commercially available DNA isolation kit following the manufacturer's protocol⁷.

2. Generation of an OMV Packaging E. coli Culture

1. Plasmid Isolation
   1. 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's protocol⁷. Determine DNA concentration and purity by measuring absorbance at 260 nm and 280 nm.
2. Co-transformation
   1. 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.
   2. 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's protocol for the competent cells chosen.
   3. 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 µg/ml.
   4. Place the culture dish at 37 °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.

3. OMV Production

1. Colony Expansion
   1. 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.
   2. 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.
   3. Maintain the culture at 37 °C shaking at 250 rpm to ensure that sufficient aeration of the culture is attained.
2. Induction of Each Plasmid
   1. 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.
   2. 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.
   3. Make a 20% stock solution of L-arabinose in water and sterile filter using a 0.22 µm filter. Store L-arabinose solution at 4 °C for 2-4 weeks.
   4. 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.
   5. Make a 1 M Isopropyl β-D-1-thiogalactopyranoside (IPTG) solution in water and sterile filter using a 0.22 µm 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.
   6. Add IPTG after an additional 3 hr incubation period to a final concentration of 0.5 mM to initiate production of the OmpA-ST.
   7. Allow the culture to grow for an additional 8-14 hr at 37 °C while shaking.

4. OMV Purification

1. Removal of Intact Bacteria
   1. Centrifuge the bacterial culture media for 15 min at 7,000 x g at 4 °C to pellet intact bacteria.
   2. Decant, and save, the media off the top of the cell pellet post centrifugation being careful to not disturb the cell pellet.
   3. 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.
2. Removal of Protein Aggregates
   1. Further purify the bacterial culture media using a 0.45 µm 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.
      1. To syringe filter, draw culture medium into sterile 10-50 ml syringe. Attach 0.45 µm filter to Luer end of syringe. Depress plunger and collect flow-through in a new vessel.
      2. 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.
3. Isolation of OMV via Ultracentrifugation
   1. Add the filtered culture media into centrifugation tubes being careful to balance opposing tubes in the centrifuge.
   2. Pellet at ~150,000 x g for 3 hr at 4 °C in an ultracentrifuge using the corresponding rotor matching both the instrument and tubes being utilized.
   3. 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.
   4. Save the centrifuged supernatant for later dynamic light scattering (DLS) testing to verify that the OMV particles were fully depleted from the media.
   5. 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.
   6. Carefully pipet the purified OMV solution from the centrifuge tube, mixing gently to ensure the entire pellet has been solubilized.

5. OMV Characterization

1. Use any commercially available DLS or nanoparticle tracking software following the provided manufacturer'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.
   1. Open the software.
   2. Turn on microscope and clean all surfaces on the microscope and nanoparticle tracking unit.
   3. 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.
   4. Place the nanoparticle tracking unit on the microscope stage and turn on the laser.
   5. Click "Capture" in the software.
   6. Adjust the microscope focus and stage to visualize the particles on the preview screen present in the software window.
   7. Adjust the "Camera Shutter" and "Camera Gain" until bright particles are clearly visualized on a dark background.
   8. Adjust capture duration to 60 sec.
   9. Click "Record".
   10. 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.
   11. Keep all analysis parameters (Detection Threshold, Blur, Min Track Length, Min Expected Particle Size) on auto detect mode and click "Process Sequence."
       NOTE: Consult the manual for how each parameter can be adjusted independently to fine tune analysis results.
   12. 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.
2. Determining OMV Protein Content
   1. 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.

6. Verification of Enzyme Packaging

1. PTE Activity Assay
   1. Perform all enzymatic assays in a suitable buffer such as 100 mM CHES buffer (pH 8) at 25 °C in either a cuvette or multiplexed in a 96/384 well plate.
   2. Add 5 µl of a 1:1,000 dilution of paraoxon in CHES buffer to 90 µl of CHES and 5 µl 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.
   3. Take absorbance readings at regular intervals (every 20 sec for ~2 hr total reaction time) at 405 nm (ε = 18.1 mM^-1cm^-1) and at 348 nm (isosbestic point ε = 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.
   4. 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.
   5. Perform standard enzymatic assay protocols to determine K_M, V_max and k_cat for the OMV encapsulated PTE.
      NOTE: A Lineweaver-Burk analysis can be utilized for determination of these kinetic parameters where the y-intercept = 1/V_max and slope = K_M/V_max²⁰.
2. Transmembrane Substrate/Product Diffusion.
   1. 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.
   2. 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.
   3. 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.

7. OMV Storage

1. Freezing
   1. Place ~100 µl 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.
   2. Store the snap frozen aliquots at -80 °C.
   3. Thaw frozen aliquots from step 7.1.1 by placing them at RT being sure not to heat the samples.
   4. 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 °C if a reduction in enzyme activity is observed due to freezing.
2. Lyophilization
   1. Lyophilize the snap frozen purified OMV aliquots utilizing commercially available lyophilization equipment²¹.
   2. Store lyophilized aliquots at RT for weeks or at -80 °C for many months.
   3. 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.
   4. Add lyophilized OMV powder directly to point-of-care for applications where prior dilution/solubilization is not necessary.

Results

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 ...

Discussion

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...

Materials

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