Preparation of Bacterial Outer Membrane Vesicles for Characterisation of Periplasmic Proteins in Their Native...

Bacterial outer membrane vesicles (OMVs) are naturally formed by budding from the outer membrane of Gram-negative bacteria. OMVs consist of a lipid bilayer identical in composition to the original outer membrane and contain periplasmic content within their lumen. Enriched with specific envelope proteins, OMVs make for an excellent native-like platform to study these proteins in-situ using biophysical methods. Here, we describe in detail the preparation of OMVs from Escherichia coli, which are luminally enriched with periplasmic proteins and uniformly labeled with stable isotopes (²H and ¹⁵N), suitable for the subsequent characterisation of proteins at atomic resolution in their native environment by solution-state NMR spectroscopy. The ability to perform structural studies of periplasmic components in-situ clears the way to reaching an in-depth understanding of the functional and mechanistic details of this unique cellular compartment.