Isolation, Purification, and Characterization of Membrane Vesicles from Haloarchaea

Membrane vesicles (MVs), also described as extracellular vesicles (EVs), exosomes, or outer membrane vesicles (OMVs), are nano-sized (10-300 nm) spherical, membrane-bound structures deriving from the cell envelope. MVs have been studied extensively in both eukaryotic and prokaryotic systems, revealing a plethora of unique functions including cell-to-cell communication and protection of the cell. They are able to encapsulate specific cargos from nucleic acids to proteins, thereby concentrating cargo and providing protection from the extracellular environment. While MV production has been identified for all domains of life, with extensive investigation particularly for Bacteria and Eukaryota, it has only been studied in a few members of the archaeal domain, leaving a void of information concerning the role of MVs for the majority of Archaea. In addition, several discrepancies exist in the process of MV preparation and analysis between studies of MV production in different archaeal organisms. To further encourage the investigation of MVs in Archaea among the scientific community, we present a standardized method for the isolation, purification, and characterization of MVs based on the archaeal model organism, Haloferax volcanii. However, the described protocol can be applied to other Archaea with the appropriate modifications that are highlighted in Subheading 4.