Co-purification of the GroEL chaperone during outer membrane vesicle purification: insights from Aeromonas salmonicida subsp. salmonicida.

Abstract

Outer membrane vesicles (OMVs) are naturally produced by Gram-negative bacteria and originate from their outer membrane. They can be extracted using ultracentrifugation or ultrafiltration using concentration columns, followed by purification with a density gradient. However, these methods may co-purify contaminants with similar physical properties. Several studies have identified GroEL, a chaperonin, as a major protein in OMV preparations. Using Aeromonas salmonicida subsp. salmonicida as a model, we detected GroEL by mass spectrometry and observed it in transmission electron microscopy images as separate from OMVs. As a cytoplasmic protein complex, GroEL is more likely a contaminant resulting from bacterial lysis during growth rather than an intrinsic OMV component. The model A. salmonicida subsp. salmonicida proved valuable in reaching this conclusion because it produces high levels of extracellular GroEL and low amounts of OMVs. This study emphasizes the need for caution when interpreting the presence of GroEL in OMV preparations and highlights the importance of rigorous purification methods to ensure OMV purity.