Materials and Reagents

0.22 µm vacuum filtration unit (such as Steritop Quick Release, EMD Millipore, catalog number: S2GPT05RE )
0.45 µm vacuum filtration unit (such as Stericup-HV, EMD Millipore, catalog number: SCHVU11RE )
≥ 50 ml syringe (such as Fisherbrand Sterile Syringes, Thermo Fisher Scientific, catalog number: 15899152 )
0.45 µm syringe filter (such as Millex-HP, EMD Millipore, catalog number: SLHP033R )
Microscale NMR tubes (such as 5mm Shigemi Tube Set, Shigemi, catalog number: BMS-005B )
Tryptone (Sigma-Aldrich, catalog number: T9410 )
Yeast Extract (Sigma-Aldrich, catalog number: Y1625 )
NaCl (Sigma-Aldrich, catalog number: S7653 )
Na₂HPO₄ (Sigma-Aldrich, catalog number: S7907 )
KH₂PO₄ (Sigma-Aldrich, catalog number: P0662 )
KCl (Sigma-Aldrich, catalog number: P9333 )
CaCl₂·2H₂O (Sigma-Aldrich, catalog number: C5080 )
MgCl₂·6H₂O (Sigma-Aldrich, catalog number: M2670 )
¹⁵NH₄Cl (Sigma-Aldrich, catalog number: 299251 )
D₂O (Sigma-Aldrich, catalog number: 617385 )
D-Glucose (Sigma-Aldrich, catalog number: G7528 )
MgSO₄ (Sigma-Aldrich, catalog number: M7506 )
Thiamine (Sigma-Aldrich, catalog number: T1270 )
Isopropyl-β-D-thiogalactoside (IPTG; Sigma-Aldrich, catalog number: I6758 )
LB-medium (Lennox) (see Recipes)
M9 medium (see Recipes)
Dulbeccos phosphate buffered saline with added salts (DPBSS)/NMR sample buffer (see Recipes)

Equipment

Shaking incubator (such as New Brunswick Innova^® 42, Eppendorf AG, catalog number: M1335-0012 )
500 ml baffled culture flask (such as ROTILABO baffled flasks, Carl Roth, catalog number: LY96.1 )
5,000 ml baffled culture flask (such as ROTILABO baffled flasks, Carl Roth, catalog number: PK29.1 )
Cell density meter (such as GE Healthcare Ultrospec, Thermo Fisher Scientific, catalog number: 10704417 )
High speed centrifuge (such as Avanti J-series, Beckman Coulter, catalog number: B22989 )
High speed large-volume fixed angle rotor (such as J-LITE JLA-16.250, Beckman Coulter, catalog number: 363934) equipped with suitable centrifugation bottles (such as 250 ml Polycarbonate Bottle, Beckman Coulter, catalog number: 356013)
High speed small-volume fixed angle rotor (such as JA-25.50, Beckman Coulter, catalog number: 363055) equipped with suitable centrifugation tubes (such as 50 ml Polycarbonate Bottle, Beckman Coulter, catalog number: 357000)
Vacuum pump, sufficiently strong for use with vacuum filtration units (ultimate vacuum ~0.01 mPa)
Vortex mixer (such as Vortex-Genie 2, Scientific Industries, catalog number: SI-0246)
Microvolume spectrophotometer (such as Nanodrop One, Thermo Fisher Scientific, ND-ONE-W)
Gel electrophoresis system for SDS page (such as Mini-PROTEAN Tetra, Bio-Rad, catalog number: 1658004) including power supply, polyacrylamide gels, and necessary buffers and staining solutions (refer to manufacturers specifications for details)

Software

Any spreadsheed or scientific graphing software allowing basic calculations (Excel, Numbers, LabPlot, Origin, etc.)

Procedure

Gene cloning and transformation
The complete gene coding for the protein of interest including its preceding periplasmic export signal should be cloned into a suitable plasmid backbone of the pET expression system (such as pET21a). Thereby, additional features such as affinity tags are not necessary and should be omitted. The final plasmid should be transformed into an OMV-overproducing strain such as BL21(DE3)ompA (Fantappiè et al., 2014). These steps can be carried out following standard molecular biology techniques. The description of detailed cloning and transformation procedures lies outside the scope of this protocol.
Preparation of growth media and buffers
Per sample requirements are: 5 ml LB medium, 1 L M9 medium, 25 ml DPBSS, 0.2 ml NMR sample buffer (see Recipes for formulations). Supplement growth media with appropriate antibiotics for selection of the strain/plasmid used for expression. When using the pET expression system, use Ampicillin to a final concentration of 100 µg ml^-1 or Kanamycin to a final concentration of 50 µg ml^-1. Stock solutions of antibiotics (Ampicillin: 100 mg ml^-1; Kanamycin: 50 mg ml^-1) and IPTG (1 M) can be prepared in H₂O/D₂O, corresponding to the solvent used for growth media.
Bacterial growth
1. Inoculate 5 ml LB medium with a single colony.
2. Grow shaking at 37 °C for 10 h.
3. Inoculate 100 ml M9 in a 500 ml baffled culture flask using 1 ml of the culture grown in LB medium.
4. Grow shaking at 37 °C overnight (~80 rpm, 25 mm orbit).
5. Add the entire preculture to 900 ml M9 in a 5,000 ml baffled culture flask.
6. Grow shaking at 37 °C (~80 rpm, 25 mm orbit).
7. Check the optical density OD₆₀₀every 30 min.
8. When the optical density reaches OD₆₀₀ ~0.4 add IPTG to a final concentration of 0.2 mM to induce protein expression.
9. Continue to monitor the optical density OD₆₀₀every 30 min.
10. When bacterial growth ceases (after ~3 h in H₂O/~5 h in D₂O based media, see Figure 1) remove the cells by centrifugation at 10,000 x g for 10 min at 10 °C.
11. Filter the supernatant with 0.45 µm vacuum filtration units. The filtered supernatant can be stored at 4 °C for up to 24 h.
  
  Figure 1. Media-dependent growth behavior. Exemplary growth curves in H₂O based (dark blue) and D₂O based (light blue) M9 minimal media. Triangles mark the time of induction, squares mark the time of harvest.
OMV Preparation
1. Collect OMVs from the filtered supernatant by centrifugation at 38,400 x g for 2 h at 10 °C. After centrifugation, centrifuge bottles should contain a small but clearly visible whitish, slightly cloudy pellet (Figure 2A).
2. Immediately discard the supernatant (OMV pellets will begin to suspend shortly).
3. Resuspend OMV pellets in a total volume of 25 ml DPBSS by pipetting.
4. Filter the OMV suspension with a 0.45 µm syringe filter.
5. Collect OMVs by centrifugation using a single centrifuge tube at 74,500 x g for 45 min at 10 °C (Figure 2B).
6. Immediately discard the supernatant.
7. Resuspend the OMV pellet in 200 µl NMR sample buffer by gentle vortexing.
8. Save 2 µl of the OMV suspension for subsequent analysis and transfer the remaining suspension to a microscale NMR tube (Figure 2C). OMV suspensions are in our hands stable at 4 °C for up to 2 weeks.
  
  Figure 2. Preparation of OMVs. OMV pellets after the first (A) and second centrifugation (B), and resuspended in NMR buffer in a 5 mm Shigemi NMR tube (C).
OMV Analysis
1. Dilute 2 µl of the final OMV suspension with 18 µl DPBSS.
2. Record a UV-Vis spectrum of the dilute OMV suspension using a microvolume spectrophotometer (range: 220-360 nm) and export the spectrum data for further analysis.
3. Correct the exported spectrum for the light-scattering contribution of the OMVs and estimate the protein concentration from the absorbance at 280 nm (see Data analysis and Figure 3A). OMV preparations should contain >15 mg/ml total protein to yield NMR spectra of good quality.
4. Prepare two samples for SDS page using denaturing Laemmli Sample Buffer, containing ~6 µg total protein each.
5. Heat-denature one of the samples at 98 °C for 15 min, keep the second sample at room temperature.
6. Analyze samples by SDS page. Samples of protein-enriched OMVs should result in two prominent bands. One band corresponding to the major porins OmpF/OmpC present in the OMV membrane runs at 37 kDa in the heat-denatured sample and results in a contorted, ladder-like pattern between 60 and 70 kDa in the non-heated sample (Figure 3B). A second band, usually not subject to heat-modifiability, should be present at the weight of the overexpressed protein of choice, indicating successful enrichment in the OMVs (Figure 3B).
  
  Figure 3. Analysis of OMVs by spectrophotometry and SDS-page. A. Exemplary UV-Vis spectrum of a dilute OMV suspension before (dashed line) and after light-scattering correction (solid line). B. Exemplary SDS page gel of OMVs enriched with a periplasmic protein (dark blue triangle). Major porins OmpF/OmpC are marked by the light blue open triangles.

Data analysis

The spectrophotometric estimation of the protein concentration in a sample based on absorbance measurement at a wavelength of 280 nm is a simple, fast, and routinely used method. However, UV-Vis spectra recorded of OMVs contain a strong light-scattering component. In order to estimate the protein concentration within an OMV sample， light-scattering due to the vesicles needs to be corrected. A light-scattering corrected UV-Vis spectrum can be obtained using the following equation, originally used to estimate the nucleic acid content of viruses (Porterfield and Zlotnick, 2010):

The total protein concentration in the OMV sample can be estimated from the corrected value of A₂₈₀, assuming 1 Abs = 1 mg ml^-1 for the mixture of proteins present in OMVs. Due to the presence of nucleic acids in OMVs (Brown et al., 2015), which strongly influences the absorbance at 280 nm, it is useful to estimate the amount of nucleic acids in the sample from the A₂₆₀/A₂₈₀ ratio of the light-scattering corrected spectrum, which can be done using the following empirical equation (Glasel, 1995):

Typically, protein-enriched OMVs contain ≤ 3% nucleic acids. If nucleic acids are detected ([%N] > 0)， the total protein concentration in the OMV sample can be estimated from the corrected values of A₂₈₀ and A₂₆₀, using the following equation (Layne, 1957):

Notes

Typically a sufficient amount of enriched OMVs can be obtained from a growth culture volume of one liter using the culture conditions described above. Nevertheless, it should be noted that bacterial growth in D₂O based media can be less reproducible than in H₂O based media, occasionally resulting in irregular growth behavior and/or lower yields. Moreover, it should be noted that the overexpression of proteins in the periplasmic space can result in cytotoxicity (Schlegel et al., 2013), which might require specific adaption of the induction levels to the expression system used.

Recipes

LB-medium (Lennox)
10 g/L Tryptone
5 g/L Yeast extract
5 g/L NaCl
Dissolve in H₂O and sterilize by autoclaving
M9 medium
6 g/L Na₂HPO₄
3 g/L KH₂PO₄
1 g/L ¹⁵NH₄Cl
0.5 g/L NaCl
2 g/L D-Glucose
1 ml/L CaCl₂ (0.1 M) solution*
1 ml/L MgSO₄ (1 M) solution*
0.1 ml/L Thiamine (10 mg/ml) solution*
Dissolve solid components in H₂O/D₂O, add salt/Thiamine solutions and sterilize by filtration using 0.22 µm vacuum filtration units.
*Note: Prepare stock solutions in H₂O/D₂O, respectively.
Dulbeccos phosphate buffered saline with added salts (DPBSS)/NMR sample buffer:
1.15 g/L Na₂HPO₄
0.2 g/L KH₂PO₄
8 g/L NaCl
0.2 g/L KCl
0.45 ml/L CaCl₂ (1 M) solution
0.25 ml/L MgCl₂ (1 M) solution
Dissolve solid components in H₂O*, add salt solutions and sterilize by filtration using 0.22 µm vacuum filtration units.
*Note: For NMR sample buffer, dissolve in H₂O to 90% final volume, then add 10% D₂O.

Acknowledgments

We thank G. Grandi (University of Trento) for providing E. coli strain BL21(DE)ΔompA. J.T. was supported by an EMBO Long-Term Fellowship (ALTF 413-2018). B.M.B. gratefully acknowledges funding from the Swedish Research Council (Vetenskapsrådet Starting Grant 2016-04721) and the Knut och Alice Wallenberg Foundation through a Wallenberg Academy Fellowship (2016.0163) as well as through the Wallenberg Centre for Molecular and Translational Medicine, University of Gothenburg, Sweden.

This protocol was originally described in Thoma and Burmann (2020).

Competing interests

The authors declare no competing interest.

References

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