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

Streather, Bree, Baker, Karen, Eastwood, Tara, Mulvihill, Daniel P. (2023) Optimised production and analysis of recombinant protein-filled vesicles from E. coli. Journal of Visualized Experiments, (196). ISSN 1940-087X. (doi:10.3791/65442) (KAR id:101129)


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.

Item Type: Article
DOI/Identification number: 10.3791/65442
Additional information: For the purpose of open access, the author(s) has applied a Creative Commons Attribution (CC BY) licence to any Author Accepted Manuscript version arising.
Uncontrolled keywords: Recombinant Proteins - metabolism, Peptides - chemistry, Proteomics, Escherichia coli - genetics - metabolism, Bacterial Proteins - metabolism, Biotechnology - methods
Subjects: Q Science > QH Natural history > QH581.2 Cell Biology
Q Science > QR Microbiology
Divisions: Divisions > Division of Natural Sciences > Biosciences
Funders: Biotechnology and Biological Sciences Research Council (
Depositing User: Daniel Mulvihill
Date Deposited: 02 May 2023 15:46 UTC
Last Modified: 10 Jan 2024 13:42 UTC
Resource URI: (The current URI for this page, for reference purposes)

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