Optimised production and analysis of recombinant protein-filled vesicles from E. coli.

This protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulphide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to many areas of scientific research including applied biotechnology and medicine.

This innovative system, using a short peptide tag, that exports diverse recombinant proteins in membrane bound vesicles from E. coli 1 , provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that enables production of otherwise challenging insoluble, toxic, or disulphide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture media and allows long-term storage of active protein. This technology results in high yields of vesicle-packaged, functional proteins for efficient downstream processing for a wide range of applications from discovery science to applied biotechnology and medicine. In this article, and associated video, a detailed protocol of the method is provided, and highlights key steps in the methodology to maximise recombinant protein filled vesicle production.