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The overall goal of this procedure is to purify and to characterize enzyme-filled bacterial outer membrane vesicles for use as in vitro catalytic reagents. This enzyme isolation technique allows bacterial OMVs to be harvested from culture media and then employed to conduct cell-free catalysis of an organophosphate compound. Directed packaging within bacterial OMVs significantly improves enzyme stability over a range of reaction and storage conditions unlike traditional techniques that often result in a gradual and continuous loss of enzyme activity.

The implications of this technique extend toward the packaging of proteins with relevance in multiple fields, such as the development of therapeutics, remediation, or the development of cell-free catalytic systems. While this protocol details purification of OMVs from E.coli, the techniques with modification to the packaging strategy could readily be used for OMV purification from a variety of microbial cell cultures. After generating outer membrane vesicles or OMVs in bacteria according to the text protocol, centrifuge the intact bacterial culture medium at 7, 000 times G and four degrees Celsius for 15 minutes.

Remove and save the supernatant, taking care not to disturb the cell pellet. Then, put the supernatant through a second spin to ensure that all bacteria are removed from the medium. To remove protein aggregates and large cellular material from the saved culture medium, draw the solution into a sterile 10 to 50 milliliter syringe.

Attach a 0.45 micron filter to lure the end of the syringe. Then depress the plunger and collect the flow-through in a new tube. Alternatively, vacuum-filter the culture medium by transferring it to a sterile filtration apparatus.

Attach the unit to a vacuum pump or sink aspirator to generate suction. Transfer the filtered medium to a new vessel. To isolate the OMVs, transfer the filtered culture medium into ultracentrifugation tubes.

Spin the medium at approximately 150, 000 times G and four degrees Celsius for three hours using a rotor that corresponds to the tubes being utilized. Immediately following ultracentrifugation to avoid loss of pellet mass, decant the OMV-depleted culture medium from the slightly brown OMV pellet which may or may not be visible by eye. The OMV pellet formed post ultracentrifugation may only be loosely adhered to the centrifugation vesicle.

Care should be taken when decanting to ensure the pellet is not dislodged and lost. Save the supernatant for later dynamic light scattering to verify that the OMV particles were fully depleted from the medium. Add 0.5 to one milliliter of sterile filtered PBS, PH 7.4 or other filtered buffer as determined by experimental needs or conditions, to the pellet and allow it to incubate at room temperature for 30 minutes.

Then carefully pipette the purified OMV solution mixing gently to ensure the entire pellet has been solubilized. To determine the OMV size distribution, open the DLS nano-particle tracking software for the instrument being used. Turn on the microscope and clean all the surfaces on the microscope and nano-particle tracking unit.

Using a one milliliter sterile syringe, add approximately 0.5 milliliters of OMV sample directly to the viewing window of the particle tracking device through the lower fitting, taking care to cover the entire viewing window without injecting any air bubbles into the system. Place the nano-particle tracking unit on the microscope stage and turn on the laser. Then click Capture in the software.

Adjust the microscope focus and stage to visualize the particles on the preview screen present in the software window. Adjust the camera shutter and camera gain until bright particles are clearly visualized on a dark background. Adjust the capture duration to 60 seconds, then click Record.

When prompted, input the sample device temperature, label the sample, and save the video data at the completion of each run into a user-designated folder. Keep all analysis parameters on auto-detect mode and click Process Sequence. Record particle size distribution and concentration as particles per milliliter as determined by the software.

To determine OMV protein content, use SDS-PAGE and Western Blots to asses OMV purity enzyme production and SpyCatcher or SC to SpyTag or ST cross-link efficiency. It is imperative to confirm the expression of the target enzymes and anchor protein. Expression of recombinant proteins can often be unsuccessful for a variety of reasons and successful protein production should be confirmed before OMV characterization.

To carry out a phosphotriesterase or PTE activity assay, add five microliters of a one to 1, 000 dilution of paraoxon in CHES buffer to five microliters of purified OMVs in 90 microliters of CHES. Take absorbance readings at 405 nanometers and 348 nanometers every 20 seconds for approximately two hours of total reaction time to monitor the progression of the chromogenic paraoxon breakdown product p-nitrophenol. Finally, carry out data analysis according to the text protocol.

Shown here are examples of the morphology of purified OMVs from E.coli via SEM. OMVs range in size from 50 to 250 nanometers as determined using particle tracking instrumentation. In this graph, the absolute OMV concentration is shown.

When N terminal OMP-A-ST was co-transformed with PTE-SC in the presence of arabinose and IPTG, a significant increase in OMV production was observed compared to the control samples. The SDS-PAGE and Western Blots seen here further characterize the OMV protein content and recombinant protein expression for OMP-A-ST, native OMP-A, PTE-SC, and OMP-A-ST-PTE-SC. In this figure, overall PTE expression levels and OMV packaging efficiency in the cell pellets, UC supernatant, and purified UC OMV pellets were determined by initial velocity measurements using paraoxon as a chromogenic substrate.

Here, Triton X-100 was used to verify translocation of paraoxon across intact OMV bilayers. There was little activity difference with or without detergent, indicating that paraoxon passes freely through endogenous pores on the OMV. Finally, as seen in this experiment, PTE-SC kinetic data is fit to a standard Michaelis-Menten enzyme kinetics equation for N terminal OMP-A-ST co-transformed with PTE-SC in the presence of a arabinose and IPTG and PTE-SC in the presence of arabinose activation.

Once all genetic constructs have been made and confirmed, the enzyme packaging and OMV purification can be accomplished over a two day period. A third day is necessary to confirm and characterize OMVs whenever a new construct is developed. While attempting this procedure, it is important to remember to verify safety ratings of all materials and centrifugation equipment.

Additionally, it is important to verify the success of each step before proceeding to the next. The characterization steps described here can be supplemented with others to confirm enzyme packaging and OMV morphology. Protein concentration, lipid content, LPS concentration, and many other properties can vary with different payloads and adduction conditions and may influence downstream applications.

After watching this video, you should have a good understanding of how to purify and characterize enzyme-filled OMVs as well as conduct preliminary assays to assess the activity of the enzyme cargo. Don't forget that working with bacteria and chemical reagents can be extremely hazardous and precautions such as proper aseptic technique and personal protective equipment should always be used while performing this procedure.